Continuous Potassium Sensors and Methods of Use Thereof

The present disclosure provides an analyte sensor for use in detecting potassium. In certain embodiments, an analyte sensor of the present disclosure includes at least two asparagine-responsive active areas, where each asparagine-responsive active area includes an asparaginase that exhibits a particular potassium dependency. In certain embodiments, an analyte sensor of the present disclosure includes at least two aspartate-responsive active areas, where each aspartate-responsive active area includes an aspartate oxidase that exhibits a particular potassium dependency. The present disclosure further provides methods for monitoring potassium levels, e.g., in vivo potassium levels, using the disclosed analyte sensors.

FIELD

The subject matter described herein relates to analyte sensors for sensing potassium ions and methods of using the same.

BACKGROUND

The detection of various analytes within an individual can sometimes be vital for monitoring the condition of their health as deviations from normal analyte levels can be indicative of a physiological condition. For example, monitoring glucose levels can enable people suffering from diabetes to take appropriate corrective action including administration of medicine or consumption of particular food or beverage products to avoid significant physiological harm. Other analytes such as potassium can be desirable to monitor for certain physiological conditions. In certain instances, it can be desirable to monitor more than one analyte to monitor single or multiple physiological conditions, particularly if a person is suffering from comorbid conditions that result in simultaneous dysregulation of two or more analytes in combination with one another.

Analyte monitoring in an individual can take place periodically or continuously over a period of time. Periodic analyte monitoring can take place by withdrawing a sample of bodily fluid, such as blood or urine, at set time intervals and analyzing ex vivo. Periodic, ex vivo analyte monitoring can be sufficient to determine the physiological condition of many individuals. However, ex vivo analyte monitoring can be inconvenient or painful in some instances. Moreover, there is no way to recover lost data if an analyte measurement is not obtained at an appropriate time. Continuous analyte monitoring can be conducted using one or more sensors that remain at least partially implanted within a tissue of an individual, such as dermally, subcutaneously or intravenously, so that analyses can be conducted in vivo. Implanted sensors can collect analyte data on-demand, at a set schedule, or continuously, depending on an individual's particular health needs and/or previously measured analyte levels. Analyte monitoring with an in vivo implanted sensor can be a more desirable approach for individuals having severe analyte dysregulation and/or rapidly fluctuating analyte levels, although it can also be beneficial for other individuals as well. Since implanted analyte sensors often remain within a tissue of an individual for an extended period of time, it can be highly desirable for such analyte sensors to be made from stable materials exhibiting a high degree of biocompatibility.

Many analytes represent intriguing targets for physiological analyses, provided that a suitable detection chemistry can be identified. To this end, enzyme-based amperometric sensors configured for assaying glucose continuously in vivo have been developed and refined over recent years to aid in monitoring the health of diabetic individuals. Other analytes commonly subject to concurrent dysregulation with glucose in diabetic individuals include, for example, potassium. It can also be desirable to monitor potassium independent of glucose dysregulation as well. For example, potassium levels can be important to monitor in people suffering various kidney or heart diseases. Implanted analyte sensors configured for detecting potassium in vivo are not currently available. Accordingly, there is a need in the art for sensors for detecting potassium in vivo.

SUMMARY

The purpose and advantages of the disclosed subject matter will be set forth in and are apparent from the description that follows, as well as will be learned by practice of the disclosed subject matter. Additional advantages of the disclosed subject matter will be realized and attained by the devices particularly pointed out in the written description and claims hereof, as well as from the appended drawings.

To achieve these and other advantages and in accordance with the purpose of the disclosed subject matter, as embodied and broadly described, the disclosed subject matter relates an analyte sensor for monitoring potassium levels in vivo.

In certain embodiments, the analyte sensor includes a sensor tail comprising at least a first working electrode and a second working electrode. In certain embodiments, the analyte sensor further includes a first analyte-responsive active area disposed upon a surface of the first working electrode, where the first analyte-responsive active area comprises a first aspartate oxidase, and a second analyte-responsive active area disposed upon a surface of the second working electrode, where the second analyte-responsive active area comprises a second aspartate oxidase. In certain embodiments, the analyte sensor further includes a first mass transport limiting membrane permeable to potassium that overcoats the first analyte-responsive active area and/or the second analyte-responsive area. In certain embodiments, the first aspartate oxidase and the second aspartate oxidase have different potassium dependencies. For example, but not by way of limitation, the first aspartate oxidase is potassium-independent and the second aspartate oxidase is potassium-dependent.

In certain embodiments, the first analyte-responsive active area further comprises a first asparaginase and the second analyte-responsive active area further comprises a second asparaginase. In certain embodiments, the first asparaginase and the second asparaginase have different potassium dependencies. For example, but not by way of limitation, the first asparaginase is potassium-independent and the second asparaginase is potassium-dependent. In certain embodiments, the first aspartate oxidase and the second aspartate oxidase are both potassium-independent. In certain embodiments, the first analyte-responsive active area comprises a first enzymatic layer comprising the first aspartate oxidase and a second enzymatic layer comprising the first asparaginase disposed upon the first enzymatic layer. In certain embodiments, the second analyte-responsive active area comprises a first enzymatic layer comprising the second aspartate oxidase and a second enzymatic layer comprising the second asparaginase disposed upon the first enzymatic layer. In certain embodiments, the first analyte-responsive active area comprises a first enzymatic layer comprising the first aspartate oxidase and the first asparaginase. In certain embodiments, the second analyte-responsive active area comprises a first enzymatic layer comprising the aspartate oxidase and the second asparaginase.

In certain embodiments, the first analyte-responsive active area and/or the second analyte-responsive active area further comprises an electron transfer agent and/or a stabilizing agent. In certain embodiments, the first mass transport limiting membrane comprises a polyvinylpyridine-based polymer, a polyvinylimidazole, a polyacrylate, a polyurethane, a polyether urethane, a silicone or a combination thereof.

In certain embodiments, the analyte sensor includes a sensor tail comprising at least a first working electrode and a second working electrode. In certain embodiments, a first aspartate-responsive active area is disposed upon a surface of the first working electrode and comprises a potassium-dependent aspartate oxidase. In certain embodiments, a second aspartate-responsive active area is disposed upon a surface of the second working electrode and comprises a potassium-independent aspartate oxidase. In certain embodiments, a first mass transport limiting membrane permeable to aspartate and potassium overcoats the first aspartate-responsive active area and/or the second aspartate-responsive active area. In certain embodiments, the first and/or second aspartate-responsive active area further comprises an electron-transfer agent. For example, but not by way of limitation, the first aspartate-responsive active area includes a first electron transfer agent and the second aspartate-responsive active area includes a second electron transfer agent. In certain embodiments, the potassium-dependent aspartate oxidase and/or the first electron transfer agent are covalently bonded to a polymer in the first aspartate-responsive active area. In certain embodiments, the potassium-independent aspartate oxidase and/or the second electron transfer agent are covalently bonded to a polymer in the second aspartate-responsive active area. In certain embodiments, the first and/or second aspartate-responsive active area further comprises a stabilizer, e.g., a serum albumin. In certain embodiments, the first mass transport limiting membrane comprises a polyvinylpyridine-based polymer, a polyvinylimidazole, a polyacrylate, a polyurethane, a polyether urethane, a silicone or a combination thereof.

In certain embodiments, the analyte sensor includes a sensor tail comprising at least a first working electrode and a second working electrode. In certain embodiments, the analyte sensor further includes a first asparagine-responsive active area disposed upon a surface of the first working electrode, where the first asparagine-responsive active area comprises a first enzyme system comprising a first aspartate oxidase and a first asparaginase, and a second asparagine-responsive active area disposed upon a surface of the second working electrode, where the second asparagine-responsive active area comprises a second enzyme system comprising a second aspartate oxidase and a second asparaginase. In certain embodiments, the analyte sensor further includes a first mass transport limiting membrane permeable to asparagine that overcoats the first asparagine-responsive active area and/or the second asparagine-responsive area. In certain embodiments, the first aspartate oxidase and the second aspartate oxidase are potassium-independent. In certain embodiments, the first asparaginase and the second asparaginase have different potassium dependencies. For example, but not byway of limitation, the first asparaginase is potassium-independent and the second asparaginase is potassium-dependent.

In certain embodiments, the first asparagine-responsive active area and/or the second asparagine-responsive active area further comprises an electron-transfer agent. In certain embodiments, one or more of the enzymes in the first enzyme system are covalently bonded to a polymer in the first asparagine-responsive active area and/or one or more of the enzymes in the second enzyme system are covalently bonded to a polymer in the second asparagine-responsive area. In certain embodiments, the first mass transport limiting membrane comprises a polyvinylpyridine-based polymer, a polyvinylimidazole, a polyacrylate, a polyurethane, a polyether urethane, a silicone or a combination thereof.

In certain embodiments, the first asparagine-responsive active area includes a first enzyme layer comprising the aspartate oxidase and a second layer comprising the first asparaginase disposed upon the first enzyme layer. In certain embodiments, the first asparagine-responsive active area includes an enzyme layer comprising the aspartate oxidase and the first asparaginase. Alternatively or additionally, the second asparagine-responsive active area includes a first enzyme layer comprising the aspartate oxidase and a second layer comprising the second asparaginase disposed upon the first enzyme layer. In certain embodiments, the second asparagine-responsive active area includes an enzyme layer comprising the aspartate oxidase and the second asparaginase.

In certain embodiments, an analyte sensor of the present disclosure includes a third working electrode and an active area disposed upon a surface of the third working electrode and responsive to a second analyte differing from potassium, wherein the active area comprises at least one enzyme responsive to the second analyte. In certain embodiments, the second analyte is glutamate, glucose, ketones, lactate, oxygen, hemoglobin AIC, albumin, alcohol, alkaline phosphatase, alanine transaminase, aspartate aminotransferase, bilirubin, blood urea nitrogen, calcium, carbon dioxide, chloride, creatinine, hematocrit, lactate, magnesium, oxygen, pH, phosphorus, potassium, sodium, aspartate, asparagine, total protein and/or uric acid. In certain embodiments, a second portion of the mass transport limiting membrane or a second mass transport limiting membrane overcoats the active area on the third working electrode. Alternatively, a second mass transport limiting membrane overcoats the active area on the third working electrode and/or overcoats the active area on the third working electrode and one of more of the asparagine-responsive active areas.

The present disclosure further provides methods for detecting potassium, e.g., potassium ions, in a fluid, e.g., biological fluid. In certain embodiments, any analyte sensor of the present disclosure can be used for detecting potassium, e.g., potassium ions, in a fluid, e.g., biological fluid of a subject. In certain embodiments, the analyte sensor is implanted in a subject at risk of or having a neurological condition or diabetes. In certain embodiments, the analyte sensor is implanted in a subject for about 15 days.

In certain embodiments, the method includes providing an analyte sensor, where the analyte sensor includes a sensor tail comprising at least a first working electrode and a second working electrode. In certain embodiments, the analyte sensor further includes a first analyte-responsive active area disposed upon a surface of the first working electrode, where the first analyte-responsive active area comprises a first aspartate oxidase, and a second analyte-responsive active area disposed upon a surface of the second working electrode, where the second analyte-responsive active area comprises a second aspartate oxidase. In certain embodiments, the analyte sensor further includes a first mass transport limiting membrane permeable to potassium that overcoats the first analyte-responsive active area and/or the second analyte-responsive area. In certain embodiments, the first aspartate oxidase and the second aspartate oxidase have different potassium dependencies. For example, but not by way of limitation, the first aspartate oxidase is potassium-independent and the second aspartate oxidase is potassium-dependent. In certain embodiments, the first analyte-responsive active area further comprises a first asparaginase and the second analyte-responsive active area further comprises a second asparaginase. In certain embodiments, the first asparaginase and the second asparaginase have different potassium dependencies. For example, but not by way of limitation, the first asparaginase is potassium-independent and the second asparaginase is potassium-dependent. In certain embodiments, the method further includes applying a potential to the first working electrode and the second working electrode, obtaining a first signal at or above an oxidation-reduction potential of the first analyte-responsive active area; obtaining a second signal at or above an oxidation-reduction potential of the second analyte-responsive active and correlating the first signal and the second signal to the concentration of potassium in the fluid.

In certain embodiments, the method can include providing an analyte sensor that includes a sensor tail comprising at least a first working electrode and a second working electrode; a first aspartate-responsive active area disposed upon a surface of the first working electrode comprising a potassium-dependent aspartate oxidase; a second aspartate-responsive active area disposed upon a surface of the second working electrode comprising a potassium-independent aspartate oxidase; and a first mass transport limiting membrane permeable to aspartate and potassium that overcoats the first aspartate-responsive active area and/or the second aspartate-responsive active area. In certain embodiments, the method further includes applying a potential to the first working electrode and the second working electrode, obtaining a first signal at or above an oxidation-reduction potential of the first aspartate-responsive active area; obtaining a second signal at or above an oxidation-reduction potential of the second aspartate-responsive active and correlating the first signal and the second signal to the concentration of potassium in the fluid.

In certain embodiments, the method includes providing an analyte sensor, where the analyte sensor includes a sensor tail comprising at least a first working electrode and a second working electrode. In certain embodiments, the analyte sensor further includes a first asparagine-responsive active area disposed upon a surface of the first working electrode, where the first asparagine-responsive active area includes a first enzyme system comprising an aspartate oxidase and a first asparaginase, and a second asparagine-responsive active area disposed upon a surface of the second working electrode, where the second asparagine-responsive active area includes a second enzyme system comprising an aspartate oxidase and a second asparaginase. In certain embodiments, the first aspartate oxidase and the second aspartate oxidase are potassium-independent. In certain embodiments, the first asparaginase and the second asparaginase have different potassium dependencies. For example, but not byway of limitation, the first asparaginase is potassium-independent and the second asparaginase is potassium-dependent. In certain embodiments, the analyte sensor further includes a first mass transport limiting membrane permeable to asparagine that overcoats the first asparagine-responsive active area and/or the second asparagine-responsive area. In certain embodiments, the method further includes applying a potential to the first working electrode and the second working electrode. In certain embodiments, the method further includes obtaining a first signal at or above an oxidation-reduction potential of the first asparagine-responsive active area. In certain embodiments, the method further includes obtaining a second signal at or above an oxidation-reduction potential of the second asparagine-responsive active area. In certain embodiments, the method further includes correlating the first signal and the second to the concentration of potassium ions in the fluid.

In certain embodiments, the first asparagine-responsive active area and the second asparagine-responsive active area of the analyte sensor for use in the disclosed methods further comprises an electron-transfer agent. In certain embodiments, one or more of the enzymes in the first enzyme system are covalently bonded to a polymer in the first asparagine-responsive active area and/or wherein one or more of the enzymes in the second enzyme system are covalently bonded to a polymer in the second asparagine-responsive area. In certain embodiments, the first mass transport limiting membrane comprises a polyvinylpyridine-based polymer, a polyvinylimidazole, a polyacrylate, a polyurethane, a polyether urethane, a silicone or a combination thereof.

In certain embodiments, the first asparagine-responsive active area of the analyte sensor for use in the disclosed methods comprises a first enzyme layer comprising the aspartate oxidase and a second layer comprising the first asparaginase disposed upon the first enzyme layer. In certain embodiments, the first asparagine-responsive active area includes an enzyme layer comprising the aspartate oxidase and the first asparaginase. In certain embodiments, the second asparagine-responsive active area of the analyte sensor for use in the disclosed methods comprises a first enzyme layer comprising the aspartate oxidase and a second layer comprising the second asparaginase disposed upon the first enzyme layer. In certain embodiments, the second asparagine-responsive active area includes an enzyme layer comprising the aspartate oxidase and the second asparaginase.

DETAILED DESCRIPTION

The present disclosure generally describes analyte sensors employing an enzyme, e.g., one or more enzymes for the detection of an analyte. For example, but not by way of limitation, the present disclosure provides analyte sensors employing one or more enzymes for the detection of potassium. In certain embodiments, the present disclosure provides multiple enzymes for detection of an analyte, e.g., potassium. The present disclosure further provides methods of detecting one or more analytes, e.g., potassium, using the disclosed analyte sensors to monitor a physiological condition.

The present disclosure provides sensor chemistries suitable for monitoring potassium levels over a range of physiologically relevant potassium concentrations. In certain embodiments, the present disclosure provides sensor chemistries utilizing an enzyme to monitor potassium concentrations, e.g., potassium ion concentrations, in a sample. In certain embodiments, the present disclosure provides sensor chemistries utilizing enzyme systems including at least two enzymes that are capable of acting in concert to monitor potassium concentrations, e.g., potassium ion concentrations, in a sample. In certain embodiments, two enzyme systems can be used to facilitate indirect measurement of a single analyte such as potassium. As used herein, the term “in concert” refers to a coupled enzymatic reaction, in which a product of a first enzymatic reaction becomes a substrate for a second enzymatic reaction, and the second enzymatic reaction serves as the basis for measuring the concentration of the substrate (e.g., analyte) reacted during the first enzymatic reaction. In certain embodiments, the product and/or substrate of a reaction can be the reduced and/or oxidized form of a cofactor or coenzyme of an enzyme of the enzyme system, e.g., FAD or NAD. Although defined in terms of two coupled enzymatic reactions, it is to be appreciated that more than two enzymatic reactions can be coupled as well in some instances. For example, a product of a first enzymatic reaction can become a substrate of a second enzymatic reaction, and a product of the second enzymatic reaction can become a substrate for a third enzymatic reaction, with the third enzymatic reaction serving as the basis for measuring the concentration of the substrate (e.g., analyte) reacted during the first enzymatic reaction. Discussion of suitable enzyme systems for detecting, e.g., indirectly detecting, potassium according to the disclosure herein follows below.

For clarity, but not by way of limitation, the detailed description of the presently disclosed subject matter is divided into the following subsections:I. Definitions; andII. Analyte Sensors;1. General Structure of Analyte Sensor Systems;2. Enzymes;3. Redox Mediators;4. Polymeric Backbone;5. Mass Transport Limiting Membrane;6. Interference Domain; and7. Manufacturing;III. Methods of Use; andIV. Exemplary Embodiments.

The terms used in this specification generally have their ordinary meanings in the art, within the context of this disclosure and in the specific context where each term is used. Certain terms are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner in describing the compositions and methods of the present disclosure and how to make and use them.

The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms or words that do not preclude additional acts or structures. The present disclosure also contemplates other embodiments “comprising,” “consisting of” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.

As used herein, “analyte sensor” or “sensor” can refer to any device capable of receiving sensor information from a user, including for purpose of illustration but not limited to, body temperature sensors, blood pressure sensors, pulse or heart-rate sensors, glucose level sensors, analyte sensors, physical activity sensors, body movement sensors, or any other sensors for collecting physical or biological information. Analytes measured by the analyte sensors can include, by way of example and not limitation, glutamate, glucose, ketones, lactate, oxygen, hemoglobin A1C, albumin, alcohol, alkaline phosphatase, alanine transaminase, aspartate aminotransferase, bilirubin, blood urea nitrogen, calcium, carbon dioxide, chloride, creatinine, hematocrit, lactate, magnesium, oxygen, pH, phosphorus, potassium, asparagine, aspartate, sodium, total protein, uric acid, etc.

The term “biological fluid,” as used herein, refers to any bodily fluid or bodily fluid derivative in which the analyte can be measured. Non-limiting examples of a biological fluid include dermal fluid, interstitial fluid, plasma, blood, lymph, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage, amniotic fluid, sweat, tears, or the like. In certain embodiments, the biological fluid is dermal fluid or interstitial fluid.

The term “electrolysis,” as used herein, refers to electrooxidation or electroreduction of a compound either directly at an electrode or via one or more electron transfer agents (e.g., redox mediators or enzymes).

As used herein, the term “homogenous membrane” refers to a membrane including a single type of membrane polymer.

As used herein, the term “multi-component membrane” refers to a membrane including two or more types of membrane polymers.

As used herein, the term “potassium-independent aspartate oxidase” refers to an aspartate oxidase that does not exhibit any change in catalytic activity in the presence of potassium, e.g., potassium ions (K+).

As used herein, the term “potassium-dependent aspartate oxidase” refers to an aspartate oxidase that exhibits increased or decreased catalytic activity in the presence of potassium, e.g., potassium ions (K+). In certain embodiments, potassium-dependent aspartate oxidases include aspartate oxidases that require different concentrations of potassium, e.g., potassium ions (K+), for maximum catalytic activity.

As used herein, the term “potassium-independent asparaginase” refers to an asparaginase that does not exhibit any change in catalytic activity in the presence of potassium, e.g., potassium ions (K+).

As used herein, the term “potassium-dependent asparaginase” refers to an asparaginase that exhibits increased or decreased catalytic activity in the presence of potassium, e.g., potassium ions (K+). In certain embodiments, potassium-dependent asparaginases include asparaginases that require different concentrations of potassium, e.g., potassium ions (K+), for maximum catalytic activity.

As used herein, the term “polyvinylpyridine-based polymer” refers to a polymer or copolymer that comprises polyvinylpyridine (e.g., poly(2-vinylpyridine) or poly(4-vinylpyridine)) or a derivative thereof.

As used herein, the term “redox mediator” refers to an electron transfer agent for carrying electrons between an analyte or an analyte-reduced or analyte oxidized enzyme and an electrode, either directly, or via one or more additional electron transfer agents. In certain embodiments, redox mediators that include a polymeric backbone can also be referred to as “redox polymers.”

The term “reference electrode” as used herein, can refer to either reference electrodes or electrodes that function as both, a reference and a counter electrode. Similarly, the term “counter electrode,” as used herein, can refer to both, a counter electrode and a counter electrode that also functions as a reference electrode.

As used herein, the term “single-component membrane” refers to a membrane including one type of membrane polymer.

1. General Structure of Analyte Sensor Systems

Before the present subject matter is described in detail, it is to be understood that this disclosure is not limited to the particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

Generally, embodiments of the present disclosure include systems, devices and methods for the use of analyte sensor insertion applicators for use with in vivo analyte monitoring systems. An applicator can be provided to the user in a sterile package with an electronics housing of the sensor control device contained therein. According to some embodiments, a structure separate from the applicator, such as a container, can also be provided to the user as a sterile package with a sensor module and a sharp module contained therein. The user can couple the sensor module to the electronics housing, and can couple the sharp to the applicator with an assembly process that involves the insertion of the applicator into the container in a specified manner. In other embodiments, the applicator, sensor control device, sensor module, and sharp module can be provided in a single package. The applicator can be used to position the sensor control device on a human body with a sensor in contact with the wearer's bodily fluid. The embodiments provided herein are improvements to reduce the likelihood that a sensor is improperly inserted or damaged, or elicits an adverse physiological response. Other improvements and advantages are provided as well. The various configurations of these devices are described in detail by way of the embodiments which are only examples.

Furthermore, many embodiments include in vivo analyte sensors structurally configured so that at least a portion of the sensor is, or can be, positioned in the body of a user to obtain information about at least one analyte of the body. It should be noted, however, that the embodiments disclosed herein can be used with in vivo analyte monitoring systems that incorporate in vitro capability, as well as purely in vitro or ex vivo analyte monitoring systems, including systems that are entirely non-invasive.

Furthermore, for each and every embodiment of a method disclosed herein, systems and devices capable of performing each of those embodiments are covered within the scope of the present disclosure. For example, embodiments of sensor control devices are disclosed and these devices can have one or more sensors, analyte monitoring circuits (e.g., an analog circuit), memories (e.g., for storing instructions), power sources, communication circuits, transmitters, receivers, processors and/or controllers (e.g., for executing instructions) that can perform any and all method steps or facilitate the execution of any and all method steps. These sensor control device embodiments can be used and can be capable of use to implement those steps performed by a sensor control device from any and all of the methods described herein.

Furthermore, the systems and methods presented herein can be used for operations of a sensor used in an analyte monitoring system, such as but not limited to wellness, fitness, dietary, research, information or any purposes involving analyte sensing over time. As used herein, “analyte sensor” or “sensor” can refer to any device capable of receiving sensor information from a user, including for purpose of illustration but not limited to, body temperature sensors, blood pressure sensors, pulse or heart-rate sensors, glucose level sensors, analyte sensors, physical activity sensors, body movement sensors, or any other sensors for collecting physical or biological information. In certain embodiments, an analyte sensor of the present disclosure can further measure analytes including, but not limited to, glucose, ketones, lactate, oxygen, hemoglobin AIC, albumin, alcohol, alkaline phosphatase, alanine transaminase, aspartate aminotransferase, bilirubin, blood urea nitrogen, calcium, carbon dioxide, chloride, creatinine, hematocrit, lactate, magnesium, oxygen, pH, phosphorus, potassium, sodium, aspartate, asparagine, total protein, uric acid, etc.

As mentioned, a number of embodiments of systems, devices, and methods are described herein that provide for the improved assembly and use of dermal sensor insertion devices for use with in vivo analyte monitoring systems. In particular, several embodiments of the present disclosure are designed to improve the method of sensor insertion with respect to in vivo analyte monitoring systems and, in particular, to prevent the premature retraction of an insertion sharp during a sensor insertion process. Some embodiments, for example, include a dermal sensor insertion mechanism with an increased firing velocity and a delayed sharp retraction. In other embodiments, the sharp retraction mechanism can be motion-actuated such that the sharp is not retracted until the user pulls the applicator away from the skin. Consequently, these embodiments can reduce the likelihood of prematurely withdrawing an insertion sharp during a sensor insertion process; decrease the likelihood of improper sensor insertion; and decrease the likelihood of damaging a sensor during the sensor insertion process, to name a few advantages. Several embodiments of the present disclosure also provide for improved insertion sharp modules to account for the small scale of dermal sensors and the relatively shallow insertion path present in a subject's dermal layer. In addition, several embodiments of the present disclosure are designed to prevent undesirable axial and/or rotational movement of applicator components during sensor insertion. Accordingly, these embodiments can reduce the likelihood of instability of a positioned dermal sensor, irritation at the insertion site, damage to surrounding tissue, and breakage of capillary blood vessels resulting in fouling of the dermal fluid with blood, to name a few advantages. In addition, to mitigate inaccurate sensor readings which can be caused by trauma at the insertion site, several embodiments of the present disclosure can reduce the end-depth penetration of the needle relative to the sensor tip during insertion.

Before describing these aspects of the embodiments in detail, however, it is first desirable to describe examples of devices that can be present within, for example, an in vivo analyte monitoring system, as well as examples of their operation, all of which can be used with the embodiments described herein.

There are various types of in vivo analyte monitoring systems. “Continuous Analyte Monitoring” systems (or “Continuous Glucose Monitoring” systems), for example, can transmit data from a sensor control device to a reader device continuously without prompting, e.g., automatically according to a schedule. “Flash Analyte Monitoring” systems (or “Flash Glucose Monitoring” systems or simply “Flash” systems), as another example, can transfer data from a sensor control device in response to a scan or request for data by a reader device, such as with a Near Field Communication (NFC) or Radio Frequency Identification (RFID) protocol. In vivo analyte monitoring systems can also operate without the need for finger stick calibration.

In vivo analyte monitoring systems can be differentiated from “in vitro” systems that contact a biological sample outside of the body (or “ex vivo”) and that typically include a meter device that has a port for receiving an analyte test strip carrying bodily fluid of the user, which can be analyzed to determine the user's blood analyte level.

In vivo monitoring systems can include a sensor that, while positioned in vivo, makes contact with the bodily fluid of the user and senses the analyte levels contained therein. The sensor can be part of the sensor control device that resides on the body of the user and contains the electronics and power supply that enable and control the analyte sensing. The sensor control device, and variations thereof, can also be referred to as a “sensor control unit,” an “on-body electronics” device or unit, an “on-body” device or unit, or a “sensor data communication” device or unit, to name a few.

In vivo monitoring systems can also include a device that receives sensed analyte data from the sensor control device and processes and/or displays that sensed analyte data, in any number of forms, to the user. This device, and variations thereof, can be referred to as a “handheld reader device,” “reader device” (or simply a “reader”), “handheld electronics” (or simply a “handheld”), a “portable data processing” device or unit, a “data receiver,” a “receiver” device or unit (or simply a “receiver”), or a “remote” device or unit, to name a few. Other devices such as personal computers have also been utilized with or incorporated into in vivo and in vitro monitoring systems.

Sensor104is adapted to be at least partially inserted into a tissue of interest, such as within the dermal or subcutaneous layer of the skin. Sensor104can include a sensor tail of sufficient length for insertion to a desired depth in a given tissue. The sensor tail can include at least one working electrode. In certain configurations, the sensor tail can include at least one active area for detecting an analyte disposed upon the working electrode. A counter electrode can be present in combination with the at least one working electrode. Particular electrode configurations upon the sensor tail are described in more detail below.

The active area can be configured for monitoring a particular analyte, e.g., potassium, e.g., potassium ions. In certain embodiments, the active area can be configured for indirectly monitoring potassium levels in a sample by detecting asparagine. For example, but not by way of limitation, the active area can be configured for indirect measurement of potassium, e.g., potassium ions, by detecting asparagine using enzyme systems that include potassium-independent and/or potassium-dependent asparaginases. In certain embodiments, the active area can be configured for indirectly monitoring potassium levels in a sample by detecting aspartate. For example, but not by way of limitation, the active area can be configured for indirect measurement of potassium, e.g., potassium ions, by detecting aspartate using enzyme systems that include potassium-independent and/or potassium-dependent aspartate oxidases.

In certain embodiments, the active area can be configured for detecting two or more analytes. In certain embodiments, the active area can be configured for detecting a second analyte. In certain embodiments, the sensor tail can include at least two active areas, where one active area is configured to detect potassium and a second active area is configured to detect a second analyte. Non-limiting examples of second analytes can be glucose, ketones, lactate, oxygen, hemoglobin AIC, albumin, alcohol, alkaline phosphatase, alanine transaminase, aspartate aminotransferase, bilirubin, blood urea nitrogen, calcium, carbon dioxide, chloride, creatinine, hematocrit, lactate, magnesium, oxygen, pH, phosphorus, potassium, sodium, aspartate, asparagine, total protein, uric acid, etc.

In certain embodiments of the present disclosure, one or more analytes can be monitored in any biological fluid of interest such as dermal fluid, interstitial fluid, plasma, blood, lymph, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage, amniotic fluid, or the like. In certain particular embodiments, analyte sensors of the present disclosure can be adapted for assaying dermal fluid or interstitial fluid to determine a concentration of one or more analytes in vivo. In certain embodiments, the biological fluid is interstitial fluid.

An introducer can be present transiently to promote introduction of sensor104into a tissue. In certain illustrative embodiments, the introducer can include a needle or similar sharp. As would be readily recognized by a person skilled in the art, other types of introducers, such as sheaths or blades, can be present in alternative embodiments. More specifically, the needle or other introducer can transiently reside in proximity to sensor104prior to tissue insertion and then be withdrawn afterward. While present, the needle or other introducer can facilitate insertion of sensor104into a tissue by opening an access pathway for sensor104to follow. For example, and not by the way of limitation, the needle can facilitate penetration of the epidermis as an access pathway to the dermis to allow implantation of sensor104to take place, according to one or more embodiments. After opening the access pathway, the needle or other introducer can be withdrawn so that it does not represent a sharps hazard. In certain embodiments, suitable needles can be solid or hollow, beveled or non-beveled, and/or circular or non-circular in cross-section. In more particular embodiments, suitable needles can be comparable in cross-sectional diameter and/or tip design to an acupuncture needle, which can have a cross-sectional diameter of about 250 microns. However, suitable needles can have a larger or smaller cross-sectional diameter if needed for certain particular applications.

In certain embodiments, a tip of the needle (while present) can be angled over the terminus of sensor104, such that the needle penetrates a tissue first and opens an access pathway for sensor104. In certain embodiments, sensor104can reside within a lumen or groove of the needle, with the needle similarly opening an access pathway for sensor104. In either case, the needle is subsequently withdrawn after facilitating sensor insertion.

B. Exemplary Reader Device

FIG. 2Ais a block diagram depicting an example embodiment of a reader device configured as a smartphone. Here, reader device120can include a display122, input component121, and a processing core206including a communications processor222coupled with memory223and an applications processor224coupled with memory225. Also included can be separate memory230, RF transceiver228with antenna229, and power supply226with power management module238. Further included can be a multi-functional transceiver232which can communicate over Wi-Fi, NFC, Bluetooth, BTLE, and GPS with an antenna234. As understood by one of skill in the art, these components are electrically and communicatively coupled in a manner to make a functional device.

C. Exemplary Data Receiving Device Architecture

For purpose of illustration and not limitation, reference is made to the exemplary embodiment of a data receiving device120for use with the disclosed subject matter as shown inFIG. 2B. The data receiving device120, and the related multi-purpose data receiving device130, includes components germane to the discussion of the analyte sensor110and its operations and additional components can be included. In particular embodiments, the data receiving device120and multi-purpose data receiving device130can be or include components provided by a third party and are not necessarily restricted to include devices made by the same manufacturer as the sensor110.

As illustrated inFIG. 2B, the data receiving device120includes an ASIC4000including a microcontroller4010, memory4020, and storage4030and communicatively coupled with a communication module4040. Power for the components of the data receiving device120can be delivered by a power module4050, which as embodied herein can include a rechargeable battery. The data receiving device120can further include a display4070for facilitating review of analyte data received from an analyte sensor110or other device (e.g., user device140or remote application server150). The data receiving device120can include separate user interface components (e.g., physical keys, light sensors, microphones, etc.).

The communication module4040can include a BLE module4041and an NFC module4042. The data receiving device120can be configured to wirelessly couple with the analyte sensor110and transmit commands to and receive data from the analyte sensor110. As embodied herein, the data receiving device120can be configured to operate, with respect to the analyte sensor110as described herein, as an NFC scanner and a BLE end point via specific modules (e.g., BLE module4042or NFC module4043) of the communication module4040. For example, the data receiving device120can issue commands (e.g., activation commands for a data broadcast mode of the sensor; pairing commands to identify the data receiving device120) to the analyte sensor110using a first module of the communication module4040and receive data from and transmit data to the analyte sensor110using a second module of the communication module4040. The data receiving device120can be configured for communication with a user device140via a Universal Serial Bus (USB) module4045of the communication module4040.

As another example, the communication module4040can include, for example, a cellular radio module4044. The cellular radio module4044can include one or more radio transceivers for communicating using broadband cellular networks, including, but not limited to third generation (3G), fourth generation (4G), and fifth generation (5G) networks. Additionally, the communication module4040of the data receiving device120can include a Wi-Fi radio module4043for communication using a wireless local area network according to one or more of the IEEE 802.11 standards (e.g., 802.11a, 802.11b, 802.11g, 802.11n (aka Wi-Fi 4), 802.11ac (aka Wi-Fi 5), 802.1 lax (aka Wi-Fi 6)). Using the cellular radio module4044or Wi-Fi radio module4043, the data receiving device120can communicate with the remote application server150to receive analyte data or provide updates or input received from a user (e.g., through one or more user interfaces). Although not illustrated, the communication module5040of the analyte sensor120can similarly include a cellular radio module or Wi-Fi radio module.

As embodied herein, the on-board storage4030of the data receiving device120can store analyte data received from the analyte sensor110. Further, the data receiving device120, multi-purpose data receiving device130, or a user device140can be configured to communicate with a remote application server150via a wide area network. As embodied herein, the analyte sensor110can provide data to the data receiving device120or multi-purpose data receiving device130. The data receiving device120can transmit the data to the user computing device140. The user computing device140(or the multi-purpose data receiving device130) can in turn transmit that data to a remote application server150for processing and analysis.

As embodied herein, the data receiving device120can further include sensing hardware4060similar to, or expanded from, the sensing hardware5060of the analyte sensor110. In particular embodiments, the data receiving device120can be configured to operate in coordination with the analyte sensor110and based on analyte data received from the analyte sensor110. As an example, where the analyte sensor110glucose sensor, the data receiving device120can be or include an insulin pump or insulin injection pen. In coordination, the compatible device130can adjust an insulin dosage for a user based on glucose values received from the analyte sensor.

D. Exemplary Sensor Control Devices

FIGS. 2C and 2Dare block diagrams depicting example embodiments of sensor control device102having analyte sensor104and sensor electronics160(including analyte monitoring circuitry) that can have the majority of the processing capability for rendering end-result data suitable for display to the user. InFIG. 2C, a single semiconductor chip161is depicted that can be a custom application specific integrated circuit (ASIC). Shown within ASIC161are certain high-level functional units, including an analog front end (AFE)162, power management (or control) circuitry164, processor166, and communication circuitry168(which can be implemented as a transmitter, receiver, transceiver, passive circuit, or otherwise according to the communication protocol). In this embodiment, both AFE162and processor166are used as analyte monitoring circuitry, but in other embodiments either circuit can perform the analyte monitoring function. Processor166can include one or more processors, microprocessors, controllers, and/or microcontrollers, each of which can be a discrete chip or distributed amongst (and a portion of) a number of different chips.

A memory163is also included within ASIC161and can be shared by the various functional units present within ASIC161, or can be distributed amongst two or more of them. Memory163can also be a separate chip. Memory163can be volatile and/or non-volatile memory. In this embodiment, ASIC161is coupled with power source170, which can be a coin cell battery, or the like. AFE162interfaces with in vivo analyte sensor104and receives measurement data therefrom and outputs the data to processor166in digital form, which in turn processes the data to arrive at the end-result glucose discrete and trend values, etc. This data can then be provided to communication circuitry168for sending, by way of antenna171, to reader device120(not shown), for example, where minimal further processing is needed by the resident software application to display the data.

FIG. 2Dis similar toFIG. 2Cbut instead includes two discrete semiconductor chips162and174, which can be packaged together or separately. Here, AFE162is resident on ASIC161. Processor166is integrated with power management circuitry164and communication circuitry168on chip174. AFE162includes memory163and chip174includes memory165, which can be isolated or distributed within. In one example embodiment, AFE162is combined with power management circuitry164and processor166on one chip, while communication circuitry168is on a separate chip. In another example embodiment, both AFE162and communication circuitry168are on one chip, and processor166and power management circuitry164are on another chip. It should be noted that other chip combinations are possible, including three or more chips, each bearing responsibility for the separate functions described, or sharing one or more functions for fail-safe redundancy.

For purpose of illustration and not limitation, reference is made to the exemplary embodiment of an analyte sensor110for use with the disclosed subject matter as shown inFIG. 2E.FIG. 2Eillustrates a block diagram of an example analyte sensor110according to exemplary embodiments compatible with the security architecture and communication schemes described herein.

As embodied herein, the analyte sensor110can include an Application-Specific Integrated Circuit (“ASIC”)5000communicatively coupled with a communication module5040. The ASIC5000can include a microcontroller core5010, on-board memory5020, and storage memory5030. The storage memory5030can store data used in an authentication and encryption security architecture. The storage memory5030can store programming instructions for the sensor110. As embodied herein, certain communication chipsets can be embedded in the ASIC5000(e.g., an NFC transceiver5025). The ASIC5000can receive power from a power module5050, such as an on-board battery or from an NFC pulse. The storage memory5030of the ASIC5000can be programmed to include information such as an identifier for the sensor110for identification and tracking purposes. The storage memory5030can also be programmed with configuration or calibration parameters for use by the sensor110and its various components. The storage memory5030can include rewritable or one-time programming (OTP) memory. The storage memory5030can be updated using techniques described herein to extend the usefulness of the sensor110.

As embodied herein, the communication module5040of the sensor100can be or include one or more modules to support the analyte sensor110communicating with other devices of the analyte monitoring system100. As an example only and not by way of limitation, example communication modules5040can include a Bluetooth Low-Energy (“BLE”) module5041As used throughout this disclosure, Bluetooth Low Energy (“BLE”) refers to a short-range communication protocol optimized to make pairing of Bluetooth devices simple for end users. The communication module5040can transmit and receive data and commands via interaction with similarly-capable communication modules of a data receiving device120or user device140. The communication module5040can include additional or alternative chipsets for use with similar short-range communication schemes, such as a personal area network according to IEEE 802.15 protocols, IEEE 802.11 protocols, infrared communications according to the Infrared Data Association standards (IrDA), etc.

To perform its functionalities, the sensor100can further include suitable sensing hardware5060appropriate to its function. As embodied herein, the sensing hardware5060can include an analyte sensor transcutaneously or subcutaneously positioned in contact with a bodily fluid of a subject. The analyte sensor can generate sensor data containing values corresponding to levels of one or more analytes within the bodily fluid.

E. Exemplary Assembly Processes for Sensor Control Devices

The components of sensor control device102can be acquired by a user in multiple packages requiring final assembly by the user before delivery to an appropriate user location.FIGS. 3A-3Ddepict an example embodiment of an assembly process for sensor control device102by a user, including preparation of separate components before coupling the components in order to ready the sensor for delivery.FIGS. 3E-3Fdepict an example embodiment of delivery of sensor control device102to an appropriate user location by selecting the appropriate delivery location and applying device102to the location.

FIG. 3Ais a proximal perspective view depicting an example embodiment of a user preparing a container810, configured here as a tray (although other packages can be used), for an assembly process. The user can accomplish this preparation by removing lid812from tray810to expose platform808, for instance by peeling a non-adhered portion of lid812away from tray810such that adhered portions of lid812are removed. Removal of lid812can be appropriate in various embodiments so long as platform808is adequately exposed within tray810. Lid812can then be placed aside.

FIG. 3Bis a side view depicting an example embodiment of a user preparing an applicator device150for assembly. Applicator device150can be provided in a sterile package sealed by a cap708. Preparation of applicator device150can include uncoupling housing702from cap708to expose sheath704(FIG. 3C). This can be accomplished by unscrewing (or otherwise uncoupling) cap708from housing702. Cap708can then be placed aside.

FIG. 3Cis a proximal perspective view depicting an example embodiment of a user inserting an applicator device150into a tray810during an assembly. Initially, the user can insert sheath704into platform808inside tray810after aligning housing orienting feature1302(or slot or recess) and tray orienting feature924(an abutment or detent). Inserting sheath704into platform808temporarily unlocks sheath704relative to housing702and also temporarily unlocks platform808relative to tray810. At this stage, removal of applicator device150from tray810will result in the same state prior to initial insertion of applicator device150into tray810(i.e., the process can be reversed or aborted at this point and then repeated without consequence).

Sheath704can maintain position within platform808with respect to housing702while housing702is distally advanced, coupling with platform808to distally advance platform808with respect to tray810. This step unlocks and collapses platform808within tray810. Sheath704can contact and disengage locking features (not shown) within tray810that unlock sheath704with respect to housing702and prevent sheath704from moving (relatively) while housing702continues to distally advance platform808. At the end of advancement of housing702and platform808, sheath704is permanently unlocked relative to housing702. A sharp and sensor (not shown) within tray810can be coupled with an electronics housing (not shown) within housing702at the end of the distal advancement of housing702. Operation and interaction of the applicator device150and tray810are further described below.

FIG. 3Dis a proximal perspective view depicting an example embodiment of a user removing an applicator device150from a tray810during an assembly. A user can remove applicator150from tray810by proximally advancing housing702with respect to tray810or other motions having the same end effect of uncoupling applicator150and tray810. The applicator device150is removed with sensor control device102(not shown) fully assembled (sharp, sensor, electronics) therein and positioned for delivery.

FIG. 3Eis a proximal perspective view depicting an example embodiment of a patient applying sensor control device102using applicator device150to a target area of skin, for instance, on an abdomen or other appropriate location. Advancing housing702distally collapses sheath704within housing702and applies the sensor to the target location such that an adhesive layer on the bottom side of sensor control device102adheres to the skin. The sharp is automatically retracted when housing702is fully advanced, while the sensor (not shown) is left in position to measure analyte levels.

FIG. 3Fis a proximal perspective view depicting an example embodiment of a patient with sensor control device102in an applied position. The user can then remove applicator150from the application site.

System100, described with respect toFIGS. 3A-3Fand elsewhere herein, can provide a reduced or eliminated chance of accidental breakage, permanent deformation, or incorrect assembly of applicator components compared to prior art systems. Since applicator housing702directly engages platform808while sheath704unlocks, rather than indirect engagement via sheath704, relative angularity between sheath704and housing702will not result in breakage or permanent deformation of the arms or other components. The potential for relatively high forces (such as in conventional devices) during assembly will be reduced, which in turn reduces the chance of unsuccessful user assembly.

F. Exemplary Sensor Applicator Devices

FIG. 4Ais a side view depicting an example embodiment of an applicator device150coupled with screw cap708. This is an example of how applicator150is shipped to and received by a user, prior to assembly by the user with a sensor.FIG. 4Bis a side perspective view depicting applicator150and cap708after being decoupled.FIG. 4Cis a perspective view depicting an example embodiment of a distal end of an applicator device150with electronics housing706and adhesive patch105removed from the position they would have retained within sensor carrier710of sheath704, when cap708is in place.

Referring toFIG. 4D-Gfor purpose of illustration and not limitation, the applicator device20150can be provided to a user as a single integrated assembly.FIGS. 4D and 4Eprovide perspective top and bottom views, respectively, of the applicator device20150,FIG. 4Fprovides an exploded view of the applicator device20150andFIG. 4Gprovides a side cut-away view. The perspective views illustrate how applicator20150is shipped to and received by a user. The exploded and cut-away views illustrate the components of the applicator device20150. The applicator device20150can include a housing20702, gasket20701, sheath20704, sharp carrier201102, spring205612, sensor carrier20710(also referred to as a “puck carrier”), sharp hub205014, sensor control device (also referred to as a “puck”)20102, adhesive patch20105, desiccant20502, cap20708, serial label20709, and tamper evidence feature20712. As received by a user, only the housing20702, cap20708, tamper evidence feature20712, and label20709are visible. The tamper evidence feature20712can be, for example, a sticker coupled to each of the housing20702and the cap20708, and tamper evidence feature20712can be damaged, for example, irreparably, by uncoupling housing20702and cap20708, thereby indicating to a user that the housing20702and cap20708have been previously uncoupled. These features are described in greater detail below.

G. Exemplary Tray and Sensor Module Assembly

FIG. 5is a proximal perspective view depicting an example embodiment of a tray810with sterilization lid812removably coupled thereto, which may be representative of how the package is shipped to and received by a user prior to assembly.

FIG. 6Ais a proximal perspective cutaway view depicting sensor delivery components within tray810. Platform808is slidably coupled within tray810. Desiccant502is stationary with respect to tray810. Sensor module504is mounted within tray810.

FIG. 6Bis a proximal perspective view depicting sensor module504in greater detail. Here, retention arm extensions1834of platform808releasably secure sensor module504in position. Module2200is coupled with connector2300, sharp module2500and sensor (not shown) such that during assembly they can be removed together as sensor module504.

H. Exemplary Applicators and Sensor Control Devices for One Piece Architectures

Referring briefly again toFIGS. 1A and 3A-3G, for the two-piece architecture system, the sensor tray202and the sensor applicator102are provided to the user as separate packages, thus requiring the user to open each package and finally assemble the system. In some applications, the discrete, sealed packages allow the sensor tray202and the sensor applicator102to be sterilized in separate sterilization processes unique to the contents of each package and otherwise incompatible with the contents of the other. More specifically, the sensor tray202, which includes the plug assembly207, including the sensor110and the sharp220, may be sterilized using radiation sterilization, such as electron beam (or “e-beam”) irradiation. Suitable radiation sterilization processes include, but are not limited to, electron beam (e-beam) irradiation, gamma ray irradiation, X-ray irradiation, or any combination thereof. Radiation sterilization, however, can damage the electrical components arranged within the electronics housing of the sensor control device102. Consequently, if the sensor applicator102, which contains the electronics housing of the sensor control device102, needs to be sterilized, it may be sterilized via another method, such as gaseous chemical sterilization using, for example, ethylene oxide. Gaseous chemical sterilization, however, can damage the enzymes or other chemistry and biologics included on the sensor110. Because of this sterilization incompatibility, the sensor tray202and the sensor applicator102are commonly sterilized in separate sterilization processes and subsequently packaged separately, which requires the user to finally assemble the components for use.

FIGS. 7A and 7Bare exploded top and bottom views, respectively, of the sensor control device3702, according to one or more embodiments. The shell3706and the mount3708operate as opposing clamshell halves that enclose or otherwise substantially encapsulate the various electronic components of the sensor control device3702. As illustrated, the sensor control device3702may include a printed circuit board assembly (PCBA)3802that includes a printed circuit board (PCB)3804having a plurality of electronic modules3806coupled thereto. Example electronic modules3806include, but are not limited to, resistors, transistors, capacitors, inductors, diodes, and switches. Prior sensor control devices commonly stack PCB components on only one side of the PCB. In contrast, the PCB components3806in the sensor control device3702can be dispersed about the surface area of both sides (i.e., top and bottom surfaces) of the PCB3804.

Besides the electronic modules3806, the PCBA3802may also include a data processing unit3808mounted to the PCB3804. The data processing unit3808may comprise, for example, an application specific integrated circuit (ASIC) configured to implement one or more functions or routines associated with operation of the sensor control device3702. More specifically, the data processing unit3808may be configured to perform data processing functions, where such functions may include but are not limited to, filtering and encoding of data signals, each of which corresponds to a sampled analyte level of the user. The data processing unit3808may also include or otherwise communicate with an antenna for communicating with the reader device106(FIG. 1A).

A battery aperture3810may be defined in the PCB3804and sized to receive and seat a battery3812configured to power the sensor control device3702. An axial battery contact3814aand a radial battery contact3814bmay be coupled to the PCB3804and extend into the battery aperture3810to facilitate transmission of electrical power from the battery3812to the PCB3804. As their names suggest, the axial battery contact3814amay be configured to provide an axial contact for the battery3812, while the radial battery contact3814bmay provide a radial contact for the battery3812. Locating the battery3812within the battery aperture3810with the battery contacts3814a,bhelps reduce the height H of the sensor control device3702, which allows the PCB3804to be located centrally and its components to be dispersed on both sides (i.e., top and bottom surfaces). This also helps facilitate the chamfer3718provided on the electronics housing3704.

The sensor3716may be centrally located relative to the PCB3804and include a tail3816, a flag3818, and a neck3820that interconnects the tail3816and the flag3818. The tail3816may be configured to extend through the central aperture3720of the mount3708to be transcutancously received beneath a user's skin. Moreover, the tail3816may have an enzyme or other chemistry included thereon to help facilitate analyte monitoring.

The flag3818may include a generally planar surface having one or more sensor contacts3822(three shown inFIG. 7B) arranged thereon. The sensor contact(s)3822may be configured to align with and engage a corresponding one or more circuitry contacts3824(three shown inFIG. 7A) provided on the PCB3804. In some embodiments, the sensor contact(s)3822may comprise a carbon impregnated polymer printed or otherwise digitally applied to the flag3818. Prior sensor control devices typically include a connector made of silicone rubber that encapsulates one or more compliant carbon impregnated polymer modules that serve as electrical conductive contacts between the sensor and the PCB. In contrast, the presently disclosed sensor contacts(s)3822provide a direct connection between the sensor3716and the PCB3804connection, which eliminates the need for the prior art connector and advantageously reduces the height H. Moreover, eliminating the compliant carbon impregnated polymer modules eliminates a significant circuit resistance and therefor improves circuit conductivity.

The sensor control device3702may further include a compliant member3826, which may be arranged to interpose the flag3818and the inner surface of the shell3706. More specifically, when the shell3706and the mount3708are assembled to one another, the compliant member3826may be configured to provide a passive biasing load against the flag3818that forces the sensor contact(s)3822into continuous engagement with the corresponding circuitry contact(s)3824. In the illustrated embodiment, the compliant member3826is an elastomeric O-ring, but could alternatively comprise any other type of biasing device or mechanism, such as a compression spring or the like, without departing from the scope of the disclosure.

The sensor control device3702may further include one or more electromagnetic shields, shown as a first shield3828aand a second shield The shell3706may provide or otherwise define a first clocking receptacle3830a(FIG. 7B) and a second clocking receptacle3830b(FIG. 7B), and the mount3708may provide or otherwise define a first clocking post3832a(FIG. 7A) and a second clocking post3832b(FIG. 7A). Mating the first and second clocking receptacles3830a,bwith the first and second clocking posts3832a,b, respectively, will properly align the shell3706to the mount3708.

Referring specifically toFIG. 7A, the inner surface of the mount3708may provide or otherwise define a plurality of pockets or depressions configured to accommodate various component parts of the sensor control device3702when the shell3706is mated to the mount3708. For example, the inner surface of the mount3708may define a battery locator3834configured to accommodate a portion of the battery3812when the sensor control device3702is assembled. An adjacent contact pocket3836may be configured to accommodate a portion of the axial contact3814a.

Moreover, a plurality of module pockets3838may be defined in the inner surface of the mount3708to accommodate the various electronic modules3806arranged on the bottom of the PCB3804. Furthermore, a shield locator3840may be defined in the inner surface of the mount3708to accommodate at least a portion of the second shield3828bwhen the sensor control device3702is assembled. The battery locator3834, the contact pocket3836, the module pockets3838, and the shield locator3840all extend a short distance into the inner surface of the mount3708and, as a result, the overall height H of the sensor control device3702may be reduced as compared to prior sensor control devices. The module pockets3838may also help minimize the diameter of the PCB3804by allowing PCB components to be arranged on both sides (i.e., top and bottom surfaces).

Still referring toFIG. 7A, the mount3708may further include a plurality of carrier grip features3842(two shown) defined about the outer periphery of the mount3708. The carrier grip features3842are axially offset from the bottom3844of the mount3708, where a transfer adhesive (not shown) may be applied during assembly. In contrast to prior sensor control devices, which commonly include conical carrier grip features that intersect with the bottom of the mount, the presently disclosed carrier grip features3842are offset from the plane (i.e., the bottom3844) where the transfer adhesive is applied. This may prove advantageous in helping ensure that the delivery system does not inadvertently stick to the transfer adhesive during assembly. Moreover, the presently disclosed carrier grip features3842eliminate the need for a scalloped transfer adhesive, which simplifies the manufacture of the transfer adhesive and eliminates the need to accurately clock the transfer adhesive relative to the mount3708. This also increases the bond area and, therefore, the bond strength.

Referring toFIG. 7B, the bottom3844of the mount3708may provide or otherwise define a plurality of grooves3846, which may be defined at or near the outer periphery of the mount3708and equidistantly spaced from each other. A transfer adhesive (not shown) may be coupled to the bottom3844and the grooves3846may be configured to help convey (transfer) moisture away from the sensor control device3702and toward the periphery of the mount3708during use. In some embodiments, the spacing of the grooves3846may interpose the module pockets3838(FIG. 7A) defined on the opposing side (inner surface) of the mount3708. As will be appreciated, alternating the position of the grooves3846and the module pockets3838ensures that the opposing features on either side of the mount3708do not extend into each other. This may help maximize usage of the material for the mount3708and thereby help maintain a minimal height H of the sensor control device3702. The module pockets3838may also significantly reduce mold sink, and improve the flatness of the bottom3844that the transfer adhesive bonds to.

Still referring toFIG. 7B, the inner surface of the shell3706may also provide or otherwise define a plurality of pockets or depressions configured to accommodate various component parts of the sensor control device3702when the shell3706is mated to the mount3708. For example, the inner surface of the shell3706may define an opposing battery locator3848arrangeable opposite the battery locator3834(FIG. 7A) of the mount3708and configured to accommodate a portion of the battery3812when the sensor control device3702is assembled. The opposing battery locator3848extends a short distance into the inner surface of the shell3706, which helps reduce the overall height H of the sensor control device3702.

A sharp and sensor locator3852may also be provided by or otherwise defined on the inner surface of the shell3706. The sharp and sensor locator3852may be configured to receive both the sharp (not shown) and a portion of the sensor3716. Moreover, the sharp and sensor locator3852may be configured to align and/or mate with a corresponding sharp and sensor locator2054(FIG. 7A) provided on the inner surface of the mount3708.

According to embodiments of the present disclosure, an alternative sensor assembly/electronics assembly connection approach is illustrated inFIGS. 8A to 8C. As shown, the sensor assembly14702includes sensor14704, connector support14706, and sharp14708. Notably, a recess or receptacle14710may be defined in the bottom of the mount of the electronics assembly14712and provide a location where the sensor assembly14702may be received and coupled to the electronics assembly14712, and thereby fully assemble the sensor control device. The profile of the sensor assembly14702may match or be shaped in complementary fashion to the receptacle14710, which includes an elastomeric sealing member14714(including conductive material coupled to the circuit board and aligned with the electrical contacts of the sensor14704). Thus, when the sensor assembly14702is snap fit or otherwise adhered to the electronics assembly14712by driving the sensor assembly14702into the integrally formed recess14710in the electronics assembly14712, the on-body device14714depicted inFIG. 8Cis formed. This embodiment provides an integrated connector for the sensor assembly14702within the electronics assembly14712.

Additional information regarding sensor assemblies is provided in U.S. Publication No. 2013/0150691 and U.S. Publication No. 2021/0204841, each of which is incorporated by reference herein in its entirety.

According to embodiments of the present disclosure, the sensor control device102may be modified to provide a one-piece architecture that may be subjected to sterilization techniques specifically designed for a one-piece architecture sensor control device. A one-piece architecture allows the sensor applicator150and the sensor control device102to be shipped to the user in a single, sealed package that does not require any final user assembly steps. Rather, the user need only open one package and subsequently deliver the sensor control device102to the target monitoring location. The one-piece system architecture described herein may prove advantageous in eliminating component parts, various fabrication process steps, and user assembly steps. As a result, packaging and waste are reduced, and the potential for user error or contamination to the system is mitigated.

FIGS. 9A and 9Bare side and cross-sectional side views, respectively, of an example embodiment of the sensor applicator102with the applicator cap210coupled thereto. More specifically,FIG. 9Adepicts how the sensor applicator102might be shipped to and received by a user, andFIG. 9Bdepicts the sensor control device4402arranged within the sensor applicator102. Accordingly, the fully assembled sensor control device4402may already be assembled and installed within the sensor applicator102prior to being delivered to the user, thus removing any additional assembly steps that a user would otherwise have to perform.

The fully assembled sensor control device4402may be loaded into the sensor applicator102, and the applicator cap210may subsequently be coupled to the sensor applicator102. In some embodiments, the applicator cap210may be threaded to the housing208and include a tamper ring4702. Upon rotating (e.g., unscrewing) the applicator cap210relative to the housing208, the tamper ring4702may shear and thereby free the applicator cap210from the sensor applicator102.

According to the present disclosure, while loaded in the sensor applicator102, the sensor control device4402may be subjected to gaseous chemical sterilization4704configured to sterilize the electronics housing4404and any other exposed portions of the sensor control device4402. To accomplish this, a chemical may be injected into a sterilization chamber4706cooperatively defined by the sensor applicator102and the interconnected cap210. In some applications, the chemical may be injected into the sterilization chamber4706via one or more vents4708defined in the applicator cap210at its proximal end610. Example chemicals that may be used for the gaseous chemical sterilization4704include, but are not limited to, ethylene oxide, vaporized hydrogen peroxide, nitrogen oxide (e.g., nitrous oxide, nitrogen dioxide, etc.), and steam.

Since the distal portions of the sensor4410and the sharp4412are sealed within the sensor cap4416, the chemicals used during the gaseous chemical sterilization process do not interact with the enzymes, chemistry, and biologics provided on the tail4524and other sensor components, such as membrane coatings that regulate analyte influx.

Once a desired sterility assurance level has been achieved within the sterilization chamber4706, the gaseous solution may be removed and the sterilization chamber4706may be aerated. Aeration may be achieved by a series of vacuums and subsequently circulating a gas (e.g., nitrogen) or filtered air through the sterilization chamber4706. Once the sterilization chamber4706is properly aerated, the vents4708may be occluded with a seal4712(shown in dashed lines).

In some embodiments, the seal4712may comprise two or more layers of different materials. The first layer may be made of a synthetic material (e.g., a flash-spun high-density polyethylene fiber), such as Tyvek® available from DuPont. Tyvek® is highly durable and puncture resistant and allows the permeation of vapors. The Tyvek® layer can be applied before the gaseous chemical sterilization process, and following the gaseous chemical sterilization process, a foil or other vapor and moisture resistant material layer may be sealed (e.g., heat sealed) over the Tyvek® layer to prevent the ingress of contaminants and moisture into the sterilization chamber4706. In other embodiments, the seal4712may comprise only a single protective layer applied to the applicator cap210. In such embodiments, the single layer may be gas permeable for the sterilization process, but may also be capable of protection against moisture and other harmful elements once the sterilization process is complete.

With the seal4712in place, the applicator cap210provides a barrier against outside contamination, and thereby maintains a sterile environment for the assembled sensor control device4402until the user removes (unthreads) the applicator cap210. The applicator cap210may also create a dust-free environment during shipping and storage that prevents the adhesive patch4714from becoming dirty.

FIGS. 10A and 10Bare isometric and side views, respectively, of another example sensor control device5002, according to one or more embodiments of the present disclosure. The sensor control device5002may be similar in some respects to the sensor control device102ofFIG. 1Aand therefore may be best understood with reference thereto. Moreover, the sensor control device5002may replace the sensor control device102ofFIG. 1Aand, therefore, may be used in conjunction with the sensor applicator102ofFIG. 1A, which may deliver the sensor control device5002to a target monitoring location on a user's skin.

Unlike the sensor control device102ofFIG. 1A, however, the sensor control device5002may comprise a one-piece system architecture not requiring a user to open multiple packages and finally assemble the sensor control device5002prior to application. Rather, upon receipt by the user, the sensor control device5002may already be fully assembled and properly positioned within the sensor applicator ISO (FIG. 1A). To use the sensor control device5002, the user need only open one barrier (e.g., the applicator cap708ofFIG. 3B) before promptly delivering the sensor control device5002to the target monitoring location for use.

As illustrated, the sensor control device5002includes an electronics housing5004that is generally disc-shaped and may have a circular cross-section. In other embodiments, however, the electronics housing5004may exhibit other cross-sectional shapes, such as ovoid or polygonal, without departing from the scope of the disclosure. The electronics housing5004may be configured to house or otherwise contain various electrical components used to operate the sensor control device5002. In at least one embodiment, an adhesive patch (not shown) may be arranged at the bottom of the electronics housing5004. The adhesive patch may be similar to the adhesive patch105ofFIG. 1A, and may thus help adhere the sensor control device5002to the user's skin for use.

As illustrated, the sensor control device5002includes an electronics housing5004that includes a shell5006and a mount5008that is matable with the shell5006. The shell5006may be secured to the mount5008via a variety of ways, such as a snap tit engagement, an interference fit, sonic welding, one or more mechanical fasteners (e.g., screws), a gasket, an adhesive, or any combination thereof. In some cases, the shell5006may be secured to the mount5008such that a sealed interface is generated therebetween.

The sensor control device5002may further include a sensor5010(partially visible) and a sharp5012(partially visible), used to help deliver the sensor5010transcutaneously under a user's skin during application of the sensor control device5002. As illustrated, corresponding portions of the sensor5010and the sharp5012extend distally from the bottom of the electronics housing5004(e.g., the mount5008). The sharp5012may include a sharp hub5014configured to secure and carry the sharp5012. As best seen inFIG. 10B, the sharp hub5014may include or otherwise define a mating member5016. To couple the sharp5012to the sensor control device5002, the sharp5012may be advanced axially through the electronics housing5004until the sharp hub5014engages an upper surface of the shell5006and the mating member5016extends distally from the bottom of the mount5008. As the sharp5012penetrates the electronics housing5004, the exposed portion of the sensor5010may be received within a hollow or recessed (arcuate) portion of the sharp5012. The remaining portion of the sensor5010is arranged within the interior of the electronics housing5004.

The sensor control device5002may further include a sensor cap5018, shown exploded or detached from the electronics housing5004inFIGS. 10A-10B. The sensor cap5016may be removably coupled to the sensor control device5002(e.g., the electronics housing5004) at or near the bottom of the mount5008. The sensor cap5018may help provide a sealed barrier that surrounds and protects the exposed portions of the sensor5010and the sharp5012from gaseous chemical sterilization. As illustrated, the sensor cap5018may comprise a generally cylindrical body having a first end5020aand a second end5020bopposite the first end5020a. The first end5020amay be open to provide access into an inner chamber5022defined within the body. In contrast, the second end5020bmay be closed and may provide or otherwise define an engagement feature5024. As described herein, the engagement feature5024may help mate the sensor cap5018to the cap (e.g., the applicator cap708ofFIG. 3B) of a sensor applicator (e.g., the sensor applicator150ofFIGS. 1 and 3A-3G), and may help remove the sensor cap5018from the sensor control device5002upon removing the cap from the sensor applicator.

The sensor cap5018may be removably coupled to the electronics housing5004at or near the bottom of the mount5008. More specifically, the sensor cap5018may be removably coupled to the mating member5016, which extends distally from the bottom of the mount5008. In at least one embodiment, for example, the mating member5016may define a set of external threads5026a(FIG. 10B) matable with a set of internal threads5026b(FIG. 10A) defined by the sensor cap5018. In some embodiments, the external and internal threads5026a, bmay comprise a flat thread design (e.g., lack of helical curvature), which may prove advantageous in molding the parts. Alternatively, the external and internal threads5026a,bmay comprise a helical threaded engagement. Accordingly, the sensor cap5018may be threadably coupled to the sensor control device5002at the mating member5016of the sharp hub5014. In other embodiments, the sensor cap5018may be removably coupled to the mating member5016via other types of engagements including, but not limited to, an interference or friction fit, or a frangible member or substance that may be broken with minimal separation force (e.g., axial or rotational force).

In some embodiments, the sensor cap5018may comprise a monolithic (singular) structure extending between the first and second ends5020a, b. In other embodiments, however, the sensor cap5018may comprise two or more component parts. In the illustrated embodiment, for example, the sensor cap5018may include a seal ring5028positioned at the first end5020aand a desiccant cap5030arranged at the second end5020b. The seal ring5028may be configured to help seal the inner chamber5022, as described in more detail below. In at least one embodiment, the seal ring5028may comprise an elastomeric O-ring. The desiccant cap5030may house or comprise a desiccant to help maintain preferred humidity levels within the inner chamber5022. The desiccant cap5030may also define or otherwise provide the engagement feature5024of the sensor cap5018.

FIGS. 11A-11Care progressive cross-sectional side views showing assembly of the sensor applicator102with the sensor control device5002, according to one or more embodiments. Once the sensor control device5002is fully assembled, it may then be loaded into the sensor applicator102. With reference toFIG. 11A, the sharp hub5014may include or otherwise define a hub snap pawl5302configured to help couple the sensor control device5002to the sensor applicator102. More specifically, the sensor control device5002may be advanced into the interior of the sensor applicator102and the hub snap pawl5302may be received by corresponding arms5304of a sharp carrier5306positioned within the sensor applicator102.

InFIG. 11B, the sensor control device5002is shown received by the sharp carrier5306and, therefore, secured within the sensor applicator102. Once the sensor control device5002is loaded into the sensor applicator102, the applicator cap210may be coupled to the sensor applicator102. In some embodiments, the applicator cap210and the housing208may have opposing, matable sets of threads5308that enable the applicator cap210to be screwed onto the housing208in a clockwise (or counter-clockwise) direction and thereby secure the applicator cap210to the sensor applicator102.

As illustrated, the sheath212is also positioned within the sensor applicator102, and the sensor applicator102may include a sheath locking mechanism5310configured to ensure that the sheath212does not prematurely collapse during a shock event. In the illustrated embodiment, the sheath locking mechanism5310may comprise a threaded engagement between the applicator cap210and the sheath212. More specifically, one or more internal threads5312amay be defined or otherwise provided on the inner surface of the applicator cap210, and one or more external threads5312bmay be defined or otherwise provided on the sheath212. The internal and external threads5312a,bmay be configured to threadably mate as the applicator cap210is threaded to the sensor applicator102at the threads5308. The internal and external threads5312a,bmay have the same thread pitch as the threads5308that enable the applicator cap210to be screwed onto the housing208.

InFIG. 11C, the applicator cap210is shown fully threaded (coupled) to the housing208. As illustrated, the applicator cap210may further provide and otherwise define a cap post5314centrally located within the interior of the applicator cap210and extending proximally from the bottom thereof. The cap post5314may be configured to receive at least a portion of the sensor cap5018as the applicator cap210is screwed onto the housing208.

With the sensor control device5002loaded within the sensor applicator102and the applicator cap210properly secured, the sensor control device5002may then be subjected to a gaseous chemical sterilization configured to sterilize the electronics housing5004and any other exposed portions of the sensor control device5002. Since the distal portions of the sensor5010and the sharp5012are sealed within the sensor cap5018, the chemicals used during the gaseous chemical sterilization process are unable to interact with the enzymes, chemistry, and biologics provided on the tail5104, and other sensor components, such as membrane coatings that regulate analyte influx.

FIGS. 12A-12Care progressive cross-sectional side views showing assembly and disassembly of an alternative embodiment of the sensor applicator102with the sensor control device5002, according to one or more additional embodiments. A fully assembled sensor control device5002may be loaded into the sensor applicator102by coupling the hub snap pawl5302into the arms5304of the sharp carrier5306positioned within the sensor applicator102, as generally described above.

In the illustrated embodiment, the sheath arms5604of the sheath212may be configured to interact with a first detent5702aand a second detent5702bdefined within the interior of the housing208. The first detent5702amay alternately be referred to a “locking” detent, and the second detent5702bmay alternately be referred to as a “firing” detent. When the sensor control device5002is initially installed in the sensor applicator102, the sheath arms5604may be received within the first detent5702a. As discussed below, the sheath212may be actuated to move the sheath arms5604to the second detent5702b, which places the sensor applicator102in firing position.

InFIG. 12B, the applicator cap210is aligned with the housing208and advanced toward the housing208so that the sheath212is received within the applicator cap210. Instead of rotating the applicator cap210relative to the housing208, the threads of the applicator cap210may be snapped onto the corresponding threads of the housing208to couple the applicator cap210to the housing208. Axial cuts or slots5703(one shown) defined in the applicator cap210may allow portions of the applicator cap210near its threading to flex outward to be snapped into engagement with the threading of the housing208. As the applicator cap210is snapped to the housing208, the sensor cap5018may correspondingly be snapped into the cap post5314.

Similar to the embodiment ofFIGS. 11A-11C, the sensor applicator102may include a sheath locking mechanism configured to ensure that the sheath212does not prematurely collapse during a shock event. In the illustrated embodiment, the sheath locking mechanism includes one or more ribs5704(one shown) defined near the base of the sheath212and configured to interact with one or more ribs5706(two shown) and a shoulder5708defined near the base of the applicator cap210. The ribs5704may be configured to inter-lock between the ribs5706and the shoulder5708while attaching the applicator cap210to the housing208. More specifically, once the applicator cap210is snapped onto the housing208, the applicator cap210may be rotated (e.g., clockwise), which locates the ribs5704of the sheath212between the ribs5706and the shoulder5708of the applicator cap210and thereby “locks” the applicator cap210in place until the user reverse rotates the applicator cap210to remove the applicator cap210for use. Engagement of the ribs5704between the ribs5706and the shoulder5708of the applicator cap210may also prevent the sheath212from collapsing prematurely.

InFIG. 12C, the applicator cap210is removed from the housing208. As with the embodiment ofFIGS. 12A-12C, the applicator cap210can be removed by reverse rotating the applicator cap210, which correspondingly rotates the cap post5314in the same direction and causes sensor cap5018to unthread from the mating member5016, as generally described above. Moreover, detaching the sensor cap5018from the sensor control device5002exposes the distal portions of the sensor5010and the sharp5012.

As the applicator cap210is unscrewed from the housing208, the ribs5704defined on the sheath212may slidingly engage the tops of the ribs5706defined on the applicator cap210. The tops of the ribs5706may provide corresponding ramped surfaces that result in an upward displacement of the sheath212as the applicator cap210is rotated, and moving the sheath212upward causes the sheath arms5604to flex out of engagement with the first detent5702ato be received within the second detent5702b. As the sheath212moves to the second detent5702b, the radial shoulder5614moves out of radial engagement with the carrier arm(s)5608, which allows the passive spring force of the spring5612to push upward on the sharp carrier5306and force the carrier arm(s)5608out of engagement with the groove(s)5610. As the sharp carrier5306moves upward within the housing208, the mating member5016may correspondingly retract until it becomes flush, substantially flush, or sub-flush with the bottom of the sensor control device5002.

At this point, the sensor applicator102in firing position. Accordingly, in this embodiment, removing the applicator cap210correspondingly causes the mating member5016to retract.

I. Exemplary Firing Mechanism of One-Piece and Two-Piece Applicators

FIGS. 13A-13Fillustrate example details of embodiments of the internal device mechanics of “firing” the applicator216to apply sensor control device222to a user and including retracting sharp1030safely back into used applicator216. All together, these drawings represent an example sequence of driving sharp1030(supporting a sensor coupled to sensor control device222) into the skin of a user, withdrawing the sharp while leaving the sensor behind in operative contact with interstitial fluid of the user, and adhering the sensor control device to the skin of the user with an adhesive. Modification of such activity for use with the alternative applicator assembly embodiments and components can be appreciated in reference to the same by those with skill in the art. Moreover, applicator216may be a sensor applicator having one-piece architecture or a two-piece architecture as disclosed herein.

Turning now toFIG. 13A, a sensor1102is supported within sharp1030, just above the skin1104of the user. Rails1106(optionally three of them) of an upper guide section1108may be provided to control applicator216motion relative to sheath318. The sheath318is held by detent features1110within the applicator216such that appropriate downward force along the longitudinal axis of the applicator216will cause the resistance provided by the detent features1110to be overcome so that sharp1030and sensor control device222can translate along the longitudinal axis into (and onto) skin1104of the user. In addition, catch arms1112of sensor carrier1022engage the sharp retraction assembly1024to maintain the sharp1030in a position relative to the sensor control device222.

InFIG. 13B, user force is applied to overcome or override detent features1110and sheath318collapses into housing314driving the sensor control device222(with associated parts) to translate down as indicated by the arrow L along the longitudinal axis. An inner diameter of the upper guide section1108of the sheath318constrains the position of carrier arms1112through the full stroke of the sensor/sharp insertion process. The retention of the stop surfaces1114of carrier arms1112against the complimentary faces1116of the sharp retraction assembly1024maintains the position of the members with return spring1118fully energized. According to embodiments, rather than employing user force to drive the sensor control device222to translate down as indicated by the arrow L along the longitudinal axis, housing314can include a button (for example, not limitation, a push button) which activates a drive spring (for example, not limitation, a coil spring) to drive the sensor control device222.

InFIG. 13C, sensor1102and sharp1030have reached full insertion depth. In so doing, the carrier arms1112clear the upper guide section1108inner diameter. Then, the compressed force of the coil return spring1118drives angled stop surfaces1114radially outward, releasing force to drive the sharp carrier1102of the sharp retraction assembly1024to pull the (slotted or otherwise configured) sharp1030out of the user and off of the sensor1102as indicated by the arrow R inFIG. 13D.

With the sharp1030fully retracted as shown inFIG. 13E, the upper guide section1108of the sheath318is set with a final locking feature1120. As shown inFIG. 13F, the spent applicator assembly216is removed from the insertion site, leaving behind the sensor control device222, and with the sharp1030secured safely inside the applicator assembly216. The spent applicator assembly216is now ready for disposal.

Operation of the applicator216when applying the sensor control device222is designed to provide the user with a sensation that both the insertion and retraction of the sharp1030is performed automatically by the internal mechanisms of the applicator216. In other words, the present invention avoids the user experiencing the sensation that he is manually driving the sharp1030into his skin. Thus, once the user applies sufficient force to overcome the resistance from the detent features of the applicator216, the resulting actions of the applicator216are perceived to be an automated response to the applicator being “triggered.” The user does not perceive that he is supplying additional force to drive the sharp1030to pierce his skin despite that all the driving force is provided by the user and no additional biasing/driving means are used to insert the sharp1030. As detailed above inFIG. 13C, the retraction of the sharp1030is automated by the coil return spring1118of the applicator216.

With respect to any of the applicator embodiments described herein, as well as any of the components thereof, including but not limited to the sharp, sharp module and sensor module embodiments, those of skill in the art will understand that said embodiments can be dimensioned and configured for use with sensors configured to sense an analyte level in a bodily fluid in the epidermis, dermis, or subcutaneous tissue of a subject. In some embodiments, for example, sharps and distal portions of analyte sensors disclosed herein can both be dimensioned and configured to be positioned at a particular end-depth (i.e., the furthest point of penetration in a tissue or layer of the subject's body, e.g., in the epidermis, dermis, or subcutaneous tissue). With respect to some applicator embodiments, those of skill in the art will appreciate that certain embodiments of sharps can be dimensioned and configured to be positioned at a different end-depth in the subject's body relative to the final end-depth of the analyte sensor. In some embodiments, for example, a sharp can be positioned at a first end-depth in the subject's epidermis prior to retraction, while a distal portion of an analyte sensor can be positioned at a second end-depth in the subject's dermis. In other embodiments, a sharp can be positioned at a first end-depth in the subject's dermis prior to retraction, while a distal portion of an analyte sensor can be positioned at a second end-depth in the subject's subcutaneous tissue. In still other embodiments, a sharp can be positioned at a first end-depth prior to retraction and the analyte sensor can be positioned at a second end-depth, wherein the first end-depth and second end-depths are both in the same layer or tissue of the subject's body.

Additionally, with respect to any of the applicator embodiments described herein, those of skill in the art will understand that an analyte sensor, as well as one or more structural components coupled thereto, including but not limited to one or more spring-mechanisms, can be disposed within the applicator in an off-center position relative to one or more axes of the applicator. In some applicator embodiments, for example, an analyte sensor and a spring mechanism can be disposed in a first off-center position relative to an axis of the applicator on a first side of the applicator, and the sensor electronics can be disposed in a second off-center position relative to the axis of the applicator on a second side of the applicator. In other applicator embodiments, the analyte sensor, spring mechanism, and sensor electronics can be disposed in an off-center position relative to an axis of the applicator on the same side. Those of skill in the art will appreciate that other permutations and configurations in which any or all of the analyte sensor, spring mechanism, sensor electronics, and other components of the applicator are disposed in a centered or off-centered position relative to one or more axes of the applicator are possible and fully within the scope of the present disclosure.

Additional details of suitable devices, systems, methods, components and the operation thereof along with related features are set forth in International Publication No. WO 2018/136898 to Rao et al., International Publication No. WO 2019/236850 to Thomas et al., International Publication No. WO 2019/236859 to Thomas et al., International Publication No. WO 2019/236876 to Thomas et al., and U.S. Patent Publication No. 2020/0196919, filed Jun. 6, 2019, each of which is incorporated by reference in its entirety herein. Further details regarding embodiments of applicators, their components, and variants thereof, are described in U.S. Patent Publication Nos. 2013/0150691, 2016/0331283, and 2018/0235520, all of which are incorporated by reference herein in their entireties and for all purposes. Further details regarding embodiments of sharp modules, sharps, their components, and variants thereof, are described in U.S. Patent Publication No. 2014/0171771, which is incorporated by reference herein in its entirety and for all purposes.

J. Exemplary Methods of Calibrating Analyte Sensors

Biochemical sensors can be described by one or more sensing characteristics. A common sensing characteristic is referred to as the biochemical sensor's sensitivity, which is a measure of the sensor's responsiveness to the concentration of the chemical or composition it is designed to detect. For electrochemical sensors, this response can be in the form of an electrical current (amperometric) or electrical charge (coulometric). For other types of sensors, the response can be in a different form, such as a photonic intensity (e.g., optical light). The sensitivity of a biochemical analyte sensor can vary depending on a number of factors, including whether the sensor is in an in vitro state or an in vivo state.

FIG. 14is a graph depicting the in vitro sensitivity of an amperometric analyte sensor. The in vitro sensitivity can be obtained by in vitro testing the sensor at various analyte concentrations and then performing a regression (e.g., linear or non-linear) or other curve fitting on the resulting data. In this example, the analyte sensor's sensitivity is linear, or substantially linear, and can be modeled according to the equation y=mx+b, where y is the sensor's electrical output current, x is the analyte level (or concentration), m is the slope of the sensitivity and b is the intercept of the sensitivity, where the intercept generally corresponds to a background signal (e.g., noise). For sensors with a linear or substantially linear response, the analyte level that corresponds to a given current can be determined from the slope and intercept of the sensitivity. Sensors with a non-linear sensitivity require additional information to determine the analyte level resulting from the sensor's output current, and those of ordinary skill in the art are familiar with manners by which to model non-linear sensitivities. In certain embodiments of in vivo sensors, the in vitro sensitivity can be the same as the in vivo sensitivity, but in other embodiments a transfer (or conversion) function is used to translate the in vitro sensitivity into the in vivo sensitivity that is applicable to the sensor's intended in vivo use.

Calibration is a technique for improving or maintaining accuracy by adjusting a sensor's measured output to reduce the differences with the sensor's expected output. One or more parameters that describe the sensor's sensing characteristics, like its sensitivity, are established for use in the calibration adjustment.

Certain in vivo analyte monitoring systems require calibration to occur after implantation of the sensor into the user or patient, either by user interaction or by the system itself in an automated fashion. For example, when user interaction is required, the user performs an in vitro measurement (e.g., a blood glucose (BG) measurement using a finger stick and an in vitro test strip) and enters this into the system, while the analyte sensor is implanted. The system then compares the in vitro measurement with the in vivo signal and, using the differential, determines an estimate of the sensor's in vivo sensitivity. The in vivo sensitivity can then be used in an algorithmic process to transform the data collected with the sensor to a value that indicates the user's analyte level. This and other processes that require user action to perform calibration are referred to as “user calibration.” Systems can require user calibration due to instability of the sensor's sensitivity, such that the sensitivity drifts or changes over time. Thus, multiple user calibrations (e.g., according to a periodic (e.g., daily) schedule, variable schedule, or on an as-needed basis) can be required to maintain accuracy. While the embodiments described herein can incorporate a degree of user calibration for a particular implementation, generally this is not preferred as it requires the user to perform a painful or otherwise burdensome BG measurement, and can introduce user error.

Some in vivo analyte monitoring systems can regularly adjust the calibration parameters through the use of automated measurements of characteristics of the sensor made by the system itself (e.g., processing circuitry executing software). The repeated adjustment of the sensor's sensitivity based on a variable measured by the system (and not the user) is referred to generally as “system” (or automated) calibration, and can be performed with user calibration, such as an early BG measurement, or without user calibration. Like the case with repeated user calibrations, repeated system calibrations are typically necessitated by drift in the sensor's sensitivity over time. Thus, while the embodiments described herein can be used with a degree of automated system calibration, preferably the sensor's sensitivity is relatively stable over time such that post-implantation calibration is not required.

Some in vivo analyte monitoring systems operate with a sensor that is factory calibrated. Factory calibration refers to the determination or estimation of the one or more calibration parameters prior to distribution to the user or healthcare professional (HCP). The calibration parameter can be determined by the sensor manufacturer (or the manufacturer of the other components of the sensor control device if the two entities are different). Many in vivo sensor manufacturing processes fabricate the sensors in groups or batches referred to as production lots, manufacturing stage lots, or simply lots. A single lot can include thousands of sensors.

Sensors can include a calibration code or parameter which can be derived or determined during one or more sensor manufacturing processes and coded or programmed, as part of the manufacturing process, in the data processing device of the analyte monitoring system or provided on the sensor itself, for example, as a bar code, a laser tag, an RFID tag, or other machine readable information provided on the sensor. User calibration during in vivo use of the sensor can be obviated, or the frequency of in vivo calibrations during sensor wear can be reduced if the code is provided to a receiver (or other data processing device). In embodiments where the calibration code or parameter is provided on the sensor itself, prior to or at the start of the sensor use, the calibration code or parameter can be automatically transmitted or provided to the data processing device in the analyte monitoring system.

Some in vivo analyte monitoring system operate with a sensor that can be one or more of factory calibrated, system calibrated, and/or user calibrated. For example, the sensor can be provided with a calibration code or parameter which can allow for factory calibration. If the information is provided to a receiver (for example, entered by a user), the sensor can operate as a factory calibrated sensor. If the information is not provided to a receiver, the sensor can operate as a user calibrated sensor and/or a system calibrated sensor.

In a further aspect, programming or executable instructions can be provided or stored in the data processing device of the analyte monitoring system, and/or the receiver/controller unit, to provide a time varying adjustment algorithm to the in vivo sensor during use. For example, based on a retrospective statistical analysis of analyte sensors used in vivo and the corresponding glucose level feedback, a predetermined or analytical curve or a database can be generated which is time based, and configured to provide additional adjustment to the one or more in vivo sensor parameters to compensate for potential sensor drift in stability profile, or other factors.

In accordance with the disclosed subject matter, the analyte monitoring system can be configured to compensate or adjust for the sensor sensitivity based on a sensor drift profile. A time varying parameter β(t) can be defined or determined based on analysis of sensor behavior during in vivo use, and a time varying drift profile can be determined. In certain aspects, the compensation or adjustment to the sensor sensitivity can be programmed in the receiver unit, the controller or data processor of the analyte monitoring system such that the compensation or the adjustment or both can be performed automatically and/or iteratively when sensor data is received from the analyte sensor. In accordance with the disclosed subject matter, the adjustment or compensation algorithm can be initiated or executed by the user (rather than self-initiating or executing) such that the adjustment or the compensation to the analyte sensor sensitivity profile is performed or executed upon user initiation or activation of the corresponding function or routine, or upon the user entering the sensor calibration code.

In accordance with the disclosed subject matter, each sensor in the sensor lot (in some instances not including sample sensors used for in vitro testing) can be examined non-destructively to determine or measure its characteristics such as membrane thickness at one or more points of the sensor, and other characteristics including physical characteristics such as the surface area/volume of the active area can be measured or determined. Such measurement or determination can be performed in an automated manner using, for example, optical scanners or other suitable measurement devices or systems, and the determined sensor characteristics for each sensor in the sensor lot is compared to the corresponding mean values based on the sample sensors for possible correction of the calibration parameter or code assigned to each sensor. For example, for a calibration parameter defined as the sensor sensitivity, the sensitivity is approximately inversely proportional to the membrane thickness, such that, for example, a sensor having a measured membrane thickness of approximately 4% greater than the mean membrane thickness for the sampled sensors from the same sensor lot as the sensor, the sensitivity assigned to that sensor in one embodiment is the mean sensitivity determined from the sampled sensors divided by 1.04. Likewise, since the sensitivity is approximately proportional to active area of the sensor, a sensor having measured active area of approximately 3% lower than the mean active area for the sampled sensors from the same sensor lot, the sensitivity assigned to that sensor is the mean sensitivity multiplied by 0.97. The assigned sensitivity can be determined from the mean sensitivity from the sampled sensors, by multiple successive adjustments for each examination or measurement of the sensor. In certain embodiments, examination or measurement of each sensor can additionally include measurement of membrane consistency or texture in addition to the membrane thickness and/or surface are or volume of the active sensing area.

Additional information regarding sensor calibration is provided in U.S. Publication No. 2010/00230285 and U.S. Publication No. 2019/0274598, each of which is incorporated by reference herein in its entirety.

K. Exemplary Bluetooth Communication Protocols

The storage memory5030of the sensor110can include the software blocks related to communication protocols of the communication module. For example, the storage memory5030can include a BLE services software block with functions to provide interfaces to make the BLE module5041available to the computing hardware of the sensor110. These software functions can include a BLE logical interface and interface parser. BLE services offered by the communication module5040can include the generic access profile service, the generic attribute service, generic access service, device information service, data transmission services, and security services. The data transmission service can be a primary service used for transmitting data such as sensor control data, sensor status data, analyte measurement data (historical and current), and event log data. The sensor status data can include error data, current time active, and software state. The analyte measurement data can include information such as current and historical raw measurement values, current and historical values after processing using an appropriate algorithm or model, projections and trends of measurement levels, comparisons of other values to patient-specific averages, calls to action as determined by the algorithms or models and other similar types of data.

According to aspects of the disclosed subject matter, and as embodied herein, a sensor110can be configured to communicate with multiple devices concurrently by adapting the features of a communication protocol or medium supported by the hardware and radios of the sensor110. As an example, the BLE module5041of the communication module5040can be provided with software or firmware to enable multiple concurrent connections between the sensor110as a central device and the other devices as peripheral devices, or as a peripheral device where another device is a central device.

Connections, and ensuing communication sessions, between two devices using a communication protocol such as BLE can be characterized by a similar physical channel operated between the two devices (e.g., a sensor110and data receiving device120). The physical channel can include a single channel or a series of channels, including for example and without limitation using an agreed upon series of channels determined by a common clock and channel- or frequency-hopping sequence. Communication sessions can use a similar amount of the available communication spectrum, and multiple such communication sessions can exist in proximity. In certain embodiment, each collection of devices in a communication session uses a different physical channel or series of channels, to manage interference of devices in the same proximity.

For purpose of illustration and not limitation, reference is made to an exemplary embodiment of a procedure for a sensor-receiver connection for use with the disclosed subject matter. First, the sensor110repeatedly advertises its connection information to its environment in a search for a data receiving device120. The sensor110can repeat advertising on a regular basis until a connection established. The data receiving device120detects the advertising packet and scans and filters for the sensor120to connect to through the data provided in the advertising packet. Next, data receiving device120sends a scan request command and the sensor110responds with a scan response packet providing additional details. Then, the data receiving device120sends a connection request using the Bluetooth device address associated with the data receiving device120. The data receiving device120can also continuously request to establish a connection to a sensor110with a specific Bluetooth device address. Then, the devices establish an initial connection allowing them to begin to exchange data. The devices begin a process to initialize data exchange services and perform a mutual authentication procedure.

During a first connection between the sensor110and data receiving device120, the data receiving device120can initialize a service, characteristic, and attribute discovery procedure. The data receiving device120can evaluate these features of the sensor110and store them for use during subsequent connections. Next, the devices enable a notification for a customized security service used for mutual authentication of the sensor110and data receiving device120. The mutual authentication procedure can be automated and require no user interaction. Following the successful completion of the mutual authentication procedure, the sensor110sends a connection parameter update to request the data receiving device120to use connection parameter settings preferred by the sensor110and configured to maximum longevity.

The data receiving device120then performs sensor control procedures to backfill historical data, current data, event log, and factory data. As an example, for each type of data, the data receiving device120sends a request to initiate a backfill process. The request can specify a range of records defined based on, for example, the measurement value, timestamp, or similar, as appropriate. The sensor110responds with requested data until all previously unsent data in the memory of the sensor110is delivered to the data receiving device120. The sensor110can respond to a backfill request from the data receiving device120that all data has already been sent. Once backfill is completed, the data receiving device120can notify sensor110that it is ready to receive regular measurement readings. The sensor110can send readings across multiple notifications result on a repeating basis. As embodied herein, the multiple notifications can be redundant notifications to ensure that data is transmitted correctly. Alternatively, multiple notifications can make up a single payload.

For purpose of illustration and not limitation, reference is made to an exemplary embodiment of a procedure to send a shutdown command to the sensor110. The shutdown operation is executed if the sensor110is in, for example, an error state, insertion failed state, or sensor expired state. If the sensor110is not in those states, the sensor110can log the command and execute the shutdown when sensor110transitions into the error state or sensor expired state. The data receiving device120sends a properly formatted shutdown command to the sensor110. If the sensor110is actively processing another command, the sensor110will respond with a standard error response indicating that the sensor110is busy. Otherwise, the sensor110sends a response as the command is received. Additionally, the sensor110sends a success notification through the sensor control characteristic to acknowledge the sensor110has received the command. The sensor110registers the shutdown command. At the next appropriate opportunity (e.g., depending on the current sensor state, as described herein), the sensor110will shut down.

L. Exemplary Sensor States and Activation

For purpose of illustration and not limitation, reference is made to the exemplary embodiment of a high-level depiction of a state machine representation6000of the actions that can be taken by the sensor110as shown inFIG. 15. After initialization, the sensor enters state6005, which relates to the manufacture of the sensor110. In the manufacture state6005the sensor110can be configured for operation, for example, the storage memory5030can be written. At various times while in state6005, the sensor110checks for a received command to go to the storage state6015. Upon entry to the storage state6015, the sensor performs a software integrity check. While in the storage state6015, the sensor can also receive an activation request command before advancing to the insertion detection state6025.

Upon entry to state6025, the sensor110can store information relating to devices authenticated to communicate with the sensor as set during activation or initialize algorithms related to conducting and interpreting measurements from the sensing hardware5060. The sensor110can also initialize a lifecycle timer, responsible for maintaining an active count of the time of operation of the sensor110and begin communication with authenticated devices to transmit recorded data. While in the insertion detection state6025, the sensor can enter state6030, where the sensor110checks whether the time of operation is equal to a predetermined threshold. This time of operation threshold can correspond to a timeout function for determining whether an insertion has been successful. If the time of operation has reached the threshold, the sensor110advances to state6035, in which the sensor110checks whether the average data reading is greater than a threshold amount corresponding to an expected data reading volume for triggering detection of a successful insertion. If the data reading volume is lower than the threshold while in state6035, the sensor advances to state6040, corresponding to a failed insertion. If the data reading volume satisfies the threshold, the sensor advances to the active paired state6055.

The active paired state6055of the sensor110reflects the state while the sensor110is operating as normal by recording measurements, processing the measurements, and reporting them as appropriate. While in the active paired state6055, the sensor110sends measurement results or attempts to establish a connection with a receiving device120. The sensor10also increments the time of operation. Once the sensor110reaches a predetermined threshold time of operation (e.g., once the time of operation reaches a predetermined threshold), the sensor110transitions to the active expired state6065. The active expired state6065of the sensor110reflects the state while the sensor110has operated for its maximum predetermined amount of time.

While in the active expired state6065, the sensor110can generally perform operations relating to winding down operation and ensuring that the collected measurements have been securely transmitted to receiving devices as needed. For example, while in the active expired state6065, the sensor110can transmit collected data and, if no connection is available, can increase efforts to discover authenticated devices nearby and establish and connection therewith. While in the active expired state6065, the sensor110can receive a shutdown command at state6070. If no shutdown command is received, the sensor110can also, at state6075, check if the time of operation has exceeded a final operation threshold. The final operation threshold can be based on the battery life of the sensor110. The normal termination state6080corresponds to the final operations of the sensor110and ultimately shutting down the sensor110.

Before a sensor is activated, the ASIC5000resides in a low power storage mode state. The activation process can begin, for example, when an incoming RF field (e.g., NFC field) drives the voltage of the power supply to the ASIC5000above a reset threshold, which causes the sensor110to enter a wake-up state. While in the wake-up state, the ASIC5000enters an activation sequence state. The ASIC5000then wakes the communication module5040. The communication module5040is initialized, triggering a power on self-test. The power on self-test can include the ASIC5000communicating with the communication module5040using a prescribed sequence of reading and writing data to verify the memory and one-time programmable memory are not corrupted.

When the ASIC5000enters the measurement mode for the first time, an insertion detection sequence is performed to verify that the sensor110has been properly installed onto the patient's body before a proper measurement can take place. First, the sensor110interprets a command to activate the measurement configuration process, causing the ASIC5000to enter measurement command mode. The sensor110then temporarily enters the measurement lifecycle state to run a number of consecutive measurements to test whether the insertion has been successful. The communication module5040or ASIC5000evaluates the measurement results to determine insertion success. When insertion is deemed successful, the sensor110enters a measurement state, in which the sensor110begins taking regular measurements using sensing hardware5060. If the sensor110determines that the insertion was not successful, sensor110is triggered into an insertion failure mode, in which the ASIC5000is commanded back to storage mode while the communication module5040disables itself.

FIG. 1Bfurther illustrates an example operating environment for providing over-the-air (“OTA”) updates for use with the techniques described herein. An operator of the analyte monitoring system100can bundle updates for the data receiving device120or sensor110into updates for an application executing on the multi-purpose data receiving device130. Using available communication channels between the data receiving device120, the multi-purpose data receiving device130, and the sensor110, the multi-purpose data receiving device130can receive regular updates for the data receiving device120or sensor110and initiate installation of the updates on the data receiving device120or sensor110. The multi-purpose data receiving device130acts as an installation or update platform for the data receiving device120or sensor110because the application that enables the multi-purpose data receiving device130to communicate with an analyte sensor110, data receiving device120and/or remote application server150can update software or firmware on a data receiving device120or sensor110without wide-area networking capabilities.

As embodied herein, a remote application server150operated by the manufacturer of the analyte sensor110and/or the operator of the analyte monitoring system100can provide software and firmware updates to the devices of the analyte monitoring system100. In particular embodiments, the remote application server150can provides the updated software and firmware to a user device140or directly to a multi-purpose data receiving device. As embodied herein, the remote application server150can also provide application software updates to an application storefront server160using interfaces provided by the application storefront. The multi-purpose data receiving device130can contact the application storefront server160periodically to download and install the updates.

After the multi-purpose data receiving device130downloads an application update including a firmware or software update for a data receiving device120or sensor110, the data receiving device120or sensor110and multi-purpose data receiving device130establish a connection. The multi-purpose data receiving device130determines that a firmware or software update is available for the data receiving device120or sensor110. The multi-purpose data receiving device130can prepare the software or firmware update for delivery to the data receiving device120or sensor110. As an example, the multi-purpose data receiving device130can compress or segment the data associated with the software or firmware update, can encrypt or decrypt the firmware or software update, or can perform an integrity check of the firmware or software update. The multi-purpose data receiving device130sends the data for the firmware or software update to the data receiving device120or sensor110. The multi-purpose data receiving device130can also send a command to the data receiving device120or sensor110to initiate the update. Additionally or alternatively, the multi-purpose data receiving device130can provide a notification to the user of the multi-purpose data receiving device130and include instructions for facilitating the update, such as instructions to keep the data receiving device120and the multi-purpose data receiving device130connected to a power source and in close proximity until the update is complete.

The data receiving device120or sensor110receives the data for the update and the command to initiate the update from the multi-purpose data receiving device130. The data receiving device120can then install the firmware or software update. To install the update, the data receiving device120or sensor110can place or restart itself in a so-called “safe” mode with limited operational capabilities. Once the update is completed, the data receiving device120or sensor110re-enters or resets into a standard operational mode. The data receiving device120or sensor110can perform one or more self-tests to determine that the firmware or software update was installed successfully. The multi-purpose data receiving device130can receive the notification of the successful update. The multi-purpose data receiving device130can then report a confirmation of the successful update to the remote application server150.

In particular embodiments, the storage memory5030of the sensor110includes one-time programmable (OTP) memory. The term OTP memory can refer to memory that includes access restrictions and security to facilitate writing to particular addresses or segments in the memory a predetermined number of times. The memory5030can be prearranged into multiple pre-allocated memory blocks or containers. The containers are pre-allocated into a fixed size. If storage memory5030is one-time programming memory, the containers can be considered to be in a non-programmable state. Additional containers which have not yet been written to can be placed into a programmable or writable state. Containerizing the storage memory5030in this fashion can improve the transportability of code and data to be written to the storage memory5030. Updating the software of a device (e.g., the sensor device described herein) stored in an OTP memory can be performed by superseding only the code in a particular previously-written container or containers with updated code written to a new container or containers, rather than replacing the entire code in the memory. In a second embodiment, the memory is not prearranged. Instead, the space allocated for data is dynamically allocated or determined as needed. Incremental updates can be issued, as containers of varying sizes can be defined where updates are anticipated.

FIG. 16is a diagram illustrating an example operational and data flow for over-the-air (OTA) programming of a storage memory5030in a sensor device100as well as use of the memory after the OTA programming in execution of processes by the sensor device110according to the disclosed subject matter. In the example OTA programming500illustrated inFIG. 5, a request is sent from an external device (e.g., the data receiving device130) to initiate OTA programming (or re-programming). At511, a communication module5040of a sensor device110receives an OTA programming command. The communication module5040sends the OTA programming command to the microcontroller5010of the sensor device110.

At531, after receiving the OTA programming command, the microcontroller5010validates the OTA programming command. The microcontroller5010can determine, for example, whether the OTA programming command is signed with an appropriate digital signature token. Upon determining that the OTA programming command is valid, the microcontroller5010can set the sensor device into an OTA programming mode. At532, the microcontroller5010can validate the OTA programming data. At533, The microcontroller5010can reset the sensor device110to re-initialize the sensor device110in a programming state. Once the sensor device110has transitioned into the OTA programming state, the microcontroller5010can begin to write data to the rewriteable memory540(e.g., memory5020) of the sensor device at534and write data to the OTP memory550of the sensor device at535(e.g., storage memory5030). The data written by the microcontroller5010can be based on the validated OTA programming data. The microcontroller5010can write data to cause one or more programming blocks or regions of the OTP memory550to be marked invalid or inaccessible. The data written to the free or unused portion of the OTP memory can be used to replace invalidated or inaccessible programming blocks of the OTP memory550. After the microcontroller5010writes the data to the respective memories at534and535, the microcontroller5010can perform one or more software integrity checks to ensure that errors were not introduced into the programming blocks during the writing process. Once the microcontroller5010is able to determine that the data has been written without errors, the microcontroller5010can resume standard operations of the sensor device.

In execution mode, at536, the microcontroler5010can retrieve a programming manifest or profile from the rewriteable memory540. The programming manifest or profile can include a listing of the valid software programming blocks and can include a guide to program execution for the sensor110. By following the programming manifest or profile, the microcontroller5010can determine which memory blocks of the OTP memory550are appropriate to execute and avoid execution of out-of-date or invalidated programming blocks or reference to out-of-date data. At537, the microcontroller5010can selectively retrieve memory blocks from the OTP memory550. At538, the microcontroller5010can use the retrieved memory blocks, by executing programming code stored or using variable stored in the memory.

N. Exemplary Security and Other Architecture Features

As embodied herein a first layer of security for communications between the analyte sensor110and other devices can be established based on security protocols specified by and integrated in the communication protocols used for the communication. Another layer of security can be based on communication protocols that necessitate close proximity of communicating devices. Furthermore, certain packets and/or certain data included within packets can be encrypted while other packets and/or data within packets is otherwise encrypted or not encrypted. Additionally or alternatively, application layer encryption can be used with one or more block ciphers or stream ciphers to establish mutual authentication and communication encryption with other devices in the analyte monitoring system100.

The ASIC5000of the analyte sensor110can be configured to dynamically generate authentication and encryption keys using data retained within the storage memory5030. The storage memory5030can also be pre-programmed with a set of valid authentication and encryption keys to use with particular classes of devices. The ASIC5000can be further configured to perform authentication procedures with other devices using received data and apply the generated key to sensitive data prior to transmitting the sensitive data. The generated key can be unique to the analyte sensor110, unique to a pair of devices, unique to a communication session between an analyte sensor110and other device, unique to a message sent during a communication session, or unique to a block of data contained within a message.

Both the sensor110and a data receiving device120can ensure the authorization of the other party in a communication session to, for example, issue a command or receive data. In particular embodiments, identity authentication can be performed through two features. First, the party asserting its identity provides a validated certificate signed by the manufacturer of the device or the operator of the analyte monitoring system100. Second, authentication can be enforced through the use of public keys and private keys, and shared secrets derived therefrom, established by the devices of the analyte monitoring system100or established by the operator of the analyte monitoring system100. To confirm the identity of the other party, the party can provide proof that the party has control of its private key.

The manufacturer of the analyte sensor110, data receiving device120, or provider of the application for multi-purpose data receiving device130can provide information and programming necessary for the devices to securely communicate through secured programming and updates. For example, the manufacturer can provide information that can be used to generate encryption keys for each device, including secured root keys for the analyte sensor110and optionally for the data receiving device120that can be used in combination with device-specific information and operational data (e.g., entropy-based random values) to generate encryption values unique to the device, session, or data transmission as need.

Analyte data associated with a user is sensitive data at least in part because this information can be used for a variety of purposes, including for health monitoring and medication dosing decisions. In addition to user data, the analyte monitoring system100can enforce security hardening against efforts by outside parties to reverse-engineering. Communication connections can be encrypted using a device-unique or session-unique encryption key. Encrypted communications or unencrypted communications between any two devices can be verified with transmission integrity checks built into the communications. Analyte sensor110operations can be protected from tampering by restricting access to read and write functions to the memory5020via a communication interface. The sensor can be configured to grant access only to known or “trusted” devices, provided in a “whitelist” or only to devices that can provide a predetermined code associated with the manufacturer or an otherwise authenticated user. A whitelist can represent an exclusive range, meaning that no connection identifiers besides those included in the whitelist will be used, or a preferred range, in which the whitelist is searched first, but other devices can still be used. The sensor110can further deny and shut down connection requests if the requestor cannot complete a login procedure over a communication interface within a predetermined period of time (e.g., within four seconds). These characteristics safeguard against specific denial of service attacks, and in particular against denial of service attacks on a BLE interface.

As embodied herein, the analyte monitoring system100can employ periodic key rotation to further reduce the likelihood of key compromise and exploitation. A key rotation strategy employed by the analyte monitoring system100can be designed to support backward compatibility of field-deployed or distributed devices. As an example, the analyte monitoring system100can employ keys for downstream devices (e.g., devices that are in the field or cannot be feasibly provided updates) that are designed to be compatible with multiple generations of keys used by upstream devices.

For purpose of illustration and not limitation, reference is made to the exemplary embodiment of a message sequence diagram600for use with the disclosed subject matter as shown inFIG. 17and demonstrating an example exchange of data between a pair of devices, particularly a sensor110and a data receiving device120. The data receiving device120can, as embodied herein, be a data receiving device120or a multi-purpose data receiving device130. At step605, the data receiving device120can transmit a sensor activation command605to the sensor110, for example via a short-range communication protocol. The sensor110can, prior to step605be in a primarily dormant state, preserving its battery until full activation is needed. After activation during step610, the sensor110can collect data or perform other operations as appropriate to the sensing hardware5060of the sensor110. At step615the data receiving device120can initiate an authentication request command615. In response to the authentication request command615, both the sensor110and data receiving device120can engage in a mutual authentication process620.

The mutual authentication process620can involve the transfer of data, including challenge parameters that allow the sensor110and data receiving device120to ensure that the other device is sufficiently capable of adhering to an agreed-upon security framework described herein. Mutual authentication can be based on mechanisms for authentication of two or more entities to each other with or without on-line trusted third parties to verify establishment of a secret key via challenge-response. Mutual authentication can be performed using two-, three-, four-, or five-pass authentication, or similar versions thereof.

Following a successful mutual authentication process620, at step625the sensor110can provide the data receiving device120with a sensor secret625. The sensor secret can contain sensor-unique values and be derived from random values generated during manufacture. The sensor secret can be encrypted prior to or during transmission to prevent third-parties from accessing the secret. The sensor secret625can be encrypted via one or more of the keys generated by or in response to the mutual authentication process620. At step630, the data receiving device120can derive a sensor-unique encryption key from the sensor secret. The sensor-unique encryption key can further be session-unique. As such, the sensor-unique encryption key can be determined by each device without being transmitted between the sensor110or data receiving device120. At step635, the sensor110can encrypt data to be included in payload. At step640, the sensor110can transmit the encrypted payload640to the data receiving device120using the communication link established between the appropriate communication models of the sensor110and data receiving device120. At step645, the data receiving device120can decrypt the payload using the sensor-unique encryption key derived during step630. Following step645, the sensor110can deliver additional (including newly collected) data and the data receiving device120can process the received data appropriately.

As discussed herein, the sensor110can be a device with restricted processing power, battery supply, and storage. The encryption techniques used by the sensor110(e.g., the cipher algorithm or the choice of implementation of the algorithm) can be selected based at least in part on these restrictions. The data receiving device120can be a more powerful device with fewer restrictions of this nature. Therefore, the data receiving device120can employ more sophisticated, computationally intense encryption techniques, such as cipher algorithms and implementations.

The analyte sensor110can be configured to alter its discoverability behavior to attempt to increase the probability of the receiving device receiving an appropriate data packet and/or provide an acknowledgement signal or otherwise reduce restrictions that can be causing an inability to receive an acknowledgement signal. Altering the discoverability behavior of the analyte sensor110can include, for example and without limitation, altering the frequency at which connection data is included in a data packet, altering how frequently data packets are transmitted generally, lengthening or shortening the broadcast window for data packets, altering the amount of time that the analyte sensor110listens for acknowledgement or scan signals after broadcasting, including directed transmissions to one or more devices (e.g., through one or more attempted transmissions) that have previously communicated with the analyte sensor110and/or to one or more devices on a whitelist, altering a transmission power associated with the communication module when broadcasting the data packets (e.g., to increase the range of the broadcast or decrease energy consumed and extend the life of the battery of the analyte sensor), altering the rate of preparing and broadcasting data packets, or a combination of one or more other alterations. Additionally, or alternatively, the receiving device can similarly adjust parameters relating to the listening behavior of the device to increase the likelihood of receiving a data packet including connection data.

As embodied herein, the analyte sensor110can be configured to broadcast data packets using two types of windows. The first window refers to the rate at which the analyte sensor110is configured to operate the communication hardware. The second window refers to the rate at which the analyte sensor110is configured to be actively transmitting data packets (e.g., broadcasting). As an example, the first window can indicate that the analyte sensor110operates the communication hardware to send and/or receive data packets (including connection data) during the first 2 seconds of each 60 second period. The second window can indicate that, during each 2 second window, the analyte sensor110transmits a data packet every 60 milliseconds. The rest of the time during the 2 second window, the analyte sensor110is scanning. The analyte sensor110can lengthen or shorten either window to modify the discoverability behavior of the analyte sensor110.

In particular embodiments, the discoverability behavior of the analyte sensor can be stored in a discoverability profile, and alterations can be made based on one or more factors, such as the status of the analyte sensor110and/or by applying rules based on the status of the analyte sensor110. For example, when the battery level of the analyte sensor110is below a certain amount, the rules can cause the analyte sensor110to decrease the power consumed by the broadcast process. As another example, configuration settings associated with broadcasting or otherwise transmitting packets can be adjusted based on the ambient temperature, the temperature of the analyte sensor110, or the temperature of certain components of communication hardware of the analyte sensor110. In addition to modifying the transmission power, other parameters associated with the transmission capabilities or processes of the communication hardware of the analyte sensor110can be modified, including, but not limited to, transmission rate, frequency, and timing. As another example, when the analyte data indicates that the subject is, or is about to be, experiencing a negative health event, the rules can cause the analyte sensor110to increase its discoverability to alert the receiving device of the negative health event.

As embodied herein, certain calibration features for the sensing hardware5060of the analyte sensor110can be adjusted based on external or interval environment features as well as to compensate for the decay of the sensing hardware5060during expended period of disuse (e.g., a “shelf time” prior to use). The calibration features of the sensing hardware5060can be autonomously adjusted by the sensor110(e.g., by operation of the ASIC5000to modify features in the memory5020or storage5030) or can be adjusted by other devices of the analyte monitoring system100.

As an example, sensor sensitivity of the sensing hardware5060can be adjusted based on external temperature data or the time since manufacture. When external temperatures are monitored during the storage of the sensors, the disclosed subject matter can adaptively change the compensation to sensor sensitivity over time when the device experiences changing storage conditions. For purpose of illustration not limitations, adaptive sensitivity adjustment can be performed in an “active” storage mode where the analyte sensor110wakes up periodically to measure temperature. These features can save the battery of the analyte device and extend the lifespan of the analyte sensors. At each temperature measurement, the analyte sensor110can calculate a sensitivity adjustment for that time period based on the measured temperature. Then, the temperature-weighted adjustments can be accumulated over the active storage mode period to calculate a total sensor sensitivity adjustment value at the end of the active storage mode (e.g., at insertion). Similarly, at insertion, the sensor110can determine the time difference between manufacture of the sensor110(which can be written to the storage5030of the ASIC5000) or the sensing hardware5060and modify sensor sensitivity or other calibration features according to one or more known decay rates or formulas.

Additionally, for purpose of illustration and not limitation, as embodied herein, sensor sensitivity adjustments can account for other sensor conditions, such as sensor drift. Sensor sensitivity adjustments can be hardcoded into the sensor110during manufacture, for example in the case of sensor drift, based on an estimate of how much an average sensor would drift. Sensor110can use a calibration function that has time-varying functions for sensor offset and gain, which can account for drift over a wear period of the sensor. Thus, sensor110can utilize a function used to transform an interstitial current to interstitial glucose utilizing device-dependent functions describing sensor110drift over time, and which can represent sensor sensitivity, and can be device specific, combined with a baseline of the glucose profile. Such functions to account for sensor sensitivity and drill can improve sensor110accuracy over a wear period and without involving user calibration.

The sensor110detects raw measurement values from sensing hardware5060. On-sensor processing can be performed, such as by one or more models trained to interpret the raw measurement values. Models can be machine learned models trained off-device to detect, predict, or interpret the raw measurement values to detect, predict, or interpret the levels of one or more analytes. Additional trained models can operate on the output of the machine learning models trained to interact with raw measurement values. As an example, models can be used to detect, predict, or recommend events based on the raw measurements and type of analyte(s) detected by the sensing hardware5060. Events can include, initiation or completion of physical activity, meals, application of medical treatment or medication, emergent health events, and other events of a similar nature.

Models can be provided to the sensor110, data receiving device120, or multi-purpose data receiving device130during manufacture or during firmware or software updates. Models can be periodically refined, such as by the manufacturer of the sensor110or the operator of the analyte monitoring system100, based on data received from the sensor110and data receiving devices of an individual user or multiple users collectively. In certain embodiments, the sensor110includes sufficient computational components to assist with further training or refinement of the machine learned models, such as based on unique features of the user to which the sensor110is attached. Machine learning models can include, by way of example and not limitation, models trained using or encompassing decision tree analysis, gradient boosting, ada boosting, artificial neural networks or variants thereof, linear discriminant analysis, nearest neighbor analysis, support vector machines, supervised or unsupervised classification, and others. The models can also include algorithmic or rules-based models in addition to machine learned models. Model-based processing can be performed by other devices, including the data receiving device120or multi-purpose data receiving device130, upon receiving data from the sensor110(or other downstream devices).

R. Exemplary Alarm Features

Data transmitted between the sensor110and a data receiving device120can include raw or processed measurement values. Data transmitted between the sensor110and data receiving device120can further include alarms or notification for display to a user. The data receiving device120can display or otherwise convey notifications to the user based on the raw or processed measurement values or can display alarms when received from the sensor110. Alarms that may be triggered for display to the user include alarms based on direct analyte values (e.g., one-time reading exceeding a threshold or failing to satisfy a threshold), analyte value trends (e.g., average reading over a set period of time exceeding a threshold or failing to satisfy a threshold; slope); analyte value predictions (e.g., algorithmic calculation based on analyte values exceeds a threshold or fails to satisfy a threshold), sensor alerts (e.g., suspected malfunction detected), communication alerts (e.g., no communication between sensor110and data receiving device120for a threshold period of time; unknown device attempting or failing to initiate a communication session with the sensor110), reminders (e.g., reminder to charge data receiving device120; reminder to take a medication or perform other activity), and other alerts of a similar nature. For purpose of illustration and not limitation, as embodied herein, the alarm parameters described herein can be configurable by a user or can be fixed during manufacture, or combinations of user-settable and non-user-settable parameters.

Sensor configurations featuring a single active area that is configured for the detection of a corresponding single analyte can employ two-electrode or three-electrode detection motifs, as described further herein in reference toFIGS. 18A-18C. Sensor configurations featuring two different active areas for detection of the same or separate analytes, either upon separate working electrodes or upon the same working electrode, are described separately thereafter in reference toFIGS. 19A-21C. Sensor configurations having multiple working electrodes can be particularly advantageous for incorporating two different active areas within the same sensor tail, since the signal contribution from each active area can be determined more readily.

When a single working electrode is present in an analyte sensor, three-electrode sensor configurations can include a working electrode, a counter electrode, and a reference electrode. Related two-electrode sensor configurations can include a working electrode and a second electrode, in which the second electrode can function as both a counter electrode and a reference electrode (i.e., a counter/reference electrode). The various electrodes can be at least partially stacked (layered) upon one another and/or laterally spaced apart from one another upon the sensor tail. Suitable sensor configurations can be substantially flat in shape or substantially cylindrical in shape or any other suitable shape. In any of the sensor configurations disclosed herein, the various electrodes can be electrically isolated from one another by a dielectric material or similar insulator.

Analyte sensors featuring multiple working electrodes can similarly include at least one additional electrode. When one additional electrode is present, the one additional electrode can function as a counter/reference electrode for each of the multiple working electrodes. When two additional electrodes are present, one of the additional electrodes can function as a counter electrode for each of the multiple working electrodes and the other of the additional electrodes can function as a reference electrode for each of the multiple working electrodes.

FIG. 18Ashows a diagram of an illustrative two-electrode analyte sensor configuration, which is compatible for use in the disclosure herein. As shown, analyte sensor200includes substrate30212disposed between working electrode214and counter/reference electrode30216. Alternately, working electrode214and counter/reference electrode30216can be located upon the same side of substrate30212with a dielectric material interposed in between (configuration not shown). Active area218is disposed as at least one layer upon at least a portion of working electrode214. Active area218can include multiple spots or a single spot configured for detection of an analyte, as discussed further herein. In certain embodiments, active area218is configured to detect potassium as described herein.

Referring still toFIG. 18A, membrane220overcoats at least active area218. In certain embodiments, membrane220can also overcoat some or all of working electrode214and/or counter/reference electrode30216, or the entirety of analyte sensor200. One or both faces of analyte sensor200can be overcoated with membrane220. Membrane220can include one or more polymeric membrane materials having capabilities of limiting analyte flux to active area218(i.e., membrane220is a mass transport limiting membrane having some permeability for the analyte of interest). According to the disclosure herein, and further described below, membrane220can be crosslinked with a branched crosslinker in certain particular sensor configurations. For example, but not by way of limitation, membrane220is crosslinked with a crosslinking agent described herein, e.g., a branched glycidyl ether. The composition and thickness of membrane220can vary to promote a desired analyte flux to active area218, thereby providing a desired signal intensity and stability. Analyte sensor200can be operable for assaying an analyte, e.g., potassium, by any of coulometric, amperometric, voltammetric, or potentiometric electrochemical detection techniques.

FIGS. 18B and 18Cshow diagrams of illustrative three-electrode analyte sensor configurations, which are also compatible for use in the disclosure herein. Three-electrode analyte sensor configurations can be similar to that shown for analyte sensor200inFIG. 18A, except for the inclusion of additional electrode217in analyte sensors201and202(FIGS. 18B and 18C). With additional electrode217, counter/reference electrode30216can then function as either a counter electrode or a reference electrode, and additional electrode217fulfills the other electrode function not otherwise accounted for. Working electrode214continues to fulfill its original function. Additional electrode217can be disposed upon either working electrode214or electrode30216, with a separating layer of dielectric material in between. For example, and not by the way of limitation, as depicted inFIG. 18B, dielectric layers219a,219band219cseparate electrodes214,30216and217from one another and provide electrical isolation. Alternatively, at least one of electrodes214,30216and217can be located upon opposite faces of substrate30212, as shown inFIG. 18C. Thus, in certain embodiments, electrode214(working electrode) and electrode30216(counter electrode) can be located upon opposite faces of substrate30212, with electrode217(reference electrode) being located upon one of electrodes214or30216and spaced apart therefrom with a dielectric material. Reference material layer230(e.g., Ag/AgCl) can be present upon electrode217, with the location of reference material layer230not being limited to that depicted inFIGS. 18B and 18C. As with sensor200shown inFIG. 18A, active area218in analyte sensors201and202can include multiple spots or a single spot. Additionally, analyte sensors201and202can be operable for assaying an analyte, e.g., potassium, by any of coulometric, amperometric, voltammetric or potentiometric electrochemical detection techniques.

Like analyte sensor200, membrane220can also overcoat active area218, as well as other sensor components, in analyte sensors201and202, thereby serving as a mass transport limiting membrane. In certain embodiments, the additional electrode217can be overcoated with membrane220. AlthoughFIGS. 18B and 18Chave depicted electrodes214,30216and217as being overcoated with membrane220, it is to be recognized that in certain embodiments only working electrode214is overcoated. Moreover, the thickness of membrane220at each of electrodes214,30216and217can be the same or different. As in two-electrode analyte sensor configurations (FIG. 18A), one or both faces of analyte sensors201and202can be overcoated with membrane220in the sensor configurations ofFIGS. 18B and 18C, or the entirety of analyte sensors201and202can be overcoated. Accordingly, the three-electrode sensor configurations shown inFIGS. 18B and 18Cshould be understood as being non-limiting of the embodiments disclosed herein, with alternative electrode and/or layer configurations remaining within the scope of the present disclosure.

FIG. 19Ashows an illustrative configuration for sensor203having a single working electrode with two different active areas disposed thereon.FIG. 19Ais similar toFIG. 18A, except for the presence of two active areas upon working electrode214: first active area218aand second active area218b, which are responsive to the same or different analytes and are laterally spaced apart from one another upon the surface of working electrode214. Active areas218aand218bcan include multiple spots or a single spot configured for detection of each analyte. The composition of membrane220can vary or be compositionally the same at active areas218aand218b. First active area218aand second active area218bcan be configured to detect their corresponding analytes at working electrode potentials that differ from one another, as discussed further below. In certain embodiments, any one of active areas218aand218b, or both, can be configured to detect potassium. In certain embodiments, any one of active areas218aand218b, or both, can be configured to detect potassium by using aspartate oxidase. In certain embodiments, any one of active areas218aand218b, or both, can be configured to detect potassium by using aspartate oxidase and asparaginase. In certain embodiments, only one active area of218aand218bis configured to detect potassium. In certain embodiments, the other active area is configured to detect a second analyte that is different from potassium. Non-limiting examples of second analytes are described herein.

FIGS. 19B and 19Cshow cross-sectional diagrams of illustrative three-electrode sensor configurations for sensors204and205, respectively, each featuring a single working electrode having first active area218aand second active area218bdisposed thereon.FIGS. 19B and 19Care otherwise similar toFIGS. 18B and 18Cand can be better understood by reference thereto. As withFIG. 19A, the composition of membrane220can vary or be compositionally the same at active areas218aand218b.

Illustrative sensor configurations having multiple working electrodes, specifically two working electrodes, are described in further detail in reference toFIGS. 20-21C. Although the following description is primarily directed to sensor configurations having two working electrodes, it is to be appreciated that more than two working electrodes can be incorporated through extension of the disclosure herein. Additional working electrodes can be used to impart additional sensing capabilities to the analyte sensors beyond just a first analyte and a second analyte, e.g., for the detection of a third and/or fourth analyte.

FIG. 20shows a cross-sectional diagram of an illustrative analyte sensor configuration having two working electrodes, a reference electrode and a counter electrode, which is compatible for use in the disclosure herein. As shown, analyte sensor300includes working electrodes304and306disposed upon opposite faces of substrate302. First active area310ais disposed upon the surface of working electrode304, and second active area310bis disposed upon the surface of working electrode306. Counter electrode320is electrically isolated from working electrode304by dielectric layer322, and reference electrode321is electrically isolated from working electrode306by dielectric layer323. Outer dielectric layers30230and332are positioned upon reference electrode321and counter electrode320, respectively. Membrane340can overcoat at least active areas310aand310b, according to various embodiments, with other components of analyte sensor300or the entirety of analyte sensor300optionally being overcoated with membrane340. In certain embodiments, membrane340can be continuous but vary compositionally within first membrane portion340aand second membrane portion340b(i.e., upon active areas310aand310b) in order to afford different permeability values for differentially regulating the analyte flux at each location. For example, but not by way of limitation, a first membrane portion340acan overcoat at least active area310aand a second membrane portion340bcan overcoat at least active area310b, according to various embodiments, with other components of analyte sensor300or the entirety of analyte sensor300.

In certain embodiments, different membrane formulations can be sprayed and/or printed onto the opposing faces of analyte sensor300. Dip coating techniques can also be appropriate, particularly for depositing at least a portion of a bilayer membrane upon one of active areas310aand310b. In certain embodiments, membrane340can be the same or vary compositionally at active areas310aand310b. For example, but not by way of limitation, membrane340can include a bilayer overcoating active area310aand be a homogeneous membrane overcoating active area310b, or membrane340can include a bilayer overcoating active areas310band be a homogeneous membrane overcoating active area310a. In certain embodiments, one of the first membrane portion340aand the second membrane portion340bcan comprise a bilayer membrane and the other of the first membrane portion340aand the second membrane portion340bcan comprise a single membrane polymer, according to particular embodiments of the present disclosure. In certain embodiments, an analyte sensor can include more than one membrane340, e.g., two or more membranes. For example, but not by way of limitation, an analyte sensor can include a membrane that overcoats the one or more active areas, e.g.,310aand310b, and an additional membrane that overcoats the entire sensor as shown inFIG. 20. In such configurations, a bilayer membrane can be formed over the one or more active areas, e.g.,310aand310b.

In certain embodiments, any one of active areas310aand310b, or both, can be configured to detect potassium, e.g., by using an aspartate oxidase. In certain embodiments, any one of active areas310aand310b, or both, can further comprise an asparaginase. In certain embodiments, only one active area of310aand310bis configured to detect potassium, e.g., by using an aspartate oxidase. In certain embodiments, only one active area of310aand310bcan further comprise an asparaginase. In certain embodiments, the other active area is configured to detect a second analyte.

Alternative sensor configurations having multiple working electrodes and differing from the configuration shown inFIG. 20can feature a counter/reference electrode instead of separate counter and reference electrodes320,321, and/or feature layer and/or membrane arrangements varying from those expressly depicted. For example, and not by the way of limitation the positioning of counter electrode320and reference electrode321can be reversed from that depicted inFIG. 20. In addition, working electrodes304and306need not necessarily reside upon opposing faces of substrate302in the manner shown inFIG. 20.

Although suitable sensor configurations can feature electrodes that are substantially planar in character, it is to be appreciated that sensor configurations featuring non-planar electrodes can be advantageous and particularly suitable for use in the disclosure herein. In particular, substantially cylindrical electrodes that are disposed concentrically with respect to one another can facilitate deposition of a mass transport limiting membrane, as described hereinbelow. In particular, concentric working electrodes that are spaced apart along the length of a sensor tail can facilitate membrane deposition through sequential dip coating operations, in a similar manner to that described above for substantially planar sensor configurations.FIGS. 21A-21Cshow perspective views of analyte sensors featuring two working electrodes that are disposed concentrically with respect to one another. It is to be appreciated that sensor configurations having a concentric electrode disposition but lacking a second working electrode are also possible in the present disclosure.

FIG. 21Ashows a perspective view of an illustrative sensor configuration in which multiple electrodes are substantially cylindrical and are disposed concentrically with respect to one another about a central substrate. As shown, analyte sensor400includes central substrate402about which all electrodes and dielectric layers are disposed concentrically with respect to one another. In particular, working electrode410is disposed upon the surface of central substrate402, and dielectric layer412is disposed upon a portion of working electrode410distal to sensor tip404. Working electrode420is disposed upon dielectric layer412, and dielectric layer422is disposed upon a portion of working electrode420distal to sensor tip404. Counter electrode430is disposed upon dielectric layer422, and dielectric layer432is disposed upon a portion of counter electrode430distal to sensor tip404. Reference electrode440is disposed upon dielectric layer432, and dielectric layer442is disposed upon a portion of reference electrode440distal to sensor tip404. As such, exposed surfaces of working electrode410, working electrode420, counter electrode430, and reference electrode440are spaced apart from one another along longitudinal axis B of analyte sensor400.

Referring still toFIG. 21A, first active areas414aand second active areas414b, which are responsive to different analytes or the same analyte, are disposed upon the exposed surfaces of working electrodes410and420, respectively, thereby allowing contact with a fluid to take place for sensing. Although active areas414aand414bhave been depicted as three discrete spots inFIG. 21A, it is to be appreciated that fewer or greater than three spots, including a continuous layer of active area, can be present in alternative sensor configurations as described herein. In certain embodiments, any one of active areas414aand414b, or both, can be configured to detect potassium. In certain embodiments, any one of active areas414aand414b, or both, can be configured to detect potassium by using an enzyme system comprising an aspartate oxidase and, optionally, an asparaginase. In certain embodiments, only one active area of414aand414bis configured to detect potassium. In certain embodiments, only one active area of414aand414bis configured to detect potassium by using an aspartate oxidase and, optionally, an asparaginase. In certain embodiments, the other active area is configured to detect a second analyte.

InFIG. 21A, sensor400is partially coated with membrane450upon working electrodes410and420and active areas414aand414bdisposed thereon.FIG. 21Bshows an alternative sensor configuration in which the substantial entirety of sensor401is overcoated with membrane450. Membrane450can be the same or vary compositionally at active areas414aand414b. For example, membrane450can include a bilayer overcoating active areas414aand be a homogeneous membrane overcoating active areas414b.

It is to be further appreciated that the positioning of the various electrodes inFIGS. 21A and 21Bcan differ from that expressly depicted. For example, the positions of counter electrode430and reference electrode440can be reversed from the depicted configurations inFIGS. 21A and 21B. Similarly, the positions of working electrodes410and420are not limited to those that are expressly depicted inFIGS. 21A and 21B.FIG. 21Cshows an alternative sensor configuration to that shown inFIG. 21B, in which sensor405contains counter electrode430and reference electrode440that are located more proximal to sensor tip404and working electrodes410and420that are located more distal to sensor tip404. Sensor configurations in which working electrodes410and420are located more distal to sensor tip404can be advantageous by providing a larger surface area for deposition of active areas414aand414b(five discrete sensing spots illustratively shown inFIG. 21C), thereby facilitating an increased signal strength in some cases. Similarly, central substrate402can be omitted in any concentric sensor configuration disclosed herein, wherein the innermost electrode can instead support subsequently deposited layers.

In certain embodiments, one or more electrodes of an analyte sensor described herein is a wire electrode, e.g., a permeable wire electrode. In certain embodiments, the sensor tail comprises a working electrode and a reference electrode helically wound around the working electrode. In certain embodiments, an insulator is disposed between the working and reference electrodes. In certain embodiments, portions of the electrodes are exposed to allow reaction of the one or more enzymes with an analyte on the electrode. In certain embodiments, each electrode is formed from a line wire with a diameter of from about 0.001 inches or less to about 0.010 inches or more. In certain embodiments, the working electrode has a diameter of from about 0.001 inches or less to about 0.010 inches or more, e.g., from about 0.002 inches to about 0.008 inches, and more preferably from about 0.004 inches to about 0.005 inches. In certain embodiments, an electrode is formed from a plated insulator, a plated wire or bulk electrically conductive material. In certain embodiments, the working electrode comprises a wire formed from a conductive material, such as platinum, platinum-iridium, palladium, graphite, gold, carbon, conductive polymer, alloys or the like. In certain embodiments, the conductive material is a permeable conductive material. In certain embodiments, the electrodes can be formed by a variety of manufacturing techniques (e.g., bulk metal processing, deposition of metal onto a substrate or the like), the electrodes can be formed from plated wire (e.g., platinum on steel wire) or bulk metal (e.g., platinum wire). In certain embodiments, the electrode is formed from tantalum wire, e.g., coated in a conductive material.

In certain embodiments, the reference electrode, which can function as a reference electrode alone, or as a dual reference and counter electrode, is formed from silver, silver/silver chloride or the like. In certain embodiments, the reference electrode is juxtaposed and/or twisted with or around the working electrode. In certain embodiments, the reference electrode is helically wound around the working electrode. In certain embodiments, the assembly of wires can be coated or adhered together with an insulating material so as to provide an insulating attachment.

In certain embodiments, additional electrodes, e.g., wire electrodes, can be included in the sensor tail. For example, but not by way of limitation, a three-electrode system (a working electrode, a reference electrode and a counter electrode) and/or an additional working electrode (e.g., an electrode for detecting a second analyte) can be included in the sensor tail. In certain embodiments where the sensor comprises two working electrodes, the two working electrodes can be juxtaposed around which the reference electrode is disposed upon (e.g., helically wound around the two or more working electrodes). In certain embodiments, the two or more working electrodes can extend parallel to each other. In certain embodiments, the reference electrode is coiled around the one or more working electrodes and extends towards the distal end (i.e., in vivo end) of the sensor tail. In certain embodiments, the reference electrode extends (e.g., helically) to the exposed region of the one or more working electrodes.

In certain embodiments, one or more working electrodes are helically wound around a reference electrode. In certain embodiments where two or more working electrodes are provided, the working electrodes can be formed in a double-, triple-, quad-, etc. helix configuration along the length of the sensor tail (for example, surrounding a reference electrode, insulated rod or other support structure). In certain embodiments, the electrodes, e.g., two or more working electrodes, are coaxially formed. For example, but not by way limitation, the electrodes all share the same central axis.

In certain embodiments, the working electrode comprises a tube with a reference electrode disposed or coiled inside, including an insulator therebetween. Alternatively, the reference electrode comprises a tube with a working electrode disposed or coiled inside, including an insulator therebetween. In certain embodiments, a polymer (e.g., insulating) rod is provided, wherein the one or more electrodes (e.g., one or more electrode layers) are disposed upon (e.g., by electro-plating). In certain embodiments, a metallic (e.g., steel or tantalum) rod or wire is provided, coated with an insulating material (described herein), onto which the one or more working and reference electrodes are disposed upon. For example, but not by way of limitation, the present disclosure provides a sensor, e.g., a sensor tail, that comprises one or more tantalum wires, where a conductive material is disposed upon a portion of the one or more tantalum wires to function as a working electrode. In certain embodiments, the platinum-clad tantalum wire is covered with an insulating material, where the insulating material is partially covered with a silver/silver chloride composition to function as a reference and/or counter electrode.

In certain embodiments where an insulator is disposed upon the working electrode (e.g., upon the platinum surface of the electrode), a portion of the insulator can be stripped or otherwise removed to expose the electroactive surface of the working electrode. For example, but not by way of limitation, a portion of the insulator can be removed by hand, excimer lasing, chemical etching, laser ablation, grit-blasting or the like. Alternatively, a portion of the electrode can be masked prior to depositing the insulator to maintain an exposed electroactive surface area. In certain embodiments, the portion of the insulator that is stripped and/or removed can be from about 0.1 mm (about 0.004 inches) or less to about 2 mm (about 0.078 inches) or more in length, e.g., from about 0.5 mm (about 0.02 inches) to about 0.75 mm (0.03 inches) in length. In certain embodiments, the insulator is a non-conductive polymer. In certain embodiments, the insulator comprises parylene, fluorinated polymers, polyethylene terephthalate, polyvinylpyrrolidone, polyurethane, polyimide and other non-conducting polymers. In certain embodiments, glass or ceramic materials can also be used in the insulator layer. In certain embodiments, the insulator comprises parylene. In certain embodiments, the insulator comprises a polyurethane. In certain embodiments, the insulator comprises a polyurethane and polyvinylpyrrolidone.

Several parts of the sensor, including the active areas, are further described below.

An active area of a presently disclosed analyte sensor can be configured for detecting an analyte. In certain embodiments, the presently disclosed analyte sensors are configured to measure potassium, e.g., potassium ions, in a sample. For example, but not by way of limitation, an active area of a presently disclosed analyte sensor is configured to detect potassium ions.

In certain embodiments, the presently disclosed analyte sensors are configured to indirectly detect potassium ions by comparing signals obtained from at least one, e.g., at least two, aspartate-responsive active areas. In certain embodiments, the current signal from the two aspartate-responsive active areas can be correlated to the concentration of potassium ions, as discussed below.

In certain embodiments, the presently disclosed analyte sensors are configured to indirectly detect potassium ions by comparing signals obtained from at least one, e.g., at least two, asparagine-responsive active areas. In certain embodiments, the current signal from the two asparagine-responsive active areas can be correlated to the concentration of potassium ions, as discussed below.

In certain embodiments, an active area of the present disclosure is disposed upon a portion of a working electrode. For example, but not by way of limitation, an active area is disposed upon a portion of the working electrode in a spotted pattern, e.g., two or more spots on the working electrode. In certain embodiments, an active area is disposed upon a portion of the working electrode in a slotted pattern. In certain embodiments, an active area is disposed upon the entire length of the working electrode or in a continuous pattern on the working electrode.

In certain embodiments, an active area of the present disclosure can have a thickness from about 0.1 μm to about 100 μm, e.g., from about 1 μm to about 90 μm, from about 1 μm to about 80 μm, from about 1 μm to about 70 μm, from about 1 μm to about 60 μm, from about 1 μm to about 50 μm, from about 1 μm to about 40 μm, from about 1 μm to about 30 μm, from about 1 μm to about 20 μm, from about 0.5 μm to about 10 μm, from about 1 μm to about 10 μm, from about 1 μm to about 5 μm or from about 0.1 μm to about 5 μm. In certain embodiments, an active area of the present disclosure has an area of about 0.01 mm to about 2.0 mm=, e.g., about 0.1 mm2to about 1.0 mm2or about 0.2 mm2to about 0.5 mm2.

It is to be appreciated that the sensitivity (output current) of the analyte sensors toward each analyte can be varied by changing the coverage (area or size) of the active areas, the area ratio of the active areas with respect to one another, the identity, thickness and/or composition of a mass transport limiting membrane overcoating the active areas. Variation of these parameters can be conducted readily by one having ordinary skill in the art once granted the benefit of the disclosure herein.

In certain embodiments, an analyte sensor of the present disclosure includes at least one active area, e.g., two active areas, configured to detect aspartate to facilitate indirect measurement of potassium ion concentration in a sample. A particular enzyme that can be used for detecting aspartate is shown inFIG. 27. In certain embodiments, the enzyme present in the aspartate-responsive active area is an aspartate oxidase. As shown inFIG. 27, aspartate oxidase can catalyze the oxidation of aspartate to oxaloacetate and the reduction of its coenzyme flavin adenine dinucleotide (FAD) to FADH2. An electron transfer agent can then mediate the electron transfer from FADH2to the working electrode. The electrochemical signal obtained at the working electrode can then be correlated to the amount of potassium ions that was initially present in the sample.

Particular examples of aspartate oxidases suitable for use in the analyte sensors disclosed herein include, but are not limited to, potassium-dependent and potassium-independent aspartate oxidases. In certain embodiments, the aspartate oxidase used in the enzyme system is potassium-dependent. For example, but not by way of limitation, the activity of the aspartate oxidase is dependent on potassium ion concentration thereby allowing the indirect determination of potassium levels in sample. In certain other embodiments, the aspartate oxidase used in the enzyme system is potassium-independent. Non-limiting examples of an aspartate oxidase for use in the present disclosure include L-aspartate oxidases from a species of the generaThermococcus, Pyrococcus, Sulfolobusand Halobacteria. In certain embodiments, the L-aspartate oxidase is fromSulfolobus tokodaiiorThermococcus litoralis. For example, but not by way of limitation, L-aspartate oxidases for use in the potassium sensors of the present disclosure are disclosed in Nasu et al., J. of Biological Chemistry 257(2):626-32 (1982); Bifulco et al., Appl. Microbiol. Biotechnol. 97(16):7285-95 (2013); Washio et al., Extremophiles 22(1):59-71 (2018); and Hao et al., Plant Science 271:133-142 (2018), the contents of each of which are incorporated herein by reference in their entireties.

In certain embodiments, the aspartate-responsive active area can include an aspartate oxidase, e.g., a potassium-independent aspartate oxidase or a potassium-independent aspartate oxidase. In certain embodiments, the aspartate-responsive active area active area can include an enzyme system consisting essentially of an aspartate oxidase. In certain embodiments, the aspartate-responsive active area active area can include an enzyme system consisting of an aspartate oxidase. In certain embodiments, an aspartate-responsive active area can include by weight from about 10% to about 80%, e.g., from about 15% to about 75%, from about 20% to about 70%, from about 25% to about 65%, from about 20% to about 60%, from about 20% to about 55%, from about 20% to about 50%, from about 20% to about 45%, from about 20% to about 40%, from about 20% to about 35% or from about 20% to about 30% of an aspartate oxidase. In certain embodiments, an active area can include by weight from about 10% to about 40% of an aspartate oxidase. In certain embodiments, an aspartate-responsive active area can include by weight from about 15% to about 35% of an aspartate oxidase. In certain embodiments, an asparagine-responsive active area can include by weight from about 20% to about 30% of an aspartate oxidase.

In certain embodiments, the aspartate-responsive active area can further include a stabilizer. In certain embodiments, the stabilizer is used for enzyme stabilization. For example, but not byway of limitation, the stabilizer can be an albumin, e.g., a serum albumin. Non-limiting examples of serum albumins include a bovine serum albumin and a human serum albumin. In certain embodiments, the stabilizer is a human serum albumin. In certain embodiments, the stabilizer is a bovine serum albumin. In certain embodiments, the aspartate-responsive active area can include a ratio of stabilizer to aspartate oxidase from about 40:1 to about 1:40, e.g., from about 35:1 to about 1:35, from about 30:1 to about 1:30, from about 25:1 to about 1:25, from about 20:1 to about 1:20, from about 15:1 to about 1:15, from about 10:1 to about 1:10, from about 9:1 to about 1:9, from about 8:1 to about 1:8, from about 7:1 to about 1:7, from about 6:1 to about 1:6, from about 5:1 to about 1:5, from about 4:1 to about 1:4, from about 3:1 to about 1:3, from about 2:1 to about 1:2 or about 1:1. In certain embodiments, the aspartate-responsive active area can include a ratio of stabilizing agent to aspartate oxidase from about 5:1 to about 1:5. In certain embodiments, the aspartate-responsive active area can include a ratio of stabilizing agent to aspartate oxidase from about 4:1 to about 1:4. In certain embodiments, the aspartate-responsive active area can include a ratio of stabilizing agent to aspartate oxidase from about 3:1 to about 1:3. In certain embodiments, the aspartate-responsive active area can include a ratio of stabilizing agent to aspartate oxidase from about 2:1 to about 1:2.

In certain embodiments, an analyte sensor of the present disclosure for indirect measurement of potassium can include a first potassium-dependent channel comprising a first aspartate-responsive active area and a second potassium-dependent channel comprising a second aspartate-responsive active area. In certain embodiments, each channel is a working electrode. In certain embodiments, the first potassium-dependent channel includes a first potassium-dependent aspartate oxidase. In certain embodiments, the second potassium-dependent channel includes a second potassium-dependent aspartate oxidase. In such embodiments, the first potassium-dependent aspartate oxidase and the second potassium-dependent aspartate oxidase exhibit different dependencies on potassium and would, therefore, result in different signals in the presence of the same potassium concentration. In certain embodiments, the difference in signals obtained from the first potassium-dependent channel and the second potassium-dependent channel can be correlated to the concentration of the potassium ions in the sample.

In certain embodiments, an analyte sensor of the present disclosure for indirect measurement of potassium can include a first potassium-dependent channel and a second potassium-dependent channel. In certain embodiments, each channel is a working electrode. In certain embodiments, the first potassium-dependent channel801includes a first potassium-dependent aspartate oxidase. In certain embodiments, the second potassium-dependent channel includes a second potassium-dependent aspartate oxidase. In such embodiments, the first potassium-dependent aspartate oxidase and the second potassium-dependent aspartate oxidase exhibit different dependencies on potassium and would, therefore, result in different signals in the presence of the same potassium concentration. In certain embodiments, the difference in signals obtained from the two channels can be correlated to the concentration of the potassium ions in the sample.

In certain embodiments, an analyte sensor of the present disclosure can include a sensor tail including at least two working electrodes. In certain embodiments, an aspartate-responsive active area (e.g., a first aspartate-responsive active area) is disposed upon the surface of the first working electrode and an aspartate-responsive active area (e.g., a second aspartate-responsive active area) is disposed upon the surface of the second working electrode, where each aspartate-responsive active area includes an aspartate oxidase. In certain embodiments, the first aspartate-responsive active area includes a potassium-independent aspartate oxidase and the second aspartate-responsive active area includes a potassium-dependent aspartate oxidase. Alternatively, the first aspartate-responsive active area includes a first potassium-dependent aspartate oxidase and the second aspartate-responsive active area includes a second potassium-dependent aspartate oxidase, where the aspartate oxidases have different dependencies on potassium ion concentration.

In certain embodiments, an analyte sensor of the present disclosure includes at least one active area, e.g., two active areas, configured to detect asparagine to facilitate indirect measurement of potassium ion concentration in a sample. A particular enzyme system that can be used for detecting asparagine is shown inFIG. 22. As shown inFIG. 22, asparaginase catalyzes the hydrolysis of asparagine to produce aspartate. Aspartate oxidase can then catalyze the oxidation of aspartate to oxaloacetate and the reduction of its coenzyme flavin adenine dinucleotide (FAD) to FADH2. An electron transfer reagent can then mediate the electron transfer from FADH2to the working electrode.

Particular examples of asparaginases suitable for use in the analyte sensors disclosed herein include, but are not limited to, potassium-dependent and potassium-independent asparaginases. In certain embodiments, the asparaginase used in the enzyme system is potassium-dependent. In certain other embodiments, the asparaginase used in the enzyme system is potassium-independent.

Non-limiting examples of potassium-independent and potassium-dependent asparaginases are disclosed in Ajewole et al., FEBS Journal 285(8):1528-1539 (2018) and Bejger et al., Acta Crystallogr. D. Biol. Crystallogr. 70(Pt 7):1854-72 (2014), the contents of which are incorporated herein by reference in their entireties. In certain embodiments, an asparaginase for use in the present disclosure is the potassium-dependent (PvAspG1) and/or potassium-independent (PvAspG-T2) asparaginases fromPhaseolus vulgaris. In certain embodiments, the asparaginase includes a substitution of the amino acid at position 118 or position 117 of PvAspG1 and PvAspG-T2, respectively.

In certain embodiments, the asparagine-responsive active area can further include a stabilizer, e.g., for stabilizing the enzyme. For example, but not by way of limitation, the stabilizer can be an albumin, e.g., a serum albumin. Non-limiting examples of serum albumins include bovine serum albumin and human serum albumin. In certain embodiments, the stabilizer is a human serum albumin. In certain embodiments, the stabilizer is a bovine serum albumin. In certain embodiments, the asparagine-responsive active area can include a ratio of stabilizing agent to asparaginase and/or aspartate oxidase from about 40:1 to about 1:40, e.g., from about 35:1 to about 1:35, from about 30:1 to about 1:30, from about 25:1 to about 1:25, from about 20:1 to about 1:20, from about 15:1 to about 1:15, from about 10:1 to about 1:10, from about 9:1 to about 1:9, from about 8:1 to about 1:8, from about 7:1 to about 1:7, from about 6:1 to about 1:6, from about 5:1 to about 1:5, from about 4:1 to about 1:4, from about 3:1 to about 1:3, from about 2:1 to about 1:2 or about 1:1. In certain embodiments, the asparagine-responsive active area can include a ratio of stabilizing agent to asparaginase and/or aspartate oxidase from about 5:1 to about 1:5. In certain embodiments, the asparagine-responsive active area can include a ratio of stabilizing agent to asparaginase and/or aspartate oxidase from about 4:1 to about 1:4. In certain embodiments, the asparagine-responsive active area can include a ratio of stabilizing agent to asparaginase and/or aspartate oxidase from about 3:1 to about 1:3. In certain embodiments, the asparagine-responsive active area can include a ratio of stabilizing agent to asparaginase and/or aspartate oxidase from about 2:1 to about 1:2. In certain embodiments, the asparagine-responsive active area can include a ratio of stabilizing agent to asparaginase and/or aspartate oxidase of about 1:1. In certain embodiments, an active area, e.g., an aspartate-responsive active area and/or an asparagine-responsive active area, can include by weight from about 10% to about 50%, e.g., from about 15% to about 45%, from about 20% to about 40%, from about 20% to about 35%, from about 20% to about 30%, of the stabilizing agent. In certain embodiments, asparagine-responsive active area can include by weight from about 15% to about 35% of the stabilizer.

In certain embodiments, an analyte sensor of the present disclosure for indirect measurement of potassium is shown inFIG. 23. As shown inFIG. 23, the analyte sensor can include a potassium-dependent channel701and a potassium-independent channel702. In certain embodiments, each channel is a working electrode. In certain embodiments, the potassium-dependent channel701includes an enzyme system that comprises a potassium-dependent asparaginase. In certain embodiments, the potassium-independent channel702includes an enzyme system that comprises a potassium-independent asparaginase. In certain embodiments, the potassium-dependent asparaginase is a wild-type form of an asparaginase and the potassium-independent asparaginase is a mutant form of the same asparaginase. In certain embodiments, the current signals obtained from channels701and702can be compared to obtain the concentration of the potassium ions in the sample.

In certain embodiments, an analyte sensor of the present disclosure for indirect measurement of potassium is shown inFIG. 24. As shown inFIG. 24, the analyte sensor can include a first potassium-dependent channel801and a second potassium-dependent channel802. In certain embodiments, each channel is a working electrode. In certain embodiments, the first potassium-dependent channel801includes an enzyme system that comprises a first potassium-dependent asparaginase. In certain embodiments, the second potassium-dependent channel802includes an enzyme system that comprises a second potassium-dependent asparaginase. In such embodiments, the first potassium-dependent asparaginase and the second potassium-dependent asparaginase exhibit different dependencies on potassium and would, therefore, result in different signals in the presence of the same potassium concentration. In certain embodiments, the difference in signals obtained from channels801and802can be correlated to the concentration of the potassium ions in the sample.

In certain embodiments, the potassium ion concentration using an analyte sensor comprising the enzyme systems ofFIG. 23orFIG. 24can be determined as shown inFIGS. 25A and 25B. For example, but not by way of limitation, in the absence of potassium ions, each of the channels (e.g., each of channels801and802) has a linear response to increasing asparagine concentrations, as shown in25A, which can be described by the following equations:

where I1and I2correspond to the current output, C is asparagine concentration and k1and k2correspond to the slope of the lines of the two channels. The two constants k1and k2can be obtained from a standard dose-responding calibration in a beaker with different levels of asparagine.

FIG. 25Billustrates the sensor response in the presence of increasing potassium ion concentration. As shown inFIG. 25B, the current response is non-linear and can be described by the following equations:

where I′1and I′2correspond to the current output, I1-0and I2-0correspond to the sensor response when potassium ion concentration is zero. It should also be noted that:

In order to determine the concentration of potassium ions in the sample, the system of equations 3-5 have to be solved. This system consists of three (3) equations with three (3) unknowns, including potassium concentration, I1-0and I2-0.

In certain embodiments, the asparagine-responsive active area can include an asparaginase, e.g., a potassium-independent asparaginase or a potassium-dependent asparaginase. In certain embodiments, the asparagine-responsive active area can further include an aspartate oxidase, e.g., a potassium-independent aspartate oxidase. Alternatively, an asparagine-responsive active area comprising an asparaginase, e.g., a potassium-independent asparaginase or a potassium-dependent asparaginase, can be disposed upon an aspartate-responsive active area as described above in Section 2A.

In certain embodiments, an analyte sensor of the present disclosure can include a sensor tail including at least two working electrodes. In certain embodiments, an asparagine-responsive active area (e.g., a first asparagine-responsive active area) is disposed upon the surface of the first working electrode and an asparagine-responsive active area (e.g., a second asparagine-responsive active area) is disposed upon the surface of the second working electrode, where each asparagine-responsive active area includes an enzyme system including an aspartate oxidase and an asparaginase. In certain embodiments, the first asparagine-responsive active area includes a potassium-independent asparaginase and the second asparagine-responsive active area includes a potassium-dependent asparaginase. Alternatively, the first asparagine-responsive active area includes a first potassium-dependent asparaginase and the second asparagine-responsive active area includes a second potassium-dependent asparaginase, where the asparaginases have different dependencies on potassium ion concentration.

In certain embodiments, an asparagine-responsive active area includes a first enzymatic layer that includes the aspartate oxidase and a second layer disposed upon the first enzymatic layer that includes an asparaginase. Alternatively or additionally, the aspartate oxidase and asparaginase are retained within the same enzymatic layer.

C. Active Areas Responsive to Additional Analytes

In certain embodiments, an analyte sensor of the present disclosure can include an active area for detecting a second analyte. For example, but not by way of limitation, an analyte sensor can be configured to detect potassium and a second analyte. Non-limiting examples of second analytes include glutamate, glucose, ketones, lactate, oxygen, hemoglobin A1C, albumin, alcohol, alkaline phosphatase, alanine transaminase, aspartate aminotransferase, bilirubin, blood urea nitrogen, calcium, carbon dioxide, chloride, creatinine, hematocrit, lactate, magnesium, oxygen, pH, phosphorus, potassium, sodium, aspartate, asparagine, total protein, uric acid, etc. In certain embodiments, such analyte sensors can include a third working electrode configured to detect a different analyte by comprising an analyte-responsive active area disposed upon a surface of a third working electrode.

In certain embodiments, the active site present on a third working electrode of an analyte sensor of the present disclosure can include one or more enzymes that can be used to detect glucose. For example, but not by way of limitation, an analyte sensor of the present disclosure can include an active area that comprises one or more enzymes for detecting glucose, e.g., disposed on a third working electrode. In certain embodiments, the analyte sensor can include an active site comprising a glucose oxidase and/or a glucose dehydrogenase for detecting glucose.

In certain embodiments, the active site present on a third working electrode of an analyte sensor of the present disclosure can include one or more enzymes that can be used to detect ketones. For example, but not by way of limitation, an analyte sensor of the present disclosure can include an active area that comprises one or more enzymes, e.g., an enzyme system, for detecting ketones, e.g., disposed on a third working electrode. In certain embodiments, the analyte sensor can include an active site comprising β-hydroxybutyrate dehydrogenase for detecting ketones. In certain embodiments, the analyte sensor can include an active site comprising β-hydroxybutyrate dehydrogenase and diaphorase for detecting ketones.

In certain embodiments, the active site present on a third working electrode of an analyte sensor of the present disclosure can include one or more enzymes that can be used to detect lactate. For example, but not by way of limitation, an analyte sensor of the present disclosure can include an active area that comprises one or more enzymes, e.g., an enzyme system, for detecting lactate, e.g., disposed on a third working electrode. In certain embodiments, the analyte sensor can include an active site comprising a lactate dehydrogenase and/or a lactate oxidase.

In certain embodiments, the active site present on a third working electrode of an analyte sensor of the present disclosure can include one or more enzymes that can be used to detect alcohol. For example, but not by way of limitation, an analyte sensor of the present disclosure can include an active area that comprises one or more enzymes, e.g., an enzyme system, for detecting alcohol, e.g., disposed on a third working electrode. In certain embodiments, the analyte sensor can include an active site comprising an alcohol dehydrogenase.

) In certain embodiments, when the sensor is configured to detect two or more analytes on two different working electrodes, detection of each analyte can include applying a potential to each working electrode separately, such that separate signals are obtained from each analyte. The signal obtained from each analyte can then be correlated to an analyte concentration through use of a calibration curve or function, or by employing a lookup table. In certain particular embodiments, correlation of the analyte signal to an analyte concentration can be conducted through use of a processor.

In certain embodiments, an analyte sensor disclosed herein can include an electron transfer agent. In certain embodiments, one or more active areas of a presently disclosed analyte sensor can include an electron transfer agent. For example, but not by way of limitation, an analyte sensor disclosed herein can include two active areas, which both include an electron transfer agent. Alternatively, an analyte sensor of the present disclosure can include two or more active areas, where only one active area includes an electron transfer agent.

In certain embodiments, an analyte sensor of the present disclosure can include at least one aspartate-responsive active area that includes an electron transfer agent. In certain embodiments, an analyte sensor of the present disclosure can include at least one asparagine-responsive active area that includes an electron transfer agent. In certain embodiments, an analyte sensor of the present disclosure can include two aspartate-responsive active areas, where each aspartate-responsive active area includes an electron transfer agent. In certain embodiments, an analyte sensor of the present disclosure can include two asparagine-responsive active areas, where each asparagine-responsive active area includes an electron transfer agent. In certain embodiments, the electron transfer agents in the two asparagine-responsive active areas or the two aspartate-responsive active areas can be the same or different. In certain embodiments, an analyte sensor of the present disclosure can include two or more active areas, where only one active area includes an electron transfer agent, e.g., the asparagine-responsive active area or aspartate-responsive active area.

Suitable electron transfer agents can facilitate conveyance of electrons to the adjacent working electrode after an analyte undergoes an enzymatic oxidation-reduction reaction within the corresponding active area, thereby generating a current that is indicative of the presence of that particular analyte. The amount of current generated is proportional to the quantity of analyte that is present.

In certain embodiments, suitable electron transfer agents can include electroreducible and electrooxidizable ions, complexes or molecules (e.g., quinones) having oxidation-reduction potentials that are a few hundred millivolts above or below the oxidation-reduction potential of the standard calomel electrode (SCE). In certain embodiments, the redox mediators can include osmium complexes and other transition metal complexes, such as those described in U.S. Pat. Nos. 6,134,461 and 6,605,200, which are incorporated herein by reference in their entirety. Additional examples of suitable redox mediators include those described in U.S. Pat. Nos. 6,736,957, 7,501,053 and 7,754,093, the disclosures of each of which are also incorporated herein by reference in their entirety. Other examples of suitable redox mediators include metal compounds or complexes of ruthenium, osmium, iron (e.g., polyvinylferrocene or hexacyanoferrate), or cobalt, including metallocene compounds thereof, for example. Suitable ligands for the metal complexes can also include, for example, bidentate or higher denticity ligands such as, for example, bipyridine, biimidazole, phenanthroline or pyridyl(imidazole). Other suitable bidentate ligands can include, for example, amino acids, oxalic acid, acetylacetone, diaminoalkanes, or o-diaminoarenes. Any combination of monodentate, bidentate, tridentate, tetradentate or higher denticity ligands can be present in a metal complex, e.g., osmium complex, to achieve a full coordination sphere. In certain embodiments, the electron transfer agent is an osmium complex. In certain embodiments, the electron transfer agent is osmium complexed with bidentate ligands.

In certain embodiments, electron transfer agents disclosed herein can include suitable functionality to promote covalent bonding to a polymer (also referred to herein as a polymeric backbone) within the active areas as discussed further below. For example, but not by way of limitation, an electron transfer agent for use in the present disclosure can include a polymer-bound electron transfer agent. Suitable non-limiting examples of polymer-bound electron transfer agents include those described in U.S. Pat. Nos. 8,444,834, 8,268,143 and 6,605,201, the disclosures of which are incorporated herein by reference in their entirety. In certain embodiments, the electron transfer agent is a bidentate osmium complex bound to a polymer described herein. In certain embodiments, the electron transfer agent is a bidentate osmium complex bound to a polymer described herein, e.g., a polymeric backbone described in Section 4 below. In certain embodiments, the polymer-bound electron transfer agent shown in FIG. 3 of U.S. Pat. No. 8,444,834 can be used in a sensor of the present disclosure.

In certain embodiments, an analyte sensor of the present disclosure for monitoring potassium, e.g., potassium ion, levels can include a sensor tail including at least two or more working electrodes, e.g., a first working electrode and a second working electrode. In certain embodiments, a first asparagine-responsive active area comprising an asparaginase (e.g., a first asparaginase), an aspartate oxidase and an electron transfer agent is disposed upon a surface of the first working electrode. In certain embodiments, a second asparagine-responsive active area comprising an asparaginase (e.g., a second asparaginase), an aspartate oxidase and an electron transfer agent is disposed upon a surface of the second working electrode. In certain embodiments, the two asparaginases differ in potassium dependency. For example, but not by way of limitation, the first asparaginase can be potassium-dependent and the second asparaginase can be potassium-independent. In certain embodiments, the first asparaginase can be potassium-independent and the second asparaginase can be potassium-dependent. In certain embodiments, the first and second asparaginases are both potassium-dependent but differ in potassium dependency. In certain embodiments, the signals measured from the two asparagine-responsive active areas can be correlated to the concentration of potassium ions in the sample analyzed.

In certain embodiments, the asparagine-responsive active area can include a ratio of aspartate oxidase and/or asparaginase to redox mediator from about 100:1 to about 1:100, e.g., from about 95:1 to about 1:95, from about 90:1 to about 1:90, from about 85:1 to about 1:85, from about 80:1 to about 1:80, from about 75:1 to about 1:75, from about 60:1 to about 1:60, from about 55:1 to about 1:55, from about 50:1 to about 1:50, from about 45:1 to about 1:45, from about 40:1 to about 1:40, from about 35:1 to about 1:35, from about 30:1 to about 1:30, from about 25:1 to about 1:25, from about 20:1 to about 1:20, from about 15:1 to about 1:15, from about 10:1 to about 1:10, from about 9:1 to about 1:9, from about 8:1 to about 1:8, from about 7:1 to about 1:7, from about 6:1 to about 1:6, from about 5:1 to about 1:5, from about 4:1 to about 1:4, from about 3:1 to about 1:3, from about 2:1 to about 1:2 or about 1:1. In certain embodiments, the asparagine-responsive active area can include a ratio of aspartate oxidase and/or asparaginase to redox mediator from about 5:1 to about 1:5. In certain embodiments, the asparagine-responsive active area can include a ratio of aspartate oxidase and/or asparaginase to redox mediator from about 4:1 to about 1:4. In certain embodiments, the asparagine-responsive active area can include a ratio of aspartate oxidase and/or asparaginase to redox mediator from about 3:1 to about 1:3. In certain embodiments, the asparagine-responsive active area can include a ratio of aspartate oxidase and/or asparaginase to redox mediator from about 2:1 to about 1:2. In certain embodiments, the asparagine-responsive active area can include a ratio of aspartate oxidase and/or asparaginase to redox mediator of about 1:1.

In certain embodiments, an analyte sensor of the present disclosure for monitoring potassium, e.g., potassium ion, levels can include a sensor tail including at least two or more working electrodes, e.g., a first working electrode and a second working electrode. In certain embodiments, a first aspartate-responsive active area comprising an aspartate oxidase (e.g., a first aspartate oxidase) and an electron transfer agent (e.g., a first electron transfer agent) is disposed upon a surface of the first working electrode. In certain embodiments, a second aspartate-responsive active area comprising an aspartate oxidase (e.g., a second aspartate oxidase) and an electron transfer agent (e.g., a second electron transfer agent) is disposed upon a surface of the second working electrode. In certain embodiments, the two aspartate oxidases differ in potassium dependency. For example, but not by way of limitation, the first aspartate oxidase can be potassium-dependent and the second aspartate oxidase can be potassium-independent. In certain embodiments, the first aspartate oxidase can be potassium-independent and the second aspartate oxidase can be potassium-dependent. In certain embodiments, the first and second aspartate oxidases are both potassium-dependent but differ in potassium dependency. In certain embodiments, the signals measured from the two aspartate-responsive active areas can be correlated to the concentration of potassium ions in the sample analyzed.

In certain embodiments, the aspartate-responsive active area can include a ratio of aspartate oxidase to redox mediator from about 100:1 to about 1:100, e.g., from about 95:1 to about 1:95, from about 90:1 to about 1:90, from about 85:1 to about 1:85, from about 80:1 to about 1:80, from about 75:1 to about 1:75, from about 60:1 to about 1:60, from about 55:1 to about 1:55, from about 50:1 to about 1:50, from about 45:1 to about 1:45, from about 40:1 to about 1:40, from about 35:1 to about 1:35, from about 30:1 to about 1:30, from about 25:1 to about 1:25, from about 20:1 to about 1:20, from about 15:1 to about 1:15, from about 10:1 to about 1:10, from about 9:1 to about 1:9, from about 8:1 to about 1:8, from about 7:1 to about 1:7, from about 6:1 to about 1:6, from about 5:1 to about 1:5, from about 4:1 to about 1:4, from about 3:1 to about 1:3, from about 2:1 to about 1:2 or about 1:1. In certain embodiments, the aspartate-responsive active area can include a ratio of aspartate oxidase to redox mediator from about 5:1 to about 1:5. In certain embodiments, the aspartate-responsive active area can include a ratio of aspartate oxidase to redox mediator from about 4:1 to about 1:4. In certain embodiments, the aspartate-responsive active area can include a ratio of aspartate oxidase to redox mediator from about 3:1 to about 1:3. In certain embodiments, the aspartate-responsive active area can include a ratio of aspartate oxidase to redox mediator from about 2:1 to about 1:2. In certain embodiments, the aspartate-responsive active area can include a ratio of aspartate oxidase to redox mediator of about 1:1.

In certain embodiments, one or more active sites for promoting analyte detection can include a polymer to which an enzyme and/or redox mediator is covalently bound. Any suitable polymeric backbone can be present in the active area for facilitating detection of an analyte through covalent bonding of the enzyme and/or redox mediator thereto. Non-limiting examples of suitable polymers within the active area include polyvinylpyridines, e.g., poly(4-vinylpyridine) or poly(2-vinylpyridine), and polyvinylimidazoles, e.g., poly(N-vinylimidazole) and poly(1-vinylimidazole), or a copolymer thereof, for example, in which quaternized pyridine groups serve as a point of attachment for the redox mediator or enzyme thereto. Illustrative copolymers that can be suitable for inclusion in the active areas include those containing monomer units such as styrene, acrylamide, methacrylamide, or acrylonitrile, for example. In certain embodiments, the polymer is a polyvinylpyridine-based polymer. In certain embodiments, the polymer is a polyvinylpyridine or a copolymer thereof. In certain embodiments, the polymer is a co-polymer of vinylpyridine and styrene. In certain embodiments, polymers that can be present in an active area include a polyurethane or a copolymer thereof, and/or polyvinylpyrrolidone. Additional non-limiting examples of polymers that can be present in the active area include, but are not limited to, those described in U.S. Pat. No. 6,605,200, incorporated herein by reference in its entirety, such as poly(acrylic acid), styrene/maleic anhydride copolymer, methylvinylether/maleic anhydride copolymer (GANTREZ polymer), poly(vinylbenzylchloride), poly(allylamine), polylysine, poly(4-vinylpyridine) quaternized with carboxypentyl groups, and poly(sodium 4-styrene sulfonate). In certain embodiments where the analyte sensor includes two active sites, the polymer within each active area can be the same or different.

In certain embodiments, when an enzyme system with multiple enzymes is present in a given active area, all of the multiple enzymes can be covalently bonded to the polymer. In certain other embodiments, only a portion of the multiple enzymes is covalently bonded to the polymer. For example, and not by the way of limitation, one or more enzymes within an enzyme system can be covalently bonded to the polymer and at least one enzyme can be non-covalently associated with the polymer, such that the non-covalently bonded enzyme is physically retained within the polymer. In certain embodiments, an aspartate oxidase and/or an asparaginase can be covalently bonded to a polymer within an analyte-responsive active area of the disclosed analyte sensors. In certain embodiments, aspartate oxidase can be covalently bonded to a polymer within an asparagine-responsive active area of the disclosed analyte sensors. In certain embodiments, asparaginase can be covalently bonded to a polymer within an asparagine-responsive active area of the disclosed analyte sensors. In certain embodiments, aspartate oxidase can be covalently bonded to the polymer and asparaginase can be non-covalently associated with the polymer. Alternatively, asparaginase can be covalently bonded to the polymer and aspartate oxidase can be non-covalently associated with the polymer. In certain embodiments where one or more enzymes is not covalently bonded, it can be physically retained within the asparagine-responsive active area. In certain embodiments, a membrane overcoating the asparagine-responsive active area can aid in retaining the one or more enzymes within the asparagine-responsive active area while still permitting sufficient inward diffusion of asparagine to permit detection thereof. Suitable membrane polymers for overcoating the analyte-responsive active area are discussed further herein.

In certain embodiments, when a stabilizer is present in an active area, one or more enzymes within the area can be covalently bonded to the stabilizer. For example, and not by the way of limitation, one or more enzymes, e.g., aspartate oxidase and/or asparaginase, can be covalently bonded to the stabilizer, e.g., albumin, present in the active area. In certain embodiments, an aspartate oxidase present in an active area of the present disclosure can be covalently bonded to the stabilizer. In certain embodiments, an asparaginase present in an active area of the present disclosure can be covalently bonded to the stabilizer.

In certain particular embodiments, covalent bonding of the one or more enzymes and/or redox mediators to the polymer and/or stabilizer in a given active area can take place via crosslinking introduced by a suitable crosslinking agent. In certain embodiments, crosslinking of the polymer and/or stabilizer to the one or more enzymes and/or redox mediators can reduce the occurrence of delamination of the enzyme compositions from an electrode. Suitable crosslinking agents can include one or more crosslinkable functionalities such as, but not limited to, vinyl, alkoxy, acetoxy, enoxy, oxime, amino, hydroxyl, cyano, halo, acrylate, epoxide and isocyanato groups. In certain embodiments, the crosslinking agent comprises one or more, two or more, three or more or four or more epoxide groups. For example, but not by way of limitation, a crosslinker for use in the present disclosure can include mono-, di-, tri- and tetra-ethylene oxides. In certain embodiments, crosslinking agents for reaction with free amino groups in the enzyme (e.g., with the free side chain amine in lysine) can include crosslinking agents such as, for example, polyethylene glycol dibutyl ethers, polypropylene glycol dimethyl ethers, polyalkylene glycol allyl methyl ethers, polyethylene glycol diglycidyl ether (PEGDGE) or other polyepoxides, cyanuric chloride, N-hydroxysuccinimide, imidoesters, epichlorohydrin, or derivatized variants thereof. In certain embodiments, the crosslinking agent is PEGDGE, e.g., having an average molecular weight (Mn) from about 200 to 1,000, e.g., about 400. In certain embodiments, the crosslinking agent is PEGDGE 400. In certain embodiments, the crosslinking agent can be glutaraldehyde. In certain embodiments, the crosslinking of the enzyme to the polymer is generally intermolecular. In certain embodiments, the crosslinking of the enzyme to the polymer is generally intramolecular.

In certain embodiments, an active area can include a ratio of crosslinking agent to one or more enzymes, e.g., aspartate oxidase, asparaginase or both, from about 50:1 to about 1:50, e.g., from about 45:1 to about 1:45, from about 40:1 to about 1:40, from about 35:1 to about 1:35, from about 30:1 to about 1:30, from about 25:1 to about 1:25, from about 20:1 to about 1:20, from about 15:1 to about 1:15, from about 10:1 to about 1:10, from about 9:1 to about 1:9, from about 8:1 to about 1:8, from about 7:1 to about 1:7, from about 6:1 to about 1:6, from about 5:1 to about 1:5, from about 4:1 to about 1:4, from about 3:1 to about 1:3, from about 2:1 to about 1:2 or about 1:1. In certain embodiments, an active area can include a ratio of crosslinking agent to one or more enzymes, e.g., aspartate oxidase, asparaginase or both, from about 50:1 to about 1:50. In certain embodiments, an active area can include a ratio of crosslinking agent to one or more enzymes, e.g., aspartate oxidase, asparaginase or both, from about 50:1 to about 30:1. In certain embodiments, an active area can include a ratio of crosslinking agent to one or more enzymes, e.g., aspartate oxidase, asparaginase or both, from about 50:1 to about 1:1. In certain embodiments, an active area can include a ratio of crosslinking agent to one or more enzymes, e.g., aspartate oxidase, asparaginase or both, from about 2:1 to about 1:2. In certain embodiments, an active area can include a ratio of crosslinking agent to one or more enzymes, e.g., aspartate oxidase, asparaginase or both, of about 1:1. In certain embodiments, an active area can include a ratio of crosslinking agent to one or more enzymes, e.g., aspartate oxidase, asparaginase or both, from about 5:1 to about 1:5. In certain embodiments, an active area can include a ratio of crosslinking agent to one or more enzymes, e.g., aspartate oxidase, asparaginase or both, from about 2:1 to about 1:2. In certain embodiments, an active area can include a ratio of crosslinking agent to one or more enzymes, e.g., aspartate oxidase, asparaginase or both, of about 1:1. In certain embodiments, an active area, e.g., aspartate-responsive active area and/or asparagine-responsive active area, can include by weight from about 5% to about 20%, e.g., from about 10% to about 20% or from about 10% to about 15%, of the crosslinking agent. In certain embodiments, the glutamate-responsive active area can include from about 10% to about 20% of the crosslinking agent.

5. Mass Transport Limiting Membranes

In certain embodiments, the analyte sensors disclosed herein further include a membrane permeable to an analyte that overcoats at least an active area, e.g., a first active area and/or a second active area, present on a working electrode of the analyte sensor. In certain embodiments, the analyte sensors disclosed herein further include a membrane permeable to potassium that overcoats at least an active area, e.g., a first active area and/or a second active area, present on a working electrode of the analyte sensor.

In certain embodiments, a membrane overcoating an analyte-responsive active area can function as a mass transport limiting membrane and/or to improve biocompatibility. A mass transport limiting membrane can act as a diffusion-limiting barrier to reduce the rate of mass transport of the analyte. For example, but not by way of limitation, limiting access of an analyte, e.g., aspartate, asparagine and/or potassium, to the analyte-responsive active area with a mass transport limiting membrane can aid in avoiding sensor overload (saturation), thereby improving detection performance and accuracy.

In certain embodiments, the mass transport limiting membrane can be homogeneous and can be single-component (contain a single membrane polymer). Alternatively, the mass transport limiting membrane can be multi-component (contain two or more different membrane polymers. In certain embodiments, the multi-component membrane can be present as a multilayered membrane or as a homogeneous admixture of two or more membrane polymers. A homogeneous admixture can be generated by combining the two or more membrane polymers in a solution and then depositing the solution upon a working electrode, e.g., dip coating. A multilayered membrane can be generated by sequentially depositing membrane polymers upon a working electrode, e.g., dip coating.

In certain embodiments, the mass transport limiting membrane can include two or more layers, e.g., a bilayer or trilayer membrane. In certain embodiments, each layer can comprise a different polymer or the same polymer at different concentrations or thicknesses. In certain embodiments, the first analyte-responsive active area can be covered by a multi-layered membrane, e.g., a bilayer membrane, and the second analyte-responsive active area can be covered by a single membrane. In certain embodiments, the first analyte-responsive active area can be covered by a multi-layered membrane, e.g., a bilayer membrane, and the second analyte-responsive active area can be covered by a multi-layered membrane, e.g., a bilayer membrane. In certain embodiments, the first analyte-responsive active area can be covered by a single membrane and the second analyte-responsive active area can be covered by a multi-layered membrane, e.g., a bilayer membrane be covered by a single membrane. In certain embodiments, the first analyte-responsive active area can be covered by a single membrane and the second analyte-responsive active area can be covered by a single membrane.

In certain embodiments, the composition of the mass transport limiting membrane disposed upon an analyte sensor that has two active areas can be the same or different where the mass transport limiting membrane overcoats each active area. For example, but not by way of limitation, the portion of the mass transport limiting membrane overcoating an asparagine-responsive active area or aspartate-responsive active area used for indirect measurement of potassium can be multi-component and/or the portion of the mass transport limiting membrane overcoating a second analyte-responsive active area can be single-component. Alternatively, the portion of the mass transport limiting membrane overcoating an asparagine-responsive active area or aspartate-responsive active area used for indirect measurement of potassium ions can be single-component and/or the portion of the mass transport limiting membrane overcoating a second analyte-responsive active area can be multi-component.

In certain embodiments, a mass transport limiting membrane can include polymers containing heterocyclic nitrogen groups. In certain embodiments, a mass transport limiting membrane can include a polyvinylpyridine-based polymer. Non-limiting examples of polyvinylpyridine-based polymers are disclosed in U.S. Patent Publication No. 2003/0042137 (e.g., Formula 2b), the contents of which are incorporated by reference herein in its entirety.

In certain embodiments, a mass transport limiting membrane can include a polyvinylpyridine (e.g., poly(4-vinylpyridine) or poly(4-vinylpyridine)), a polyvinylimidazole, a polyvinylpyridine copolymer (e.g., a copolymer of vinylpyridine and styrene), a polyacrylate, a polyurethane, a polyether urethane, homopolymers, copolymers or terpolymers of polyurethanes, a silicone, a polytetrafluoroethylene, a polyethylene-co-tetrafluoroethylene, a polyolefin, a polyester, a polycarbonate, a biostable polytetrafluoroethylene, a polypropylene, a polyvinylchloride, a polyvinylidene difluoride, a polybutylene terephthalate, a polymethylmethacrylate, a polyether ether ketone, cellulosic polymers, polysulfones and block copolymers thereof including, for example, di-block, tri-block, alternating, random and graft copolymers or a chemically related material and the like.

In certain embodiments, a membrane for use in the present disclosure, e.g., a single-component membrane, can include a polyvinylpyridine (e.g., poly(4-vinylpyridine) and/or poly(2-vinylpyridine)). In certain embodiments, a membrane for use in the present disclosure, e.g., a single-component membrane, can include poly(4-vinylpyridine). In certain embodiments, a membrane for use in the present disclosure, e.g., a single-component membrane, can include a copolymer of vinylpyridine and styrene. In certain embodiments, the membrane can comprise a polyvinylpyridine-co-styrene copolymer. For example, but not by way of limitation, a polyvinylpyridine-co-styrene copolymer for use in the present disclosure can include a polyvinylpyridine-co-styrene copolymer in which a portion of the pyridine nitrogen atoms were functionalized with a non-crosslinked polyethylene glycol tail and a portion of the pyridine nitrogen atoms were functionalized with an alkylsulfonic acid, e.g., a propylsulfonic acid, group. In certain embodiments, a derivatized polyvinylpyridine-co-styrene copolymer for use as a membrane polymer can be the 10Q5 polymer as described in U.S. Pat. No. 8,761,857, the contents of which are incorporated by reference herein in its entirety. In certain embodiments, the polyvinylpyridine-based polymer has a molecular weight from about 50 Da to about 500 kDa.

A suitable copolymer of vinylpyridine and styrene can have a styrene content ranging from about 0.01% to about 50% mole percent, or from about 0.05% to about 45% mole percent, or from about 0.1% to about 40% mole percent, or from about 0.5% to about 35% mole percent, or from about 1% to about 30% mole percent, or from about 2% to about 25% mole percent, or from about 5% to about 20% mole percent. Substituted styrenes can be used similarly and in similar amounts. A suitable copolymer of vinylpyridine and styrene can have a molecular weight of 5 kDa or more, or about 10 kDa or more, or about 15 kDa or more, or about 20 kDa or more, or about 25 kDa or more, or about 30 kDa or more, or about 40 kDa or more, or about 50 kDa or more, or about 75 kDa or more, or about 90 kDa or more, or about 100 kDa or more. In non-limiting examples, a suitable copolymer of vinylpyridine and styrene can have a molecular weight ranging from about 5 kDa to about 150 kDa, or from about 10 kDa to about 125 kDa, or from about 15 kDa to about 100 kDa, or from about 20 kDa to about 80 kDa, or from about 25 kDa to about 75 kDa, or from about 30 kDa to about 60 kDa.

In certain embodiments, the membrane includes a polyurethane membrane that includes both hydrophilic and hydrophobic regions. In certain embodiments, a hydrophobic polymer component is a polyurethane, a polyurethane urea or poly(ether-urethane-urea). In certain embodiments, a polyurethane is a polymer produced by the condensation reaction of a diisocyanate and a difunctional hydroxyl-containing material. In certain embodiments, a polyurethane urea is a polymer produced by the condensation reaction of a diisocyanate and a difunctional amine-containing material. In certain embodiments, diisocyanates for use herein include aliphatic diisocyanates, e.g., containing from about 4 to about 8 methylene units, or diisocyanates containing cycloaliphatic moieties. Additional non-limiting examples of polymers that can be used for the generation of a membrane of a presently disclosed sensor include vinyl polymers, polyethers, polyesters, polyamides, inorganic polymers (e.g., polysiloxanes and polycarbosiloxanes), natural polymers (e.g., cellulosic and protein based materials) and mixtures (e.g., admixtures or layered structures) or combinations thereof. In certain embodiments, the hydrophilic polymer component is polyethylene oxide and/or polyethylene glycol. In certain embodiments, the hydrophilic polymer component is a polyurethane copolymer. For example, but not by way of limitation, a hydrophobic-hydrophilic copolymer component for use in the present disclosure is a polyurethane polymer that comprises about 10% to about 50%, e.g., 20%, hydrophilic polyethylene oxide.

In certain embodiments, the membrane includes a silicone polymer/hydrophobic-hydrophilic polymer blend. In certain embodiments, the hydrophobic-hydrophilic polymer for use in the blend can be any suitable hydrophobic-hydrophilic polymer such as, but not limited to, polyvinylpyrrolidone, polyhydroxyethyl methacrylate, polyvinylalcohol, polyacrylic acid, polyethers such as polyethylene glycol or polypropylene oxide, and copolymers thereof, including, for example, di-block, tri-block, alternating, random, comb, star, dendritic and graft copolymers. In certain embodiments, the hydrophobic-hydrophilic polymer is a copolymer of poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO). Non-limiting examples of PEO and PPO copolymers include PEO-PPO diblock copolymers, PPO-PEO-PPO triblock copolymers, PEO-PPO-PEO triblock copolymers, alternating block copolymers of PEO-PPO, random copolymers of ethylene oxide and propylene oxide and blends thereof. In certain embodiments, the copolymers can be substituted with hydroxy substituents.

In certain embodiments, hydrophilic or hydrophobic modifiers can be used to “fine-tune” the permeability of the resulting membrane to an analyte of interest. In certain embodiments, hydrophilic modifiers such as poly(ethylene) glycol, hydroxyl or polyhydroxyl modifiers and the like, and any combinations thereof, can be used to enhance the biocompatibility of the polymer or the resulting membrane.

In certain embodiments where multiple active areas are present, the mass transport limiting membrane can overcoat each active area, including the option of compositional variation upon differing active areas, which can be achieved through sequential dip coating operations to produce a bilayer membrane portion upon a working electrode located closer to the sensor tip.

In certain embodiments where multiple active areas are present, a separate mass transport limiting membrane can overcoat each active area. For example, but not by way of limitation, a mass transport limiting membrane can be disposed on the first active area, e.g., the aspartate-responsive active area or asparagine-responsive active area, and a separate, second mass transport limiting membrane can overcoat the second active area. In certain embodiments, the two mass transport limiting membranes are spatially separated and do not overlap each other. In certain embodiments, the first mass transport limiting membrane does not overlap the second mass transport limiting membrane and the second mass transport limiting membrane does not overlap the first mass transport limiting membrane. Alternatively, the second mass transport limiting membrane overlaps the first mass transport limiting membrane. In certain embodiments, the first mass transport limiting membrane comprises different polymers than the second mass transport limiting membrane. Alternatively, the first mass transport limiting membrane comprises the same polymers as the second mass transport limiting membrane. In certain embodiments, the first mass transport limiting membrane comprises the same polymers as the second mass transport limiting membrane but comprise different crosslinking agents.

In certain embodiments of the present disclosure, an asparagine-responsive active area used for indirect measurement of potassium ions can be overcoated with a multi-component membrane including a polyvinylpyridine and a polyvinylpyridine-co-styrene copolymer, either as a bilayer membrane or a homogeneous admixture, and a second analyte-responsive active area can be overcoated with a single-component membrane that includes polyvinylpyridine or a polyvinylpyridine-co-styrene copolymer. Alternatively, an asparagine-responsive active area used for indirect measurement of potassium ions can be overcoated with a single-component membrane that includes polyvinylpyridine or a polyvinylpyridine-co-styrene copolymer and a second analyte-responsive active area can be overcoated with a multi-component membrane including a polyvinylpyridine and a polyvinylpyridine-co-styrene copolymer, either as a bilayer membrane or a homogeneous admixture.

In certain embodiments of the present disclosure, an aspartate-responsive active area used for indirect measurement of potassium ions can be overcoated with a multi-component membrane including a polyvinylpyridine and a polyvinylpyridine-co-styrene copolymer, either as a bilayer membrane or a homogeneous admixture, and a second analyte-responsive active area can be overcoated with a single-component membrane that includes polyvinylpyridine or a polyvinylpyridine-co-styrene copolymer. Alternatively, an aspartate-responsive active area used for indirect measurement of potassium ions can be overcoated with a single-component membrane that includes polyvinylpyridine or a polyvinylpyridine-co-styrene copolymer and a second analyte-responsive active area can be overcoated with a multi-component membrane including a polyvinylpyridine and a polyvinylpyridine-co-styrene copolymer, either as a bilayer membrane or a homogeneous admixture.

Polydimethylsiloxane (PDMS) can be incorporated in any of the mass transport limiting membranes disclosed herein.

In certain embodiments, the mass transport limiting membrane can comprise a membrane polymer crosslinked with a crosslinking agent disclosed herein and above in Section 4. In certain embodiments where there are two mass transport limiting membranes, e.g., a first mass transport limiting membrane and a second mass transport limiting membrane, each membrane can be crosslinked with a different crosslinking agent. For example, but not by way of limitation, the crosslinking agent can result in a membrane that is more restrictive to diffusion of certain compounds, e.g., analytes within the membrane, or less restrictive to diffusion of certain compounds, e.g., by affecting the size of the pores within the membrane. For example, but not by way of limitation, in a sensor that is configured to detect potassium, the mass transport limiting membrane overcoating the analyte-responsive area can have a pore size that restricts the diffusion of compounds larger than potassium through the membrane.

In certain embodiments, crosslinking agents for use in the present disclosure can include polyepoxides, carbodiimide, cyanuric chloride, triglycidyl glycerol, N-hydroxysuccinimide, imidoesters, epichlorohydrin or derivatized variants thereof. In certain embodiments, a membrane polymer overcoating one or more active areas can be crosslinked with a branched crosslinker, e.g., which can decrease the amount of extractables obtainable from the mass transport limiting membrane. Non-limiting examples of a branched crosslinker include branched glycidyl ether crosslinkers, e.g., including branched glycidyl ether crosslinkers that include two or three or more crosslinkable groups. In certain embodiments, the branched crosslinker can include two or more crosslinkable groups, such as polyethylene glycol diglycidyl ether. In certain embodiments, the branched crosslinker can include three or more crosslinkable groups, such as polyethylene glycol tetraglycidyl ether. In certain embodiments, the mass transport limiting membrane can include polyvinylpyridine or a copolymer of vinylpyridine and styrene crosslinked with a branched glycidyl ether crosslinker including two or three crosslinkable groups, such as polyethylene glycol tetraglycidyl ether or polyethylene glycol diglycidyl ether. In certain embodiments, the epoxide groups of a polyepoxides, e.g., polyethylene glycol tetraglycidyl ether or polyethylene glycol diglycidyl ether, can form a covalent bond with pyridine or an imidazole via epoxide ring opening resulting in a hydroxyalkyl group bridging a body of the crosslinker to the heterocycle of the membrane polymer.

In certain embodiments, the crosslinking agent is polyethylene glycol diglycidyl ether (PEGDGE). In certain embodiments, the PEGDGE used to promote crosslinking (e.g., intermolecular crosslinking) between two or more membrane polymer backbones can exhibit a broad range of suitable molecular weights. In certain embodiments, the molecular weight of the PEGDGE can range from about 100 g/mol to about 5,000 g/mol. The number of ethylene glycol repeat units in each arm of the PEGDGE can be the same or different, and can typically vary over a range within a given sample to afford an average molecular weight. In certain embodiments, the PEGDGE for use in the present disclosure has an average molecular weight (Mn) from about 200 to 1,000, e.g., about 400. In certain embodiments, the crosslinking agent is PEGDGE 400.

In certain embodiments, the polyethylene glycol tetraglycidyl ether used to promote crosslinking (e.g., intermolecular crosslinking) between two or more membrane polymer backbones can exhibit a broad range of suitable molecular weights. Up to four polymer backbones may crosslinked with a single molecule of the polyethylene glycol tetraglycidyl ether crosslinker. In certain embodiments, the molecular weight of the polyethylene glycol tetraglycidyl ether can range from about 1,000 g/mol to about 5,000 g/mol. The number of ethylene glycol repeat units in each arm of the polyethylene glycol tetraglycidyl ether can be the same or different, and can typically vary over a range within a given sample to afford an average molecular weight. In certain embodiments, the mass transport limiting membrane can be deposited directly onto the active area.

In certain other embodiments, a membrane polymer overcoating one or more active areas can be crosslinked with a branched crosslinker including three or more crosslinkable groups, such as polyethylene glycol tetraglycidyl ether, which can decrease the amount of extractables obtainable from the mass transport limiting membrane, as referenced above. In certain embodiments, the mass transport limiting membrane can include polyvinylpyridine or a copolymer of vinylpyridine and styrene crosslinked with a branched glycidyl ether crosslinker including three crosslinkable groups, such as polyethylene glycol tetraglycidyl ether. In certain embodiments, the epoxide groups of the polyethylene glycol tetraglycidyl ether can form a covalent bond with pyridine or an imidazole via epoxide ring opening resulting in a hydroxyalkyl group bridging a body of the crosslinker to the heterocycle of the membrane polymer.

In certain embodiments, an analyte sensor described herein for monitoring potassium, e.g., potassium ion, levels can include a sensor tail including at least one working electrode, an asparagine-responsive active area disposed upon a surface of the working electrode and a mass transport limiting membrane permeable to asparagine and potassium that overcoats at least the first active area. In certain embodiments, the asparagine-responsive active area includes an asparaginase, an aspartate oxidase, an electron transfer agent and, optionally, a polymer. In certain embodiments, one or both enzymes are covalently bonded to the polymer.

In certain embodiments, an analyte sensor of the present disclosure for monitoring potassium, e.g., potassium ion, levels can include a sensor tail including at least two or more working electrodes, e.g., a first working electrode and a second working electrode. In certain embodiments, a first asparagine-responsive active area comprising an asparaginase, an aspartate oxidase and an electron transfer agent is disposed upon a surface of the first working electrode. In certain embodiments, a second asparagine-responsive active area comprising an asparaginase, an aspartate oxidase and an electron transfer agent is disposed upon a surface of the second working electrode. In certain embodiments, the analyte sensor further includes a mass transport limiting membrane permeable to asparagine and/or potassium that overcoats at least one of the asparagine-responsive active area. In certain embodiments, the mass transport limiting membrane permeable to asparagine and/or potassium overcoats both of the asparagine-responsive active areas. Alternatively, a first mass transport limiting membrane permeable to asparaginase overcoats the first asparagine-responsive active area and a second mass transport limiting membrane permeable to asparaginase overcoats the second asparagine-responsive active area. In certain embodiments, the first mass transport limiting membrane and the second mass transport limiting membrane comprise the same polymers. Alternatively, the first mass transport limiting membrane and the second mass transport limiting membrane comprise different polymers.

In certain embodiments, an analyte sensor described herein for monitoring potassium, e.g., potassium ion, levels can include a sensor tail including at least one working electrode, an aspartate-responsive active area disposed upon a surface of the working electrode and a mass transport limiting membrane permeable to aspartate and potassium that overcoats at least the first active area. In certain embodiments, the aspartate-responsive active area includes an aspartate oxidase, an electron transfer agent and, optionally, a polymer. In certain embodiments, the enzyme is covalently bonded to the polymer.

In certain embodiments, an analyte sensor of the present disclosure for monitoring potassium, e.g., potassium ion, levels can include a sensor tail comprising at least two or more working electrodes, e.g., a first working electrode and a second working electrode, where each working electrode has an aspartate-responsive active area. For example, but not by way of limitation, a first aspartate-responsive active area comprising an aspartate oxidase and an electron transfer agent is disposed upon a surface of the first working electrode. In certain embodiments, a second aspartate-responsive active area comprising an aspartate oxidase and an electron transfer agent is disposed upon a surface of the second working electrode. In certain embodiments, the analyte sensor further includes a mass transport limiting membrane permeable to aspartate and/or potassium that overcoats at least one of the aspartate-responsive active area. In certain embodiments, the mass transport limiting membrane permeable to aspartate and/or potassium overcoats both of the aspartate-responsive active areas. Alternatively, a first mass transport limiting membrane permeable to aspartate and/or potassium overcoats the first aspartate-responsive active area and a second mass transport limiting membrane permeable to aspartate and/or potassium overcoats the second aspartate-responsive active area. In certain embodiments, the first mass transport limiting membrane and the second mass transport limiting membrane comprise the same polymers. Alternatively, the first mass transport limiting membrane and the second mass transport limiting membrane comprise different polymers.

In certain embodiments, an analyte sensor of the present disclosure for monitoring potassium, e.g., potassium ion, levels can include a sensor tail including at least three or more working electrodes, e.g., a first working electrode, a second working electrode and a third working electrode, where the two of the three working electrodes are configured to detect different analytes. In certain embodiments, a first aspartate-responsive active area comprising a potassium-dependent aspartate oxidase and an electron transfer agent is disposed upon a surface of the first working electrode. In certain embodiments, a second aspartate-responsive active area comprising a potassium-independent aspartate oxidase and an electron transfer agent is disposed upon a surface of the second working electrode. In certain embodiments, an additional analyte-responsive active area comprising an enzyme for detecting a second analyte is disposed upon a surface of the third working electrode. In certain embodiments, the analyte sensor further includes a mass transport limiting membrane permeable to aspartate and/or potassium that overcoats at least one, e.g., both, of the aspartate-responsive active areas. In certain embodiments, the mass transport limiting membrane permeable to aspartate and/or potassium overcoats one or more aspartate-responsive active areas and the second-analyte responsive active area. Alternatively, a first mass transport limiting membrane permeable to aspartate and/or potassium overcoats at least one, e.g., both, of the aspartate-responsive active areas and a second mass transport limiting membrane permeable to the second analyte overcoats the second-analyte responsive active area. In certain embodiments, the second mass transport limiting membrane permeable to the second analyte overcoats at least a portion of at least one, or both, of the aspartate-responsive active areas.

In certain embodiments, an analyte sensor of the present disclosure for monitoring potassium, e.g., potassium ion, levels can include a sensor tail including at least three or more working electrodes, e.g., a first working electrode, a second working electrode and a third working electrode, where the two of the three working electrodes are configured to detect different analytes. In certain embodiments, a first asparagine-responsive active area comprising an aspartate oxidase, potassium-dependent asparaginase and an electron transfer agent is disposed upon a surface of the first working electrode. In certain embodiments, a second asparagine-responsive active area comprising an aspartate oxidase, a potassium-independent asparaginase and an electron transfer agent is disposed upon a surface of the second working electrode. In certain embodiments, an additional analyte-responsive active area comprising an enzyme for detecting a second analyte is disposed upon a surface of the third working electrode. In certain embodiments, the analyte sensor further includes a mass transport limiting membrane permeable to asparagine and/or potassium that overcoats at least one, e.g., both, of the asparagine-responsive active areas. In certain embodiments, the mass transport limiting membrane permeable to asparagine and/or potassium overcoats one or more asparagine-responsive active areas and the second-analyte responsive active area. Alternatively, a first mass transport limiting membrane permeable to asparagine and/or potassium overcoats at least one, e.g., both, of the asparagine-responsive active areas and a second mass transport limiting membrane permeable to the second analyte overcoats the second-analyte responsive active area. In certain embodiments, the second mass transport limiting membrane permeable to the second analyte overcoats at least a portion of at least one, or both, of asparagine-responsive active areas.

In certain embodiments, the mass transport limiting membrane has a thickness, e.g., dry thickness, ranging from about 0.1 μm to about 1,000 μm, e.g., from about 1 μm to about 500 μm, about 10 μm to about 100 μm or about 10 μm to about 100 μm. In certain embodiments, the mass transport limiting membrane can have a thickness from about 0.1 μm to about 100 μm, e.g., from about 1 μm to about 90 μm, from about 1 μm to about 80 μm, from about 1 μm to about 70 μm, from about 1 μm to about 60 μm, from about 1 μm to about 50 μm, from about 1 μm to about 40 μm, from about 1 μm to about 30 μm, from about 1 μm to about 20 μm, from about 0.5 μm to about 10 μm, from about 1 μm to about 10 μm, from about 1 μm to about 5 μm or from about 0.1 μm to about 5 μm. In certain embodiments, the mass transport limiting membrane can have a thickness from about 1 μm to about 100 μm. In certain embodiments, the sensor can be dipped in the mass transport limiting membrane solution more than once. For example, but not by way of limitation, a sensor (or working electrode) of the present disclosure can be dipped in a mass transport limiting membrane solution at least twice, at least three times, at least four times or at least five times to obtain the desired mass transport limiting membrane thickness.

6. Interference Domain

In certain embodiments, the sensor of the present disclosure, e.g., sensor tail, can further comprise an interference domain. In certain embodiments, the interference domain can include a polymer domain that restricts the flow of one or more interferants, e.g., to the surface of the working electrode. In certain embodiments, the interference domain can function as a molecular sieve that allows analytes and other substances that are to be measured by the working electrode to pass through, while preventing passage of other substances such as interferents. In certain embodiments, the interferents can affect the signal obtained at the working electrode. Non-limiting examples of interferents include acetaminophen, ascorbate, ascorbic acid, bilirubin, cholesterol, creatinine, dopamine, ephedrine, ibuprofen, L-dopa, methyldopa, salicylate, tetracycline, tolazamide, tolbutamide, triglycerides, urea and uric acid.

In certain embodiments, the interference domain is located between the working electrode and one or more active areas, e.g., asparagine-responsive active area or aspartate-responsive active area. In certain embodiments, non-limiting examples of polymers that can be used in the interference domain include polyurethanes, polymers having pendant ionic groups and polymers having controlled pore size. In certain embodiments, the interference domain is formed from one or more cellulosic derivatives. Non-limiting examples of cellulosic derivatives include polymers such as cellulose acetate, cellulose acetate butyrate, 2-hydroxyethyl cellulose, cellulose acetate phthalate, cellulose acetate propionate, cellulose acetate trimellitate and the like.

In certain embodiments, the interference domain is part of the mass transport limiting membrane and not a separate membrane. In certain embodiments, the interference domain is disposed between the mass limiting membrane and one or more analyte-responsive active areas.

In certain embodiments, the interference domain includes a thin, hydrophobic membrane that is non-swellable and restricts diffusion of high molecular weight species. For example, but not by way of limitation, the interference domain can be permeable to relatively low molecular weight substances while restricting the passage of higher molecular weight substances.

In certain embodiments, the interference domain can be deposited directly onto the working electrode, e.g., onto the surface of the permeable working electrode. In certain embodiments, the interference domain has a thickness, e.g., dry thickness, ranging from about 0.1 μm to about 1,000 μm, e.g., from about 1 μm to about 500 μm, about 10 μm to about 100 μm or about 10 μm to about 100 μm. In certain embodiments, the interference domain can have a thickness from about 0.1 μm to about 100 μm, e.g., from about 1 μm to about 90 μm, from about 1 μm to about 80 μm, from about 1 μm to about 70 μm, from about 1 μm to about 60 μm, from about 1 μm to about 50 μm, from about 1 μm to about 40 μm, from about 1 μm to about 30 μm, from about 1 μm to about 20 μm, from about 0.5 μm to about 10 μm, from about 1 μm to about 10 μm, from about 1 μm to about 5 μm or from about 0.1 μm to about 5 μm. In certain embodiments, the sensor can be dipped in the interference domain solution more than once. For example, but not by way of limitation, a sensor (or working electrode) of the present disclosure can be dipped in an interference domain solution at least twice, at least three times, at least four times or at least five times to obtain the desired interference domain thickness.

The present disclosure further provides methods for manufacturing the presently disclosed analyte sensors that includes one or more active sites. In certain embodiments, the method includes screen printing a working electrode, e.g., a carbon working electrode, e.g., by using a carbon ink.

In certain embodiments, the method can further include adding a composition including an enzyme onto a surface of the working electrode to generate an analyte-responsive active area on the working electrode. For example, but not by way of limitation, the composition can include an aspartate oxidase, e.g., a potassium-dependent aspartate oxidase or a potassium-independent aspartate oxidase. In certain embodiments, the composition can further include an asparaginase, e.g., a potassium-dependent asparaginase or a potassium-independent asparaginase. In certain embodiments, the composition can further include a redox mediator. In certain embodiments, the composition can further include a crosslinking agent, e.g., polyethylene glycol diglycidyl ether, and a stabilizing agent, e.g., an albumin such as BSA. In certain embodiments, the method can further include curing the enzyme composition.

Alternatively, a first enzyme composition including an aspartate oxidase can be initially deposited onto a surface of the working electrode to generate an analyte-responsive active area on the working electrode. In certain embodiments, the first enzyme composition can further include a redox mediator, a crosslinking agent, e.g., polyethylene glycol diglycidyl ether, and/or a stabilizing agent, e.g., an albumin such as BSA. In certain embodiments, the method can include curing the first enzyme composition to generate a first enzyme layer. In certain embodiments, the method can include depositing a second enzyme composition including an asparaginase onto a surface of the first enzyme layer, and curing the second enzyme composition to generate a first second layer. In certain embodiments, the second enzyme composition can include, a crosslinking agent, e.g., polyethylene glycol diglycidyl ether, and/or a stabilizing agent, e.g., an albumin such as BSA.

In analyte sensors with two working electrodes, a second analyte-responsive active area can be generated on a second working electrode using the methods described herein. In certain embodiments, in analyte sensors with two working electrodes, a second aspartate-responsive active area or a second asparagine-responsive active area can be generated on a second working electrode using the methods described herein. In certain embodiments, the first aspartate-responsive active area comprises a potassium-independent aspartate oxidase and the second aspartate-responsive active area comprises a potassium-dependent aspartate oxidase. In certain embodiments, the first asparagine-responsive active area comprises a potassium-independent asparaginase and the second asparagine-responsive active area comprises a potassium-dependent asparaginase.

In certain embodiments, the method can further include adding a membrane composition on top of the cured enzyme composition(s). In certain embodiments, the membrane composition can include a polymer, e.g., polyvinylpyridine-based polymer, e.g., a polyvinylpyridine, and/or a crosslinking agent, e.g., polyethylene glycol diglycidyl ether. In certain embodiments, the method can include curing the polymer composition.

Generally, the thickness of the membrane is controlled by the concentration of the membrane solution, by the number of droplets of the membrane solution applied, by the number of times the sensor is dipped in or sprayed with the membrane solution, by the volume of membrane solution sprayed on the sensor, and the like, and by any combination of these factors. In certain embodiments, the membrane described herein can have a thickness ranging from about 0.1 micrometers (μm) to about 1,000 μm, e.g., from about 1 μm to and about 500 μm, about 10 μm to about 100 μm or about 10 μm to about 100 μm. In certain embodiments, the sensor can be dipped in the membrane solution more than once. For example, but not by way of limitation, a sensor (or working electrode) of the present disclosure can be dipped in a membrane solution at least twice, at least three times, at least four times or at least five times to obtain the desired membrane thickness.

In certain embodiments, the membrane can overlay one or more active areas, and in certain embodiments, the active areas can have a thickness from about 0.1 μm to about 100 μm, e.g., from about 1 μm to about 90 μm, from about 1 μm to about 80 μm, from about 1 μm to about 70 μm, from about 1 μm to about 60 μm, from about 1 μm to about 50 μm, from about 1 μm to about 40 μm, from about 1 μm to about 30 μm, from about 1 μm to about 20 μm, from about 0.5 μm to about 10 μm, from about 1 μm to about 10 μm, from about 1 μm to about 5 μm or from about 0.1 μm to about 5 μm. In certain embodiments, a series of droplets can be applied atop of one another to achieve the desired thickness of the active area and/or membrane, without substantially increasing the diameter of the applied droplets (i.e., maintaining the desired diameter or range thereof). In certain embodiments, each single droplet can be applied and then allowed to cool or dry, followed by one or more additional droplets. For example, but not by way of limitation, at least one droplet, at least two droplets, at least three droplets, at least four droplets or at least five droplets are added atop of one another to achieve the desired thickness of the active area.

III. Methods of Use

The present disclosure further provides methods of using the analyte sensors disclosed herein. In certain embodiments, the present disclosure provides methods for monitoring potassium, e.g., potassium ion, levels in a sample. For example, but not by way of limitation, the subject in need of potassium monitoring can be a subject that is a risk of developing or has developed one or more disorders and/or conditions associated with dysregulated levels of potassium as described herein. In certain embodiments, the subject in need of potassium monitoring can be a subject that is at risk of developing or has developed one or more disorders and/or conditions associated with a potassium deficiency as described herein. In certain embodiments, the subject in need of potassium monitoring can be a subject that is at risk of developing or has developed one or more disorders and/or conditions associated with elevated levels of potassium as described herein.

In certain embodiments, a potassium sensor of the present disclosure can be used to continuously monitor potassium levels in a subject at risk of having or has a neurological disorder, e.g., Parkinson's disease, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis or Lou Gehrig's disease (ALS) and multiple sclerosis (MS). Additional examples of diseases and disorders associated with potassium dysregulation include autism, cancer, heart failure, myocardial infarction and atherosclerosis as disclosed in Udensi and Tchounwou, Int. J. Clin. Exp. Physiol. 4(3):111-122 (2017), the contents of which is hereby incorporated by reference in its entirety.

In certain embodiments, a potassium sensor of the present disclosure can be implanted in a subject at risk of developing or has developed one or more disorders and/or conditions associated with a potassium deficiency. In certain embodiments, a potassium sensor of the present disclosure can be implanted in a subject for up to about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days or about 20 days. In certain embodiments, the potassium sensors of the present disclosure can be implanted in a subject for up to about 15 days.

In certain embodiments, a method for monitoring potassium includes: (i) providing an analytic sensor including: (a) a sensor tail including at least a first working electrode and a second electrode; (b) a first asparagine-responsive active area disposed upon a surface of the first working electrode, where the asparagine-responsive active area includes an enzyme system including an aspartate oxidase and a first asparaginase and, optionally, a first polymer. (c) a second asparagine-responsive active area disposed upon a surface of the second working electrode, where the asparagine-responsive active area includes an enzyme system including aspartate oxidase and a second asparaginase and, optionally, a second polymer and (d) a mass transport limiting membrane permeable to asparagine that overcoats the first and/or second asparagine-responsive active areas. In certain embodiments, the method further includes (ii) applying a potential to the first working electrode; (iii) obtaining a first signal at or above an oxidation-reduction potential of the first asparagine-responsive active area; (iv) applying a potential to the second working electrode; (v) obtaining a second signal at or above an oxidation-reduction potential of the second asparagine-responsive active area; and (vi) correlating the first signal and the second signal to the concentration of potassium, e.g., potassium ions, in the fluid. In certain embodiments, the first and/or second asparagine-responsive active areas further include an electron-transfer agent. In certain embodiments, the asparaginase of the first asparagine-responsive active area is potassium-dependent and the asparaginase of the second asparagine-responsive active area is potassium-independent. Alternatively, both asparaginases are potassium-dependent but exhibit different potassium dependencies.

In certain embodiments, methods of the present disclosure for monitoring potassium can include: (i) exposing an analyte sensor to a fluid including potassium ions and asparagine, wherein the analyte sensor includes: (a) a sensor tail including at least a first working electrode and a second working electrode; (b) a first asparagine-responsive active area disposed upon a surface of the first working electrode, where the first asparagine-responsive active area includes an enzyme system comprising an aspartate oxidase and an asparaginase and, optionally, a first polymer. (c) a second aspartate-responsive active area disposed upon a surface of the second working electrode, where the second asparagine-responsive active area includes an enzyme system comprising an aspartate oxidase and an asparaginase and, optionally, a second polymer; and (d) a mass transport limiting membrane permeable to asparagine and potassium ions that overcoats the first and/or second asparagine-responsive active areas. In certain embodiments, the method further includes (ii) applying a potential to the first working electrode and the second working electrode; (iii) obtaining a first signal at or above an oxidation-reduction potential of the first asparagine-responsive active area; (iv) obtaining a second signal at or above an oxidation-reduction potential of the second asparagine-responsive active area; and (v) correlating the first signal and the second signal to the concentration of potassium ions in the fluid. In certain embodiments, the first and/or second asparagine-responsive active areas further include an electron-transfer agent. In certain embodiments, the asparaginase of the first asparagine-responsive active area is potassium-dependent and the asparaginase of the second asparagine-responsive active area is potassium-independent. Alternatively, both asparaginases are potassium-dependent but exhibit different potassium dependencies.

In certain embodiments, a method for monitoring potassium includes: (i) providing an analyte sensor including: (a) a sensor tail including at least a first working electrode and a second electrode; (b) a first aspartate-responsive active area disposed upon a surface of the first working electrode, where the first aspartate-responsive active area includes a first aspartate oxidase (e.g., a potassium-dependent aspartate oxidase) and, optionally, a first polymer; (c) a second aspartate-responsive active area disposed upon a surface of the second working electrode, where the second aspartate-responsive active area includes a second aspartate oxidase (e.g., a potassium-independent aspartate oxidase) and, optionally, a second polymer and (d) a mass transport limiting membrane permeable to aspartate that overcoats the first and/or second aspartate-responsive active areas. In certain embodiments, the method further includes (ii) applying a potential to the first working electrode; (iii) obtaining a first signal at or above an oxidation-reduction potential of the first aspartate-responsive active area; (iv) applying a potential to the second working electrode; (v) obtaining a second signal at or above an oxidation-reduction potential of the second aspartate-responsive active area; and (vi) correlating the first signal and the second signal to the concentration of potassium, e.g., potassium ions, in the fluid. In certain embodiments, the first and/or second aspartate-responsive active areas further include an electron-transfer agent. In certain embodiments, the aspartate oxidase of the first aspartate-responsive active area is potassium-dependent, and the aspartate oxidase of the second aspartate-responsive active area is potassium-independent. Alternatively, both aspartate oxidases are potassium-dependent but exhibit different potassium dependencies. In certain embodiments, the membrane polymer comprises a polyvinylpyridine or a polyvinylimidazole. In certain embodiments, the membrane polymer comprises a copolymer of vinylpyridine and styrene. In certain embodiments, the mass transport limiting membrane of the analyte sensor comprises a membrane polymer crosslinked with a branched crosslinker comprising three or more crosslinkable groups. In certain embodiments, the branched crosslinker comprises polyethylene glycol tetraglycidyl ether.

In certain embodiments, methods of the present disclosure for monitoring potassium can include: (i) exposing an analyte sensor to a fluid including potassium ions and aspartate, wherein the analyte sensor includes: (a) a sensor tail including at least a first working electrode and a second working electrode; (b) a first aspartate-responsive active area disposed upon a surface of the first working electrode, where the first aspartate-responsive active area includes a first aspartate oxidase and, optionally, a first polymer; (c) a second aspartate-responsive active area disposed upon a surface of the second working electrode, where the second aspartate-responsive active area includes a second aspartate oxidase and, optionally, a second polymer; and (d) a mass transport limiting membrane permeable to aspartate and potassium ions that overcoats the first and/or second aspartate-responsive active areas. In certain embodiments, the method further includes (ii) applying a potential to the first working electrode and the second working electrode; (iii) obtaining a first signal at or above an oxidation-reduction potential of the first aspartate-responsive active area, e.g., where the first signal being proportional to a concentration of aspartate in the fluid; (iv) obtaining a second signal at or above an oxidation-reduction potential of the second aspartate-responsive active area, e.g., where the second signal being proportional to a concentration of aspartate in the fluid; and (v) correlating the first signal and the second signal to the concentration of potassium ions in the fluid. In certain embodiments, the first and/or second aspartate-responsive active areas further include an electron-transfer agent. In certain embodiments, the aspartate oxidase of the first aspartate-responsive active area is potassium-dependent, and the aspartate oxidase of the second aspartate-responsive active area is potassium-independent. Alternatively, both aspartate oxidases are potassium-dependent but exhibit different potassium dependencies.

In certain embodiments, the present disclosure further provides methods for detecting potassium and a second analyte. For example, but not by way of limitation, the method of the present disclosure can further include detecting a second analyte by providing an analyte sensor that includes an active area and/or exposing an analyte sensor that includes an active area to a fluid, e.g., bodily fluid, comprising aspartate, potassium and the second analyte. In certain embodiments, the analyte sensor for use in a method for detecting potassium and a second analyte can further include a third working electrode; and an active area disposed upon a surface of the third working electrode and responsive to the second analyte differing from the first analyte, where the third active area comprises at least one enzyme responsive to the second analyte and, optionally, a second polymer and/or an electron transfer agent; wherein a portion, e.g., second portion, of the mass transport limiting membrane overcoats the active area. Alternatively, the active area can be covered by a second mass transport limiting membrane that is separate and/or different than the mass transport limiting membrane that overcoats at least one of the aspartate-responsive active areas. In certain embodiments, the second mass transport limiting membrane can overcoat a portion of at least one of the aspartate-responsive active areas.

A. In certain non-limiting embodiments, the presently disclosed subject matter provides analyte sensors for detecting potassium levels comprising:

(i) a sensor tail comprising at least a first working electrode; and

(ii) a first analyte-responsive active area disposed upon a surface of the first working electrode comprising a first aspartate oxidase.

A1. The analyte sensor of A, further comprising a first mass transport limiting membrane permeable to potassium that overcoats the first analyte-responsive active area.

A2. The analyte sensor of A or A1, wherein the first aspartate oxidase is a potassium-dependent aspartate oxidase.

A3. The analyte sensor of A or A1, wherein the first aspartate oxidase is a potassium-independent aspartate oxidase.

A4. The analyte sensor of any one of A-A3, wherein the first analyte-responsive active area further comprises a first asparaginase.

A5. The analyte sensor of A4, wherein the first asparaginase is a potassium-dependent aspartate oxidase.

A6. The analyte sensor of A4, wherein the first asparaginase is a potassium-independent aspartate oxidase.

A7. The analyte sensor of any one of A-A6, further comprising a second working electrode and a second analyte-responsive active area disposed upon a surface of the second working electrode, wherein the second analyte-responsive active area comprises a second aspartate oxidase.

A8. The analyte sensor of A7, wherein the second aspartate oxidase is a potassium-dependent aspartate oxidase.

A9. The analyte sensor of A7, wherein the second aspartate oxidase is a potassium-independent aspartate oxidase.

A10. The analyte sensor of any one of A7-A9, wherein the first aspartate oxidase and the second aspartate oxidase exhibit different potassium dependencies.

A11. The analyte sensor of any one of A7-A10, wherein the second analyte-responsive active area further comprises a second asparaginase.

A12. The analyte sensor of A11, wherein the first asparaginase is a potassium-dependent aspartate oxidase.

A13. The analyte sensor of A11, wherein the second asparaginase is a potassium-independent aspartate oxidase.

A14. The analyte sensor of any one of A1 I-A13, wherein the first asparaginase and the second asparaginase exhibit different potassium dependencies.

A15. The analyte sensor of any one of A-A14, wherein the first analyte-responsive active area and/or the second analyte-responsive active area further comprises an electron transfer agent.

A16. The analyte sensor of any one of A-A15, wherein the first analyte-responsive active area and/or the second analyte-responsive active area further comprises a stabilizing agent.

A17. The analyte sensor of A16, wherein the stabilizing agent is an albumin.

A18. The analyte sensor of A17, wherein the albumin is bovine serum albumin.

A19. The analyte sensor of any one of A-A17, wherein the first analyte-responsive active area and/or the second analyte-responsive active area further comprises a polymer.

A20. The analyte sensor of A19, wherein (i) the first aspartate oxidase and/or first asparaginase is covalently bonded to the polymer and/or (ii) the second aspartate oxidase and/or second asparaginase is covalently bonded to the polymer.

A21. The analyte sensor of any one of A19-A20, wherein the first and/or second electron transfer agent is covalently bonded to the polymer.

A22. The analyte sensor of A15-A21, wherein the first and second electron transfer agent are the same.

A23. The analyte sensor of any one of A-A22, wherein the first mass transport limiting membrane overcoats the second analyte-responsive active area.

A24. The analyte sensor of any one of A-A23, wherein the first mass transport limiting membrane comprises a polyvinylpyridine-based polymer, a polyvinylimidazole, a polyacrylate, a polyurethane, a polyether urethane, a silicone or a combination thereof.

A25. The analyte sensor of A24, wherein the first mass transport limiting membrane comprises a polyvinylpyridine-based polymer.

A26. The analyte sensor of A24, wherein the first mass transport limiting membrane comprises a polyurethane.

A27. The analyte sensor of A24, wherein the first mass transport limiting membrane comprises a silicone.

A28. The analyte sensor of A24, wherein the first mass transport limiting membrane comprises polyvinylpyridine.

A29. The analyte sensor of A24, wherein the first mass transport limiting membrane comprises a copolymer of vinylpyridine and styrene.

A30. The analyte sensor of A-A29, further comprising a third working electrode and a third analyte-responsive active area disposed upon a surface of the third working electrode and responsive to a second analyte differing from potassium, wherein the third analyte-responsive active area comprises at least one enzyme responsive to the second analyte.

B. In certain non-limiting embodiments, the presently disclosed subject matter provides for analyte sensors comprising:

(i) a sensor tail comprising at least a first working electrode and a second working electrode;

(ii) a first aspartate-responsive active area disposed upon a surface of the first working electrode comprising a first aspartate oxidase; and

(iii) a second aspartate-responsive active area disposed upon a surface of the second working electrode comprising a second aspartate oxidase.

B1. The analyte sensor of B, further comprising a first mass transport limiting membrane permeable to aspartate and potassium that overcoats the first aspartate-responsive active area and/or the second aspartate-responsive active area.

B2. The analyte sensor of B or B1, wherein the first aspartate oxidase is a potassium-dependent aspartate oxidase.

B3. The analyte sensor of any one of B-B2, wherein the second aspartate oxidase is a potassium-independent aspartate oxidase.

B4. The analyte sensor of B or B1, wherein the first aspartate oxidase and second aspartate oxidase are potassium-dependent aspartate oxidases.

B5. The analyte sensor of B4, wherein the first aspartate oxidase and the second asparaginase exhibit different potassium dependencies.

B6. The analyte sensor of any one of B-B5, wherein the first aspartate-responsive active area further comprises an electron transfer agent.

B7. The analyte sensor of any one of B-B6, wherein the first aspartate-responsive active area further comprises a stabilizing agent.

B8. The analyte sensor of B7, wherein the stabilizing agent is an albumin.

B9. The analyte sensor of B8, wherein the albumin is bovine serum albumin.

B10. The analyte sensor of any one of B-B9, wherein the second aspartate-responsive active area further comprises an electron transfer agent.

B11. The analyte sensor of any one of B-B10, wherein the second aspartate-responsive active area further comprises a stabilizing agent.

B12. The analyte sensor of B1, wherein the stabilizing agent is an albumin.

B13. The analyte sensor of B12, wherein the albumin is bovine serum albumin.

B14. The analyte sensor of any one of B-B13, wherein the first and/or second aspartate-responsive active areas further comprise a polymer.

B15. The analyte sensor of B14, wherein the first aspartate oxidase is covalently bonded to the polymer and/or the second aspartate oxidase is covalently bonded to the polymer.

B16. The analyte sensor of B14 or B15, wherein the first and/or second electron transfer agent is covalently bonded to the polymer.

B17. The analyte sensor of any one of B10-B16, wherein the first and second electron transfer agent are the same.

B18. The analyte sensor of any one of B1-B17, wherein the first mass transport limiting membrane comprises a polyvinylpyridine-based polymer, a polyvinylimidazole, a polyacrylate, a polyurethane, a polyether urethane, a silicone or a combination thereof.

B19. The analyte sensor of B18, wherein the first mass transport limiting membrane comprises a polyvinylpyridine-based polymer.

B20. The analyte sensor of B18, wherein the first mass transport limiting membrane comprises a polyurethane.

B21. The analyte sensor of B18, wherein the first mass transport limiting membrane comprises a silicone.

B22. The analyte sensor of B18, wherein the first mass transport limiting membrane comprises polyvinylpyridine.

B23. The analyte sensor of B18, wherein the first mass transport limiting membrane comprises a copolymer of vinylpyridine and styrene.

B24. The analyte sensor of B-B23, further comprising:

(iv) a third working electrode; and

(v) a third active area disposed upon a surface of the third working electrode and responsive to a second analyte differing from potassium, wherein the third active area comprises at least one enzyme responsive to the second analyte.

B25. The analyte sensor of B24, further comprising a second portion of the mass transport limiting membrane that overcoats the third active area.

B26. The analyte sensor of B24, further comprising a second mass transport limiting membrane that overcoats the third active area.

B27. The analyte sensor of B26, wherein the second mass transport limiting membrane further overcoats the first aspartate-responsive active area and/or the second aspartate-responsive active area.

B28. The analyte sensor of B26 or B27, wherein the second mass transport limiting membrane comprises a polymer that is not present in the first mass transport limiting membrane.

C. In certain non-limiting embodiments, the presently disclosed subject matter provides for analyte sensors comprising:

(i) a sensor tail comprising at least a first working electrode and a second working electrode;

(ii) a first asparagine-responsive active area disposed upon a surface of the first working electrode comprising a first aspartate oxidase and a first asparaginase; and

(iii) a second asparagine-responsive active area disposed upon a surface of the second working electrode comprising a second aspartate oxidase and a second asparaginase.

C1. The analyte sensor of C, further comprising a first mass transport limiting membrane permeable to aspartate and potassium that overcoats the first asparagine-responsive active area and/or the second asparagine-responsive active area.

C2. The analyte sensor of C or C1, wherein the first asparaginase is a potassium-dependent asparaginase.

C3. The analyte sensor of any one of C-C2, wherein the second asparaginase is a potassium-independent asparaginase.

C4. The analyte sensor of C or C1, wherein the first asparaginase and second asparaginase are potassium-dependent asparaginases.

C5. The analyte sensor of C4, wherein the first asparaginase and the second asparaginase exhibit different potassium dependencies.

C6. The analyte sensor of any one of C-C5, wherein the first asparagine-responsive active area further comprises an electron transfer agent.

C7. The analyte sensor of any one of C-C6, wherein the first asparagine-responsive active area further comprises a stabilizing agent.

C8. The analyte sensor of C7, wherein the stabilizing agent is an albumin.

C9. The analyte sensor of C8, wherein the albumin is bovine serum albumin.

C10. The analyte sensor of any one of C-C9, wherein the second asparagine-responsive active area further comprises an electron transfer agent.

C11. The analyte sensor ofany one of C-C10, wherein the second asparagine-responsive active area further comprises a stabilizing agent.

C12. The analyte sensor of C11, wherein the stabilizing agent is an albumin.

C13. The analyte sensor of C12, wherein the albumin is bovine serum albumin.

C14. The analyte sensor of any one of C-C13, wherein the first and/or second asparagine-responsive active areas further comprise a polymer.

C15. The analyte sensor of C14, wherein the first asparaginase is covalently bonded to the polymer and/or the second asparaginase is covalently bonded to the polymer.

C16. The analyte sensor of C14 or C15, wherein the first and/or second electron transfer agent is covalently bonded to the polymer.

C17. The analyte sensor of any one of C10-C16, wherein the first and/or second electron transfer agent are the same.

C18. The analyte sensor of any one of C1-C17, wherein the first mass transport limiting membrane comprises a polyvinylpyridine-based polymer, a polyvinylimidazole, a polyacrylate, a polyurethane, a polyether urethane, a silicone or a combination thereof.

C9. The analyte sensor of C18, wherein the first mass transport limiting membrane comprises a polyvinylpyridine-based polymer.

C20. The analyte sensor of C18, wherein the first mass transport limiting membrane comprises a polyurethane.

C21. The analyte sensor of C18, wherein the first mass transport limiting membrane comprises a silicone.

C22. The analyte sensor of C18, wherein the first mass transport limiting membrane comprises polyvinylpyridine.

C23. The analyte sensor of C18, wherein the first mass transport limiting membrane comprises a copolymer of vinylpyridine and styrene.

C24. The analyte sensor of C-C23, further comprising:

(iv) a third working electrode; and

(v) a third active area disposed upon a surface of the third working electrode and responsive to a second analyte differing from potassium, wherein the third active area comprises at least one enzyme responsive to the second analyte.

C25. The analyte sensor of C24, further comprising a second portion of the mass transport limiting membrane overcoats the third active area.

C26. The analyte sensor of C24, further comprising a second mass transport limiting membrane overcoats the third active area.

C27. The analyte sensor of C26, wherein the second mass transport limiting membrane further overcoats the first asparagine-responsive active area and/or the second asparagine-responsive active area.

C28. The analyte sensor of C26 or C27, wherein the second mass transport limiting membrane comprises a polymer that is not in the first mass transport limiting membrane.

C29. The analyte sensor of any one of C-C28, wherein the first asparagine-responsive active area comprises a first enzymatic layer comprising the aspartate oxidase and a second enzymatic layer comprising the first asparaginase disposed upon the first enzyme layer.

C30. The analyte sensor of any one of C-C29, wherein the second asparagine-responsive active area comprises a first enzymatic layer comprising the aspartate oxidase and a second enzymatic layer comprising the second asparaginase disposed upon the first enzyme layer.

C31. The analyte sensor of any one of C-C28, wherein the first asparagine-responsive active area includes an enzymatic layer comprising the first aspartate oxidase and the first asparaginase.

C32. The analyte sensor of any one of C-C29 and C31, wherein the second asparagine-responsive active area includes an enzymatic layer comprising the second aspartate oxidase and the second asparaginase.

D. In certain non-limiting embodiments, the presently disclosed subject matter provides for analyte sensors for measuring potassium levels comprising:

(i) a sensor tail comprising at least a first working electrode and a second working electrode;

(ii) a first analyte-responsive active area disposed upon a surface of the first working electrode comprising a first enzymatic layer comprising a first aspartate oxidase and a second enzymatic layer comprising a first asparaginase; and

(iii) a second analyte-responsive active area disposed upon a surface of the second working electrode comprising a first enzymatic layer comprising a second aspartate oxidase and a second enzymatic layer comprising a second asparaginase.

D1. The analyte sensor of D, wherein the second enzymatic layer of the first analyte-responsive active area is disposed upon the first enzymatic layer of the first analyte-responsive active area and/or wherein the second enzymatic layer of the second analyte-responsive active area is disposed upon the first enzymatic layer of the second analyte-responsive active area.

D2. The analyte sensor of D or D1, wherein the first asparaginase is a potassium-dependent asparaginase.

D3. The analyte sensor of any one of D-D2, wherein the second asparaginase is a potassium-independent asparaginase.

D4. The analyte sensor of D or D1, wherein the first asparaginase and second asparaginase are potassium-dependent asparaginases.

D5. The analyte sensor of D4, wherein the first asparaginase and the second asparaginase exhibit different potassium dependencies.

D6. The analyte sensor of any one of D-D5, wherein the first asparagine-responsive active area further comprises an electron transfer agent.

D7. The analyte sensor of any one of D-D6, wherein the first asparagine-responsive active area further comprises a stabilizing agent.

D8. The analyte sensor of D7, wherein the stabilizing agent is an albumin.

D9. The analyte sensor of D8, wherein the albumin is bovine serum albumin.

D10. The analyte sensor of any one of D-D9, wherein the second asparagine-responsive active area further comprises an electron transfer agent.

D11. The analyte sensor of any one of D-D10, wherein the second asparagine-responsive active area further comprises a stabilizing agent.

D12. The analyte sensor of D11, wherein the stabilizing agent is an albumin.

D13. The analyte sensor of D12, wherein the albumin is bovine serum albumin.

D14. The analyte sensor of any one of D-D13, wherein the first and/or second asparagine-responsive active areas further comprise a polymer.

D15. The analyte sensor of D14, wherein the first asparaginase is covalently bonded to the polymer and/or the second asparaginase is covalently bonded to the polymer.

D16. The analyte sensor of D14 or D15, wherein the first and/or second electron transfer agent is covalently bonded to the polymer.

D17. The analyte sensor of any one of D10-D16, wherein the first and second electron transfer agent are the same.

D18. The analyte sensor of any one of D1-D17, further comprising a first mass transport limiting membrane permeable to potassium that overcoats the first analyte-responsive active area and/or the second analyte-responsive active area.

D19. The analyte sensor of D18, wherein the first mass transport limiting membrane comprises a polyvinylpyridine-based polymer, a polyvinylimidazole, a polyacrylate, a polyurethane, a polyether urethane, a silicone or a combination thereof.

D20. The analyte sensor of D19, wherein the first mass transport limiting membrane comprises a polyvinylpyridine-based polymer.

D21. The analyte sensor of D19, wherein the first mass transport limiting membrane comprises a polyurethane.

D22. The analyte sensor of D19, wherein the first mass transport limiting membrane comprises a silicone.

D23. The analyte sensor of D19, wherein the first mass transport limiting membrane comprises polyvinylpyridine.

D24. The analyte sensor of D19, wherein the first mass transport limiting membrane comprises a copolymer of vinylpyridine and styrene.

D25. The analyte sensor of D-D24, further comprising:

(iv) a third working electrode; and

(v) a third active area disposed upon a surface of the third working electrode and responsive to a second analyte differing from potassium, wherein the third active area comprises at least one enzyme responsive to the second analyte.

D26. The analyte sensor of D25, further comprising a second portion of the mass transport limiting membrane overcoats the third active area.

D27. The analyte sensor of D25, further comprising a second mass transport limiting membrane overcoats the third active area.

D28. The analyte sensor of D27, wherein the second mass transport limiting membrane further overcoats the first asparagine-responsive active area and/or the second asparagine-responsive active area.

D29. The analyte sensor of D27 or D28, wherein the second mass transport limiting membrane comprises a polymer that is not in the first mass transport limiting membrane.

E. In certain non-limiting embodiments, the presently disclosed subject matter provides for methods for detecting potassium ions in a fluid using the analyte sensors of any one of A-D29.

F. In certain non-limiting embodiments, the presently disclosed subject matter provides for methods for detecting potassium ions in a fluid comprising:

(i) providing an analyte sensor of any one of A-D29;

(ii) applying a potential to the first working electrode and the second working electrode;

(iii) obtaining a first signal at or above an oxidation-reduction potential of the first analyte-responsive active area, first aspartate-responsive active area or first asparagine-responsive active area:

(iv) obtaining a second signal at or above an oxidation-reduction potential of the second analyte-responsive active area, second aspartate-responsive active area or second asparagine-responsive active area; and

(v) correlating the first signal and the second signal to the concentration of the potassium ions in the fluid.

F1. The method of F, wherein the fluid is interstitial fluid.

F2. The method of F or F1, wherein the analyte sensor is implanted in a subject at risk of or having a neurological condition or diabetes.

F3. The method of any one of F-F2, wherein the sensor tail is configured to be implanted in a subject.

F4. The method of any one of F-F3, wherein the analyte sensor is implanted in a subject for at least about 15 days.

EXAMPLES

The presently disclosed subject matter will be better understood by reference to the following Examples, which are provided as exemplary of the presently disclosed subject matter, and not by way of limitation.

Example 1: Potassium Sensor with an Enzyme System Comprising Aspartate Oxidase and Asparaginase

The present example provides sensors for indirectly detecting potassium ion concentrations by analysis of asparagine levels in a sample. The enzyme system ofFIG. 22was used to indirectly detect potassium ions. Each sensor includes an aspartate sensing layer that is disposed upon a working electrode and an asparaginase layer disposed upon the aspartate sensing layer. Each sensor includes a different asparaginase, where each asparaginase differs in their dependency on potassium.

The chemical composition for the aspartate sensing layer is shown in Table 1, which includes an L-aspartate oxidase, a redox mediator, bovine serum albumin (BSA) and the crosslinker, polyethylene glycol diglycidyl ether 400 (PEGDGE400). The chemical compositions for four different types of asparaginase layers are shown in Tables 2-5, each of which include an asparaginase, BSA and PEGDGE400. The asparaginases in Table 2 and Table 4 were prepared according to procedures in Ajewole et al., FEBS Journal 285(8):1528-1539 (2018) and Bejger et al., Acta Crystallogr. D. Biol. Crystallogr. 70(Pt 7):1854-72 (2014). The wild type was PvAspG1 and the mutant asparaginase was a mutant version of PvAspG1 at amino acid position 118. The asparaginases in Tables 3 and 5 were purchased from Sigma (Catalogue No. A3809) and ProSpec (Catalogue No. ENZ-287).

To make the sensors, the components of the aspartate sensing layer were first mixed and incubated for 30 minutes at room temperature in 10 mM PBS buffer and deposited twice on a working electrode. The resulting sensing layer was cured. Asparaginase layers were formed by depositing the asparagine sensing chemistry formulations from Tables 2-5 onto the aspartate sensing layer and cured.

The sensors were then tested in 100 mM TRIS-HCl buffer at pH 7.5 at 33° C. As shown inFIG. 26, asparagine was first added at a concentration of 100 μM and then at a concentration of 400 μM. All sensors responded to the additions of asparagine. Subsequently, 1 mM, 3 mM and 7 mM of potassium nitrate were added to the solution. As shown inFIG. 26, the sensor response spiked immediately after addition of a new concentration of potassium nitrate (KNO3) followed by increasing over the course of several minutes before stabilizing thereafter. Table 6 summarizes the sensor signal increase in percent at different potassium concentrations relative to the sensor current at 400 μM asparagine with no potassium present. The data shown inFIG. 26and in Table 6 are the average of 6 sensors for each type of asparaginase. It can be seen that all four types of sensors responded to potassium differently, indicating that such asparaginases can be included in a two-channel sensor as shown inFIG. 23orFIG. 24for detecting potassium levels in a sample.

Example 2: Potassium Sensor Comprising Aspartate Oxidase

The present example provides sensors for indirectly detecting potassium ion concentrations by analysis of aspartate levels in a sample using a potassium-dependent enzyme. Each sensor includes an aspartate sensing layer disposed upon a carbon working electrode that includes the enzyme system ofFIG. 27to indirectly detect potassium ions. As shown inFIG. 27, the aspartate sensing layer includes a potassium-dependent aspartate oxidase and a redox mediator.

To make the sensors, the components of the aspartate sensing layer were mixed and incubated at room temperature in 10 mM PBS buffer for 30 minutes. Then 30 nL of the mixture was deposited twice on a working electrode, and cured overnight at 25° C. under 60% relative humidity. The components for the aspartate sensing layer are shown in Table 7, which include an L-aspartate oxidase, a redox mediator, bovine serum albumin (BSA) and the crosslinker, polyethylene glycol diglycidyl ether 400 (PEGDGE400).

The sensors were then tested in 20 mM TRIS buffer at pH 7.5 at 33° C. As shown inFIG. 28, the sensor was tested in the presence of 100 μM aspartate and 120 mM NaCl. Subsequently, 1 mM, 3 mM, 5 mM, 7 mM and 10 mM of potassium chloride (KCl) was added to the solution. As shown inFIG. 28, the sensor response spiked immediately after addition of a new KCl concentration and increased over the course of several minutes before stabilizing thereafter.

The sensors were tested in the presence of different concentrations of potassium and aspartate at a stable concentration of NaCl (130 mM).FIG. 29shows the current response of a sensor in the presence of various concentrations of aspartate and potassium, andFIG. 30provides the percentage increase in current response of the sensor in the presence of various concentrations of aspartate and potassium. As shown inFIGS. 29 and 30, the sensors can detect different potassium levels in the presence of different concentrations of aspartate, and that higher relative responses are observed at lower levels of aspartate.

Various patents, patent applications, publications, product descriptions, protocols, and sequence accession numbers are cited throughout this application, the inventions of which are incorporated herein by reference in their entireties for all purposes.