ANALYTE SENSORS AND METHODS OF MANUFACTURING ANALYTE SENSORS

A method of manufacturing laminate structure including the steps of providing a waveguide structure having a plurality of waveguide cores and including a first surface, creating an oxygen sensing polymer cavity in the first surface of the waveguide structure to receive an oxygen sensing polymer, filling the oxygen sensing polymer cavity with the oxygen sensing polymer and curing the oxygen sensing polymer, adding a first layer material on top of the first surface of the waveguide structure, where the first layer material includes a reaction chamber cavity that is contiguous with the oxygen sensing polymer, filling the reaction chamber cavity with an enzymatic hydrogel and curing the enzymatic hydrogel; adding a second layer material on top of the first layer material, where the second layer material includes a conduit cavity to receive a conduit hydrogel, filling the conduit cavity with a conduit hydrogel and curing the conduit hydrogel, and adding a top cap on top of the second layer of material.

BACKGROUND

Field

The disclosed and described technology relates generally to optical enzymatic analyte sensors, such as, for example, glucose sensors, using waveguides with separate emission and excitation paths to a target material and methods of manufacturing such optical enzymatic analyte sensors.

Description of the Related Technology

Diabetes is a disease of insufficient blood glucose regulation. In non-diabetic people, the body's beta cells monitor glucose and deliver just the right amount of insulin on, for example, a minute-by-minute basis for tissues in the body to uptake the right amount of glucose, keeping blood glucose at healthy levels. In diabetic patients, this regulation primarily fails due to: 1) insufficient insulin production and secretion, and/or 2) a lack of normal sensitivity to insulin by the tissues of the body. Glucose sensors can be used to monitor glucose levels in diabetic patients allowing proper dosing of diabetic treatments, including, for example, insulin.

More generally, analyte tracking and monitoring enable improved monitoring, diagnosis, and treatment of diseases, including diabetes. Existing methods to measure, monitor, and track analyte levels, may include sampling a bodily fluid, preparing the sample for measurement, and estimating the analyte level in the sample. For example, a diabetic may prick a finger to obtain a blood sample to measure glucose in a glucose monitoring unit. Such existing methods may be painful, unpleasant or inconvenient for the patient, resulting in lower compliance with physician orders to, for example, take glucose readings at certain times each day or based on patient activity. Moreover, effective monitoring, diagnosis, and treatment may benefit from fusing multiple sensor readings that measure different aspects of a patient's state.

Readings from one or more analyte sensors, as well as other bio sensor systems and/or activity sensors may be combined with past readings to determine results that characterize a patient's state, and may be used to monitor, diagnose, and treat a patient. For example, an alarm may be triggered if a patient's glucose level exceeds a threshold.

Accordingly, there is a need for analyte sensors (1) that do not require unpleasant blood draws or sample preparation if measurements are to be taken multiple times each day, (2) to be sufficiently selective, sensitive, and to provide repeatable and reproducible measurements, and (3) that are stable with low drift. There is also a need for controllers that may interrogate sensors based on protocols that define sampling timing, duration, and frequency.

Moreover, there is a need for analysis engines or tools (1) to analyze raw sensor readings and determine various results including, for example, sensor readings including analyte levels, trends, and alarms, (2) to incorporate past readings and patient history from a knowledge base, (3) to incorporate patient activity data, so that sensor readings may be correlated with and analyzed based on activities, enabling, for example, alarm conditions that vary with patient activity levels, and (4) to incorporate and fuse data from other bio sensors, which measure other aspects of a patient's condition.

Additionally, there is a need for analysis engines to (1) receive and accept orders and instructions from a physician, via a network, so that the orders and instructions may be converted to protocols that set sensor operating parameters and reading requirements (for example, a protocol to a controller that increases frequency or reduces the duration of a reading), (2) accept queries from physicians over a network, or from patients over a portable computing device (for example, a smart phone), for results of data that has been taken, or to modify a protocol, and (3) transmit results to a physician, as well as to a patient or caretaker.

Analyte sensors, such as glucose sensors, can produce a digital electronic signal that depends on the concentration of a specific chemical or set of chemicals (analyte) in bodily fluid or tissue. The sensor usually includes two main components, (1) a chemical or biological part that reacts or complexes with the analyte in question to form new chemical or biological products or changes in energy that can be detected by means of the second component and (2) a transducer. The first component (chemical or biological) can be said to act as a receptor/indicator for the analyte. For the second component, a variety of transduction methods can be used including, for example, electrochemical (such as potentiometric, amperometric, conductimetric, impedimetric), optical, calorimetric, and acoustic. After transduction, the signal is usually converted to an electronic digital signal that corresponds to a concentration of a particular analyte. Example analytes that can be measured using the embodiments of the inventions disclosed and escribed herein include, and are not limited to, glucose, galactose, fructose, lactose, peroxide, cholesterol, amino acids, alcohol, lactic acid, and mixtures of the foregoing.

The disclosed technology integrates an innovative analyte sensor, controlled by a controller, with an analysis engine that incorporates historical data and protocols from a knowledge base, bio sensor data from a biological sensor system, and activity data from an activity sensor system/database to generate results for measuring, monitoring and diagnosing a patient. The disclosed technology details embodiments of a laminate optical analyte sensor, methods for manufacturing the sensor, systems and methods for inserting it, and systems and methods for adhering a medical device on the skin of a patient, such as a controller in communications with the sensor.

SUMMARY

Methods and apparatuses or devices being disclosed herein each have several aspects, no single one of which is solely responsible for its desirable attributes. Without limiting the scope of this disclosure, for example, as expressed by the claims which follow, its more prominent features will now be discussed briefly. After considering this discussion, and particularly after reading the section entitled “Detailed Description,” one will understand how the features being disclosed and described provide advantages that include monitoring, diagnosing, and treating a patient using results obtained from an analyte sensor.

In various embodiments described herein pertain to continuous analyte monitors, components thereof, and methods of making the same. In some embodiments, methods of preparing a component layers for a sensor tip for a analyte monitoring device are described. In some embodiments, the methods pertain to fabricating a sensor tip that is small enough to be inserted subcutaneously into a patient. In some embodiments, the sensor tip comprises an oxygen conduit, an enzymatic layer, and a sensing layer.

Certain embodiments described herein pertain to continuous glucose monitors, components thereof, and methods of making the same. In some embodiments, methods of preparing a component layers for a sensor tip for a glucose monitoring device are described. In some embodiments, the methods pertain to fabricating a sensor tip that is small enough to be inserted subcutaneously into a patient. In some embodiments, the sensor tip comprises an oxygen conduit, an enzymatic layer, and a sensing layer.

Embodiments of adispensable, curable reversible oxygen binding molecule-albumin nanogel solution, configured to form a hydrogel upon curing are disclosed. The dispensable, thermal curable reversible oxygen binding molecule-albumin nanogel comprises a reversible oxygen binding molecule albumin nanoparticles, wherein the reversible oxygen binding molecule and albumin are interconnected with a bifunctional linkers, wherein the reversible oxygen binding molecule albumin nanoparticles are coupled to poly(ethylene glycol) (PEG) through a thio-linkage, and wherein the reversible oxygen binding molecule albumin nanoparticles are functionalized to a nanogel matrix via a PEG-based linker.

In addition certain embodiments are directed to a method of making a dispensable, curable reversible oxygen binding molecule-albumin nanogel solution, where the method comprises covalently linking reversible oxygen binding molecule to albumin by incubation with a bifunctional linker to form reversible oxygen binding molecule albumin nanoparticles of Formula (I);

thiolating the reversible oxygen binding molecule albumin nanoparticles with a thiolating agent; conjugating the thiolated reversible oxygen binding molecule albumin nanoparticles using maleimide poly(ethylyene glycol)-methacrylate (PEG-MA); and crosslinking the pegylated reversible oxygen binding molecule albumin nanoparticles with a first diacrylate crosslinker to form the dispensable, thermal-curable reversible oxygen binding molecule-albumin nanogel solution.

Embodiments are disclosed for a crosslinked reversible oxygen binding molecule-based material that comprises a hydrogel matrix and a reversible oxygen binding molecule-albumin nanoparticle having an albumin molecule covalently linked to at least one reversible oxygen binding molecule molecule by a bifunctional linker of Formula (I), wherein the reversible oxygen binding molecule-albumin nanoparticle is PEGylated; and wherein the reversible oxygen binding molecule-albumin nanoparticle is functionalized to the hydrogel matrix via a PEG-based linker.

Methods are disclosed for making a dispensable, curable enzyme-albumin nanogel solution, where the method comprises covalently linking an enzyme to albumin by incubation of the enzyme with albumin and a bifunctional linker to form an enzyme albumin nanoparticle of Formula (IV));

thiolating the enzyme albumin nanoparticle with a thiolating agent to form a thiolated enzyme albumin nanoparticle of Formula (V);

conjugating the thiolated enzyme albumin nanoparticle to poly(ethylene glycol) methacrylate to form a pegylated enzyme-albumin nanoparticle of Formula (VI);

thiolating the reversible oxygen binding molecule albumin nanoparticles with a thiolating agent; conjugating the thiolated reversible oxygen binding molecule albumin nanoparticles to poly(ethylene glycol) methacrylate to form a pegylated glucose oxidase-albumin nanoparticle; mixing the pegylated enzyme-albumin nanoparticle, and a first diacrylate to form a pre-nanogel solution; crosslinking the pre-nanogel solution to form a crosslinked enzymatic nanogel; and adding the crosslinked enzymatic nanogel to a solution to form the dispensable, thermal-curable enzyme-albumin nanogel solution wherein the bifunctional liker (L) is a homobifunctional linker, a heterobifunctional linker, or a direct connection between enzyme and albumin; i is selected from the group consisting of —C(O)(CH2)p-and N═CH(CH2)p-, wherein p is an integer ranging from 1 to 10; j is —(CH2—)q-, wherein q is an integer ranging from 1 to 10; n is an integer ranging from 1 to 1000; and R6is selected from the group consisting of —C1-4alkyl and H; wherein the enzyme is selected from a group consisting of glucose oxidase, glutamate oxidase, alcohol oxidase, lactate oxidase, ascorbate oxidase, cholesterol oxidase, choline oxidase, laccase, and tyrosinase.

Methods are also provided for making a dispensible, curable enzymatic nanogel solution, where the method comprises complexing enzyme and CAT to albumin by incubation with bifunctional linker, at low temperature, low oxygen concentration, pH of between about 7.0 and 8.0, for at least about 24 hours to form enzymatic nanoparticles; adding sulfhydryl groups to the nanoparticles to form thiolated enzymatic nanoparticles; conjugating the thiolated enzymatic nanoparticles with poly(ethylyene glycol)-methacrylate (PEG-MA) to form pegylated enzymatic nanoparticles; and crosslinking the pegylated enzymatic nanoparticles with methacrylate hydrogel monomers to form the dispensable, thermal-curable enzymatic nanogel solution.

Moreover, a dispensable, curable enzyme-albumin nanogel solution, configured to form a hydrogel upon thermal curing, the enzyme-albumin nanogel is disclosed. The enzyme-albumin nanogel solution comprises a nanogel matrix comprising:

wherein e is an integer ranging from 1 to 10 and R5is selected from the group consisting of —C1-4alkyl and H; a enzyme albumin nanoparticle, wherein the enzyme and albumin are interconnected with bifunctional linkers, wherein the enzyme albumin nanoparticle is coupled to poly(ethylene glycol) (PEG) through a thio-linkage, and wherein the enzyme albumin nanoparticle is functionalized to the nanogel matrix via a PEG-based linker; and enzyme albumin nanoparticles, wherein the enzyme and albumin are interconnected with bifunctional linkers, wherein the enzyme albumin nanoparticle is coupled to poly(ethylene glycol) (PEG) through a thio-linkage, and wherein the enzyme albumin nanoparticles is functionalized to the nanogel matrix via a PEG-based linker.

Also disclosed is a crosslinked, enzymatic-nanoparticle-based material, comprising a hydrogel matrix; an enzyme-functionalized albumin nanoparticle having an albumin molecule covalently linked to at least one enzyme via a bifunctional-based linker, wherein the enzyme-albumin nanoparticles are PEGylated, and wherein the enzyme-albumin nanoparticles are functionalized to a hydrogel matrix; and a reversible oxygen binding molecule-albumin nanoparticle having an albumin molecule covalently linked to at least one reversible oxygen binding molecule molecule via a bifunctional linker, wherein the reversible oxygen binding molecule-albumin nanoparticle is PEGylated, and wherein the reversible oxygen binding molecule-albumin nanoparticles are functionalized to the hydrogel matrix via a PEG-based linker.

Embodiments of a dispensible, curable oxygen-sensing mixture that comprises an oxygen detecting luminescent dye configured to reversibly bind oxygen and emit light when oxygen is bound, wherein the luminescent dye is distributed within a co-polymer matrix comprising a blend of polystyrene and polysiloxane, are disclosed.

Further disclosed is an oxygen-sensing polymer that comprises an oxygen detecting luminescent dye distributed within a polymer matrix, where the polymer matrix comprises a blend of polystyrene and polystyrene acrylonitrile distributed within a polysiloxane matrix, wherein the oxygen detecting luminescent dye is configured to reversibly bind oxygen and is configured to emit light when oxygen is bound.

Embodiments of analyte sensors are disclosed in some embodiments, the analyte sensors comprise a first layer having crosslinked reversible oxygen binding material that comprise: a first reversible oxygen binding material-albumin nanoparticle configured to transport O2and having albumin and reversible oxygen binding material interconnected by a difunctional linker,wherein the reversible oxygen binding material-albumin nanoparticles are PEGylated, wherein the reversible oxygen binding material-albumin nanoparticles are functionalized within a first hydrogel matrix; a second layer comprising: a first enzymatically-active nanoparticle and a second enzymatically-active nanoparticle and a second reversible oxygen binding material-albumin nanoparticle configured to transport O2; the first enzymatically-active nanoparticle comprising albumin interconnected to an enzyme; the second enzymatically-active nanoparticle comprising albumin interconnected to catalase (CAT); and the second reversible oxygen binding material-albumin nanoparticle comprising albumin and reversible oxygen binding material interconnected by a difunctional linker wherein the second reversible oxygen binding material-albumin nanoparticle is PEGylated, wherein the first enzymatically-active nanoparticle, the second enzymatically-active nanoparticle, and the second reversible oxygen binding material-albumin nanoparticle are functionalized within a second hydrogel matrix; and a sensing region in communication with the second layer, the sensing region comprising a luminescent dye covalently or non-covalently linked to a polymer matrix.

Additional embodiments of analyte sensors are disclosed where the analyte sensors comprise a first layer having crosslinked reversible oxygen binding material, comprising: a first reversible oxygen binding material-albumin nanoparticle configured to transport o2 and having albumin and reversible oxygen binding material interconnected by a difunctional linker, wherein the reversible oxygen binding material-albumin nanoparticles are PEGylated; wherein the reversible oxygen binding material-albumin nanoparticles are functionalized within a first hydrogel matrix; a second layer comprising: a first enzymatically-active nanoparticle and a second enzymatically-active nanoparticle and a second reversible oxygen binding material-albumin nanoparticle configured to transport O2; the first enzymatically-active nanoparticle comprising albumin interconnected to glucose oxidase (GOx); the second enzymatically-active nanoparticle comprising albumin interconnected to catalase (CAT); and the second reversible oxygen binding material-albumin nanoparticle comprising albumin and reversible oxygen binding material interconnected by a difunctional linker wherein the second reversible oxygen binding material-albumin nanoparticle is PEGylated; wherein the first enzymatically-active nanoparticle, the second enzymatically-active nanoparticle, and the second reversible oxygen binding material-albumin nanoparticle are functionalized within a second hydrogel matrix; and a sensing region in communication with the second layer, the sensing region comprising a luminescent dye covalently or non-covalently linked to a polymer matrix.

Also disclosed is an active hydrogel composition, prepared by the steps of: dispersing a nanogel in a liquid medium, the nanogel comprising a nanostructure covalently linked to a macromer and conjugated to a polymer network; adding a crosslinker to the nanogel dispersed in the liquid medium; and performing a crosslinking step to form the active hydrogel composition.

Embodiments of a glucose sensor, comprising: a first layer having crosslinked hemoglobin-based material, comprising: a first hemoglobin-albumin nanoparticle configured to transport O2 and having albumin and hemoglobin interconnected by a difunctional linker, wherein the hemoglobin-albumin nanoparticles are PEGylated; wherein the hemoglobin-albumin nanoparticles are functionalized within a first hydrogel matrix; a second layer comprising: a first enzymatically-active nanoparticle and a second enzymatically-active nanoparticle and a second hemoglobin-albumin nanoparticle configured to transport O2; the first enzymatically-active nanoparticle comprising albumin interconnected to glucose oxidase (GOx); the second enzymatically-active nanoparticle comprising albumin interconnected to catalase (CAT); and the second hemoglobin-albumin nanoparticle comprising albumin and hemoglobin interconnected by a difunctional linker wherein the second hemoglobin-albumin nanoparticle is PEGylated; wherein the first enzymatically-active nanoparticle, the second enzymatically-active nanoparticle, and the second hemoglobin-albumin nanoparticle are functionalized within a second hydrogel matrix; and a sensing region in communication with the second layer, the sensing region comprising a porphyrin dye covalently or non-covalently linked to a polymer matrix, are disclosed

Embodiments of the invention are directed to a method of manufacturing a polymer laminate thin film waveguide structure comprising the steps of: providing a first material to be embossed, wherein the first material has a first index of refraction; embossing at least one waveguide structure into the first material; filling the embossed waveguide structure with a second material having a second index of refraction; and applying a third material on top of the first material and second material, wherein the third material has a third index of refraction.

Also disclosed are methods of manufacturing a laminate structure for use in an analyte sensor comprising the steps of: constructing a waveguide laminate structure comprising the steps of: providing a waveguide first material to be embossed, wherein the waveguide first material has a first index of refraction; embossing at least one waveguide structure into the waveguide first material, wherein the at least one waveguide structure comprises four waveguide cores and wherein at least one of the waveguide cores is an oxygen reference waveguide core; filling the embossed waveguide structure with a waveguide second material having a second index of refraction; and applying a waveguide third material on top of the waveguide first material and the waveguide second material, wherein the waveguide third material has a third index of refraction. In some embodiments, the method also includes constructing a reaction chamber laminate structure comprising the steps of: providing a reaction chamber first material structure comprising a first PSA having a first PSA first liner and a first PSA second liner; cutting a first feature into the reaction chamber first material structure; providing a reaction chamber second material structure comprising a reaction chamber material and a reaction chamber material liner; removing the first PSA first liner; and attaching the reaction chamber second material to the reaction chamber first material structure forming the reaction chamber laminate structure having a thickness.

A method of manufacturing a laminate structure for use in an analyte sensor is disclosed where the method comprises the steps of: providing waveguide laminate structure comprising at least one waveguide structure; providing a reaction chamber laminate structure comprising: a reaction chamber first material structure comprising a first PSA having a PSA liner; a first feature included in the reaction chamber first material structure; and a reaction chamber second material structure comprising a reaction chamber material and a reaction chamber material liner; removing the PSA liner from the reaction chamber first material structure exposing the first PSA; and attaching the first PSA to the waveguide laminate structure forming the laminate structure.

Additional methods of manufacturing a laminate structure comprising the following steps is disclosed: providing a waveguide structure comprising a plurality of waveguide cores and having a first surface; creating an oxygen sensing polymer cavity in the first surface of the waveguide structure to receive an oxygen sensing polymer; filling the oxygen sensing polymer cavity with the oxygen sensing polymer and curing the oxygen sensing polymer; adding a first layer material on top of the first surface of the waveguide structure, wherein the first layer material comprises a reaction chamber cavity that is contiguous with the oxygen sensing polymer; filling the reaction chamber cavity with an enzymatic hydrogel and curing the enzymatic hydrogel; adding a second layer material on top of the first layer material, wherein the second layer material includes a conduit cavity to receive a conduit hydrogel; filling the conduit cavity with a conduit hydrogel and curing the conduit hydrogel; and adding a top cap on top of the second layer of material.

Moreover, embodiments of the invention are directed to a method of manufacturing laminate structure comprising the steps of: providing a waveguide structure comprising a plurality of waveguide cores filled with a core material and a first surface having a clad coating layer with a clad liner thereon; laser cutting an oxygen sensing polymer cavity in the first surface of the waveguide structure to receive an oxygen sensing polymer, wherein the oxygen sensing polymer cavity is contiguous with the waveguide cores; filling the oxygen sensing polymer cavity with the oxygen sensing polymer and curing the oxygen sensing polymer; removing the clad liner from the clad coating layer; attaching a PEEK material layer on top of the clad coating layer, wherein the PEEK material layer comprises: a PSA on a first surface to adhere to the clad coating layer; a PEEK liner on a second surface; and a reaction chamber cavity that is contiguous with the oxygen sensing polymer; filling the reaction chamber cavity in the PEEK material layer with an enzymatic hydrogel and curing the enzymatic hydrogel; removing the PEEK liner from the PEEK material layer; attaching a conduit layer material on top of the PEEK material layer, wherein the conduit layer material comprises a PVDF material having a first surface, a second surface and a conduit hydrogel cavity, wherein a layer of silicone PSA is includes on the first surface and the second surface; filling the conduit hydrogel cavity with a conduit hydrogel and curing the conduit hydrogel; and attaching a top cap comprising a plurality of perforations therein on top of the conduit layer of material.

Embodiments of the invention are also directed to a laminate structure that comprises: a waveguide structure comprising a plurality of waveguide cores filled with a core material and a clad coating layer; an oxygen sensing polymer cavity in the waveguide structure filled with an oxygen sensing polymer, wherein the oxygen sensing polymer cavity is contiguous with the waveguide cores and wherein the oxygen sensing polymer is in optical communication with the waveguide cores; a PEEK material layer on top of the clad coating layer, wherein the PEEK material layer comprises: a PSA on a first surface to adhere to the clad coating layer; a PEEK liner on a second surface; and a reaction chamber cavity that is contiguous with the oxygen sensing polymer and that is filled with an enzymatic hydrogel; a conduit layer material on top of the PEEK material layer, wherein the conduit layer material comprises a PVDF material having a first surface, a second surface and a conduit hydrogel cavity that is filled with a conduit hydrogel, wherein a layer of silicone PSA is included on the first surface and the second surface; and a top cap comprising a plurality of perforations therein on top of the conduit layer of material.

Embodiments are directed to a method of manufacturing a thin film sensing element are disclosed where the method comprises: creating a polymer laminate thin film waveguide structure comprising the steps of: providing a first material to be embossed, wherein the material has a first index of refraction; embossing at least one waveguide structure into the material; filling the embossed waveguide structure with a second material having a second index of refraction; and applying a third material on top of the first material, wherein the third material has a third index of refraction; creating a reaction chamber laminate structure comprising the steps of: providing a first layer comprising an adhesive; providing a second layer comprising a PEEK material; joining the first layer to the second layer; cutting at least a portion of the first layer away from the second layer; joining the polymer laminate thin film waveguide structure to the first layer of the reaction chamber laminate structure; cutting a reaction chamber through the reaction chamber laminate structure at least partially into the filled waveguide structure; and microfluidically filling the reaction chamber with an oxygen sensing polymer and an enzymatic hydrogel.

The disclosure also relates to methods of manufacturing a thin film sensing element comprising: creating a polymer laminate thin film waveguide structure comprising the steps of: providing a first material to be embossed, wherein the material has a first index of refraction; embossing at least one waveguide structure into the material; filling the embossed waveguide structure with a second material having a second index of refraction; and applying a third material on top of the first material, wherein the third material has a third index of refraction; creating a reaction chamber laminate structure comprising the steps of: providing a first layer comprising an adhesive; providing a second layer comprising a PEEK material; joining the first layer to the second layer; cutting at least a portion of the first layer away from the second layer; joining the polymer laminate thin film waveguide structure to the first layer of the reaction chamber laminate structure; cutting a reaction chamber through the reaction chamber laminate structure at least partially into the filled waveguide structure; and microfluidically filling the reaction chamber with an oxygen sensing polymer and an enzymatic hydrogel.

Embodiments are also directed to an active hydrogel composition, prepared by the steps of: dispersing a nanogel in a liquid medium, the nanogel comprising a nanostructure covalently linked to a macromer and conjugated to a polymer network; adding a crosslinker to the nanogel dispersed in the liquid medium; and performing a crosslinking step to form the active hydrogel composition.

Methods and systems are disclosed for an inserter system for a minimally invasive tissue implant. As will be readily apparent to those skilled in the art, the methods and inserted systems disclosed herein are equally applicable for use with, for example, biosensors, micro catheters and drug eluting implants. In some embodiments, the inserter system is for use with as continuous glucose monitoring system. In one example, the system for sensor implantation can include an inserter and a sensor. The inserter can include a lancet tip that includes a convex feature attached to a first surface of the lancet tip. The inserter can also include an inset on either side of the lancet tip. The sensor can include a distal end that is configured to form a loop. The loop is configured to pass around the insets of the lancet tip, with a portion of the loop positioned adjacent the convex feature.

DETAILED DESCRIPTION

The disclosed and described technology relates to continuous analyte monitoring systems that may include an opto-enzymatic sensor, a controller, an analysis engine, a knowledge base, a smart card, and a portable computing device. Example analytes that can be measured using the embodiments of the invention disclosed and described herein include, and are not limited to, glucose, galactose, fructose, lactose, peroxide, cholesterol, amino acids, alcohol, lactic acid, and mixtures of the foregoing. Although much of the disclosure contained herein is directed to a glucose monitoring system that may include an opto-enzymatic sensor, a controller, an analysis engine, a knowledge base, a smart card, and a portable computing device, the embodiments of the present invention can be used to monitor many different analytes, including and not limited to, the ones listed in this paragraph.

In some embodiments, the system communicates with and incorporates data from activity sensor systems and bio sensor systems. In some embodiments, the system communicates over the cloud or internet with health care providers including doctors and nurses via a health provider network and may also communicate with a patient's caregiver. The disclosed technology provides interconnected care that supports the patient directly, and provides their immediate caregivers, as well as their physician and health provider network, with timely information to support the patient and health care provider goal of sustained glycemic control.

Continuous Health Monitoring System

FIG. 1Ais a block diagram illustrating an embodiment of a continuous health monitoring system100, including a sensor110, a controller120, and an analysis engine130. At least a portion of the sensor110is implanted in a patient. A controller120on the skin of the patient is optically connected to the sensor110. The controller120is in electronic communication with an analysis engine130, via a wireless or wired connection. The analysis engine130may be packaged separately from the controller120. The analysis engine130transmits protocols to the controller120, which optically interrogates the sensor110that senses real-time biological conditions in a patient. In response to the interrogation, sensor110optically transmits sensed data to the controller120. The controller120collects one or more analyte readings included in the sensed data, and transmits the collected analyte readings to the analysis engine130. The readings may be transmitted from the controller to the analysis engine130in a burst. For example, the analysis engine130may transmit a protocol to controller120requesting sensor readings every 30 seconds, and/or bursts of readings every 5 minutes. The controller120may interrogate the sensor110every 30 seconds, and record the sensed data. Every 5 minutes, corresponding to every 10 sensed readings, the controller120may transmit the 10 sensed readings to the analysis engine130.

In an embodiment, the sensor110is an opto-enzymatic (optical-enzymatic) sensor that provides interstitial fluid measurements of an analyte when optically interrogated with visible light. The sensor110may be implanted subcutaneously so that the sensor is in contact with interstitial bodily fluid containing analytes. The sensor transduces a concentration of an analyte to determine a measure of the analyte concentration. The sensor110communicates the measure of the analyte concentration to the controller120over a communication channel between the controller120on the skin of the patient and the subcutaneous sensor110when the sensor is interrogated with visible light. In an embodiment, the communication channel between the control120and the sensor110is an optical channel. In an embodiment, the analyte concentration is indicative of a blood sugar condition, such as a blood glucose level.

The controller120interrogates the sensor110with visible light from a compact laser source124or other light source and measures the glucose dependent luminescent emissions from the percutaneous sensing element (sensor)110. The on-body controller120may interrogate the sensor frequently (for example, each minute) and then transmit sensor measurements in bursts (for example, every five minutes). Controller120converts the received raw optical signals into glucose measurements and transmits the measurements via a protocol to an external receiver using a wireless communication protocol. In an embodiment, the wireless communication protocol is a Bluetooth, low energy protocol.

The sensor measurements may be analyzed by analysis engine130and then displayed or transmitted for display. Analysis engine130may be housed in a dedicated computing device, in an insulin pump, or an artificial pancreas device equipped with a Bluetooth receiver and a processor for interpreting the sensor data and converting it into calibrated glucose measurements. By housing the analysis engine130in, for example, an insulin pump or artificial pancreas, the disclosed technology enables a closed loop solution for the patient for sensing interstitial glucose levels, and modifying outputs from the insulin pump or artificial pancreas to the patient based at least in part on the sensed glucose levels. Analysis engine130transmits protocols to the controller that defines the duration, frequency and timing of sensor interrogation. The analysis engine130receives bursts of analyte (for example, glucose) readings from which it determines results, including individual or time series of analyte levels, trends, patterns, graphs, and alerts. The analysis engine130may include a processor or processing circuit. The analysis engine130may communicate with the controller via a wired or wireless connection

FIG. 1Bis an illustration101of the sensor ofFIG. 1Aand the controller120ofFIG. 1Abefore they are connected to each other. Sensor110ofFIG. 1Ais housed in sensor assembly110A, which also houses transducer111and at least one waveguide119(seeFIG. 2B) in a sensor subassembly with a connector103for connecting to controller120, which is housed in controller assembly120A. As used herein, a “waveguide” is an optical path for light based on internal reflection due to a higher index of refraction in the light path than the volume surrounding the light path. A waveguide, or light pipe, is preferably made of polymers. The controller120is affixed to the patient's skin and is in optical communication with the sensor110. The controller (on-body transmitter) may be enclosed in assembly120A, an ergonomically shaped, low profile, waterproof assembly designed to allow unobtrusive body wear. The on-body transmitter in assembly120A may be cleanable.

After at least the distal portion of the implantable percutaneous sensor110is implanted, the on-body controller120is attached to the sensor assembly110A.FIG. 1Cis a corresponding illustration102of the sensor ofFIG. 1B, and the controller ofFIG. 1Bconnected to each to each other. The controller120is not visible because the controller housing is not transparent. The controller assembly120A is affixed to the patient's skin using, for example, an adhesive system disclosed and described in more detail herein, and the sensor110is implanted percutaneously in the patient. The sensor110and controller120communicate optically through the connector103.

FIG. 2Ais a functional block diagram of the sensor110inFIG. 1A. Sensor110includes a transducer111that transduces an interstitial analyte level, such as, for example, a blood glucose level in the bodily fluid/tissue into which the sensor110is implanted. A waveguide119receives optical interrogation signals, and transmits analyte readings. In an embodiment, the optically received signals and optically transmitted signals may be received and transmitted via an optical pathway through a connector and via optical fiber and/or a waveguide from and to controller120. Transducer111determines interstitial measurements of glucose when the sensing element is optically interrogated with visible light. The sensor provides a measurement of the interstitial glucose based on the difference between an interstitial reference oxygen measurement and measurements of the oxygen remaining after a two stage enzymatic reaction of glucose and oxygen as described in more detail below.

FIG. 2Bis an illustration200of an exemplary sensor110ofFIG. 2A. Illustration200depicts a sensor subassembly110A. As described in more detail below, the sensor assembly110A may include three layers, including middle layer112, which houses transducer111and waveguide119. The middle layer112may be approximately 7 mm long and 0.4 mm wide. An enzymatic hydrogel channel113includes hydrogel that reacts with interstitial glucose that enters in glucose in1et114on one side of the middle layer112, along the width dimension. An oxygen sensing polymer115forms a band or channel along the width dimension of the middle layer112starting in proximity to the glucose in1et114but not necessarily extending across the entire width of middle layer112. The oxygen sensing polymer band/channel115forms a continuous band/channel, but may be considered to be divided into distinct regions, for example, the first region117A closest to the glucose in1et114, the second region117B next closest to the first region114, and the third region116farthest from the glucose in1et. Glucose interacts with the oxygen sensing polymer in the presence of the hydrogel in the enzymatic hydrogel channel113, and diffuses along the continuous oxygen sensing polymer band115starting at the glucose in1et114in the first region117A, then onto the second region117B, and finally onto the third region117C, at increasing distances from the glucose in1et114. When the sensor110is interrogated with visible light, the waveguides119transmit sensor readings for regions117A-C and for the oxygen reference116, which are used to estimate analyte (glucose) concentration. The sensor110readings provide oxygen levels, which are an indication of oxygen consumption levels in the oxygen sensing polymer115in regions117A-C. In an embodiment, the oxygen sensing polymer115is divided into two regions, three regions (as in the embodiment inFIG. 2B), four regions, 5 regions, or more regions. Dividing the oxygen sensing polymer band115into regions corresponds to sampling the oxygen sensing polymer band115at different distances from the glucose in1et114. This sampling makes it possible to estimate a profile along the oxygen sensing polymer band115. Each “sensor reading” includes a vector of readings—one for each region117A-C, and an oxygen reference reading116.

FIG. 2Cis a series of curves of oxygen consumption vs. distance (in mm) from the glucose in1et114, for steady state glucose concentrations of 100 mg/dL, 200 mg/dL, and 300 mg/dL. Close to the glucose in1et114, there is good discrimination between glucose concentrations 100 mg/dL and 200 mg/dL, but poor discrimination between glucose concentrations 200 mg/dL and 300 mg/dL. In contrast, at distances farther from the glucose in1et114, there is poor discrimination between glucose concentrations 100 mg/dL and 200 mg/dL, but good discrimination between glucose concentrations 200 mg/dL and 300 mg/dL. Therefore, in this embodiment, there is good sensitivity for lower glucose concentrations closer to the glucose in1et114, and good sensitivity for higher glucose concentrations farther from the glucose in1et114. This is analogous to taking pictures in bright sun1ight with short exposures to avoid saturation, and taking pictures in dark rooms with long exposures to enable discrimination at low light levels. By taking oxygen consumption readings or glucose concentration readings via multiple waveguides at different distances from the glucose in1et114, analogous to different camera exposures, the raw sensor readings may be used to determine glucose concentrations over a greater range of glucose levels than would be possible with a single sensor reading.

The four flexible waveguides119along the vertical dimension of middle layer112transmit sensor readings from regions117A-C and oxygen reference116through sensor subassembly110A to controller120. In the case of a zero interstitial glucose concentration, the reference oxygen concentration and the working oxygen concentration are the same. In the case of a low glucose concentration, the majority of the glucose and oxygen consumption by the enzymatic reaction occurs in the first reaction region117A volume of the enzymatic hydrogel113proximal to the glucose in1et114. As the interstitial glucose concentration increases, the enzymatic reaction moves further into the second and third reaction region117B,117C volumes of the enzymatic hydrogel113.

This progressive reaction to differing glucose concentrations depicted inFIG. 2Callows for high sensitivity to low glucose concentrations by monitoring the first reaction region117A volume for oxygen concentration, and wide dynamic range by monitoring the second and third reaction region117B,117C volumes for oxygen concentration as well. When the interstitial glucose concentration is low and limited glucose diffuses through the glucose in1et114into the first reaction region117A volume, the oxygen consumption in the enzymatic hydrogel113is primarily proximal to the glucose in1et114. The interstitial glucose concentration is readily calculable from a set of oxygen concentration measurements. Given a reference oxygen level and three oxygen concentration measurements in the enzymatic hydrogel113in regions117A-C, the glucose concentration is a linear function of the sum of the differences between each of the three oxygen concentration measurements and the reference oxygen concentration measurement. For each glucose concentration, there is a reference oxygen concentration measurement and a corresponding set of oxygen concentrations in the enzymatic hydrogel113and a corresponding oxygen concentration difference. There is a direct relationship of the net oxygen concentration difference measured from the enzymatic reaction chamber compared to the oxygen reference measurement versus the steady state glucose concentration. This direct relationship allows the sensor to be calibrated with a parameterized equation that yields a calculated glucose concentration based on the measured oxygen differences.

Depending on the parameterization, the calculated glucose concentration can be the concentration of glucose in the environment of the sensor. This can be an in vitro glucose concentration if the sensor is calibrated using in vitro glucose solutions, or an interstitial glucose concentration if the sensor is an implanted glucose biosensor. Alternatively, the parameterized equation may provide a direct calculation of blood glucose concentration, such as when the relationship between the blood and the interstitial tissue is assumed to be linear, and the parameterized equation is determined using a linear regression with blood glucose measurements as inFIG. 26. Alternatively, a second parameterized calculation may be used to calculate a blood glucose measurement from sensor calculated interstitial glucose measurements. For example, an enhanced Bayesian calibration method can be implemented using the Extended Kalman Filter to account for the existence of blood glucose-to-interstitial glucose kinetics by incorporating a population convolution model [Andrea Facchinetti, Giovanni Sparacino, and Claudio Cobelli. Enhanced Accuracy of Continuous Glucose Monitoring by On1ine Extended Kalman Filtering. Diabetes Technology & Therapeutics. May 2010, Vol. 12, No. 5: 353-363].

As the interstitial glucose concentration increases and the amount of glucose diffusing through the glucose in1et114increases, and more glucose is reacted in the second and third regions117B,117C, the oxygen consumption occurs farther within each reaction region117B,117C volume. The net oxygen consumed for a given glucose concentration is determined from the set of oxygen concentration differences. The total oxygen concentration difference is the sum of the net oxygen differences (reference-working as measured in regions117A-C) from the three volumes compared to the reference oxygen concentration. The interstitial glucose concentration can therefore be determined from net oxygen consumption by means of a linear calibration.

The oxygen concentration measurement is based on the luminescence lifetime (τ) of an oxygen-sensitive luminescent dye. The lifetime (τ) expresses the amount of time the luminescent dye (or luminophore) remains in an excited state following excitation by light of a suitable frequency. The sensor110oxygen-sensitive luminescent dye lifetime measurement is made using a time domain approach in which the oxygen sensing polymer sample is excited with a pulse of light and then the time-dependent intensity is measured. The lifetime is calculated from the slope of the log of intensity versus time.

In another embodiment, the sensor110is pre-interrogated with an optical signal at a wavelength that does not excite the luminescent dye but with a known lifetime decay to calibrate the on body transmitter and optical system before each glucose measurement is made. The light is reflected by the dye instead of inducing a luminescent signal. In addition, the pre-interrogation pulse of light ensures that proper optical connections have been maintained before each measurement.

The difference in the reference and working oxygen concentrations are used to calculate the interstitial glucose concentration. In the case of a zero interstitial glucose concentration, the reference oxygen concentration and the working oxygen concentration are the same. In the case of a low glucose concentration, the majority of the glucose and oxygen consumption by the enzymatic reaction occur in the first reaction volume of the enzymatic hydrogel proximal to the glucose in1et. As the interstitial glucose concentration increases, the enzymatic reaction moves further into the second and third reaction volumes of the enzymatic hydrogel.

The relationship of the interstitial glucose concentration to oxygen consumed in the enzymatic reaction is a function of the distance from the glucose in1et114. For example, a first reaction region117A volume close to the glucose in1et114will be sensitive to low concentrations of glucose, and exhibit high dynamic range when differentiating among different, low glucose concentrations.

FIG. 3Ais a functional block diagram of the controller120inFIG. 1A.FIG. 3Billustrates the controller housing120A that is affixed to the patient's skin, and is connected via a connector and an optical pathway to waveguides119of sensor110. The controller120includes a processing circuit121, a controller memory circuit123, a laser source125, a battery126, a detector127, a transmitter128, and a receiver129, and may also include a temperature sensor124. The controller120is embedded within a flexible housing120A configured to be affixed on a patient's skin, and connected via an optical channel to sensor110.

Processing circuit (processor)121converts the received raw optical signals into glucose measurements using the methods disclosed herein. Transmitter128transmits the measurements via a protocol to an external receiver using a wireless communication protocol. In an embodiment, the wireless communication protocol is a Bluetooth low energy protocol. Laser source125is an optical excitation source. In an embodiment, laser source125is a single stage laser diode. In an embodiment, laser source125emits light at a wavelength of substantially 405 nm, corresponding substantially to the peak absorption wavelength of the luminescent dye. The detector127is a multipixel, miniaturized silicon photomultiplier chip. The optical source emitter (laser source)125and the detector127silicon components are mounted in a high precision polymer housing within the durable transmitter120.

The receiver129receives protocols, described below, from analysis engine130. The controller processing circuit121determines timing, duration, and frequency to interrogate the sensor110via the optical pathway between the controller120and sensor110. The laser source125interrogates the sensor110via the optical pathway (waveguide), and the detector127receives the sensed data via the optical pathway. The sensed data is stored in controller memory circuit124. For example, based on the protocol, the optical transmitter128may interrogate sensor110every30seconds. The optical receiver may store sensed data in memory unit124and every five minutes while sensing an analyte level, the controller transmitter129transmits the sensed data stored since the previous burst transmission to analysis engine130. This transmission may be over a wireless communication channel or any other communication means.

The processor121estimates glucose or other analyte levels based on the detections optically received by detector127. The relationship of the interstitial glucose concentration to oxygen consumed in the enzymatic reaction is a function of the distance from the glucose in1et114.

Processor121may monitor system components and trigger alarms. For example, processor121may trigger sensor status alarms, battery level alarms, controller connection to sensor alarms, and controller performance alarms. Processor122may command the laser source125to emit light to the sensor110, and analyze the return light detected by the detector127to inspect the optical connection to the sensor, as well as the sensor status. The processor may also monitor battery126status and performance, including battery level.

The processor121may conduct calibration operations independently or in conjunction with the analysis engine130. Calibration operations may include calibrating glucose measurements from raw sensor data and factory calibration factors, updating calibration based on self-monitoring of blood glucose (SMBG) data, determining when the user should recalibrated based on oxygen sensor data, and determining when the implanted sensor110should be replaced based on oxygen sensor data and gain. Calibration operations may trigger alerts related to calibration, such as “replace the sensor110,” or “time to recalibrate with SMBG data.”

The processor121calibrates sensor readings detected by detector127. The processor121may use factory calibration data to calibrate sensor readings. The factory calibration data may be retrieved from a smart card by reading 2D barcodes or by using near field communications or radio frequency ID to transmit the factory calibration data from the smart card to processor121. In an embodiment, processor121may use linear calibration to calibrate raw sensor readings by multiplying the raw sensor reading by a scale factor and adding an offset factor to determine a calibrated sensor measurement. In an embodiment, processor121may use non1inear calibration to calibrate raw sensor readings. In an embodiment, calibration may include modifying a calibrating factor (such as the scale factor, offset factor, or a coefficient for a non1inear calibration factor) based on the measured temperature. Processor121may use self-monitoring of blood glucose (SMBG) data to update the calibration scale factors and calibration offset factory.

The linear calibration required to convert net oxygen consumed to interstitial glucose concentration will be determined by a factory calibration. Calibration data may be read from a smart tag. The factory calibration will be determined from the luminescent signals of the oxygen sensing polymer while the sensors are exposed to a well-mixed aqueous glucose solution under known conditions at the final stage of the manufacturing process.

Temperature sensor124measures temperature to ensure that the temperature is in the operating range of the sensor110, since the enzymatic reactions in sensor110are temperature sensitive and temperature can impact the sensor calibration.

The controller120includes battery126which powers controller120. In an embodiment, battery126may power controller120for period of time between charges. In an embodiment, the period of time between charges is 5 days, 7 days, or two weeks. In an embodiment, battery126may be recharged using inductive power transfer. In an embodiment, battery126may be recharged using a battery charger. In an embodiment, battery126is not rechargeable and may be replaced with a new battery.

FIG. 4is a functional block diagram illustrating an example of a continuous health monitoring system400, including a sensor110, a controller120, an analysis engine130, a knowledge base140, a smart card150, and/or a portable computing device160. In an embodiment, the sensor110, controller120, and analysis engine130are described above with reference toFIG. 1A. The analysis engine130is in wired or wireless communication with a knowledge base140. The analysis engine130is in wireless communication with the smart card150. The analysis engine is in wireless communication with a personal computing device.

In an embodiment, the knowledge base140may be implemented in a memory block or in a memory unit, for example, as a relational database. The knowledge base140may be included in the same housing as the analysis engine130(for example, in a handheld or laptop computing device or smartphone or any other portable device). In an implementation, the knowledge base140may be included in a memory block or memory unit in a computing device separate from the analysis engine. In an embodiment, the knowledge base140may be accessible to the analysis engine130over a network, such as a wired or wireless local area network, via a router (not shown), or over the internet. The knowledge base140may include patient specific information that identifies the patient, as well as patient data relevant to analyses performed by the analysis engine130, and which may impact analyte monitoring, including patient conditions and patient history. Past sensed data such as, for example, glucose levels—sensed by the opto-enzymatic sensor110, or other sensors, may also be included in the knowledge base140. Data for trends, patterns and analysis, bounds to determine whether readings are within normal limits, and alert conditions may also be stored in the knowledge base140.

The knowledge base140may include the detailed mapping of standard orders from a doctor, received via a health provider network and over the internet/cloud, to timing, frequency, and type of interrogations of the sensor, as well as other sensors. The knowledge base140may also track activity data and other bio sensed data, to enable multi-sensor fusion and analyses as well as to provide a health care provider or caregiver, a more complete picture of a patient's health status. The knowledge base140may include data that support analyses performed by the analysis engine. In some embodiments, the knowledge base140may be implemented in a distributed database. In an embodiment, the knowledge base140may, in addition to being in communication with the analysis engine, be in communication with the controller120, the portable computing device160, the smart card150, one or more activity sensor systems, and one or more biosensor systems.

In an embodiment, the trends and graphs determined by the analysis engine130may include glucose measurements, glycemic history, a glycemic dynamics envelope, insulin on board/insulin levels, and normative glycemic profiles with an insulin overlay. Example profiles include a 24 hour average, based on 7 days, 24 hour averages on a daily basis based on the last 49 days, or a basal profile overlay with24hour average.

The analysis engine130may estimate whether a patient missed a meal bolus using piezo data, insulin data, time of day, and/or prior identified meal periods. The analysis engine130may use an algorithm to determine the likelihood of a missed bolus using the likelihood of an activity state, insulin bolus data, insulin data entered by a patient or caregiver, monitored data readings, and prior readings.

In an embodiment, the analysis engine130generates alerts when an analyte level, trend, statistic, or other measure falls outside normal limits, exceeds a threshold, or is less than a threshold. Alerts are state dependent, for example based on activity, time of day, and/or user inputs. Example alert conditions include: missed bolus if likely eating a meal (or not eating a meal), sustained hyperglycemia if during or after eating a meal (or not during or after a meal), developing and or severe hypoglycemia, (dependent on activity and/or time of day), or near hypoglycemia for an extended time period. The smart card150provide a visual monitor of analyte (for example, glucose) readings. The smart card150may be carried in the patient's wallet. The patient, or an aide or health care provider with the patient, interacts with the system via a smart card150and/or a portable computing device160. The analysis engine130may transmit results to the smart card150and/or one or more portable computing devices160.

FIG. 5is a functional block diagram of the smart card150inFIG. 4. The smart card150communicates queries and results to and from the analysis engine130using a transmitter158and receiver159. In an embodiment, the transmitter158and receiver159may communicate over short distances using RFID and/or NFC technology with a smart card. In an embodiment, receiver159may include more than one receiver. For example, one for short distance reception using RFID or NFC, and another to receive results from analysis engine130over a distance varying from centimeters to meters. In an embodiment, transmitter158may include more than one transmitter. For example, one for short distance transmission using RFID or NFC, and another to transmit queries to analysis engine130over a distance varying from centimeters to meters. In an embodiment, transmitter158and receiver159may be combined in a transceiver (not shown).

The smart card150includes a processor circuit (processor)151in wired communication with memory circuit (memory)153, transmitter158, and receiver159. The smart card150receives inputs via a touchscreen155aand/or a camera155b,each in wired communication with the processor151. The smart card150includes a display157a,speaker157b,and/or actuator157c,each in wired communication with the processor151. The touchscreen155aand display157amay be integrated so that a user may select an item on display157aby touching touchscreen155aat one or more corresponding points on the touchscreen155a.The display157aoutputs visual data and information, the speaker outputs audio data and information, and the actuator157coutputs tactile data and information. Smart card150displays/transmits—numerically and/or graphically—analyte readings using display157a,speaker157b,and/or actuator157c.

In an embodiment, the smart card150uses lights, sound, vibration, or its visual display157ato “display” alarms when readings or trends are not within normal or preset/pre-identified limits. The processor151may be an embedded chip, such as a microcontroller circuit chip. In an embodiment, the microcontroller chip conforms to the ISO/IEC 14443 standard. The ISO/IEC 14443 standard is an international standard for contactless smart chips and cards that operate (i.e., can be read from or written to) at a distance of less than 10 centimeters (4 inches). This standard operates at 13.56 MHz and includes specifications for the physical characteristics, radio frequency power and signal interface, initialization and anti-collision protocols and transmission protocol. In an embodiment, the smart card may conform to the ISE/IEC7816standard for contact smart cards.

A smart tag (not shown) may use bar codes read by camera155b,near field communications received by receiver159, or RFID received by receiver159. The smart tag may store sensor identity, sensor expiration, factory calibration data, and/or other device data. The smart tag may be read by other computing devices with a camera, an NFC receiver, and/or an RFID receiver, such as a smart phone, wearable computer, desktop computer, tablet, portable receiver, or charging platform.

FIG. 6Ais a functional block diagram of the portable computing device160inFIG. 4.FIG. 6Billustrates an example portable computing device160A. The portable computing device160may be a cell phone, wearable computing device, tablet, personal digital assistant, or other computing device. Portable computing device160may include an application that enables viewing of results from the analysis engine130and/or knowledge base140, as well as sending queries. For example, a query may include a request for trend data or a protocol to take additional data. Alerts may be viewed on the portable computing device160, as well as system alarms. System alarms may include sensor status alarms, battery level alarms, controller connection to sensor alarms, and controller performance alarms.

A patient or health care provider may view results from the analysis engine130on one or more portable computing devices160, using an application (app) that communicates queries and results to and from the analysis engine130using transmitter168and receiver169. In an embodiment, transmitter168and receiver169may communicate over short distances using RFID and/or NFC with a smart card. In an embodiment, receiver168may include more than one receiver. For example, one for short distance reception using RFID or NFC, and another to receive results from analysis engine130over a distance varying from centimeters to meters. In an embodiment, transmitter168may include more than one transmitter. For example, one for short distance transmission using RFID or NFC, and another to transmit queries to analysis engine130over a distance varying from centimeters to meters. In an embodiment, transmitter168and receiver169may be combined in a transceiver (not shown).

The portable computing device160includes a processor circuit (processor)161in wired communication with memory circuit (memory)163, transmitter168, and receiver169. The portable computing device160receives inputs via a touchscreen165a,a keypad165b,a camera165c,and/or a motion sensor165d,each in wired communication with the processor161. A patient may enter a query using the touchscreen165a,keypad165b,or by speech entry via a microphone (not shown). The portable computing device160includes display167a,speaker167b,and/or actuator167c,each in wired communication with the processor161. The touchscreen165aand display167amay be integrated so that a user may select an item on display167aby touching touchscreen165aat one or more corresponding points on the touchscreen165a.The display167aoutputs visual data and information, the speaker167boutputs audio data and information, and the actuator167coutputs tactile data and information. In an embodiment, portable computing device160may display a trend line on display167a,output a high glucose reading over speaker167b,and/or output tactile data using actuator167cin case of an alarm or alert. The tactile alert may, for example, correspond to a tapping of a patient's wrist when the portable computing device160is a wearable computer worn on a patient's wrist, or a vibration when the portable computing device160is a phone or tablet.

The processor circuit161on portable computing device160may run a software application (app) to certain continuous health monitoring operations described herein, including displaying results, accepting user input, and communicating with other system components. The software application may include validation checks, tests, or other operations to validate data elements that are communicated, processed, stored, retrieved, displayed, or otherwise operated upon. For example, each function call may use cyclic redundancy checks (CRC), checksums, or other methods to detect errors and ensure data integrity. For example, cyclic redundancy checks may be applied for each function call. The CRC and/or checksum of each function may be determined in a preprocessing or software compilation step. These data integrity measures may be hard coded into a read on1y memory (ROM) image of the application. During runtime of the application, each function call may calculate a cyclic redundancy check of the function. The calculated value may be compared to the previously determined (and, possible, hard coded) value, and compared to see if they match. If they match, the function is validated and it is acceptable to run the function call, If not, the application may capture diagnostic data, report the validation error, mark the data for the process as invalid (and/or discard the data), and restart the process. If there are multiple errors in a row, or a particular error that repeats over time, system alerts may be recorded for diagnostic purposes by the system, as well as to the user. By including validation checking within the application itself, the mobile health software application may be validated independent of the operating system hosting the mobile health software application.

Such self-validation may be applied not on1y to the portable computing device160, but to smart card150, analysis engine130, controller120, and an application hosted on health provider network/monitor210(seeFIG. 10). The knowledge base140may incorporate data integrity or validation testing when conducting database transactions.

A smart tag (not shown) may use bar codes read by camera165b,near field communications received by receiver169, or RFID received by receiver169. The smart tag may store sensor identity, sensor expiration, factory calibration data, and/or other device data.

FIG. 7is a functional block diagram illustrating an example of a continuous health monitoring system700, including a sensor110, a controller120, an analysis engine130, a knowledge base140, a smart card150, a portable computing device160, a bio sensor system170, and/or an activity sensor system180. In an embodiment, the sensor110, controller120, and analysis engine130are described above with reference toFIG. 1A. In an embodiment, the knowledge base140, smart card150, and portable device160are described above with reference toFIG. 4.

The analysis engine130sends protocols to, and/or receives data from activity sensor system180and/or bio sensor system170. Activity sensor systems include sensors, such as gyros or motion sensors, which enable estimation of patient activity (sleeping, resting, eating, strenuous exercising, etc.). In an embodiment, activity sensor system may be included in portable computing device160, which include motion sensor165d.Bio sensor systems170measure aspects of the patient's condition, such as pulse rate, temperature, respiration rate, pulse oximetry, or other analyte readings. The analysis engine130can also be configured to receive data from a third party activity sensor system such as, for example, a Fitbit® activity tracker.

The protocols indicate two types of information. The first type of information includes parameters, settings, and preferences for sensing, and the device for taking the data that are typically independent of sampling rate, duration, and timing. The second type of information includes sampling type, timing, rate, and duration. These protocols, and the two types of information, are used for analyte sensing (including glucose level), other bio sensors, and activity sensors. The activity data and bio sensor data that are communicated to the analysis engine130from activity sensor180and bio sensor system170, respectively, may be stored in the knowledge base140, and used to generate results (trends, patterns, alerts, sensor levels). The analysis engine may fuse the data from sensor110, bio sensor system170, activity sensor system180, and data from knowledge base140to generate results.

For example, the analysis engine130may trigger an alarm when sensor110senses a blood glucose reading that is sustained over 150 mg/dl for 30 minutes when an activity sensor determines that a patient is not sleeping based on a reading from motion sensor165dor data received from an activity sensor180, which can be indicative of patient activity other than sleeping. However, the analysis engine130may not trigger an alarm when the sensor110senses a blood glucose reading that is sustained over 150 mg/dl for 30 minutes when an analysis engine determines that a patient is at rest based on a reading from motion sensor165dor an activity sensor180in conjunction with the time of day and ambient light level; but the analysis engine130can be configured to trigger an alarm when sensor110senses a blood glucose reading has been sustained over150mg/dl for2hours if the analysis engine determines a patient is at rest.

In an embodiment, analysis engine130communicates with and/or interfaces to one or more bio sensor systems170and/or one or more activity sensor systems180.

FIG. 8is a functional block diagram of the bio sensor system170inFIG. 7. The bio sensor system170communicates bio sensor data and bio sensor protocols to and from the analysis engine130using transmitter178and receiver179. In an embodiment, transmitter178and receiver179may be combined in a transceiver (not shown). The bio sensor170includes a processor circuit (processor)171in wired communication with memory circuit (memory)173, transmitter178, and receiver179. The bio sensor170measures/monitors an aspect of a patient's health/biology that may relate to a medical condition or otherwise characterize a patient, and communicates data based on these measures to the processor171. Example data that can be obtained by these measurements or monitoring may include an analyte level, pulse rate, temperature, respiration rate, or pulse oximetry.

FIG. 9is a functional block diagram of the activity sensor system180inFIG. 7. The activity sensor system180communicates activity sensor data and activity sensor protocols to and from the analysis engine130using transmitter188and receiver189. In an embodiment, transmitter188and receiver189may be combined in a transceiver (not shown). The activity sensor180includes a processor circuit (processor)181in wired communication with memory circuit (memory)183, transmitter188, and receiver189. The activity sensor180measures an aspect of a patient's activity based on, for example, movement related to whether a patient is stationary, walking, running, or climbing stairs, and communicates data based on these measures to the processor171. The activity sensor system180may, for example, use sensors and algorithms similar to the sensors and algorithms used by commercially available fitness tracking systems such as, for example, the Fitbit® activity tracker.

FIG. 10is a functional block diagram illustrating an example of a continuous health monitoring system1000, including a sensor110, a controller120, an analysis engine130, a knowledge base140, a smart card150, a portable computing device160, a bio sensor system170, an activity sensor system180, a network200, and/or a health provider network/monitor210. In an embodiment, the sensor110, controller120, and analysis engine130are described above with reference toFIG. 1A. In an embodiment, the knowledge base140, smart card150, and portable device160are described above with reference toFIG. 4. In an embodiment, the bio sensor system170and activity sensor system180are described above with respect toFIG. 7.

In addition to communicating results and data generated by analysis engine130to smart card150and/or portable computing device160, the analysis engine130may communicate results and data to a network200, and on to health provider network/monitor210. Network200is connected by wire or wirelessly to the analysis engine130. Network200is in wired or wireless communication with health provided network/monitor210. In an embodiment, network200is an internetworking network (internet) enabling communication with a doctor via health provider network/monitor210. In an embodiment, health provider network/monitor210includes electronic patient records (not shown) such as, for example, electronic health records and electronic medical records, medical databases (not shown), desktop physician workstations, and/or portable computing devices.

FIG. 11is a functional block diagram of a health provider network/monitor210. Health provider network/monitor210may include a computing device used by a physician or other provider. The health provider network/monitor210may run a software application (app) directed to monitoring the results of analysis engine130, providing these results to a physician or another caregiver (nurse, spouse, etc.), recording results in a medical database, and/or enabling the physician to generate orders (such as the need for office visits, hospitalizations, changes in medications, etc.), based on the patient's history, condition, and/or results. The health provider network/monitor210includes a receiver219and transmitter218to receive results and transmit orders to and from the analysis engine130via network200. The receiver219and transmitter219are in communication with processor211.

The health provider network/monitor210includes a processor211in wired communication with memory213, transmitter218, and receiver219. The health provider network/monitor210receives inputs via a touchscreen215a,a keypad215b(keyboard215b), and/or a microphone215ceach in wired communication with the processor161. A physician may enter a query using the touchscreen215a,keypad/keyboard215b,or by speech entry via microphone215c.The health provider network/monitor210includes display217a,speaker217b,and/or actuator217c,each in wired communication with the processor211. The touchscreen215aand display217amay be integrated so that a physician may select an item on display217aby touching touchscreen215aat one or more corresponding points on the touchscreen215a.The display217aoutputs visual data and information, the speaker217boutputs audio data and information, and the actuator217coutputs tactical data and information. In an embodiment, portable computing device210may display a trend line on display217a,output a high glucose reading over speaker217b,and/or output tactile data using actuator217c(that can be, for example, by vibration), in case of an alarm or alert. The tactile alert may correspond to a tapping of a doctor's wrist when the health provider network/monitor210is a wearable computer worn on a doctor's or other healthcare provider's wrist, or a vibration when the health provider network/monitor210is a phone, tablet or other device.

A doctor may monitor a patient's progress by viewing results from the analysis engine via network200. The network200(internet, cloud) may include a health provider network and/or monitoring station used by the physician. This enables communications of results, including glucose levels, trends, patterns, and alerts. Data may be stored in the patient's electronic medical record (not shown).

A doctor may also submit orders. These orders may impact alarm thresholds, and may set alarms or thresholds with respect to different patient activities. For example, an order may request frequent glucose readings at a predefined frequency, during and after a meal, or lower a glycemic alarm during strenuous exercise detected by an activity sensor

The orders may be transmitted from the health provider network/monitor210via the network200to the analysis engine130. The knowledge base140maps the order from the doctor to both types of protocol information that indicates, for example, when and how often to interrogate the sensor, as well as relationships with activity levels and/or other reading (from a bio sensor, etc.). The knowledge base140may store the mappings from order to protocol, as well as the form of analysis to perform on the sensed data. The doctor may query for data from the knowledge base140.

FIG. 12is a functional block diagram illustrating an example of a continuous health monitoring system1200, including a sensor110, a controller120, an analysis engine130, a knowledge base140, a smart card150, a portable computing device160, a bio sensor system170, an activity sensor system180, a router190, a network200, and/or a health provider network/monitor210. In an embodiment, the sensor110, controller120, and analysis engine130are described above with reference toFIG. 1A.

In an embodiment, the knowledge base140, smart card150, and portable device160are described above with reference toFIG. 4. In an embodiment, the bio sensor system170and activity sensor system180are described above with respect toFIG. 7. In an embodiment, the network190and health provider network/monitor210are described above with reference toFIG. 10. The router190is in wireless or wired communication with the analysis engine130, portable computing device160, biosensor system170, activity sensor system180, and/or network200.

The router190processes and routes information. The router190transmits orders, queries, activity data, and bio sensor data to the analysis engine130. The router190receives results, activity protocols, and bio sensor protocols from the analysis engine130. The router190receives queries from portable computing device160for analysis by analysis engine130and transmits results from analysis engine130to portable computing device160. In an embodiment, the router receives queries from smart card150and sends results to smart card150. In an embodiment, the router190transmits bio sensor protocols to bio sensor system170, and receives bio sensor data from bio sensor system170. In an embodiment, the router190receives orders from the network200, and transmits results to the network200.

In an embodiment, the router190is a smart card150. In an embodiment, the router190includes multiple network elements and/or routers.

FIG. 13is a flowchart that illustrates an example of a method1300of continuous health monitoring. In some embodiments, the method1300may be performed by the system100inFIG. 1A. In some embodiments, the method1300may be performed by the system400inFIG. 4. In some embodiments, the method1300may be performed by the system700inFIG. 7. In some embodiments, the method1300may be performed by the system1000inFIG. 10. In some embodiments, the method1300may be performed by the system1200inFIG. 12.

In block1305, method1300transduces, by a sensor implanted in a patient, a concentration of an analyte to a measure of the analyte concentration. In an embodiment, the analyte is glucose. In some implementations, the functionality of block1305is performed by the transducer111of the sensor110illustrated inFIGS. 1A, 2A, 4, 7, 10, and 12.

In block1310, method1300interrogates, by a controller affixed to skin of the patient, the sensor with visible light. In some embodiments, the functionality of block1310is performed by optical transmitter125of the controller120illustrated inFIGS. 1A, 3A, 4, 7, 10, and12.

In block1315, method1300communicates, by the sensor, the measure of the analyte concentration in response to the interrogating with visible light. In some embodiments, the functionality of block1315is performed by an optical transmitter118of the sensor110illustrated inFIGS. 1A, 2, 4, 7, 10, and 12.

In block1320, method1300receives, by the controller, the measure of the analyte concentration. In some embodiments, the functionality of block1320is performed by optical receiver127of the controller120illustrated inFIGS. 1A, 3A, 4, 7, 10, and 12.

In block1325, method1300determines, by the controller, a frequency, a timing, and/or a duration of interrogating the sensor to determine a measure of the analyte concentration in response to a protocol. In some implementations, the functionality of block1325is performed by processor121of the controller120illustrated inFIGS. 1A, 3A, 4, 7, 10, and12.

In block1330, method1300stores, by the controller, a plurality of measures of the analyte concentration. In some embodiments, the functionality of block1330is performed by memory circuit (memory)123of the controller120illustrated inFIGS. 1A, 3A, 4, 7, 10, and 12.

In block1335, method1300transmits, by the controller, the plurality of measures of the analyte concentration. In some embodiments, the functionality of block1335is performed by transmitter128of the controller120illustrated inFIGS. 1A, 3A, 4, 7, 10, and 12.

In block1340, method1300stores, by a knowledge base, the plurality of measures of the analyte concentration. In some embodiments, the functionality of block1340is performed by knowledge base140illustrated inFIGS. 4, 7, 10, and 12.

[In block1345, method1300transmits, by an analysis engine, the protocol to the controller. In some embodiments, the functionality of block1345is performed by analysis engine130illustrated inFIGS. 1A, 4, 7, 10, and 12.

In block1350, method1300receives, by the analysis engine, the plurality of measures of the analyte concentration. In some implementations, the functionality of block1350is performed by analysis engine130illustrated inFIGS. 1A, 4, 7, 10, and 12.

In block1355, method1300determines, by the analysis engine, a result in response to the plurality of measures of the analyte concentration and the protocol. In some embodiments, the functionality of block1350is performed by analysis engine130illustrated inFIGS. 1A, 4, 7, 10, and 12. In an embodiment, the result is a glucose level, a glycemic history, a glycemic dynamics envelope, insulin levels, and/or normative glycemic profiles with insulin overlay.

FIG. 14is a flowchart that illustrates an example of a workflow1400of continuous health monitoring by a sensor, a controller, and an analysis engine. In some aspects, the workflow1400may be performed by the system100inFIG. 1A, the system400inFIG. 4, the system700inFIG. 7, the system1000inFIG. 10, and/or the system1200inFIG. 12. In block1405, analysis engine130sends a protocol to controller120. In block1410, the controller120interrogates sensor110based on the protocol. In block1415, sensor110senses measures associated with glucose levels in response to each interrogation. In block1420, controller120determines glucose level concentration estimates based on the sensor measures over a time period. In block1425, analysis engine130analyzes bursts of glucose level readings to determine trends, patterns, and trigger alerts.

FIG. 15is a flowchart that illustrates an example of a workflow1500of continuous health monitoring incorporating doctor orders. In some aspects, the workflow1500may be performed by the system1000inFIG. 10and/or the system1200inFIG. 12. In block1505, a doctor views results and patient history at health provider network/monitor210. In block1510, the doctor issues an order in response to results and patient history at health provider network/monitor210(FIGS. 10 and 12). In block1515, analysis engine130receives the order. In block1520, analysis engine130requests mapping of the order to a protocol, the mapping included in the knowledge base140. In block1525, the analysis engine130sends the protocol associated with the order to the controller120. In block1530, the controller120interrogates the sensor110based on the new protocol. In block1535, the sensor110senses glucose concentrations associated with glucose levels in the interstitial fluid into which the sensor110is implanted in response to each interrogation. In block1540, the controller120determines glucose level estimates based on the sensor measurements over a time period. In block1545the controller120transmits a time series burst of glucose readings to the analysis engine130. In block1550, the analysis engine130analyzes burst(s) of glucose readings to determine trends, patterns, and trigger alerts.

FIG. 16is a flowchart that illustrates an example of a workflow1600of continuous health monitoring incorporating activity data. In some aspects, the workflow1600may be performed by the system700inFIG. 7, the system1000inFIG. 10, and/or the system1200inFIG. 12. In block1605, the analysis engine130receives activity data from the activity sensor system180, and estimates the patient's level of activity. In this embodiment, the analysis engine130determines that the patient is sleeping. In block1610, the analysis engine130issues protocols for the sleeping patient to the controller120. In block1615, the controller120interrogates the sensor110based on a sleeping patient protocol that is included in the controller120. In block1620, the controller120determines glucose level estimates based on the sensor measures over a time period. In block1625, the controller120transmits a time series burst of glucose readings to the analysis engine. In block1630, the analysis engine130analyzes burst(s) of glucose readings to determine trends, patterns, and trigger alerts. The alerts are dependent on the protocol. For example, a patient who is sleeping may have the low glucose alert set to a lower threshold value than a patient that is not sleeping, but exercising. For example, in a sleeping patient, the alarm for a high glucose level may not be triggered if the glucose measurement is slowly climbing above a primary threshold but has not yet crossed a secondary threshold.

Other workflows may include incorporation of bio sensed data or inputs/queries from the patient.

FIG. 17is a flowchart that illustrates an example of a method1700of continuous health monitoring. In some aspects, the method1700may be performed by the controller120inFIGS. 1A, 3A, 4, 7, 10, and 12.

In block1705, method1700emits, by a laser source, a plurality of optical interrogation signals via an optical pathway to a sensor implanted percutaneously in a patient. In an embodiment, the analyte is glucose and the optical pathway is a waveguide. In some embodiments, the functionality of block1705is performed by the laser source emitter125of the controller120illustrated inFIGS. 1A, 3A, 4, 7, 10, and 12.

In block1710, method1700measures, by a detector, a plurality of luminescent emissions from the sensor, the luminescent emissions indicative of an interstitial analyte concentration of the patient. In some embodiments, the functionality of block1710is performed by the detector127of the controller120illustrated inFIGS. 1A, 3A, 4, 7, 10, and 12.

In block1715, method1700determines, by a processor circuit, a measure of analyte concentration based on the detected luminescent emissions. In some embodiments, the functionality of block1715is performed by the processor (processor circuit)121of the controller120illustrated inFIGS. 1A, 3A, 4, 7, 10, and 12.

In block1720, method1700stores, by a memory circuit, the determined measure of analyte concentration. In some embodiments, the functionality of block1720is performed by the memory (memory circuit)123of the controller120illustrated inFIGS. 1A, 3A, 4, 7, 10, and 12.

In block1725, method1700transmits, by a transmitter, the measure of analyte concentration. In some embodiments, the functionality of block1725is performed by the transmitter128of the controller120illustrated inFIGS. 1A, 3A, 4, 7, 10, and 12.

Analyte Sensor and Method of Manufacturing an Analyte Sensor

Disclosed and described herein are embodiments of a layered optical sensor such as, for example, sensor110, that can be used to measure different analytes in a patient. A non-exhaustive list of example analytes that can be measured with embodiments of the present invention include, and are not limited to, glucose, galactose, lactose, peroxide, cholesterol, amino acids, fructose, alcohol, lactic acid, and mixtures of the preceding analytes. In particular, disclosed herein is a unique method for forming a layered optical sensor through a layering technique and capillary filling, as well as a method of mass manufacturing optical sensors. The disclosed sensors can advantageously be quickly and easily manufactured, allowing for mass production for embodiments of the sensor.

Laminate Structure

Accordingly,FIG. 18illustrates an example embodiment of a layered optical sensor for measuring an analyte. The disclosure can relate to a sensor subassembly, and can be incorporated with other sensor features. The analyte can be, for example, glucose, galactose, lactose, peroxide, cholesterol, amino acids, fructose, alcohol, lactic acid, and mixtures of the preceding analytes, but the particular analyte to be measured is not limiting.

As shown, the layered optical sensor from sensor subassembly110A can be composed of a plurality of different layers, where the layers can be located on top of one another. Each of the layers can provide a specific structure or purpose, though other types of layers can be used as well. While the below disclosure discusses the specifics of a three-layer configuration, it will be understood that other numbers of layers could be used (e.g., 2, 4, 5 or more), and the number of layers can vary depending on the internal components of the sensor and the requirements or functions of the sensor.

In some embodiments, a bottom layer1802can be generally stiff, thus allowing for mechanical modulation. Specifically, the bottom layer1802can provide for mechanical integrity of the layered optical sensor, and thus can be the strongest of the layers in some embodiments. Further, the bottom layer1802can have structural support features sufficient to mate with a lancet, or other implantation devices. For example, the bottom layer1802can include protrusions, notches, or attachment mechanisms. In some embodiments, the bottom layer1802can have a particular stiffness to provide durability to the layered optical sensor.

In some embodiments, the bottom layer1802may be formed from a structural polymer, such as a robust biocompliant polymer film of polyether ether ketone (PEEK). However, other materials can be used as well to form the bottom layer1802, such as metals (e.g., Nitinol), plastics, rubbers, and the particular material is not limiting. Preferably, the material forming the bottom layer1802can be biocompatible in order to reduce a patient's response to implantation of the layered optical sensor. However, in some embodiments the material may not be biocompatible, such as if the sensor will on1y be inserted into a patient for a short period of time or if the sensor will be coated with a biocompatible coating.

In some embodiments, the bottom layer1802can be formed of a single piece of material formed in a generally rectangular shape. Thus, in some embodiments there are no cutouts, apertures, holes, or protrusions into the bottom layer1802, un1ike the other below disclosed layers, and the bottom layer1802can be generally flat on the top and bottom. In some embodiments, the bottom layer1802can have beveled and/or tapered edges, which can be advantageous for fitting of layers together.

In some embodiments, the bottom layer1802can have a width of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 mm. In some embodiments, the bottom layer1802can have a length of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mm. However, the particular dimensions of the bottom layer1802are not limiting.

Next, at least one middle layer, or optical sensing layer,1804can be formed on top of bottom layer1802. As mentioned, a plurality of middle layers can be used, each having the same or different configurations, though the use of a single middle layer1804is discussed herein.

The middle layer1804can include a distal section1806and a proximal section1808. The distal section1806can be generally flat, and can be shaped similarly to the distal section of bottom layer1802. In some embodiments, the distal section1806may not have any apertures cut out of it, and thus can be generally the same thickness throughout.

The proximal end1808can include a number of features for the construction of the layered optical sensor. A close-up view of the proximal end1808is shown inFIG. 19. As shown, the proximal end1808can include an enzymatic hydrogel cavity1902and an oxygen sensing polymer cavity1904. WhileFIG. 19shows the discussed features filled with the respective polymers, during construction of the layered optical sensor, and specifically, middle layer1804, these portions are left as empty cavities, and will be filled in a manner as discussed in detail below. The middle layer1804can include other cavities as well, such as the oxygen reference cavity1908and a glucose in1et cavity1906, which can be in fluid communication with the enzymatic hydrogel cavity1902and the oxygen sensing polymer cavity1904. The particular amount and type of cavity in the middle layer1804is not limiting.

Further, as shown inFIG. 19, the proximal end1808can include a number of optical circuits or waveguides1910allowing for optical radiation, such as light, to pass into the oxygen sensing polymer cavity1904and oxygen reference cavity1908.

In some embodiments, the middle layer1804may be formed from polymer, such as a polymer laminate. However, other materials can be used as well, such as metals (e.g., Nitinol), plastics, rubbers, and the particular material is not limiting. Preferably, the material forming the middle layer1804can be biocompatible in order to reduce a patient's response to implantation/insertion. However, in some embodiments the material may not be biocompatible, such as if the sensor will on1y be inserted for a short period of time. In some embodiments, the material of the middle layer1804is the same as the material of the bottom layer1802. In some embodiments, the material of the middle layer1804is the different from the material of the bottom layer1802.

In some embodiments, the dimensions of the middle layer1804are generally the same as that of the bottom layer1802. In some embodiments, the middle layer1804is bigger than the bottom layer1802. In some embodiments, the middle layer1804is smaller than the bottom layer1802. In some embodiments, the middle layer1804can have a width of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 mm. In some embodiments, the middle layer1804can have a length of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mm. However, the particular dimensions of the middle layer1804are not limiting.

Next, as depicted inFIG. 18, a top layer1810can be formed on top of middle layer1804, or plurality of middle layers. The top layer1810can be generally flat, and can be shaped similarly to the bottom layer1802and/or middle layer1804. In some embodiments, portions of the top layer1810may not have any apertures cut out of it, and thus can be generally the same thickness throughout. In some embodiments, the top layer1810may have portions cut out of it to form an oxygen conduit cavity1812. Similar to the middle layer1804, during construction of the layered optical sensor, these oxygen conduit cavities1812are left as empty cavities, and will be filled in a manner as discussed in detail below. In some embodiments, other cavities can be included in the top layer1810. For example, the oxygen reference cavity1908can be moved from the middle layer1804to the top layer1810.

In some embodiments, the top layer1810may be formed from a polymer, such as, for example, a polymer laminate. However, other materials can be used as well, such as metals (e.g., Nitinol), plastics, rubbers, and the particular material is not limiting. Preferably, the material forming the top layer1810can be biocompatible in order to reduce a patient's response to implantation/insertion. However, in some embodiments the material may not be biocompatible, such as if the sensor will on1y be inserted for a short period of time. In some embodiments, the material of the top layer1810is the same as the material of the bottom layer1802and/or the middle layer1804. In some embodiments, the material of the top layer1810is the different from the material of the bottom layer1802and/or the middle layer1804.

In some embodiments, the dimensions of the top layer1810are generally the same as that of the bottom layer1802and/or middle layer1804. In some embodiments, the top layer1810is bigger than the bottom layer1802and/or middle layer1804. In some embodiments, the top layer1810is smaller than the bottom layer1802and/or middle layer1804. In some embodiments, the top layer1810can have a width of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 mm. In some embodiments, the top layer1810can have a length of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mm. However, the particular dimensions of the top layer1810are not limiting.

Further, a top cap layer can be used to seal the top layer1810. For example, the top cap layer can be formed from a silicone pressure sensitive adhesive (PSA). This can be oxygen permeable and glucose impermeable, thus allowing for oxygen to pass through the top cap layer and into the oxygen conduit cavity and preventing glucose or other analytes from passing through . In some embodiments, a conduit hydrogel is dispensed into shaped region in conduit structure. In some embodiments, PSA is directly shaped by embossing to create shaped region. In some embodiments, a punched structure is laminated to the PSA to create shaped region.

FIG. 20Aillustrates an embodiment of a layered optical sensor incorporating all layers discussed above and being filled by the respective polymers. However, as shown inFIG. 20A, the structures can have a slightly different configuration than discussed above. For example, the oxygen conduit cavity1812may not be generally rectangular shaped as discussed above, but can instead take a different configuration. In some embodiments, the oxygen conduit cavity1812may extend into and/or through the middle layer1804.

FIG. 20Bis a cross-section of the layered optical sensor ofFIG. 20Aincorporating all layers previously discussed and being filled by the respective polymers. As depicted, included is a base layer1802, an optical sensing layer1804, which includes a plurality of waveguides/optical circuits1910, an oxygen sensing polymer1904and an enzymatic hydrogel1902, and a conduit layer1810, which includes a reversible oxygen binding protein hydrogel1908.

As mentioned above, the different layers1802,1804, and1810can be bonded together to form a layered optical sensor. In some embodiments, adhesives can be used to bond the layers together. In some embodiments, the layers can be heated in order for the layers to adhere to one another.

FIGS. 20C to 20Eillustrate another embodiment of a layered optical sensor according to embodiments of the present invention.FIG. 20Cis a partial top view of the layered optical sensor andFIGS. 20D and 20Eare cross-sectional views, which are identified inFIG. 20C.

As depicted inFIG. 20C, the layered optical sensor1950includes multiple waveguide cores1952. A reaction chamber1954is formed adjacent to the distal ends of select waveguide cores1952.FIG. 20Dis a cross-sectional view of the reaction chamber1954taken along line A-A inFIG. 20CandFIG. 20Dis a cross-sectional view of the reaction chamber1954taken along line B-B isFIG. 20D.

The layered optical sensor of this embodiment includes a plurality of waveguide cores1952located in an optical sensing layer1956, an oxygen sensing polymer region1958, which is contiguous with and in direct communication with select waveguide cores1952in the optical sensing layer1956, (i.e., the oxygen sensing polymer1958contacts at least a portion of the select waveguide cores1952), an enzymatic reaction region1960, where the region is geometrically defined by the contiguous portions of the enzymatic reaction layer1968and is in direct communication with the oxygen sensing polymer region1958, an oxygen permeable polymer layer1962, an oxygen transport layer1964and a capping layer1966. In some embodiments, the optical sensing layer1956and/or the capping layer1966provide a biocompliant tissue interface.

As can be seen inFIGS. 20D and 20E, the oxygen sensing polymer region1958is constructed to contact select waveguide cores1952and to extend into and between the optical sensing layer1956and the enzymatic reaction layer1968of the sensor body such that the oxygen sensing polymer region1958contacts and is in communication with both the waveguide cores1952and the enzymatic hydrogel in the enzymatic reaction region1960. Prior to filling the oxygen sensing polymer region1958with the oxygen sensing polymer, the waveguide cores1952are exposed to allow direct contact with the oxygen sensing polymer in the oxygen sensing polymer region1958. The enzymatic hydrogel reaction region1960is formed such that a portion of the oxygen sensing polymer in the oxygen sensing polymer region1958will be contiguous with the enzymatic hydrogel in the enzymatic hydrogel reaction region1960, such that the oxygen sensing polymer in the oxygen sensing polymer region1958will define part of the geometric boundary for the enzymatic hydrogel reaction region1960.

In one embodiment, the oxygen sensing polymer region1958is formed and filled prior to the creation of the enzymatic reaction layer1968, such that the oxygen sensing polymer region1958intersects with the plurality of waveguide cores1952. The shape of the enzymatic hydrogel reaction region1960is defined in part by the shaping of the oxygen sensing polymer region1958. The oxygen sensing polymer region1958can be filled with the oxygen sensing polymer using any filling methods disclosed herein, for example, see the capillary action filling section below. As can be seen inFIG. 20E, the oxygen sensing polymer region1958includes a surface1972(which may be an ablated or an embossed portion of the oxygen sensing polymer region1958), which forms a contiguous portion of the enzymatic hydrogel reaction region1960.

In some embodiments, the surface1972is formed along with the enzymatic hydrogel reaction region1960. A gross opening larger than the desired shape for the enzymatic hydrogel reaction region1960is formed in the enzymatic reaction layer1968using a low tolerance method (such as CO2laser cutting), and then the enzymatic reaction layer1968is laminated to the optical sensing layer1956. The oxygen sensing polymer is then dispensed into the gross opening in the enzymatic reaction layer1968and into the contiguous space of the oxygen sensing polymer region1958, using any of the filling methods disclosed herein. In this embodiment, the surface1972, which forms the base of the enzymatic hydrogel reaction region1960and the remainder of the enzymatic hydrogel reaction region1960in the enzymatic reaction layer1968are created by shaping the oxygen sensing polymer that fills the enzymatic reaction layer1968and oxygen sensing polymer region1958.

In some embodiments, the enzymatic hydrogel reaction region1960along with surface1972are created by material displacement of the oxygen sensing polymer while uncured, using the embossing method discussed below, by placement of an embossing insert with a shape to create the enzymatic hydrogel reaction region1960with surface1972, thus forming the enzymatic hydrogel reaction region1960and surface1972upon curing of the polymer.

In some embodiments, the enzymatic hydrogel reaction region1960and surface1972are formed by material removal of cured oxygen sensing polymer in the oxygen sensing polymer region1958. The material removal of the oxygen sensing polymer may be accomplished by laser ablation using, for example, femtosecond, nanosecond, or UV laser systems.

In some embodiments, the surface1972is formed along with enzymatic hydrogel reaction region1960. For this, a gross opening larger than the desired shape of the enzymatic hydrogel reaction region1960is formed in the lower portion of the enzymatic reaction layer1968. In this embodiment, the upper portion of the enzymatic reaction layer1968above the enzymatic hydrogel reaction region1960remains intact, while the adhesive layer that comprises the lower portion of the enzymatic reaction layer1968is modified to form a gross opening larger than the desired enzymatic hydrogel reaction region1960using a low tolerance method (such as CO2 laser cutting). The enzymatic reaction layer1968is laminated to the optical sensing layer1956. The oxygen sensing polymer is dispensed into the lower portion of the gross opening in the enzymatic reaction layer1968and into the contiguous space of the oxygen sensing polymer region1958by means of microfluidic filling from an adjacent filling well and filling vent, i.e., capillary filling. In this embodiment, the enzymatic hydrogel reaction region1960and the surface1972in the oxygen sensing polymer are formed by ablation of the upper portion of the enzymatic reaction layer1968and the lower portion of enzymatic reaction layer1968, which forms the walls of the enzymatic hydrogel reaction region1960and surface1972, which forms the base of the enzymatic hydrogel reaction region1960, which is contiguous with the oxygen sensing polymer in the oxygen sensing polymer region1958. As can be seen inFIG. 20E, forming the surface1972in the oxygen sensing polymer in the oxygen sensing polymer layer1958, ensures that the oxygen sensing polymer in the oxygen sensing polymer region1958and the enzymatic hydrogel in the enzymatic hydrogel reaction region1960are in physical contact with each other and therefore, in communication with each other.

In some embodiments, the surface1972is formed along with the enzymatic hydrogel reaction region1960. The oxygen sensing polymer is dispensed into oxygen sensing polymer region1958. The enzymatic reaction layer1968is then laminated to the optical sensing layer1956without first forming the enzymatic hydrogel reaction region1960. In this embodiment, the enzymatic hydrogel reaction region1960and the surface1972in the oxygen sensing polymer are formed by ablation of select regions of the enzymatic reaction layer1968and the oxygen sensing polymer region1958to ensure that the base of the enzymatic hydrogel reaction region1960is contiguous with the oxygen sensing polymer by forming surface1972. As can be seen inFIG. 20E, forming the surface1972in the oxygen sensing polymer in the oxygen sensing polymer layer1958, ensures that the oxygen sensing polymer in the oxygen sensing polymer region1958and the enzymatic hydrogel in the enzymatic hydrogel reaction region1960are in physical contact with each other and therefore, in communication with each other.

In some embodiments, the oxygen sensing polymer region1958is formed along with the enzymatic hydrogel reaction region1960. The enzymatic reaction layer1968is laminated to the optical sensing layer1956without first forming the enzymatic hydrogel reaction region1960or oxygen sensing polymer region1958. In this embodiment, the enzymatic hydrogel reaction region1960is created by ablation of select regions of the enzymatic reaction layer1968, and the oxygen sensing polymer region1958is created by ablation through the enzymatic hydrogel reaction region1960. In this embodiment, the shape of the oxygen sensing polymer region1958does not intersect with the side walls of the enzymatic hydrogel reaction region1960. The oxygen sensing polymer is then dispensed into the oxygen sensing polymer region1958. The surface of the oxygen sensing polymer then serves as the direct surface1972that interfaces with the enzymatic hydrogel in the enzymatic hydrogel reaction region1960.

After the oxygen sensing polymer is cured, the enzymatic hydrogel reaction region1960can now be filled with the enzymatic hydrogel using any of the filling methods disclosed herein. The enzymatic hydrogel is then crosslinked. In some embodiments, the enzymatic hydrogel is dehydrated prior to application of a subsequent contiguous polymer layer.

Next, an oxygen permeable polymer layer1962is laminated to the enzymatic hydrogel reaction layer1968. The polymer for this oxygen permeable polymer layer1962must be one that is permeable to oxygen and impermeable to the analyte that is being sensed, which in some embodiments, is glucose. This creates an oxygen permeable, analyte impermeable membrane. In some embodiments, the oxygen permeable polymer layer1962is laminated along with an oxygen transport layer1964. In some embodiments, the oxygen transport layer1964contains a reversible oxygen binding molecule. In some embodiments, the oxygen transport layer1964contains a hydrogel that includes a reversible oxygen binding molecule.

In some embodiments, a capping layer1966is laminated to the oxygen transport layer1964. In some embodiments, the capping layer1966provides mechanical stabilization to oxygen transport layer1964

After the lamination and filling of the polymer laminate structure of this embodiment with active hydrogels and the oxygen sensing polymer, the physical structure of individual optical sensors is attained by laser cutting the final shape of the individual sensors from the upper exposed layer through the bottom exposed layer.

In some embodiments, the enzymatic reaction layer1968also serves as a mechanical support for the sensor1950to enable implantation into and extraction from tissue. In some embodiments, the lower portion of the enzymatic reaction layer1968(the adhesive layer) in the region of the sensor tip is removed and this region used to form a looped sensor lancet interface3140as described below. In some embodiments, the oxygen permeable polymer layer1962in the region of the sensor tip is removed and this region used to form a looped sensor lancet interface3140. In some embodiments, the oxygen permeable polymer layer1962, and the oxygen transport layer1964in the region of the sensor tip are removed and this region used to form a looped sensor lancet interface3140.

In some embodiments, the oxygen permeable polymer layer1962, the oxygen transport layer1964and the capping layer1966are removed in the region of the optical input to form the optical sensing layer1956. In some embodiments, the region of the optical input to the optical sensing layer is an optical microlens array.

In some embodiments, the layers comprising the optical sensors1950are laminated to create a plurality of optical sensors1950in a card, where the laminate layers each comprise at least10,20,50, or at least100optical sensors1950.

Embossing

As discussed above, the layered optical sensor can be formed by the combination of a number of different layers. Specifically, embossing can be used to produce precise internal structures by leveraging techniques from silicon wafer manufacturing.

During the manufacturing of the layers discussed above, inserts can be used to form specific cavities, such as those discussed above. Thus, the polymer of the particular layer will pass around the outside of the insert. For example, a rectangular mold can be used to form the top layer1810. An insert can then be placed on the mold in the desired shape and desired location of the oxygen conduit cavity1812. Then, when the layer1810is solidified, such as through curing, and the insert is removed, the oxygen conduit cavity1812will remain in the solidified layer. This can be done for all of the layers and cavities discussed above.

In some embodiments, embossing can also be used to fill specific cavities located within or next to other cavities. Thus, for example, an insert can be placed into the enzymatic hydrogel cavity1902in the shape of the oxygen sensing polymer cavity1904while the enzymatic hydrogel is filled. Once the hydrogel is solidified, for example through UV curing, the insert can be removed, and the oxygen sensing polymer can be filled in the oxygen sensing polymer cavity1904remaining adjacent to the enzymatic hydrogel cavity1902. Thus, the enzymatic hydrogel and oxygen sensing polymer can be adjacent and in communication with one another. Further, a second insert can be used in a similar fashion to form the glucose in1et cavity1906. Thus, the oxygen sensing polymer can be filled, followed by the enzymatic hydrogel, while still leaving the glucose in1et cavity1906in communication outside the sensor.

The embossing technique described is shown inFIG. 21. As shown, a portion of a hydrogel2102in the sensor can be embossed through placement of an insert, thus leaving a cavity2104formed. This cavity2104can then be filled with another type of hydrogel2106, thus forming adjacent hydrogels in communication with one another.

Further, in some embodiments, embossing can be used to form the cavities for waveguides, ink well, and registration markings embossed into an ultraviolet curable optical polymer, such as, but not limited to, UV curable acrylate (bottom clad). In some embodiments, ink is deposited into the ink well and flows into the ink registration markings. Next, UV curable acrylate with a higher index of refraction than a base clad index of refraction is coated to fill the embossed cavities in the bottom CLAD (CORE). The CORE material may also fill the remainder of the ink well and registration markings that were not filled with ink. Next, the core material is cured. Next, the top clad material with an index of refraction lower than the CORE material is coated over the bottom clad and core material. In some embodiments, the top CLAD material may be embossed with a pattern for luminescent oxygen sensing dye or other registration marks. Next, the top clad material is cured.

In some embodiments, once the embossing procedures are performed, the different layers can be laminated together to form a layered optical sensor with empty cavities to be filled with the oxygen sensing polymer, etc.

Capillary Filling Methodology

In some embodiments, capillary action (e.g., wicking, microfluidic filling) can be used to fill the different cavities in the layered optical sensor. This action allows liquid to flow in narrow spaces without the assistance of (or in opposition to) external forces, such as gravity. Capillary action can occur as the combination of surface tension and adhesive force between the liquid and the surfaces contacting the liquid can act to move the liquid from one location into a narrower location or cavity.

In some embodiments, the oxygen sensing polymer cavity1904and the enzymatic hydrogel cavity1902can be accessible from the surface of the middle layer1804through the glucose in1et cavity1906. In some embodiments, the oxygen sensing polymer cavity1904and the enzymatic hydrogel cavity1902can be shaped so that the accessible surface area of the oxygen sensing polymer cavity1904and the enzymatic hydrogel cavity1902is less than the cross-sectional area of the oxygen sensing polymer cavity1904and the enzymatic hydrogel cavity1902in at least one substantially orthogonal dimension.

In some embodiments, the cavities discussed above (e.g., oxygen sensing polymer cavity1904, enzymatic hydrogel cavity1902, oxygen reference cavity1908, and oxygen conduit cavity1812) can be filled through the use of capillary action. For example, a larger volume of hydrogel/polymer, depending on what is to be filled, can be located adjacent to outlets of the different cavities, such as the glucose in1et cavity1906. Capillary action can force and/or draw in a portion of the hydrogel/polymer from the larger volume of hydrogel/polymer1931into the particular cavity, as shown inFIG. 22. In some embodiments, the larger body volume of hydrogel/polymer1931can be a milliliter volume while the volume of cavities to be filled can be measured in picoliters.

In some embodiments, the larger volume can be pre-treated in order to fill the cavities. For example, for hydrophobic or amphipathic surfaces, an amphipathic pretreatment solution is dispensed to allow hydrogel filling by capillary action. In some embodiments, the dispensing solution can be Hydroxyethylmethacrylate (HEMA) in water and ethanol. In some embodiments, the dispensing solution can be HEMA in water and isopropyl alcohol. In some embodiments, the dispensing solution is volatilized. In some embodiments, the dispensing solution is not volatilized.

In some embodiments, the cavities can be filled simultaneously. In some embodiments, the cavities can be filled one after another.

In some embodiments, the cavities can be laterally filled into picoliter volumes in hydrophobic, amphipathic, or hydrophilic surfaces from nanoliter or microliter adjacent volumes.

Methods of Manufacturing

Advantageously, embodiments of the disclosed layered optical sensor can be mass manufactured, thus allowing the layered optical sensor to be produced cheaply as compared to other sensors in the art. Thus, consumers can experience the benefit of the mass production by being able to purchase and use sensors, particularly glucose sensors, without having to pay significant sums of money. Thus, low income users, such as elderly patients, will not have to worry as much about their ability to purchase high priced medical devices.

FIG. 23illustrates an example of a method of manufacturing the layered optical sensor. First, raw optical sheets, which can be layers, can be produced into a sheet. As shown, a significant amount of the sensor can be formed at once from a single sheet. For example, 10, 20, 100, 200, 250, 300, 350, 400, 500, or 1000 sensor cards can be formed per sheet. The sensor cards can be semi-individuated, thus allowing for ease of splitting apart all the sensors on the sheet. A top layer can be attached to the raw optical sheets, thus forming ready to fill sheets shown inFIG. 24.

These ready to fill sheets can be filled with different hydrogels/polymers, such as described in detail above, to form a plurality of semi-individuated filled sensor cards.

Further, electronic components can be attached to the plurality of filled sensor cards. The sensor cards can be calibrated while they are in a semi-individuated form in an array. The sensor cards can be calibrated by exposing each of them to fluids under fixed test conditions with sterile glucose, or other analyte, and oxygen of known concentrations and monitoring each sensor card response. In some embodiments, semi-individuated sensors can be fully functional and can be optically interrogated to test the devices and to generate individual calibration parameters for each sensor at the card level.

Each sensor card in the array can have a unique identity that can be registered during calibration. Thus, calibration parameters for each sensor can be generated from these optical measurements associated with the specific card and stored for subsequent retrieval. In some embodiments, the use of retrieving calibration information from 2D barcodes, near field communications (NFC), and radio frequency identification (RFID) can be used to transmit and receive the calibration data and information.

After calibration, the sensor cards can be assembled with other devices, such as delivery devices. In some embodiments, the sensor cards are not assembled with delivery devices. The sensor cards can then be packaged as desired, and can be sterilized for use in a patient. In some embodiments, the sensor cards are sterilized before packaging. In some embodiments, the sensor cards are not sterilized.

Thus, as shown inFIG. 23and described herein, hundreds of sensor cards can be quickly and easily manufactured and calibrated. Thus, the cost of the layered optical sensors can be drastically reduced, allowing easier access to patients.

Manufacturing Method Embodiments

In some embodiments, the herein disclosed sensors can be manufactured using a reel to reel manufacturing process. In some embodiments, the first step in this reel to reel manufacturing process is to create or form polymer laminate thin film waveguides to be used in the sensors. In some embodiments, the waveguides are formed as a multilayer laminate structure.

Waveguide formation begins with the embossing of a plurality of waveguide structures into a sheet material included on a roll. Depicted inFIG. 51is an embodiment of a process7000to create the plurality of waveguides. First, an embossing plate7002is created. The embossing plate7002is a positive feature tool/plate, typically in metal, however, other materials may be used, that is used to emboss the negative features of the waveguide into a polymer material.

Depicted inFIG. 52is an embodiment of an embossing plate7002, which includes108positive waveguide structures7004. As used herein, each set of108waveguide structures7004that are embossed with an embossing plate will be referred to as a card7005. As can be seen in the figure, each positive waveguide structure7004includes a unique barcode7006and a set of fiducials7008, both of which are depicted inFIG. 53. The fiducials7008are markings for the waveguides7004that allow the position of the waveguides to be seen/identified throughout the manufacturing process. The fiducials, which include a plurality of cross-hairs, are essentially registration marks that provide for optical positioning alignment of the waveguides throughout the sensor manufacturing process. For example, as depicted inFIG. 54, the fiducials7008are used to properly position and mount the optical engine7010(the optical interconnect, optical interface, etc. as discussed in more detail below) onto the completed laminate structure that forms the sensor.

The barcodes7006are included such that each waveguide on the embossing plate7002has a unique identifier. Although the barcodes will be repeated in the material that is embossed with embossing plate7002as the same embossing plate7002is used to emboss multiple cards7005, as discussed below, each time the embossing plate7002embosses another card7005of waveguide structures7004, a unique barcode associated with the embossing of that card7005, is embossed as well. That is, the barcode associated with each card7005is changed between subsequent embossing of cards7005. Thus, when the individual barcodes7006for each waveguide structure7004on the embossing plate7002are combined with the unique barcode for each embossed card7005, each waveguide structure7004produced and hence, each sensor that incorporates a waveguide structure7004, has a unique identification number that can be tracked and used as part of the design history record.

Referring again toFIG. 51, once the embossing plate(s)7002are constructed, they are loaded onto a heated roller7012. Although multiple embossing plates7002are depicted, a single embossing plate7002may sufficient. Additional embossing plates7002can be added to the heated roller7012to increase waveguide structure7004production rates. After the embossing plates7002are loaded onto the heated roller7012, the embossing process can begin.

The material to be embossed (the embossing layer7014, which is the main component in the optical layer) must be a polymer material that is (1) bio compliant, (2) can accept, receive and maintain the micro patterns and textures (i.e., the required bevels for the waveguide structures7004, etc.) from the embossing plate7002and (3) has the required optical properties (i.e., cladding properties including the index of refraction (n)) to prevent/reduce light from exiting the waveguides at unintended locations. In one embodiment, this polymer material7014is polyvinylidene fluoride (PVDF).

FIG. 51depicts four embossed cards7005. As depicted inFIG. 55, each card7005has an individual set of108embossed waveguide structures7004with each waveguide structure7004having a unique barcode7006on that card7005and each card7005having a unique barcode7018as discussed above. Thus, this barcode7018changes each time the embossing plate7002embosses a new waveguide card7005. The heated roller7012combined with the embossing plate7002embosses the waveguide structures7004to a depth within the embossing layer7014of approximately 40 μm. In some embodiments, the waveguide structures7004are embossed to a depth of approximately 20 μm, 30 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm or even deeper depending on the thickness of the embossing layer7014.

After the waveguide structures7004and associated barcodes7006and fiducials7008are embossed as depicted inFIG. 51, ink7020is dispensed into the barcodes7006and fiducials7008. In some embodiments, because the barcodes7006and fiducials7008are micro structures, these structures can be filled microfluidically as discussed above, by adding ink to one of the circular areas in either fiducial7008and allowing the ink to “wick” by capillary action, into the remaining portions of the fiducials708and the barcodes7006. In some embodiments, the barcodes7006and fiducials7008are filled with ink with a knife coating process as will be readily understood by those of skill in the art. In some embodiments, after the ink7020is dispensed into the barcodes7006and fiducials7008, the ink dispense is inspected7022to insure the barcodes7006and fiducials7008are satisfactorily filled with ink7020.

As depicted inFIG. 51, the next step in the waveguide formation process7000is the waveguide structure7004filling process7024. In this step, the waveguide structures7004are filled with a core UV curable material7026. While UV curable materials are discussed herein for the core material and adhesives, these materials are not so limited and can include thermal curable materials as well. In some embodiments, the core UV curable material7026is a polymer that has the requisite high index of refraction (n) to direct excitation light and emission light (as discussed in more detail below) along and through the waveguides. In some embodiments, the core UV curable material7026is an epoxy that has the requisite high index of refraction (n) to direct excitation light and emission light (as discussed in more detail below) along and through the waveguides. In some embodiments, the core UV curable material7026is applied with a knife coating process as will be readily understood by those of skill in the art. In some embodiments, after the waveguide structures7004are filled with the core UV curable material7026, the filled waveguide structures7004are inspected7028. After the filled waveguide structures7004pass inspection, a clad coating layer7030is applied on top of and cured to the embossing layer7014that contains cards7005with the filled waveguide structures7004. The clad coating layer7030can be attached/cured to the embossing layer with, for example, a UV curable adhesive Similar to the requirements for the embossing layer7014, the clad coating layer7030must have the required optical properties (i.e., cladding properties including the index of refraction (n)) to prevent/reduce light from exiting the waveguides at unintended locations. With the application of the clad coating layer7030, the laminate structure for the waveguides and hence, the optical layer, is complete. The completed length of the multilayer waveguide laminate structure, which can include a plurality of completed cards7005, can be rolled onto a reel for use in the next steps of the sensor manufacturing process.

Depicted inFIG. 56is a cross-section of an embodiment of a multilayer waveguide laminate structure7032constructed in accordance with the disclosed embodiments. In this embodiment, the embossing layer7014is PVDF with a thickness of approximately 75 μm and an index of refraction n=1.42. The depth of the embossed waveguide structures7004is approximately 40 μm. The waveguide structures7004are filled with core UV curable epoxy7026that has an index of refraction n=1.5037. The top clad coating layer7030is epoxy with a thickness of approximately 25 μm and an index of refraction n=1.42. In all embodiments, in order to prevent light from exiting the waveguides7004, the index of refraction of the core UV curable epoxy7026needs to be higher than the index of refraction of both the embossing layer7014material and the top clad coating layer7030material. The embossing layer7014and the top clad coating layer7030are attached to each other with a UV curable adhesive7034. As can be seen in the embodiment depicted inFIG. 56, in some embodiments, the embossed waveguide structures7004can have beveled or angled sidewalls7036, which allows the embossing plate7002to be clean1y removed from the embossed polymer material.

The next component in the manufacturing process is the reaction chamber (“RC”) laminate structure. Similar to the manufacturing of the multilayer waveguide laminate structure, the RC laminate structure can be manufactured using a reel to reel manufacturing process. Depicted inFIG. 57is an embodiment of a reel to reel process to manufacture an RC laminate structure8000. In some embodiments, the RC laminate structure8000is a multilayer structure that includes at least the following layers: (1) a bottom pressure sensitive adhesive (PSA) layer8002(which is preferably a bio compliant adhesive that is preferably a hydrophobic and which, in some embodiments, is a synthetic rubber), which can include a bottom release liner8004, which can be, for example, a polyethylene terephthalate (PET) liner and/or a top release liner8006, which can be, for example, a PET liner, both of which protect the PSA, (2) a middle polyether ether ketone (PEEK) layer8008, which provides a mechanical core to the sensor, and (3) a top removeable liner8010that protects the RC laminate structure during the manufacturing process. The top removeable liner8010is important to a successful manufacturing process for a couple of additional reasons. When the resulting composite laminate structure that includes both the multilayer waveguide laminate structure and the RC laminate structure is filled with polymers and hydrogels, it is inevitable that there will be over splash. Any over splash included on the top surface, which will be laminated to the conduit layer as discussed below, will result in poor bonding strength between the structures and hence, possible delamination of the final laminated structure. Thus, prior to laminating the RC laminate structure to the conduit layer, the top removeable liner8010can be removed, exposing a clean surface for bonding to the conduit layer. Additionally, the top removeable liner8010increases the thickness of the RC laminate structure. Accordingly, the cavities that are created in the RC laminate structure (as discussed in more detail below), will be deeper and will have a higher volume. Higher volume cavities allow for a more diluted material to be flowed into the cavities because a higher volume of a diluted material can have the same effectiveness as a lower volume of a less diluted material. Materials that are diluted have a lower viscosity, which allows them to flow with less resistance, which is important when relying on microfluidics and capillary action to fill the cavities as is the case with the embodiments of the present invention.

As discussed in more detail below, constructing the RC laminate structure8000and hence, the sensor, as a multilayer laminate structure allows certain features that are necessary in the manufacturing of the sensor and operation of the sensor, to be included (typically, laser cut) into certain layers during the manufacturing process. Constructing the sensor in this manner, permits a very reproducible, high-speed, high tolerance, automated manufacturing process that allows for high volume production at a reduced cost.

Because having the PSA in the PSA layer8002contacting the PEEK layer, which is laminated to the PSA layer8002in a later step and into which the loop portion of the sensor (discussed in more detail below) will be laser cut, can have a detrimental effect on the loop portion, certain areas in the PSA layer8004are laser cut to remove the PSA in these areas. Thus, in some embodiments, laser cutting of a nose feature8012occurs to remove the PSA in the area8014of the laminate structure where the sensor loop8016will eventually be laser cut out of the PEEK material (seeFIG. 54). Thus, once the RC laminate structure8000is laminated to the multilayer waveguide laminate structure7032as discussed in more detail below, a void will be created in the completed laminated structure where the nose feature8012was laser cut.

This initial laser cutting of the nose features8012may be a non-registered laser cut. That is, there are no previous laser cuts or other registration marks/fiducials in the RC laminate structure to use as a reference for the nose feature8012laser cuts. However, once the nose feature8012laser cuts are performed, these laser cuts can now be used as reference/registration marks for any subsequent laser cuts/cutting in the laminate structure. Thus, all subsequent laser cuts will now be registered laser cuts that all relate back to the nose feature8012laser cuts. This is helpful because in all new laminate structures that are manufactured, all laser cuts will have the same positioning in relation to the nose feature8012laser cuts, which results in a very high quality manufacturing process because it is reproducible and has very little variations.

This laser cutting of features that are “sandwiched” between adjacent layers is not limited to cutting of completed laminate structures but can also be performed on the individual layers that make up the laminate structure before the individual layers are laminated together to form a laminated structure. Manufacturing the laminate structures in this manner, allows voids and fill channels to be created in the different laminate layers, where the voids can be filled with liquids such as, for example, the oxygen sensing polymer and the enzymatic hydrogel, though fill ports that are laser cut into the different laminate layers, after the laminate structure is assembled. Thus, when the individual layers are laminated together, the features cut into the individual layers line up and combine to form the required voids, flow channels and fill wells in the assembled laminate structure.

Based upon which areas in the laminate structure need to be filled with certain liquids, the fill wells can be created in the laminate structure accordingly. Constructing the laminate structure in this manner permits the voids to be filled microfluidically, which results in completely filling the voids with accurate volumes of material. Because the fill wells are being filled with picoliter or microliter volumes of liquids in order to fill nanoliter volume voids, once the liquids are deposited into the fill wells, they “wick” into the voids through capillary action and fill the associated volumes within the laminate structure.

Turning back toFIG. 57, after the nose feature8012is laser cut into the PSA layer8002, the top release liner8006is removed at8018and the PEEK layer8008and top removeable liner8010are laminated to the PSA layer8002. Because, as discussed above, the nose feature8012was laser cut into the PSA layer8002in the area8014of the laminate structure where the sensor loop8016will eventually be laser cut into the PEEK layer8008, the PSA in this area does not contact the PEEK layer8008. Next, any features that need to be laser cut through all of the layers of the RC laminate structure8000are laser cut at8020.

Depicted inFIG. 58is a bottom view of an RC laminate structure8000constructed in accordance with the disclosed embodiments. Similar to the arrangement of the waveguide structures7004on the multilayer waveguide laminate structure, the elements of the RC laminate structure8000are arranged in groups of108to correspond to the108waveguide structures7004on each waveguide card7005. Depicted inFIG. 58are the elements that have been laser cut through all three RC laminate structure8000layers as well as elements that have on1y been laser cut into the PSA layer8002. The elements that have been laser cut through all three RC laminate structure8000layers include the optical chip openings8022, oxygen sensitive/sensing polymer filling ports/wells8024and vent openings8026, which allow air to escape when the oxygen sensitive/sensing polymer is being added to the laminate structure. In this embodiment, laser cut on1y into the PSA layer8002are the nose features8012for the sensor loops8016. Although on1y a few of the nose features8012are shown cut into the PSA layer8002, each chip opening8022will have a corresponding laser cut nose feature8012.

After construction of the RC laminate structure8000is completed, the RC laminate structure8000can be laser cut to form individual RC laminate cards8030, as depicted inFIG. 58, similar in size to the waveguide cards7005for laminating to the waveguide cards7005. These cards8030are kiss cut through all layers except the bottom release liner8004so they can remain together on the reel of material/release liner8004for laminating to the waveguide cards7005either in a later reel to reel process or by manually peeling each RC laminate card8030away from the release liner8004for laminating to the waveguide cards7005.

With the RC laminate structure8000completed, the RC laminate structure8000can now be laminated to the multilayer waveguide laminate structure. For this lamination process, the individual waveguide cards7005that make up the multilayer waveguide laminate structure are individuated from one another and placed into a card or metal frame8032as depicted inFIG. 59. The individuating and placement of the waveguide cards7005into the metal frame can be done manually or with an automated reel to reel process. Once the waveguide cards7005are placed into the metal frame8032, the bottom release liner8004can be peeled off of the RC laminate cards8030exposing the PSA layer8002and placed on top of the waveguide cards7005in the metal frame8030, thereby laminating the RC laminate card8030to the top of the waveguide cards7005with the PSA layer8002.

With the RC laminate card8030laminated to the waveguide card7005, the reaction chambers8050can now be laser cut into the composite laminate structure.FIG. 60is a magnified view of the distal portion8053(seeFIG. 61, which shows the distal portion8053of the waveguides7004, which is the portion of the waveguide that will be inserted into a patient's tissue and the proximal portion8054of the waveguides7004, which will be coupled to the optical chip as depicted inFIG. 43A) of a waveguide structure7004. A control port8056is laser cut above the oxygen reference waveguide core8060to expose the oxygen reference waveguide core8060and the reaction chamber8050is laser cut above the three remaining waveguide cores8062to expose these waveguide cores8062. In order to expose the tops of the waveguide cores, the laser cuts through the top removeable liner8010, the PEEK layer8008and the PSA layer8002of the RC laminate card8030. Additionally, a dispensing port8064is laser cut adjacent to and contiguous with, the reaction chamber8050. After the waveguide cores8060,8062are exposed, as depicted inFIG. 62, an open groove8070is laser cut across the tops of the waveguide cores8060,8062thereby connecting all four waveguide cores8060,8062with the dispensing port8064. Lastly, bevel8072or stepped8074surfaces are laser cut into each of the waveguide cores8060,8062. The beveled and stepped surfaces8072,8074direct light into and out of the waveguide cores8060,8062.FIGS. 63A and 63Bdepict the beveled cuts/surfaces8072and the stepped cuts/surfaces8074where arrows8073indicate the direction that light travels into and along the waveguide cores8060,8062. A stepped surface8074redirects more light into the waveguide core than the beveled surface8072for oxygen sensing polymers with a lower index of refraction than the waveguide core index of refraction because the faces of the step are perpendicular to the light path of the waveguide channel, while a flat bevel face would orient the light from oxygen sensing polymer and into the waveguide core away from the waveguide core rather than through the waveguide core. In some embodiments, in order to permit easy detection of the depth of the stepped waveguide cuts/surfaces, the surface of the clad layer or embossing layer7014may have an opaque or color layer.

After the bevel8072or stepped8074surfaces are laser cut into each of the waveguide cores8060,8062, the oxygen sensitive/sensing polymer8080is dispensed into the dispensing port8064. Due to microfluidics, the oxygen sensitive/sensing polymer8080is wicked along the open groove8070and fills all four beveled or stepped surfaces8072,8074created in the waveguide cores8060,8062. After the oxygen sensitive/sensing polymer8080is cured, the enzymatic hydrogel8082is dispensed into the dispensing port8064and due to microfluidics, flows into the reaction chamber8050forming a layer on top of the oxygen sensitive/sensing polymer8080.FIG. 64Adepicts a cross-section taken along line A-A inFIG. 62andFIG. 64Bdepicts a cross-section taken along line B-B inFIG. 64A. Both figures show the waveguide cores8060,8062, the oxygen sensitive/sensing polymer8080, and the enzymatic hydrogel8082after being dispensed and cured in the reaction chamber8050. The figures depict a filled stepped cut8074.

In some embodiments for manufacturing the composite laminate structure that comprises the multilayer waveguide laminate structure and the RC laminate structure8000, the waveguide cores8060,8062are laser cut to include the beveled or stepped surfaces8072,8074prior to the multilayer waveguide laminate structure being laminated to the RC laminate structure. In these embodiments, a lateral oxygen sensitive/sensing polymer fill channel (as discussed below), will also be laser cut into the PSA layer8002at the same time the nose features8012are laser cut into the PSA layer8002. These embodiments where the waveguide cores8060,8062are laser cut to include the beveled or stepped surfaces8072,8074prior to the multilayer waveguide laminate structure being laminated to the RC laminate structure, will now be described in detail.

FIGS. 65A and 65Bdepict top and bottom views, respectively, of an RC laminate structure8000constructed in accordance with the embodiment where the waveguide cores8060,8062are laser cut to include the beveled or stepped surfaces8072,8074prior to the multilayer waveguide laminate structure being laminated to the RC laminate structure. In the embodiment depicted, in addition to the nose feature8012being laser cut into the PSA layer8002, lateral oxygen sensitive/sensing polymer fill channels8028are also laser cut into the PSA layer8002. Similar to the previous embodiment of the RC laminate structure8000, the elements of the RC laminate structure are arranged in groups of108to correspond to the108waveguide structures7004on each waveguide card7005. Depicted inFIG. 65Aare the elements that have been laser cut through all three RC laminate structure8000layers. Included are the optical chip openings8022, oxygen sensitive/sensing polymer filling ports/wells8024and vent openings8026that allow air to escape when the oxygen sensitive/sensing polymer and the enzymatic hydrogel are being added to the laminate structure. As can be seen inFIG. 65B, the optical chip openings8022, oxygen sensitive/sensing polymer filling ports/wells8024and vent openings8026extend through the laminate structure and through the PSA layer8002and any respective liners. As can also be seen inFIG. 65B, in this embodiment, laser cut on1y into the PSA layer8002are the nose features8012for the sensor loops8016and the lateral oxygen sensitive/sensing polymer fill channels8028, which connect the oxygen sensitive/sensing polymer fill wells8024with the areas of the reaction chamber into which the oxygen sensitive/sensing polymer must fill.

After construction of the RC laminate structure8000is completed, the RC laminate structure8000can be laser cut to form individual RC laminate cards8030, as depicted inFIGS. 65A and 65B, similar in size to the waveguide cards7005for laminating to the waveguide cards7005. These RC laminate cards8030are kiss cut through all layers except the bottom release liner8004so they can remain on the reel of material/release liner8004for laminating to the waveguide cards7005in a later reel to reel process.

With the RC laminate structure8000completed, the RC laminate structure8000can now be laminated to the multilayer waveguide laminate structure (the waveguide cards7005), which was previously laser cut such that the waveguide cores8060,8062include the beveled or stepped surfaces8072,8074. For this lamination process, the individual waveguide cards7005that make up the multilayer waveguide laminate structure are individuated from one another and placed into a card or metal frame8032as depicted inFIG. 59. The individuating and placement of the cards7005into the metal frame can be done manually or with an automated reel to reel process. Once the cards7005are placed into the metal frame8032, the RC laminate cards8030can be peeled off of the bottom release liner8004exposing the PSA layer8002and placed on top of the waveguide card7005in the metal frame8032, thereby laminating the RC laminate card8030to the top of the waveguide card7005with the PSA layer8002. Depicted inFIG. 66is a cross-section of the completed composite laminate structure8090mounted in metal frame8030.

With the RC laminate card8030laminated to the waveguide card7005, the reaction chambers8050can now be laser cut into the composite laminate structure8090.FIG. 67is a blow up of a portion9000of the composite laminate structure8090. Shown inFIG. 67are the fiducials7008, a partial view of the optical chip opening8022, the nose feature8012cut into the PSA layer8002, oxygen sensitive/sensing polymer fill ports8024, vent opening8026, the lateral oxygen sensitive/sensing polymer fill channels8028that are laser cut into the PSA layer8002, the proximal portions8054of the waveguides structures7004and distal portions8053of the waveguide structures7004, which extend into the area where the lateral oxygen sensitive/sensing polymer fill channels8028are located and which include the beveled or stepped surfaces8072,8074that were laser cut into the waveguide cores8060,8062. Thus, the lateral oxygen sensitive/sensing polymer fill channels8028are located below the top surface (i.e., below the top removeable liner8010of the RC laminate structure) depicted inFIG. 67.FIG. 68is a perspective rendering of a portion of the composite laminate structure8090depicted inFIG. 67

With the composite laminate structure8090being fully assembled, the composite laminate structure8090can now be filled with the oxygen sensitive/sensing polymer. To fill the beveled or stepped surfaces8072,8074that were laser cut into the waveguide cores8060,8062prior to laminating the waveguide cards7005and the RC laminate cards8030together, the oxygen sensitive/sensing polymer is dispensed into the oxygen sensitive/sensing polymer fill ports8024. Due to microfluidics, the oxygen sensitive/sensing polymer is wicked into the lateral oxygen sensitive/sensing polymer fill channels8028and flows across the beveled or stepped surfaces8072,8074in the waveguide cores8060,8062until it reaches the vent opening8026where due to contact with air, it stops flowing.FIG. 69shows a portion of the composite laminate structure8090, which includes the nose features8012, the oxygen sensitive/sensing polymer fill ports8024, vent openings8026, the lateral oxygen sensitive/sensing polymer fill channels8028and distal portions8053of the waveguide structures7004, after the composite laminate structure8090has been filled with the oxygen sensitive/sensing polymer. Thus, after the oxygen sensing polymer is cured, the beveled or stepped surfaces8072,8074remain filled with oxygen sensitive/sensing polymer.

Next, after the oxygen sensing polymer is cured, as can be seen inFIGS. 60 and 68, reaction chambers8050, reference ports8056and an associated enzymatic hydrogel dispensing port/well8058are laser cut into the composite laminate structure8090into the lateral oxygen sensitive/sensing polymer fill channels8028which is the area where the oxygen sensitive/sensing polymer has filled the beveled or stepped surfaces8072,8074in the waveguide cores8060,8062. In this embodiment in which the oxygen sensing polymer is filled to intersect with the waveguide cores prior to reaction chamber formation, the reaction chambers8050are laser cut to a depth within the cured oxygen sensing polymer that is deep enough to receive a sufficient amount of enzymatic hydrogel but not deep enough to destroy the interface of the oxygen sensing polymer and the waveguide cores8060,8062or to expose the waveguide cores8060,8062. In some embodiments, laser cutting into the oxygen sensitive/sensing polymer layer can form a surface such as surface1972as described herein with respect toFIG. 20E.

After the reaction chambers8050are formed, including cutting into the oxygen sensitive/sensing polymer, the composite laminate structure8090can now be filled with enzymatic hydrogel. Thus, the enzymatic hydrogel is dispensed into the enzymatic hydrogel dispensing ports/wells8058where it then wicks by capillary action into the reaction chambers8050and into the cavities that were laser cut into the oxygen sensing polymer. Depicted inFIG. 70is a drawing showing the relationship between the oxygen sensitive/sensing polymer fill ports8024, the oxygen sensitive/sensing lateral fill channels8028, the vent openings8026, the waveguide cores8060,8062, the reaction chambers8050and the enzymatic hydrogel dispensing port/wells8058

In the disclosed embodiments, after the reaction chamber is filled with the oxygen sensing polymer and cured and the enzymatic hydrogel and cured, the conduit laminate9050(which is the transport layer/region), can be applied to the composite laminate structure8090. As depicted inFIG. 71, in some embodiments, the conduit laminate9050includes a bottom PET easy release liner9052, a silicon PSA layer9054, a medical grade PET layer9056, another silicon PSA layer9058and top PET tight release liner9060. Similar to the RC laminate cards8030, the conduit laminate9050is manufactured to have a layout that matches the layout on the waveguide cards7005. Thus the conduit structures/openings (optical chip openings8022, fill wells8024, etc.) are laid out in groups of108. Also similar to the RC laminate cards8030, the conduit laminate9050can be laser cut to form individual conduit laminate cards similar in size to the waveguide cards7005and the RC laminate cards8030for laminating to the waveguide cards7005and the RC laminate cards8030. The conduit cards are kiss cut through all layers except the bottom PET easy release liner9052so they can remain together on the reel of material/release liner9052for laminating to the composite laminate structure8090either in a later reel to reel process or by manually peeling each conduit laminate card away from the easy release liner9052for laminating to the composite laminate structure8090.

To laminate the conduit laminate card9062to the RC laminate card8030of the composite laminate structure8090, as depicted inFIG. 72, the top removeable liner8010is removed from the RC laminate card8030and the bottom easy release liner9052is removed from the conduit laminate card9062exposing the silicon PSA layer9054. The conduit laminate card9062is then placed on top of the RC laminate card8030in the metal frame8032, thereby laminating the conduit laminate card9062to the top of the RC laminate card8030and hence, the composite laminate structure8090with the silicon PSA layer9054. As can be seen inFIG. 72, in some embodiments, the conduit laminate9050includes a conduit hydrogel fill well9064.

FIG. 73depicts a completed laminated structure9080(except for a capping layer). The completed laminated structure9080has been filled with the conduit hydrogel9066. To complete the sensor loop laminated structure, as depicted inFIGS. 54, 72 and 73, the sensor loop8016is laser cut into the PEEK layer8008in the area9068above the nose feature8012. After the sensor loop8016is laser cut, the108individual sensors that are included on a completed card9070(seeFIG. 74), are laser cut to create individual sensors9072. With the laminated structure being completed, except for any capping layer, and laser cut, the optical chip/engine7010can be added to the optical chip opening8022as depicted inFIG. 75.

Depicted inFIGS. 76-86is a laminate structure manufacturing method according to another embodiment of the invention.FIG. 76depicts a waveguide structure7004constructed in accordance with any of the embodiments described herein. The waveguide structure7004includes an embossing layer material7014and a plurality of waveguide cores8060,8062. After the waveguide structure7004is constructed, a top clad coating layer and liner7030(not shown inFIG. 76but described with respect toFIG. 56and shown inFIG. 77) is added on top of the embossing layer material7014and the plurality of waveguide cores8060,8062. This top clad coating layer and liner7030is added to keep the waveguide structures7004clean during the laser cutting step and the oxygen sensing polymer filling step.

Next, the waveguide structure7004is laser cut to form the oxygen sensing polymer fill cavity9082. As can be seen inFIGS. 76-78, the oxygen sensitive/sensing polymer fill cavity9082includes a control port8056that is contiguous or in optical communication with the oxygen reference waveguide core8060. The laser cutting provides a cavity9082that allows the oxygen sensitive/sensing polymer8080to contact and optically communicate with the waveguide cores8060,8062. Although in some embodiments, beveled or stepped surfaces are laser cut at the interface9083of the waveguide cores8060,8062and the oxygen sensitive/sensing polymer8080, in some embodiments, these beveled or stepped surfaces are not required.FIG. 77depicts a waveguide structure7014that includes a top clad coating layer and liner7030that was laser cut to include the oxygen sensitive/sensing polymer fill cavity9082and that is now ready to be filled with the oxygen sensitive/sensing polymer8080.

FIG. 78shows the oxygen sensitive/sensing polymer fill cavity9082filled with the oxygen sensitive/sensing polymer8080. In some embodiments, the oxygen sensitive/sensing polymer fill cavity9082is filled with the oxygen sensitive/sensing polymer8080with a knife coating process. As will be readily understood by those of skill in the art, other filling methods such as, for example, microfluidic filling, may be used to fill the oxygen sensitive/sensing polymer fill cavity9082. Once filling is completed and the oxygen sensitive/sensing polymer8080is cured, the liner on the top clad coating layer7030can be removed leaving the clad coating7030in place.

Next, as depicted inFIG. 79, another layer comprising a PEEK material9085with a PSA9099on its bottom surface and a liner (not shown in the figures) on its top surface, is placed on top of the waveguide structure7004that has been filled with the oxygen sensitive/sensing polymer8080. This layer, known as the reaction chamber (“RC”) laminate structure, includes the reaction chamber9086, which is located on top of the oxygen sensitive/sensing polymer8080that communicates with waveguide cores8062and the control port8056, which is located on top of the oxygen sensitive/sensing polymer8080in control port8056that communicates with the oxygen reference waveguide core8060. In some embodiments, the RC laminate structure is in the form of a sticker that has the reaction chamber9086and the control port8056pre-cut such that the sticker can be positioned over the filled waveguide structure7004and “stuck” or adhered in place on the filled waveguide structure7004. In some embodiments, placement of the RC laminate structure sticker is by way of an automated machine that precisely places the RC laminate structure sticker in place such that all structures (cavities, fill areas, etc.) are in alignment. With the RC laminate structure in place, the reaction chamber cavity9086can now be filled with enzymatic hydrogel8082(seeFIG. 80). In some embodiments, the reaction chamber9086is filled with the enzymatic hydrogel8082with a knife coating process. Once filling is completed and the enzymatic hydrogel8082is cured, the liner on the top of the PEEK material9085can be removed leaving a clean surface for the next layer (the conduit layer), to adhere to.

In some embodiments instead of using a knife coating process to fill the reaction chamber9086, microfluidics will be used to fill the reaction chamber9086. In these embodiments, the RC laminate structure or sticker needs to include additional structures to aid in the filling process. Depicted inFIG. 81is an embodiment of an RC laminate structure or sticker9087that can be used when microfluidics will be used to fill the reaction chamber9086. As previously disclosed, the RC laminate structure or sticker9087can comprise a PEEK material with a PSA on its bottom surface and a liner on its top surface. The RC laminate structure or sticker9087includes a reaction chamber9086, a control port8056, an enzymatic hydrogel fill well9088and a RC entrance9089that connects the reaction chamber9086with the enzymatic hydrogel fill well9088. In some embodiments, the reaction chamber9086, control port8056, enzymatic hydrogel fill well9088and RC entrance9089are laser cut into the RC laminate structure or sticker9087. Thus, after the RC laminate structure or sticker9087is placed on the filled waveguide structure7004, enzymatic hydrogel8082is dispensed into the enzymatic hydrogel fill well9088and flows as a result of microfluidics through the RC entrance9089and into the reaction chamber9086, thereby precisely filling the reaction chamber9086. After the enzymatic hydrogel8082is cured, the liner on the top of the PEEK material can be removed leaving a clean surface for the next layer (the conduit layer), to adhere to.

In some embodiments, as depicted inFIG. 82a plurality of RC laminate structures or stickers9087, which may or may not include the enzymatic hydrogel fill well9088, are laser cut into a sheet material forming108individual RC laminate structures or stickers9087that correspond to the structures of the previous embodiments laid out in a card configuration. Thus, layered optical sensors constructed in accordance with these embodiments, can be mass manufactured and assembled using the methods described and disclosed with respect to the previous embodiments.

With the RC laminate structure or sticker9087in place and the reaction chamber9086filled with the cured enzymatic hydrogel8082and the top liner removed, the conduit laminate9090, which comprises a PVDF material9091that is sandwiched between top and bottom silicone PSA layers9092(seeFIG. 86), is applied. As depicted inFIG. 83, the conduit laminate9090includes a cavity9093, which can be laser cut, to receive the conduit hydrogel9094. As depicted inFIG. 84, the cavity9093is then filled with the conduit hydrogel9094using a knife coating process, for example, and cured. After curing, as depicted inFIG. 85, top cap9095, which can be a PVDF material and which can include a plurality of micro perforations9096is applied and laminated to the top of the conduit laminate9090. The plurality of micro perforations9096in the top cap9095allow oxygen contained in the interstitial fluid (blood) into which the layered optical sensor is implanted/inserted, to enter the conduit hydrogel9094for sensing/measuring by the analyte sensor. With construction of the sensor laminate structure complete, an optical chip/engine can be added.

Depicted inFIG. 86is an exploded view a sensor constructed in accordance with the disclosed embodiments.

While most of the card cross-sections with laminate structures therein on1y include a single waveguide structure or single sensor, as supported by the disclosed embodiments, a plurality of sensors are included on each card.

From the foregoing description, it will be appreciated that an inventive product and manufacturing method for a laminated optical sensor are disclosed. While several components, techniques and aspects have been described with a certain degree of particularity, it is manifest that many changes can be made in the specific designs, constructions and methodology herein above described without departing from the spirit and scope of this disclosure. As will be readily understood by those of skill in the art, various components, methods and processes from the manufacturing embodiments disclosed and described herein, can be combined and used with other manufacturing method embodiments disclosed and described herein to arrive at new manufacturing method embodiments that may include methods and processes from multiple embodiments disclosed and described herein.

Adhering a Medical Device to the Skin of a Patient

Disclosed herein are embodiments of a multilayer composite adhesive system, configured to adhere, in some embodiments, to an body wearable device, such as, for example, the opto-enzymatic analyte sensors disclosed and described herein, to the surface of skin. The multilayer composite adhesive systems disclosed herein can attach to the bottom of the body wearable device housing thereby allowing the device to be attached to the skin for an extended period of time, for example, 4 to 7 days, 7 to 10 days, 10 to 14 days or 14 to 21 days.

Current adhesive systems have difficulty remaining on the skin for extended periods of time because they do not address the differences in mechanical properties between the skin and the adhesive, i.e., stress/strain differentials that exist between skin and the adhesive systems. Skin typically has a low stress strain relationship that may be approximated as0.05MPa for strains of 1.0 or 0.02 MPa for strains of 0.4. The skin is viscoelastic and current adhesive systems are typically highly elastic. Because of the mechanical mismatch between the skin and current adhesive systems, when current adhesive systems are in place on skin and the skin moves (stretches/tension and compresses/compression), these adhesive systems do not move to the same extent as the skin and therefore, experience stress/strain mismatch between the adhesive system material and the skin. This mismatch results in high shear forces at the interface between the adhesive system adhesive layer and the skin upon which it is adhered. As a result of these shear forces, current adhesive systems experience edge peel, which eventually leads to peel off of the entire adhesive system.

Another issue with current adhesive systems is that they suffer from moisture loading (moisture trapped between the skin and the adhesive system) because they have an inadequate moisture vapor transmission rate (“MVTR”), which results in “float off” of the system. MVTR is a measure of the passage of water vapor through a substance and/or barrier. Because perspiration naturally occurs on the skin, if the MVTR of a material or adhesive system is low, this can result in moisture accumulation between the skin and the adhesive system that can promote bacterial growth, cause skin irritation, and can cause the adhesive system to peel away or “float off” from the skin.

Thus, adhesive systems must be designed to (1) address the mismatch of mechanical properties that exist between skin and the adhesive systems and (2) have a high MVTR. Prior adhesive systems have attempted to address the issue of mismatch of mechanical properties and the resulting edge peel, by using aggressive adhesives, i.e., adhesives that have high adhesion to skin. An adhesive's aggressiveness is defined by its initial bond strength and its sustained bond strength. However, these aggressive adhesives do not address the main problem of strain mismatch and the high shear forces that result between the skin and the adhesive and therefore, result in systems that do not expand and contract to the same extent as the skin and remain strongly attached to the skin resulting in very high shear forces leading to pain to the wearer, and which will eventually lead to edge peel and peel off. Additionally, using an aggressive adhesive is very difficult and painful to remove from the skin when a wearer desires to remove the adhesive system. However, an adhesive that is not sufficiently aggressive will not maintain attachment to the skin as the skin expands and contracts and will result in edge peel and peel off.

Accordingly, adhesive system embodiments of the present invention have been designed to address these deficiencies of prior adhesive systems.

In order to achieve the required sustained attachment to the skin while allowing the adhesive system have a high MVTR and to be easily removed from the skin when desired, embodiments of the present invention are directed to multilayer composite adhesive systems where the properties of the layers combine to form a system with a high MVTR that addresses the mismatch of mechanical properties and that uses a skin adhesive that provides sufficient adhesion to skin while allowing the adhesive system to be easily removed with little pain. Thus, each layer of the present adhesive systems can have different mechanical and material properties but when the properties of all layers are combined, they address the issues with prior systems by mimicking skin mechanics in order to address the strain mismatch between the skin and the adhesive system while providing a high MVTR.

To satisfy these requirements, the multilayer composite adhesive systems of the embodiments of the present invention have been designed to have a high MVTR and a low, effective Young's/elastic modulus. Further, the system can plastically deform when worn on the skin and has good adhesion to skin while being easily removed from the skin when desired. The MVTR of a material can be an inherent property of the material or a material's MVTR can be changed/adjusted by altering the material to include, for example, openings, slits, cuts or other perforations (collectively, “perforations”) therein, resulting in a material that has a higher effective MVTR, thereby providing a pathway for moisture to escape through the material. As used herein, (1) “inherent” shall mean a property of an unmodified material and (2) “effective” shall mean the resulting property after a material or layer or multilayer adhesive system has been modified, for example, as disclosed herein to include modifications such as perforations or the resulting properties of a multilayer adhesive system constructed in accordance with the embodiments disclosed herein.

A material typically plastically deforms when its linear elastic force is exceeded as stress is developed in the material. Similar to a material's MVTR, a material's elastic modulus can be an inherent property of the material or it can be changed/adjusted by modifying the material to include, for example, perforations therein, resulting in a material that has an effective elastic modulus that is lower than its inherent elastic modulus. The shape, orientation, size and spacing of these perforations, can also be used to change a material's elastic in different directions, i.e., the web and cross-web directions of the material, depending on the size, orientation and spacing of the perforations.

For example, as discussed in detail below, a material that includes perforations that are longer in length than the gap/spacing between adjacent perforations will have a lower effective elastic modulus than a material that includes perforations that are shorter in length than the gap/spacing between adjacent perforations. Using perforations that have different lengths and spacing between in different directions allows tuning of the modulus of elasticity in the different directions, i.e. a first modulus of elasticity in a first direction and a second modulus of elasticity in a second direction where the first and second elastic modulus's can be the same or different. As discussed in more detail below, the length of the perforations and the spacing between adjacent perforations can be adjusted to tune the effective elastic modulus of the materials/layers and hence, the effective modulus of the embodiments of the adhesive systems disclosed and described herein. For example, the effective elastic modulus of an individual layer or the constructed multilayer adhesive system can be tuned/adjusted to be less than approximately 100 Kpa, 90 Kpa, 70 Kpa, 60 Kpa, 50 Kpa, 40 Kpa, 30 Kpa, 20 Kpa, and 10 Kpa, at 100% strain.

Thus, embodiments of the present adhesive systems have been designed to have a high MVTR and low elastic modulus, i.e., designed to have low elasticity, that undergo plastic deformation at low strains. Having an adhesive system that plastically deforms when attached skin, allows the system to use a less aggressive adhesive to attach the adhesive system to the skin as the shear forces between the adhesive and the skin are significantly reduced after the adhesive system plastically deforms. Adhesive systems that plastically deform when worn on the skin, solves the issue of edge peel and results in an adhesive system that remains attached to the skin for an extended period of time, for example, five (5) weeks.

The multilayer, composite adhesive system embodiments disclosed herein are also advantageous as they permit different system designs based on the intended use of the system while allowing one to design the system to have the required MVTR and elastic modulus properties. For example, one may desire to have an adhesive system with moisture wicking properties, or one may desire to have an adhesive system to absorb bodily fluid such as in the form of a bandage, or one may desire to an adhesive system with sufficient strength to attach medical devices and other medical items to the body. Different uses may require different properties or a combination of properties, which can be achieved through the use of layers of different materials, which individually may not meet the intended use requirements but when modified as discussed herein and combined, provide the required properties.

Material properties to consider in designing adhesive system embodiments of the present invention include, and are not limited to, Young's modulus, MVTR, hydrophobicity, hydrophilicity and moisture wicking, adhesive strength, adhesive hypoalgernicity and intact adhesive system removal.

FIGS. 28A-Cillustrate exploded and side views of an embodiment of the adhesive system2800. The adhesive system2800is a multilayer adhesive system that provides a high MVTR in general, especially under the housing of the attached device. In some examples, the adhesive system2800includes a first layer composed of a device adhesive2830, a second layer composed of the outer ring2820, and a top layer composed of the coin standard2810. The adhesive system2800can be oriented such that the first layer device adhesive2830is attached to the bottom of the device and the third layer coin standard2810is attached to the surface of the skin.

Turning first to the coin standard2810, in some examples the coin standard2810is attached to the skin. The surface of the coin standard2810can be composed of an acrylate pressure adhesive on a PET release. The pressure sensitive adhesive allows the coin standard2810to adhere to the skin when pressure is applied—thereby activating the adhesive without the use of a solvent, water or heat. The material of the coin standard2810can be composed of a spun lace non-woven material with a high MVTR. In some examples, the coin standard2810can have a thickness of 4 mm.

As illustrated inFIGS. 28A to 28C, the coin standard2810can include an opening2812that extends through the coin standard2810. In some examples, the opening2812can have a diameter of 3 mm and can be placed a distance of10mm from the narrow end of the coin standard2810.

Turning next to the outer ring2820, in some examples, the outer ring2820is composed of a re-attachable pressure sensitive adhesive. The outer ring2820can be composed of a lined silicon/silicon pressure sensitive adhesive on a PTFE release.

In some examples, the outer ring2820can be joined to the coin standard2810. The attachment between the two layers can form a gap2822. The outer ring2820can be attached to the coin standard2810with acrylate pressure sensitive adhesive. In some examples, the acrylate pressure sensitive adhesive can be a polyurethane acrylate (P-UR acrylate). In some embodiments, the release liner of the outer ring2820is formed from a patterned PET and PTFE pattern. The PET can be bonded to the PTFE below the coin and the PTFE below the silicon. In some examples, the outer ring2820can have a base width of 30 mm and a length of 40 mm. In some embodiments, outer ring2820can have a width of 7 mm and a thickness of 6 mm.

FIGS. 29A-Billustrate a top and side view of another embodiment of the adhesive system2860. The adhesive system2860illustrated inFIGS. 29A-Bis a multi-layered system that includes a top layer2840with a top layer adhesive2842and a bottom layer2844with a bottom layer adhesive2846. The top layer2840can be formed from a material having a low intrinsic elastic modulus or it can be made from a material that has been modified (as discussed in more detail below) to have a low effective elastic modulus. Example materials for the top layer include polyurethane and a silicone elastomer. The bottom layer2844includes an outer ring2850, a middle ring2852, a central portion layer2854, and gaps2856, which can be continuous or discontinuous. The outer ring2850can include a number of variations. In some examples, the outer ring2850is a high strength bio-compliant skin adhesive that can be connected to the top layer2840of the adhesive system2860. The bottom layer2844can include a middle ring2854and a central portion2854of spun lace, non-woven material, which can be a material that wicks moisture, such as perspiration, away from under the device.

In other examples, the bottom layer2844can be a spun lace, non-woven material that includes a plurality of cuts or gaps2856therein that divide the bottom layer2844into an outer ring2850, a middle ring2852and a central portion2854. In this embodiment, the bottom layer adhesive2844can be more aggressive than the top layer adhesive2842.

In another embodiment, the outer annular region2850can be a re-attachable bio-compliant skin adhesive connected to the top layer2840of the adhesive system2860. The outer annular region2850can have a central portion2854of spun lace, non-woven material. The outer annular region2850may also have an additional adhesive layer above the central portion2854of spun lace, non-woven material. In other examples, the outer annular region2850can have the same materials as the central portion2854. As well, the outer annular region2850can have an adhesive connected to the top layer2840of the adhesive system2860.

In some examples, the adhesive system2860includes a top layer2850that can be a backing material that has a high MVTR, such as polyurethane. In some examples, the backing material is thin and complaint. In some embodiments, as illustrated inFIG. 29B, one or more layers can include one or more physical gaps2856. In some examples, these gaps2856can be in the spun lace, non-woven material of the bottom layer2844and adhesive layer below the backing of the top layer2852creating discontinuous segments. The physical gaps2856provide strain relief in the adhesive system2860as the adhesive system2860is stretched, allowing the discontinuous segments of the annular region to move independently of one another. In some examples, additional gaps through the entire adhesive system2860can provide further strain relief. In some examples, these additional gaps in the spun lace and skin adhesive can provide further strain relief. While in the figures, these gaps2854are shown as extending completely through the material, it should be noted that these gaps can also be recessed, indented or embossed portions of the material, which create failure lines in the material that are designed to fail and hence, cause gaps to form in the material, when stress is applied to the material, thereby providing the required strain relief.

In another embodiment of the adhesive system2860depicted inFIGS. 29C and29D, instead of the bottom layer being divided into ring-shaped discontinuous portions, the bottom layer2844can be divided into polygonal-shaped discontinuous portions2870. The top layer2840can be formed from a material having a low intrinsic elastic modulus or it can be made from a material that has been modified (as discussed in more detail below) to have a low effective elastic modulus. The top layer2840may be attached to the bottom layer2844with an adhesive. The bottom layer2844can be a spun lace, non-woven material that includes an adhesive for attaching to the skin2872.FIG. 29Cdepicts the adhesive system2860adhered to skin2872when the skin is in a relaxed state. When adhered to the skin2872, the discontinuous portions2870form discrete attachment points to the skin2872. As depicted inFIG. 29D, when the skin2872is stressed/stretched as indicated by arrows2874, because the top layer2840has a low elastic modulus either inherently or through modification as discussed herein, the discontinuous portions2870that are adhered to the skin2872easily move with the skin in the direction of arrows2874. The combination of the bottom layer2844having discrete attachment points between the discontinuous portions2870and the skin2872and the top layer2840having a low elastic modulus that stretches and/or plastically deforms under stress, provides the required strain relief between the skin2872and the adhesive system2860.

In the herein disclosed embodiments, dividing the bottom layer of the adhesive system into multiple annular regions or other discontinuous portions, helps to minimize the strain on the inner or central regions of the adhesive system by distributing stress across the annular regions or discontinuous portions. Adhesive systems constructed in this manner, create a stress-strain gradient between the inner or central regions and the ring or discontinuous portions that extend away from the inner or central regions. For example, the embodiment of the adhesive system depicted inFIGS. 29A and 29Bincludes a bottom layer2844with discontinuous portions (annular regions2850,2852) that are detached from a central portion (central portion2854). In this embodiment, a device, such as an opto-enzymatic device as disclosed herein, may be included on the adhesive system in the area above central portion2854(a loaded portion). Thus, designing an adhesive system that has a central loaded portion with discontinuous portions extending away from the central loaded portion (see for example,FIGS. 29C and 29D), allows for the stresses on the loaded central portion to be distributed across the exterior discontinuous portions.

In some examples, the adhesive system2800is re-sealable and provides for comfortable adhesion. The illustrated adhesive system2800can include two zones of attached materials. In some embodiments, the outer layer can be elastic, with a low durimetry. The outer layer can allow the adhesive system2800and attached device to be re-sealable to the skin. In some embodiments, the inner layer can be composed of a material that is less elastic but has a high MVTR. As will be discussed in further detail below, the material properties of the inner layer can allow the skin to breath by allowing water and/or water vapor to evaporate off the surface of the skin.

Depicted inFIGS. 29E to 29Jis another embodiment of the present adhesive system. The adhesive system6000is a two-layer system that includes a top layer6004and a bottom layer6006. The top layer6004can be made from a material having an intrinsic low elastic modulus and an intrinsic high MVTR or it can be made from a material that is modified to have an effective lower elastic modulus and/or an effective higher MVTR. The top layer6004can include an adhesive for attaching the top layer6004to the bottom layer6006. Thus, a material having a higher elastic modulus and/or a lower MVTR than desired may be used but may be modified mechanically, for example, to include a plurality of modifications, such as, for example, perforations6008, along a first direction6010, and/or a plurality of modifications, such as, for example, perforations6012, along a second direction6014(as depicted inFIGS. 29G and 291, that extend through the thickness of the top layer6004and which can also extend through the adhesive.

The plurality of perforations6008,6012transform the top layer material from a material having a high or first intrinsic elastic modulus and/or a low intrinsic MVTR into a material having an effective lower or second elastic modulus and/or an effective higher MVTR. The effective low elastic modulus is achieved by creating stress relaxing perforations that expand as the material is stretched. As the perforations expand, a plurality of concentrated areas of stress6016develop between adjacent perforations6008,6010, that undergo plastic deformation when stress is applied to the top layer6004. Because any stress that is applied to the top layer6004is concentrated in areas6016, these concentrated areas of stress6016plastically deform under external loads that are lower than stress that would cause an unmodified top layer6004material to plastically deform. This plastic deformation provides further strain relief between the top layer6004and the skin. The stress becomes lower for a given strain after deformation. Although the perforations6008,6012in this embodiment are shown in a cross-hatch orthogonal pattern, the perforations6008,6012can have any shape or pattern as long as they allow the material to separate creating a low elastic modulus response and preferentially create concentrated areas of stress6016between adjacent perforations. Additionally, in some embodiments, the plurality of perforations6008,6012may extend completely through the top layer6004material while in other embodiments, they may not extend completely through the thickness of the material/layer and instead may be recessed, indented or embossed portions that fail when under stress and create the concentrated areas of stress6016between adjacent indentations causing the material layer to plastically deform under stress when applied to skin. In some embodiments, the top layer6004is a polyurethane material. In some embodiments, the top layer is a silicone elastomer.

The bottom layer6006can comprise any material (wicking materials, adhesives, etc.) and the material should be chosen based on the intended use of the adhesive system. In some embodiments, the material for the bottom layer6006is a wicking material such as, for example, a spun lace non-woven material, that includes an adhesive for adhering the bottom layer6006to skin. The wicking material of the bottom layer6006, which contacts the skin, transports moisture laterally from areas of high moisture to areas of low moisture. As illustrated inFIGS. 29E, 29F, 29H and 29J, the bottom layer6006includes a plurality of perforations6018therein that form a plurality of discontinuous portions6020. These perforations6018can be continuous or discontinuous. Accordingly, when the bottom layer6006is adhered to skin and is stressed, the plurality of discontinuous portions6020separate from each other, thereby providing strain relief in the bottom layer6006. Because the discontinuous portions6020are adhered to the skin, as they separate and move away from the adjacent discontinuous portions6020, they move with the skin, independently of one another. Although, in some embodiments, the plurality of perforations6018may extend completely through the bottom layer6006material, they may also be recessed, indented or embossed portions of the material, which create failure lines in the material that are designed to fail under stress and hence, cause adjacent discontinuous portions6020to separate from one another, when stress is applied to the material, thereby providing the required strain relief. In the present embodiment, the plurality of perforations6018that form a plurality of curvilinear discontinuous portions6020are depicted as curvilinear, however, the plurality of perforations6018need not be curvilinear and instead can be any geometry such as, for example, polygonal- square or rectangular, which form correspondingly-shaped discontinuous portions6020, see for example, discontinuous portions2870inFIGS. 29C and 29D. It is on1y required that the plurality of perforations6018result in a plurality of discontinuous portions6020being formed in the bottom layer6006material that separate from each other and move with the skin, independent of one another.

As illustrated in the figures, the top layer6004is attached to the bottom layer6006with the first layer adhesive thereby sandwiching the bottom layer6006between the top layer6004and the skin when the adhesive system6000is attached to the skin. In this embodiment, because the perforations6018extend through the entire thickness of the bottom layer6006, which create discontinuous portions6020that are adjacent to one another, the bottom layer6006typically has a lower effective elastic modulus than the top layer6004. Therefore, the top layer6004provides structural reinforcement for the bottom layer6004and holds the adhesive system6000together.

As depicted inFIG. 29J, which is a bottom view of the adhesive system6000, the top layer6004has a first perimeter6022that defines a first area and the bottom layer6006has a second perimeter6024that defines a second area. In some embodiments, the first area is greater than the second area, which results in portions6026of the first perimeter6022extending beyond the second perimeter6024. Thus, when the adhesive system6000is attached to the skin, in addition to the bottom layer6006adhering to the skin with the bottom layer adhesive, the portions6026of the top layer6004that extend beyond the perimeter6022of the bottom layer6006(i.e., overhang the bottom layer6006), result in a portion of the top layer6004also adhering to the skin with the top layer adhesive. In some embodiments, the bottom layer adhesive can be less aggressive than the top layer adhesive. In the present embodiment, a less aggressive adhesive may be used to adhere the bottom layer6006to the skin as the plurality of discontinuous portions6020transform the bottom layer into a very low elastic modulus layer. Because the discontinuous portions6020separate under low stress and therefore, move with the skin independently of one another, the bottom layer adhesive can be less aggressive as the shear forces between the discontinuous portions6020and the skin, are low. The lower shear forces result from the smaller contact area between the bottom layer adhesive on the discontinuous portions6020and the skin. Thus, smaller area discontinuous portions6020allow less aggressive adhesives to be used resulting in reduced skin irritation and easier and less painful removal from the skin. In this embodiment, the top layer6004and the bottom layer6006, are attached to the skin with an adhesive.

In some embodiments, the top layer adhesive used to attach the top layer6004to the bottom layer6006and the portions6026of the top layer that extend beyond the perimeter6022of the bottom layer6006to the skin, is a more aggressive adhesive than the bottom layer adhesive. This more aggressive adhesive is necessary to keep the top layer attached to the bottom layer6006and the skin when stress is applied to the adhesive system6000due to movement (expansion and contraction) of the skin. That is, the top layer6004must expand and contract to the same extent as the skin in order to cause the perforations6008,6012to open and preferentially induce formation of the concentrated areas of stress6016and hence, plastic deformation of the top layer6004, thereby minimizing stress in the top layer6004. Thus, the top layer6004must remain attached to the skin.

In addition to using an aggressive adhesive to impart a higher initial and sustained bond strength between the portions6026of the top layer6004that extend beyond the perimeter6024of the bottom layer6006that attach to the skin with the top layer adhesive, the area of the portions6026of the top layer6004that extend beyond the perimeter6024of the bottom layer6006can be increased such that a larger area of the top layer6004is attached to the skin with the top layer adhesive. The increased area of the top layer6004that adheres to the skin allows a less aggressive adhesive to be used while keeping the adhesive system6000attached to the skin and causing the adhesive system6000to plastically deform under the stress imparted due to movement of the skin.

In additional embodiments of a two-layer adhesive system according to the present invention, as depicted inFIGS. 29K and 29L, the adhesive system6000includes a top layer6004, which can be constructed in accordance with embodiments herein to include, for example, a plurality of perforations6008, along a first direction, and/or a plurality of perforations6012, along a second direction that create openings in the material and concentrated areas of stress6016between adjacent perforations as depicted inFIG. 291. The bottom layer6006can comprise a hydrocolloid. Because hydrocolloids are low elastic modulus materials with high MVTRs, in these embodiments, the bottom layer6006may (FIG. 29L) or may not (FIG. 29K) include the plurality of perforations6004,6008therein that the top layer6004includes.

Depicted inFIGS. 29M to 29Rare additional embodiments of the present multilayer adhesive system. The adhesive systems6500,6600are three-layer systems that include a top layer6504,6604, middle layer6508,6608and bottom layer6512,6612. The top layer6504can be made from a material having an intrinsic low elastic modulus and an intrinsic high MVTR or it can be formed of a material that is modified to have an effective lower elastic modulus and/or an effective higher MVTR. The modifications can be, for example, a plurality of perforations6008along a first direction, and/or a plurality of perforations6012along a second direction that create concentrated areas of stress6016between adjacent perforations as depicted inFIG. 29F. In some embodiments, the top layer is a polyurethane material. In some embodiments, the top layer is a silicone elastomer.

In the embodiment depicted inFIGS. 29N, the middle layer6508can be a separate adhesive to attach the top layer6505to the bottom layer6512. In some embodiments, the middle layer6508can be a fiber reinforced adhesive, such as, for example, a polyester fiber reinforced acrylate adhesive. Because fiber reinforced adhesives typically have a higher elastic modulus than desired, as depicted inFIGS. 290 and 29PwhereFIG. 29Pis a bottom view of the adhesive system6500, the middle layer6508in these embodiments can also include the plurality of perforations6008along a first direction, and/or the plurality of perforations6012along a second direction, similar to the top layer6504, in order to reduce the elastic modulus of the middle layer6508. In some embodiments, as depicted inFIG. 29N, the middle layer6508is unmodified.

As depicted inFIGS. 29N to29P, the bottom layer6512can comprise a hydrophobic material or a wicking material such as, for example, a spun lace non-woven material, that includes and adhesive for adhering the bottom layer6512to skin. As illustrated in the figures, the bottom layer6512in these embodiments, can be constructed in a similar manner with similar properties as the bottom layer6006for the two layer embodiments of the present adhesive system (see for example.FIG. 29H), to include a plurality of perforations6018therein that form a plurality of discontinuous portions6020. Accordingly, when the bottom layer6512is adhered to skin and is stressed, the plurality of discontinuous portions6020separate from each other, thereby providing strain relief in the bottom layer6512. Because the discontinuous portions6020are adhered to the skin, once they separate from the adjacent discontinuous portions6020, they move with the skin, independently of one another. Thus, the same wicking material designs disclosed above for the bottom layer6006of the two-layer adhesive system embodiments, can be used for the three-layer adhesive system embodiments.

In another embodiment of the three-layer adhesive system6600, as depicted inFIGS. 29Q and 29R, the system includes a top layer6604, middle layer6608and bottom layer6612. The top layer6604can be, similar to previous embodiments, made from a material having an intrinsic low elastic modulus and an intrinsic high MVTR or it can be formed of a material that is modified to have an effective lower elastic modulus and/or an effective higher MVTR. The modifications can be, for example, a plurality of perforations6008along a first direction, and/or a plurality of perforations6012along a second direction that create concentrated areas of stress6016between adjacent perforations as depicted inFIG. 291. In some embodiments, the top layer is a polyurethane material. In some embodiments, the top layer is a silicone elastomer.

In the embodiment depicted inFIG. 29Q, the middle layer6608can comprise a hydrophobic material or a wicking material such as, for example, a spun lace non-woven material. As illustrated, the middle layer6608in these embodiments, can be constructed in a similar manner with similar properties as the bottom layer6006for the two layer embodiments of the present adhesive system depicted inFIG. 291, to include a plurality of perforations6018therein that form a plurality of discontinuous portions6020. In this embodiment, the bottom layer6612can comprise a hydrocolloid, which attaches to the middle layer6608and the skin. Accordingly, when the three-layer adhesive system6600is adhered to skin and is stressed, the plurality of discontinuous portions6020of the middle layer6608to move with the hydrocolloid, which moves with the skin because it is a low elastic modulus material, and separate from each other, thereby providing strain relief in the middle layer6608. Because the discontinuous portions6020are adhered to the skin through the hydrocolloid, once they separate from the adjacent discontinuous portions6020, they move with the skin, independently of one another. Thus, the same wicking material designs disclosed above for the bottom layer6006of the two-layer adhesive system embodiments, can be used for the middle layer6608in this embodiment of the three-layer adhesive system.

In the three-layer adhesive system embodiments6500,6600depicted inFIGS. 29M-29R, the top layer6504,6604has a first perimeter6522,6622that defines a first area, the middle layer6508,6608has a second perimeter6524,6624that defines a second area and the bottom layer6512,6612has a third perimeter6526,6626that defines a third area. In some embodiments, the first area is greater than the second and third areas, which results in portions6528,6628of the first perimeter6522,6622extending beyond the second and third perimeters6524,6624,6526,6626(seeFIGS. 29P and 29R). Thus, when the adhesive systems6500,6600are attached to the skin, in addition to the bottom layer6512,6612adhering to the skin, the portions6528,6628of the top layer6504,6604that extend beyond the perimeters6524,6624,6526,6626of the middle layer6508,6608and bottom layer6512,6612(i.e., overhang the middle layer6508,6608and bottom layer6512,6612), result in a portion of the top layer6504,6604also adhering to the skin. Accordingly, adhesives with similar properties to those disclosed above for the two-layer adhesive system embodiments, can be used to attach the three-layer adhesive system embodiments to skin.

As previously disclosed, the length of the perforations6008,6012and the spacing between adjacent perforations in the embodiments of the adhesive systems disclosed herein, can be changed/adjusted to tune the effective elastic modulus of the materials/layers and hence, the effective modulus of the completed multilayer adhesive systems.

As illustrated inFIG. 29S, embodiments of the present adhesive systems can include layers that have been modified to include a plurality of first perforations6008along a first direction6010and a plurality of second perforations6012along a second direction6014. In some embodiments, (a) the plurality of first perforations6008have a length L1and adjacent first perforations6008are separated by a distance L2and (b) the plurality of second perforations6012have a length L3and adjacent second perforations6012are separated by a distance L4. The lengths L1and L3and the distances L2and L4can be chosen to change the size of the concentrated areas of stress6016that are created between adjacent first perforations6008and adjacent second perforations6012, which changes the effective elastic modulus of the layer that includes the first and second perforations6008,6012. Thus, for example, when L1and L3have lengths that are longer than the distances L2and L4, the layer will have an effective elastic modulus that is lower than a layer having an L1and L3with lengths that are shorter than the distances L2and L4. Accordingly, adhesive system layer embodiments that include first and second perforations6008,6012having lengths L1and L3, respectively, that are significantly longer than the distances L2and L4, will have a much lower elastic modulus than adhesive system layer embodiments that include first and second perforations6008,6012having lengths L1and L3, respectively, that are not significantly longer than the distances L2and L4. In some embodiments, L1is substantially equal to L3and L2is substantially equal to L4, which results in a layer/adhesive system having an effective elastic modulus that is substantially the same in both the first and second directions6010,6014. In some embodiments, L1is not substantially equal to L3and L2is not substantially equal to L4, which results in a layer/system having an effective elastic modulus that is not substantially the same in both the first and second directions6010,6014. In some embodiments, L1and L3can range from approximately 1.0 mm to 3.0 mm and L2and L4can range from approximately 0.25 mm to 1.0 mm. Also, in some embodiments, adhesive system layers may on1y include perforations along one direction so as to on1y substantially change the effective elastic modulus of the layer/material in one direction.

Although the plurality of perforations in the disclosed embodiments are shown in a cross-hatch pattern or are orthogonal to one another, any pattern of a plurality of perforations that create concentrated areas of stress in a layer or multilayer adhesive system, may be used. The type of patterned perforations used will affect the effective elastic modulus of the layer and/or adhesive system.

Modifying L1, L2, L3, and L4as outlined above, allows the effective elastic modulus of an individual layer or the constructed multilayer adhesive system to be tuned/adjusted to be less than approximately 100 Kpa, 90 Kpa, 70 Kpa, 60 Kpa, 50 Kpa, 40 Kpa, 30 Kpa, 20 Kpa, and 10 Kpa, at 100% strain. Thus, modifying the individual layers or the constructed multilayer adhesive system as outlined above, allows the effective elastic modulus to be maintained for strains up to 0.4 and preferably, up to 1.0.

In some embodiments of the two-layer adhesive systems disclosed herein, the top layer can have an effective elastic modulus less than 0.02 Mpa (20 Kpa) that is maintained for strains up to 0.4 and preferably, for strains up to 1.0. In some embodiments, the bottom layer can have an effective elastic modulus less than 0.02 Mpa (20 Kpa) that is maintained for strains up to 0.4 and preferably, for strains up to 1.0. In some embodiments, the two-layer adhesive system can have an effective elastic modulus less than 0.02 Mpa (20 Kpa) that is maintained for strains up to 0.4 and preferably, for strains up to 1.0. In some embodiments, the concentrated areas of stress plastically deform when an external load is applied to achieve a net strain of up to 0.4 in the two-layer adhesive system. In some embodiments, when the multilayer adhesive system is deformed by an external load to a strain of up to 0.4, the multilayer adhesive system deforms resulting in >90% of the achieved strain being retained when the external load is removed.

In some embodiments of the three-layer adhesive systems disclosed herein, the top layer can have an effective elastic modulus less than 0.02 Mpa (20 Kpa) that is maintained for strains up to 0.4 and preferably, for strains up to 1.0. In some embodiments, the middle layer can have an effective elastic modulus less than 0.02 Mpa (20 Kpa) that is maintained for strains up to 0.4 and preferably, for strains up to 1.0. In some embodiments, the bottom layer can have an effective elastic modulus less than 0.02 Mpa (20 Kpa) that is maintained for strains up to 0.4 and preferably, for strains up to 1.0. In some embodiments, the three-layer adhesive system can have an effective elastic modulus less than 0.02 Mpa (20 Kpa) that is maintained for strains up to 0.4 and preferably, for strains up to 1.0. In some embodiments, the concentrated areas of stress plastically deform when an external load is applied to achieve a net strain of up to 0.4 in the two-layer adhesive system. In some embodiments, when the multilayer adhesive system is deformed by an external load to a strain of up to 0.4, the multilayer adhesive system deforms resulting in >90% of the achieved strain being retained when the external load is removed.

Depicted inFIG. 29Tis a chart showing the results of strain tests that were performed on adhesive systems constructed in accordance with the embodiments disclosed herein. As used in the description ofFIG. 29T, unmodified means that the layer was not modified as disclosed herein to include any perforations therein and modified means that the layer was modified to include either a plurality of perforations in the first and second directions (for the polyurethane (PU) top layer and the adhesive middle layer) or a plurality of perforations that form a plurality of discontinuous portions therein (the adhesive-backed spun lace, non-woven bottom layer). It should be noted that the adhesive systems identified in the chart started to plastically deform at 40% strain, reducing the slope calculation of the modulus.

The following seven adhesive systems were tested. Set1comprised an adhesive system having an unmodified polyurethane top layer. At 25% strain, the elastic modulus was approximately 15 Kpa and at 40% strain, the elastic modulus was approximately 14 Kpa. Set2acomprised an unmodified polyurethane top layer and an unmodified hydrocolloid bottom layer. At 25% strain, the elastic modulus was approximately 15 Kpa and at 40% strain, the elastic modulus was approximately 16 Kpa. Set2bcomprised a modified polyurethane top layer and an unmodified hydrocolloid bottom layer. At 25% strain, the elastic modulus was approximately 10 Kpa and at 40% strain, the elastic modulus was approximately 10 Kpa. Set3acomprised an unmodified polyurethane top layer and an unmodified adhesive backed spun lace, non-woven bottom layer. At 25% strain, the elastic modulus was approximately 44 Kpa and at 40% strain, the elastic modulus was approximately 38 Kpa. Set3bcomprised an unmodified polyurethane top layer, an unmodified adhesive middle layer and an unmodified adhesive backed spun lace, non-woven bottom layer. At 25% strain, the elastic modulus was approximately 64 Kpa and at 40% strain, the elastic modulus was approximately 51 Kpa. Set4acomprised a modified polyurethane top layer and a modified adhesive backed spun lace, non-woven bottom layer. At 25% strain, the elastic modulus was approximately 25 Kpa and at 40% strain, the elastic modulus was approximately 0 Kpa. Set4bcomprised a modified polyurethane top layer, a modified adhesive middle layer and a modified adhesive backed spun lace, non-woven bottom layer. At 25% strain, the elastic modulus was approximately 22 Kpa and at 40% strain, the elastic modulus was approximately 19 Kpa.

As can clearly be seen inFIG. 29T. modifying the adhesive layers as disclosed herein, reduces the materials and hence, the adhesive system's elastic modulus.

Depicted inFIGS. 29U to 29Wis an illustration of how adhesive systems according to embodiments of the present invention react and respond when attached to skin.FIGS. 29U to 29Ware cross-sectional views through a two-layer adhesive system according to embodiments of the present invention, for example, the embodiments associated withFIGS. 29E to 29I. Although a two-layer system adhesive system is depicted, three-layer adhesive systems of the embodiments of the present invention, will react and respond in a similar manner.

FIG. 29Udepicts the adhesive system6000when initially attached to the skin6001. As can be seen in the figure, the adhesive system6000includes a top layer6004with a plurality of perforations6008along a first direction that is attached to a middle layer6006with a top layer adhesive6005. The bottom layer6006attaches to the skin6001with a bottom layer adhesive6007and includes a plurality of perforations6018that form a plurality of discontinuous portions6020in the bottom layer6006.

As depicted inFIG. 29V, when the skin6001stretches in the direction indicated by arrows6021, the discontinuous portions6020of the bottom layer6006, which are attached to the skin6001with bottom layer adhesive6007, also move in direction6021causing any discontinuous portions6020that are connected to adjacent discontinuous portions6020to separate. Accordingly, movement of the discontinuous portions6020away from each other causes the material of the top layer6004, which is attached to the bottom layer6006with top layer adhesive6005, to move in a corresponding manner. This movement imparts stress on the top layer6004, which causes the concentrated areas of stress6016to form in the areas between adjacent perforations6008in the top layer6004. These concentrated areas of stress6016plastically deform and elongate under the stress applied by movement of the skin6001as a result of the top layer6004being stretched beyond its elastic limit. This plastic deformation provides strain relief between the adhesive system6000and the skin6001.

Once the skin6001is unstressed or returned to its relaxed state, which is depicted inFIG. 29W, the concentrated areas of stress6016in the top layer6004that plastically deformed and hence, elongated, now form wrinkles6025in the adhesive system6000. As a result of top layer's6004plastic deformation and the discontinuous portions6020separating from each other, the shear forces/stress between the skin6001and bottom layer adhesive6007is reduced. In subsequent movement/stretching of the skin6001and the adhesive system6000, the discontinuous portions6020of the bottom layer6006and the material of the top layer6004can now move freely with the skin as the wrinkles6025or elongated material of the top layer6004, freely elongate allowing the adhesive system6000to move with the skin6001with very minimal shear forces between the adhesive system6000and skin6001. Thus, there is minimal “pulling” on the adhesive system, which drastically reduces the occurrence of edge peel. If the wrinkled portions6025are elongated past there previously deformed length, these wrinkled portions6025again undergo plastic deformation and elongate, thereby creating larger wrinkles6025, which again reduce shear forces between the adhesive system6000and skin6001.

In addition, this reduction in shear forces/stress after plastic deformation, permits the use of an adhesive that has a high initial bond strength with a lower sustained bond strength, which results in an adhesive system that is easy to remove with less pain and that is able to be removed as an intact system (in one piece).

In some examples, the bottom of the device housing can have channels or other disruptions2845that allow air flow under the device housing and also allow moisture to flow away from the skin and adhesive system6000. The device can therefore be bonded to the underlying adhesive system6000in a disrupted manner. The device can be attached to the adhesive system6000in a plurality of ways. For example, the device housing2832can be attached to the adhesive system6000using heat staking, an adhesive layer (e.g. device adhesive2830discussed above or any other type of adhesive) or through ultrasonic welding.

FIG. 30illustrates a schematic view of the device2832attached to the skin6001with the adhesive system6000. As discussed above, the material layers of the adhesive system6000can provide a high MVTR under the housing of the device2832such that water does not accumulate under the device2832.

FIG. 30includes a plurality of arrows that illustrate the movement of moisture from the skin6001and through the adhesive system6000. As denoted by the arrow, the skin6001can perspire, generating sweat2844that moves to the surface of the skin6001. The high MVTR material of the adhesive system6000can transfer the sweat2844the bottom layer6006, which can be a wicking material. The wicking material of the adhesive system6000can pull the moisture away from the skin6001. The adhesive system6000can then allow the water vapor2840to evaporate from the skin6001by causing it to travel laterally through the wicking material of the adhesive system6000. In some embodiments, the material of the adhesive system6000can also serve to repel water from the top surface of the adhesive system6000. Additionally, any disruptions2845on the bottom of the device housing2832also helps aid sweat and other water vapor to evaporate from under the adhesive system6000and device housing2832.

Turning briefly to the embodiments of the adhesive systems illustrated inFIG. 29, in some examples, the moisture will wick through the layer of spun lace non-woven material and will evaporate through the top layer, which, in some embodiments, is a modified polyurethane. Evaporation may occur through the plurality of perforations in the top layer of the adhesive systems. In some examples, the moisture will evaporate form the top of the adhesive system and diffuse out from under the sensor housing2832,3110through the disruptions2845on the bottom of the sensor housing2832,3110.

Implanting a Sensor in a Patient

Disclosed is an inserter system and associated methods for transdermally inserting a sensor for a continuous glucose monitoring system.

The sensor inserter system is a single-use device that can allow the patient to safely and reliably place the sensing element of the sensor assembly into the skin with little or no pain. The sensor inserter system can be sterile packaged such that it can provide a simple and safe way to handle the sensor assembly during sensor insertion. In some examples, the sensor inserter is preassembled with the disposable sensor and sterilized as a system. The disposable sensor is ready for insertion when the sensor inserter is removed from its packaging.

In some examples, the disposable sensor can be inserted on the abdomen or the dorsal upper arm. The sensor insertion process is simple and reliably inserts the sensor. The sensor inserter system can enable the proper depth placement of the percutaneous sensor. The sensor insertion process using the sensor inserter system can be simple, intuitive, and brief. After the sensor is attached to the skin of the patient, the sensor inserter can be withdrawn and disposed. In some embodiments, the sensor inserter may be reusable—up to 20 times, with replaceable, one-time-use lancets.

As will be described in more detail below, the sensing element of the sensor assembly is inserted into the subcutaneous tissue using the sensor inserter system. The sensor inserter system is preassembled with the sensor assembly and can be provided to the user using a sterile sensor inserter assembly to facilitate easy sensor placement. The percutaneous sensing element of the sensor assembly is inserted into the tissue by means of an insertion lancet. The sensor inserter assembly can be removed after the sensor assembly is placed and discarded. As discussed in previous sections above, the on-body transmitter can be connected to the sensor assembly after the sensor is placed. The on-body transmitter can interrogate the sensor assembly in order to obtain sensor measurements that can be transmitted to the primary display. The primary display can contain a receiver and microprocessor to convert the transmitted measurements into calibrated glucose measurements.

FIGS. 31A and 31Bprovide a schematic illustration of the interaction between the sensor assembly, the inserter system, and the interaction with the tissue of a patient. Turning first to the sensor assembly, in some embodiments, the sensor assembly can include a sensor housing3110. The sensor housing3110can include a sensor mechanical optical interconnect (OIC)3120. As discussed above, the sensor mechanical optical interconnect3120can be mechanically connected to a transmitter mechanical optical interconnect3300. In some embodiments, a surface of the sensor housing3110can include an adhesive system2800that can allow the sensor assembly to be attached to a surface of the patient's skin3400.

In order to deliver the percutaneous portion of the device, such the sensing element of the sensor assembly into the skin, an inserter system2900can be provided. The inserter system2900can include a lancet hub3020that includes a lancet3000or other insertion structure. As will be described in more detail below, the lancet3000can include a lancet sensor interface3010that is configured to retain a portion of the looped sensor lancet interface3140. As shown inFIG. 31A, the sensor housing3110can include a body laminate3130with looped sensor lancet interface3140that can be retained on the lancet sensor interface3010. The lancet3000is configured to insert a portion of the sensor assembly (at least the sensor looped distal portion4004as disclosed and described below), into the interstitial fluid/tissue interface3500. As will be described in more detail below, the inserter system2900can be configured to allow the lancet3000to be removed from the patient's tissue while leaving a portion of the sensor assembly (e.g. the sensing element) implanted in the tissue of the patient.

FIG. 32illustrates a schematic illustration of the inserter system2900that are further illustrated inFIGS. 33A-D.FIGS. 33A-Cillustrate an embodiment of the inserter system2900and sensor assembly3100. In some embodiments, the inserter system2900can include an inserter housing2910and a cap2940. The cap2940can be provided to prevent unintentional contact of the patient with the lancet3000.FIG. 33Dillustrates a perspective view of the complete inserter system2900and a perspective view of the internal sensor assembly3100removed from its internal location within the inserter housing2910.

The sensor assembly3100consists of a sensor housing3110, a lancet3000, an adhesive system2800, and a sensor subassembly3160. As noted above, in some embodiments, the sensor subassembly3160can include the sensing element described above. As well, in some embodiments, the sensor subassembly3160does not contain any electronics.

As will be described in more detail below, the inserter system2900can include a housing and a rail system2920. To insert the sensor subassembly3160into the tissue, the inserter system2900can include a lancet assembly3170that can include a lancet3000and a lancet hub3010(FIG. 33D). The sensor assembly3100can include a sensor housing3110, the sensor subassembly3160, the adhesive system (described in more detail above), and the lancet assembly3170.

As illustrated inFIG. 33B, the sensor subassembly3160can be adhered to the upper surface of the sensor housing3110. In some embodiments, the insertion lancet3000can be adhered to the bottom surface of the sensor housing3110. As will be described in more detail below, the tip of the lancet3000can be mechanically mated to the tip of the sensor subassembly3160. The tip of the lancet3000can be shaped like a suture cutting needle so as to allow the sensor subassembly to be clean1y inserted into the patient's tissue with minimal trauma and little or no pain. With such a shape, the tip of the lancet3000cuts skin and other body tissue instead of tearing through skin and body tissue. Embodiments of the lancet3000design will be discussed in more detail below. After the lancet3000is delivered through the skin, upon withdrawal of the lancet3000from the skin, the sensor is released from the tip of the lancet3000and remains implanted. Embodiments of the lancet3000disclosed herein can be used to deliver and implant sensors for analyte monitors, including the glucose monitors disclosed herein as well as to deliver and implant micro catheters and drug eluting implants. The micro catheters can be for infusion pumps to deliver, for example, insulin, therapeutic agents and other treatments (chemotherapy, for example) to a patient.

As depicted inFIG. 34AandFIG. 34B, lancets/insertion structures3000according to embodiments of the present invention comprise a substantially planar, non-rigid, non-frangible, elongate member having a proximal portion3003, an intermediate portion3004, a distal portion3005for piercing the skin for subcutaneous insertion and a longitudinal axis3051. In some embodiments, the elongate member may not be planar and may be rigid and/or frangible. The elongate member can have a thickness “T” ranging between approximately 100 μm to approximately 400 μm depending on the material used and the depth of insertion (as discussed below). This thickness can be uniform along the length of the elongate member or the thickness can vary. The thickness “T” of the elongate member can be chosen to ensure that the elongate member remains in a configuration that permits successful insertion through skin and into subcutaneous tissue and this thickness may be dependent on the Young's modulus of the material from which the elongate member is constructed as well as other properties of the material. That is, the Young's modulus of the elongate member material will correspond to the thickness of the material required to ensure successful insertion through the skin. In some embodiments, the elongate member is constructed from fully tempered stain1ess steel such as, for example, stain1ess steel (SS) 1.4028. Stain1ess steel 1.4028 is a martensitic stain1ess steel. Martensitic stain1ess steels are ones with high hardness and high carbon content. These steels are generally fabricated using methods that require hardening and tempering treatments is used in the quenched and tempered condition in a host of constructional where corrosion resistance is required. Due to its higher carbon content, SS 1.4028 is more hardenable than SS 1.4021, with a 50 HRC and a Young's modulus of 200 GPa. As for other martensitic grades, optimal corrosion resistance is attained when the steel is in the hardened condition and the surface is finely ground or polished.

Also, the thickness “T” of the material used and which material (Young's modulus) used for the elongate member may be dependent on the depth of insertion of the elongate member's distal portion3005into subcutaneous tissue, i.e., the distance that the tip3030of the elongate member's distal portion3005is inserted into the subcutaneous tissue as measured from the surface of the tissue to the deepest point of the tip3030within the tissue. This distance is also known as the insertion length of the elongate member.

In some embodiments, for an insertion length of the elongate member ranging between approximately 5 mm to approximately 9 mm, the thickness “T” of the elongate member is approximately 200 μm. In some embodiments, for an insertion length of the elongate member of approximately 9 mm, the thickness “T” of the elongate member is approximately 180 μm. In some embodiments, for an insertion length of the elongate member of approximately 9 mm, the thickness “T” of the elongate member is approximately 250 μm. In some embodiments, for an insertion length of the elongate member ranging between approximately 4 mm to approximately 10 mm, the thickness “T” of the elongate member ranges between approximately 180 μm and approximately 250 μm.

The elongate member includes a first surface3001and a second surface3002. As depicted in the figures, the first surface3001and the second surface3002are opposite each other and can be a top and a bottom surface of the elongate member. The proximal portion3003of the elongate member provides a mechanical interconnect between the lancet3000and the sensor assembly3100for attaching the lancet3000to the sensor assembly3100.

FIGS. 35A-35Qdepict various embodiments of the distal portion3005of the elongate member. The distal portion3005includes a first surface3006, a second surface3007that is substantially opposite the first surface3006and a tip3030. In order to cut through the skin and subcutaneous tissue during insertion, the distal portion3005includes at least one cutting surface/edge3050. This cutting surface3050can be, for example, a positive convex surface that forms a cutting surface/edge. In some embodiments, the distal tip portion includes a plurality of cutting surfaces3050that can be adjacent to the distal portion first surface3006and/or the distal portion second surface3007or that can be disposed between the distal portion first surface3006and the distal portion second surface3007.

In some embodiments, as depicted inFIGS. 36A-36E, the cutting surface3050extends from the tip3030along at least a portion of the length of the distal portion3005thereby creating a cutting portion3011having a cutting surface length3012. This cutting surface length3012may be dependent on the angle (α) of the cutting surface3050and the desired width of the cutting surface3050. In some embodiments, the cutting surface3050forms an acute angle (α) that is defined by the intersection of a plane that is substantially parallel to the first surface3006and a line that is tangent to the cutting surface3050. In some embodiments, the acute angle (α) ranges between approximately 15° and 45°. In some embodiments, the cutting surface length3012ranges between approximately 300 μm and 1,000 μm.

The location and the design of the cutting surfaces3050allow the lancet3000to be inserted into the skin and subcutaneous tissue of the patient with low trauma and/or pain as these surfaces cause the distal portion3005to cut through the skin and subcutaneous tissue instead of tearing through the tissue. In some embodiments, the cutting surfaces3050can be formed by chemical etching, laser milling, mechanical grinding or micro electric discharge machining (EDM).

In some embodiments, the perimeter of the distal portion3005can be sized for the sensor package and elongate member with a tissue stretch that can be 20%, 30%, 40%, or 50%.

In some embodiments of the lancet3000, as depicted in the figures, the distal portion3005can include one or more insets or recessed portions3040that extend between the first surface3006of the distal portion3005and the second surface3007of the distal portion3005. The one or more insets or recesses3040are designed to receive at least a portion of a looped sensor lancet interface3140located in the percutaneous portion of the sensor (discussed further below) to be inserted/implanted into the skin and can be, for example, circular or curvilinear. In some embodiments, the one or more insets or recessed portions3040form an area on the distal portion3005that has a narrower width than other portions of the distal portion3005. This narrower area provides a recess to receive portions of the looped sensor lancet interface3140. In addition to the one or more insets or recessed portions3040that extend between the first surface3006of the distal portion3005and the second surface3007, in some embodiments, the distal portion3005can include a recessed area3009on each side of the distal portion3005that extend along at least a portion of the length of the distal portion3005. These recessed areas3009can also receive a portion of the sensor lancet interface3140.

In some embodiments, as depicted inFIG. 34A, the distal portion first surface3006can include surface recesses3041, which can also receive at least a portion of the looped sensor lancet interface3140. Because the looped sensor lancet interface3140can be received in insets/recessed portions3040, recessed areas3009and surface recesses3041, these elements help retain the sensing element on the distal portion and can also help to reduce the profile of the distal portion3005during insertion, which aids in reducing pain and trauma during implantation.

In order to help retain the looped sensor lancet interface3140on the distal portion3005prior to and during insertion of the distal portion3005into subcutaneous tissue, a retaining element/structure3060is included. In some embodiments, the retaining element/structure3060is on the first surface3006and in some embodiments the retaining element3060is on the second surface3007. The retaining element/structure3060is designed to retain the looped sensor lancet interface3140on the distal portion3005during insertion into the tissue and to release the sensing element3140from the distal portion3005upon removal of the distal portion3005from the tissue, thereby leaving the looped sensor lancet interface3140implanted within the subcutaneous tissue along with the percutaneous portion of the sensor. Retaining of the looped sensor lancet interface3140on the distal portion3005before and during subcutaneous tissue insertion (i.e., when there is no movement of the distal portion3005and when there is forward movement of the distal portion3005) and release of the looped sensor lancet interface3140upon removal of the distal portion3005from the skin (backward movement of the distal portion3005), can be achieved by (1) designing the distal facing front surface3008of the retaining element/structure3060to have a certain shape/geometry and/or (2) a combination of the geometry of the distal facing front surface3008of the retaining element/structure3060and the orientation of the looped sensor lancet interface3140with respect to the distal facing front surface3008.

FIGS. 36A-36Edepict various embodiments of the distal portion3005of the elongate member having retaining elements/structures3060with different shapes/geometries for the distal facing front portion3008. As used herein, a “substantially forward facing front portion” of the engagement/retaining structure3060is defined by the below description and depicted inFIGS. 36A-36E.FIG. 36Adepicts a distal facing front portion3008having a curved geometry with an angle θ1of between approximately 20° and approximately 90° formed between a tangent of the curved distal facing front portion3008and a plane that is parallel to the distal portion second surface3007.FIG. 36Bdepicts a distal facing front portion3008having a curved geometry with an angle θ1of between approximately 90° and approximately 160° formed between a tangent of the curved distal facing front portion3008and a plane that is parallel to the distal portion second surface3007.FIG. 36Cdepicts a distal facing front portion3008having an acute angled geometry where the acute angle (α) is defined by the intersection of (1) a plane tangent to a first portion3008aof the distal facing front portion3008that forms an angle θ1with the distal portion second surface3007of between approximately 20° and approximately 90° and (2) a plane that forms an angle θ2of up to ±20° with the first surface3006a.FIG. 36Ddepicts a distal facing front portion3008having an obtuse angled geometry where the obtuse angle (α) is defined by the intersection of (1) a plane tangent to a first portion3008aof the distal facing front portion3008that forms an angle θ1with the distal portion second surface3007of between approximately 90° and approximately 160° and (2) a plane that forms an angle θ2of up to ±20° with the first surface3006a.FIG. 36Edepicts a distal facing front portion3008having an obtuse angled geometry where the obtuse angle a is defined by the intersection of (1) a plane tangent to a first portion3008aof the distal facing front portion3008that forms an angle θ1with the distal portion second surface3007of between approximately 90° and approximately 160° and (2) a plane tangent to a second portion3008bof the distal facing front portion3008that forms an angle θ2with the distal portion first surface3006of between approximately 10° and approximately 45°.

Depicted inFIGS. 36F-36Mare top and bottom views (top views shown inFIGS. 36F, 36H, 36J and 36Land bottom views shown inFIGS. 36G, 36I, 36K and 36M), of various embodiments of the distal portion3005of the elongate member. As can be seen in the figures, the embodiments include a leading tip portion3031and a trailing portion3032with each portion having cutting edges and cutting surfaces as discussed below. As can also be seen inFIGS. 36G, 36I, 36K and 36M, the tip portion3031has a cutting angle Ω. In some embodiments, the cutting angle Ω ranges from between approximately 30° to approximately 40°. It is important that the cutting angle Ω remain narrow such that the tip portion3031does not tear skin/flesh as it is inserted into skin/flesh to deliver the sensing element. In preferred embodiments, the cutting angle Ω is 30°, 31°, 32°, 33°, 34°, 35°, 36°, 37°, 38°, 39°, or 40°.

As depicted inFIGS. 36F-36M, the leading tip portion3031includes tip portion cutting edges3051and tip portion cutting surfaces3052. In the embodiments shown inFIGS. 36G, 36I, 36K and 36M, the tip portion cutting surfaces3052(shaded in the figures) are located on the second side/surface3007and extend from the second side/surface3007to the first side/surface3006. The trailing portion3032includes trailing portion cutting edges3053and trailing portion cutting surfaces3054. In the embodiments shown inFIGS. 36F, 36H and 36J, the trailing portion cutting edges3053(shaded in the figures) are located on the first side/surface3006and extend from the first side/surface3006to the second side/surface3007. As can be seen inFIG. 36M, in some embodiments, both the tip portion cutting surfaces3052and the trailing portion cutting surfaces3054are located on the second side/surface3007and extend from the second side/surface3007to the first side/surface3006. As discussed in more detail below, the width WTipof the leading tip portion3031and the width WTrailof the trailing portion3032and the associated cutting edges3051and3053, respectively, are important for delivering the looped sensor lancet interface3140into the skin.

Depicted inFIG. 36Nis a looped sensor lancet interface3140of a sensor assembly3100, according to an embodiment of the invention. The looped sensor lancet interface3140includes an elongate sensing portion4000and a sensor looped distal portion4004that is defined and bounded by a sensor transmission element4006. As depicted inFIG. 36N, the elongate sensing portion4000extends to a proximal end4008of the sensor looped distal portion4004where it divides into two legs of the sensor transmission element4006, which form the sensor looped distal portion4004. The sensor looped distal portion4004includes a first opening4010that is adjacent to the loop tip portion3143with a maximum first width4012and a second opening4014disposed between the proximal end4008and the first opening4010. The second opening4014has a maximum second width4016that is greater than the maximum first width4012. The first opening4010and the second opening4014are contiguous. As can be seen inFIG. 36N, the sensor transmission element4006includes sensor looped transition portions4018(a) between the first opening4010and the second opening4014and (b) between proximal end4008and the second opening4014of the sensor looped distal portion4004, that are thicker than the other portions of the sensor transmission element4006. As discussed in more detail below, the thicker portions of the sensor looped transition portions4018aid in the un1oading of the sensor looped distal portion4004from the distal portion3005and also helps in anchoring of the sensor in subcutaneous tissue. After implantation, the looped sensor lancet interface3140along with the percutaneous portion of the sensor, provides the required interstitial fluid information to the sensor assembly110A and hence, the analyte sensors of the embodiments of the present invention.

Depicted inFIG. 35BandFIGS. 35H-35K, are embodiments of a looped sensor lancet interface3140loaded in place on the distal portion3005of an elongate member. As can be seen in the figures, the elongate sensing portion4000extends along the distal portion first surface3006and the sensor looped distal portion4004loops over the tip3030in order for the loop tip portion3143to engage the retaining structure3060. Once the loop tip portion3143is engaged on the retaining structure3060, portions of the sensor transmission element4006that define the first opening4010of the sensor looped distal portion4004are received within the insets/recessed portions3040. To cause the portions of the sensor transmission element4006that define the first opening4010of the sensor looped distal portion4004to be received within the insets/recessed portions3040, once the sensor looped distal portion4004is looped over the tip3030, the elongate sensing portion4000is tensioned or pulled proximally away from the tip3030, causing (1) the loop tip portion3143to engage the retaining structure3060and (2) the portions of the sensor transmission element4006that define the first opening4010of the sensor looped distal portion4004to seat or be received within the insets/recessed portions3040. Further proximal movement/tensioning of the elongate sensing portion4000, causes the sensor transition element4006portions that define the second opening4014of the sensor looped distal portion4004to collapse inwards, reducing the width of the second opening4014. Thus, when sensor looped distal portion4004is loaded onto the elongate member, the width of the second opening4014is reduced causing the sensor transition element4006to deform. This deformation, however, is elastic and therefore, once the sensor looped distal portion4004is un1oaded from the distal portion3005, the sensor transition element4006springs back to its original shape, which causes the second opening4014to return to its original shape and width that is wider than its width during the insertion/implantation process, which helps anchor the looped sensor lancet interface3140and hence, the sensor assembly3100, in tissue because when the second opening4014returns to its original shape and width (its pre-loaded width), the width of the second opening4014and hence, the width of the looped sensor lancet interface3140, is now wider than the width of the tissue that was cut by the cutting edges3051,3053and cutting surfaces3052,3054of the distal portion3005of an elongate member during insertion/implantation. The thicker sensor looped transition portions4018on the sensor transition element4006, aid in helping the second opening4014return to its original shape and width.

Depicted inFIG. 350is another embodiment of a looped sensor lancet interface3140loaded in place on the distal portion3005of an elongate member. In this embodiment, the retaining structure3060is disposed on the same surface as the elongate sensing portion4000. As can be seen inFIG. 350, the elongate sensing portion4000extends along the distal portion first surface3006and the sensor looped distal portion4004is placed over the retaining structure3060such that the loop tip portion3143is positioned distally of the retaining structure3060. Once the loop tip portion3143is positioned distally of the retaining structure3060, the elongate sensing portion4000is tensioned or pulled proximally away from the tip3030, causing, as discussed above, (a) the loop tip portion3143to engage the retaining structure3060and (b) the sensor transition element4006portions that define the second opening4014of the sensor looped distal portion4004to elastically deform and collapse inwards. As also discussed above, once the sensor looped distal portion4004is un1oaded from the distal portion3005, the sensor transition element4006springs back to its original shape, which causes the second opening4014to return to its original shape and width.

It is important that in most embodiments, the width WTipof the leading tip portion3031and the width WTrailof the trailing portion3032and the associated cutting edges3051and3053, respectively, be sufficiently wide in order to “shield” or cut tissue wide enough such that leading portions of the looped sensor lancet interface3140are passing through “cut” skin during delivery and are not tearing skin during delivery. That is, after the loop tip portion3143is positioned distally of the retaining structure3060and the elongate sensing portion4000is tensioned or pulled proximally away from the tip3030, causing, as discussed above, (a) the loop tip portion3143to engage the retaining structure3060and (b) the sensor transition element4006portions that define the second opening4014of the sensor looped distal portion4004to elastically deform and collapse inwards and therefore, ready for insertion into skin, with the sensor looped distal portion4004elastically deformed and collapsed inwards, the cutting edges3051of the leading tip portion3031and the cutting edges3053of the trailing portion3032, extend past at least the leading edges of the looped sensor lancet interface3140.

As can best be seen inFIG. 360, which shows a looped sensor lancet interface3140loaded in place on the distal portion3005of an elongate member, the width WTipof the leading tip portion3031and hence, the cutting edge3051, is greater than the width of the first opening4010. Further, the width WTrailof the trailing portion3032and hence, the cutting edges3053of the trailing portion3032, is greater than at least the width of the sensor looped transition portions4018between the first opening4010and the second opening4014. Thus, during insertion/delivery of the looped sensor lancet interface3140into skin/tissue, the cutting edges3051of the leading tip portion3031and the cutting edges3053of the trailing portion3032cut a wide enough opening in the skin/tissue for the looped sensor lancet interface3140to be implanted into the skin without tearing of the skin/tissue thereby reducing pain and resistance during delivery and implantation.

Although in the embodiments of the lancet3000disclosed and described herein, all of the features associated with retaining and releasing the looped sensor lancet interface3140, i.e., the insets/recesses3040, recessed area3009, surface recesses3041and the retaining element/structure3060, are depicted as being on the distal portion3005of the elongate member, these need not be limited to the distal portion3005. Instead, these features can be located at any location along the elongate member, for example, they can be located at the intermediate portion3004of the elongate member, such that the looped sensor lancet interface3140can be loaded onto and delivered into subcutaneous tissue from this portion of the elongate member.

FIG. 37illustrates an embodiment of a method3700of inserting/implanting a sensing element into subcutaneous tissue. Prior to insertion/implantation of the sensing element3141into subcutaneous tissue, the sensing element is loaded onto the lancet3000(block3710). The sensing element3141and hence, the sensor looped distal portion4004are loaded in the manner described above such that the sensor transition element4006portions that define the second opening4014of the sensor looped distal portion4004are elastically deformed and collapse inwards. During insertion, the distal portion3005of the elongate member is advanced distally or forward into the subcutaneous tissue (block3720). After the distal portion3005/ tip3030is delivered to its desired depth within the subcutaneous tissue, i.e., the depth of insertion for the sensing element3141, the distal portion3005/tip3030is retracted proximally or backwards away from or out of the subcutaneous tissue (block3730). Because, as illustrated in the figures, the looped sensor lancet interface3140is engaged with the distal portion3005/tip3030in a manner that on1y restricts backward movement of the looped sensor lancet interface3140on the elongate member, backward movement of the distal portion3005/tip3030causes the loop tip portion3143to disengage from the retaining structure3060, which permits the sensor looped distal portion4004to disengage and un1oad from the distal portion3005of the elongate member (block3740). As the sensor looped distal portion4004disengages and un1oads from the distal portion3005, the inwardly-tensioned sensor transition element4006portions that define the second opening4014, spring back outwardly to substantially assume their original shape and width, which now help to anchor the sensor looped distal portion4004and hence, the sensing element3141, at the correct depth within the subcutaneous tissue. As the distal portion3005/tip3030continues to retract from the skin or body tissue, the remaining components of the sensing element3141disengage from the elongate member leaving the sensing element3141implanted in the subcutaneous tissue.

Although, embodiments of the lancet3000disclosed herein have been described for delivering/implanting a sensing element in body tissue, embodiments of the lancet3000can be used for other medical applications. For example, embodiments of the lancet3000can be used to implant drug delivery cannulas (micro catheters) or other delivery lumens for infusion pumps to deliver, for example, insulin and other therapeutic agents/treatments to a patient. Additionally, items that can be delivered with the embodiments of the lancet300disclosed herein include, and are not limited to, drug eluting implants. In some embodiments, these delivery lumens and other implants can be combined with the sensor looped distal portion4004to allow the delivery lumens and other implants to be implanted in a similar manner to how embodiments of the looped sensor lancet interface3140are implanted.

FIGS. 35P-35Qdepict additional embodiments of the sensor assembly3100and the sensor assembly3100retained on the lancet3000.FIGS. 35P and 35Rillustrate the sensor assembly3100. In some embodiments, the sensor assembly3100can include an opening3150that extends along the length of the body of the sensor assembly3100. As illustrated inFIG. 35Q, the lancet3000can include a corresponding convex horn3070that extends from the surface of the lancet3000. In some examples, the convex horn3070of the lancet3000can engage the opening3150such that the opening3150is disposed about the convex horn3070. This configuration can help to properly retain the sensor assembly3100along the lancet3000.

Analyte Sensor and its Operation

The bio sensor of the present invention does not utilize an electrochemical sensing modality and does not require the immobilization of the enzyme to an electrode. Rather the present biosensor requires the formation of active hydrogels within the sensor. There is a need to consistently formulate an active hydrogel impregnated with controlled active macromers; for example, the formulation of a hydrogel with the desired permeability properties containing an activated enzyme (e.g. GOx macromere). Preferably, the active hydrogel material is formulated so that it can be characterized during sensor manufacturing without destructive sensor testing.

In some embodiments, methods of preparing a sensor tip for a glucose monitoring device are described. In some embodiments, the methods pertain to fabricating a sensor tip that is small enough to be inserted subcutaneously into a patient with little or no pain. In some embodiments, the sensor tip and its components are adapted and configured to be mass-produced at small-length scale.

In some embodiments, the sensor tip (e.g., sensing system) comprises one or more components (e.g., regions, layers, sections, etc.). In some embodiments, as shown inFIG. 38, the individual components of the glucose sensor tip3800include an oxygen conduit3820, an oxygen in1et surface3821, an enzymatic region3830, and a sensor region3840(e.g., an oxygen sensing polymer). In some embodiments, the oxygen conduit3820, enzymatic region3830, and sensor region3840can be combined to provide the sensing portion of a glucose sensor system. In some embodiments, the glucose sensor tip further comprises a base support3860. In some embodiments, the base support3860is configured to provide a substrate on which one or more components of the glucose sensor tip3800can reside.

In some embodiments, each region (e.g., the oxygen conduit, the enzymatic region, and/or the oxygen sensing polymer region) is a distinct layer within a glucose sensing device. In some embodiments, a region can be embedded within, or supported by another region. In some embodiments, multiple regions serving each function can be provided. For example, in some embodiments, there are multiple oxygen conduit regions, enzymatic regions, and/or sensor regions. In some embodiments, there 1, 2, 3, 4, 5, or more oxygen conduit regions, enzymatic regions, and/or sensor regions. In some embodiments, each region serves a discrete function (e.g., one region acts as an oxygen conduit, one acts as the enzymatic region, and one acts as a sensor). In some implementations, regions can serve similar, overlapping, or the same function.

In some embodiments, as shown inFIGS. 39 and 40, the oxygen conduit region3820comprises a species that binds and releases oxygen, transporting it through or within the region. In some embodiments, also as shown inFIGS. 39 and 40, the enzymatic region3830comprises one or more enzymes that catalyze a reaction to convert one or more species within the enzymatic region into identifiable products. As shown inFIGS. 39 and 40, glucose oxidase (GOx) and catalase (CAT) can be used together in the enzymatic region3830. While GOx and CAT used as exemplary enzymes throughout this disclosure, other enzymes or enzyme combinations can be employed keeping in mind the goal of the enzymatic layer is to produce a measurable species for analytical data. Non-limiting examples of other suitable enzymes may be glycollate oxidase, lactate oxidase, galactose oxidase, xanthine oxidase, pyruvate oxidase, D-aspartate oxidase. monoamine oxidase, carbohydrate oxidase, cholesterol oxidase, and alcohol oxidase.

As shown inFIGS. 38, 39, and 40, in some embodiments, the oxygen conduit is configured to receive environmental oxygen (e.g., from the tissue of a patient or some other environment proximal to the tip) and to transport it. In some embodiments, as shown, the enzymatic region3830(i.e., enzymatic hydrogel), is configured to receive oxygen from a portion of the oxygen conduit3820through an enzymatic region oxygen in1et3831. Also as shown, in some embodiments, the enzymatic region3830is configured to receive environmental glucose (e.g., from the tissue of a patient) via a glucose in1et3832.

As shown inFIGS. 39 and 40, the one or more enzymes can, for example, catalyze reactions to convert reactants (e.g., analytes) into identifiable products. In some embodiments, the enzymatic region comprises combinations of enzymes that catalyze reactions to convert analytes and other enzymes that catalyze reactions to convert the byproducts of the primary reaction. For example, as shown inFIGS. 38 and 39, in some embodiments, the GOx can convert glucose and oxygen into gluconolactone and H2O2:

H2O2can then be converted back to oxygen and water in the presence of water and CAT to afford product oxygen:

As shown above, this reaction scheme causes a net decrease in the amount of oxygen (by ½ of a mole compared to environmental oxygen). This decrease in oxygen can be detected using an oxygen sensing polymer3840and by comparing the amount of product oxygen to the amount of oxygen in a reference sample.

As shown inFIGS. 38, 39, and 40, a reference oxygen sensing polymer3845is provided to provide a measure of the amount of environmental oxygen present. In some embodiments, the difference between the oxygen present at the reference oxygen sensing polymer3845and the oxygen sensing polymer3840can be used to provide an indirect measure of glucose. In some embodiments, this indirect measurement allows, highly sensitive glucose monitoring.

In some embodiments, as discussed elsewhere herein, the oxygen sensing polymer regions3840and3845comprise an oxygen detecting dye. In some embodiments, the dye is a luminescent dye. Generally, luminescent dyes used to probe for oxygen may be polyaromatic hydrocarbons, fullerenes, phosphorescent organic probes, metal ligand complexes such as Pt complexes, PD complexes, Ru(II) complexes, Ir complexes, Os complexes, Re complex, lanthanide complexes, and a like, porphyrins, metalloporphyrins, and luminescent nanomaterials. Non-limiting examples of suitable luminescent dyes may be metallo derivatives of octaethylporphyrin, tetraphenylporphyrin, tetrabenzoporphyrin, or the chlorins, bacteriochlorins, or isobacteriochlorins and their partially or fully fluorinated analogs. Other suitable con/pounds include palladium coproporphyrin (PdCPP), platinum and palladium octaethylporphyrin (PtOEP, PdOEP), platinum and palladium tetraphenylporphyrin (PtTPP, PdTPP), camphorquinone (CQ), and xanthene type dyes such as erythrosin B (EB). Other suitable compounds include ruthenium, osmium and iridium complexes with ligands such as 2,2′-bipyridine, 1,10-phenanthroline, 4,7-diphenyl-1,10-phenanthroline and the like. Suitable examples of these include, tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(II) perchlorate, tris(2,2′-bipyridine)ruthenium(II) perchlorate, and tris(1,10-phenanthroline)ruthenium(II) perchlorate. While the perchlorate salts are particularly useful, other counterions that do not interfere with the luminescence may be used. In some embodiments, the porphyrin dye is platinum tetrakis pentafluorophenyol porphyrin (PtTFPP).

In some embodiments, the luminescent dye emits a measurable signal dependent on the amount of oxygen present. Thus, interrogating the oxygen in the oxygen sensing polymer of the reaction region3840and the reference oxygen sensing polymer3845give a measure of the amount of glucose present.

In some embodiments, the working oxygen sensing polymer3840and the reference oxygen sensing polymer3845are interrogated by test waveguides3850and reference waveguides3855, respectively, as shown inFIGS. 38, 39, 40, and 41. Information gathered by these waveguides can be gathered, processed, and used to provide information to a patient or doctor regarding glucose levels in the patient.

Certain embodiments disclosed herein provide methods for making glucose monitoring device components and methods of combining components to yield devices that provide a convenient means of continuous glucose monitoring. In some embodiments, the methods disclosed herein are especially suited for preparing devices that have very small dimensions. For example, in some embodiments, a given sensor feature comprises a three dimensional shape having an x-dimension, a y-dimension, and a z-dimension. In some embodiments, the smallest dimension of the x-, y-, and z-dimensions feature is less than about 0.05 mm. In some embodiments, the glucose sensor tip3800shown inFIG. 41Ahas dimensions of about 0.05 mm by about 0.3 mm by about 1.5 mm in the x-, y-, and z-directions. In some embodiments, the glucose sensor tip has dimensions less than about 0.05 mm by about 0.3 mm by about 1.5 mm. In some embodiments, the small features of the sensor tip minimize the size of the device and maximize the efficiency and accuracy at which these devices can measure analytes.

In some embodiments, these small dimensions can be achieved by the unique polymer systems and fabrication methods disclosed herein (as shown inFIG. 41B). For instance, these small features can be provided by supplying solutions of crosslinkable (or crosslinked) materials that can be taken-up by spaces (e.g., channels, tunnels, paths, etc.) in molds (e.g., dye casts, lithography plates, knife coated, etc.) by capillary action to produce features of less than about 0.05 mm, in some cases as small as about 10 μm, at their smallest dimension (as shown in41C). For example, as shown inFIG. 42, solutions can be taken up through capillary action into mold4200ports4210. These ports are configured to distribute the material solutions via channels throughout sensing tip3800. These solutions, as discussed in more detail elsewhere herein, can then be cured (e.g., crosslinked with crosslinkers) and/or concentrated to afford individual sensor components (e.g., an oxygen conduit3820, an enzymatic region3830, and/or an oxygen sensing polymer region3840,3845).

In the mass production of the present biosensor, active hydrogels are preferably consistently prepared and located within a specified region inside the sensor. The volume of the regions for locating the active hydrogels for oxygen transport or enzymatic reduction of an analyte are small for devices that will be minimally invasive. For example, an active hydrogel region may be <200 pL, <500 pL, <1 nL, <5 nL, <10 nL, or <50 nL. The controlled immobilization of a target macromer (e.g., oxygen binding molecule or enzyme) and incorporation of the target macromer into the hydrogel polymer network is difficult to consistently accomplish in such small reaction volumes using prior art methods, and direct placement of a membrane or hydrogel that must be cut to size is difficult. Further, the characterization of the extent of crosslinking of the hydrogel and macromer to be immobilized is difficult to assay in the sensor given the small volumes present, particularly in a non-destructive manner. The methods for making the present biosensor disclosed herein address these manufacturing issues.

According to the present invention, in order for the active hydrogel to have stable properties and to prevent immobilized macromer from diffusing from the sensor, the immobilized macromer is preferably retained by a stable linkage in a hydrogel, rather than being entrapped passively into a hydrogel as is typically the case in prior art biosensors. In some embodiments of the present methonds, this process of macromer stabilization and immobilization may be facilitated by crosslinking the target macromer to a nano structure (e.g., a carrier protein such as albumin) and conjugation of the macromer-nanostructure complex to a polymer network to form a nanogel particle. According to the present invention, the nanogel particle is used as a precursor or interim form from which the active hydrogel regions (e.g., the oxygen conduit region and the enzymatic region) may be formulated within a bio sensor. In contrast to prior hydrogel formation methods, the macromer-nanostructure complex and the resulting nanogel particles of the present invention may be more fully characterized and formulated in a controlled and consistent manner. Moreover, it has been found that the characteristics of the nanogel particles primarily determine the properties of the active hydrogel.

Thus, embodiments of the present methods are able to overcome the complex challenges of consistently crosslinking a macromer to a nanostructure while conjugating the complex to a polymer network in very small volumes inside individual sensors amenable to minimally invasive application. Furthermore, embodiments of the present methods may be used to execute multistep formulation chemistry while maintaining quality control of the resulting active hydrogels.

In some embodiments, in order to improve quality control in a biosensor application, the extent of the target macromer crosslinking with the nanostructure is preferably controlled to achieve consistent crosslinking to form a reproducible nanogel particle with the desired stability and activity. For example, enzymatic activity is inversely proportional to the concentration of linker used to link enzyme to the nanostructure because extensive crosslinking may result in a distortion of the enzyme structure (i.e., the active site conformation) [Chui, W. K. and L. S. Wan. 1997. Prolonged retention of cross-linked trypsin in calcium alginate microspheres. J. Microencapsulation 14:51-61]. With this distortion, the accessibility and accommodation of the active substrate may be reduced, thus affecting the retention of biological activity. In some embodiments of the present methods, for example where the nanostructure is a protein with a given number of crosslinking sites (such as lysine (Lys) residues on albumin), extent of the crosslinking between the nanostructure and the target macromer may be controlled by reducing the number of available crosslinking sites on the protein available to the crosslinking reaction between the target macromer and the protein.

For example, in some embodiments, an oxygen conduit component is prepared using a dispensable, UV-curable nanogel solution. In some embodiments, the dispensable, UV-curable nanogel solution can be prepared by first interconnecting (e.g., covalently bonding, complexing, etc.) a nanostructure with one or more reversible oxygen binding molecules thereby forming a reversible oxygen binding nanoparticle. In some embodiments, the oxygen conduit nanostructure comprises a macromolecular structure capable of supporting one or more oxygen binding molecules. In some embodiments, the nanostructure is albumin and the oxygen binding molecule is hemoglobin. For purposes of summarizing the disclosure, certain features have been described herein using albumin (with Lys residues that act as the crosslinking sites) and hemoglobin. While albumin and hemoglobin used to describe features herein, these molecules are exemplary and other nanostructures or oxygen binding molecules are envisioned. Sources of the nanostructures and oxygen binding molecules may be derived from a natural source (e.g., a human, an animal, or a plant) or synthesized. For example, in some embodiments, the nanostructure is any suitable protein. In some embodiments the reversible oxygen binding molecule comprises any suitable oxygen binding protein (e.g., hemoglobin, myoglobin, a synthetic oxygen carrier, etc.).

In some embodiments, the nanoparticle comprises a plurality of hemoglobin molecules functionalized to each albumin molecule. In some embodiments, the nanoparticle comprises less than one hemoglobin molecules per albumin molecule. In some embodiments, the ratio of hemoglobin to albumin is at least about 0.5:1, about 1:1, about 2:1, about 5:1, about 10:1, or about 15:1.

In some embodiments, hemoglobin is bound to albumin covalently. In some embodiments, the covalent link between hemoglobin is formed using a bifunctional linker. Suitable linkers include amino acids, peptides, nucleotides, nucleic acids, organic linker molecules, disulfide linkers, and polymer linkers (e.g., PEG). The linker can include one or more spacing groups including, but not limited to alkylene, alkenylene, alkynylene, alkyl, alkenyl, alkynyl, alkoxy, aryl, heteroaryl, aralkyl, aralkenyl, aralkynyl and the like. The linker can be neutral, or carry a positive or negative charge. In some embodiments the bifunctional linker is an organic linker molecule and selected from a dialdehyde, a dicarboxylic acid, a diepoxide, or the like. In some embodiments, the bifunctional linker is represented by one or more of the following structures:

where R is selected from the group consisting of —CH2—, —(CH2O)CH2, —CH(R—OH)—, —(CH2CH2O)—CH2CH2—, —(CF2CF2O)—CF2CF2——(CH2CH2CH2O)—CH2CH2CH2—, —(CF2CF2O)—CF2CF2—, —(CF2CF2CF2O)—CF2CF2CF2—, “a” is an integer between 0 and 1000, and LG is a leaving group. Non-limiting examples of leaving groups may be chloride, bromide, iodide, imidazole, benztriazole, triflate, tosylate, mesylate, or combinations thereof. In some embodiments, the hemoglobin and albumin are functionalized via amine groups residing on the hemoglobin and albumin molecules. In some embodiments, when a dialdehyde, a dicarboxylic acid, or a diepoxide are used as the bifunctional linkers, diimines, diamides, and diamines, respectively, result through reaction with the reversible oxygen binding molecule (e.g., hemoglobin) and albumin amines. In some embodiments, combinations of bifunctional linkers can be used.

There are additional amine-reactive groups below, which may be located on the termini of bifunctional linkers and in this configuration, used as crosslinking agents of primary amine groups. Bifunctional primary amine linkers may consist of a combination of the same (homobifunctional crosslinkers) or different (heterobifunctional crosslinkers) reactive groups. The scheme below denotes some non-limiting examples of suitable reactive groups.

The crosslinking of hemoglobin and albumin may involve multiple site reactions. For example, albumin is rich in Lys residues. One common and versatile technique for crosslinking or labeling peptides and proteins such as antibodies involves the use of chemical groups that react with primary amines (—NH2). Primary amines exist at the N-terminus of each polypeptide chain and in the side-chain of lysine (Lys) amino acid residues. These primary amines are positively charged at physiologic pH; therefore, they occur predominantly on the outside surfaces of native protein tertiary structures where they are readily accessible to conjugation reagents introduced into the aqueous medium. Furthermore, among the available functional groups in typical biological or protein samples, primary amines are especially nucleophilic; this makes them easy to target for conjugation with several reactive groups. Formaldehyde and glutaraldehyde are aggressive carbonyl (—CHO) reagents that condense amines via Mannich reactions and/or reductive amination.

The following represents a hemoglobin molecule linked to albumin using a dialdehyde (i.e., via a diimine linker):

In some embodiments, as shown above, the difunctional linker is glutaraldehyde (or another dialdehyde) and forms a diimine link via the aldehydes of glutaraldehyde and amines from hemoglobin and albumin. That configuration is also represented by the depiction:

In some embodiments, the hemoglobin is covalently linked to albumin by incubation with gluteraldehyde, at low temperature, low to no oxygen concentration, pH of between about 7.0 and 8.0, for an incubation time to complete the reaction, which is preferably at least about 2 hours or more, to form hemoglobin-albumin nanoparticles.

In some embodiments, the incubation time with glutaraldehyde is at least about 10 hours, about 24 hours, about 36 hours, or about 48 hours. In some embodiments, the glutaraldehyde (or other bifunctional linker) is provided to the albumin/hemoglobin solution at a low concentration. In some embodiments, the reaction is performed at low temperature and is below about 30° C., about 20° C., about 10° C., about 5° C., about 2° C.

In some embodiments, after incubation with glutaraldehyde and formation of the diimine linker, the hemoglobin-albumin nanoparticles are subjected to a reduction to convert the diimine linkages to diamine linkages. Non-limiting examples of reducing agents may be sodium borohydride, sodium cyanoborohydride, sodium triacetoxyborohydride, lithium aluminum hydride, and transition metal catalysis in the presence of hydrogen gas. For example, the hemoglobin-albumin nanoparticle is diluted with a coupling buffer (e.g., 0.1 M sodium phosphate, 0.15 M NaCl, or standard phosphate buffer solution) and a borohydride (e.g., sodium cyanoborohydride, or sodium borohydride) is added. Unreacted aldehyde sites are blocked by the addition of a quenching buffer solution (e.g., 1 M Tris-HCl, pH 7.4), and the reaction solution filtered to remove unreacted borohydride. The resulting reduced nanoparticles may be characterized using SDS Page.

In some embodiments, mixed bifunctional linkers (heterobifunctional crosslinkers) can be used (for example a linker having an aldehyde and a carboxylic acid). For example, in some embodiments, the hemoglobin (or albumin) can first be decorated with a linker under a first set of reaction conditions. This decorated molecule can then be exposed to albumin (or hemoglobin) under a set of second reaction conditions to create a bond through the linker.

In some embodiments, the reversible oxygen binding molecules are not covalently bound to the nanostructure and instead are bound via electrostatic interactions or complexation.

In some embodiments, after functionalization of the hemoglobin to the albumin via, e.g., a diimine linker, the reversible oxygen binding nanoparticle is further functionalized and/or decorated nucleophilic species (e.g., —NH2, —OH, —SH, etc.). In some embodiments, the functionalization of the albumin nucleophilic species (e.g., —NH2, —OH, —SH, etc.) to form an albumin carrier may occur prior to the functionalization of the hemoglobin to the albumin carrier. For purposes of the following discussion, the hemoglobin is shown having already been functionalized to the albumin, though the discussion may encompass functionalization of albumin to form an albumin carrier prior to functionalization of the hemoglobin to albumin.

In some embodiments, the nucleophilic species is a thiol (i.e., —SH) and the nanoparticle is thiolated. In some embodiments, the nanoparticle (e.g., the nanostructure, the reversible oxygen binding molecule, or both) is thiolated using a thiolating agent. A wide array of thiolating agents may be used in this capacity, In some embodiments, the thiolating agent is selected from the group consisting of:

where R1is selected from the group consisting of —CH2—, —(CH2O)CH2—, —(CH2CH2O)—CH2CH2—, and —(CH2CH2CH2O)—CH2CH2CH2—, and “b” is an integer between 0 and 10. In some embodiments, Traut's reagent (2-iminothiolane) is used as the thiolating agent.

wherein c is selected from the group consisting of —C(O)(CH2)p— and —N═CH(CH2)p—, wherein p is an integer ranging from 1 to 10. Other non-limiting examples of suitable thiolating agents may be N-succinimididyl S-acrylthioacetate or succinimidyl acetyl-thiopropropionate. [Hermanson, G. T. Bioconjugate Techniques; Academic Press: New York, 2013].

In various embodiments, the nanoparticle is contacted with Traut's reagent (2-iminothiolane). Traut's reagent reacts with primary amines (—NH2) to introduce sulfhydryl (—SH) groups while maintaining charge properties similar to the original amino group. Once added, sulfhydryl groups can be specifically targeted for reaction in a variety of useful labeling, crosslinking and immobilization procedures.

Preferably, the 2-iminothiolane reacts with primary amines at pH 7 to 10, creating aminidine compounds with a sulfhydryl group. More preferably, the 2-iminothiolane reaction is at pH 7 to 9. This allows for crosslinking or labeling of molecules such as proteins through use of disulfide or thioether conjugation. In some embodiments, thiol-ene polymerization conditions are typically chosen to minimize side reactions. In particular, disulfide formation can present a challenge in the consistent formation of thiol-ne hydrogels. For example, thiol-functionalized macromers can react with each other to form disulfide linkages, making them inaccessible for subsequent reaction with alkenes. Additionally, thiols on macromers can react with various functional groups that are present on biologics (i.e., off-target reactions leading to oxidation of cysteine residues on proteins).

According to some embodiments of the present methods, the extent of the nucleophilic functional groups (e.g., sulfhydryls) introduced onto the lysine (Lys) residues of albumin can be controlled by the availability of an initiator, such as 2-iminothiolane (Traut's reagent). For example, in embodiments where the functionalization of the albumin with nucleophilic species (e.g., —NH2, —OH, —SH, etc.) occurs prior to crosslinking the hemoglobin to the albumin, depending on the reaction of the initiator and the albumin, the remaining unreacted lysine residues on the albumin are then available for crosslinking with hemoglobin for stabilization. In some embodiments, a bifunctional linker chemistry may then be selected to allow an alternative crosslinking approach for crosslinking of the hemoglobin to albumin, such as a reaction using glutaraldehyde, so that the nucleophilic group functionalized Lys residues are excluded from the crosslinking reaction and may alter the conformation of binding between the albumin and hemoglobin.

The functionalization of the Lys residues is a process that can be monitored by methods known to the skilled artisan (e.g., by1H NMR or by fluorescence-based assay) and tuned to achieve the desired number of lysine residues to be excluded from a subsequent crosslinking reaction between the hemoglobin and albumin. The extent of the lysine residues that are converted to nucleophilic groups can be monitored as can the conjugation of a linker to the nucleophilic group. This allows the crosslinking reaction between the hemoglobin and albumin to be regulated.

For purposes of summarizing the discussion that follows, certain features of the present methods are described using Traut's reagent and sulfhydryls (thiol groups). While Traut's reagent and sulfhydryls are used herein to discuss certain features, these molecules and groups are exemplary and other initiators and nucleophilic groups, as well as other nanostructures and oxygen binding agents, are envisioned within the scope of the present invention.

In some embodiments, the number of lysine residues that are converted into thiol functional groups (sulfhydryls) may be set by the molar ratio of the primary amines (e.g., Lys residues on albumin) to 2-iminothiolane (Traut's reagent). In some embodiments, for example where the nanostructure has many lysine residues, adjusting the molar ratio of Traut's reagent in the reaction allows one to control the level of thiolation. For example, for IgG molecules (150 kDa), reaction with a10-fold molar excess of Traut's reagent ensures that all antibody molecules will be modified with at least 3-7 sulfhydryl groups. By comparison, nearly all available primary amines (˜20 in the typical IgG) could be thiolated using a 50-fold molar reagent excess.

The extent of the thiolation may be monitored using any method known in the art so that the desired level of thiolation is achieved in the bulk reaction. In some embodiments, the active thiol groups on the protein surface may be assayed by the disulfide exchange reaction with 2,2′-dithiopyridine (2,2′-DTP) to produce 2-thiopyridinone (2-TP) with an absorption at 343 nm (molar absorption coefficient: 8.1×103M−1cm−1) [Pedersen, A. O., and Jacobsen, J. (1980) Reactivity of the thiol group in human and bovine albumin at pH 3-9, as measured by exchange with 2,2-dithiodipyridine. Eur. J. Biochem. 106, 291-295].

In some embodiments, quantitative spectroscopic measurements may be used to conveniently provide the thiol concentration. For example, the parent protein may show a small absorption band in this range, which should be subtracted from the spectrum after the disulfide exchange reaction, where the difference in the thiol groups per protein before and after the modification corresponds to the mean of the sulfhydryl-functionalized chains on the protein surface.

In some embodiments, a fluorescence-based assay may be used, such as the method described by Udenfriend [Udenfriend, S., Stein, S., Bohlen, P., Dairman, W., Leimgruber, W. & Weigele, M. Fluorescamine: A Reagent for Assay of Amino Acids, Peptides, Proteins, and Primary Amines in the Picomole Range Science 178 871-872 (1972)], which is based on the rapid reaction of fluorescamine (4-phenyl-spiro [furan-2(3H), 1′-phthalan]-3,3′-dione) with primary amines in proteins, such as the terminal amino group of peptides and the e-amino group of lysine, to form highly fluorescent moieties

Fluorescamine reacts with the primary amino groups found in terminal amino acids and the e-amine of lysine to form fluorescent pyrrolinone type moieties.

In some embodiments, the protein assay of Udenfriend [Udenfriend, S., Stein, S., Bohlen, P., Dairman, W., Leimgruber, W. & Weigele, M. Fluorescamine: A Reagent for Assay of Amino Acids, Peptides, Proteins, and Primary Amines in the Picomole Range Science 178 871-872 (1972)], may be modified for microplates as described by Lorenzen [Lorenzen, A. & Kennedy, S. W. A Fluorescence-Based Protein Assay for Use with a Microplate Reader Anal. Biochem. 214 346-348 (1993)]. For example, a series of dilutions of Bovine Serum Albumin (BSA) ranging from 0 to 500 g/ml was made using phosphate buffered saline (PBS) pH 7.4 as the diluent. After dilution, 150 μl aliquots of samples and standards were pipetted into microplate wells in replicates of eight. The microplate was placed on a microplate shaker and 50 μl of 1.08 mM (3 mg/ml) fluorescamine dissolved in acetone was added to each well. Following the addition of fluorescamine the plate was shaken for one minute. The fluorescence was then determined using a FL600 fluorescence plate reader (BioTek Instruments, Inc., Winooski, Vt.) with a 400 nm, 30 nm bandwidth, excitation filter and a 460 nm, 40 nm bandwidth emission filter. The sensitivity setting was at 29, and the data collected from the bottom with a 5 mm probe using static sampling with a 0.35 second delay, 50 reads per well. When lower protein concentrations (0-500 μg/ml) were examined, the reaction was found to be linear. Using a least means squared regression analysis, a straight line was generated and utilized for the determination of protein concentrations. This allowed for determination of an equation describing the standard curve.

Various buffers may be used for thiolation with Traut's reagent. Representative examples of suitable buffering agents include, but are not limited to, phosphates, carbonates, citrates, tris buffers, and buffered saline salts (e.g., tris buffered saline or phosphate buffered saline). In some embodiments, the buffer is preferably a phosphate buffered saline (PBS) solution (PBS, Thermo Fisher). In other embodiments, a 0.1M borate buffer adjusted to pH 8 may be used for thiolation. Other buffers devoid of primary amines that maintain solubility of the nanostructure (e.g., carrier protein) may also be used. Traut's reagent is very stable in acidic or neutral buffers that are devoid of primary amino groups. Even in alkaline conditions, hydrolysis is slow compared to the rate of reaction with primary amines. Because hydrolysis is slow relative to the amine reaction rate, thiolation with Traut's reagent does not require as large a molar excess of reagent as other types of modification reagents, such as SATA.

In some embodiments, the nucleophilic species (e.g., the thiol) may be used to further functionalize a portion of the nanoparticle (e.g., the nanostructure, the reversible oxygen binding molecule, or both) with a hydrophilic species. In some embodiments, the nucleophile of the nanoparticle is used to attack an electrophilic group (e.g., a carboxylic acid, epoxide, succinimidyl group, maleimide, etc.) situated on a hydrophilic species thereby coupling the hydrophilic species to the nanoparticle. In some embodiments, this functionalization can be performed in the presence of various coupling reagents to facilitate coupling (e.g., EDC, DCC, etc.).

In some embodiments, the hydrophilic species is coupled to albumin via a thiol of the albumin and a maleimide of the hydrophilic species, as shown below:

wherein c is selected from the group consisting of —C(O)(CH2)p— and —N═CH(CH2)p—, wherein p is an integer ranging from 1 to 10 and wherein d is —(CH2)q—, wherein q is an integer ranging from 1 to 10.

Thiol-maleimide reactions offer a number of advantages: (1) at neutral pH, maleimides react with high selectivity for thiols; (2) thiol-maleimide reactions occur rapidly under physiological conditions; and (3) the thiol-maleimide linkage formed with aryl thiols can undergo retro-Michael reaction under reducing conditions for controlled degradation and release applications. However, it is important to note that maleimide groups undergo ring hydrolysis under aqueous conditions, yielding maleamic acid that is not reactive with thiols. Solution pH, temperature, neighboring functional groups, and hydroxyl ion concentration affect the rate of ring hydrolysis (k=500-1600 M-1 s-1).[ref78] Although maleimide ring hydrolysis after formation of succinimide thioether linkages will not significantly change the properties of an existing hydrogel, ring hydrolysis in the precursor solution before hydrogel preparation can significantly increase network defects; such defects typically increase mesh size and reduce network retention of loaded therapeutics, affecting release characteristics. In addition, because unreacted small-molecule maleimides can be cytotoxic, so thorough purification of maleimide-functionalized macromers after synthesis is typically preferred.

In some embodiments, the nanostructure may be decorated with one or more hydrophilic polymers. Non-limiting examples of hydrophilic polymers may be polyethylene glycol (PEG, e.g., PEGylated), poly(N-isopropylacrylamide) (PNIPAM), polyacrylamide (PAM), polyvinyl alcohol (PVA), polyacrylic acid, polyethyleneimine (PEI), poly(2-oxazoline), poly(vinylpyrrolidone), and copolymers thereof.

In some embodiments, as shown below, the nanostructure is PEGylated. In some embodiments, as shown below, the nanostructure is PEGylated using a maleimide of PEG. For example, human serum albumin may be modified by reacting 2-iminothiolane (IMT) with the amino groups of Lys to create active thiol groups and then binding the active thiol groups with maleimide-terminated poly(ethylene glycol) (PEG).

In some embodiments, the quantitative fluorescence-based assays discussed above may be utilized to tune the number of free lysine residues remaining and the number of sulfhydryls ready for functionalization, e.g., with MAL-PEG or MAL-PEG-ACRYL conjugation. After functionalization, the number of unreacted sulfhydryls can be determined by labeling them with fluorescein-5-maleimide in excess and filtering unreacted fluorescein-5-maleimide prior to quantitation. The degree of labeling with fluorescein-5-maleimide can be determined either by absorption using (ε′=fluor molar extinction coefficient: 68,000 M-1 cm-1) or by fluorescence emission (excitation at 491 nm and emission at 518 nm).

For example, albumin (0.25 mM) (BSA Sigma-Aldrich, St. Louis, Mo.) was incubated overnight with 5 mM 2-iminothiolane (BioAffinity Systems, Rockford, Ill.) and 7.5 mM maleimide PEG-5000 in phosphate buffer saline (PBS). The surface amino groups were thiolated, and thiol groups generated on the protein in situ were derivatized by the maleimide-PEG in the reaction mixture. The single step reaction limited the oxidation of the thiols of the thiolated protein to generate dimers and polymers of BSA, and is the preferred approach to generate PEGylated proteins. Excess reagents were removed by tangential flow filtration using the Minim System (Pall Life Sciences, Ann Arbor, Mich.) after overnight incubation. A 70 kDa membrane was used for diafiltration for removal of unreacted PEG and excess iminothiolane, and PEG-BSA was concentrated to 2.5 gms/dL (protein based). This example yielded an average of 12 copies of PEG 5K chains conjugated to a BSA molecule, a molecular weight of 130 kDa and a molecular radius of 8-9 nm.

In order to retain the hemoglobin-albumin complex in a polymer network, the bonds between the linker and the hemoglobin-albumin complex and within the polymer network preferably have little to no biodegradation. In some embodiments of the present invention, acrylate bonds are preferably used within the polymer network, and a stable thioether linkage between a polymer linker and the hemoglobin-albumin complex is preferably used to immobilize the complex in the polymer network. In some embodiments, a maleimide-activated PEG, which may be reacted with the thiols of cysteine residues or the sulfhydryls derived from Lys residues, is preferably used to form stable thioether linkages because it exhibits a much higher stability against hydrolysis than an NHS ester of PEG acid.

Accordingly, in some embodiments, the one or more of the hydrophilic species further comprises a polymerizable unit (e.g., an acrylate, methacrylate, etc.). In some embodiments, the hydrophilic species and polymerizable unit are functionalized to the nanoparticle using maleimide-PEG-methacrylate (mal-PEG-MA) as shown below:

wherein c is selected from the group consisting of —C(O)(CH2)p— and —N═CH(CH2)p—, wherein p is an integer ranging from 1 to 10, wherein d is —(CH2)q—, wherein q is an integer ranging from 1 to 10, wherein n is an integer ranging from 1 to 1000 and wherein R5is selected from the group consisting of —C1-4alkyl and H.

The extent of acryl group coupling to the macromer complex may be monitored using any monitoring method known in the art, e.g.,1H NMR. Alternatively, an iodine (Wijs solution) assay, as disclosed in Lubrizol Test Procedure, TP-TM-005C, may be used to determine the number of acrylate groups coupled to the macromer complex through double bond quantization. For example, a 10 mg sample may be dissolved in water and an excess of Wijs solution (0.1M iodine monochloride, Sigma Aldrich), for example, 50-60% excess of titrateable double bonds, added. The resulting solution then may be incubated in the dark for about30minutes at room temperature. After further dilution with deionized water, 4-20 mL aqueous 1 M potassium iodide solution may be added, and the resulting solution immediately titrated using 0.1 N sodium thiosulfate. 1-2 mL 1% aqueous starch indicator solution may be added and the titration continued till completion. The iodine value then may be calculated to indicate the number of acrylate groups present in the sample.

In some embodiments, the polymerizable group of the hydrophilic species unit can be co-polymerized in a first crosslinking solution (which can contain one or more crosslinkers) to form a nanogel:

In some embodiments, the first crosslinking solution comprises the following structure (Formula I):

where e is an integer ranging from 1 to 10 and R5is selected from the group consisting of —C1-4alkyl and H. In some embodiments, a plurality of differing crosslinkers having the Formula I structure can be used to form the nanogel. Non-limiting examples of the first cross linking solution may be ethylene glycol diacrylate, diethylene glycol diacrylate, triethylene glycol diacrylate, tetraethylene glycol diacrylate, polyethylene glycol diacrylate, trimethylol propane triacrylate, pentaerythritol triacrylate, diethylene glycol dimethacrylate, triethylene glycol dimethacrylate, tetraethylene glycol dimethacrylate, polyethylene glycol dimethacrylate, dipropylene glycol dimethacrylate, polypropylene glycol dimethacrylate, trimethylol propane trimethacrylate and glycerine trimethacrylate.

In some embodiments, the first crosslinking solution comprises tetraethyleneglycol diacrylate (TEGDA). In some embodiments, the crosslinking solution comprises TEGDA at a weight % (weight of TEGDA/weight of solution) ranging from about 0% to about 5%, about 5% to about 15%, about 15% to about 25%, about 25% to about 50%, about 50% to about 75%, or about 75% to about 100%.

In some embodiments, the first crosslinking solution comprises the hemoglobin-albumin nanoparticle at a weight % (weight of nanoparticle/weight of solution) ranging from about 0% to about 0.5%, about 0.5% to about 1.5%, about 1.5% to about 2.5%, about 2.5% to about 5.0%, about 5.0% to about 7.5%, or about 7.5% to about 10.0%.

In some embodiments, the nanogel can further be diffused in a liquid medium (i.e., an oxygen conduit fluid) to provide an emulsion, suspension, mixture, or solution. In some embodiments, the liquid of the oxygen conduit fluid comprises one or more of crosslinking agents and water. In some embodiments, the oxygen conduit fluid comprises a second crosslinker (or a second combination of crosslinkers). In some embodiments, the second crosslinker is also represented by Formula I above. In some embodiments, the second crosslinker is ethylene glycol dimethacrylate (EGDMA). In some embodiments, the EGDMA is present at a weight % (weight of EGDMA/weight of liquid medium) ranging from about 0% to about 5%, about 5% to about 15%, about 15% to about 25%, about 25% to about 50%, about 50% to about 75%, or about 75% to about 100%. In some embodiments, the second crosslinker is (TEGDA). In some embodiments, the TEGDA is present at a weight % (weight of TEGDA/weight of liquid solution) ranging from about 0% to about 5%, about 5% to about 15%, about 15% to about 25%, about 25% to about 50%, about 50% to about 75%, or about 75% to about 100%.

In some embodiments, the liquid medium and nanogel are configured to flow into the glucose sensor tip via capillary action. In some embodiments, the viscosity of the liquid medium and nanogel is sufficiently low to allow this capillary uptake. In some embodiments, the nanogel solution is introduced to a template via a port4210, as shown inFIG. 42.

In some embodiments, when the nanogel is dispersed in ethylene glycol dimethacrylate at about 0.25 g gel wt./1 mL, the oxygen conduit fluid has a viscosity of less than about 2000 cP, about 1000 cP, about 500 cP, about 250 cP, about 100 cP, about 50 cP, about 25 cP, about 10 cP, about 5 cP, about 1 cP, or about 0.5 cP. In some embodiments, when the nanogel is dispersed in ethylene glycol dimethacrylate at about 0.25 g gel wt./1 mL, the oxygen conduit fluid is characterized by an ability to pass through a 20 g needle using less than 60 N pressure.

In some embodiments, as discussed above, the oxygen conduit fluid is configured to be dispensed as a solution into sub-millimeter features of the glucose sensor tip. Small features of the glucose sensor tip can be provided by supplying solutions of the nanogels which are taken-up by spaces (e.g., channels, tunnels, paths, etc.) in molds (e.g., dye casts) by capillary action. The oxygen conduit fluid can fill these device features and, upon filling, be cured using UV light (in the presence of a second crosslinker) and/or concentrated (to remove any volatile liquids) to afford the oxygen conduit3820.

In some embodiments, where applicable, the second crosslinking step is performed while the nanogel is suspended in an oxygen conduit fluid (e.g., the second crosslinker, water, combinations thereof, etc.). In some embodiments, the second crosslinking step affords a hydrogel capable of rapidly transporting oxygen (e.g., diffusion controlled) from the oxygen conduit to other regions of the sensor tip.

Some embodiments pertain to a crosslinked hemoglobin-based material represented by the following structure:

where “” represents a hydrogel or nanogel matrix and m is an integer from 0 and 20. In some embodiments, these materials are used as an oxygen conduit. In some embodiments, the hemoglobin-albumin material comprises PEG-based linker and is represented by the following structure:

wherein m is an integer between 0 and 8.

In some embodiments, the crosslinked hemoglobin-based material is represented by the following structure:

wherein c is selected from the group consisting of —C(O)(CH2)p— and —N═CH(CH2)p—, wherein p is an integer ranging from 1 to 10; wherein d is —(CH2)q—, —(CF2)q— wherein q is an integer ranging from 1 to 10; wherein n is an integer ranging from 1 to 1000; and wherein R5is selected from the group consisting of —C1-4alkyl and H, or F.

In some embodiments, the nanogel or hydrogel matrix of the crosslinked hemoglobin-based material comprises:

wherein e is an integer ranging from 1 to 10; and wherein R5is selected from the group consisting of —C1-4alkyl and H. In some embodiments, the nanogel or hydrogel matrix of the crosslinked hemoglobin-based material comprises:

In some embodiments, after curing or concentrating, the crosslinked hemoglobin-based material is dense. In some embodiments, the crosslinked material has a modulus of at least about 8 GPa at a total material concentration of less than about 10 mg/mL. In some embodiments, after curing or concentrating, the crosslinked hemoglobin-based material has a storage modulus of at least about 0.01 GPa, about 0.1 GPa, 0.5 GPa, 1.0 GPa, 2.0 GPa, 4.0 GPa, or about 6.0 GPa at a total material concentration of about 10 mg/mL.

In some embodiments, the crosslinked hemoglobin-based material has a water content of at least about 70%, about 80%, about 90%, about 95%, about 97.5%, about 99%, or about 99.5% of the total dry weight of the crosslinked hemoglobin-based material.

Some embodiments pertain to a method of making a dispensable, UV-curable enzyme-albumin nanogel solution. In some embodiments, the method of making a UV-curable enzyme-albumin nanogel comprises linking a nanostructure to an enzyme. In some embodiments, the nanostructure is as described above. In some embodiments, the nanostructure is albumin. In some embodiments, the enzyme is GOx or CAT. In some embodiments, like the oxygen conduit described above, the method of making a UV-curable enzyme-albumin nanogel comprises incorporating a hemoglobin-albumin nanostructure. In some embodiments, the hemoglobin-albumin nanostructure is provided using the methods previously described to afford a crosslinkable nanostructure.

In some embodiments, the nanogel of the enzyme-albumin nanogel further comprises GOx linked to an albumin molecule and/or CAT linked to an albumin nanostructure. In some embodiments, GOx-albumin nanoparticles and CAT-albumin nanoparticles are provided (with the separate GOx-albumin and CAT-albumin molecules). In some embodiments, GOx and CAT enzymes are functionalized to the same albumin molecule. In some embodiments, where present, hemoglobin-albumin nanoparticles are also provided prior to nanogel formation. In some embodiments, each of GOx, CAT, and/or hemoglobin are functionalized to a single albumin nanostructure prior to nanogel formation.

As stated above, for purposes of summarizing the disclosure, certain features of enzymatic-albumin nanogels have been described herein using albumin and GOx or CAT. While albumin and GOx and albumin and CAT nanoparticles are described herein, any nanostructure or enzymatic molecule is envisioned. Similarly, when the more general term enzyme is used, both GOx and CAT are envisioned.

Similar to the hemoglobin-albumin nanoparticles above, in some embodiments, the nanoparticle comprises one or more enzyme molecules functionalized to each albumin molecule. In some embodiments, the nanoparticle comprises less than one enzyme molecule per albumin molecule. In some embodiments, the ratio of enzyme molecules to albumin is at least about 0.5:1, about 1:1, about 2:1, about 5:1, or about 10:1.

In some embodiments, the enzyme is bound to albumin covalently. In some embodiments, the covalent link to the enzyme is formed using a bifunctional linker. Suitable linkers include amino acids, peptides, nucleotides, nucleic acids, organic linker molecules, disulfide linkers, and polymer linkers (e.g., PEG). The linker can include one or more spacing groups including, but not limited to alkylene, alkenylene, alkynylene, alkyl, alkenyl, alkynyl, alkoxy, aryl, heteroaryl, aralkyl, aralkenyl, aralkynyl and the like. The linker can be neutral, or carry a positive or negative charge. In some embodiments the bifunctional linker is an organic linker and is selected from a dialdehyde, a dicarboxylic acid, a diepoxide, or the like. In some embodiments, the bifunctional linker is represented by one or more of the following structures:

where R3is selected from the group consisting of —CH2—, —CF2—, —(CH(R—OH)—, —(CH2O)CH2—, —(CF2CF2O)—CF2CF2—, —(CH2CH2CH2O)—CH2CH2CH2, —(CF2CF2CF2O)-CF2CF2CF2, f is an integer ranging from 0 and 1000, and LG is a leaving group. Non-limiting examples of leaving groups may be chloride, bromide, iodide, imidazole, benztriazole, triflate, tosylate, mesylate, or combinations thereof.

There are additional amine-reactive groups below, which may be located on the termini of bifunctional linkers and in this configuration, used as crosslinking agents of primary amine groups. Bifunctional primary amine linkers may consist of a combination of the same (homobifunctional crosslinkers) or different (heterobifunctional crosslinkers) reactive groups. The scheme below denotes some non-limiting examples of useful reactive groups.

In some embodiments, mixed bifunctional linkers can be used (for example a linker having an aldehyde and a carboxylic acid). For example, in some embodiments, the enzyme (or albumin) can first be decorated with a linker under a first set of reaction conditions. This decorated molecule can then be exposed to albumin (or enzyme) under a set of second reaction conditions to create a bond through the linker.

The crosslinking of enzyme and albumin may involve multiple site reactions. For example, albumin is rich in Lys residues. One common and versatile technique for crosslinking or labeling peptides and proteins such as antibodies involves the use of chemical groups that react with primary amines (—NH2). Primary amines exist at the N-terminus of each polypeptide chain and in the side-chain of lysine (Lys) amino acid residues. These primary amines are positively charged at physiologic pH; therefore, they occur predominantly on the outside surfaces of native protein tertiary structures where they are readily accessible to conjugation reagents introduced into the aqueous medium. Furthermore, among the available functional groups in typical biological or protein samples, primary amines are especially nucleophilic; this makes them easy to target for conjugation with several reactive groups.

In some embodiments, the enzyme and albumin are functionalized via amine groups from each of the albumin and enzyme molecules. For example, in some embodiments, when a dialdehyde, a dicarboxylic acid, or a diepoxide is used as the bifunctional linkers, diimines, diamides, and diamines, respectively, result from coupling of the enzyme to the albumin. In some embodiments, combinations of bifunctional linkers can be used. The following represents an enzyme molecule linked to albumin using a dialdehyde (i.e., via a diimine linker):

In some embodiments, the difunctional linker is glutaraldehyde and forms a diimine link via the aldehydes of the linker and amines from enzyme and albumin (where g is an integer ranging from 0 and 20). A glutaraldehyde-based linker configuration is represented by the depiction:

In some embodiments, the enzyme is covalently linked to albumin by incubation with gluteraldehyde, at low temperature, low oxygen concentration, pH of between about 7.0 and 8.0, for at least about 24 hours to form enzymatic nanoparticles.

In some embodiments, the incubation time with glutaraldehyde is at least about 1 hour, about 24 hours, about 36 hours, or about 48 hours. In some embodiments, the incubation time is at least about 10 hours, about 24 hours, about 36 hours, or about 48 hours. In some embodiments, the glutaraldehyde (or other bifunctional linker) is provided to the albumin/hemoglobin or albumin/enzyme solution at a low concentration, e.g., at a wt % below about 0.0001 wt. % or at a molar ratio below about 0.1. In some embodiments, the temperature is below about 30° C., about 20° C., about 10° C., about 5° C., about 0° C. or lower than −5° C.

Glutaraldehyde has been widely used as a mild crosslinking agent for the immobilization of enzymes because the reaction proceeds in aqueous buffer solution under conditions close to physiological pH, ionic strength, and temperature. Essentially, two methods have been used: (i) the formation of a three-dimensional network as a result of intermolecular crosslinking and (ii) the binding to an insoluble carrier (e.g., nylon, fused silica, controlled pore glass, crosslinked proteins such as gelatin and bovine serum albumin (BSA), and polymers with pendant amino groups).

In some embodiments, after incubation with glutaraldehyde and formation of the diimine linker, the enzyme-albumin nanoparticles may be subjected to a reduction to convert the diimine linkages to diamine linkages. Non-limiting examples of reducing agents may be sodium borohydride, sodium cyanoborohydride, sodium triacetoxyborohydride, lithium aluminum hydride, and transition metal catalysis in the presence of hydrogen gas. For example, the enzyme-albumin nanoparticle may be diluted with a coupling buffer (e.g., 0.1 M sodium phosphate, 0.15 M NaCl, or standard phosphate buffer solution) and a borohydride (e.g., sodium cyanoborohydride, or sodium borohydride) may be added. Unreacted aldehyde sites may be blocked by the addition of a quenching buffer solution (e.g., 1 M Tris-HCl, pH 7.4), and the reaction solution filtered to remove unreacted borohydride. The resulting reduced nanoparticles may be characterized using, e.g., SDS-polyacrylamide (SDS Page) electrophoresis, as illustrated inFIG. 49.

FIG. 49shows an example of SDS Page of the reduced nanoparticles after EDC coupling reaction with GOx and amine. Using the values obtained for the protein standards, a graph of log Molecular Weight (MW) vs. Rfis plotted inFIG. 50.

The plot should be linear for most proteins, provided that the proteins are fully denatured and the gel percentage is appropriate for the MW range of the sample. The reaction efficiency is demonstrated in going from 1 to 8 with no coupling reagent present in 1 and increased amounts of reagent from 2 to 8, thus showing an increase in molecular weight as the coupling of the amine occurs.

In some embodiments, the enzyme molecules are not covalently bound to the nano structure and instead are bound via electrostatic interactions or complexation.

In some embodiments, after functionalization of the enzyme to the albumin via, e.g., a diimine linker, the enzymatic nanoparticle is further functionalized and/or decorated nucleophilic species (e.g., —NH2, —OH, —SH, etc.). In some embodiments, the functionalization of the albumin with nucleophilic species (e.g., —NH2, —OH, —SH, etc.) form an albumin carrier may occur prior to the functionalization of the enzyme to the albumin carrier. For purposes of the following discussion, the enzyme is shown having already been functionalized to the albumin, though the discussion may encompass functionalization of albumin to form an albumin carrier prior to functionalization of the hemoglobin to albumin.

In some embodiments, the nucleophilic species is a thiol (i.e., —SH) and the nanoparticle is thiolated. In some embodiments, the nanoparticle (e.g., the nanostructure, the enzyme, or both) is thiolated using a thiolating agent. A wide variety of thiolating agents may be used in this capacity. In some embodiments, the thiolating agent is selected from the group consisting of:

where R4is selected from the group consisting of —CH2—, —(CH2O)CH2—, —(CH2CH2O)—CH2CH2—, and —(CH2CH2CH2O)—CH2CH2CH2—, and “h” is an integer between 0 and 10.

In some embodiments, Traut's reagent (2-iminothiolane) is used as the thiolating agent.

wherein i is selected from the group consisting of —C(O)(CH2)r— and —N═CH(CH2)r—, wherein r is an integer ranging from 1 to 10. Other non-limiting examples of suitable thiolating agents may be , N-succinimididyl S-acrylthioacetate or succinimidyl acetyl-thiopropropionate. [Hermanson, G. T. Bioconjugate Techniques; Academic Press: New York, 2013].

Traut's reagent reacts with primary amines (—NH2) to introduce sulfhydryl (—SH) groups while maintaining charge properties similar to the original amino group. Once added, sulfhydryl groups can be specifically targeted for reaction in a variety of useful labeling, crosslinking and immobilization procedures.

Preferably, the 2-iminothiolane reacts with primary amines at pH 7 to 10, creating aminidine compounds with a sulfhydryl group. More preferably, the 2-iminothiolane reaction is at pH 7 to 9. This allows for crosslinking or labeling of molecules such as proteins through use of disulfide or thioether conjugation. Thiol-ene polymerization conditions are typically chosen to minimize side reactions. In particular, disulfide formation can present a challenge in the consistent formation of thiol-ene hydrogels. For example, thiol-functionalized macromers can react with each other to form disulfide linkages, making them inaccessible for subsequent reaction with alkenes. Additionally, thiols on macromers can react with various functional groups that are present on biologics (i.e., off-target reactions leading to oxidation of cysteine residues on proteins).

According to some embodiments of the present methods, the extent of the nucleophilic functional groups (e.g., sulfhydryls) introduced onto the lysine (Lys) residues of albumin can be controlled by the availability of an initiator, such as 2-iminothiolane (Traut's reagent). In embodiments where the functionalization of the albumin with a nucleophilic species (e.g., —NH2, —OH, —SH, etc.) occurs prior to crosslinking the enzyme to the albumin, depending on the reaction of the initiator and the albumin, the remaining unreacted lysine residues on the albumin are then available for crosslinking with the enzyme for stabilization. In some embodiments, a bifunctional linker chemistry may then be selected to allow an alternative crosslinking approach for crosslinking of the enzyme to albumin, such as a reaction using glutaraldehyde, so that the nucleophilic group functionalized Lys residues are excluded from the crosslinking reaction and may alter the conformation of binding between the albumin and enzyme.

The functionalization of the Lys residues is a process that can be monitored by methods known to the skilled artisan (e.g., by1H NMR or by fluorescence-based assay) and tuned to achieve the desired number of lysine residues to be excluded from a subsequent crosslinking reaction with the enzyme and albumin. The extent of the lysine residues that are converted to nucleophilic groups can be monitored as can the conjugation of a linker to the nucleophilic group. This allows the crosslinking reaction between the enzyme and albumin to be regulated.

For purposes of summarizing the discussion that follows, certain features of the present methods are described using Traut's reagent and sulfhydryls (thiol groups). While Traut's reagent and sulfhydryls are used herein to discuss certain features, these molecules and groups are exemplary and other initiators and nucleophilic groups, as well as other nanostructures and enzymes, are envisioned within the scope of the present invention. Example enzymes include, and are not limited to: dehydrogenase, oxidases, esterases, transaminases, etc. In addition, general enzyme groups enabled with appropriate dyes sensitive to products of enzyme substrate RXN5may be used. Oxygen consuming or producing enzymes such as, for example, enzymes in the class of oxidoreductases, should be used.

In some embodiments, the number of lysine residues that are converted into thiol functional groups (sulfhydryls) may be set by the molar ratio the primary amines (e.g., Lys residues on albumin) and 2-iminothiolane (Traut's reagent). In some embodiments, for example where the nanostructure has many lysine residues, adjusting the molar ratio of Traut's reagent in the reaction allows one to control the level of thiolation. For example, for IgG molecules (150 kDa), reaction with a 10-fold molar excess of Traut's reagent ensures that all antibody molecules will be modified with at least 3-7 sulfhydryl groups. By comparison, nearly all available primary amines (˜20 in the typical IgG) could be thiolated using a 50-fold molar reagent excess of Traut's reagent.

The extent of the thiolation may be monitored using any method known in the art so that the desired level of thiolation is achieved in the bulk reaction. In some embodiments, the active thiol groups on the protein surface may be assayed by the disulfide exchange reaction with 2,2′-dithiopyridine (2,2′-DTP) to produce 2-thiopyridinone (2-TP) with an absorption at 343 nm (molar absorption coefficient: 8.1×103M−1cm−1) [Pedersen, A. O., and Jacobsen, J. (1980) Reactivity of the thiol group in human and bovine albumin at pH 3-9, as measured by exchange with 2,2-dithiodipyridine. Eur. J. Biochem. 106, 291-295].

In some embodiments, quantitative spectroscopic measurements may be used to conveniently provide the thiol concentration. For example, the parent protein may show a small absorption band in this range, which is subtracted from the spectrum after the disulfide exchange reaction, where the difference in the thiol groups per protein before and after the modification corresponds to the mean of the sulfhydryl-functionalized chains on the protein surface.

In some embodiments, a fluorescence-based assay may be used, such as the method described by Udenfriend [Udenfriend, S., Stein, S., Bohlen, P., Dairman, W., Leimgruber, W. & Weigele, M. Fluorescamine: A Reagent for Assay of Amino Acids, Peptides, Proteins, and Primary Amines in the Picomole Range Science 178 871-872 (1972)], which is based on the rapid reaction of fluorescamine (4-phenyl-spiro [furan-2(3H), 1′-phthalan]-3,3′-dione) with primary amines in proteins, such as the terminal amino group of peptides and the e-amino group of lysine, to form highly fluorescent moieties

Fluorescamine reacts with the primary amino groups found in terminal amino acids and the e amine of lysine to form fluorescent pyrrolinone type moieties. In some embodiments, the protein assay of Udenfriend [Udenfriend, S., Stein, S., Bohlen, P., Dairman, W., Leimgruber, W. & Weigele, M. Fluorescamine: A Reagent for Assay of Amino Acids, Peptides, Proteins, and Primary Amines in the Picomole Range Science 178 871-872 (1972)], may be modified for microplates as described by Lorenzen [Lorenzen, A. & Kennedy, S. W. A Fluorescence-Based Protein Assay for Use with a Microplate Reader Anal. Biochem. 214 346-348 (1993)] and as discussed previously.

Various buffers may be used for thiolation with Traut's reagent. Representative examples of suitable buffering agents include, but are not limited to, phosphates, carbonates, citrates, tris buffers, and buffered saline salts (e.g., tris buffered saline or phosphate buffered saline). In some embodiments, the buffer is preferably a phosphate buffered saline (PBS) solution (PBS, Thermo Fisher). In other embodiments, a 0.1M borate buffer adjusted to pH 8 may be used for thiolation. Other buffers devoid of primary amines that maintain solubility of the nanostructure (e.g., carrier protein) may also be used. Traut's reagent is very stable in acidic or neutral buffers that are devoid of primary amino groups. Even in alkaline conditions, hydrolysis is slow compared to the rate of reaction with primary amines. Because hydrolysis is slow relative to the amine reaction rate, thiolation with Traut's reagent does not require as large a molar excess of reagent as other types of modification reagents, such as SATA.

In some embodiments, the nucleophilic species (e.g., the thiol) may be used to further functionalize the nanoparticle (e.g., the nanostructure, the enzymatic molecule, or both) with a hydrophilic species. In some embodiments, the nucleophile of the nanoparticle is used to attack an electrophilic group (e.g., a carboxylic acid, epoxide, succinimidyl group, etc.) situated on a hydrophilic species thereby coupling the hydrophilic species to the nanoparticle. In some embodiments, this functionalization can be performed in the presence of various coupling reagents to facilitate coupling (e.g., EDC, DCC, etc.).

In some embodiments, the hydrophilic species is coupled to albumin via a thiol of the albumin and a maleimide of the hydrophilic species, as shown below:

wherein i is selected from the group consisting of —C(O)(CH2)r— and —N═CH(CH2)r—, wherein r is an integer ranging from 1 to 10 and wherein j is —(CH2)s—, wherein s is an integer ranging from 1 to 10.

In some embodiments, the nanostructure may be decorated with one or more hydrophilic polymers. Non-limiting examples of hydrophilic polymers may be polyethylene glycol (PEG,e.g., PEGylated), poly(N-isopropylacrylamide) (PNIPAM), polyacrylamide (PAM), polyvinyl alcohol (PVA), polyacrylic acid, polyethyleneimine (PEI), poly(2-oxazoline), poly(vinylpyrrolidone), and copolymers thereof.

In some embodiments, as shown below, the nanostructure is PEGylated. In some embodiments, as shown below, the nanostructure is PEGylated using a maleimide of PEG. For example, human serum albumin may be modified by reacting 2-iminothiolane (IMT) with the amino groups of Lys to create active thiol groups and then binding the active thiol groups with maleimide-terminated poly(ethylene glycol) (PEG).

In some embodiments, the quantitative fluorescence-based assays discussed above may be utilized to tune the number of free lysine residues remaining and the number of sulfhydryls ready for functionalization, e.g., with MAL-PEG or MAL-PEG-ACRYL conjugation. After functionalization, the number of unreacted sulfhydryls may be determined by labeling them with fluorescein-5-maleimide in excess and filtering unreacted fluorescein-5-maleimide prior to quantitation. The degree of labeling with fluorescein-5-maleimide may be determined either by absorption using (ε′=fluor molar extinction coefficient: 68,000 M-1 cm-1) or by fluorescence emission (excitation at 491 nm and emission at 518 nm).

In order to retain the enzyme-albumin complex in a polymer network, the bonds between the linker and the enzyme-albumin complex and within the polymer network preferably have little to no biodegradation. In some embodiments of the present invention, acrylate bonds are preferably used within the polymer network, and a stable thioether linkage between a polymer linker and the enzyme-albumin complex is preferably used to immobilize the complex in the polymer network. In some embodiments, a maleimide-activated PEG, which may react with the thiols of cysteine residues or the sulfhydryls derived from Lys residues, is preferably used to form stable thioether linkages because it exhibits a much higher stability against hydrolysis than an NHS ester of PEG acid.

Catalytic activity (e.e., units of enzyme activity/nmole of protein, and Kmand kcatenzymatic parameters) of a single enzyme or multiple enzymes in an enzyme0carrier protein nanoparticle complex, described above, may be stabilized, in some instances, by increasing the carrier protein to enzyme ratio during conjugation with bifunctional linkers, such as with glutaraldehyde. The carrier protein may be human serum albumin, other types of suitable carrier proteins, peptides, or other molecular structures rich in primary amine, sulfhydryl or carboxyl groups, that are available for conjugation with bifunctional linkers to enzymes. Enzymes that are applicable to the embodiments of the present invention are included in an enzyme class known as Oxidoreductases. Oxidoreductases either consume or produce oxygen during the catalytic reaction with an analyte. The ratio of carrier proteins or carrier peptides to enzymes, in an enzyme-carrier protein nanoparticle complex, may range from 0.1:1, to about 1:1, to about 5:1, to about 10:1, to about 100:1, to about 1000:1.

The enzyme activity stabilized enzyme-carrier protein nanoparticles are used as building blocks to construct the corresponding nanogel. The stabilized enzyme activity properties of the nanoparticle will be imparted to the corresponding nanogel. The stabilized enzyme activity properties of the nanogel precursor will be imparted to the constructed active hydrogel. Stabilization of enzyme activities in nanoparticles, nanogels, and active hydrogels, through the above described general methods and formulations, or other suitable methods and formulations, are crucial for the proper design, fabrication, assembly, testing, and sehlf-life properties of an active enzymatic hydrogel-based commercial analyte sensor such as, for example, a glucose sensor.

In some embodiments, the one or more of the hydrophilic species further comprises a polymerizable unit (e.g., an acrylate, methacrylate, etc.). In some embodiments, the hydrophilic species and polymerizable unit are functionalized to the nanoparticle using maleimide-PEG-methacrylate (mal-PEG-MA) as shown below:

wherein n is an integer ranging from 1 to 1000 and wherein R6is selected from the group consisting of —C1-4alkyl and H.

The extent of acryl group coupling to the macromer complex may be monitored using, e.g.,1H NMR. Alternatively, an iodine (Wijs solution) assay, as disclosed in Lubrizol Test Procedure, TP-TM-005C, may be used to determine the number of acrylate groups coupled to the macromer complex. For example, a 10 mg sample may be dissolved in water and an excess of Wijs solution (0.1M iodine monochloride, Sigma Aldrich), for example, 50-60% excess of titrateable double bonds, added. The resulting solution is then incubated in the dark for about 30 minutes at room temperature. After further dilution with deionized water, 4-20 mL aqueous 1 M potassium iodide solution is added, and the resulting solution immediately titrated using 0.1 N sodium thiosulfate. 1-2 mL 1% aqueous starch indicator solution is added and the titration continued till completion. The iodine value then may be calculated to indicate the number of acrylate groups present in the sample.

In some embodiments, the polymerizable group of the hydrophilic species unit can be co-polymerized with a first enzymatic crosslinking solution to form an enzymatic nanogel:

wheredenotes an attachment to the nanogel matrix.

In some embodiments, the first enzymatic crosslinking solution comprises the following structure (Formula II):

where k is an integer ranging from 1 to 10 and R6is selected from the group consisting of —C1-4alkyl and H. In some embodiments, the first crosslinking solution comprises a plurality of differing crosslinkers having the Formula II structure. Non-limiting examples of the first crosslinking solution may be diethylene glycol diacrylate, triethylene glycol diacrylate, tetraethylene glycol diacrylate, polyethylene glycol diacrylate, trimethylol propane triacrylate, pentaerythritol triacrylate, diethylene glycol dimethacrylate, triethylene glycol dimethacrylate, tetraethylene glycol dimethacrylate, polyethylene glycol dimethacrylate, dipropylene glycol dimethacrylate, polypropylene glycol dimethacrylate, trimethylol propane trimethacrylate and glycerine trimethacrylate.

In some embodiments, the first crosslinking solution comprises TEGDA. In some embodiments, the crosslinking solution comprises TEGDA at a weight % (weight of TEGDA/weight of solution) ranging from about 0% to about 5%, about 5% to about 15%, about 15% to about 25%, about 25% to about 50%, about 50% to about 75%, or about 75% to about 100%.

In some embodiments, the first enzymatic crosslinking solution comprises a diamine represented by the following structure:

where l is an integer ranging from 1 to 10. The diamine compound useful in the first enzymatic crosslinking solution may be linear, branched, or cyclic. Additionally, the diamine may be chiral or achiral. Non-limiting examples of diamines may be ethylenediamine, 1,1-dimethylethylenediamine, tetramethylethylenediamine, 1,3-diaminopropane, putrescine, cadaverine, hexamethylenediamine, 1,2-diaminopropane, and 1,2-diaminocyclohexane. In some embodiments, the first crosslinking solution comprises hexamethylenediamine (HMDA).

In some embodiments, the crosslinking solution comprises HMDA at a weight % (weight of HMDA/weight of solution) ranging from about 0% to about 5%, about 5% to about 15%, about 15% to about 25%, about 25% to about 50%, about 50% to about 75%, or about 75% to about 100%.

In some embodiments, the first enzymatic crosslinking solution comprises polymer additives. In some embodiments, the polymer additives are added to the crosslinking milieu to afford various copolymer enzymatic nanogels. For instance in some embodiments, the following monomer is added to the enzymatic nanoparticle and crosslinking solution:

where R7is is selected from the group consisting of —C1-4alkyl and H and t is an integer ranging from 1 to 1000. Among hydroxyacrylates useful in the invention are such compounds as hydroxyalkylacrylates and hydroxymethacrylates. Non-limiting examples of hydroxyalkylacrylates and hydroxymethacrylates may be hydroxypropylacrylate (HPA), hydroxyethylacrylate (HEA), hydroxypropylmethacrylate, hydroxyethylmethacrylate (HEMA), hydroxyl, n-butyl acrylate, hydroxy n-octyl acrylate, hydroxy iso-butyl acrylate, PEG acrylates, and PEG methacrylates.

In some embodiments, the first enzymatic crosslinking solution comprises PEG methacrylate (PEGMA). In some embodiments, the crosslinking solution comprises PEGMA at a weight % (weight of PEGMA/weight of solution) ranging from about 0% to about 5%, about 5% to about 15%, about 15% to about 25%, about 25% to about 50%, about 50% to about 75%, or about 75% to about 100%.

In some embodiments, the first enzymatic crosslinking solution comprises hydroxyethyl methylacrylate (HEMA). In some embodiments, the crosslinking solution comprises HEMA at a weight % (weight of HEMA/weight of solution) ranging from about 0% to about 5%, about 5% to about 15%, about 15% to about 25%, about 25% to about 50%, about 50% to about 75%, or about 75% to about 100%.

In some embodiments, the first enzymatic crosslinking solution comprises: HEMA, TEGDA, and PEGMA. In some embodiments, the first enzymatic crosslinking solution comprises: HMDA, TEGDA, and PEGMA. In some embodiments, the first enzymatic crosslinking solution comprises: HMDA, TEGDA, HEMA, and PEGMA.

In some embodiments, the first enzymatic crosslinking solution comprises the hemoglobin-albumin nanoparticle at a weight % (weight of nanoparticle/weight of solution) ranging from about 0% to about 0.5%, about 0.5% to about 1.5%, about 1.5% to about 2.5%, about 2.5% to about 5.0%, about 5.0% to about 7.5%, or about 7.5% to about 10.0%.

In some embodiments, the first enzymatic crosslinking solution comprises the enzyme-albumin nanoparticles at a weight % (weight of nanoparticle/weight of solution) ranging from about 0% to about 0.5%, about 0.5% to about 1.5%, about 1.5% to about 2.5%, about 2.5% to about 5.0%, about 5.0% to about 7.5%, or about 7.5% to about 10.0%.

In some embodiments, the enzymatic nanogel can further be diffused in a liquid medium (i.e., an enzymatic nanogel fluid) to provide an emulsion, suspension, mixture, or solution. In some embodiments, the liquid of the enzymatic nanogel fluid comprises one or more of crosslinking agents and/or water. In some embodiments, the enzymatic nanogel fluid comprises a second crosslinker (or a second combination of crosslinkers). In some embodiments, the second crosslinker is also represented by Formula II. In some embodiments, the second crosslinker is ethylene glycol dimethacrylate (EGDMA). In some embodiments, the second crosslinker is TEGDA. In some embodiments, the enzymatic nanogel fluid comprises EGDMA dissolved in TEGDA. In some embodiments, the enzymatic nanogel liquid with the nanogel is configured to flow into the glucose sensor tip via capillary action (see, e.g.,FIG. 42). In some embodiments, the viscosity of the liquid medium and enzymatic nanogel is sufficiently low to allow this capillary uptake.

In some embodiments, the EGDMA is present at a weight % (weight of EGDMA/weight of solution) ranging from about 0% to about 5%, about 5% to about 15%, about 15% to about 25%, about 25% to about 50%, about 50% to about 75%, or about 75% to about 100%. In some embodiments, the TEGDA is present at a weight % (weight of TEGDA/weight of solution) ranging from about 0% to about 5%, about 5% to about 15%, about 15% to about 25%, about 25% to about 50%, about 50% to about 75%, or about 75% to about 100%.

In some embodiments, when the enzymatic nanogel is dispersed in dispensing solution at about 0.25 g gel wt./1 mL, the enzymatic nanogel fluid has a viscosity of less than about 2000 cP, about 1000 cP, about 500 cP, about 250 cP, about 100 cP, about 50 cP, about 25 cP, about 10 cP, about 5 cP, about 1 cP. In some embodiments, when the enzymatic nanogel is dispersed in the dispensing solution at about 0.25 g gel wt./1 mL, the enzymatic nanogel fluid is characterized by an ability to pass through a 20 g needle using less than 60 N pressure.

In some embodiments, as discussed above, the enzymatic nanogel fluid is configured to be dispensed as a solution into sub-millimeter features of the glucose sensor tip. Small features of the glucose sensor tip can be provided by supplying solutions of the enzymatic nanogel which are taken-up by spaces (e.g., channels, tunnels, paths, etc.) in molds (e.g., dye casts) by capillary action. The enzymatic nanogel fluid can fill these device features and, upon filling, be cured using UV light (in the presence of a second crosslinker) and/or concentrated (to remove any volatile liquids) to afford the enzymatic region3830.

In some embodiments, where applicable, the second crosslinking step is performed while the enzymatic nanogel is suspended in the enzymatic nanogel fluid (e.g., comprising a second crosslinker, water, combinations thereof, etc.). In some embodiments, the second crosslinking step affords a hydrogel capable of rapidly transporting oxygen (e.g., diffusion controlled) from the oxygen conduit to other regions of the sensor tip.

Some embodiments pertain to forming a crosslinked enzymatic material using the methods disclosed above. In some embodiments, the enzymatic material comprises one or more of the following structures:

where the variables are as defined above and wherein “” represents a hydrogel or nanogel matrix.

In some embodiments, the enzymatic material comprises one or more enzymatic nanostructures and hemoglobin-albumin nanostructures. In some embodiments, the enzymatic material comprises one or more of the following structures:

where the variables are as defined above and wherein “” represents a hydrogel or nanogel matrix.

In some embodiments, the hydrogel or nanogel matrix of the enzymatic material is represented by one or more of the following:

where u is an integer ranging from 1 to 10 and R7is selected from the group consisting of —C1-4alkyl and H.

[In some embodiments, the hydrogel or nanogel matrix of the enzymatic material is represented by each of the following:

where the variables are as defined above.

In some embodiments, the hydrogel or nanogel matrix of the enzymatic material is represented by each of the following:

where the variables are as defined above.

In some embodiments, the hydrogel or nanogel matrix of the enzymatic material is represented by each of the following:

where the variables are as defined above.

In some embodiments, the enzymatic material comprises one of the above combinations where n is as described above, u is 4, and R11is H.

Thus, according to the present invention, controlling the extent of the crosslinking between the target macromer and nanostructure, and so the number of polymerization sites available to build the polymer network around the macromer-nanostructure complex, may be achieved by setting the number of residues that are available for crosslinking and by the molar ratios of target macromer and nanostructure and the amount of linker (such as glutaraldehyde).

For example, assume 59 lysine residues are available on the albumin. For a solution prepared with 1.244 μmols albumin, 0.050 mmols Traut's reagent, and 0.0376 mmol Acryl-PEG-MAL, the ratio of Lys residues to be converted with a sulfhydryl is 0.050 mmol/(59*1.244 mols), which is approximately 68%. The reaction is allowed to proceed overnight. Assuming a theoretically complete reaction, the percentage of the stilaiydryi sites that are PEGyiateal is 0.0376 mmol/0.050 mmols, or approximately 75%. Therefore, 40 of the 59 Lys residues will be converted to sulfhydryls, 30 of the 59 Lys residues will be PEGylated. As discussed previously, the actual number of Lys residues converted to sulfydryls can be assayed, and the remainder of non-PEGylated sulfyhydryls can be assayed, as the reactions proceed. These measurements allow one of ordinary skill in the art to adjust the reaction conditions to achieve a desired degree of Lys residues that are either converted to sulfhydryls or capped with a linker such as PEG.

The extent of the crosslinking of the nanostructure to the target macromer can be dictated then by the number of free sites on the nanostructure and the amount of the target macromer. For example, hemoglobin (Hb) may be added to the carrier albumin at a 3:1 molar ratio (e.g., 3.733 μmol Hb with 1.244 μmol Alb-MAL-PEG-Acryl) with excess glutaraldehyde. If, continuing with the example above, the number of free Lys residues on the carrier albumin is 19, and the number of free Lys residues on Hb that are modified by glutaraldehyde is 14 [Michael P. Doyle, Izydor Apostol and Bruce A. Kerwin, Glutaraldehyde Modification of Recombinant Human Hemoglobin Alters Its Hemodynamic Properties. Journal of biologic chemistry 274, 2583-2591. Jan. 22, 1999], then the average number of binding sites between Hb and the carrier albumin is approximately 6, or approximately 45% of the available sites. Adjusting the stoichiometric ratio of Hb to carrier albumin allows the percentage of the sites of the Hb that are crosslinked to the carrier albumin to be controlled. For example, increasing the molar ratio of Hb to carrier albumin to 5:1 would decrease the extent of Hb crosslinking by glutaraldehyde to approximately 27%.

This approach thus allows one to control the extent of crosslinking of a target macromer (e.g., hemoglobin, GOx, CAT) with a nanostructure (e.g., albumin) using available crosslinking sites (e.g., Lys residues on carrier albumin) and the number of target macromers that are crosslinked to a nanostructure. The PEGyiated (hydrophilic polymer species functionalized) crosslinking sites include a polymerizable unit (e,g,, Acryl) to which additional monomers may be linked and crosslinked, so the number of polymerization sites available for building a polymer network around a macromer-nanostructure complex may also be controlled using the approach of the present invention.

The macromer-nanostructure complex may be polymerized with a network of biocompliant (linear) monomers (such as HEMA and PEGMA) and crosslinker monomers (such as TEGDA and EDGMA). Additionally, the polymer network can be modified by incorporation of a hydrophilic compounds, such as methacrylic acid (MAA) or acrylic acid (AA). The resulting polymer network around the macromer-nanostructure complex primarily determines the bulk properties of the active hydrogel regions of the biosensor. For example, the polymerization of HEMA may be realized in the presence of acrylic acid (AA) in order to enhance the hydrophilicity of the active hydrogel; however, incorporation of a hydrophilic compound may also decrease the mechanical strength of the active hydrogel. In order to avoid the hydrosolubilization of the hydrogel, a crosslinker, such as TEGDA, that can form stable, non-biodegradable bonds, may be incorporated with the crosslinking solution.

Typically, each linear monomer, crosslinker and/or hydrophilic compound incorporated is first purified, for example by passing through the exchange ion columns, to remove any impurities that may inhibit the polymerization/crosslinking reaction. A hydrophilic compound may be incorporated into a crosslinking solution at a weight % (weight of hydrophilic compound/weight of solution) up to about 5%, to about 10%, to about 15%, to about 20%, to about 25%, to about 30% or to about 35%. A crosslinker may be incorporated in a molar % of up to about 0.5% (mol/mol linear monomer). Other components, such as an initiator (e.g. tetramethylethylenediamine (TEMED)) and/or an activator (e.g., ammonium persulfate (APS), may be added into the crosslinking solution.

The characteristics of the polymer network around the macromer-carrier complex, and so the characteristics of the final active hydrogel, can be adjusted by the ratios of linear and crosslinking monomers. The ratio of the monomers to the macromer-carrier complex increases the extent of the polymer network that can encompass the macromer-carrier complex. By adjusting the relative amounts of linear and crosslinking monomers, the porosity and permeability of the active hydrogel matrix may be adjusted. In general, increasing the relative amount of crosslinker will decrease the pore size in the active hydrogel and so decrease its permeability to solutes. With more extensive crosslinking, the extent of water absorption and swelling be limited, and an increase in hydration time will also be observed. Thus, the relative ratios of monomers (linear and crosslinker), as well as the relative amount of hydrophilic compounds, can be used to adjust the permeability of the hydrogel network formed from the nanogel particles.

For example, a nanogel particle may be formed by crosslinking Albumin-GOx-CAT-PEG-Acryl (this chemical formula is intended to include multiple repeats of GOx, CAT, PEG-Acryl on a single albumin molecule) with HEMA, PEGMA and TEGDA. In some embodiments, the nanogel particle may comprise: GOx:Albumin in a molar ratio range of about 10 to 0.5:1, or about 5 to 1:1; CAT:Albumin in a molar ratio range of about 2 to 0.02:1, or about 1.5 to 0.05:1; PEG-Acryl:Albumin in a molar ratio range of about 30 to 2:1, or about 10 to 2:1; HEMA:Albumin in a molar ratio range of about 400 to 40:1, or about 200 to 40:1; PEGMA:HEMA in a molar ratio range of about 10 to 2:1, or about 10 to 4:1; and (HEMA+PEGMA):TEGDA in a molar ratio range of about 200 to 20:1, or 150 to 50:1.

In another example, a nanogel particle may be formed by crosslinking Albumin-Hb-PEG-Acryl (this chemical formula is intended to include multiple repeats of Hb and PEG-Acryl on a single albumin molecule) with TEGDA. In some embodiments, the nanogel particle may comprise: Hb:Albumin in a molar ratio range of about 20 to 1:1, or about 10 to 1:1; PEG-Acryl:Albumin in a molar ratio range of about 40 to 4:1, or about 30 to 10:1; and TEGDA:PEG in a molar ratio range of about 3 to 0.1:1, or about 2 to 0.5:1.

Nanogel particles according to the present invention are used as a precursor, or interim, to form the active hydrogel on the sensor. One advantage to the use of the nanogel particles according to the present invention is that the activity and chemical and structural properties (e.g., particle size, number of available acryl-terminus sites, etc.) of the nanogel particle can be assayed and characterized in a consistent manner prior to the formation of the active hydrogel areas on the sensor. Moreover, the activity of the active hydrogel areas may be adjusted in a consistent, measurable way by manipulating and characterizing the polymer network around the macromer-nano structure complex. For example, the bulk enzymatic reaction of glucose oxidase follows the ping-pong kinetics, while alternative effective reaction kinetics can be achieved by incorporation of a diffusion limiting polymer network around a core enzymatic-carrier complex to limit substrate availability to the enzymatic reaction.

Some embodiments pertain to a dispensable, UV-curable enzyme-albumin nanogel solution, configured to form a hydrogel upon UV curing, the enzyme-albumin nanogel comprising a hemoglobin-albumin nanoparticle, wherein the hemoglobin and albumin are interconnected with diimine linkers, wherein the hemoglobin-albumin nanoparticle is coupled to poly(ethylene glycol) (PEG) through a thio-linkage, and wherein the hemoglobin-albumin nanoparticle is functionalized to the nanogel matrix via a PEG-based linker and glucose oxidase-albumin nanoparticles, wherein the glucose oxidase and albumin are interconnected with diimine linkers, wherein the glucose oxidase-albumin nanoparticle is coupled to poly(ethylene glycol) (PEG) through a thio-linkage, and wherein the glucose oxidase-albumin nanoparticles is functionalized to the nanogel matrix via a PEG-based linker.

In some embodiments the dispensable, UV-curable enzyme-albumin nanogel solution further comprises a catalase-albumin nanoparticle, wherein catalase and albumin are interconnected via diimine linkers, wherein the catalase-albumin nanoparticle is coupled to poly(ethylene glycol) (PEG) through a thio-linkage, and wherein the catalase-albumin nanoparticle is functionalized to the nanogel matrix via a PEG-based linker.

In some embodiments, the crosslinked, enzymatic-nanoparticle-based material, comprising a hydrogel matrix; an enzyme-functionalized albumin nanoparticle having an albumin molecule covalently linked to at least one enzyme via a diimine-based linker, wherein the enzyme-albumin nanoparticles are PEGylated, and wherein the enzyme-albumin nanoparticles are functionalized to a hydrogel matrix and a hemoglobin-albumin nanoparticle having an albumin molecule covalently linked to at least one hemoglobin molecule via a diimine linker, wherein the hemoglobin-albumin nanoparticle is PEGylated, and wherein the hemoglobin-albumin nanoparticles are functionalized to the hydrogel matrix via a PEG-based linker.

In some embodiments, the crosslinked, enzymatic-nanoparticle-based material described above, have a p50 of at least about 0.1 kPa, about 1.0 kPa, about 1.5 kPa, about 2.0 kPa, about 2.5 kPa, or about 3.5 kPa.

In some embodiments, after curing or concentrating, the crosslinked, enzymatic-nanoparticle-based material has a storage modulus of at least about 8 GPa at a total material concentration of less than about 10 mg/mL. In some embodiments, after curing or concentrating, the crosslinked hemoglobin-based material has a storage modulus of storage modulus of at least about 0.01 GPa, about 0.1 GPa, 0.5 GPa, 1.0 GPa, 2.0 GPa, 4.0 GPa, or about 6.0 GPa at a total material concentration of about 10 mg/mL.

In some embodiments, the crosslinked, enzymatic-nanoparticle-based material has a water content of at least about 70%, about 80%, about 90%, about 95%, about 97.5%, about 99%, about 99.5%, or about 99.9% of the total dry weight of the crosslinked hemoglobin-based material.

Some embodiments pertain to preparing a dispensable, UV-curable oxygen-sensing mixture, comprising an analyte detecting dye. In some embodiments, the analyte is oxygen and the dye is an oxygen detecting dye. In some embodiments, the dye is luminescent. Non-limiting examples of suitable luminescent dyes may be metallo derivatives of octaethylporphyrin, tetraphenylporphyrin, tetrabc,nzoporph:,Tin, or the chlorins, bacteriochlorins, Of isobacteriochlorins and their partially Of fully fluorinated analogs. Other suitable compounds include palladium coproporphyrin (PdCPP), platinum and palladium octaethylporphyrin (PtOEP. PdOEP), platinum and palladium tetraphenylporplwin (PtTPP, PdTPP), camphorquinone (CQ), and xanthene type dyes such as erythrosin B (EB), Other suitable compounds include ruthenium, osmium and iridium complexes with ligands such as 2,2′-bipyridine, 1,10-phenanthroline, diphenyl-1,10-phenanthroline and the like. Suitable examples of these include, tris(4,7,-diphenyl-1,10-phenanthroline)ruthenium(H) perchlorate, tris(2,2′-bipyridine)ruthenium(II) perehlorate, and tris(1,10-phenanthroline)ruthenium(II) perchlorate. While the perchlorate salts are particularly useful, other counterions that do not interfere with the luminescence may be used. In some embodiments, the porphyrin dye is platinum tetrakis pentafluorophenyol porphyrin (PtTFPP). In some embodiments, the porphyrin dye is configured to reversibly bind oxygen and to emit light when oxygen is bound. In some embodiments, the porphyrin dye is platinum tetrakis pentafluorophenyl porphyrin.

In some embodiments, the dye is prepared in a crosslinkable solution that can be distributed adjacent to or within the enzymatic layer of the glucose sensing tip. In some embodiments, the dye is distributed within a dispensable solution of polymer precursors. In some embodiments, the dispensable solution of polymer precursors is configured to crosslink or polymerize when exposed to UV light, or ambient conditions (room temperature and humidity). In some embodiments, the solution comprises a polymerization initiator.

In some embodiments, the dispensable polymer precursor solution comprises one or more vinyl containing monomers. In some embodiments, the vinyl containing monomer may be aliphatic or aromatic. Non-limiting examples of vinyl monomers may be isobornyl acrylate, vinyl chloride, vinyl fluoride, vinylidene chloride, vinyl alcohol, dichloroethylene, styrene, methylstyrene, dimethylstyrene, ethylstyrene, vinylstyrene, chlorostyrene, indene, vinylnaphthalene, vinylfuran, acrylic acid, acryl chloride, acrylonitrile, acryl amide, methacrylic acid, methacrylonitrile, methyl acrylate, ethyl acrylate, vinyl acrylate, allyl acrylate, methyl methacrylate, ethyl methacrylate, vinyl methacrylate, allyl methacrylate, benzyl methacrylate, vinyl acetate, vinyl chloroacetate, vinyl stearate, and ethylvinylethe r. In some embodiments, the vinyl containing monomer is selected from the group consisting of: vinyl alcohol and vinyl acrylate. In some embodiments, the dispensable polymer precursor solution comprises styrene. In some embodiments, the styrene monomer (or other vinylic monomer or mixture of monomers) is present in the polymer precursor solution at a weight % (e.g., wt styrene/wt precursor solution) ranging from about 0% to about 5%, about 5% to about 15%, about 15% to about 25%, about 25% to about 50%, about 50% to about 75%, or about 75% to about 100%. In some embodiments, the dispensable polymer precursor solution comprises acrylonitrile. In some embodiments, the acrylonitrile monomer is present in the polymer precursor solution at a weight % (e.g., wt acrylonitrile/wt precursor solution) ranging from about 0% to about 5%, about 5% to about 15%, about 15% to about 25%, about 25% to about 50%, about 50% to about 75%, or about 75% to about 100%.

In some embodiments, the dispensable polymer precursor solution comprises a silanol. In some embodiments, mixtures of silanols are used. Non-limiting examples of silanols may be trimethylsilanol, tert-butyldimethylsilanol, dimethylphenylsilanol, triisopropylsilanol, diphenylsilanediol, trimethoxyvinylsilane, and triethylsilanol. In some embodiments, the silanol is present in the polymer precursor solution at a weight % ranging from about 0% to about 5%, about 5% to about 15%, about 15% to about 25%, about 25% to about 50%, about 50% to about 75%, or about 75% to about 100%.

In some embodiments, the dispensable polymer precursor comprises an acrylate monomer selected from the group consisting of: HMDA, TEGDA, HEMA, and PEGMA. In some embodiments, mixtures of multiple acrylates are used. In some embodiments, the acrylate(s) are present in the polymer precursor solution at a weight % ranging from about 0% to about 5%, about 5% to about 15%, about 15% to about 25%, about 25% to about 50%, about 50% to about 75%, or about 75% to about 100%.

In some embodiments, the dye is present in the polymer precursor solution at a weight % ranging from about 0% to about 0.5%, about 0.5% to about 1.5%, about 1.5% to about 2.5%, about 2.5% to about 5.0%, about 5.0% to about 7.5%, or about 7.5% to about 10.0%.

In some embodiments, the dispensable polymer precursor solution comprises one or more of the porphyrin dye, styrene, the silanol, and acrylonitrile.

In some embodiments, the dispensable polymer precursor solution (or emulsion) is of low viscosity. In some embodiments, the precursor solution has a viscosity of less than about 2000 cP, about 1000 cP, about 500 cP, about 250 cP, about 100 cP, about 50 cP, about 25 cP, about 10 cP, about 5 cP, about 1 cP, or about 0.5 cP.

In some embodiments, after curing the oxygen sensing material has a storage modulus of at least about 8 GPa at a total material concentration of less than about 10 mg/mL. In some embodiments, after curing or concentrating, the oxygen sensing material has a storage modulus of at least about 0.01 GPa, about 0.1 GPa, 0.5 GPa, 1.0 GPa, 2.0 GPa, 4.0 GPa, or about 6.0 GPa at a total material concentration of about 10 mg/mL.

In some embodiments, an oxygen sensor polymer system formed using one of the polymer precursor solutions described above has high quantum efficiency. In some embodiments, the quantum efficiency is greater than about 50%, about 40%, about 20%, or about 10% of the polymer system. In some embodiments, the quantum efficiency is between about 20% and about 40%.

In some embodiments, the polymer precursor solution is rapidly curable. In some embodiments, the polymer precursor solution cures in less than about 60, about 40, about 30, about 20, about 15, about 10, or about 5 seconds upon exposure to UV light.

In some embodiments, the resultant polymer is a composite of one or more of the following repeat units:

Any solution disclosed herein is UV curable and thermal curable. For thermal curing, a water soluble thermal initiator must be used. Non-limiting examples of useful thermal initiators may be VA-080 (2,2′-azobis(2-methyl-N-(1,1-bis(hydroxymethyl)-2-hydroxyethyl)propionamide)), VA-086 (2,2′-azobis(2-methyl-N-(2-hydroxyethyl)-propionamide)), VA-044 (2,2′-azobis[2-(2-imidazolin-2-yl)propane]dihydrochloride), VA-057 (2,2′-azobis(2-(N-(2-carboxyethyl)amidino)propane)), VA-058 (2,2′-azobis(2-(3,4,5,6-tetrahydropyrimidin-2-yl)propane)dihydrochloride), VA-060 (2,2′-azobis(2-(1-(2-hydroxyethyl)-2-imidazolin-2-yl)propane)dihydrochloride), V-50 (2,2′-azobis(2-amidinopropane)dihydrochloride) and V-501 (4,4′-azobis(4-cyanopentanoic acid)) (all supplied by Wako Pure Chemical Industries). If thermal curing is used, a thermal source is provided.

Any methods of manufacturing the oxygen conduit, enzymatic region, and oxygen sensing region can include a variety of different steps discussed above. For purposes of summarizing the disclosure, certain aspects, advantages and features of the inventions have been described herein. It is to be understood that not necessarily any or all such advantages are achieved in accordance with any particular embodiment of the inventions disclosed herein. No aspects of this disclosure are essential or indispensable.

Each reference cited in the discussion above is hereby incorporated by reference in its entirety.

Optical Enzymatic Sensor

Disclosed herein are example embodiments of optical glucose sensors. At least one advantageous feature of the disclosed optical glucose sensors is that they are configured to reduce mechanical tolerance requirements in manufacture and operation. The disclosed sensors include a plurality of waveguides configured to direct light to and from a target material, such as an oxygen sensing polymer. Excitation waveguides can receive light from an excitation source in a transmitter that is housed separately from the sensor. Similarly, emission waveguides can deliver light from the sensor to a detector on the transmitter. Proper alignment of such a sensor with the transmitter can determine whether excitation light enters the sensor and reaches the target material as well as whether light emitted from the target material reaches a detector. Accordingly, the sensors disclosed herein are configured to increase the tolerances for achieving proper alignment through the use of total internal reflections at boundaries of materials. The orientation of these boundaries is such that the transmitter with the light sources and detectors can be attached to the sensor without being precisely aligned while still maintaining optical connection with the sensor. This can reduce costs and complexity associated with manufacture of such sensors.

Also disclosed herein are example systems that include a disposable sensor and a separately housed transmitter with an emitter array and an emission detector. An optical interconnect couples the optics of the disposable sensor to the optics of the transmitter. The transmitter is configured to couple to a portion of the sensor that extends out of a patient when in use. Alignment pins on the transmitter can facilitate correct alignment with the optics of the sensor. The sensor and optical interconnect are configured so that the transmitter can be aligned with relatively large variations in position while still achieving suitable optical alignment. Accordingly, optical connections conveying excitation and emission signals between the transmitter and the sensor can be readily made without precise alignment of the optical pathways.

The disclosed optical glucose sensors are advantageously configured to operate using luminescent lifetime measurements. Luminescent lifetime measurements provide advantages relative to other optical sensors such as intensity- or amplitude-based measurements. For example, lifetime measurements can be relatively immune to background fluorescence or luminescence. As another example, lifetime measurements can be relatively immune to intensity or amplitude variations associated with changes in optical coupling or photobleaching of a target sensing molecule. Lifetime measurements may be challenging, however, due at least in part to nanosecond lifetimes making it difficult to perform such measurements with small, inexpensive instrumentation. However, the disclosed optical glucose sensors utilize target materials that have lifetimes on the order of microseconds rather than nanoseconds, making reliable measurements possible using relatively small and inexpensive materials, such as optical sources and detectors. Furthermore, lifetime measurements of oxygen also make possible factory calibration and potentially calibration-free optical sensors for oxygen sensing due at least in part to the lifetime of the relevant materials being based on fixed, quantum chemistry properties of the material (e.g., an oxygen sensing polymer).

Other advantages of the disclosed optical glucose sensors include a relatively high sensitivity to low glucose concentrations. The signal to noise ratio of the lifetime measurement does not generally diminish with decreasing glucose concentrations. The disclosed oxygen sensing polymer, for example, can enable oxygen levels to be measured with relatively high sensitivity from ambient oxygen tissue concentrations to relatively small oxygen concentrations. This is due at least in part to the optical glucose sensor being a differential oxygen sensing device. For example, for low glucose levels, a difference between reference and working oxygen concentrations is small, but the optical lifetime measurements from the oxygen sensing polymer for the set of oxygen measurements is not generally diminished due to lower glucose concentration.

Other advantages of the disclosed optical glucose sensors include an ability to perform self-assessment tests prior to measurements. For example, the optical glucose sensors can include a relatively low power light source and a high-power light source. The low power light source can be used to interrogate the sensor to determine whether a proper optical connection exists. If no proper optical connection exists, the transmitter can be configured to not emit light from the high-power light source. This can increase the safety of a user by reducing or preventing the high-power light source from potentially shining in the eye of a person when the transmitter is disconnected from the sensor. The optical glucose sensor can also advantageously be configured to provide an optical signal from the low power light source with a known lifetime decay to calibrate the transmitter and optical system before glucose measurements are made. The lower power light source can be configured so that the light from the light source is reflected by the target material instead of inducing a luminescent signal.

Optical Glucose Sensor Overview

The optical glucose sensors described herein are a part of a continuous glucose monitoring system. The monitoring system is generally an opto-enzymatic, percutaneous sensing system that utilizes a disposable sensor. The system includes an implantable optical sensor, a transmitter optically coupled to the sensor, an analysis engine, and a computing device. The disposable sensor contains a small percutaneous sensing element that is inserted/implanted into the tissue. The sensor is an optical-enzymatic sensor that provides interstitial fluid measurements of analytes, glucose for example, when optically interrogated with visible light. The sensor provides a measurement of the interstitial glucose based on the difference between an interstitial reference oxygen measurement and measurements of the oxygen remaining after a two-stage enzymatic reaction of glucose and oxygen. When implanted in the patient, the optical sensor can be in optical communication with the transmitter.

The optical sensor can include a sensor subassembly that is a polymer laminate structure connected to an optical interconnect component that interfaces with the transmitter. The top layer of the polymer laminate structure contains an oxygen conduit (e.g., a hemoglobin polymer matrix embedded within siloxane) to transport oxygen. The middle layer contains an enzymatic hydrogel to transduce glucose into changes in oxygen partial pressure, an oxygen sensing polymer (e.g., platinum-porphyrin immobilized in a hydrophobic oxygen permeable polymer) to transduce oxygen partial pressure into luminescent lifetime signals, and an optical circuit to direct light to interrogate the oxygen sensing polymer to obtain the luminescent signals. The optical circuit includes a miniaturized, structured waveguide with a plurality of optical channels connected to a plurality of contiguous oxygen sensing polymer volumes adjacent to the enzymatic hydrogel and at least one spatially-distinct oxygen sensing polymer volume adjacent to the oxygen conduit. The bottom layer of the sensor subassembly is a structural polymer (e.g., a robust biocompliant polymer film) for mechanical integrity.

The monitoring system generally works by determining lifetimes (e.g., decay rates) of luminescent emissions from the oxygen sensing polymer. For example, when the oxygen sensing polymer is excited with a suitable frequency of light, the porphyrin dye in the polymer matrix produces a strong luminescent emission. The lifetimes of the optical emissions are quantitatively correlated with the partial pressure of oxygen in the oxygen sensing polymer. The net oxygen consumed by the diffusion-limited reaction of glucose and oxygen is quantitatively correlated with the interstitial glucose concentration. The net oxygen consumed by the reaction is calculated as the difference in the oxygen concentration remaining after the reaction (in the presence of glucose) and a reference oxygen concentration (in the absence of glucose) (O2 reference-O2 remaining).

In use, the transmitter can be affixed to a patient's skin so that it is in optical communication with the sensor. The transmitter can provide one or more of the functions of: (1) optically interrogating the sensor, (2) processing the received optical sensor signals, (3) having the capabilities to control, power, and communicate, and (4) being configured to form a mechanical optical interconnect with the sensor. The transmitter of the monitoring system contains instrumentation to optically interrogate the optical sensor, a microprocessor to convert the raw optical signals into measurements, and a wireless transceiver to transmit the measurements to an external receiver. In some embodiments, the transmitter enables real-time data communication with other electronic devices such as smartphones. The transmitter includes optical excitation sources such as single stage laser diodes that emit 405 nm light corresponding to the peak absorption wavelength of a luminescent dye in the target material. The detector on the transmitter can be a multi-pixel, miniaturized silicon photomultiplier chip. The transmitter is configured to form a mechanical optical interconnect with the optical sensor. The transmitter is also configured to optically interrogate the sensor and to receive emitted light from the sensor to determine analyte concentrations. The transmitter can be configured to take measurements at any time interval, for example, every 30 seconds, or each minute and can therefore, provide real-time monitoring. The transmitter can be configured to transmit bursts of glucose readings to an analysis engine or other computing device. For example, the transmitter may transmit bursts of glucose readings every five minutes to an analysis engine. The analysis engine receives bursts of glucose readings from which it determines results, including time series glucose levels, trends, patterns, and alerts.

A portable computing device, such as a cell phone, wearable computing device, tablet, personal digital assistant, or other computing device may include an application that enables viewing of results from the analysis engine as well as sending queries. Alerts may be viewed on the portable computing device as well as system alarms (such as low battery).

Example Optics of Glucose Sensor

FIG. 43Aillustrates an example optical glucose sensor4300configured to couple to an optical interconnect4302(e.g., housed in a transmitter) and configured to deliver light to and from a target material for glucose measurements. The optical glucose sensor4300mechanically and optically couples to the transmitter (not shown) by coupling to the optical interconnect4302using a sensor optical interface4310attached to a sensor body4320. In some embodiments, the sensor optical interface4310is a chip bonded to the sensor body4320.

The transmitter mechanically couples to the sensor4300through alignment pins4308on the optical interconnect4302that are configured to mate with alignment receptacles4318on the sensor optical interface4310. The sensor optical interface4310can include features4314and4316configured to mate with or be complementary with optical features (such as lenses) on the optical interconnect4302. In some embodiments, these features can assist in aligning the optical interconnect4302relative to the sensor optical interface4310as well. In some embodiments, the sensor optical interface4310includes optical elements (e.g., lenses) instead of or in addition to the excitation sources and optics4304and/or detector and optics4306. The transmitter optically couples to the sensor4300through excitation sources and optics4304on the optical interconnect4302that are configured to transmit excitation light to waveguides4330on the sensor body4320. The transmitter also optically couples to the sensor4300through a detector and optics4306on the optical interconnect4302that is configured to detect emission light from the waveguides4330on the sensor body4320.

When interrogating the sensor4300, the transmitter can produce excitation light4311and deliver that light to the sensor using the excitation sources and optics4304. The excitation light4311is received at the sensor optical interface4310where it undergoes total internal reflection at an internal boundary between materials in the sensor optical interface4310, as described in greater detail herein with reference toFIGS. 45A and 45B. The reflected excitation light4321arrives at the waveguide4330at an excitation light receiving element4322, where it again undergoes total internal reflection to enter the waveguide4330. In response to the interrogation, the transmitter can receive emitted light that can be analyzed to determine glucose levels. The emitted light4323exits the waveguide4330at an emission transmission element4324where it undergoes total internal reflection from the waveguide to the sensor optical interface4310. Within the sensor optical interface4310, the emitted light4323undergoes internal total reflection once again where the redirected emitted light4313is incident on the optics and detector4306on the optical interconnect4302. As illustrated, the excitation optical path and the emission optical path are separate entering and leaving the sensor body4320through the sensor optical interface4310. The optical paths are combined and separated in the sensor4300using the waveguides4330. The waveguides4330can be made to be flexible so that when the sensor body4320bends (e.g., during and after insertion into a patient), the optical signals (e.g., excitation and emission light) are not substantially diminished.

As described in greater detail elsewhere herein, the sensor4300can be configured to have a low-mechanical tolerance optical interface between the sensor optical interconnect4302and the sensor body4320through the sensor optical interface4310. Asymmetric geometries can be used at the optical interfaces between elements (e.g., the optical interconnect4302, the sensor optical interface4310, and the sensor body4320) to decrease the sensitivity of the optical transmission efficiency on mechanical positioning of the optical interconnect4302with respect to the sensor elements (e.g., the excitation receiving elements4322and/or emission receiving elements4324).

To decrease the effects of misalignment between the optical interconnect4302and the sensor body4320, the sensor optical interface4310, the excitation receiving elements4322, and the emission receiving elements4324can be configured to have an increasing physical dimension orthogonal to the direction of light travel in at least one axis. This can decrease mechanical sensitivity in the axis of the change in the physical dimension. For example, to decrease sensitivity in the direction parallel to the optical axis in the waveguides4330, the excitation receiving elements4322can be configured to have a wide collection aperture in the sensor body4320compared to the aperture of the light transmitted from the sensor optical interface4310. Similarly, the sensor emission path can be configured to have a narrow emission aperture in the sensor body4320compared to the emission path receiving the light in the sensor optical interface4310.

In some embodiments, the light path from the sensor optical interconnect4310into the sensor body4320is relatively shallow to decrease the sensitivity of positioning in at least one axis parallel to the direction of light travel in the waveguides4330. For example, the angle of total internal reflection in the sensor body4320at the emission receiving element4324can be less than or equal to about 10 degrees, less than or equal to about 20 degrees, or less than or equal to about 30 degrees. The angle of total internal reflection in the sensor optical interface4310can be configured to be complementary to the total internal reflection in the emission receiving element4324to induce a targeted total angle change through sensor body4320and the sensor optical interface4310. In some embodiments, the total change in direction of the optical path from the sensor body4320(e.g., from the waveguides4330) to the optical interconnect4302can be about 90 degrees. A similar configuration can be implemented for the excitation pathways as well so that the total change in optical path direction is about 90 degrees while also achieving a relatively shallow angle of incidence entering the sensor body4320through the excitation receiving element4322. In some embodiments, misalignment in the direction perpendicular to the optical path in the waveguides4330can be achieved using lenslets in the optical interconnect4302and/or on the sensor optical interface4310. For example, these lenslets (e.g., the lenses that are part of the excitation sources and optics4304and/or the detector and optics4306) can focus or collimate the light to and from the sensor body4320. By reducing the sensitivity to mechanical misalignment, manufacturing costs and complexity can be reduced.

In some embodiments, the excitation receiving elements4322and/or the emission receiving elements4324can be wider than the waveguide4330. For example, the receiving elements4322,4324can be about 5 mm wide. In certain implementations, the receiving elements4322,4324can be larger than the waveguides (e.g., wider and/or deeper), thereby having a relatively large volume making them easier to manufacture. In some implementations, the receiving elements4322,4324can have an index of refraction that is the same or substantially the same as the waveguide4330. In some embodiments, the optical interconnect4302has a relatively small exit aperture for excitation light4311that is delivered to the sensor optical interface4310. In certain implementations, the excitation light4311is configured to enter the sensor optical interface4310collimated. In some embodiments, the optical interconnect4302has a relatively large exit aperture for emission light4323that leaves the sensor optical interface4310. In certain implementations, the emission light4311is configured to enter the sensor optical interface4310collimated.

FIG. 43Billustrates the sensory body4320and waveguides4330of the example optical glucose sensor4300illustrated inFIG. 43A. For the illustrated sensor4300, excitation light travels from the top of the page in the waveguides4330towards the target materials4340a,4340b,which in some embodiments is an oxygen sensing polymer in the reaction region (4340a) and the reference region (4340b). Emitted light travels from the target materials4340a,4340bin the waveguides towards the top of the page. The waveguides4330each include an excitation path4330a,an emission path4330b,and a transmission path4330cthat all meet at a branching point4333. An advantageous feature of the waveguides4330is that, at the branching point4333, the cross-sectional area of the emission path4330bis greater than the cross-sectional area of the excitation path4330cso that a majority of the emitted light enters the emission path4330bfrom the transmission path4330c.In addition, the cross-sectional area of the emission path4330bdecreases while the cross-sectional area of the excitation path4330aincreases from the branching point4333towards the sensor optical interface4310(towards the top of the page). This allows for a larger target for excitation light entering the waveguides4330, making it easier to sufficiently mechanically align the sensor optical interface4310and the optical interconnect4302.

In use, the sensor4300and the optical interconnect4302operate to excite a target material4340a,4340bwith excitation light. The target material can be, for example, a reaction chamber4340acomprising an oxygen sensing polymer, a glucose in1et, and an enzymatic hydrogel with oxygen conduit; or a reference chamber4340bcomprising an oxygen sensing polymer with an oxygen conduit), as described in greater detail elsewhere herein with reference toFIGS. 38 and 40, for example. The excitation light/signal travels within the excitation path4330aand the transmission path4330cto an optrode or other optical sensing device to excite the target materials4340a,4340b(e.g., an oxygen sensing polymer). The target material4340a,4340bproduce an emission or luminescent light signal that travels from the optrode to the emission path4330bvia the transmission path4330c,some of which is described in greater detail herein with reference toFIGS. 20 and 40.

The reaction chamber4340aincludes an enzymatic hydrogel with three contiguous glucose reaction volumes (as previously described in detail herein with reference toFIG. 2B, for example), where an in1et regulates glucose entering into the first reaction volume. The three contiguous glucose reaction volumes inside the enzymatic hydrogel each have a dimension of approximately 0.1 mm×0.1 mm×0.1 mm, respectively. All three glucose reaction volumes contain the same enzymatic hydrogel material. In some embodiments, glucose diffuses through the in1et into the first reaction volume and undergoes a reaction with the glucose oxidase enzyme in the hydrogel. The unreacted glucose diffuses into the second reaction volume and undergoes another reaction with the glucose oxidase enzyme in the hydrogel, and the remaining unreacted glucose diffuses into the third glucose reaction volume where it is reacted. The rate of diffusion of glucose in each volume is determined by the permeability of the hydrogel. The oxygen conduit supplies the same oxygen flux to each progressive volume from a homogeneous oxygen concentration within the oxygen conduit that is transported through an oxygen permeable, hydrophobic membrane. The glucose oxidase and catalase enzymatic reactions consume oxygen in proportion to the amount of glucose in each reaction volume. The total oxygen remaining in the entire enzymatic hydrogel depends on the interstitial oxygen concentration that is supplied by the oxygen conduit and the diffusion limited oxygen consumption that is dependent on the interstitial glucose concentration.

To measure the oxygen concentration remaining in the enzymatic hydrogel, all three reaction volumes of the enzymatic hydrogel are in physical contact with an adjacent oxygen sensing polymer layer operating as a reference volume for oxygen measurements. The oxygen conduit is also in physical contact with an adjacent oxygen sensing polymer layer. The three glucose reaction portions of the target material4340areaction volume and the reference material4340breaction volume are interrogated optically through separate optrodes to excite the luminescent dye in each volume and to obtain oxygen measurements for the illuminated region of each one. For each optrode, there is a dedicated waveguide and optical source that generates and delivers the excitation pulse of light to each optical sensing polymer in in each volume in target material4340areference material4340b.Each of these waveguides returns the luminescent emission signal from the oxygen sensing polymer in each volume, i.e., each of the three reaction volumes in the target material4340areference material4340b,to a single common detector. The four oxygen sensing polymer volumes are each interrogated with a short 100 microsecond light pulse temporally-multiplexed with 400 microsecond luminescent emission observation periods after each pulse.

FIG. 43Cillustrates a portion of the waveguides4330of the example optical glucose sensor4300embodiment ofFIG. 43Awhere excitation paths4330aand emission paths4330bmerge. The branching point4333in each of the waveguides4330can act as an efficient beam splitter/combiner system. The excitation path4330aand the emission path4330bare separate entering and leaving the sensor body4320with respect to the sensor optical interface4310. The excitation path4330ais tapered, having its widest cross-sectional area at the sensor optical interface4310and its narrowest cross-sectional area moving towards the target materials4340aand4340b,in order to inject into a transmission path4330cin the sensor body4320optical circuit. The waveguides4330can be configured to maintain the multimode light characteristics in the transition between transmission paths4330cand excitation paths4330aor between transmission paths4330cand emission paths4330b.The transmission path4330csplits into two paths at the branching point4333, the excitation path4330aand the emission path4330bwhere the width of the emission path4330bis greater than the width of the excitation branch4330aat the branching point4333in order to bias a majority of the emitted light4323into entering the emission path4330b.In some embodiments, the ratio of the widths is approximately 4 to 1. In certain implementations, this beam splitter arrangement can result in about 81% efficiency in dividing light into appropriate paths, compared to about 50% efficiency for dichroic mirrors.

As can be seen inFIG. 43C, the geometry of the excitation path4330aand emission path4330bdirects a majority of the excitation light4321into the excitation path4330aand a majority of the emission light4323into the emission path4330b.

FIGS. 44A and 44Brespectively illustrate a cut-away side view and a top view of an example sensor4300with a sensor optical interface4310. The sensor4300can include a sensor waveguide system4330that is part of the sensor body4320, the sensor waveguide system4330having a plurality of measurement waveguides. As illustrated in the cut-away side view ofFIG. 44A, materials can be arranged and selected to direct excitation light4311(or emission light) through a sensor optical interface4310through two or more total internal reflections at boundaries between materials. For example, the sensor optical interface4310can include a first redirecting element4315comprising a first material with a first index of refraction n1, the first material adjacent to another material with a larger index of refraction. In certain implementations, the first index of refraction can be about 1 and the material of the first redirection element4315can be air. The index of refraction of the adjacent material can be configured to be approximately the same as for cladding4332to reduce reflections (and signal loss) at the boundary between the sensor body4320and the sensor optical interface4310. The boundary between the first redirecting element4315and the adjacent material in the sensor optical interface4310can be configured so that incident light from the optical interconnect of the transmitter undergoes total internal reflection at the boundary.

The reflected or redirected excitation light4321can then enter the sensor body4320. Within the sensor body4320, materials can be arranged so that boundaries between materials are configured such that the redirected excitation light4321undergoes another total internal reflection to be redirected into the excitation path4330aof the waveguide4330. For example, a second material4334with a second index of refraction, n2, can be arranged with an included planar surface that is adjacent to a third material4335with a third index of refraction, n3, where n3>n2. Due to the combination of the difference in indices of refraction and the inclination of the surfaces, the redirected excitation light4321undergoes total internal reflection to be redirected into the core4336of the waveguide4330, the core4336being surrounded by cladding4332. The core4336can have a fourth index of refraction, n4, that is close to but greater than the index of refraction of the cladding4332(e.g., n3<n4) so that light is maintained within and directed along the waveguide by undergoing total internal reflection at the boundary between the cladding4332and the core4336. Another advantage of the inclination of the boundary between the second material4334and the third material4335is that it relaxes mechanical alignment requirements by providing a larger acceptable range of positions for the optical interconnect4302along a direction parallel to the optical path down the waveguide4330.

By way of example, the first material4315can be air with an index of refraction of 1 (n1=1.0). The adjacent material (cladding4332in this example) in the sensor optical interconnect can have an index of refraction of 1.53. The second material4334in the sensor body4320can be a UV-cured material (e.g., an adhesive) with an index of refraction of about1.32(e.g., an acrylate). The third material can be cladding4332, such as an acrylate, with an index of refraction of about 1.53. The core4336can also be an acrylate with an index of refraction of about 1.56.

As described above, the sensor4300can include a plurality of measurement waveguides in a sensor waveguide system4330. An individual measurement waveguide can include a transmission path4330chaving a transmission aperture at a first end of the measurement waveguide (e.g., at the target material4340a,4340b) and a branching point4333.

As depicted inFIG. 44B, the individual measurement waveguide can include an excitation path4330ahaving an excitation aperture4322at a second end of the measurement waveguide opposite the first end, the excitation path4330aextending from the branching point4333to the excitation aperture4322. The excitation aperture4322can be a boundary between different materials where excitation light4321undergoes total internal reflection to be redirected into the excitation path4330aof the waveguide. For example, the excitation aperture4322can be where the second material4334and third material4332meet.

The individual measurement waveguide can include an emission path4330bhaving an emission aperture4324at the second end of the measurement waveguide, the emission path4330bextending from the branching point4333to the emission aperture4324. The emission aperture4324can be constructed in a fashion similar to the excitation aperture4322, where two materials form a boundary; the indices of refraction of the materials and the inclination of the boundary configured to redirect the emitted light4323by way of total internal reflection into the sensor optical interconnect4310. In some embodiments, individual emission paths4330bjoin together at a combined emission aperture4324such that emitted light4323from a plurality of emission paths is redirected at the emission aperture4324into the sensor optical interconnect4310.

The individual measurement waveguides can include a core4336comprising a core material having a core index of refraction n4and cladding material4332having a cladding index of refraction n3less than the core index of refraction (n3<n4), the cladding material4332surrounding the core material4336to form the excitation path4330a,the emission path4330b,and the transmission path4330c.In some embodiments, a boundary between the cladding4332and the core4336is configured to be inclined in such a way as to form the emission aperture4324and/or the excitation apertures4322, as described in greater detail herein with reference toFIGS. 45A and 45B.

As depicted inFIG. 44B, individual measurement waveguides4330are configured to receive excitation light4321at the excitation aperture4322, guide the excitation light4321along the excitation path4330afrom the excitation aperture4322to the branching point4333, and guide the excitation light4321along the transmission path4330cfrom the branching point4333to the transmission aperture (in the direction towards the right side ofFIG. 44B) for excitation of a target material4340a,4340b.The individual waveguides4330are further configured to receive emitted light4323from the target material4340a,4340b(coming from the right side inFIG. 44B) at the transmission aperture, guide the emitted light4323into and along the transmission path4330cfrom the transmission aperture to the branching point4333, and guide a majority of the emitted light4323into and along the emission path4330b(because of its widest cross-sectional area at the branching point4333) from the branching point4333to the emission aperture4324. The individual waveguides can be configured to direct emitted light4323from a plurality of emission paths4330bto a combined emission aperture4324of the sensor waveguide system4330.

The excitation and emission apertures4322,4324can be configured such that the excitation aperture4322has a first interface material with a first index of refraction and the emission aperture4324has a second interface material with a second index of refraction lower than the first optical interface index of refraction, the apertures having an interface between the first interface material and the second interface material. The optical path of excitation light4321through the sensor optical interface to a measurement waveguide begins in a first direction, experiences total internal reflection within the sensor optical interface and then again at the interface between the first optical interface material and the second optical interface material, thereby experiencing total internal reflection to end in a second direction substantially perpendicular to the first direction. Similarly, the optical path of emitted light from a measurement waveguide through the sensor optical interface begins in the second direction, experiences total internal reflection at the interface between the first optical interface material and the second optical interface material, enters the sensor optical interface4310and is totally internally reflected again to be redirected to the optical interconnect4302, for a total redirection of about 90 degrees.

FIGS. 45A and 45Billustrate additional embodiments of sensors4300with sensor optical interfaces4310configured to relay excitation light4321and emission light4323from a waveguide4330. The excitation and emission apertures illustrated respectively inFIGS. 45A and 45Brepresent apertures having fewer materials and being simpler to manufacture than the aperture configuration illustrated inFIG. 44A.

In the illustrated exemplary embodiment, the core4336and cladding4332are cut to form an inclined boundary to reflect light with little or no light lost in the reflection. For example, excitation light4311enters the sensor optical interface4310and encounters a boundary between a first material4315(e.g., air, n1=1) and a second material4316(e.g., acrylate, n2=1.53). Due at least in part to the shallow angle of incidence of the light relative to the angle of the boundary, the excitation light4311is reflected at the boundary and enters the sensor body4320. After reflection at the boundary in the sensor optical interface4310, the optical path of the light forms an angle, θ1of about 15°, relative to the optical axis of the waveguide. The reflected excitation light4321then crosses a boundary between cladding4332and the core4336. At this boundary, a small fraction (e.g., less than or equal to about 5%, less than or equal to about 3%, or less than or equal to about 2%) of light4335is reflected out of the waveguide4330and the light is refracted so that its angle, θ2, relative to the optical axis of the waveguide4330increases to about 20 degrees. The light encounters a boundary between the core4336and the cladding4332, and because of the shallow angle of incidence of the light relative to the angle of the boundary (e.g., θ3is about 10 degrees, but θ3can be less than or equal to about 30 degrees, less than or equal to about 20 degrees, less than or equal to about 10 degrees, or less than or equal to about 5 degrees relative to a planar surface of the sensor body4320or optical axis of the waveguide4330) and because the difference in indices of refraction (e.g., n3>n2), the reflected light4321undergoes total internal reflection so that its optical path is redirected to be substantially parallel with the optical/longitudinal axis of the waveguide4330. As depicted inFIG. 45B, the emission light path is similarly configured. The angle θ1that the emitted light entering the sensor optical interface4310makes with the optical axis of the waveguide4330can be about 19 degrees, whereas for the excitation light leaving the sensor optical interface4310, the angle θ1was about 15 degrees. The differences in angles are due at least in part to the geometries of the system. For example, at the boundary between the core4336(n=n3) and the cladding4332(n=n2), a small fraction (e.g., less than or equal to about 3%, less than or equal to about 2%, or less than or equal to about 1%) of light4337is reflected out of the waveguide4330and the remaining light is refracted so that its angle, θ1, relative to the optical axis of the waveguide4330is about 19 degrees.

The core4336can be shaped to have a relatively shallow inclination relative to a plane of the waveguide. Generally, a redirection optical element is positioned at about 45 degrees to redirect an optical path about 90 degrees. However, the size of the target for the incident light is about the same distance as the height of the core4336, which can be a relatively small target. The problems in these cases is that a relatively small misalignment in light source may result in a complete loss of optical signal in the waveguide4330. The sensors disclosed herein solve this problem by using a combination of redirection elements to achieve a total redirection of the optical path of about90degrees. In particular, redirection within the sensor body4320, e.g., at the boundary between the cladding4332and the core4336, can be accomplished using a planar surface that is shallower or more acute than a 45 degree optical redirection element. This can increase the effective size of the target for the light. As illustrated inFIG. 45A, the size, w, of the target for the reflected excitation light4321is about 280 μm with a waveguide thickness, h, of about 50 μm (e.g., a thickness of the core4336). In general, the target size, w, of the redirection element increases with decreasing angle (e.g., w=h*cot(θ3)). By making the target size, w, larger, greater allowances for misalignment can be made without significant or complete signal loss relative to systems that use a 45 degree redirection element, for example.

Method of Contouring Refractive Index

The 4×1 optical architecture described herein, injects light at the input port of each channel of the waveguides by tuning the refractive index of the top clad layer. Each channel core is surrounded on the bottom, right, and left sides by a low index material such as PVDF for instance (embossing layer). The output from each optical source (i.e. LEDs or Laser Diodes, for example), is focused onto the input port of each channel arriving within a tailored angular profile. This cone of light is incident onto a top clad layer and refracted towards the beveled facet of the input channel core. The light illuminating the shallow angle bevel facet (i.e. 8° as an example) undergoes total internal reflection and subsequently gets launched into the input channel of the waveguide. The light within the focal spot, which does not illuminate the bevel facet is directed away from the waveguide sensor by contouring the refractive index of the bottom optical adhesive layer and choice of housing material. The refractive index prescription for the different core and clad optical layers defining the waveguide controls how much light propagates down the full length of the waveguide as this transmitted light undergoing multiple reflections at each core/clad interface.

A non-sequential macro script was written for execution within Zemax to investigate the waveguide coupling efficiency over a broad range of core and clad refractive index values. The coupling efficiency of the incident light at the input channel and arriving at the tips of each channel for both LED and laser diode optical sources are presented in the following set of plots. The stepwise contoured refractive index profile for one embodiment of the optical layer stack of the embodiments of the present invention is included in Table 1 below.

FIGS. 46A and 46Billustrate an example embodiment of an optical glucose sensor4600with two excitation sources4604a,4604bper waveguide4630. The waveguide4630employs a similar configuration to the waveguide4330described herein with reference toFIGS. 45A and 45B. For example, the waveguide4630includes a tapered planar bevel design to decrease positional sensitivity along the optical axis of the waveguide4630for coupling into the planar waveguide structure. As described herein, this exemplary design provides a positional window of about 283.5 μm along the optical axis of the waveguide4630corresponding to a thickness of about 50 μm for the core4636a,4636bof the waveguide4630. For comparison, for a 50 μm waveguide thickness, a 45 degree redirection element would have a positional window of about 50 μm along the optical axis of the waveguide4630.

The sensor4600can include two light sources per waveguide to provide integrated fault detection of the sensor optical circuit and/or to calibrate the sensor4600. A first light source4604acan be configured to provide red excitation light4611athat is redirected at boundary4615aand redirected at a boundary between core4636aand cladding4632, with a small portion of light4635abeing reflected out of the sensor body4620. The first light source4604acan be configured to be relatively low-powered for safety concerns. The first light source4604acan be configured to provide light having a color or wavelength spectrum tailored to not excite the target material (e.g., so as to not induce fluorescence in the target material, which in some embodiments is an oxygen sensing polymer).

A second light source4604bcan be configured to provide blue excitation light4611bthat is redirected at boundary4615band redirected at a boundary between core4636band the cladding4632, with a small portion of light4635bbeing reflected out of the sensor body4620. The second light source4604bcan be configured to be relatively high-powered for performing glucose measurements. The second light source4604bcan be configured to provide light having a color or wavelength spectrum tailored to excite the target material (e.g., so as to induce fluorescence in the target material).

The sensor4600can be configured to include integrated fault detection of the sensor optical circuit (e.g., to verify the connection between the optical interconnect4302, the sensor optical interface4310, and the sensor body4620). To do so, the sensor4600transmits an optical signal(s) having a known temporal decay (e.g., lifetime) with a tailored wavelength configured to not cause fluorescence in the oxygen sensing polymer of the target4640. Accordingly, the light is substantially reflected by the target material4640(e.g., the oxygen sensing polymer). By detecting the signal sufficiently corresponding to the known excitation signal, the sensor4600can determine: (1) whether a proper optical connection exists, (2) that the operation of the detection system is proper, (3) that operation of the optics of the sensor4600through the sensor optical interface4310is proper, (4) verify temporal stability of lifetime measurements, and/or (5) determine noise of measurements.

The sensor4600can be configured to include integrated calibration of lifetime measurements from a luminescent source. For example, the sensor4600can use the first light source4604ato transmit a signal(s) of a known temporal decay (lifetime) with a proper wavelength to not excite the oxygen sensing polymer in the target material4640. Accordingly, the excitation signal is substantially reflected by the target material4640, e.g., oxygen sensing polymer. By measuring the lifetime of the return optical signal, and because the light is reflected from the target material4640rather than exciting it, the measured lifetime can be calibrated so as to correspond to the known lifetime of the excitation signal. For example, this data can be acquired for a number of data points and a map of the measured lifetime as a function of known lifetime can be generated. Similarly, a map of the known lifetime as a function of measured lifetime can be generated. These maps can be used to determine transfer functions of lifetime measurements to account for potential biases in the detection system. These signals can also be used to determine dark noise interference and/or system non-linearity.

In some embodiments, the first light source4604ais used to verify satisfactory connection conditions and to provide calibration information prior to using the second light source4604b.For example, for each waveguide, the first light source4604acan provide excitation light having a wavelength that does not excite the target material. If a suitable or acceptable signal is seen in return, then the sensor4600can fire the second light source4604bto excite the target material (oxygen sensing polymer in some embodiments) and detect fluorescence decay lifetime to determine glucose concentrations. Thus, the second light source4604bcan be configured to be fired in a particular waveguide after the first light source4604aif the measured signal from the excitation provided by the first light source4604aindicates that proper operating conditions are present. In addition, the first and second light sources4604a,4604bcan be fired multiple times per waveguide per measurement to improve a signal to noise ratio of the response.

FIGS. 47A-47Cillustrate an example of optical routing of different optical signals in an example optical glucose sensor4700. The optical routing of sensor4700with sensor body4720includes directing light using excitation paths4730a,emission paths4730b,and transmission paths to deliver excitation light4721to a target4740and to deliver emission light4723from the target4740. As described elsewhere herein, excitation light4721can be delivered to the target material4740using a combination of an excitation path4730aand a transmission path of a waveguide4730. Similarly, emission light4723can be delivered from the target material4740to the sensor optical interface for measurements. As depicted inFIG. 47C, the sizes of the excitation path4730aand emission path4730bin the waveguide4730can be configured to change along the optical axis of the waveguide4730so that a majority of emission light4723enters the emission path4730band/or to provide a relatively large target for excitation light4321from the sensor optical interface to enter the excitation path4730a.At a point where the transmission path4730branches into the excitation path4730aand emission path4730b(branching point4333), the width of the emission path4730bcan be greater than the width of the excitation path such that a majority of the emission light4723enters the emission path4730b.Similarly, at a point at an end of the emission path4730band at a beginning of the excitation path4730a,the width of the excitation path can be greater than the width of the excitation path such that a majority of the excitation light4721enters the excitement path4730a.

Example Signals in an Optical Glucose Sensor

FIGS. 48A and 48Billustrate examples of signals in an optical glucose sensor, the signals used to verify proper optical connections, to calibrate the sensor, and to measure glucose concentrations. The lifetime (temporal decay) obtained from the emission of the oxygen sensing polymer is quantitatively correlated with the oxygen partial pressure in the oxygen sensing polymer. For example, the relationship of lifetime to oxygen concentration in the oxygen sensing polymer follows the Stern Volmer equation.

The oxygen measurement is based on the luminescence lifetime of an oxygen-sensitive luminescent dye in the oxygen sensing polymer or target material. The lifetime expresses the amount of time the luminescent dye (or luminophore) remains in an excited state following excitation by light of a suitable frequency. To measure the lifetime, a time-domain approach is used in which the target material is excited with a pulse of light and then the time-dependent intensity is measured. The lifetime is calculated from the slope of the log of intensity versus time. The target material is first illuminated with an optical signal at a wavelength that does not excite the luminescent dye but with a known lifetime decay to calibrate the transmitter and optical system before each glucose measurement is made. The light is reflected by the dye instead of inducing a luminescent signal. Accordingly, a transfer function, F1(λ), can be determined that maps a measured lifetime, λ′, to a known lifetime, λ. In addition, the pre-interrogation pulse ensures that proper optical connections have been maintained before each measurement. Once this transfer function is known, the target material can be interrogated with an optical signal that excites the luminescent dye and the fluorescence signal can be measured as a function of time. Using this measured signal, a lifetime can be determined, λ*, and mapped to a fluorescence lifetime, kc, of the target material using the transfer function, F1(λ), determined using the first light source.

As previously discussed, the red signal light source can be a low-intensity light source of a red wavelength. The blue signal light source can be a higher intensity class 3 source of a blue wavelength. The excitation light is guided to a red luminescent dye, in some implementations. The red dye can be configured to have a high quantum efficiency for converting the blue excitation into a red emission with a lifetime decay signal. The red luminescent dye does not have a high quantum efficiency for converting the red excitation source into an emission with a lifetime decay signal, but reflects some of the red excitation light as a return emission.

In some embodiments, the red signal is provided for a tailored period and modulated (with a desired amplitude signal characteristic), while the blue source will be pulsed. The return signal can be detected by the same emitter as a higher power blue light source. When the low power red light source is detected with appropriate signal characteristics, this indicates that it is safe to energize the higher intensity light source.

The return signal from the red source can be detected by the same emitter as for a higher power blue source. The red source signal can be modulated with a known lifetime decay. When the low power red light source is detected it will have a measured lifetime decay. This known versus measured signal will allow the sensor to be calibrated for lifetime decay when appropriate. This method allows individual channels to be assessed for quality, operation, and calibrated for lifetime decay, when the channel is excited by the dual source approach.

In certain implementations, the blue signal can be a modulated light that is similar to a digital signal that is turned on and off intermittently. In various implementations, the blue signal can be a sinusoidal signal used in a phase-based method to determine lifetime. To create the red light decay signal for calibration purposes, a digital method may be used to decrease the amplitude of the source signal at specified times from a digital source.

As described herein, the sensor can be configured to have a dual source configuration for each waveguide to provide fault or integrity checks for each channel of the sensor. The sampling of the excitation with the emission response may be repeated multiple times for each channel to improve the signal to noise of the response. After the one or more series of measurements are made, the sensor system can be configured to pause until a subsequent measurement cycle begins (e.g., 30 sec later, 1 min later, 5 min later, etc.).

The foregoing disclosure provides for embodiments of optical analyte sensors with innovative features. These optical analyte sensors are generally described in the context of glucose measurements. However, it should be understood that features of the disclosed sensors can be applicable to other analyte measurements. Moreover, while several components, techniques and aspects have been described with a certain degree of particularity, it is manifest that many changes can be made in the specific designs, constructions and methodology herein above described without departing from the spirit and scope of this disclosure.

It is to be understood that the embodiments of the invention described herein are not limited to particular variations set forth herein as various changes or modifications may be made to the embodiments of the invention described and equivalents may be substituted without departing from the spirit and scope of the embodiments of the invention. As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features that may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the embodiments of the present invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process act(s) or step(s) to the objective(s), spirit or scope of the embodiments of the present invention. All such modifications are intended to be within the scope of the claims made herein.

Language of degree used herein, such as the terms “approximately,” “about,” “generally,” and “substantially” as used herein represent a value, amount, or characteristic close to the stated value, amount, or characteristic that still performs a desired function or achieves a desired result. For example, the terms “approximately”, “about”, “generally,” and “substantially” may refer to an amount that is within less than or equal to 10% of, within less than or equal to 5% of, within less than or equal to 1% of, within less than or equal to 0.1% of, and within less than or equal to 0.01% of the stated amount. If the stated amount is 0 (e.g., none, having no), the above recited ranges can be specific ranges, and not within a particular % of the value. Additionally, numeric ranges are inclusive of the numbers defining the range, and any individual value provided herein can serve as an endpoint for a range that includes other individual values provided herein. For example, a set of values such as 1, 2, 3, 8, 9, and 10 is also a disclosure of a range of numbers from 1-10, from 1-8, from 3-9, and so forth.

While a number of embodiments and variations thereof have been described in detail, other modifications and methods of using the same will be apparent to those of skill in the art. Accordingly, it should be understood that various applications, modifications, materials, and substitutions can be made of equivalents without departing from the unique and inventive disclosure herein or the scope of the claims.