Patent Description:
Periodic, ex vivo analyte monitoring using a withdrawn bodily fluid can be sufficient to observe a given physiological condition for many individuals. However, ex vivo analyte monitoring may be inconvenient or painful for some persons, particularly if bodily fluid withdrawal needs to occur fairly frequently (e. , several times per day). Continuous analyte monitoring using an implanted in vivo analyte sensor may 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 due to the convenience offered. Subcutaneous, interstitial, or dermal analyte sensors can provide sufficient measurement accuracy in many cases while affording minimal user discomfort.

Many analytes represent intriguing targets for physiological analyses, provided that a suitable detection chemistry can be identified. To this end, amperometric sensors configured for assaying glucose 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, lactate, oxygen, pH, Ale, ketones, and the like. Sensors configured for detecting analytes commonly dysregulated in combination with glucose are known but are considerably less refined at present.

In vivo analyte sensors typically are configured to analyze for a single analyte in order to provide specific analyses, oftentimes employing an enzyme to provide high specificity for a given analyte. Because of such analytical specificity, current in vivo analyte sensors configured for assaying glucose are generally ineffective for assaying other analytes that are frequently dysregulated in combination with glucose or resulting from dysregulated glucose levels. At best, current analyte monitoring approaches require a diabetic individual to wear two different in vivo analyte sensors, one configured for assaying glucose and the other configured for assaying another analyte of interest, such as lactate or ketones. Analyte monitoring approaches employing multiple in vivo analyte sensors may be highly inconvenient for a user. Moreover, when multiple in vivo analyte sensors are used, there is an added cost burden for equipment and an increased statistical likelihood for failure of at least one of the individual in vivo analyte sensors.

<CIT> discloses a method for sensing an analyte utilizing a sensor having a working electrode, the method includes providing the working electrode with an analyte-specific enzyme and a redox mediator, providing the working electrode to the analyte, accumulating charge derived from the analyte reacting with the analyte-specific enzyme and the redox mediator for a set period of time, connecting the working electrode to circuit after the set period of time, and measuring the signal from the accumulated charge.

The following figures are included to illustrate certain aspects of the present disclosure, and should not be viewed as exclusive embodiments.

The invention is as specified in the claims. The present disclosure generally describes analyte sensors employing multiple enzymes for detection of two different analytes and, more specifically, analyte sensors employing multiple enzymes for detection of glucose and ketones and corresponding methods for use thereof.

As discussed above, analyte sensors employing an enzyme are commonly used to detect a single analyte, such as glucose or a related analyte, due to the enzyme's frequent specificity for a particular substrate or class of substrate. However, the monitoring of multiple analytes may be complicated by the need to employ a corresponding number of analyte sensors to facilitate the separate detection of each analyte. This approach may be problematic or undesirable, especially when monitoring multiple analytes in vivo, due to issues such as, for example, the cost of multiple analyte sensors, user discomfort when wearing multiple analyte sensors, and an increased statistical likelihood for failure of an individual analyte sensor.

The present disclosure provides analyte sensors that are responsive to both glucose and ketones, two analytes that are commonly dysregulated in diabetic individuals. Since glucose and ketones concentrations may not directly correlate with each other in a diabetic individual also exhibiting ketoacidosis (ketone dysregulation), it may be advantageous to monitor both analytes concurrently using the analyte sensors disclosed herein, potentially leading to improved health outcomes. In addition to providing health benefits for diabetic individuals, the analyte sensors may be beneficial for other individuals who wish to monitor their ketones levels, such as individuals practicing a ketogenic diet. Ketogenic diets may be beneficial for promoting weight loss as well as helping epileptic individuals manage their condition. Concurrent glucose monitoring during ketogenic diet monitoring may offer related advantages.

In particular, the present disclosure provides analyte sensors in which a glucose-responsive active area and a ketones-responsive active area are present within the tail of a single analyte sensor, thereby allowing both analytes to be monitored concurrently for identifying potential dysregulation thereof using the single analyte sensor. As evident from the description above, the concurrent detection of glucose and ketones using a single analyte sensor may provide several advantages over monitoring approaches employing separate analyte sensors. Various physical dispositions of the glucose-responsive active area and the ketones-responsive active area are possible within the analyte sensors, as discussed hereinafter. Particular implementations of the present disclosure include sensor architectures in which the glucose-responsive active area and the ketones-responsive active area may be interrogated separately to determine the concentration of each analyte, such as through disposing the active areas upon separate working electrodes. As discussed hereinafter, there are challenges associated with incorporating active areas featuring different detection chemistries upon a single analyte sensor, which are addressed by the present disclosure.

Glucose-responsive analyte sensors are a well-studied and still developing field to aid diabetic individuals in better managing their health. Despite the prevalence of comorbid analyte dysregulation in diabetic individuals, sensor chemistries suitable for detecting ketones and other analytes commonly dysregulated in combination with glucose have significantly lagged behind the more well-developed glucose detection chemistries. The present disclosure alleviates this deficiency by providing sensor chemistries suitable for detecting ketones with good response stability over a range of ketones concentrations, particularly detection chemistries utilizing enzyme systems comprising at least two enzymes that are capable of acting in concert to facilitate detection of ketones. As used herein, the term "in concert" refers to a coupled enzymatic reaction, in which the product of a first enzymatic reaction becomes the substrate for a second enzymatic reaction, and the second enzymatic reaction serves as the basis for measuring the concentration of the substrate (analyte) reacted during the first enzymatic reaction. Although defined in terms of two coupled enzymatic reactions, it is to be appreciated that more than two enzymatic reactions may be coupled as well in some instances. For example, the product of a first enzymatic reaction may become the substrate of a second enzymatic reaction, and the product of the second enzymatic reaction may become the substrate for a third enzymatic reaction, with the third enzymatic reaction serving as the basis for measuring the concentration of the substrate (analyte) reacted during the first enzymatic reaction. Discussion of suitable enzyme systems for detecting ketones according to the disclosure herein follows hereinbelow.

It may be desirable to utilize two or more enzymes acting in concert with one another to detect a given analyte of interest when a single enzyme is unable to facilitate detection. Situations in which a single enzyme may be ineffective for promoting analyte detection include, for example, those in which the enzyme is inhibited by one or more products of the enzymatic reaction or is unable to cycle between an oxidized state and reduced state when disposed within an analyte sensor. Some products produced by a single enzyme may not be electrochemically detectable.

Even having suitable detection chemistries in hand, combining a glucose-responsive active area and a ketones-responsive active area upon a single analyte sensor is not a straightforward matter. Glucose-responsive analyte sensors commonly employ a membrane overcoating the glucose-responsive active area to function as a mass transport limiting membrane and/or to improve biocompatibility. Limiting glucose access to the glucose-responsive active area with a mass transport limiting membrane can aid in avoiding sensor overload (saturation), thereby improving detection performance and accuracy. A mass transport limiting membrane may act as a diffusion-limiting barrier to reduce the rate of mass transport of glucose to accomplish the foregoing. The mass transport limiting membrane may be homogeneous and comprise a single membrane polymer in conventional glucose-responsive sensors. Unfortunately, glucose and ketones exhibit significantly different permeability values through a given membrane material, such that if a single mass transport limiting membrane overcoats the active areas of an analyte sensor capable of detecting both glucose and ketones, significantly different sensitivities may be realized for each analyte, thereby complicating one's ability to detect glucose and ketones concurrently and accurately. Although analyte sensitivity issues may be addressed, in principle, by adjusting the membrane thickness and/or altering the size of the active areas with respect to one another, these solutions may be difficult to implement in practice.

In response to the foregoing, the present disclosure also provides membrane compositions and methods for deposition thereof that are suitable to facilitate concurrent detection of glucose and ketones. Specifically, the present disclosure provides membrane compositions having different permeability values that may be disposed separately as distinct compositions upon the glucose-responsive active area and the ketones-responsive active area. Surprisingly, a membrane polymer suitable for use as a mass transport limiting membrane in a glucose-responsive analyte sensor may also be suitably incorporated in a multi-component mass transport limiting membrane for overcoating the active area in a ketones-responsive analyte sensor, even when the membrane polymer alone is otherwise unsuitable for use with ketones due to poor performance (e.g., undesired permeability values). Advantageously, the architectures of the analyte sensors disclosed herein allow a continuous membrane having a homogenous membrane portion to be disposed upon the glucose-responsive active area of the analyte sensors and a multi-component membrane portion to be disposed upon the ketones-responsive active area of the analyte sensors, thereby levelizing the permeabilities of each analyte concurrently to afford improved sensitivity and detection accuracy. As used herein, the term "homogenous membrane" refers to a membrane comprising a single type of membrane polymer, and the term "multi-component membrane" refers to a membrane comprising two or more types of membrane polymers. Both bilayer and admixed membranes may be suitable for use as the multi-component membrane in the disclosure herein. By utilizing a multi-component membrane in conjunction with the sensor architectures disclosed herein, manufacturing advantages may be realized when combining glucose-responsive and ketones-responsive detection chemistries with one another, as compared to manufacturing approaches that alter the membrane thickness and/or the size of the active areas for adjusting the sensitivity of one of the analytes.

Before describing the analyte sensors of the present disclosure in further detail, a brief overview of suitable in vivo analyte sensor configurations and sensor systems employing the analyte sensors will be provided first so that the embodiments of the present disclosure may be better understood. <FIG> shows a diagram of an illustrative sensing system that may incorporate an analyte sensor of the present disclosure, specifically an analyte sensor comprising a glucose-responsive active area and a ketones-responsive active area. As shown, sensing system <NUM> includes sensor control device <NUM> and reader device <NUM> that are configured to communicate with one another over a local communication path or link, which may be wired or wireless, uni- or bi-directional, and encrypted or non-encrypted. Reader device <NUM> may constitute an output medium for viewing analyte concentrations and alerts or notifications determined by sensor <NUM> or a processor associated therewith, as well as allowing for one or more user inputs, according to some embodiments. Reader device <NUM> may be a multi-purpose smartphone or a dedicated electronic reader instrument. While only one reader device <NUM> is shown, multiple reader devices <NUM> may be present in certain instances. Reader device <NUM> may also be in communication with remote terminal <NUM> and/or trusted computer system <NUM> via communication path(s)/link(s) <NUM> and/or <NUM>, respectively, which also may be wired or wireless, uni- or bi-directional, and encrypted or non-encrypted. Reader device <NUM> may also or alternately be in communication with network <NUM> (e.g., a mobile telephone network, the internet, or a cloud server) via communication path/link <NUM>. Network <NUM> may be further communicatively coupled to remote terminal <NUM> via communication path/link <NUM> and/or trusted computer system <NUM> via communication path/link <NUM>. Alternately, sensor <NUM> may communicate directly with remote terminal <NUM> and/or trusted computer system <NUM> without an intervening reader device <NUM> being present. For example, sensor <NUM> may communicate with remote terminal <NUM> and/or trusted computer system <NUM> through a direct communication link to network <NUM>, according to some embodiments, as described in <CIT>. Any suitable electronic communication protocol may be used for each of the communication paths or links, such as near field communication (NFC), radio frequency identification (RFID), BLUETOOTH® or BLUETOOTH® Low Energy protocols, WiFi, or the like. Remote terminal <NUM> and/or trusted computer system <NUM> may be accessible, according to some embodiments, by individuals other than a primary user who have an interest in the user's analyte levels. Reader device <NUM> may comprise display <NUM> and optional input component <NUM>. Display <NUM> may comprise a touch-screen interface, according to some embodiments.

Sensor control device <NUM> includes sensor housing <NUM>, which may house circuitry and a power source for operating sensor <NUM>. Optionally, the power source and/or active circuitry may be omitted. A processor (not shown) may be communicatively coupled to sensor <NUM>, with the processor being physically located within sensor housing <NUM> or reader device <NUM>. Sensor <NUM> protrudes from the underside of sensor housing <NUM> and extends through adhesive layer <NUM>, which is adapted for adhering sensor housing <NUM> to a tissue surface, such as skin, according to some embodiments.

Sensor <NUM> is adapted to be at least partially inserted into a tissue of interest, such as within the dermal or subcutaneous layer of the skin. Sensor <NUM> may comprise a sensor tail of sufficient length for insertion to a desired depth in a given tissue. The sensor tail may comprise at least one working electrode and a glucose-responsive active area and a ketones-responsive active area upon a surface of the at least one working electrode to facilitate detection of these analytes. A counter electrode may be present in combination with the at least one working electrode. Particular electrode configurations upon the sensor tail are described in more detail below in reference to <FIG>.

One or more mass transport limiting membranes may overcoat the glucose-responsive active area and the ketones-responsive active area upon the at least one working electrode, as also described in further detail below. The glucose-responsive active area may comprise a glucose-responsive enzyme and the ketones-responsive active area may comprise an enzyme system comprising at least two enzymes that are capable of acting in concert to facilitate detection of ketones. Suitable enzyme systems are further described below in reference to <FIG>. The glucose-responsive active area and the ketones-responsive active area may each include a polymer to which at least some of the enzymes are covalently bonded, according to various embodiments. In various embodiments of the present disclosure, glucose and ketones may 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 particular embodiments, analyte sensors of the present disclosure may be adapted for assaying dermal fluid or interstitial fluid to determine concentrations of glucose and/or ketones in vivo.

Referring still to <FIG>, sensor <NUM> may automatically forward data to reader device <NUM>. For example, analyte concentration data (i.e., glucose and/or ketones concentrations) may be communicated automatically and periodically, such as at a certain frequency as data is obtained or after a certain time period has passed, with the data being stored in a memory until transmittal (e.g., every minute, five minutes, or other predetermined time period). In other embodiments, sensor <NUM> may communicate with reader device <NUM> in a non-automatic manner and not according to a set schedule. For example, data may be communicated from sensor <NUM> using RFID technology when the sensor electronics are brought into communication range of reader device <NUM>. Until communicated to reader device <NUM>, data may remain stored in a memory of sensor <NUM>. Thus, a user does not have to maintain close proximity to reader device <NUM> at all times, and can instead upload data at a convenient time. In yet other embodiments, a combination of automatic and non-automatic data transfer may be implemented. For example, data transfer may continue on an automatic basis until reader device <NUM> is no longer in communication range of sensor <NUM>.

An introducer may be present transiently to promote introduction of sensor <NUM> into a tissue. In illustrative embodiments, the introducer may comprise a needle or similar sharp. It is to be recognized that other types of introducers, such as sheaths or blades, may be present in alternative embodiments. More specifically, the needle or other introducer may transiently reside in proximity to sensor <NUM> prior to tissue insertion and then be withdrawn afterward. While present, the needle or other introducer may facilitate insertion of sensor <NUM> into a tissue by opening an access pathway for sensor <NUM> to follow. For example, the needle may facilitate penetration of the epidermis as an access pathway to the dermis to allow implantation of sensor <NUM> to take place, according to one or more embodiments. After opening the access pathway, the needle or other introducer may be withdrawn so that it does not represent a sharps hazard. In illustrative embodiments, suitable needles may be solid or hollow, beveled or non-beveled, and/or circular or non-circular in cross-section. In more particular embodiments, suitable needles may be comparable in cross-sectional diameter and/or tip design to an acupuncture needle, which may have a cross-sectional diameter of about <NUM> microns. It is to be recognized, however, that suitable needles may have a larger or smaller cross-sectional diameter if needed for particular applications.

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

Referring now to <FIG>, particular enzyme systems that may be used for detecting ketones according to the disclosure herein will be described in further detail. In the depicted enzymatic reactions, β-hydroxybutyrate serves as a surrogate for ketones formed in vivo. As shown in <FIG>, one pair of concerted enzymes that may be used for detecting ketones according to the disclosure herein is β-hydroxybutyrate dehydrogenase (HBDH) and diaphorase, which may be deposited within a ketones-responsive active area upon the surface of at least one working electrode, as described further herein. When a ketones-responsive active area contains this pair of concerted enzymes, β-hydroxybutyrate dehydrogenase may convert β-hydroxybutyrate and oxidized nicotinamide adenine dinucleotide (NAD+) into acetoacetate and reduced nicotinamide adenine dinucleotide (NADH), respectively. The enzyme cofactors NAD+ and NADH aid in promoting the concerted enzymatic reactions disclosed herein. The NADH may then undergo reduction under diaphorase mediation, with the electrons transferred during this process providing the basis for ketone detection at the working electrode. Thus, there is a <NUM>:<NUM> molar correspondence between the amount of electrons transferred to the working electrode and the amount of β-hydroxybutyrate converted, thereby providing the basis for ketones detection and quantification based upon the measured amount of current at the working electrode. Transfer of the electrons resulting from NADH reduction to the working electrode may take place through an electron transfer agent, such as an osmium (Os) compound, as described further below. Albumin may be present as a stabilizer with this pair of concerted enzymes. According to particular embodiments, the β-hydroxybutyrate dehydrogenase and the diaphorase may be covalently bonded to a polymer within the ketones-responsive active area of the analyte sensors. The NAD+ may or may not be covalently bonded to the polymer, but if the NAD+ is not covalently bonded, it may be physically retained within the ketones-responsive active area. A membrane overcoating the ketones-responsive active area may aid in retaining the NAD+ within the ketones-responsive active area while still permitting sufficient inward diffusion of ketones to permit detection thereof. Suitable membrane polymers for overcoating the ketones-responsive active area are discussed further herein.

Other suitable chemistries for enzymatically detecting ketones are shown in <FIG>. In both instances, there is again a <NUM>:<NUM> molar correspondence between the amount of electrons transferred to the working electrode and the amount of β-hydroxybutyrate converted, thereby providing the basis for ketones detection.

As shown in <FIG>, β-hydroxybutyrate dehydrogenase (HBDH) may again convert β-hydroxybutyrate and NAD+ into acetoacetate and NADH, respectively. Instead of electron transfer to the working electrode being completed by diaphorase (see <FIG>) and a transition metal electron transfer agent, the reduced form of NADH oxidase (NADHOx (Red)) undergoes a reaction to form the corresponding oxidized form (NADHOx (Ox)). NADHOx (Red) may then reform through a reaction with molecular oxygen to produce superoxide, which may undergo subsequent conversion to hydrogen peroxide under superoxide dismutase (SOD) mediation. The hydrogen peroxide may then undergo reduction at the working electrode to provide a signal that may be correlated to the amount of ketones that were initially present. The SOD may be covalently bonded to a polymer in the ketones-responsive active area, according to various embodiments. Like the enzyme system shown in <FIG>, the β-hydroxybutyrate dehydrogenase and the NADH oxidase may be covalently bonded to a polymer in the ketones-responsive active area, and the NAD may or may not be covalently bonded to a polymer in the ketones-responsive active area. If the NAD+ is not covalently bonded, it may be physically retained within the ketones-responsive active area, with a membrane polymer promoting retention of the NAD+ within the ketones-responsive active area.

As shown in <FIG>, another enzymatic detection chemistry for ketones may utilize β-hydroxybutyrate dehydrogenase (HBDH) to convert β-hydroxybutyrate and NAD+ into acetoacetate and NADH, respectively. The electron transfer cycle in this case is completed by oxidation of poly-<NUM>,<NUM>-phenanthroline-<NUM>,<NUM>-dione at the working electrode to reform NAD. The poly-<NUM>,<NUM>-phenanthroline-<NUM>,<NUM>-dione may or may not be covalently bonded to a polymer within the ketones-responsive active area. Like the enzyme system shown in <FIG>, the β-hydroxybutyrate dehydrogenase may be covalently bonded to a polymer in the ketones-responsive active area, and the NAD may or may not be covalently bonded to a polymer in the ketones-responsive active area. Inclusion of an albumin in the active area may provide a surprising improvement in response stability. A suitable membrane polymer may promote retention of the NAD+ within the ketones-responsive active area.

The glucose-responsive active areas in the analyte sensors disclosed herein may be physically adsorbed to a working electrode surface and may comprise a glucose-responsive enzyme, such as glucose oxidase or glucose dehydrogenase. The glucose-responsive active area may comprise a polymer that is covalently bound to the glucose-responsive enzyme, according to various embodiments. Suitable polymers for inclusion in the active areas are described below.

The analyte sensors disclosed herein may feature active areas of different types (i.e., a glucose-responsive active area and a ketones-responsive active area) upon a single working electrode or upon two or more separate working electrodes. Single working electrode sensor configurations may employ two-electrode or three-electrode detection motifs, according to various embodiments of the present disclosure and as described further herein. Sensor configurations featuring a single working electrode are described hereinafter in reference to <FIG>. Each of these sensor configurations may suitably incorporate a glucose-responsive active area and a ketones-responsive active area according to various embodiments of the present disclosure. Sensor configurations featuring multiple working electrodes are described thereafter in reference to <FIG> and <FIG>. When multiple working electrodes are present, a ketones-responsive active area may be disposed upon a first working electrode and a glucose-responsive active area may be disposed upon a second working electrode. Sensor configurations employing multiple working electrodes may be particularly advantageous for incorporating both a glucose-responsive active area and a ketones-responsive active area according to the disclosure herein, since mass transport limiting membranes having differing compositions and/or different permeability values may be deposited more readily during manufacturing when the active areas are separated and/or spaced apart in this manner. Particular sensor configurations featuring multiple working electrodes disposed in a manner to facilitate deposition of mass transport limiting membranes having differing compositions, particularly by dip coating, upon each working electrode are shown in <FIG>. Suitable techniques for depositing the mass transport limiting membranes disclosed herein include, for example, spray coating, painting, inkjet printing, stenciling, roller coating, dip coating, or the like, and any combination thereof.

When a single working electrode is present in an analyte sensor, three-electrode sensor configurations may comprise a working electrode, a counter electrode, and a reference electrode. Related two-electrode sensor configurations may comprise a working electrode and a second electrode, in which the second electrode may function as both a counter electrode and a reference electrode (i.e., a counter/reference electrode). In both two-electrode and three-electrode sensor configurations, both the glucose-responsive active area and the ketones-responsive active area may be disposed upon the single working electrode. In some embodiments, the various electrodes may be at least partially stacked (layered) upon one another and/or laterally spaced apart from one another upon the sensor tail. Suitable sensor configurations may be substantially flat in shape or substantially cylindrical in shape, with the glucose-responsive active area and the ketones-responsive active area being laterally spaced apart upon the working electrode. In all of the sensor configurations disclosed herein, the various electrodes may be electrically isolated from one another by a dielectric material or similar insulator.

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

Analyte sensor configurations having a single working electrode will now be described in further detail. <FIG> shows a cross-sectional diagram of an illustrative two-electrode analyte sensor configuration having a single working electrode, which is compatible for use in some embodiments of the disclosure herein. As shown, analyte sensor <NUM> comprises substrate <NUM> disposed between working electrode <NUM> and counter/reference electrode <NUM>. Alternately, working electrode <NUM> and counter/reference electrode <NUM> may be located upon the same side of substrate <NUM> with a dielectric material interposed in between (configuration not shown). Active areas 218a and 218b (i.e., a glucose-responsive active area and a ketones-responsive active area) are laterally spaced apart from one another upon the surface of working electrode <NUM>. In the various sensor configurations shown herein, active areas 218a and 218b may comprise multiple spots or a single spot configured for detection of each analyte. Analyte sensor <NUM> may be operable for assaying glucose and ketones by any of coulometric, amperometric, voltammetric, or potentiometric electrochemical detection techniques.

Referring still to <FIG>, membrane <NUM> overcoats at least active areas 218a and 218b and may optionally overcoat some or all of working electrode <NUM> and/or counter/reference electrode <NUM>, or the entirety of analyte sensor <NUM>. One or both faces of analyte sensor <NUM> may be overcoated with membrane <NUM>. Membrane <NUM> may comprise one or more polymeric membrane materials (membrane polymers) having suitable capabilities for limiting analyte flux to active areas 218a and 218b. Although not readily apparent in <FIG>, the composition of membrane <NUM> may vary at active areas 218a and 218b in order to differentially regulate the analyte flux at each location, as described further herein. For example, membrane <NUM> may be sprayed and/or printed onto active areas 218a and 218b, such that the composition of membrane <NUM> differs at each location. In another alternative, membrane <NUM> may be deposited by dip coating starting from end A of analyte sensor <NUM>. Specifically, end A of analyte sensor <NUM> may be dipped in a first coating formulation to overcoat active area 218a. After partially curing the first coating formulation upon active area 218a, end A of analyte sensor <NUM> may be dipped in a second coating formulation to overcoat both active areas 218a and 218b with the second coating formulation. As such, membrane <NUM> may be continuous and feature a bilayer at active area 218a and be homogeneous at active area 218b.

<FIG> show cross-sectional diagrams of illustrative three-electrode sensor configurations having a single working electrode, which are compatible for use in some embodiments of the disclosure herein. Three-electrode sensor configurations featuring a single working electrode may be similar to that shown for analyte sensor <NUM> in <FIG>, except for the inclusion of additional electrode <NUM> in analyte sensors <NUM> and <NUM> (<FIG>). With additional electrode <NUM>, electrode <NUM> may then function as either a counter electrode or a reference electrode, and additional electrode <NUM> may fulfill the other electrode function not otherwise accounted for. Working electrode <NUM> continues to fulfill its original function in either case. Additional electrode <NUM> may be disposed upon either working electrode <NUM> or electrode <NUM>, with a separating layer of dielectric material in between each. For example, as depicted in <FIG>, electrodes <NUM>, <NUM> and <NUM> are located upon the same face of substrate <NUM> and are electrically isolated from one another by dielectric layers 219a, 219b and 219c in between. Alternately, at least one of electrodes <NUM>, <NUM> and <NUM> may be located upon opposite faces of substrate <NUM>, as shown in <FIG>. Thus, in some embodiments, electrode <NUM> (working electrode) and electrode <NUM> (counter electrode) may be located upon opposite faces of substrate <NUM>, with electrode <NUM> (reference electrode) being located upon one of electrodes <NUM> or <NUM> and spaced apart therefrom with a dielectric material. Reference material layer <NUM> (e.g., Ag/AgC!) may be present upon electrode <NUM>, with the location of reference material layer <NUM> not being limited to that depicted in <FIG>. As with analyte sensor <NUM> shown in <FIG>, active areas 218a and 218b in analyte sensors <NUM> and <NUM> are disposed laterally spaced apart from one another upon working electrode <NUM> in the sensor configurations of <FIG>. Like analyte sensor <NUM>, analyte sensors <NUM> and <NUM> may be operable for assaying glucose and ketones by any of coulometric, amperometric, voltammetric, or potentiometric electrochemical detection techniques.

Also like analyte sensor <NUM>, membrane <NUM> may also overcoat active areas 218a and 218b, as well as other sensor components, in analyte sensors <NUM> and <NUM>. Additional electrode <NUM> may be overcoated with membrane <NUM> in some embodiments. Although <FIG> have depicted all of electrodes <NUM>, <NUM> and <NUM> as being overcoated with membrane <NUM>, it is to be recognized that only working electrode <NUM> or active areas 218a and 218b may be overcoated in some embodiments. Although not apparent in <FIG>, the thickness of membrane <NUM> may be the same or different at various locations, such as varying thicknesses at active areas 218a and 218b. Likewise, membrane <NUM> may also vary compositionally at active areas 218a and 218b in order to differentially regulate the analyte flux at each location. For example, dip coating from end A of analyte sensors <NUM> and <NUM> may be used to deposit a continuous membrane featuring a bilayer membrane portion at active area 218a and a homogeneous membrane portion at active area 218b, as described in more detail above for <FIG>. As in two-electrode analyte sensor configurations (<FIG>), one or both faces of analyte sensors <NUM> and <NUM> may be overcoated with membrane <NUM> in the sensor configurations of <FIG>, or the entirety of analyte sensors <NUM> and <NUM> may be overcoated. Accordingly, the three-electrode sensor configurations shown in <FIG> should be understood as being illustrative and non-limiting of the disclosure herein, with alternative electrode and/or layer configurations residing within the scope of the present disclosure.

Sensor configurations having multiple working electrodes, specifically two working electrodes, will now be described in further detail in reference to <FIG> and <FIG>. 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 may be incorporated through extension of the disclosure herein. Additional working electrodes may be used to impart additional sensing capabilities to the analyte sensors beyond just glucose and ketones sensing.

<FIG> shows 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 some embodiments of the disclosure herein. As shown in <FIG>, analyte sensor <NUM> includes working electrodes <NUM> and <NUM> disposed upon opposite faces of substrate <NUM>. Active area 310a is disposed upon the surface of working electrode <NUM>, and active area 310b is disposed upon the surface of working electrode <NUM>. Active areas 310a and 310b may be glucose-responsive and ketones-responsive, according to various embodiments of the present disclosure. Counter electrode <NUM> is electrically isolated from working electrode <NUM> by dielectric layer <NUM>, and reference electrode <NUM> is electrically isolated from working electrode <NUM> by dielectric layer <NUM>. Outer dielectric layers <NUM> and <NUM> are positioned upon reference electrode <NUM> and counter electrode <NUM>, respectively. Membrane <NUM> has first membrane portion 340a and second membrane portion 340b, which separately overcoat at least active areas 310a and 310b, respectively, according to various embodiments, with other components of analyte sensor <NUM> or the entirety of analyte sensor <NUM> optionally being overcoated with first membrane portion 340a and/or second membrane portion 340b as well. Again, membrane <NUM> may be continuous but vary compositionally within first membrane portion 340a and second membrane portion 340b (i.e., upon active areas 310a and 310b) in order to afford different permeability values for differentially regulating the analyte flux at each location. For example, different membrane formulations may be sprayed and/or printed onto the opposing faces of analyte sensor <NUM>. Dip coating techniques may also be appropriate, particularly for depositing at least a portion of a bilayer membrane upon one of active areas 310a and 310b. Accordingly, one of first membrane portion 340a and second membrane portion 340b may comprise a bilayer membrane and the other of first membrane portion 340a and second membrane portion 340b may comprise a single membrane polymer, according to particular embodiments of the present disclosure. Like analyte sensors <NUM>, <NUM> and <NUM>, analyte sensor <NUM> may be operable for assaying glucose and ketones by any of coulometric, amperometric, voltammetric, or potentiometric electrochemical detection techniques.

Alternative sensor configurations having multiple working electrodes and differing from that shown in <FIG> may feature a counter/reference electrode instead of separate counter and reference electrodes <NUM>,<NUM>, and/or feature layer and/or membrane arrangements varying from those expressly depicted. For example, the positioning of counter electrode <NUM> and reference electrode <NUM> may be reversed from that depicted in <FIG>. In addition, working electrodes <NUM> and <NUM> need not necessarily reside upon opposing faces of substrate <NUM> in the manner shown in <FIG>.

Although suitable sensor configurations may feature electrodes that are substantially planar in character, it is to be appreciated that sensor configurations featuring non-planar electrodes may be advantageous and particularly suitable for use in the disclosure herein. In particular, substantially cylindrical electrodes that are disposed concentrically with one another may facilitate deposition of a mass transport limiting membrane, as described hereinbelow. <FIG> show perspective views of analyte sensors featuring substantially cylindrical electrodes that are disposed concentrically with respect to one another. Although <FIG> have depicted sensor configurations featuring two working electrodes, it is to be appreciated that similar sensor configurations having either one working electrode or more than two working electrodes are possible through extension of the disclosure herein.

<FIG> shows 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 sensor <NUM> includes central substrate <NUM> about which all electrodes and dielectric layers are disposed concentrically with respect to one another. In particular, working electrode <NUM> is disposed upon the surface of central substrate <NUM>, and dielectric layer <NUM> is disposed upon a portion of working electrode <NUM> distal to sensor tip <NUM>. Working electrode <NUM> is disposed upon dielectric layer <NUM>, and dielectric layer <NUM> is disposed upon a portion of working electrode <NUM> distal to sensor tip <NUM>. Counter electrode <NUM> is disposed upon dielectric layer <NUM>, and dielectric layer <NUM> is disposed upon a portion of counter electrode <NUM> distal to sensor tip <NUM>. Reference electrode <NUM> is disposed upon dielectric layer <NUM>, and dielectric layer <NUM> is disposed upon a portion of reference electrode <NUM> distal to sensor tip <NUM>. As such, exposed surfaces of working electrode <NUM>, working electrode <NUM>, counter electrode <NUM>, and reference electrode <NUM> are spaced apart from one another along longitudinal axis B of analyte sensor <NUM>. Spacing apart of working electrode <NUM> and working electrode <NUM> along a longitudinal axis may also be realized in substantially planar sensor configurations as well, such as those provided above.

Referring still to <FIG>, active areas 414a and 414b are disposed upon the exposed surfaces of working electrodes <NUM> and <NUM>, respectively, thereby allowing contact with a fluid to take place for sensing of glucose and/or ketones to take place. Although active areas 414a and 414b have been depicted as three discrete spots in <FIG>, it is to be appreciated that fewer or greater than three spots may be present in alternative sensor configurations. Each of active areas 414a and 414b can also be a continuous layer that is disposed as a ring upon the exposed surface of working electrodes <NUM> and <NUM>, respectively.

Similar to the sensor configuration discussed above, at least working electrodes <NUM> and <NUM> and active areas 414a and 414b thereon are overcoated with a membrane in the sensor configuration of <FIG>. Although a membrane featuring a single composition may overcoat active areas 414a and 414b, the membrane compositions may differ compositionally in each location in order to afford different permeability values, thereby levelizing the sensor response for each analyte. In the sensor configuration depicted in <FIG>, membrane portion <NUM> having a first composition overcoats working electrode <NUM> and active area 414a, along with optional overcoating of dielectric layer <NUM>, and membrane portion <NUM> having a second composition differing from the first composition overcoats working electrode <NUM> and active area 414b, along with optional overcoating of dielectric layer <NUM> and/or dielectric layer <NUM>. Although not shown in <FIG>, counter electrode <NUM>, reference electrode <NUM>, and dielectric layers <NUM> and <NUM> may also be overcoated with membrane <NUM>.

<FIG> shows an alternative sensor configuration to that depicted in <FIG>, in which all components upon the sensor tail are membrane-coated. In the sensor configuration shown in <FIG>, sensor <NUM> contains working electrode <NUM>, active area 414a, and dielectric layer <NUM> that are each overcoated with first portion 452a of membrane <NUM>. First portion 452a comprises two membrane layers, thereby defining a bilayer membrane. Second portion 452b of membrane <NUM> overcoats working electrode <NUM>, active area 414b, and the remainder of the sensor tail (i.e., counter electrode <NUM>, reference electrode <NUM>, and dielectric layers <NUM>, <NUM> and <NUM>) with a single membrane polymer. While shown as having two portions 452a and 452b, it is to be appreciated that additional portions may be present. Moreover, first portion 452a may be a bilayer membrane, as depicted, or a homogenous admixture of multiple membrane polymers. Sensor configurations having first portion 452a as a bilayer membrane may feature an active area 414a that is ketones-responsive and an active area 414b that is glucose-responsive, according to various embodiments of the present disclosure. Further details regarding suitable membrane polymers and techniques for deposition of first and second portions 452a,452b of membrane <NUM> at each location are provided hereinbelow.

It is to be further appreciated that the positioning of the various electrodes in <FIG> may differ from that expressly depicted. For example, the positions of counter electrode <NUM> and reference electrode <NUM> may be reversed from the depicted configurations in <FIG>. Similarly, the positions of working electrodes <NUM> and <NUM> are not limited to those that are expressly depicted in <FIG>. <FIG> shows an alternative sensor configuration to that shown in <FIG>, in which sensor <NUM> contains counter electrode <NUM> and reference electrode <NUM> that are located more proximal to sensor tip <NUM> and working electrodes <NUM> and <NUM> that are located more distal to sensor tip <NUM>. Sensor configurations in which working electrodes <NUM> and <NUM> are located more distal to sensor tip <NUM> may be advantageous by providing a larger surface area for deposition of active areas 414a and 414b (five discrete sensing spots illustratively shown in <FIG>), thereby facilitating an increased signal strength in some cases. The locations of the bilayer membrane defined by first portion 452a and the homogeneous membrane defined by second portion 452b have been similarly adjusted to accommodate the change in location of working electrodes <NUM> and <NUM>.

Although <FIG> have depicted sensor configurations that are each supported upon central substrate <NUM>, it is to be appreciated that alternative sensor configurations may be electrode-supported instead and lack central substrate <NUM>. In particular, the innermost concentric electrode may be utilized to support the other electrodes and dielectric layers. <FIG> shows an alternative sensor configuration to that depicted in <FIG>, in which sensor <NUM> does not contain central substrate <NUM> and counter electrode <NUM> is the innermost concentric electrode and is employed for disposing reference electrode <NUM>, working electrodes <NUM> and <NUM>, and dielectric layers <NUM>, <NUM>, <NUM>, and <NUM> sequentially thereon. In view of the disclosure herein, it is again to be appreciated that other electrode and dielectric layer configurations may be employed in sensor configurations lacking central substrate <NUM>. As such, the sensor configuration depicted in <FIG> should be considered illustrative in nature and non-limiting.

Accordingly, some embodiments of analyte sensors disclosed herein may comprise a sensor tail comprising at least a working electrode, a glucose-responsive active area comprising a glucose-responsive enzyme disposed upon a surface of the working electrode and a ketones-responsive active area disposed upon the surface of the working electrode and spaced apart from the glucose-responsive active area. The ketones-responsive active area comprises an enzyme system comprising at least two enzymes that are capable of acting in concert to facilitate detection of ketones. Each active area has an oxidation-reduction potential, and the oxidation-reduction potential of the glucose-responsive active area is sufficiently separated from the oxidation-reduction potential of the ketones-responsive active area to allow independent production of a signal from one of the glucose-responsive active area or the ketones-responsive active area.

When the glucose-responsive active area and the ketones-responsive active area are arranged upon a single working electrode in this manner, one of the active areas may be configured such that it can be interrogated separately to facilitate detection of each analyte, as described hereinafter. That is, either the glucose-responsive active area or the ketones-responsive active area may produce a signal independently of the other active area.

Some or other embodiments of analyte sensors disclosed herein may feature the glucose-responsive active area and the ketones-responsive active area upon the surface of different working electrodes. Such analyte sensors may comprise a sensor tail comprising at least a first working electrode and a second working electrode, a ketones-responsive active area disposed upon a surface of the first working electrode, a glucose-responsive active area comprising a glucose-responsive enzyme disposed upon a surface of the second working electrode, and a membrane having a first portion overcoating the ketones-responsive active area and a second portion overcoating the glucose-responsive active area, in which the first portion and the second portion have different compositions.

In particular embodiments, the first portion is multi-component and comprises at least a first membrane polymer and a second membrane polymer that differ from one another, and the second portion is homogeneous and comprises one of the first membrane polymer and the second membrane polymer.

According to various embodiments of the present disclosure, an electron transfer agent may be present in the glucose-responsive active area and the ketones-responsive active area in any of the illustrative sensor configurations disclosed herein. Suitable electron transfer agents may facilitate conveyance of electrons to the adjacent working electrode after either 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. Depending on the sensor configuration used, the electron transfer agents in the glucose-responsive active area and the ketones-responsive active area may be the same or different. For example, when the glucose-responsive active area and the ketones-responsive active area are disposed upon the same working electrode, the electron transfer agent within each active area may be different (e.g., chemically different such that the electron transfer agents exhibit different oxidation-reduction potentials). When multiple working electrodes are present, the electron transfer agent within each active area may be the same or different, since each working electrode may be interrogated separately.

According to various embodiments of the present disclosure, suitable electron transfer agents may 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). According to some embodiments, suitable electron transfer agents may include low-potential osmium complexes, such as those described in <CIT> and <CIT>. Additional examples of suitable electron transfer agents include those described in <CIT>,<CIT> and <CIT>. Other suitable electron transfer agents may comprise 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 may also include, for example, bidentate or higher denticity ligands such as, for example, bipyridine, biimidazole, phenanthroline, or pyridyl(imidazole). Other suitable bidentate ligands may include, for example, amino acids, oxalic acid, acetylacetone, diaminoalkanes, or o-diaminoarenes. Any combination of monodentate, bidentate, tridentate, tetradentate, or higher denticity ligands may be present in a metal complex to achieve a full coordination sphere.

Active areas suitable for detecting glucose and ketones may also comprise a polymer to which the electron transfer agents are covalently bound. Any of the electron transfer agents disclosed herein may comprise suitable functionality to promote covalent bonding to the polymer within the active areas. Suitable examples of polymer-bound electron transfer agents may include those described in <CIT>, <CIT> and <CIT>. Suitable polymers for inclusion in the active areas may include, but are not limited to, polyvinylpyridines (e.g., poly(<NUM>-vinylpyridine)), polyvinylimidazoles (e.g., poly(<NUM>-vinylimidazole)), or any copolymer thereof. Illustrative copolymers that may be suitable for inclusion in the active areas include those containing monomer units such as styrene, acrylamide, methacrylamide, or acrylonitrile, for example. The polymer within each active area may be the same or different.

In particular embodiments of the present disclosure, the mass transport limiting membrane overcoating each active area may comprise at least a crosslinked polyvinylpyridine homopolymer or copolymer. The composition of the mass transport limiting membrane may be the same or different where the mass transport limiting membrane overcoats each active area. In particular embodiments, the portion of the mass transport limiting membrane overcoating the glucose-responsive active area may be single-component (contain a single membrane polymer) and the portion of the mass transport limiting membrane overcoating the ketones-responsive active area may be multi-component (contain two or more different membrane polymers, one of which is a polyvinylpyridine homopolymer or copolymer). The multicomponent membrane may be present as a bilayer membrane or as a homogeneous admixture of the two or more membrane polymers. A homogeneous admixture may be deposited by combining the two or more membrane polymers in a solution and then depositing the solution upon a working electrode. In still more specific embodiments of the present disclosure, the glucose-responsive active area may be overcoated with a membrane comprising a polyvinylpyridine-co-styrene copolymer, and the ketones-responsive active area may be overcoated with a multicomponent membrane comprising polyvinylpyridine and polyvinylpyridine-co-styrene, either as a bilayer membrane or a homogeneous admixture.

The manner of covalent bonding between the electron transfer agent and the polymer comprising each active area is not considered to be particularly limited. Covalent bonding of the electron transfer agent to the polymer may take place by polymerizing a monomer unit bearing a covalently bound electron transfer agent, or the electron transfer agent may be reacted with the polymer separately after the polymer has already been synthesized. According to some embodiments, a bifunctional spacer may covalently bond the electron transfer agent to the polymer within the active area, with a first functional group being reactive with the polymer (e.g., a functional group capable of quaternizing a pyridine nitrogen atom or an imidazole nitrogen atom) and a second functional group being reactive with the electron transfer agent (e.g., a functional group that is reactive with a ligand coordinating a metal ion).

Similarly, according to some or other various embodiments of the present disclosure, one or more of the enzymes within the active areas may be covalently bonded to the polymer. When an enzyme system comprising multiple enzymes is present in a given active area, all of the multiple enzymes may be covalently bonded to the polymer in some embodiments, and in other embodiments, only a portion of the multiple enzymes may be covalently bonded to the polymer. For example, one or more enzymes comprising an enzyme system may be covalently bonded to the polymer and at least one enzyme may be non-covalently associated with the polymer, such that the non-covalently bonded enzyme is physically entrained within the polymer. According to more specific embodiments, covalent bonding of the enzyme(s) to the polymer in a given active area may take place via a crosslinker introduced with a suitable crosslinking agent. Suitable crosslinking agents for reaction with free amino groups in the enzyme (e.g., with the free side chain amine in lysine) may include crosslinking agents such as, for example, polyethylene glycol diglycidyl ether (PEGDGE) or other polyepoxides, cyanuric chloride, N-hydroxysuccinimide, imidoesters, epichlorohydrin, or derivatized variants thereof. Suitable crosslinking agents for reaction with free carboxylic acid groups in the enzyme may include, for example, carbodiimides. The crosslinking of the enzyme to the polymer is generally intermolecular, but can be intramolecular in some embodiments.

The electron transfer agent and/or the enzyme(s) may be associated with the polymer in the active area through means other than covalent bonding as well. In some embodiments, the electron transfer agent and/or the enzyme(s) may be ionically or coordinatively associated with the polymer. For example, a charged polymer may be ionically associated with an oppositely charged electron transfer agent or enzyme(s). In still other embodiments, the electron transfer agent and/or the enzyme(s) may be physically entrained within the polymer without being bonded thereto. Physically entrained electron transfer agents and/or enzyme(s) may still suitably interact with a fluid to promote analyte detection without being substantially leached from the active areas.

In particular embodiments, the glucose-responsive enzyme in the glucose-responsive active area may be covalently bonded to a polymer in the glucose-responsive active area, in combination with an electron transfer agent that is also covalently bonded to the polymer.

In other particular embodiments, at least a portion of the enzymes in the enzyme system within the ketones-responsive active area may be covalently bonded to a polymer in the ketones-responsive active area, in combination with an electron transfer agent that is also covalently bonded to the polymer. One suitable enzyme system that may be suitable to facilitate detection of ketones is β-hydroxybutyrate dehydrogenase (NADH), nicotinamide adenine dinucleotide (NAD+), and diaphorase (see <FIG>). In particular embodiments of the present disclosure, the β-hydroxybutyrate dehydrogenase and diaphorase may be covalently bonded to the polymer in the ketones-responsive active area, and the NAD+ may be non-covalently associated with the polymer. The polymer within the ketones-responsive active area may be chosen such that outward diffusion of the NAD+ is limited. The membrane polymer overcoating the ketones-responsive active area may similarly limit outward diffusion of NAD+ to promote a reasonable sensor lifetime (days to weeks) while still allowing sufficient inward ketones diffusion to promote detection. In still further embodiments, the components of the foregoing enzyme system may be covalently bonded or non-covalently associated with the polymer in the ketones-responsive active area as described previously, in combination with an electron transfer agent that is also covalently bonded to the polymer.

The glucose-responsive and ketones-responsive active areas in the analyte sensors disclosed herein may comprise one or more discrete spots (e.g., one to about ten spots, or even more discrete spots), which may range in size from about <NUM><NUM> to about <NUM><NUM>, although larger or smaller individual spots within the active areas are also contemplated herein. Active areas defined as continuous bands around a cylindrical electrode are also possible in the disclosure herein.

In more specific embodiments, analyte sensors of the present disclosure may comprise a sensor tail that is configured for insertion into a tissue. Suitable tissues are not considered to be particularly limited and are addressed in more detail above. Similarly, considerations for deploying a sensor tail at a particular position within a tissue are addressed above.

In embodiments wherein the glucose-responsive active area and the ketones-responsive active area are arranged upon a single working electrode, the oxidation-reduction potential associated with the glucose-responsive active area may be separated from the oxidation-reduction potential of the ketones-responsive active area by at least about <NUM> mV, or by at least about <NUM> mV, or by at least about <NUM> mV. The upper limit of the separation between the oxidation-reduction potentials is dictated by the working electrochemical window in vivo. By having the oxidation-reduction potentials of the two active areas sufficiently separated in magnitude from one another, an electrochemical reaction make take place within one of the two active areas (i.e., within the glucose-responsive active area or the ketones-responsive active area) without substantially inducing an electrochemical reaction within the other active area. Thus, a signal from one of the glucose-responsive active area or the ketones-responsive active area may be independently produced at or above its corresponding oxidation-reduction potential (the lower oxidation-reduction potential) but below the oxidation-reduction potential of the other of the glucose-responsive active area and the ketones-responsive active area (the higher oxidation-reduction potential). At or above the oxidation-reduction potential (the higher oxidation-reduction potential) of the other active area that was not previously interrogated, in contrast, electrochemical reactions may occur within both the glucose-responsive active area and the ketones-responsive active area. As such, the resulting signal at or above the higher oxidation-reduction potential may include a signal contribution from both the glucose-responsive active area and the ketones-responsive active area, and the observed signal is a composite signal. The signal contribution from one active area (either the glucose-responsive active area or the ketones-responsive active area) at or above its oxidation-reduction potential may then be determined by subtracting from the composite signal the signal obtained solely from either the glucose-responsive active area or the ketones-responsive active area at or above its corresponding oxidation-reduction potential.

In more specific embodiments, the glucose-responsive active area and the ketones-responsive active area may contain different electron transfer agents when the active areas are located upon the same working electrode, so as to afford oxidation-reduction potentials that are sufficiently separated in magnitude from one another. More specifically, the glucose-responsive active area may comprise a first electron transfer agent and the ketones-responsive active area may comprise a second electron transfer agent, with the first and second electron transfer agents being different. The metal center and/or the ligands present in a given electron transfer agent may be varied to provide sufficient separation of the oxidation-reduction potentials within the two active areas, according to various embodiments of the present disclosure.

Ideally, glucose-responsive active areas and ketones-responsive active areas located upon a single working electrode may be configured to attain a steady state current rapidly upon operating the analyte sensor at a given potential. Rapid attainment of a steady state current may be promoted by choosing an electron transfer agent for each active area that changes its oxidation state quickly upon being exposed to a potential at or above its oxidation-reduction potential. Making the active areas as thin as possible may also facilitate rapid attainment of a steady state current. For example, suitable thicknesses for the glucose-responsive active area and ketones-responsive active area may range from about <NUM> microns to about <NUM> microns. In some or other embodiments, combining a conductive material such as, for example, carbon nanotubes, graphene, or metal nanoparticles within one or more of the active areas may promote rapid attainment of a steady state current. Suitable amounts of conductive particles may range from about <NUM>% to about <NUM>% by weight of the active area, or from about <NUM>% to about <NUM>% by weight, or from about <NUM>% to about <NUM>% by weight, or from about <NUM>% to about <NUM>% by weight. Stabilizers may also be employed to promote response stability.

It is also to be appreciated that the sensitivity (output current) of the analyte sensors toward each analyte may be varied by changing the coverage (area or size) of the active areas, the areal 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 may be conducted readily by one having ordinary skill in the art once granted the benefit of the disclosure herein.

Detection methods for assaying glucose and ketones employing analyte sensors featuring a glucose-responsive active area and a ketones-responsive active area upon a single working electrode may comprise: exposing an analyte sensor to a fluid comprising at least one of glucose and ketones. The analyte sensor comprises a sensor tail comprising at least a working electrode, particularly a single working electrode, and at least a glucose-responsive active area and a ketones-responsive active area disposed upon a surface of the working electrode and space apart from the glucose-responsive active area. The glucose-responsive active area comprises a glucose-responsive enzyme and a polymer, and the ketones-responsive active area comprises an enzyme system comprising two or more enzymes that are capable of acting in concert to facilitate detection of ketones. Each active area has an oxidation-reduction potential, and the oxidation-reduction potential of a first active area (e.g., either the glucose-responsive active area or the ketones-responsive active area) is sufficiently separated from the oxidation-reduction potential of the other of the glucose-responsive active area or the ketones-responsive active area to allow production of a signal from the first active area independent of production of a signal from the other active area. The methods additionally comprise: obtaining a first signal at or above a lower of the oxidation-reduction potential and the second oxidation-reduction potential but below a higher of the first oxidation-reduction potential and the second oxidation-reduction potential, such that the first signal is proportional to a concentration of one of glucose or ketones in the fluid; obtaining a second signal at or above a higher of the first oxidation-reduction potential and the second oxidation-reduction potential, such that the second signal is a composite signal comprising a signal contribution from the glucose-responsive active area and a signal contribution from the ketones-responsive active area; and subtracting the first signal from the second signal to obtain a difference signal, the difference signal being proportional to a concentration of one of glucose and ketones.

In more specific embodiments, the oxidation-reduction potential associated with the first active area may be separated from the oxidation-reduction potential of the second active area by at least about <NUM> mV, or by at least about <NUM> mV, or by at least about <NUM> mV in order to provide sufficient separation for independent production of a signal from the first active area. In particular, the oxidation-reduction potentials of the first active area and the second active area may be separated by about <NUM> mV to about <NUM> mV, or about <NUM> mV to about <NUM> mV, or about <NUM> mV to about <NUM> mV.

In some embodiments, the signals associated with each active area may be correlated to a corresponding concentration of glucose or ketones by consulting a lookup table or calibration curve for each analyte. A lookup table for each analyte may be populated by assaying multiple samples having known analyte concentrations and recording the sensor response at each concentration for each analyte. Similarly, a calibration curve for each analyte may be determined by plotting the analyte sensor response for each analyte as a function of the concentration and determining a suitable calibration function over the calibration range (e.g., by regression, particularly linear regression).

A processor may determine which sensor response value in a lookup table is closest to that measured for a sample having an unknown analyte concentration and then report the analyte concentration accordingly. In some or other embodiments, if the sensor response value for a sample having an unknown analyte concentration is between the recorded values in the lookup table, the processor may interpolate between two lookup table values to estimate the analyte concentration. Interpolation may assume a linear concentration variation between the two values reported in the lookup table. Interpolation may be employed when the sensor response differs a sufficient amount from a given value in the lookup table, such as variation of about <NUM>% or greater.

Likewise, according to some or other various embodiments, a processor may input the sensor response value for a sample having an unknown analyte concentration into a corresponding calibration function. The processor may then report the analyte concentration accordingly.

Detection methods for assaying glucose and ketones employing analyte sensors featuring a glucose-responsive active area and a ketones-responsive active area upon separate working electrodes may comprise: exposing an analyte sensor to a fluid comprising at least one of glucose and ketones. The analyte sensor comprises a sensor tail comprising at least a first working electrode and second working electrode, a ketones-responsive active area disposed upon a surface of the first working electrode, a glucose-responsive active area disposed upon a surface of the second working electrode, and a membrane having a first portion overcoating the ketones-responsive active area and a second portion overcoating the glucose-responsive active area. The glucose-responsive active area comprises a glucose-responsive enzyme, and the ketones-responsive active area comprises an enzyme system comprising at least two enzymes that are capable of acting in concert to facilitate detection of ketones.

In particular embodiments, the first portion may be multi-component and comprise at least a first membrane polymer and a second membrane polymer that differ from one another, and the second portion may be homogeneous and comprise one of the first membrane polymer and the second membrane polymer. As such, the membrane overcoating the glucose-responsive active area differs in composition from the multi-component membrane overcoating the ketones-responsive active area.

The methods additionally comprise 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 glucose-responsive active area, in which the first signal is proportional to a concentration of glucose in the fluid, obtaining a second signal at or above an oxidation-reduction potential of the ketones-responsive active area, in which the second signal is proportional to a concentration of ketones in the fluid, and correlating the first signal to the concentration of glucose in the fluid and the second signal to the concentration of ketones in the fluid.

The first portion of the membrane may comprise an admixture of membrane polymers in some embodiments of the present disclosure or comprise a bilayer membrane or other membrane structure having at least two membrane layers in other embodiments of the present disclosure. When the first portion of the membrane comprises a bilayer membrane, the bilayer membrane may comprise a first membrane polymer disposed upon the ketones-responsive active area, and a second membrane polymer disposed upon the first membrane polymer. The homogeneous membrane overcoating the glucose-responsive active area may comprise the second membrane polymer. That is, the first membrane polymer may be disposed directly upon the ketones-responsive active area, and the second membrane polymer may be disposed upon the first membrane polymer and upon the glucose-responsive active area. Thus, the first portion of the membrane may be thicker than the second portion of the membrane. As discussed above, bilayer membranes and homogeneous membranes of this type may be deposited by dip coating of particular electrode configurations in some embodiments of the present disclosure. In particular embodiments of the present disclosure, the first portion of the membrane may comprise polyvinylpyridine (PVP) and polyvinylpyridine-co-styrene, and second portion of the membrane may comprise polyvinylpyridine-co-styrene.

According to more specific embodiments, the first signal and the second signal maybe measured at different times. Thus, in such embodiments, a potential may be alternately applied to the first working electrode and the second working electrode. In other specific embodiments, the first signal and the second signal may be measured simultaneously via a first channel and a second channel, in which case a potential may be applied to both electrodes at the same time. In either case, the signal associated with each active area may then be correlated to the concentration of glucose and ketones using a lookup table or a calibration function in a similar manner to that discussed above.

A poly(vinylpyridine)-bound transition metal complex having the structure shown in Formula <NUM> was prepared. Further details concerning this transition metal complex and electron transfer therewith is provided in commonly owned <CIT>. The subscripts for each monomer represent illustrative atomic ratios and are not indicative of any particular monomer ordering.

Example <NUM>: Detection of Ketones Using an Analyte Sensor Having Diaphorase and β-Hydroxybutyrate Dehydrogenase Acting in Concert. For this example, the enzyme system of <FIG> was used to facilitate detection of ketones. The spotting formulation shown in Table <NUM> below was coated onto a carbon working electrode. Deposition was performed to place six spots, each having an area of around <NUM><NUM>, upon the working electrode. Following deposition, the working electrode was cured overnight at <NUM>. Thereafter, a homogeneous PVP membrane was applied to the working electrode via dip coating using a coating solution formulated with <NUM> of <NUM>/mL PVP, <NUM> of <NUM>/mL PEGDGE400 (PEGDGE with a molecular weight of approximately <NUM>), and <NUM> of <NUM>/mL polydimethylsiloxane (PDMS). Membrane curing was performed for <NUM> hours at <NUM>, followed by <NUM> hours at <NUM> in desiccated vials.

Ketone analyses were conducted by immersing the electrode in <NUM> PBS buffer (pH = <NUM>) at <NUM> and introducing various amounts of β-hydroxybutyrate (<NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM> total β-hydroxybutyrate addition) as a ketones surrogate. <FIG> shows four replicates of the response for an electrode containing diaphorase, NAD+, and β-hydroxybutyrate dehydrogenase when exposed to varying β-hydroxybutyrate concentrations. Only two traces are apparent in <FIG> due to overlap of the signal response for the four sensors tested. As shown, the current response increased over the course of several minutes following exposure to a new β-hydroxybutyrate concentration before stabilizing thereafter. <FIG> shows an illustrative plot of average current response versus β-hydroxybutyrate concentration for the electrodes of <FIG>. The ketone sensors also exhibited a stable response over extended measurement times, as shown in <FIG> shows an illustrative plot of current response for the electrodes of <FIG> when exposed to <NUM> of β-hydroxybutyrate in <NUM> PBS at <NUM> for <NUM> weeks. The mean signal loss over the measurement period was only <NUM>%.

Example <NUM>: Detection of Glucose and Ketones Using an Analyte Sensor Having a Glucose-Responsive Active Area and a Ketones-Responsive Active Area on Separate Working Electrodes. For this example, an analyte sensor was prepared with a glucose-responsive active area comprising glucose oxidase deposited upon a first working electrode and a ketones-responsive active area comprising diaphorase and β-hydroxybutyrate dehydrogenase deposited upon a second working electrode. The two working electrodes were carbon electrodes disposed upon opposing faces of a planar dielectric substrate.

The glucose-responsive active area was deposited upon the first working electrode using the glucose oxidase formulation specified in Table <NUM> below. Active area deposition was conducted by placing five discrete, separate spots, each having an area of approximately <NUM><NUM>, upon the working electrode. Following deposition, the working electrode was cured overnight at <NUM>.

The ketones-responsive active area was deposited upon the second working electrode using the diaphorase/β-hydroxybutyrate formulation specified in Table <NUM> above. Active area deposition and curing was conducted as in Example <NUM> above, except five sensing spots were deposited in this instance. Curing of the ketones-responsive active area and the glucose-responsive active area was conducted at the same time.

Following deposition of the active areas, a bilayer membrane was deposited upon the ketones-responsive active area as follows. A PVP membrane was first deposited upon the ketones-responsive active area using via a modified slot coating procedure. The PVP membrane in this example was deposited from a coating solution formulated with <NUM> of <NUM>/mL PVP, <NUM> of <NUM>/mL PEGDGE400, and <NUM> of <NUM>/mL PDMS. Curing was then performed for <NUM> hours at <NUM>. The slot coating procedure was conducted using a syringe pump to pump the coating solution from a nozzle located a small distance above a row of sensor tails. The coating solution was pumped at a constant rate while moving the nozzle at a fixed rate across the row of sensor tails. Parameters such as flow rates, nozzle diameter, the rate of nozzle movement, the distance between the nozzle and the sensor tails, the solution viscosity, the temperature, and the solvent were varied to afford a membrane having a desired thickness.

Thereafter, the entire assembly (i.e., both working electrodes, the glucose-responsive active area, the PVP coating upon the working electrode containing the ketones-responsive active area, and the counter and reference electrodes) was dip coated to introduce a crosslinked polyvinylpyridine-co-styrene membrane polymer thereon. The membrane polymer coating the entire assembly was deposited using <NUM> of polyvinylpyridine-co-styrene in <NUM>:<NUM> ethanol:HEPES buffer (<NUM>/mL), <NUM> PEGDGE400 in <NUM>:<NUM> ethanol:HEPES buffer (<NUM>/mL), and <NUM> of aminopropyl-terminated polydimethylsiloxane (PDMS) in ethanol (<NUM>/mL). Curing was again performed for <NUM> hours at <NUM>, followed by <NUM> hours at <NUM> in a desiccated environment. Thus, a homogeneous membrane (polyvinylpyridine-co-styrene) was deposited upon the glucose-responsive active area and a bilayer membrane (inner layer of PVP and outer layer of polyvinylpyridine-co-styrene) was deposited upon the ketones-responsive active area.

The analyte sensor was used to assay for glucose and ketones simultaneously in <NUM> PBS at <NUM>. In a first experiment, the sensor was exposed for <NUM> weeks at <NUM> to a <NUM> PBS solution containing <NUM> glucose and <NUM> β-hydroxybutyrate (ketones surrogate). The sensor was held at +<NUM> mV relative to Ag/AgCl for this experiment. <FIG> shows an illustrative plot of the response for an analyte sensor containing a glucose-responsive active area and a ketones-responsive active area disposed upon separate working electrodes following exposure to <NUM> glucose and <NUM> ketones. As shown, the response of the analyte sensor remained very steady over the observation period for both analytes.

Next, glucose and β-hydroxybutyrate were added incrementally to <NUM> PBS at <NUM> to determine the response of the analyte sensor toward each analyte. The sensor was again held at +<NUM> mV relative to Ag/AgCl for this test. Glucose was added over a concentration range of <NUM>-<NUM>, and β-hydroxybutyrate was added over a concentration range of <NUM>-<NUM>. Each analyte was added simultaneously at concentrations of <NUM> or under. Above <NUM>, only additional glucose was added to the solution, with <NUM> representing the maximum ketones concentration tested. <FIG> show illustrative plots of the analyte sensor response to varying concentrations of glucose and β-hydroxybutyrate. As shown in <FIG> and <FIG>, the analyte sensor afforded a linear response toward both analytes over the tested concentration ranges. As shown in <FIG>, the sensor response was rapid for both analytes and remained stable at a given analyte concentration.

Example <NUM>: Detection of Ketones Using an Analyte Sensor Having NADH Oxidase and β-Hydroxybutyrate Dehydrogenase Acting in Concert. For this example, the enzyme system of <FIG> was used to facilitate detection of ketones. The spotting formulation shown in Table <NUM> below was coated onto either a carbon working electrode or a carbon nanotube working electrode. Coating was conducted by hand in <NUM> passes to coat the entirety of the sensor tip. The mean active area was <NUM><NUM> for the carbon working electrode and <NUM><NUM> for the carbon nanotube working electrode. Following deposition, the working electrodes were cured overnight at <NUM>. Thereafter, a PVP membrane was applied to the working electrodes via dip coating using a coating solution formulated with <NUM> of <NUM>/mL PVP and <NUM> of <NUM>/mL PEGDGE400. Membrane curing was performed for <NUM> hours at <NUM>.

Ketone analyses were conducted as set forth in Example <NUM>. <FIG> show four replicates of the response for an electrode containing NADHOx, NAD+, and β-hydroxybutyrate dehydrogenase when exposed to varying β-hydroxybutyrate concentrations. <FIG> shows the current response for a carbon working electrode, and <FIG> shows the current response for a carbon nanotube working electrode. As shown, the current response for both types of working electrode increased as the β-hydroxybutyrate concentration increased up to a concentration of <NUM>. <FIG> shows an illustrative plot of current response versus time for an electrode containing NADHOx, NAD+, and β-hydroxybutyrate dehydrogenase after exposure to increasing β-hydroxybutyrate dehydrogenase concentrations. As shown, the current increased rapidly after adding β-hydroxybutyrate dehydrogenase and stabilized thereafter.

Example <NUM>: Detection of Ketones Using an Analyte Sensor Containing Poly-<NUM>,<NUM>-phenanthroline-<NUM>,<NUM>-dione and β-Hydroxybutyrate Dehydrogenase. For this example, the enzyme system of <FIG> was used to facilitate detection of ketones. The spotting formulation shown in Table <NUM> below was coated onto either a carbon working electrode or a carbon nanotube working electrode. Coating and curing of the spotting formulation and the PVP membrane was conducted as specified in Example <NUM>. The mean active area was <NUM><NUM> for the carbon working electrode and <NUM><NUM> for the carbon nanotube working electrode.

Ketone analyses were conducted as set forth in Example <NUM>. <FIG> show four replicates of the response for an electrode containing poly-<NUM>,<NUM>-phenanthroline-<NUM>,<NUM>-dione and β-hydroxybutyrate dehydrogenase when exposed to varying β-hydroxybutyrate concentrations. <FIG> shows the current response for a carbon working electrode, and <FIG> shows the current response for a carbon nanotube working electrode. As shown, the current response for both types of working electrode increased as the β-hydroxybutyrate concentration increased up to a concentration of about <NUM> before the response began to flatten.

Vehicle fail safes, such as ignition locks, are sometimes used to prevent an operator from operating a vehicle when impaired or otherwise not in a condition to safely operate the vehicle. Operating the vehicle while impaired could potentially present significant dangers to the operator and the public. One common type of ignition lock is designed to prevent drunk driving and, more specifically, to prevent individuals from operating a vehicle while intoxicated through alcohol use. Such lock devices connect a breath-alcohol analyzer or optical sensor to the vehicle's ignition system, and the driver must successfully pass a blood alcohol level test before the vehicle can be started.

Intoxication is one type of impairment or condition that an operator may experience that renders the operator unfit or unable to operate a vehicle. However, other impairments and conditions can also afflict an operator and should also be monitored closely to ensure the operator does not operate a vehicle while impaired. For example, an operator with diabetes and driving while hypoglycemic (i.e., low blood sugar) could potentially undergo light-headedness, confusion, headache, loss of consciousness, seizures, and delayed reflexes, any of which could endanger his/her own life and those in the vehicle or in the vicinity of the vehicle.

Analyte monitoring systems, have been developed to facilitate long-term monitoring of analytes in bodily fluid (e.g., blood). Some analyte monitoring systems are designed to detect and monitor levels of blood glucose, which can be helpful in treating diabetic conditions. Other analyte monitoring systems, however, are designed to detect and monitor other analytes present in an operator's bodily fluid, and abnormal analyte levels detected in an operator may be indicative that the operator is currently unfit to safely operate a vehicle.

The following discussion describes an analyte monitoring and vehicle control system used to prevent operation of a vehicle when operator analyte levels cross a predetermined threshold. Having the sensor control device <NUM> (<FIG>) properly deployed allows a user to intelligently track and monitor bodily fluid analyte levels and trends. When some analyte levels surpass certain thresholds, physical or cognitive impairment may ensue that renders a user unfit to safely operate a vehicle. In such instances, the user should take appropriate action to bring analyte levels back into safe ranges prior to attempting to operate a vehicle. In some cases, however, a user may feel perfectly fine to operate a vehicle but nonetheless have unsafe analyte levels that could suddenly trigger the onset of a dangerous physical impairment. In such cases, it may be advantageous to have a failsafe system in place that prevents or warns the user from operating a vehicle and potentially placing self and/or others in danger.

<FIG> is a schematic diagram of an example analyte monitoring and vehicle control system <NUM>, according to one or more embodiments of the present disclosure. As illustrated, the analyte monitoring and vehicle control system <NUM> (hereafter "the system <NUM>) includes the sensor control device <NUM>, which may be deployed on a user or "operator" <NUM> and otherwise delivered to a target monitoring location on the body of the operator <NUM>, such as the back of an arm. As discussed above, the sensor control device <NUM> includes the sensor <NUM> (<FIG>), and when properly deployed, the sensor <NUM> is positioned transcutaneously within the skin to detect and monitor analytes present within a bodily fluid of the operator <NUM>. The adhesive patch <NUM> (<FIG>) applied to the bottom of the sensor control device <NUM> adheres to the skin to secure the sensor control device <NUM> in place during operation.

While the system <NUM> is described herein as including the on-body sensor control device <NUM> to detect and report analyte levels, the system <NUM> may alternatively incorporate an ex vivo analyte sensor (e.g., a self-monitoring blood glucose "SMBG" meter). Accordingly, the term "sensor control device" should be interpreted herein to include not only on-body sensor systems, as generally described above, but also traditional, hand-held sensor systems.

As illustrated, the system <NUM> may further include the reader device <NUM>, and the sensor control device <NUM> may be in communication with the reader device <NUM> via a local communication path or link to provide analyte concentration data automatically, periodically, or as desired by the operator <NUM>. The reader device <NUM> may be in communication with a control module <NUM>, which is in communication with the electrical system of a vehicle <NUM> and powered by the vehicle battery or otherwise powered by a separate battery. In such embodiments, data transmitted to the reader device <NUM> from the sensor control device <NUM> may be subsequently transmitted by the reader device <NUM> to the control module <NUM> for processing. In other embodiments, however, the sensor control device <NUM> may communicate directly with the control module <NUM> via any wireless communication protocol, such as BLUETOOTH®. In such embodiments, the reader device <NUM> may or may not be necessary in the system <NUM>.

In the illustrated embodiment, the vehicle <NUM> is depicted as an automobile. As used herein, however, the term "vehicle" is used broadly and is meant to include any kind of transportation vehicle that can be operated by a human user or "operator," but can also include autonomous vehicles used to transport humans. Examples of the vehicle <NUM> include, but are not limited to, any type of automobile, truck, sport utility vehicle, aircraft, watercraft, spacecraft, and or any other means of transportation, or combinations thereof.

The control module <NUM> may include a communications interface to communicate information to/from the sensor control device <NUM> and/or the reader device <NUM>. In the case of an exemplary BLUETOOTHD-enabled sensor control device <NUM> and/or reader device <NUM>, a pairing mode may be entered into when the sensor control device <NUM> approaches the vehicle <NUM>. Upon pairing, the control module <NUM> may be programmed and configured to automatically detect the presence of and establish communication with the sensor control device <NUM> and/or the reader device <NUM>. For example, when the operator <NUM> approaches or enters the vehicle <NUM>, the control module <NUM> may automatically detect the presence of the sensor control device <NUM> and enable communication therebetween or with the reader device <NUM>.

In some embodiments, the control module <NUM> may be in communication with a vehicle user interface <NUM> included in the vehicle <NUM>, such as an infotainment system, a touchscreen display, or an information display. In such embodiments, the control module <NUM> may visually communicate with the operator <NUM> via the vehicle user interface <NUM> and may also be able to audibly communicate with the operator <NUM> via the audio speakers included in the vehicle <NUM>. In other embodiments, however, the control module <NUM> may be configured to communicate with the reader device <NUM> to be able to communicate with the operator <NUM>.

As illustrated, the control module <NUM> may be or otherwise include a computer system <NUM> configured and otherwise programmed to control various operations and/or systems of the vehicle <NUM> based on real-time measured analyte levels of the operator <NUM> as obtained by the sensor control device <NUM>. Operation of the vehicle <NUM> is controlled, disabled, or modified by either disabling one or more critical systems of the vehicle <NUM> or by activating warning systems in the vehicle <NUM>. When the real-time measured analyte levels of the operator <NUM> are within a predetermined safe range, then it may be considered safe for the operator <NUM> to operate the vehicle <NUM>. When the real-time measured analyte levels of the operator <NUM> fall outside the predetermined safe range or cross a predetermined threshold, however, the computer system <NUM> may then be programmed to control, disable, or modify operation of the vehicle <NUM>.

In some embodiments, for example, the computer system <NUM> may be configured to disable various critical vehicle systems when detected analyte levels of the operator <NUM> fall outside of a predetermined range or otherwise cross a predetermined threshold, thus progressively and safely disabling operation of the vehicle when identifying the operator <NUM> as impaired for safe operation of the vehicle <NUM>. Critical vehicle systems of the vehicle <NUM> that may be disabled include the ignition system (e.g., energy switching/control system), the transmission system (or gear box), the fuel system, energy supply system (e.g., a battery, capacitor, conversion/reaction cell, etc.). When elevated or lowered (unsafe) analyte levels are detected, the computer system <NUM> may prevent the critical vehicle systems from functioning or operating. Consequently, the operator <NUM> will be unable to start or operate the vehicle <NUM>, thereby preventing the operator <NUM> from placing themselves and/or others in danger.

In other embodiments, or in addition thereto, the computer system <NUM> may be configured to activate various non-critical vehicle systems when detected analyte levels of the operator <NUM> surpass or cross a predetermined threshold. Non-critical vehicle systems that may be activated include, for example, the vehicle horn, the vehicle lights, or an audible warning system installed in the vehicle <NUM>. In such embodiments, activation of the non-critical vehicle systems may alert law enforcement and others (e.g., operators of adjacent vehicles, bystanders, pedestrians, etc.) of an operator <NUM> that may be driving in an impaired condition, thus allowing law enforcement to quickly address any issues related thereto and placing others on notice of a potentially dangerous situation.

In yet other embodiments, or in addition thereto, the computer system <NUM> may be configured to automatically place a phone call to one or more emergency contacts when analyte levels of the operator <NUM> fall outside of a predetermined safe operating range or otherwise cross a predetermined threshold. In such embodiments, the computer system <NUM> may operate through the reader device <NUM> (e.g., a cellular phone) or a cellular or satellite communication system incorporated into the vehicle <NUM> (e.g., OnStar®). In other embodiments, or in addition thereto, the computer system <NUM> may be configured to automatically send a message (e.g., text or SMS message, email, etc.) to an emergency contact when analyte levels of the operator <NUM> fall outside of a predetermined safe operating range or otherwise cross a predetermined threshold. Example emergency contacts include, but are not limited to, a spouse, a parent, medical personnel (e.g., a doctor), a hospital, <NUM>, or any combination thereof.

In some embodiments, the system <NUM> may further include one or more proximity sensors <NUM> configured to detect the presence of the operator <NUM> and, more particularly, the sensor control device <NUM>. In such embodiments, the proximity sensor(s) <NUM> may be configured to monitor the general area of the driver's seat <NUM> within the vehicle <NUM>. If the sensor control device <NUM> is detected within the area of the driver's seat <NUM> by the proximity sensor(s) <NUM>, that may provide a positive indication that the operator <NUM> is in the driver's seat <NUM> and potentially attempting to operate the vehicle <NUM>. In such cases, a signal may be sent to the control module <NUM> alerting the computer system <NUM> that the operator <NUM> is in the vehicle <NUM> and potentially attempting to operate the vehicle <NUM>. If the real-time measured analyte levels of the operator <NUM> are within a predetermined safe range or below a predetermined level, then the computer system <NUM> may allow the operator <NUM> to operate the vehicle <NUM>. When the real-time measured analyte levels of the operator <NUM> fall outside the predetermined safe range or cross a predetermined threshold, however, the computer system <NUM> may control, disable, or modify operation of the vehicle <NUM>, as generally described above. As will be appreciated, the proximity sensor(s) <NUM> may be advantageous in preventing operation of the vehicle <NUM> only when the impaired operator <NUM> is in the driver's seat <NUM> and ready to operate the vehicle <NUM>. Consequently, a user wearing the sensor control device <NUM> is able to ride as a passenger in the vehicle <NUM> in any state without affecting operation of the control module <NUM> or the vehicle <NUM>.

In some embodiments, the control module <NUM> may further include a vehicle status detection module <NUM> configured to detect the current status of the vehicle <NUM>, including whether the vehicle <NUM> is currently moving or is stationary. In addition, the vehicle status detection module <NUM> may be configured to determine whether or not the motor in the vehicle <NUM> is currently operating or is stopped. In one or more embodiments, the vehicle status detection module <NUM> may provide a status signal to the control module <NUM>, and the control module <NUM> can then use the status signal to determine what vehicle operations should be activated or disabled when the real-time measured analyte levels of the operator <NUM> fall outside the predetermined safe range or cross a predetermined threshold. For example, when the status signal indicates that the vehicle <NUM> is stationary, the control module <NUM> can disable the vehicle fuel system, transmission system, ignition system, or any combination thereof. In contrast, when the status signal indicates that the vehicle <NUM> is moving, the control module <NUM> can activate the vehicle horn, flash the vehicle lights, or activate an audible warning to the operator <NUM> and/or those around the operator <NUM> that the operator <NUM> is impaired.

In some embodiments, once the operator <NUM> enters the vehicle <NUM> or when the control module <NUM> pairs with the sensor control device <NUM> and/or the reader device <NUM>, an app may be launched on the reader device <NUM> or the vehicle user interface <NUM>, and a digital dashboard may appear on the reader device <NUM> and/or the vehicle user interface <NUM> that depicts current analyte levels, trend, historical data, and projected analyte levels. If the current analyte levels fall outside of a predetermined safe operating range, however, the computer system <NUM> may be programmed to disable one or more critical vehicle systems to prevent the operator <NUM> from operating the vehicle <NUM>. In such embodiments, a visual or audible alert may be issued by the control module <NUM> to inform the operator <NUM> as to why the vehicle <NUM> is not starting. More particularly, a visual alert (e.g., a written message) may be generated and displayed on the reader device <NUM> or the vehicle user interface <NUM>, or an audible alert (e.g., a vocal message) may be transmitted through the speakers in the reader device <NUM> or the vehicle <NUM>.

If not done automatically, the operator <NUM> may be prompted to obtain a current analyte level upon pairing the sensor control device <NUM> with the control module <NUM>. In some cases, the vehicle <NUM> may be prevented from being operated until a current analyte level is obtained. If the current analyte levels are within safe limits, the computer system <NUM> may allow operation of the vehicle <NUM>. In some aspects, and unless done automatically, the control module <NUM> may prompt the operator <NUM> to obtain additional current analyte levels after operating the vehicle <NUM> for a predetermined period of time (e.g., after <NUM> hour, <NUM> hours, <NUM> hours, etc.).

In some embodiments, the control module <NUM> may be configured to issue visual or audible recommendations or coaching to the operator <NUM> that may help bring measured analyte levels back into safe ranges. In such embodiments, such visual or audible recommendations may prompt the user to take some action that could result in bringing analyte levels back into safe ranges. Moreover, in some embodiments, the operator <NUM> may be able to communicate with the control module <NUM> verbally by issuing verbal responses or commands. This may prove advantageous in helping prevent distracted operation of the vehicle <NUM>.

In some embodiments, settings of the control module <NUM> may be customized by the operator <NUM> to allow the user to make informed decisions once unsafe analyte levels have been detected and a visual or audible alert has been issued by the control module <NUM>. More specifically, in at least one embodiment, the control module <NUM> may include a bypass feature that the operator <NUM> might enable to allow the operator <NUM> to operate the vehicle <NUM> even when unsafe analyte levels have been measured. In such embodiments, the operator <NUM> may operate the vehicle <NUM> by acknowledging that the operator <NUM> might be operating the vehicle <NUM> in an impaired or unsafe health state.

In some embodiments, the computer system <NUM> may be configured or otherwise programmed to calculate a predicted timeline when analyte levels of the operator <NUM> may depart from a predetermined safe range or otherwise cross a predetermined threshold. In such embodiments, the control module <NUM> may be configured to issue visual or audible alerts to the operator <NUM> indicating approximately how much time the operator <NUM> has before unsafe analyte levels may be reached and a potential unsafe medical condition may ensue. Multiple alerts may be provided to indicate when the operator has specific time increments remaining before unsafe analyte levels are reached. For example, visual or audible alerts may be issued when unsafe analyte levels will be reached within an hour, within a half hour, within <NUM> minutes, within <NUM> minutes, within <NUM> minute, and any time increment therebetween. Furthermore, a visual or audible alert may be issued once the analyte levels of the operator reach an unsafe level or cross a predetermined threshold.

In some embodiments, if unsafe analyte levels are measured while the operator <NUM> is operating the vehicle <NUM>, the control module <NUM> may be configured to issue one or more alerts (visual or audible) warning the operator <NUM> of the unsafe analyte levels. In some cases, the volume of the stereo in the vehicle <NUM> may be automatically lowered to enable the operator <NUM> to hear an audible alert. In such embodiments, the control module <NUM> may be configured to suggest one or more corrective actions to the operator <NUM>. Example corrective actions include, but are not limited to, slowing and stopping the vehicle <NUM>, locating and driving to a nearby convenience store or pharmacy, and locating a nearby hospital or medical facility. If the vehicle <NUM> is an autonomous vehicle, and the current analyte levels place the operator <NUM> in potentially dangerous conditions, the control module <NUM> may automatically direct the vehicle <NUM> to a medical facility for treatment. Alternatively, or in addition thereto, the control module <NUM> may progressively reduce or restrict the speed of the vehicle <NUM> when unsafe analyte levels are detected, thus forcing the operator <NUM> to come to a stop and remedy the issue before continuing to operate the vehicle <NUM>.

The system <NUM> may be useful in several different scenarios to protect the operator <NUM> and/or those around the operator <NUM> while driving. In some applications, the system <NUM> may be incorporated voluntarily by the operator to detect impairment in real-time. In other applications, the system <NUM> may be required by the owner of the vehicle <NUM> to detect impairment of the operator <NUM>. In such applications, the owner of the vehicle <NUM> may be a transport or trucking company. In yet other applications, the system <NUM> may be legally imposed on the operator <NUM> to detect impairment.

Unless otherwise indicated, all numbers expressing quantities and the like in the present specification and associated claims are to be understood as being modified in all instances by the term "about. " Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the embodiments of the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claim, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

One or more illustrative embodiments incorporating various features are presented herein. Not all features of a physical implementation are described or shown in this application for the sake of clarity. It is understood that in the development of a physical embodiment incorporating the embodiments of the present invention, numerous implementation-specific decisions must be made to achieve the developer's goals, such as compliance with system-related, business-related, government-related and other constraints, which vary by implementation and from time to time. While a developer's efforts might be time-consuming, such efforts would be, nevertheless, a routine undertaking for those of ordinary skill in the art and having benefit of this disclosure.

While various systems, tools and methods are described herein in terms of "comprising" various components or steps, the systems, tools and methods can also "consist essentially of" or "consist of" the various components and steps.

As used herein, the phrase "at least one of" preceding a series of items, with the terms "and" or "or" to separate any of the items, modifies the list as a whole, rather than each member of the list (i.e., each item). The phrase "at least one of" allows a meaning that includes at least one of any one of the items, and/or at least one of any combination of the items, and/or at least one of each of the items.

Claim 1:
An analyte sensor (<NUM>) comprising:
a sensor tail comprising at least a first working electrode (<NUM>) and a second working electrode (<NUM>), the sensor tail configured for insertion into a tissue;
a ketones-responsive active area (310a) disposed upon a surface of the first working electrode (<NUM>), the ketones-responsive active area (310a) comprising an enzyme system comprising at least two enzymes that are capable of acting in concert to facilitate detection of ketones;
a glucose-responsive active area (310b) comprising a glucose-responsive enzyme disposed upon a surface of the second working electrode (<NUM>); and
a membrane (<NUM>) having a first portion (340a) overcoating the ketones-responsive active area (310a) and a second portion (340b) overcoating the glucose-responsive active area (310b);
wherein the first portion (340a) and the second portion (340b) have different compositions.