Analyte sensor for detecting ketones and method of using same - Patents.com

Abstract

The present disclosure provides an analyte sensor for use in detecting ketones. In certain embodiments, the ketone-responsive active site of the disclosed analyte sensor comprises an enzyme system comprising β-hydroxybutyrate dehydrogenase and NADH oxidase disposed on a surface of a platinum working electrode. The present disclosure further provides a method for detecting ketones using the disclosed analyte sensor.

Description

[Technical field]

[0001] TECHNICAL FIELD The subject matter described herein relates to analyte sensors for detecting ketones and methods of using same. [Background technology]

[0002] The detection of various analytes in an individual may be important for monitoring the health status of an individual, since deviations from normal analyte levels may be indicative of physiological conditions.For example, monitoring ketone levels may allow a person suffering from diabetes to take appropriate corrective measures to avoid serious physiological suffering from ketoacidosis.Other analytes may also be desirable for monitoring other physiological conditions.In some cases, it may be desirable to monitor more than one analyte to monitor multiple physiological conditions, especially if the person suffers from a comorbid condition that results in simultaneous dysregulation of two or more analytes in combination with each other.

[0003] Analyte monitoring in an individual may be performed periodically or continuously over a period of time. Periodic analyte monitoring may be performed by taking samples of bodily fluids, such as blood or urine, at set time intervals and analyzing them ex vivo. Periodic ex vivo analyte monitoring may be sufficient to determine the physiological status of many individuals. However, ex vivo analyte monitoring may be inconvenient and painful in some cases. Furthermore, if analyte measurements are not obtained at the appropriate time, there is no way to recover lost data. Continuous analyte monitoring may be performed using one or more sensors that remain at least partially implanted in the tissue of the individual, such as transdermally, subcutaneously, or intravenously, thereby allowing analysis to be performed in vivo. The implanted sensors may collect analyte data on demand, on a set schedule, or continuously, depending on the individual's particular health needs and / or previously measured analyte levels. In vivo analyte monitoring using implanted sensors may be a more desirable approach for individuals with severe analyte dysregulation and / or rapidly fluctuating analyte levels, but may also be beneficial for other individuals. Because implanted analyte sensors often remain in an individual's tissues for extended periods of time, it may be highly desirable for such analyte sensors to be made from stable materials that exhibit a high degree of biocompatibility.

[0004] Many analytes represent attractive targets for physiological analysis, provided that suitable detection chemistries can be identified. To this end, enzyme-based amperometric sensors configured to continuously assay glucose in vivo have been developed and improved over the last few years to aid in monitoring the health of diabetic individuals. Other analytes that are commonly subject to simultaneous dysregulation along with glucose in diabetic individuals include, for example, lactate, oxygen, A1c, ketones, and others. It may also be desirable to monitor these and other analytes apart from glucose dysregulation. Analyte sensors configured to detect analytes other than glucose in vivo are known, but currently are not very well improved. For example, poor sensitivity to analytes with low abundance may be particularly problematic. Thus, there is a need in the art for improved sensors for detecting ketones in vivo. Summary of the Invention

[0005] The objects and advantages of the disclosed subject matter will be set forth in and become apparent from the following description and will be learned by practice of the disclosed subject matter. Additional advantages of the disclosed subject matter may be realized and obtained by the apparatus particularly pointed out in the written description and claims, as well as from the appended drawings.

[0006] To achieve these and other advantages, and in accordance with the objects of the disclosed subject matter as embodied and broadly described, the disclosed subject matter includes an analyte sensor comprising a sensor tail including at least a first working electrode, a ketone-responsive active region disposed on a surface of the first working electrode, and a ketone-permeable mass transport limiting membrane covering at least a portion of the ketone-responsive active region. In certain embodiments, the ketone-responsive active region comprises an enzyme system comprising β-hydroxybutyrate dehydrogenase and NADH oxidase. In certain embodiments, the first working electrode is a platinum electrode. In certain embodiments, the ketone-responsive active region does not comprise an electron transfer agent. In certain embodiments, the ketone-responsive active region does not comprise superoxide dismutase. 。

[0007] In certain embodiments, the ketones-responsive active region further comprises a stabilizer and / or a crosslinker. In certain embodiments, one or more enzymes in an enzyme system comprising β-hydroxybutyrate dehydrogenase and NADH oxidase are covalently bound to a stabilizer in the ketones-responsive active region. In certain embodiments, one or more enzymes in an enzyme system comprising β-hydroxybutyrate dehydrogenase and NADH oxidase are covalently bound to a polymer in the ketones-responsive active region. In certain embodiments, the mass transport limiting membrane comprises polyvinylpyridine, polyvinylimidazole, polyvinylpyridine copolymer, polyacrylate, polyurethane, polyether urethane, or combinations thereof. In certain embodiments, the mass transport limiting membrane comprises polyvinylpyridine. Alternatively or additionally, the mass transport limiting membrane comprises a copolymer of vinylpyridine and styrene.

[0008] In certain embodiments, the analyte sensor of the present disclosure further includes a second working electrode and a second active area disposed on the surface of the second working electrode and responsive to a second analyte different from ketones. In certain embodiments, the second active area includes at least one enzyme responsive to the second analyte. In certain embodiments, a second portion of the mass transport limiting membrane covers the second active area. In certain embodiments, the second analyte includes glucose.

[0009] In certain embodiments, the sensor tail is configured for insertion into tissue, e.g., for detecting levels of ketones in vivo. In certain embodiments, the ketone-responsive active region responds to ketones at a potential of about +0.2V to about +0.5V relative to an Ag / AgCl reference. In certain embodiments, the ketone-responsive active region responds to ketones at a potential of about +0.3V to about +0.4V relative to an Ag / AgCl reference. In certain embodiments, the ketone-responsive active region responds to ketones at a potential of about +0.35V relative to an Ag / AgCl reference.

[0010] The present disclosure further provides a method for detecting ketones. In certain embodiments, the method may include providing an analyte sensor including: (a) a sensor tail comprising at least a first working electrode that is a platinum electrode; (b) a ketone-responsive active region disposed on a surface of the first working electrode, the ketone-responsive active region comprising an enzyme system comprising β-hydroxybutyrate dehydrogenase and NADH oxidase; and (c) a ketone-permeable mass transport limiting membrane covering at least the ketone-responsive active region. In certain embodiments, the method further includes applying a potential to the first working electrode, obtaining a first signal that is equal to or greater than the redox potential of the ketone-responsive active region and proportional to a concentration of ketones in a fluid in contact with the ketone-responsive active region, and correlating the first signal with the concentration of ketones in the fluid.

[0011] In certain embodiments, the first working electrode is a platinum electrode. In certain embodiments, the ketones-responsive active region does not include an electron transfer agent. In certain embodiments, the ketones-responsive active region does not include superoxide dismutase. In certain embodiments, the ketones-responsive active region further includes a stabilizer and / or a crosslinker. In certain embodiments, one or more enzymes in the enzyme system comprising β-hydroxybutyrate dehydrogenase and NADH oxidase are covalently attached to a stabilizer in the ketones-responsive active region. In certain embodiments, one or more enzymes in the enzyme system comprising β-hydroxybutyrate dehydrogenase and NADH oxidase are covalently attached to a polymer in the ketones-responsive active region. In certain embodiments, the mass transport limiting membrane comprises polyvinylpyridine, polyvinylimidazole, polyvinylpyridine copolymer, polyvinylpyrrolidone, polyacrylate, polyurethane, polyether urethane, or copolymers or combinations thereof. In certain embodiments, the mass transport limiting membrane comprises polyvinylpyridine. Alternatively or additionally, the mass transport limiting membrane comprises a copolymer of vinylpyridine and styrene.

[0012] In certain embodiments, the sensor tail is configured for insertion into tissue. In certain embodiments, the ketones-responsive active region responds to ketones at a potential of about +0.2V to about +0.5V relative to an Ag / AgCl reference. In certain embodiments, the ketones-responsive active region responds to ketones at a potential of about +0.3V to about +0.4V relative to an Ag / AgCl reference. In certain embodiments, the ketones-responsive active region responds to ketones at a potential of about +0.35V relative to an Ag / AgCl reference.

[0013] In certain embodiments, an analyte sensor for use in the disclosed methods may further include a second working electrode and a second active area disposed on a surface of the second working electrode and responsive to a second analyte different from ketones. In certain embodiments, the second active area includes at least one enzyme responsive to the second analyte, and a second portion of the mass transport limiting membrane covers the second active area. In certain embodiments, the second analyte comprises glucose.

[0014] In certain embodiments, the analyte sensor of the present disclosure is for use in a subject in need thereof. For example, but not by way of limitation, the subject may be a diabetic subject. In certain embodiments, the subject may be on a ketogenic diet. In certain embodiments, the subject is in a state of ketosis. [Brief description of the drawings]

[0015] The following figures are included to illustrate certain aspects of the present disclosure and should not be considered as exclusive embodiments. The disclosed subject matter is capable of considerable modification, alteration, combinations, and equivalents in form and function without departing from the scope of the present disclosure. [Figure 1A] 1 is a system overview of a sensor applicator, a reader device, a monitoring system, a network, and a remote system. [Figure 1B] 1 is a schematic diagram illustrating the operating environment of an example analyte monitoring system for use with the technology described herein. [Figure 1C] 1 shows a schematic diagram of an exemplary sensing system that can incorporate an analyte sensor of the present disclosure. [Figure 2A] 1 is a schematic block diagram illustrating an example embodiment of a reader device. [Figure 2B] 1 is a block diagram illustrating an example of a data receiving device for communicating with a sensor according to an exemplary embodiment of the disclosed subject matter. [Figure 2C] 1 is a schematic block diagram illustrating an example embodiment of a sensor control device. [Figure 2D]1 is a schematic block diagram illustrating an example embodiment of a sensor control device. [Figure 2E] 1 is a block diagram illustrating an example of an analyte sensor according to an exemplary embodiment of the disclosed subject matter. [Figure 3A] 13A-13C are proximal perspective views illustrating an example embodiment of a user preparing a tray for assembly. [Figure 3B] 11A-11C are side views illustrating an example embodiment of a user preparing an applicator device for assembly. [Figure 3C] 13A-13C are proximal perspective views illustrating an example embodiment of a user inserting an applicator device into a tray during assembly. [Figure 3D] 11A-11C are proximal perspective views illustrating an example embodiment of a user removing the applicator device from the tray during assembly. [Figure 3E] 1A-1C are proximal perspective views illustrating an example embodiment of a patient applying a sensor with an applicator device. [Figure 3F] 1A and 1B are proximal perspective views showing an example embodiment of a patient with an applied sensor and a used applicator device. [Figure 4A] 1A-1C are side views of an example embodiment of an applicator device coupled with a cap. [Figure 4B] 1 is a side perspective view of an example embodiment of an applicator device and cap separated. [Figure 4C] 1 is a perspective view illustrating an example embodiment of an applicator device and a distal end of an electronics housing. [Figure 4D] FIG. 1 is a top perspective view of an exemplary applicator device in accordance with the disclosed subject matter. [Figure 4E] FIG. 4E is a bottom perspective view of the applicator device of FIG. 4D. [Figure 4F] FIG. 4E is an exploded view of the applicator device of FIG. 4D. [Figure 4G] 4E is a side cross-sectional view of the applicator device of FIG. 4D. [Diagram 5] FIG. 13 is a proximal perspective view showing an example embodiment of a tray with an associated sterilization lid. [Figure 6A]1A-1C are proximal perspective cross-sectional views illustrating an example embodiment of a tray having a sensor delivery component. [Figure 6B] FIG. 13 is a proximal perspective view showing the sensor delivery component. [Figure 7A] FIG. 2 illustrates an isometric exploded top view of an exemplary sensor control device. [Figure 7B] FIG. 2 illustrates an isometric exploded bottom view of an exemplary sensor control device. [Figure 8A] 1A-1C are assembly and cross-sectional views of an on-body device including an integrated connector for sensor assembly. [Figure 8B] 1A-1C are assembly and cross-sectional views of an on-body device including an integrated connector for sensor assembly. [Figure 8C] 1A-1C are assembly and cross-sectional views of an on-body device including an integrated connector for sensor assembly. [Figure 9A] 2D is a side view of an example embodiment of the sensor applicator of FIG. 1A coupled with the cap of FIG. 2C. [Figure 9B] 2D is a side cross-sectional view of an example embodiment of the sensor applicator of FIG. 1A coupled with the cap of FIG. 2C. [Figure 10A] FIG. 2 is an isometric view of another example sensor control device. [Figure 10B] FIG. 13 is a side view of another example sensor control device. [Figure 11A] 10A-10B. FIG. 11 is a side cross-sectional view showing a staged assembly of a sensor applicator having the sensor control device of FIGS. [Figure 11B] 10A-10B. FIG. 11 is a side cross-sectional view showing a staged assembly of a sensor applicator having the sensor control device of FIGS. [Figure 11C] 10A-10B. FIG. 11 is a side cross-sectional view showing a staged assembly of a sensor applicator having the sensor control device of FIGS. [Figure 12A] 10A-10B , depicting step-by-step cross-sectional side views illustrating assembly and disassembly of an example embodiment of a sensor applicator having the sensor control device of FIGS. [Figure 12B] 10A-10B , depicting step-by-step cross-sectional side views illustrating assembly and disassembly of an example embodiment of a sensor applicator having the sensor control device of FIGS. [Figure 12C] 10A-10B , depicting step-by-step cross-sectional side views illustrating assembly and disassembly of an example embodiment of a sensor applicator having the sensor control device of FIGS. [Figure 13A] 1A-1D show cross-sectional views illustrating an example embodiment of an applicator during a placement stage. [Figure 13B] 1A-1D show cross-sectional views illustrating an example embodiment of an applicator during a placement stage. [Figure 13C] 1A-1D show cross-sectional views illustrating an example embodiment of an applicator during a placement stage. [Figure 13D] 1A-1D show cross-sectional views illustrating an example embodiment of an applicator during a placement stage. [Figure 13E] 1A-1D show cross-sectional views illustrating an example embodiment of an applicator during a placement stage. [Figure 13F] 1A-1D show cross-sectional views illustrating an example embodiment of an applicator during a placement stage. [Figure 14] 1 is a graph showing an example of the in vitro sensitivity of an analyte sensor. [Figure 15] 1 is a diagram illustrating example operational states of a sensor according to an exemplary embodiment of the disclosed subject matter. [Figure 16] 1 is a diagram illustrating an example of operations and data flow for over-the-air programming of a sensor in accordance with the disclosed subject matter. [Figure 17] 1 is a diagram illustrating an example of data flow for reliable exchange of data between two devices in accordance with the disclosed subject matter. [Figure 18A] 1 shows a schematic cross-sectional view of an analyte sensor including a single active area. [Figure 18B] 1 shows a schematic cross-sectional view of an analyte sensor including a single active area. [Figure 18C] 1 shows a schematic cross-sectional view of an analyte sensor including a single active area. [Figure 19A] 1 shows a schematic cross-sectional view of an analyte sensor that includes two active areas. [Figure 19B] 1 shows a schematic cross-sectional view of an analyte sensor that includes two active areas. [Figure 19C] 1 shows a schematic cross-sectional view of an analyte sensor that includes two active areas. [Figure 20] 1 shows a schematic cross-sectional view of an analyte sensor that includes two active areas. [Figure 21A] FIG. 1 shows a perspective view of an analyte sensor including two active areas on separate working electrodes. [Figure 21B] FIG. 1 shows a perspective view of an analyte sensor including two active areas on separate working electrodes. [Figure 21C] FIG. 1 shows a perspective view of an analyte sensor including two active areas on separate working electrodes. [Figure 22] 1 shows a schematic diagram of a particular enzyme system that can be used to detect ketones according to the present disclosure. [Diagram 23] Voltammograms of NADH and NADH with hydrogen peroxide (H2O2) are shown. [Figure 24] 4 shows four replicates of the current response for an electrode containing NAD+, NADH oxidase, and β-hydroxybutyrate dehydrogenase compared to a control without NADH oxidase when exposed to varying concentrations of β-hydroxybutyrate. [Diagram 25] 25 shows an exemplary plot of sensor current response versus concentration of β-hydroxybutyrate for the electrodes of FIG. 24. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] The present disclosure generally describes analyte sensors employing one or more enzymes for the detection of an analyte. For example, without limitation, the present disclosure provides analyte sensors employing one or multiple enzymes for the detection of an analyte, such as ketones. In certain embodiments, the present disclosure further provides analyte sensors employing multiple enzymes for the detection of two different analytes, such as ketones and multiple enzymes for the detection of a second analyte, such as glucose. Depending on the sensor configuration, the analyte sensors of the present disclosure can be configured to detect one analyte or multiple analytes simultaneously or nearly simultaneously. The present disclosure further provides methods of detecting one or more analytes using the disclosed analyte sensors.

[0017] Glucose-responsive analyte sensors are a well-studied and currently evolving field to help diabetic individuals better manage their health. Despite the high prevalence of co-occurring analyte dysregulation in diabetic individuals, sensor chemistries suitable for detecting ketones and other commonly dysregulated analytes lag significantly behind better developed glucose detection chemistries. The present disclosure alleviates this deficiency by providing sensor chemistries suitable for detecting ketones with good response stability over a wide range of ketone concentrations, particularly detection chemistries that utilize enzyme systems with at least two enzymes that can act in concert to facilitate the detection of ketones. As used herein, the term "in concert" refers to coupled enzyme reactions in which the product of a first enzyme reaction becomes a substrate for a second enzyme reaction, which serves as the basis for measuring the concentration of a substrate (e.g., an analyte) reacted during the first enzyme reaction. In certain embodiments, the product and / or substrate of the reaction may be the reduced and / or oxidized form of a cofactor or coenzyme of an enzyme of the enzyme system, e.g., NAD or NADP. Although defined in terms of two coupled enzymatic reactions, it should be recognized that in some cases three or more coupled enzymatic reactions may occur. For example, the product of a first enzymatic reaction may serve as a substrate for a second enzymatic reaction, which in turn may serve as a substrate for a third enzymatic reaction, which may serve as a basis for measuring the concentration of a substrate (e.g., an analyte) reacted during the first enzymatic reaction. A discussion of enzyme systems suitable for detecting ketones according to the present disclosure is provided below.

[0018] For purposes of clarity, and not by way of limitation, the detailed description of the subject matter disclosed herein is divided into the following subsections. I. Definitions; and II. Analyte sensor; 1. General structure of the analyte sensor system; 2. Enzymes; 3. Redox mediators; 4. Polymer backbone; 5. Mass-transfer limiting membrane; 6. Interference domains; and 7. Manufacturing; III. Method of Use; and IV. Illustrative Embodiments I. Definition The terms used herein generally have their ordinary meaning in the art, within the context of this disclosure and in the specific context in which each term is used. Certain terms are discussed below or elsewhere in this specification to provide further guidance to the practitioner in describing the compositions and methods of the present disclosure and how to make and use them.

[0019] As used herein, the use of the word "a" or "an" in conjunction with the term "comprising" in the claims and / or specification may mean "one," but is not inconsistent with the meaning of "one or more," "at least one," and "one or more."

[0020] As used herein, the terms "comprise(s)," "include(s)," "having," "has," "can," "contain(s)," and variations thereof are intended to be open-ended transitional phrases, terms, or words that do not exclude additional acts or structures. The present disclosure also contemplates other embodiments that "comprise," "consist," and "consist essentially of" the embodiments or elements set forth herein, whether or not expressly stated.

[0021] The term "about" or "approximately" means within an acceptable error range of a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, i.e., on the limitations of the measurement system. For example, "about" can mean within 3 or more standard deviations as is customary in the art. Alternatively, "about" can mean within 20%, preferably within 10%, more preferably within 5%, and even more preferably within 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold of a value.

[0022] As used herein, an "analyte sensor" or "sensor" may refer, for purposes of illustration and not limitation, to any device capable of receiving sensor information from a user, including a temperature sensor, a blood pressure sensor, a pulse or heart rate sensor, a glucose level sensor, an analyte sensor, a physical activity sensor, a body movement sensor, or any other sensor for gathering physical or biological information. Analytes measured by an analyte sensor may include, by way of example and not limitation, glucose, ketones, lactate, oxygen, hemoglobin A1C, albumin, alcohol, alkaline phosphatase, alanine transaminase, aspartate aminotransferase, bilirubin, blood urea nitrogen, calcium, carbon dioxide, chloride, creatinine, hematocrit, lactate, magnesium, oxygen, pH, phosphorus, potassium, sodium, total protein, uric acid, and others.

[0023] The term "biological fluid" as used herein means any bodily fluid or derivative of a bodily fluid in which an analyte can be measured. Non-limiting examples of biological fluids include dermal fluid, interstitial fluid, plasma, blood, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, amniotic fluid, sweat, tears, etc. In certain embodiments, the biological fluid is dermal fluid or interstitial fluid.

[0024] The term "electrolysis," as used herein, means the electro-oxidation or electro-reduction of a compound either directly at an electrode or via one or more electron transfer agents (redox mediators or enzymes).

[0025] As used herein, the term "homogeneous membrane" means a membrane comprising a single type of membrane polymer. As used herein, the term "multi-component membrane" means a membrane comprising two or more types of membrane polymers.

[0026] As used herein, the term "polyvinylpyridine-based polymer" refers to a polymer or copolymer comprising polyvinylpyridine (eg, poly(2-vinylpyridine) or poly(4-vinylpyridine)) or a derivative thereof.

[0027] As used herein, the term "redox mediator" refers to an electron transfer agent that transports electrons between an analyte or an analyte reductase or an analyte oxidase and an electrode, either directly or via one or more additional electron transfer agents. In certain embodiments, a redox mediator that includes a polymer backbone may also be referred to as a "redox polymer."

[0028] As used herein, the term "reference electrode" can refer to a reference electrode or an electrode that functions as both a reference electrode and a counter electrode. Similarly, the term "counter electrode" as used herein can refer to both a counter electrode and a counter electrode that also functions as a reference electrode.

[0029] II. Test substance sensor Before describing the present subject matter in detail, it is to be understood that this disclosure is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

[0030] The publications discussed herein are provided merely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the present disclosure is not entitled to antedate such publications by virtue of prior disclosure. Further, the publication dates provided may be different from the actual publication dates, which must be independently confirmed.

[0031] In general, embodiments of the present disclosure include systems, devices, and methods for the use of an analyte sensor insertion applicator for use with an in vivo analyte monitoring system. The applicator can be provided to a user in a sterile package with the electronics housing of the sensor control unit contained therein. According to some embodiments, a structure separate from the applicator, such as a container, can also be provided to a user in a sterile package with a sensor module and a tip module contained therein. A user can connect the sensor module to the electronics housing and connect the tip to the applicator in an assembly process that includes inserting the applicator into the container in a specified manner. In other embodiments, the applicator, sensor control unit, sensor module, and tip module can be provided in a single package. The applicator can be used to position the sensor control unit on the human body to contact the sensor with the wearer's bodily fluids. The embodiments provided herein are improvements that reduce the likelihood that the sensor will be improperly inserted or damaged, or will induce an adverse physiological response. Other improvements and advantages are also provided. Various configurations of these devices are described in detail by way of embodiments that are merely examples.

[0032] Additionally, many embodiments include an in vivo analyte sensor that is structurally configured such that at least a portion of the sensor is located or can be located within a user's body to obtain information regarding at least one analyte in the body. However, it should be noted that the embodiments disclosed herein can be used with in vivo analyte monitoring systems that incorporate in vitro capabilities, as well as purely in vitro or ex vivo analyte monitoring systems, including completely non-invasive systems.

[0033] Furthermore, for each and every embodiment of the methods disclosed herein, systems and devices capable of implementing each of these embodiments are encompassed within the scope of the present disclosure. For example, sensor control device embodiments are disclosed, and these devices may have one or more sensors, analyte monitoring circuitry (e.g., analog circuitry), memory (e.g., for storing instructions), power sources, communication circuitry, transmitters, receivers, processors, and / or controllers (e.g., for executing instructions) that can perform or facilitate the execution of any method step. These sensor control device embodiments may be used, or may be capable of being used, to perform the steps performed by the sensor control device from any method described herein.

[0034] Additionally, the systems and methods presented herein can be used for the operation of sensors used in analyte monitoring systems, such as, but not limited to, wellness, fitness, diet, research, information, or any purpose including analyte detection over time. As used herein, an "analyte sensor" or "sensor" can mean, for purposes of explanation and without limitation, any device capable of receiving sensor information from a user, including a temperature sensor, a blood pressure sensor, a pulse or heart rate sensor, a glucose level sensor, an analyte sensor, a physical activity sensor, a body movement sensor, or any other sensor for gathering physical or biological information. The analyte sensor of the present disclosure measures ketones. In certain embodiments, the analyte sensors of the present disclosure can further measure analytes including, but not limited to, glucose, ketones, lactate, oxygen, hemoglobin A1C, albumin, alcohol, alkaline phosphatase, alanine transaminase, aspartate aminotransferase, bilirubin, blood urea nitrogen, calcium, carbon dioxide, chloride, creatinine, hematocrit, lactate, magnesium, oxygen, pH, phosphorus, potassium, sodium, total protein, uric acid, and the like.

[0035] As mentioned above, several embodiments of systems, devices, and methods are described herein that provide improved assembly and use of a skin sensor insertion device for use with an in vivo analyte monitoring system. In particular, several embodiments of the present disclosure are designed to improve sensor insertion methods for in vivo analyte monitoring systems, and in particular to prevent premature retraction of the insertion tip during the sensor insertion process. For example, some embodiments include a skin sensor insertion mechanism with an increased firing rate and slower tip retraction. In other embodiments, the tip retraction mechanism can be motion-activated, which prevents the tip from retracting until the user pulls the applicator off the skin. As a result, these embodiments can reduce the likelihood of premature retraction of the insertion tip during the sensor insertion process, reduce the likelihood of improper sensor insertion, and reduce the likelihood of damage to the sensor during the sensor insertion process, to name a few advantages. Some embodiments of the present disclosure also provide an improved insertion tip module that accounts for the small scale of skin sensors and the relatively shallow insertion path present in the subject's skin layer. Additionally, some embodiments of the present disclosure are designed to prevent undesired axial and / or rotational movement of the applicator parts during sensor insertion. These embodiments can thus reduce the likelihood of capillary breakage resulting in instability of the placed skin sensor, irritation at the insertion site, damage to surrounding tissue, and blood contamination of skin fluids, to name a few advantages. Additionally, to mitigate potential inaccurate sensor readings due to injury at the insertion site, some embodiments of the present disclosure can reduce penetration of the sensor tip to the distal depth of the needle during insertion.

[0036] However, before describing these aspects of the embodiments in detail, it is first desirable to describe examples of devices that may be present, for example, in an in vivo analyte monitoring system, and examples of their operation, all of which may be used in conjunction with the embodiments described herein.

[0037] There are various types of in vivo analyte monitoring systems. For example, a "continuous analyte monitoring" system (or "continuous glucose monitoring" system) can continuously transmit data from a sensor controller to a reader device without prompting, e.g., automatically, according to a schedule. A "flash analyte monitoring" system (or "flash glucose monitoring" system or simply "flash" system), as another example, can transmit data from a sensor controller in response to a scan or request for data by a reader device, e.g., via near field communication (NFC) or radio frequency identification (RFID) protocols. In vivo analyte monitoring systems can also operate without the need for fingerstick calibration.

[0038] In vivo analyte monitoring systems can be distinguished from "in vitro" systems, which contact a biological sample outside the body (i.e., "ex vivo") and generally include a measurement device having a port that accepts an analyte test strip that carries a bodily fluid that can be analyzed to determine a user's blood glucose level.

[0039] An in-vivo monitoring system may include a sensor that contacts a user's bodily fluid while positioned in-vivo and detects the level of an analyte contained therein. The sensor may be part of a sensor control device that resides on the user's body and includes electronics and a power source that enable and control the detection of the analyte. Sensor control devices and variations thereof may also be referred to as "sensor control units," "on-body electronics" devices or units, "on-body" devices or units, or "sensor data communication" devices or units, to name a few.

[0040] In-vivo monitoring systems may also include devices that receive sensed analyte data from the sensor control device and process and / or display the sensed analyte data to a user in any number of forms. This device and variations thereof may be referred to as a "handheld reader device," "reader device" (or simply "reader"), "handheld electronic device" (or simply "handheld"), "portable data processing" device or unit, "data receiver," "receiver" device or unit (or simply "receiver"), or "remote" device or unit, to name a few. Other devices, such as personal computers, are also utilized with or incorporated into in-vivo and in-vitro monitoring systems.

[0041] 1. General structure of the test substance sensor system A. Exemplary In Vivo Analyte Monitoring Systems Before describing the presently disclosed analyte sensors and components thereof in further detail, a brief overview of preferred in vivo analyte sensor configurations and sensor systems employing analyte sensors is provided so that embodiments of the present disclosure may be better understood.

[0042] FIG. 1A is a conceptual diagram illustrating an example embodiment of an analyte monitoring system 100 including a sensor applicator 150, a sensor control device 102, and a reader device 120, where the sensor applicator 150 can be used to deliver the sensor control device 102 to a monitoring location on a user's skin, where the sensor 104 is maintained in place by an adhesive patch 105 for a period of time. The sensor control device 102, further described in FIGS. 2B and 2C, can communicate with the reader device 120 via a communication path 140 using wired or wireless technology. Examples of wireless protocols include Bluetooth, Bluetooth Low Energy (BLE, BTLE, Bluetooth SMART, etc.), Near Field Communication (NFC), etc. A user can monitor applications installed in memory on the reader device 120 using a screen 122, and the input 121 and device battery can be recharged using a power port 123. Further details regarding the reader device 120 are described below with respect to FIG. 2A. The reader device 120 can communicate with the local computer system 170 via communication path 141 using wired or wireless technology. The local computer system 170 can include one or more of a laptop, desktop, tablet, phablet, smartphone, set-top box, video game console, or other computing device, and the wireless communication can include any of several applicable wireless network protocols including Bluetooth, Bluetooth Low Energy (BTLE), Wi-Fi, or others. The local computer system 170 can communicate with the network 190 via communication path 143 in the same way that the reader device 120 can communicate with the network 190 via communication path 142, using wired or wireless technology as already mentioned. The network 190 can be any of private and public networks, local or wide area networks, or some other network.The trusted computer system 180 may include a server, provide authentication services and secured data storage, and communicate with the network 190 via communication path 144 using wired or wireless technology.

[0043] FIG. 1B illustrates an operating environment for an analyte monitoring system 100a in which the technology described herein can be embodied. The analyte monitoring system 100a can include a system of parts designed to provide monitoring of parameters such as analyte levels in a human or animal body, or provide other operations based on the configuration of various parts. As embodied herein, the system can include a low-power analyte sensor 110, or simply a "sensor," worn by a user or attached to the body about which information is collected. As embodied herein, the analyte sensor 110 can be a sealed, disposable device with a predetermined active use life (e.g., 1 day, 14 days, 30 days, etc.). The sensor 110 can be attached to the skin of a user's body and can be designed to remain adhered for the duration of the sensor's life or remain functional when selectively removed and reattached. The low-power test substance monitoring system 100a may further include a data reader device 120 or a general-purpose data receiving device 130 configured as described herein to facilitate retrieval and delivery of data, including test substance data, from the test substance sensor 110.

[0044] As embodied herein, the analyte monitoring system 100a may include software or firmware libraries or applications provided to a third party, for example, via a remote application server 150 or an application storefront server 160, and embedded in a general-purpose hardware device 130, such as a mobile phone, tablet, personal computing device, or other similar computing device capable of communicating with the analyte sensor 110 via a communications link. The general-purpose hardware may further include embedded devices, including but not limited to, an insulin pump or insulin pen, having embedded libraries configured to communicate with the analyte sensor 110. While the illustrated embodiment of the analyte monitoring system 100a includes only one of each of the illustrated devices, the present disclosure contemplates that the analyte monitoring system 100a may incorporate multiple of each of the components that interact throughout the system. For example, but not limited to, as embodied herein, the data reader device 120 and / or the general-purpose data receiving device 130 may include multiple of each. As embodied herein, the multiplexed data receiving device 130 may communicate directly with the sensors 110 described herein. Additionally or alternatively, the data receiving device 130 may communicate with a secondary data receiving device 130 to provide analyte data or visualization or analysis of the data for secondary display to a user or other authorized party.

[0045] FIG. 1C shows a schematic diagram of an exemplary sensing system that may incorporate the analyte sensors of the present disclosure. As shown, the sensing system 100 includes a sensor control device 102 and a reader device 120 configured to communicate with each other via a local communication path or link 140, which may be wired or wireless, unidirectional or bidirectional, and encrypted or unencrypted. The reader device 120 may, according to certain embodiments, configure an output medium for visualizing the analyte concentration and any warnings or alerts determined by the sensor 104 or its associated processor, and for enabling one or more user inputs. The reader device 120 may be a general-purpose smartphone or a dedicated electronic reader device. Although only one reader device 120 is shown, multiple reader devices 120 may be present in certain cases. The reader device 120 may communicate with the remote terminal 90170 and / or the trusted computer system 90180 via communication path(s) / link(s) 90141 and / or 90142, which may be wired or wireless, unidirectional or bidirectional, and encrypted or unencrypted, respectively. The reader device 120 may also or instead communicate with a network 150 (e.g., a mobile telephone network, the Internet, or a cloud server) via communication path / link 151. The network 150 may further be communicatively coupled with the remote terminal 90170 via communication path / link 152 and / or the trusted computer system 90180 via communication path / link 153. Alternatively, the sensor 104 may communicate directly with the remote terminal 90170 and / or the trusted computer system 90180 without the presence of the intervening reader device 120. For example, but not by way of limitation, according to certain embodiments described in U.S. Patent Application Publication No. 2011 / 0213225, the entire contents of which are incorporated by reference herein, the sensor 104 may communicate with a remote terminal 90170 and / or a trusted computer system 90180 via a direct communications link to the network 150.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. According to certain embodiments, the remote terminal 90170 and / or the trusted computer system 90180 may be accessible by individuals other than the primary user who are interested in the user's analyte levels. The reader device 120 may include a display 122 and an optional input component 121. According to certain embodiments, the display 122 may include a touch screen interface.

[0046] The sensor control device 102 includes a sensor housing 103, which may house circuitry and a power source for operating the sensor 104. Optionally, the power source and / or active circuitry may be omitted. A processor (not shown) may be communicatively connected to the sensor 104, the processor being physically located within the sensor housing 103 or the reader device 120. According to certain embodiments, the sensor 104 protrudes from an underside of the sensor housing 103 and extends through an adhesive layer 105 adapted to adhere the sensor housing 103 to a tissue surface, such as the skin.

[0047] The sensor 104 is adapted to be inserted at least partially into a tissue of interest, such as into a skin layer or a subcutaneous layer of the skin. The sensor 104 may include a sensor tail of sufficient length for insertion to a desired depth in a given tissue. The sensor tail may include at least one working electrode. In certain configurations, the sensor tail may include an active area for detecting an analyte. There may be a counter electrode in combination with the at least one working electrode. Particular electrode configurations on the sensor tail are described in more detail below.

[0048] The active area may be configured to detect a particular analyte. In certain embodiments, the active area of ​​the presently disclosed sensors is configured to detect ketones. In certain embodiments, the active area may be configured to detect more than one analyte. In certain embodiments, additional analytes to be detected using the presently disclosed sensors include any dysregulated analyte along with ketones. For example, without limitation, the presently disclosed analyte sensors can detect ketones and additional analytes such as creatinine, oxygen, glucose, and / or lactate. In certain embodiments, the additional analyte is glucose.

[0049] In certain embodiments of the present disclosure, one or more analytes may be monitored in skin fluid, interstitial fluid, plasma, blood, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, amniotic fluid, or any other biological fluid of interest. In certain embodiments, the analyte sensor of the present disclosure may be adapted to assay skin fluid or interstitial fluid to determine the concentration of one or more analytes in vivo. In certain embodiments, the biological fluid is interstitial fluid.

[0050] Still referring to FIG. 1C, the sensor 104 may automatically send data to the reader device 120. For example, but not by way of limitation, analyte concentration data (i.e., glucose and / or ketone concentrations) may be automatically and periodically communicated, e.g., at a frequency, when the data is obtained or after a period of time has elapsed, and the data is stored in memory until transmission (e.g., every minute, every five minutes, or other predetermined period of time). In certain other embodiments, the sensor 104 may communicate with the reader device 120 in a non-automatic manner and not according to a set schedule. For example, but not by way of limitation, data may be communicated from the sensor 104 using RFID technology when the sensor electronics are within communication range of the reader device 120. The data may remain stored in the sensor 104's memory until communicated to the reader device 120. That is, the user is not required to maintain close proximity to the reader device 120 at all times, but instead may upload the data at a convenient time. In certain other embodiments, a combination of automatic and non-automatic data transfer may be performed. For example, and without limitation, data transfer may continue automatically until the reader device 120 is no longer in communication range of the sensor 104 .

[0051] An introducer may be temporarily present to facilitate the introduction of the sensor 104 into the tissue. In certain exemplary embodiments, the introducer may include a needle or similar tip. As one skilled in the art will readily appreciate, other types of introducers, such as a sheath or blade, may be present in alternative embodiments. More specifically, a needle or other introducer may be temporarily present proximal to the sensor 104 prior to insertion into the tissue and then retracted. While present, the needle or other introducer may facilitate the insertion of the sensor 104 into the tissue by opening an access path followed by the sensor 104. For example, but not by way of limitation, according to one or more embodiments, the needle may facilitate the penetration of the epidermis as an access path to the dermis to allow the implantation of the sensor 104 to occur. After opening the access path, the needle or other introducer may be retracted so as not to present injury from the tip. In certain embodiments, suitable needles may be solid or hollow, beveled or non-beveled, and / or circular or non-circular in cross section. In more specific embodiments, suitable needles may be similar in cross-sectional diameter and / or tip design to acupuncture needles, and may have a cross-sectional diameter of about 250 microns (250 μm). However, suitable needles may have a larger or smaller cross-sectional diameter if necessary for a particular application.

[0052] In certain embodiments, the tip of the needle (while present) may be angled beyond the end of the sensor 104, such that the needle penetrates the tissue first and opens an access path for the sensor 104. In certain embodiments, the sensor 104 may reside within a lumen or groove of the needle, such that the needle similarly opens an access path for the sensor 104. In either case, the needle is subsequently retracted after facilitating insertion of the sensor.

[0053] B. Exemplary Reader Device 2A is a block schematic diagram illustrating an example of an embodiment of a reader device configured as a smartphone. Here, the reader device 120 may include a display 122, an input component 121, and a processing core 206 including a communication processor 222 coupled to a memory 223 and an application processor 224 coupled to a memory 225. A separate memory 230, an RF transceiver 228 having an antenna 229, and a power supply 226 having a power management module 238 may be included. A multi-function transceiver 232 capable of communicating with an antenna 234 via Wi-Fi, NFC, Bluetooth, BTLE, and GPS may further be included. As will be appreciated by those skilled in the art, these components are electrically and communicatively connected in a manner to create a functional device.

[0054] C. Exemplary Data Receiving Device Architecture For purposes of explanation and not limitation, reference is made to an exemplary embodiment of a data receiving device 120 for use with the disclosed subject matter shown in FIG. 2B. The data receiving device 120 and associated general-purpose data receiving device 130 include components germane to the discussion of the analyte sensor 110, its operation and may include additional components. In certain embodiments, the data receiving device 120 and general-purpose data receiving device 130 may be or include components provided by third parties and may not necessarily be limited to including devices made by the same manufacturer as the sensor 110.

[0055] 2B, the data receiving device 120 includes an ASIC 4000 that includes a microcontroller 4010, memory 4020, and storage 4030, and is communicatively connected to a communication module 4040. Power for the components of the data receiving device 120 can be delivered by a power module 4050, which may include a rechargeable battery as embodied herein. The data receiving device 120 may further include a display 4070 to facilitate review of analyte data received from the analyte sensor 110 or other devices (e.g., the user device 140 or the remote application server 150). The data receiving device 120 may include separate user interface components (e.g., a physical key, a light sensor, a microphone, etc.).

[0056] The communication module 4040 may include a BLE module 4041 and an NFC module 4042. The data receiving device 120 may be wirelessly connected to the analyte sensor 110 and configured to transmit commands to and receive data from the analyte sensor 110. As embodied herein, the data receiving device 120 may be configured to operate as an NFC scanner and a BLE endpoint via a specific module (e.g., the BLE module 4042 or the NFC module 4043) of the communication module 4040 with respect to the analyte sensor 110 described herein. For example, the data receiving device 120 may issue commands to the analyte sensor 110 (e.g., an activation command for a data broadcast mode of the sensor, a pairing command to identify the data receiving device 120) using a first module of the communication module 4040, and receive data from and transmit data to the analyte sensor 110 using a second module of the communication module 4040. The data receiving device 120 may be configured for communication with the user device 140 via a Universal Serial Bus (USB) module 4045 of the communication module 4040 .

[0057] As another example, the communication module 4040 may include, for example, a cellular radio module 4044. The cellular radio module 4044 may include one or more radio transceivers for communicating using broadband cellular networks, including but not limited to third generation (3G), fourth generation (4G), and fifth generation (5G) networks. Furthermore, the communication module 4040 of the data receiving device 120 may include a Wi-Fi radio module 4043 for communicating using wireless local area networks according to one or more of the IEEE 802.11 standards (e.g., 802.11a, 802.11b, 802.11g, 802.11n (also referred to as Wi-Fi4), 802.11ac (also referred to as Wi-Fi5), and 802.11ax (also referred to as Wi-Fi6)). Using the cellular radio module 4044 or the Wi-Fi radio module 4043, the data receiving device 120 may communicate with the remote application server 150 to receive analyte data or provide updates or input received from a user (e.g., via one or more user interfaces). Although not shown, the communications module 5040 of the analyte sensor 120 may also include a cellular radio module or a Wi-Fi radio module.

[0058] As embodied herein, the on-board storage 4030 of the data receiving device 120 can store the analyte data received from the analyte sensor 110. Additionally, the data receiving device 120, the multi-purpose data receiving device 130, or the user device 140 can be configured to communicate with a remote application server 150 over a wide area network. As embodied herein, the analyte sensor 110 can provide data to the data receiving device 120 or the multi-purpose data receiving device 130. The data receiving device 120 can transmit the data to the user computing device 140. The user computing device 140 (or the multi-purpose data receiving device 130) can then transmit the data to the remote application server 150 for processing and analysis.

[0059] As embodied herein, the data receiving device 120 may further include sensing hardware 4060 similar to or extended from the sensing hardware 5060 of the analyte sensor 110. In certain embodiments, the data receiving device 120 may be configured to interface with the analyte sensor 110 and act based on the analyte data received from the analyte sensor 110. By way of example, if the analyte sensor 110 is a glucose sensor, the data receiving device 120 may be or include an insulin pump or an insulin injection pen. In conjunction, the compatible device 130 may adjust insulin dosage for the user based on the glucose value received from the analyte sensor.

[0060] D. Exemplary Sensor Control Device 2C and 2D are block schematic diagrams illustrating an example embodiment of a sensor controller 102 having an analyte sensor 104 and sensor electronics 160 (including analyte monitoring circuitry) that may have most of the processing power to make the end result data suitable for display to a user. In FIG. 2C, a single semiconductor chip 161 is shown, which may be a custom application specific integrated circuit (ASIC). Within the ASIC 161, certain high level functional units are shown, including an analog front end (AFE) 162, a power management (or control) circuit 164, a processor 166, and a communication circuit 168 (which may be implemented as a transmitter, receiver, transceiver, passive circuitry, or in other ways according to a communication protocol). In this embodiment, both the AFE 162 and the processor 166 are used as analyte monitoring circuitry, although in other embodiments, either circuitry may perform the analyte monitoring function. The processor 166 may include one or more processors, microprocessors, controllers, and / or microcontrollers, each of which may be a separate chip or distributed among many different chips (and portions thereof).

[0061] The memory 163 is also included in the ASIC 161 and may be shared by the various functional units present in the ASIC 161 or distributed between two or more of them. The memory 163 may also be a separate chip. The memory 163 may be a volatile and / or non-volatile memory. In this embodiment, the ASIC 161 is connected to a power source 170, which may be a button cell battery or the like. The AFE 162 interfaces with the in-vivo analyte sensor 104 to receive measurement data therefrom and outputs the data in digital form to the processor 166, which then processes the data to arrive at final result glucose discrete values ​​and trend values, etc. This data may then be provided to the communication circuitry 168 for transmission via the antenna 171 to the reader device 120 (not shown), for example when a resident software application requires minimal further processing to display the data.

[0062] FIG. 2D is similar to FIG. 2C, but instead includes two discrete semiconductor chips 162 and 174, which may be packaged together or separately. Here, AFE 162 resides in ASIC 161. Processor 166 is integrated with power management circuitry 164 and communication circuitry 168 on chip 174. AFE 162 includes memory 163, and chip 174 includes memory 165, which may be separate or distributed therein. In one example embodiment, AFE 162 is combined with power management circuitry 164 and processor 166 on one chip, while communication circuitry 168 is on a separate chip. In another example embodiment, both AFE 162 and communication circuitry 168 are on one chip, and processor 166 and power management circuitry 164 are on another chip. It should be noted that other chip combinations are possible, including three or more chips, each performing separate functions as described, or sharing one or more functions for fail-safe redundancy.

[0063] For purposes of illustration and not limitation, reference is made to an exemplary embodiment of an analyte sensor 110 for use with the disclosed subject matter shown in Figure 2E, which illustrates a block diagram of an example analyte sensor 110 according to an exemplary embodiment compatible with the security architecture and communication schemes described herein.

[0064] As embodied herein, the analyte sensor 110 may include an application specific integrated circuit ("ASIC") 5000 communicatively coupled to a communication module 5040. The ASIC 5000 may include a microcontroller core 5010, an on-board memory 5020, and a storage memory 5030. The storage memory 5030 may store data used in a security architecture for authentication and encryption. The storage memory 5030 may store programming instructions for the sensor 110. As embodied herein, a specific communication chipset may be embedded within the ASIC 5000 (e.g., NFC transceiver 5025). The ASIC 5000 may receive power from a power module 5050, e.g., an on-board battery or an NFC pulse. The storage memory 5030 of the ASIC 5000 may be programmed to include information such as an identifier of the sensor 110 for identification and tracking purposes. The storage memory 5030 may also be programmed with configuration or calibration parameters for use by the sensor 110 and its various components. The storage memory 5030 may include re-writable or one-time programming (OTP) memory. The storage memory 5030 may be updated using the techniques described herein to extend the usefulness of the sensor 110.

[0065] As embodied herein, the communication module 5040 of the sensor 100 may be or include one or more modules that assist the analyte sensor 110 in communicating with other devices of the analyte monitoring system 100. By way of example only and not limitation, an example of the communication module 5040 may include a Bluetooth® Low Energy ("BLE") module 5041. As used throughout this disclosure, Bluetooth® Low Energy ("BLE") refers to a short-range communication protocol optimized to simplify pairing of Bluetooth® devices for end users. The communication module 5040 may transmit and receive data and commands via interaction with a similarly capable communication module of the data receiving device 120 or user device 140. The communication module 5040 may include additional or alternative chipsets for use with personal area networks according to the IEEE 802.15 protocol, infrared communications according to the Infrared Data Association standard (IrDA), and other similar short-range communication schemes.

[0066] To perform its functionality, the sensor 100 may further include suitable sensing hardware 5060 appropriate for that function. As embodied herein, the sensing hardware 5060 may include an analyte sensor that is placed transcutaneously or subcutaneously in contact with the subject's bodily fluid. The analyte sensor may generate sensor data that includes a value corresponding to a level of one or more analytes in the bodily fluid.

[0067] E. Exemplary Assembly Process for Sensor Control Device The parts of the sensor control device 102 may be acquired by the user in multiple packages that require final assembly by the user prior to delivery to the appropriate user location. Figures 3A-3D show an example embodiment of a user assembly process for the sensor control device 102 that includes preparing the separate parts before joining the parts to prepare the sensor for delivery. Figures 3E-3F show an example embodiment of delivering the sensor control device 102 to the appropriate user location by selecting the appropriate delivery location and orienting the device 102 to that location.

[0068] 3A is a proximal perspective view showing an example embodiment of a user preparing a container 810, configured herein as a tray (although other packaging may be used) for an assembly process. The user may accomplish this preparation by, for example, removing the lid 812 from the tray 810 such that the non-adhesive portion of the lid 812 is peeled away from the tray 810 to remove the adhesive portion of the lid 812, exposing the platform 808. Removal of the lid 812 may be suitable in various embodiments, so long as the platform 808 is properly exposed in the tray 810. The lid 812 may then be set aside.

[0069] 3B is a side view illustrating an example embodiment in which a user prepares applicator device 150 for assembly. Applicator device 150 may be provided in a sterile package sealed by cap 708. Preparing applicator device 150 may include removing housing 702 from cap 708 to expose sheath 704 (FIG. 3C). This may be accomplished by unscrewing (or otherwise removing) cap 708 from housing 702. Cap 708 may then be set aside.

[0070] 3C is a proximal perspective view showing an example embodiment in which a user inserts applicator device 150 into tray 810 during assembly. First, a user can align housing orientation feature 1302 (or slot or recess) and tray orientation feature 924 (abutment or detent) before inserting sheath 704 into platform 808 inside tray 810. Inserting sheath 704 into platform 808 temporarily unlocks sheath 704 relative to housing 702, and also temporarily unlocks platform 808 relative to tray 810. At this stage, removal of applicator device 150 from tray 810 will result in the same condition as before the initial insertion of applicator device 150 into tray 810 (i.e., the process can be reversed or interrupted at this point and then repeated without consequence).

[0071] During distal advancement of the housing 702, the sheath 704 is maintained in position relative to the housing 702 within the platform 808 and couples with the platform 808 to advance the platform 808 distally relative to the tray 810. This process unlocks and folds the platform 808 within the tray 810. The sheath 704 is unlocked relative to the housing 702 and contacts and disengages a locking feature (not shown) within the tray 810 that prevents the sheath 704 from moving (relatively) while the housing 702 continues to advance the platform 808 distally. When advancement of the housing 702 and platform 808 is completed, the sheath 704 is permanently unlocked relative to the housing 702. At the end of distal advancement of the housing 702, the tip and sensor (not shown) within the tray 810 can couple with the electronics housing (not shown) within the housing 702. The operation and interaction of applicator device 150 and tray 810 are described further below.

[0072] 3D is a proximal perspective view illustrating an example embodiment in which a user removes applicator device 150 from tray 810 during assembly. A user can remove applicator 150 from tray 810 by advancing housing 702 proximally relative to tray 810, or by other action that has the same end effect as decoupling applicator 150 from tray 810. Applicator device 150 is removed with sensor control device 102 (not shown) fully assembled therein (tip, sensor, electronics) and positioned for delivery.

[0073] 3E is a proximal perspective view of an example embodiment in which a patient applies the sensor control unit 102 to a target area of ​​the skin, such as the abdomen or other suitable location, using the applicator device 150. The sensor is applied to the target location by advancing the housing 702 distally, collapsing the sheath 704 within the housing 702 so that the adhesive layer on the bottom side of the sensor control unit 102 adheres to the skin. When the housing 702 is fully advanced, the tip is automatically retracted while the sensor (not shown) is left in place to measure the analyte level.

[0074] 3F is a proximal perspective view of an example embodiment of a patient in an attached position with the sensor control unit 102. The user can then remove the applicator 150 from the attachment site.

[0075] 3A-3F and elsewhere herein, the system 100 can reduce or eliminate the chances of accidental breakage, permanent deformation, or improper assembly of applicator components compared to prior art systems. Because the applicator housing 702 directly engages the platform 808 while the sheath 704 is unlocked, rather than indirect engagement through the sheath 704, the relative angle between the sheath 704 and the housing 702 does not result in breakage or permanent deformation of the arms or other components. The likelihood of relatively large forces (e.g., in conventional devices) during assembly is reduced, thereby reducing the chances of unsuccessful user assembly.

[0076] F. Exemplary Sensor Applicator Device FIGURE 4A is a side view of an example embodiment of applicator device 150 coupled to a screw cap 708. This is an example of how applicator 150 may be shipped and received by a user prior to assembly with a sensor by the user. FIGURE 4B is a side perspective view of applicator 150 and cap 708 after coupling has been removed. FIGURE 4C is a perspective view of an example embodiment of the distal end of applicator device 150 removed from the position where electronics housing 706 and adhesive patch 105 were held within sensor carrier 710 of sheath 704 with cap 708 in place.

[0077] With reference to Figures 4D-G, for purposes of illustration and not limitation, the applicator device 20150 can be provided to a user as a single integrated assembly. Figures 4D and 4E provide top and bottom perspective views, respectively, of the applicator device 20150, Figure 4F provides an exploded view of the applicator device 20150, and Figure 4G provides a side cross-sectional view. The perspective views show how the applicator 20150 is shipped and received by a user. The exploded and cross-sectional views show the components of the applicator device 20150. The applicator device 20150 may include a housing 20702, a gasket 20701, a sheath 20704, a tip carrier 201102, a spring 205612, a sensor carrier 20710 (also referred to as a "puck carrier"), a tip hub 205014, a sensor control device (also referred to as a "puck") 20102, an adhesive patch 20105, a desiccant 20502, a cap 20708, a serial label 20709, and a tamper evidence feature 20712. When received by a user, only the housing 20702, the cap 20708, the tamper evidence feature 20712, and the label 20709 are visible. The tamper evidence feature 20712 may be, for example, a sticker coupled to each of the housing 20702 and the cap 20708, such that the tamper evidence feature 20712 may be irreparably damaged, for example, by disconnecting the housing 20702 from the cap 20708, thereby indicating to a user that the housing 20702 and the cap 20708 have previously been disconnected. These features are described in more detail below.

[0078] G. Assembly of Exemplary Trays and Sensor Modules FIG. 5 is a proximal perspective view showing an example embodiment of a tray 810 with a sterilization lid 812 removably coupled thereto, which may illustrate how the package may be shipped and received by a user prior to assembly.

[0079] 6A is a proximal perspective cross-sectional view showing the sensor delivery components in a tray 810. A platform 808 is slidably coupled within the tray 810. A desiccant 502 is fixed relative to the tray 810. A sensor module 504 is mounted within the tray 810.

[0080] 6B is a proximal perspective view showing the sensor module 504 in greater detail, where the retention arm extension 1834 of the platform 808 releasably secures the sensor module 504 in place. The module 2200 is coupled to the connector 2300, tip module 2500, and sensor (not shown) so that they can be removed together as the sensor module 504 during assembly.

[0081] H. Exemplary Applicator and Sensor Control Unit for a One-Piece Architecture Referring again briefly to FIGS. 1A and 3A-3G, for a two-piece architecture system, the sensor tray 202 and the sensor applicator 102 are provided to the user as separate packages, thus requiring the user to open each package and ultimately assemble the system. In some applications, the separate sealed packages allow the sensor tray 202 and the sensor applicator 102 to be sterilized by separate sterilization processes that are specific to the contents of each package and are not otherwise compatible with the contents of the other package. More specifically, the sensor tray 202, including the plug assembly 207, including the sensor 110 and the tip 220, may be sterilized using radiation sterilization, such as electron beam (i.e., "e-beam") irradiation. Suitable radiation sterilization processes include, but are not limited to, electron beam (e-beam) irradiation, gamma irradiation, x-ray irradiation, or any combination thereof. However, radiation sterilization may damage electrical components located within the electronics housing of the sensor control device 102. Thus, if the sensor applicator 102, including the electronics housing of the sensor control device 102, needs to be sterilized, it may be sterilized by another method, such as gas chemical sterilization using ethylene oxide, but gas chemical sterilization may damage enzymes or other chemical and biological materials contained in the sensor 110. Due to this sterilization incompatibility, the sensor tray 202 and the sensor applicator 102 are typically sterilized by separate sterilization processes and then packaged separately, thus requiring the user to ultimately assemble the parts for use.

[0082] 7A and 7B are respectively exploded top and bottom views of a sensor control device 3702 according to one or more embodiments. The shell 3706 and the mount 3708 act as opposing clamshell halves that contain or otherwise substantially encapsulate various electronic components of the sensor control device 3702. As shown, the sensor control device 3702 may include a printed circuit board assembly (PCBA) 3802 including a printed circuit board (PCB) 3804 to which a number of electronic modules 3806 are connected. Examples of electronic modules 3806 include, but are not limited to, resistors, transistors, capacitors, inductors, diodes, and switches. Conventional sensor control devices typically stack PCB components on only one side of the PCB. In contrast, the PCB components 3806 in the sensor control device 3702 may be distributed on both surface areas (i.e., top and bottom) of the PCB 3804.

[0083] In addition to the electronics module 3806, the PCBA 3802 may include a data processing unit 3808 mounted on the PCB 3804. The data processing unit 3808 may comprise, for example, an application specific integrated circuit (ASIC) configured to perform one or more functions or routines associated with the operation of the sensor controller 3702. More specifically, the data processing unit 3808 may be configured to perform data processing functions, which may include, but are not limited to, filtering and encoding data signals, each of which corresponds to a sampled analyte level of a user. The data processing unit 3808 may also include an antenna for communicating with or otherwise communicating with the reader device 106 (FIG. 1A).

[0084] A battery aperture 3810 may be defined in the PCB 3804 and sized to receive and seat a battery 3812 configured to power the sensor controller 3702. An axial battery contact 3814a and a radial battery contact 3814b may couple to the PCB 3804 and extend into the battery aperture 3810 to facilitate delivery of power from the battery 3812 to the PCB 3804. As their names suggest, the axial battery contact 3814a may be configured to provide an axial contact to the battery 3812, and the radial battery contact 3814b may provide a radial contact to the battery 3812. Positioning the battery 3812 in the battery aperture 3810 with the battery contacts 3814a and b helps reduce the height H of the sensor controller 3702, thereby allowing the PCB 3804 to be centrally located and its components to be distributed on both sides (i.e., top and bottom). This also helps facilitate mounting the chamber 3718 onto the electronics housing 3704 .

[0085] The sensor 3716 may be centrally disposed with respect to the PCB 3804 and may include a tail 3816, a flag 3818, and a neck 3820 interconnecting the tail 3816 and the flag 3818. The tail 3816 may be configured to extend through a central aperture 3720 of the mount 3708 to be transcutaneously received beneath the skin of a user. Additionally, the tail 3816 may have an enzyme or other chemical contained therein that helps facilitate monitoring of the analyte.

[0086] The flag 3818 may include a generally flat surface having one or more sensor contacts 3822 (three shown in FIG. 7B ) disposed thereon. The sensor contacts 3822 may be configured to align with and engage with one or more corresponding circuit contacts 3824 (three shown in FIG. 7A ) provided on the PCB 3804. In some embodiments, the sensor contacts 3822 may comprise a carbon-impregnated polymer printed or otherwise digitally applied to the flag 3818. Conventional sensor control devices generally include a connector made from silicone rubber encapsulating one or more compliant carbon-impregnated polymer modules that act as conductive contacts between the sensor and the PCB. In contrast, the sensor contacts 3822 of the present disclosure provide a direct connection between the sensor 3716 and the PCB 3804 connection, thereby eliminating the need for a prior art connector and advantageously reducing the height H. Additionally, by eliminating the compliant carbon-impregnated polymer modules, circuit resistance is significantly eliminated, thus improving circuit conductivity.

[0087] The sensor control device 3702 may further include a compliant member 3826, which may be disposed to be interposed between the flag 3818 and an inner surface of the shell 3706. More specifically, when the shell 3706 and the mount 3708 are assembled together, the compliant member 3826 may be configured to provide a passive biasing load to the flag 3818 that urges the sensor contacts 3822 into continuous engagement with the corresponding circuit contacts 3824. In the illustrated embodiment, the compliant member 3826 is an elastomeric O-ring, but may instead comprise any other type of biasing device or mechanism, such as a compression spring, without departing from the scope of the present disclosure.

[0088] The sensor control device 3702 may further include one or more electromagnetic shields, shown as a first shield 3828a and a second shield. The shell 3706 may include or otherwise define a first clocking receptacle 3830a (FIG. 7B) and a second clocking receptacle 3830b (FIG. 7B), and the mount 3708 may include or otherwise define a first clocking post 3832a (FIG. 7A) and a second clocking post 3832b (FIG. 7A). The shell 3706 is properly aligned to the mount 3708 by pairing the first and second clocking receptacles 3830a,b with the first and second clocking posts 3832a,b, respectively.

[0089] 7A , the inner surface of the mount 3708 may include or otherwise define a number of pockets or recesses configured to accommodate various components of the sensor control unit 3702 when the shell 3706 is mated with the mount 3708. For example, the inner surface of the mount 3708 may define a battery locator 3834 configured to accommodate a portion of the battery 3812 when the sensor control unit 3702 is assembled. An adjacent contact pocket 3836 may be configured to accommodate a portion of the axial contact 3814a.

[0090] Additionally, a number of module pockets 3838 may be defined on the inner surface of the mount 3708 to accommodate various electronic modules 3806 disposed on the bottom of the PCB 3804. Additionally, a shield locator 3840 may be defined on the inner surface of the mount 3708 to accommodate at least a portion of the second shield 3828b when the sensor control device 3702 is assembled. The battery locator 3834, contact pocket 3836, module pocket 3838, and shield locator 3840 all extend a short distance on the inner surface of the mount 3708, and as a result, the overall height H of the sensor control device 3702 may be reduced as compared to conventional sensor control devices. The module pocket 3838 may also help minimize the diameter of the PCB 3804 by locating PCB components on both sides (i.e., top and bottom).

[0091] 7A, the mount 3708 may further include a plurality of carrier grip features 3842 (two shown) defined about the periphery of the mount 3708. The carrier grip features 3842 are axially offset from a bottom 3844 of the mount 3708 where a transfer adhesive may be applied during assembly. In contrast to conventional sensor control devices that include a conical carrier grip feature that generally intersects with the bottom of the mount, the carrier grip features 3842 of the present disclosure are offset from the plane where the transfer adhesive is applied (i.e., the bottom 3844). This may prove advantageous to help ensure that the delivery system does not inadvertently adhere to the transfer adhesive during assembly. Additionally, the carrier grip features 3842 of the present disclosure eliminate the need for a wavy transfer adhesive, which simplifies the manufacture of the transfer adhesive and eliminates the need to precisely register the transfer adhesive to the mount 3708. This also increases the bonding area and therefore the bond strength.

[0092] 7B, the bottom 3844 of the mount 3708 may include or otherwise define a plurality of grooves 3846, which are defined at or near the perimeter of the mount 3708 and are equidistantly spaced from one another. A transfer adhesive (not shown) may be coupled to the bottom 3844, and the grooves 3846 may be configured to help wick (transport) moisture away from the sensor control device 3702 and towards the perimeter of the mount 3708 during use. In some embodiments, the spacing of the grooves 3846 may sandwich a module pocket 3838 (FIG. 7A) defined on the opposite (inner) side of the mount 3708. As will be appreciated, alternating the location of the grooves 3846 and the module pockets 3838 ensures that opposing features on either side of the mount 3708 do not extend into one another. This helps maximize the usage of material for the mount 3708 and may help maintain a minimum height H of the sensor control device 3702. The module pocket 3838 may also significantly reduce mold sink and improve the flatness of the bottom 3844 where the transfer adhesive adheres.

[0093] 7B, the inner surface of the shell 3706 may include or otherwise define a number of pockets or recesses configured to accommodate various components of the sensor control unit 3702 when the shell 3706 is mated with the mount 3708. For example, the inner surface of the shell 3706 may define an opposing battery locator 3848 that is positionable opposite the battery locator 3834 (FIG. 7A) of the mount 3708 and configured to accommodate a portion of the battery 3812 when the sensor control unit 3702 is assembled. The opposing battery locator 3848 extends a short distance into the inner surface of the shell 3706, thereby helping to reduce the overall height H of the sensor control unit 3702.

[0094] An inner surface of the shell 3706 may be provided with or otherwise defined by a tip and sensor locator 3852. The tip and sensor locator 3852 may be configured to receive both a tip (not shown) and a portion of the sensor 3716. Further, the tip and sensor locator 3852 may be configured to align and / or mate with a corresponding tip and sensor locator 2054 ( FIG. 7A ) provided on an inner surface of the mount 3708.

[0095] An alternative sensor assembly / electronics assembly connection approach is shown in Figures 8A-8C in accordance with an embodiment of the present disclosure. As shown, the sensor assembly 14702 includes a sensor 14704, a connector support 14706, and a tip 14708. In particular, a recess or receptacle 14710 may be defined at the bottom of a mount for the electronics assembly 14712 to provide a location for the sensor assembly 14702 to be received and coupled to the electronics assembly 14712, thereby fully assembling the sensor control device. The profile of the sensor assembly 14702 may be shaped in a manner that matches or is complementary to the receptacle 14710, which includes an elastomeric sealing member 14714 (coupled to a circuit board and including a conductive material aligned with the electrical contacts of the sensor 14704). Thus, by driving the sensor assembly 14702 into the integrally formed recess 14710 of the electronics assembly 14712, when the sensor assembly 14702 is snap-fit ​​or otherwise adhered to the electronics assembly 14712, the on-body device 14714 shown in FIG. 8C is formed. This embodiment provides an integrated connector for the sensor assembly 14702 in the electronics assembly 14712.

[0096] Further information regarding sensor assemblies is provided in U.S. Publication No. 2013 / 0150691 and U.S. Publication No. 2021 / 0204841, each of which is incorporated by reference in its entirety into this specification.

[0097] According to an embodiment of the present disclosure, the sensor control device 102 may be modified to provide a one-piece architecture that can undergo sterilization techniques specifically designed for the one-piece architecture sensor control device. The one-piece architecture allows the sensor applicator 150 and the sensor control device 102 to be shipped to the user in a single sealed package that does not require any final user assembly steps. Rather, the user only needs to open one package and subsequently deliver the sensor control device 102 to the target monitoring location. The one-piece system architecture described herein may prove advantageous in reducing parts, various manufacturing process steps, and user assembly steps. As a result, packaging materials and waste are reduced, and the possibility of user error or contamination of the system is mitigated.

[0098] Figures 9A and 9B are side and cross-sectional views, respectively, of an example embodiment of the sensor applicator 102 coupled with the applicator cap 210. More specifically, Figure 9A illustrates how the sensor applicator 102 is shipped and received by a user, and Figure 9B illustrates the sensor control unit 4402 disposed within the sensor applicator 102. Thus, a fully assembled sensor control unit 4402 may already be assembled and installed within the sensor applicator 102 prior to delivery to the user, thereby eliminating any additional assembly steps that would otherwise have to be performed by the user.

[0099] The fully assembled sensor control device 4402 may be attached to the sensor applicator 102, followed by coupling the applicator cap 210 to the sensor applicator 102. In some embodiments, the applicator cap 210 may be threadedly engaged with the housing 208 and may include a tamper ring 4702. Upon rotating (e.g., unscrewing) the applicator cap 210 relative to the housing 208, the tamper ring 4702 may shear, thereby releasing the applicator cap 210 from the sensor applicator 102.

[0100] In accordance with the present disclosure, while attached to the sensor applicator 102, the sensor control device 4402 may be subjected to gaseous chemical sterilization 4704 configured to sterilize the electronics housing 4404 and any other exposed portions of the sensor control device 4402. To accomplish this, chemicals may be injected into a sterilization chamber 4706 cooperatively defined by the sensor applicator 102 and the interconnected cap 210. In some embodiments, chemicals may be injected into the sterilization chamber 4706 via one or more vents 4708 defined by the applicator cap 210 at its proximal end 610. Examples of chemicals that may be used for gaseous chemical sterilization 4704 include, but are not limited to, ethylene oxide, vaporized hydrogen peroxide, nitric oxide (e.g., nitrous oxide, nitrogen dioxide, etc.), and water vapor.

[0101] Because the sensor 4410 and distal portion of tip 4412 are sealed within sensor cap 4416, chemicals used during the gas chemical sterilization process do not interact with the enzymes, chemicals, and biological agents provided in tail 4524 and other sensor components such as membrane coatings that regulate the influx of test substances.

[0102] Once the desired sterility assurance level has been achieved within the sterilization chamber 4706, the gas solution may be removed and the sterilization chamber 4706 vented. Venting may be accomplished by a series of vacuums followed by circulation of a gas (e.g., nitrogen) or filtered air through the sterilization chamber 4706. Once the sterilization chamber 4706 has been properly vented, the vent 4708 may be closed by a seal 4712 (shown in dotted lines).

[0103] In some embodiments, the seal 4712 may comprise two or more layers of different materials. A first layer may be made of a synthetic material (e.g., flash-spun high density polyethylene fibers), such as Tyvek® available from DuPont®. Tyvek® is highly durable and puncture resistant, and allows vapor transmission. The Tyvek® layer may be applied prior to the gas-chemical sterilization process, and following the gas-chemical sterilization process, a foil or other vapor and moisture resistant material layer may be sealed (e.g., heat sealed) over the Tyvek® layer to prevent ingress of contaminants and moisture into the sterilization chamber 4706. In other embodiments, the seal 4712 may comprise only a single protective layer applied to the applicator cap 210. In such embodiments, that single layer may be gas permeable for the sterilization process, but may also provide protection against moisture and other harmful elements once the sterilization process is complete.

[0104] With the seal 4712 in place, the applicator cap 210 provides a barrier to outside contamination, thereby maintaining a sterile environment for the assembled sensor control device 4402 until the user removes (unscrews) the applicator cap 210. The applicator cap 210 may also create a dust-free environment that prevents the adhesive patch 4714 from becoming contaminated during shipping and storage.

[0105] 10A and 10B are isometric and side views, respectively, of another example of a sensor control device 5002 in accordance with one or more embodiments of the present disclosure. The sensor control device 5002 may be similar in some respects to the sensor control device 102 of FIG. 1A and thus may be best understood by reference thereto. Moreover, the sensor control device 5002 may replace the sensor control device 102 of FIG. 1A and thus may be used in conjunction with the sensor applicator 102 of FIG. 1A, which may deliver the sensor control device 5002 to a target monitoring location on the skin of a user.

[0106] However, unlike the sensor control device 102 of FIG. 1A, the sensor control device 5002 may comprise a one-piece system architecture that does not require a user to open multiple packages and ultimately assemble the sensor control device 5002 prior to application. Rather, when received by a user, the sensor control device 5002 may already be fully assembled and properly positioned within the sensor applicator 150 (FIG. 1A). To use the sensor control device 5002, a user need only open a single barrier (e.g., applicator cap 708 of FIG. 3B) before immediately delivering the sensor control device 5002 to a target monitoring location for use.

[0107] As shown, the sensor control unit 5002 includes an electronics housing 5004 that may be generally disk-shaped and have a circular cross-section. However, in other embodiments, the electronics housing 5004 may exhibit other cross-sectional shapes, such as oval or polygonal, without departing from the scope of the present disclosure. The electronics housing 5004 may be configured to house or otherwise contain various electrical components used to operate the sensor control unit 5002. In at least one embodiment, an adhesive patch (not shown) may be disposed on the bottom of the electronics housing 5004. The adhesive patch may be similar to the adhesive patch 105 of FIG. 1A and thus aid in adhering the sensor control unit 5002 to a user's skin for use.

[0108] As shown, the sensor control device 5002 includes an electronics housing 5004 that includes a shell 5006 and a mount 5008 that can mate with the shell 5006. The shell 5006 can be secured to the mount 5008 via a variety of methods, such as a snap-fit ​​engagement, an interference fit, sonic welding, one or more mechanical fasteners (e.g., screws), a gasket, an adhesive, or any combination thereof. In some examples, the shell 5006 can be secured to the mount 5008 such that a sealed interface is created therebetween.

[0109] The sensor control device 5002 may further include a sensor 5010 (partially visible) and a tip 5012 (partially visible) that are used to aid in transdermal delivery of the sensor 5010 beneath the skin of a user during application of the sensor control device 5002. As shown, corresponding portions of the sensor 5010 and tip 5012 extend distally from a bottom (e.g., mount 5008) of the electronics housing 5004. The tip 5012 may include a tip hub 5014 configured to secure and deliver the tip 5012. As best seen in FIG. 10B, the tip hub 5014 may include or otherwise define a mating member 5016. To couple the tip 5012 to the sensor control device 5002, the tip 5012 may be advanced axially through the electronics housing 5004 until the tip hub 5014 engages the top surface of the shell 5006 and the mating member 5016 extends distally from the bottom of the mount 5008. Once the tip 5012 penetrates the electronics housing 5004, an exposed portion of the sensor 5010 may be received within a hollow or recessed portion (arch) of the tip 5012. The remainder of the sensor 5010 is disposed within the interior of the electronics housing 5004.

[0110] The sensor control device 5002 may further include a sensor cap 5018, shown exploded or separated from the electronics housing 5004 in FIGS. 10A-10B. The sensor cap 5016 may be removably coupled to the sensor control device 5002 (e.g., electronics housing 5004) at or near the bottom of the mount 5008. The sensor cap 5018 may serve to provide a sealed barrier surrounding and protecting the exposed portions of the sensor 5010 and tip 5012 from gas chemical sterilization. As shown, the sensor cap 5018 may comprise a generally cylindrical body having a first end 5020a and a second end 5020b opposite the first end 5020a. The first end 5020a may be open to provide access to an interior chamber 5022 defined within the body. In contrast, the second end 5020b may be closed and may include or otherwise define an engagement feature 5024. As described herein, the engagement feature 5024 may aid in pairing the sensor cap 5018 with a cap (e.g., applicator cap 708 of FIG. 3B) of a sensor applicator (e.g., sensor applicator 150 of FIGS. 1 and 3A-3G) and may aid in removing the sensor cap 5018 from the sensor control device 5002 when the cap is removed from the sensor applicator.

[0111] The sensor cap 5018 may be removably coupled to the electronics housing 5004 at or near the bottom of the mount 5008. More specifically, the sensor cap 5018 may be removably coupled to a mating member 5016 that extends distally from the bottom of the mount 5008. In at least one embodiment, for example, the mating member 5016 may define a set of male threads 5026a (FIG. 10B) that are matable with a set of female threads 5026b (FIG. 10A) defined by the sensor cap 5018. In some embodiments, the male and female threads 5026a,b may comprise a flat thread design (e.g., no helical curvature), which may prove advantageous in molding the part. Alternatively, the male and female threads 5026a,b may comprise a helical thread engagement. Thus, the sensor cap 5018 may be threadably coupled to the sensor control device 5002 at the mating member 5016 of the tip hub 5014. In other embodiments, the sensor cap 5018 may be removably coupled to the mating member 5016 via an interference or friction fit, or other types of engagement including, but not limited to, frangible members or materials that can be broken with minimal separation force (e.g., axial or rotational force).

[0112] In some embodiments, the sensor cap 5018 may include a monolithic (single) structure extending between the first and second ends 5020a,b. However, in other embodiments, the sensor cap 5018 may include two or more components. In the illustrated embodiment, for example, the sensor cap 5018 may include a seal ring 5028 disposed at the first end 5020a and a desiccant cap 5030 disposed at the second end 5020b. The seal ring 5028 may be configured to help seal the interior chamber 5022, as described in more detail below. In at least one embodiment, the seal ring 5028 may comprise an elastomeric O-ring. The desiccant cap 5030 may contain or comprise a desiccant that helps maintain a preferred humidity level within the interior chamber 5022. The desiccant cap 5030 may also define or otherwise comprise an engagement feature 5024 of the sensor cap 5018.

[0113] 11A-11C are staged cross-sectional side views illustrating assembly of a sensor applicator 102 with a sensor control device 5002 in accordance with one or more embodiments. Once the sensor control device 5002 is fully assembled, it may be installed within the sensor applicator 102. With reference to FIG. 11A, the tip hub 5014 may include or otherwise define a hub snap pole 5302 configured to aid in coupling the sensor control device 5002 to the sensor applicator 102. More specifically, the sensor control device 5002 may be advanced into the sensor applicator 102, and the hub snap pole 5302 may be received by a corresponding arm 5304 of a tip carrier 5306 disposed within the sensor applicator 102.

[0114] 11B, the sensor control unit 5002 is shown received by the tip carrier 5306 and thus secured within the sensor applicator 102. Once the sensor control unit 5002 is installed within the sensor applicator 102, the applicator cap 210 may be coupled to the sensor applicator 102. In some embodiments, the applicator cap 210 and the housing 208 may have opposing matable sets of threads 5308 that allow the applicator cap 210 to be threaded into the housing 208 in a clockwise (or counterclockwise) direction, thereby securing the applicator cap 210 to the sensor applicator 102.

[0115] As shown, the sheath 212 is also disposed within the sensor applicator 102, which may include a sheath fixation mechanism 5310 configured to ensure that the sheath 212 does not collapse prematurely during an impact event. In the embodiment shown, the sheath fixation mechanism 5310 may comprise a threaded engagement between the applicator cap 210 and the sheath 212. More specifically, one or more internal threads 5312a may be defined or otherwise provided on an interior surface of the applicator cap 210, and one or more external threads 5312b may be defined or otherwise provided on the sheath 212. The internal or external threads 5312a,b may be configured such that the applicator cap 210 threadably engages with the sensor applicator 102 at the threads 5308. The female and male threads 5312 a,b may have the same thread pitch as the threads 5308 that allow the applicator cap 210 to be screwed onto the housing 208 .

[0116] 11C , applicator cap 210 is shown as being fully threaded (coupled) to housing 208. As shown, applicator cap 210 may further include or otherwise define a cap post 5314 centrally located within and extending proximally from a bottom of applicator cap 210. Cap post 5314 may be configured to receive at least a portion of sensor cap 5018 as applicator cap 210 threads onto housing 208.

[0117] Once the sensor control unit 5002 is installed in the sensor applicator 102 and the applicator cap 210 is properly secured, the sensor control unit 5002 may undergo a gas chemical sterilization configured to sterilize the electronics housing 5004 and any other exposed portions of the sensor control unit 5002. Because the sensor 5010 and the distal portion of the tip 5012 are sealed within the sensor cap 5018, the chemicals used during the gas chemical sterilization process cannot interact with other sensor components, such as the enzymes, chemicals, and biologics provided in the tail 5104, and the membrane coating that regulates the influx of the analyte.

[0118] 12A-12C are step-by-step cross-sectional side views illustrating assembly and disassembly of an alternative embodiment of a sensor applicator 102 with a sensor control unit 5002, according to one or more further embodiments. As generally described above, the fully assembled sensor control unit 5002 may be attached to the sensor applicator 102 by coupling the hub snap pole 5302 into the arm 5304 of a tip carrier 5306 disposed within the sensor applicator 102.

[0119] In the illustrated embodiment, the sheath arm 5604 of the sheath 212 may be configured to interact with a first detent 5702a and a second detent 5702b defined within the housing 208. The first detent 5702a may alternatively be referred to as a "locking" detent and the second detent 5702b may alternatively be referred to as a "firing" detent. When the sensor control device 5002 is initially attached to the sensor applicator 102, the sheath arm 5604 may be received within the first detent 5702a. As discussed below, the sheath 212 may be actuated to move the sheath arm 5604 to the second detent 5702b, thereby placing the sensor applicator 102 in a firing position.

[0120] 12B, applicator cap 210 is aligned with and advanced toward housing 208 such that sheath 212 is received within applicator cap 210. Instead of rotating applicator cap 210 relative to housing 208, threads of applicator cap 210 may be snapped onto corresponding threads of housing 208 to couple applicator cap 210 to housing 208. Axial cuts or slots 5703 (one shown) defined in applicator cap 210 may allow a portion of applicator cap 210 near its threads to flex outwardly and snap into engagement with threads of housing 208. Once applicator cap 210 is snapped onto housing 208, sensor cap 5018 may correspondingly snap onto cap post 5314.

[0121] 11A-11C , the sensor applicator 102 may include a sheath fixation mechanism configured to ensure that the sheath 212 does not collapse prematurely during an impact event. In the embodiment shown, the sheath fixation mechanism includes one or more ribs 5704 (one shown) defined near a base of the sheath 212 and configured to interact with one or more ribs 5706 (two shown), and a shoulder 5708 defined near a base of the applicator cap 210. The rib 5704 may be configured to interlock between the rib 5706 and the shoulder 5708 while the applicator cap 210 is attached to the housing 208. More specifically, when applicator cap 210 is snapped onto housing 208, applicator cap 210 is rotated (e.g., clockwise), thereby positioning rib 5704 of sheath 212 between rib 5706 and shoulder 5708 of applicator cap 210, such that applicator cap 210 is "locked" in place until a user counter-rotates applicator cap 210 to remove applicator cap 210 for use. Engagement of rib 5704 between rib 5706 and shoulder 5708 of applicator cap 210 may also prevent sheath 212 from prematurely folding.

[0122] In Figure 12C, the applicator cap 210 is removed from the housing 208. Similar to the embodiment of Figures 21A-21C, and generally as described above, the applicator cap 210 can be removed by counter-rotating the applicator cap 210, which correspondingly rotates the cap post 5314 in the same direction to unthread the sensor cap 5018 from the mating member 5016. Additionally, removing the sensor cap 5018 from the sensor control device 5002 exposes the sensor 5010 and a distal portion of the tip 5012.

[0123] When applicator cap 210 is unthreaded from housing 208, rib 5704 defined on sheath 212 may slidingly engage an upper portion of rib 5706 defined on applicator cap 210. The upper portion of rib 5706 may provide a corresponding ramp surface that effects upward movement of sheath 212 as applicator cap 210 rotates, which causes sheath arm 5604 to flex out of engagement with first detent 5702a to be received in second detent 5702b. As the sheath 212 moves to the second detent 5702b, the radial shoulder 5614 moves out of radial engagement with the carrier arms 5608, thereby allowing the passive spring force of the spring 5612 to urge the tip carrier 5306 upward, causing the carrier arms 5608 to move out of engagement with the grooves 5610. As the tip carrier 5306 moves upward within the housing 208, the mating member 5016 may correspondingly retract until it is flush, substantially flush, or near-flush with the bottom of the sensor control device 5002. At this point, the sensor applicator 102 is in the fired position. Thus, in this embodiment, removing the applicator cap 210 correspondingly retracts the mating member 5016.

[0124] I. Exemplary Firing Mechanisms for One-Piece and Two-Piece Applicators 13A-13F show detailed example embodiments of internal device mechanisms for "firing" the applicator 216 to apply the sensor control unit 222 to a user, and for safely retracting the tip 1030 into the used applicator 216. Together, these figures represent an example sequence for driving the tip 1030 (carrying a sensor coupled to the sensor control unit 222) into the user's skin, retracting the tip leaving the sensor in operative contact with the user's interstitial fluid, and adhesively adhering the sensor control unit to the user's skin. Modifications of such operations for use with alternative applicator assembly embodiments and components may be understood by those skilled in the art with reference to the same. Additionally, the applicator 216 may be a sensor applicator having a one-piece or two-piece architecture as disclosed herein.

[0125] 13A, the sensor 1102 is supported in the tip 1030, immediately above the user's skin 1104. Rails 1106 (optionally three of them) of the upper guide section 1108 may be provided to control movement of the applicator 216 relative to the sheath 318. The sheath 318 is held by a detent feature 1110 in the applicator 216, such that an appropriate downward force along the longitudinal axis of the applicator 216 may overcome the resistance provided by the detent mechanism 1110 to translate the tip 1030 and sensor control unit 222 along the longitudinal axis into (and onto) the user's skin 1104. Additionally, a catch arm 1112 of the sensor carrier 1022 engages the tip retraction assembly 1024 to maintain the tip 1030 in position relative to the sensor control unit 222.

[0126] In FIG. 13B, a user force is applied over or above the detent feature 1110, causing the sheath 318 to collapse within the housing 314, translating the sensor control device 222 (with associated components) downward along the longitudinal axis as shown by arrow L. The inner diameter of the upper guide section 1108 of the sheath 318 constrains the position of the carrier arm 1112 through the entire stroke of the sensor / tip insertion process. The retention of the stop surface 1114 of the carrier arm 1112 against the complementary surface 1116 of the tip retraction assembly 1024 maintains the position of the member along with a fully biased return spring 1118. According to an embodiment, rather than employing a user force to translate the sensor control device 222 downward along the longitudinal axis as shown by arrow L, the housing 314 may include a button (e.g., but not limited to, a push button) that activates a drive spring (e.g., but not limited to, a coil spring) to actuate the sensor control device 222.

[0127] In Figure 13C, the sensor 1102 and tip 1030 have reached their fully inserted depth, causing the carrier arm 1112 to move off the inner diameter of the upper guide section 1108. The compression force of the coil return spring 1118 then drives the angled stop surface 1114 radially outward, releasing the force and driving the tip carrier 1102 of the tip retraction assembly 1024 to pull the (slotted or otherwise configured) tip 1030 outward from the user and away from the sensor 1102, as shown by arrow R in Figure 13D.

[0128] With tip 1030 fully retracted, as shown in Figure 13E, upper guide section 1108 of sheath 318 is secured by final locking feature 1120. As shown in Figure 13F, the used applicator assembly 216 is removed from the insertion site leaving behind the sensor control device 222 and tip 1030 safely secured inside the applicator assembly 216. The used applicator assembly 216 is now ready for disposal.

[0129] The operation of the applicator 216 when applying the sensor control device 222 is designed to provide the user with the sensation that both insertion and retraction of the tip 1030 are performed automatically by the internal mechanisms of the applicator 216. In other words, the present invention avoids the user experiencing the sensation of manually driving the tip 1030 into the user's skin. That is, once the user applies sufficient force to overcome the resistance of the detent features of the applicator 216, the resulting movement of the applicator 216 is perceived as an automatic response to a "triggered" applicator. Even though all of the driving force is provided by the user and no additional biasing / driving means are used to insert the tip 1030, the user does not perceive that he is providing additional force to drive the tip 1030 to pierce the skin. As described above in FIG. 13C, retraction of the tip 1030 is automated by the coil return spring 1118 of the applicator 216.

[0130] With respect to any of the applicator embodiments described herein and any of their components, including but not limited to the tip, tip module, and sensor module embodiments, one of ordinary skill in the art will understand that the embodiments may be sized and configured for use with a sensor configured to detect an analyte level in a bodily fluid in the epidermis, dermis, or subcutaneous tissue of a subject. In some embodiments, for example, the tip and distal portion of the analyte sensor disclosed herein may both be sized and configured to be located at a particular distal depth (i.e., the furthest point of penetration in a tissue or layer of the subject's body, e.g., the epidermis, dermis, or subcutaneous tissue). With respect to some applicator embodiments, one of ordinary skill in the art will recognize that certain embodiments of the tip may be sized and configured to be located at a different distal depth in the subject's body relative to the final distal depth of the analyte sensor. In some embodiments, for example, the tip may be located at a first distal depth in the subject's epidermis prior to retraction, while the distal portion of the analyte sensor may be located at a second distal depth in the subject's dermis. In other embodiments, the tip may be located at a first distal depth in the subject's dermis prior to retraction while a distal portion of the analyte sensor may be located at a second distal depth in the subject's subcutaneous tissue. In yet other embodiments, the tip may be located at a first distal depth and the analyte sensor at a second distal depth prior to retraction, where both the first distal depth and the second distal depth are within the same layer or tissue of the subject's body.

[0131] Additionally, with respect to any of the applicator embodiments described herein, one of ordinary skill in the art will appreciate that one or more structural components coupled thereto, including but not limited to the analyte sensor and one or more spring mechanisms, may be positioned within the applicator in an eccentric location relative to one or more axes of the applicator. In some applicator embodiments, for example, the analyte sensor and spring mechanism may be positioned in a first eccentric location relative to the applicator axis on a first side of the applicator, and the sensor electronics may be positioned in a second eccentric location relative to the applicator axis on a second side of the applicator. In other applicator embodiments, the analyte sensor, spring mechanism, and sensor electronics may be positioned in an eccentric location relative to the applicator axis on the same side. One of ordinary skill in the art will appreciate that other permutations and configurations in which any or all of the analyte sensor, spring mechanism, sensor electronics, and other applicator components are positioned in a central or eccentric location relative to one or more axes of the applicator are possible and fully within the scope of the present disclosure.

[0132] Further details of suitable devices, systems, methods, components, and their operation, along with associated features, are described in Rao et al., International Publication No. WO 2018 / 136898, Thomas et al., International Publication No. WO 2019 / 236850, Thomas et al., International Publication No. WO 2019 / 236859, Thomas et al., International Publication No. WO 2019 / 236876, and U.S. Patent Publication No. 2020 / 0196919, filed June 6, 2019, each of which is incorporated herein by reference in its entirety. Further details regarding embodiments of the applicators, their components, and variations thereof are described in U.S. Patent Publication Nos. 2013 / 0150691, 2016 / 0331283, and 2018 / 0235520, all of which are incorporated herein by reference in their entirety for all purposes. Further details regarding embodiments of the tip module, tips, their components, and variations thereof are described in U.S. Patent Publication No. 2014 / 0171771, which is incorporated by reference in its entirety and for all purposes.

[0133] J. Exemplary Methods for Calibrating Analyte Sensors A biochemical sensor can be described by one or more sensing characteristics. A common sensing characteristic is referred to as the sensitivity of a biochemical sensor, which is a measure of the sensor's responsiveness to the concentration of a chemical or composition it is designed to detect. For electrochemical sensors, this response may be in the form of a current (amperometric) or charge (coulometric). For other types of sensors, the response may be in a different form, such as photon intensity (e.g., optical light). The sensitivity of a biochemical analyte sensor can vary depending on several factors, such as whether the sensor is in an in vitro or in vivo condition.

[0134] FIG. 14 is a graph showing the in vitro sensitivity of an amperometric analyte sensor. The in vitro sensitivity can be obtained by testing the sensor in vitro at various analyte concentrations and performing regression (e.g., linear or nonlinear) or other curve fitting on the resulting data. In this example, the sensitivity of the analyte sensor is linear or substantially linear and can be modeled with the equation y=mx+b, where y is the electrical output current of the sensor, x is the analyte level (or concentration), m is the slope of the sensitivity, and b is the intercept of the sensitivity, where the intercept generally corresponds to the background signal (e.g., noise). For sensors with linear or substantially linear responses, the analyte level corresponding to a given current can be determined from the slope and intercept of the sensitivity. Sensors with nonlinear sensitivity require additional information to determine the analyte level resulting from the sensor's output current, and one of ordinary skill in the art is familiar with how to model nonlinear sensitivity. In certain embodiments of an in vivo sensor, the in vitro sensitivity may be the same as the in vivo sensitivity, while in other embodiments a transfer (or transformation) function is used to translate the in vitro sensitivity into an in vivo sensitivity that is applicable to the in vivo application for which the sensor is intended.

[0135] Calibration is a technique for improving or maintaining accuracy by adjusting the measured output of a sensor to reduce the difference from the expected output of the sensor. One or more parameters describing the sensing characteristics of the sensor, such as its sensitivity, are established for use in the calibration adjustment.

[0136] Some in vivo analyte monitoring systems require calibration after the sensor is implanted in a user or patient, either through user intervention or by the system itself in an automated fashion. For example, when user intervention is required, the user performs an in vitro measurement (e.g., a blood glucose (BG) measurement using a finger prick and an in vitro test strip) while the analyte sensor is implanted and inputs it into the system. The system then compares the in vitro measurement to the in vivo signal and uses the difference to determine an estimate of the in vivo sensitivity of the sensor. The in vivo sensitivity can then be used in an algorithmic process that converts the data collected by the sensor into a value indicative of the user's analyte level. This and other processes that require user action to perform the calibration are referred to as "user calibration." Systems may require user calibration due to instability in the sensitivity of sensors, where sensitivity drifts or changes over time. Thus, multiple user calibrations (e.g., on a regular (e.g., daily) schedule, a variable schedule, or on an as-needed schedule) may be required to maintain accuracy. Although the embodiments described herein may incorporate some degree of user calibration for a particular implementation, this is generally not preferred as it may require the user to perform painful or otherwise cumbersome BG measurements and may be subject to user error.

[0137] Some in vivo analyte monitoring systems can periodically adjust calibration parameters by using automated measurements of sensor characteristics made by the system itself (e.g., processing circuitry running software). Repeated adjustment of sensor sensitivity based on variables measured by the system (not the user) is generally denoted "system" (or automatic) calibration, and can be performed with or without user calibration, such as an early BG measurement. As with repeated user calibration, repeated system calibration is generally necessitated by drift in sensor sensitivity over time. Thus, although the embodiments described herein can be used with some degree of automated system calibration, preferably the sensor sensitivity is relatively stable over time and thus post-implant calibration is not required.

[0138] Some in vivo analyte monitoring systems operate with factory calibrated sensors. Factory calibration refers to the determination or estimation of one or more calibration parameters prior to distribution to users or healthcare professionals (HCPs). The calibration parameters can be determined by the manufacturer of the sensor (or the manufacturer of other parts of the sensor control device if the two parts are different). Many in vivo sensor manufacturing processes assemble sensors in groups or batches called production lots, production stage lots, or simply lots. A lot can contain thousands of sensors.

[0139] The sensor may include calibration codes or parameters that are derived or determined during one or more sensor manufacturing processes, coded or programmed into the data processing device of the analyte monitoring system as part of the manufacturing process, or provided on the sensor itself, for example as a bar code, laser tag, RFID tag, or other machine-readable information carried on the sensor. When the code is provided to a receiver (or other data processing device), user calibration during in vivo use of the sensor may be unnecessary or the frequency of in vivo calibration while the sensor is worn may be reduced. In embodiments where the calibration codes or parameters are provided on the sensor itself prior to or at the start of use of the sensor, the calibration codes or parameters may be automatically transmitted or provided to the data processing device of the analyte monitoring system.

[0140] Some in vivo analyte monitoring systems operate with sensors that may be one or more of factory calibrated, system calibrated, and / or user calibrated. For example, a sensor may be provided with a calibration code or parameters that may enable factory calibration. If the information is provided to the receiver (e.g., entered by a user), the sensor may operate as a factory calibrated sensor. If the information is not provided to the receiver, the sensor may operate as a user calibrated sensor and / or a system calibrated sensor.

[0141] In further aspects, program or executable instructions can be provided to or stored in the data processor and / or receiver / controller unit of the analyte monitoring system for providing a time-varying adjustment algorithm to the in vivo sensor during use. For example, a time-based predetermined or analytical curve or database can be generated based on retrospective statistical analysis of analyte sensors used in vivo and corresponding glucose level feedback, and can be configured to provide further adjustments to one or more in vivo sensor parameters or other factors to compensate for potential sensor drift in the stability profile.

[0142] In accordance with the disclosed subject matter, an analyte monitoring system may be configured to compensate or adjust sensor sensitivity based on a sensor drift profile. A time-varying parameter β(t) may be defined or determined based on an analysis of sensor behavior during in vivo use, and a time-varying drift profile may be determined. In certain aspects, the compensation or adjustment to the sensor sensitivity may be programmed in a receiver unit, controller, or data processor of the analyte monitoring system, such that the compensation or adjustment, or both, may be performed automatically and / or iteratively as sensor data is received from the analyte sensor. In accordance with the disclosed subject matter, the adjustment or compensation algorithm may be user-initiated or executed (rather than self-initiated or executed), such that the adjustment or compensation to the analyte sensor sensitivity profile is implemented or executed upon user initiation or activation of a corresponding function or routine, or when the user enters a sensor calibration code.

[0143] According to the disclosed subject matter, each sensor in a sensor lot (in some embodiments not including the sample sensor used for in vitro testing) can be non-destructively inspected to determine or measure its characteristics, such as the thickness of the film at one or more points on the sensor, and other characteristics can be measured or determined, including physical characteristics such as the surface area / volume of the active area. Such measurements or determinations can be performed in an automated manner, for example using an optical scanner or other suitable measurement device or system, and the determined sensor characteristics for each sensor in the sensor lot are compared to corresponding average values ​​based on the sample sensors for possible correction of the calibration parameters or codes assigned to each sensor. For example, for a calibration parameter defined as sensor sensitivity, the sensitivity is approximately inversely proportional to the film thickness, so that for example, for a sensor having a measured film thickness approximately 4% thicker than the average film thickness of sensors sampled from the same sensor lot as a given sensor, the sensitivity assigned to the sensor in one embodiment is the average sensitivity determined from the sampled sensors divided by 1.04. Similarly, sensitivity is approximately proportional to the active area of ​​a sensor, so that for a sensor with a measured active area approximately 3% smaller than the average active area of ​​sensors sampled from the same sensor lot, the assigned sensitivity for that sensor is the average sensitivity multiplied by 0.97. The assigned sensitivity may be determined from the average sensitivity from the sampled sensors by multiple successive adjustments for each inspection or measurement of the sensor. In certain embodiments, the inspection or measurement of each sensor may further include measurements of the uniformity or structure of the film in addition to the thickness of the film and / or the surface area or volume of the active sensing area.

[0144] Further information regarding sensor calibration is provided in U.S. Publication No. 2010 / 00230285 and U.S. Publication No. 2019 / 0274598, each of which is incorporated by reference in its entirety.

[0145] K. Exemplary Bluetooth Communication Protocol The storage memory 5030 of the sensor 110 may include software blocks related to the communication protocol of the communication module. For example, the storage memory 5030 may include a BLE service software block whose functions provide an interface making the BLE module 5041 available to the computing hardware of the sensor 110. These software functions may include a BLE logical interface and an interface parser. The BLE services provided by the communication module 5040 may include a generic access profile service, a generic attribute service, a generic access service, a device information service, a data transmission service, and a security service. The data transmission service may be a primary service used to transmit data such as sensor control data, sensor status data, analyte measurement data (past and present), and event log data. The sensor status data may include error data, current active time, and software state. The analyte measurement data may include information such as current and past raw measurements, current and past values ​​after processing using appropriate algorithms or models, predictions and trends of measurement levels, comparison of other values ​​to patient-specific averages, calls to action determined by algorithms or models, and other similar types of data.

[0146] In accordance with aspects of the disclosed subject matter, as embodied herein, the sensor 110 may be configured to communicate with multiple devices simultaneously by adapting the characteristics of the communication protocol or medium supported by the hardware and radio of the sensor 110. As an example, the BLE module 5041 of the communication module 5040 may comprise software or firmware to enable multiple simultaneous connections between the sensor 110 as a central device and other devices as peripheral devices, or the sensor 110 as a peripheral device when another device is the central device.

[0147] A connection and subsequent communication session between two devices using a communication protocol such as BLE may be characterized by a similar physical channel operating between the two devices (e.g., the sensor 110 and the data receiving device 120). The physical channel may include a single channel or a set of channels, including, for example and without limitation, using a common clock and channel or an agreed-upon set of channels determined by a frequency hopping sequence. The communication sessions may use a similar amount of the available communication spectrum, and multiple such communication sessions may exist in close proximity. In certain embodiments, each set of devices in a communication session uses a different physical channel or set of channels to manage interference with devices that are also in close proximity.

[0148] For purposes of illustration and not limitation, reference is made to an exemplary embodiment of a process for connection between a sensor and a receiver for use with the disclosed subject matter. First, the sensor 110 repeatedly advertises its connection information to its surroundings in search of the data receiving device 120. The sensor 110 may periodically repeat the advertisement until a connection is established. The data receiving device 120 detects the advertising packet and scans and filters for the sensor 120 to connect via the data provided in the advertising packet. Next, the data receiving device 120 sends a scan request command, and the sensor 110 responds with a scan response packet providing additional details. Next, the data receiving device 120 sends a connection request using the Bluetooth device address associated with the data receiving device 120. The data receiving device 120 may also continuously request to establish a connection to the sensor 110 with a particular Bluetooth device address. The device then establishes the initial connection to allow data exchange to begin. The device begins the process of initializing the data exchange service and performs a mutual authentication process.

[0149] During the first connection between the sensor 110 and the data sink 120, the data sink 120 may initialize a service, characteristic, and attribute discovery process. The data sink 120 may evaluate these characteristics of the sensor 110 and store them for use during the next connection. The device then enables notification of customized security services to be used for mutual authentication of the sensor 110 and the data sink 120. The mutual authentication process may be automated and does not require user intervention. After the mutual authentication process is successfully completed, the sensor 110 sends a connection parameter update requesting the data sink 120 to use connection parameter settings that are suitable for the sensor 110 and configured to maximize longevity.

[0150] The data sink 120 then executes sensor control processing to backfill historical data, current data, event logs, and factory data. As an example, for each type of data, the data sink 120 sends a request to initiate the backfill process. The request may specify a range of records defined, for example, based on measurements, timestamps, etc., as needed. The sensor 110 responds with the requested data until all previously unsent data in the memory of the sensor 110 is delivered to the data sink 120. The sensor 110 may respond to a backfill request from the data sink 120 that all data has already been sent. Once the backfill is complete, the data sink 120 may notify the sensor 110 that it is ready to receive periodic measurements. The sensor 110 may send measurements over multiple notifications results on a repeating basis. As embodied herein, the multiple notifications may be redundant notifications to ensure that the data is sent correctly. Alternatively, the multiple notifications may constitute a single payload.

[0151] For purposes of illustration and not limitation, reference is made to an exemplary embodiment of a process for sending a shutdown command to the sensor 110. The shutdown operation is performed, for example, when the sensor 110 is in an error state, an insertion failure state, or a sensor expired state. If the sensor 110 is not in those states, the sensor 110 may record the command and perform the shutdown when the sensor 110 transitions to the error state or the sensor expired state. The data sink 120 sends a properly formatted shutdown command to the sensor 110. If the sensor 110 is actively processing another command, the sensor 110 responds with a standard error response indicating that the sensor 110 is busy. Otherwise, the sensor 110 sends a response upon receiving the command. Additionally, the sensor 110 sends a success notification via the sensor control property to inform the sensor 110 that it has received the command. The sensor 110 registers the shutdown command. At the next appropriate opportunity (e.g., depending on the current sensor state, as described herein), the sensor 110 shuts down.

[0152] L. Exemplary Sensor States and Actuation For purposes of illustration and not limitation, reference is made to an exemplary embodiment of a high level depiction of a state machine representation 6000 of actions that may be taken by the sensor 110 shown in FIG. 15. After initialization, the sensor enters a state 6005 associated with manufacturing the sensor 110. In the manufacturing state 6005, the sensor 110 may be configured for operation, e.g., the storage memory 5030 may be written to. At various times while in state 6005, the sensor 110 checks for a received command to proceed to a save state 6015. Upon entering the save state 6015, the sensor performs a software integrity check. While in the save state 6015, the sensor may also receive an actuation request command before proceeding to the insertion detection state 6025.

[0153] Upon entering state 6025, the sensor 110 may store information about devices authorized to communicate with the sensor set during operation or may initialize algorithms related to performing and interpreting measurements from the sensing hardware 5060. The sensor 110 may also initialize a life cycle timer responsible for maintaining an active count of the sensor's 110 operation time and may begin communication with authorized devices to transmit recorded data. While in the insertion detection state 6025, the sensor may enter state 6030, where the sensor 110 checks whether the operation time is equal to a predefined threshold. This operation time threshold may correspond to a timeout function to determine whether the insertion is successful. If the operation time reaches the threshold, the sensor 110 proceeds to state 6035, where the sensor 110 checks whether the average data read volume is greater than a threshold corresponding to the expected data read volume to trigger the detection of a successful insertion. If the data read volume is lower than the threshold while in state 6035, the sensor proceeds to state 6040, corresponding to an insertion failure. If the data read volume meets the threshold, the sensor proceeds to active pair state 6055.

[0154] The active pair state 6055 of the sensor 110 indicates a state during which the sensor 110 is operating normally by recording measurements, processing the measurements, and reporting them as necessary. While in the active pair state 6055, the sensor 110 attempts to transmit measurements or establish a connection with the receiving device 120. The sensor 110 also increases the operating time. When the sensor 110 reaches a predefined operating time threshold (e.g., the operating time reaches a predefined threshold), the sensor 110 transitions to the active expired state 6065. The active expired state 6065 of the sensor 110 indicates a state during which the sensor 110 is operating for its maximum predefined time.

[0155] While in the active expired state 6065, the sensor 110 may perform operations generally related to the termination of operations and ensuring that collected measurements have been securely transmitted to a receiving device as necessary. For example, while in the active expired state 6065, the sensor 110 may transmit collected data and, if a connection is not feasible, may increase attempts to discover and establish a connection with a nearby authenticated device. While in the active expired state 6065, the sensor 110 may receive a shutdown command in state 6070. If a shutdown command is not received, the sensor 110 may also check in state 6075 whether the operation time has exceeded a final operation threshold. The final operation threshold may be based on the battery life of the sensor 110. The normal termination state 6080 corresponds to a final operation of the sensor 110, ultimately shutting down the sensor 110.

[0156] Before the sensor is activated, the ASIC 5000 is in a low power storage mode state. For example, the activation process may begin when an incident RF field (e.g., an NFC field) drives the voltage of the power supply to the ASIC 5000 above a reset threshold, which causes the sensor 110 to proceed to a wake-up state. While in the wake-up state, the ASIC 5000 enters an activation sequence state. The ASIC 5000 then starts the communication module 5040. The communication module 5040 is initialized and triggers a power-on self-test. The power-on self-test may include the ASIC 5000 communicating with the communication module 5040 using a predetermined sequence of reading and writing data to verify that the memory and one-time programmable memory are not corrupted.

[0157] When the ASIC 5000 enters the measurement mode for the first time, an insertion detection sequence is performed to verify that the sensor 110 is properly placed on the patient's body before a proper measurement can be made. First, the sensor 110 interprets a command to activate a measurement setup process, causing the ASIC 5000 to enter a measurement command mode. The sensor 110 then enters a measurement lifecycle state temporarily, performing several consecutive measurements to check if the insertion was successful. The communication module 5040 or the ASIC 5000 evaluates the measurement results to determine successful insertion. Once the insertion is deemed successful, the sensor 110 enters a measurement state, where the sensor 110 begins to take periodic measurements using the sensing hardware 5060. If the sensor 110 determines that the insertion was not successful, the sensor 110 is triggered into an insertion failure mode, and the ASIC 5000 is commanded to return to a storage mode while the communication module 5040 disables itself.

[0158] M. Exemplary Over-the-Air Updates 1B further illustrates an example of an operating environment for providing over-the-air ("OTA") updates for use with the techniques described herein. An operator of the analyte monitoring system 100 may bundle updates for the data sink 120 or sensor 110 into updates for applications running on the multipurpose data sink 130. Using available communication channels between the data sink 120, the multipurpose data sink 130, and the sensor 110, the multipurpose data sink 130 may receive periodic updates for the data sink 120 or sensor 110 and initiate installation of the updates to the data sink 120 or sensor 110. The multipurpose data sink 130 serves as an installation or update platform for the data sink 120 or sensor 110, since applications that enable the multipurpose data sink 130 to communicate with the analyte sensor 110, the data sink 120, and / or the remote application server 150 can update software or firmware on the data sink 120 or sensor 110 without wide area network capabilities.

[0159] As embodied herein, a remote application server 150 operated by the manufacturer of the analyte sensor 110 and / or the operator of the analyte monitoring system 100 may provide software and firmware updates to the devices of the analyte monitoring system 100. In certain embodiments, the remote application server 150 may provide updated software and firmware to the user device 140 or directly to the general-purpose data receiving device. As embodied herein, the remote application server 150 may also provide application software updates to the application storefront server 160 using an interface provided by the application storefront. The general-purpose data receiving device 130 may periodically contact the application storefront server 160 to download and install updates.

[0160] After the multipurpose data receiving device 130 downloads the application update including the firmware or software update for the data receiving device 120 or the sensor 110, the data receiving device 120 or the sensor 110 and the multipurpose data receiving device 130 establish a connection. The multipurpose data receiving device 130 determines that a firmware or software update is available for the data receiving device 120 or the sensor 110. The multipurpose data receiving device 130 may prepare the software or firmware update for delivery to the data receiving device 120 or the sensor 110. As an example, the multipurpose data receiving device 130 may compress or split data associated with the software or firmware update, may encrypt or decrypt the firmware or software update, or may perform an integrity check of the firmware or software update. The multipurpose data receiving device 130 transmits data for the firmware or software update to the data receiving device 120 or the sensor 110. The multipurpose data receiving device 130 may also transmit a command to the data receiving device 120 or the sensor 110 to initiate the update. Additionally or alternatively, the multipurpose data receiving device 130 may provide a notification to a user of the multipurpose data receiving device 130 and may include instructions to facilitate the update, such as instructions to keep the data receiving device 120 and the multipurpose data receiving device 130 connected to a power source and in close proximity until the update is complete.

[0161] The data receiving device 120 or the sensor 110 receives data for the update and a command to start the update from the multipurpose data receiving device 130. The data receiving device 120 may then install the firmware or software update. To install the update, the data receiving device 120 or the sensor 110 may put itself into a so-called "safe" mode with limited operating capabilities, or may reboot. Once the update is complete, the data receiving device 120 or the sensor 110 may re-enter a standard operating mode or be reset. The data receiving device 120 or the sensor 110 may perform one or more self-tests to determine that the firmware or software update was successfully installed. The multipurpose data receiving device 130 may receive a notification of the successful update. The multipurpose data receiving device 130 may then report confirmation of the successful update to the remote application server 150.

[0162] In a particular embodiment, the storage memory 5030 of the sensor 110 includes a one-time programmable (OTP) memory. The term OTP memory may refer to a memory that includes access restrictions and security to facilitate a predetermined number of writes to a particular address or segment in the memory. The memory 5030 may be pre-populated into a number of pre-allocated memory blocks or containers. The containers are pre-allocated to a fixed size. If the storage memory 5030 is a one-time programmable memory, the containers may be considered to be in a non-programmable state. Additional containers that have not yet been written to may be in a programmable or writable state. Containerizing the storage memory 5030 in this manner may improve the transportability of code and data to be written to the storage memory 5030. Updating the software of a device (e.g., a sensor device described herein) stored in an OTP memory may be performed by replacing only the code in one or more specific previously written containers with updated code written to a new container or containers, rather than replacing the entire code in the memory. In a second embodiment, the memory is not pre-populated. Instead, the space allocated for data is dynamically allocated or determined as needed. Containers of various sizes can be defined in which updates are expected, allowing incremental updates to be issued.

[0163] 16 is a schematic diagram illustrating example operations and data flows for over-the-air (OTA) programming of a storage memory 5030 in a sensor device 100 and use of the memory after OTA programming in execution of a process by the sensor device 110 in accordance with the disclosed subject matter. In the example of OTA programming 500 shown in FIG. 5, a request is sent from an external device (e.g., data receiving device 130) to initiate OTA programming (or reprogramming). At 511, a communication module 5040 of the sensor device 110 receives the OTA programming command. The communication module 5040 transmits the OTA programming command to the microcontroller 5010 of the sensor device 110.

[0164] At 531, after receiving the OTA programming command, the microcontroller 5010 verifies the OTA programming command. The microcontroller 5010 may, for example, determine whether the OTA programming command is signed with a proper digital signature token. Upon determining that the OTA programming command is valid, the microcontroller 5010 may set the sensor device to an OTA programming mode. At 532, the microcontroller 5010 may verify the OTA programming data. At 533, the microcontroller 5010 may reset the sensor device 110 to reinitialize the sensor device 110 to a programming state. Once the sensor device 110 transitions to the OTA programming state, the microcontroller 5010 may begin writing data to the rewritable memory 540 (e.g., memory 5020) of the sensor device at 534 and to the OTP memory 550 (e.g., storage memory 5030) of the sensor device at 535. The data written by the microcontroller 5010 may be based on the verified OTA programming data. The microcontroller 5010 may write data to mark one or more programming blocks or regions of the OTP memory 550 as invalid or inaccessible. The data written to the free or unused portions of the OTP memory may be used to replace the invalidated or inaccessible programming blocks of the OTP memory 550. After the microcontroller 5010 has written the data to the respective memories at 534 and 535, the microcontroller 5010 may perform one or more software integrity checks to ensure that no errors were introduced into the programming blocks during the writing process. Once the microcontroller 5010 is able to determine that the data was written without error, the microcontroller 5010 may resume normal operation of the sensor device.

[0165] At 536, in the execution mode, the microcontroller 5010 can retrieve a programming manifest or profile from the rewritable memory 540. The programming manifest or profile can include a list of valid software programming blocks and can include a guide to program execution for the sensor 110. By following the programming manifest or profile, the microcontroller 5010 can determine which memory blocks of the OTP memory 550 are appropriate to execute and can avoid executing out-of-date or invalidated programming blocks or referencing out-of-date data. At 537, the microcontroller 5010 can selectively retrieve memory blocks from the OTP memory 550. At 538, the microcontroller 5010 can use the retrieved memory blocks by executing programming code stored in the memory or by using variables stored in the memory.

[0166] N. Exemplary Security and Other Architectural Features As embodied herein, a first layer of security for communications between analyte sensor 110 and other devices may be established based on a security protocol dictated by and integrated into the communications protocol used for communication. Another layer of security may be based on communications protocols requiring proximity of the communicating devices. Additionally, certain packets and / or certain data contained within packets may be encrypted, while other packets and / or data within packets are otherwise encrypted or unencrypted. Additionally or alternatively, application layer encryption may be used in conjunction with one or more block or stream ciphers to establish mutual authentication and communications encryption with other devices in analyte monitoring system 100.

[0167] The ASIC 5000 of the analyte sensor 110 may be configured to dynamically generate authentication and encryption keys using data held in the storage memory 5030. The storage memory 5030 may also be pre-programmed with a set of valid authentication and encryption keys for use with a particular class of device. The ASIC 5000 may be further configured to perform an authentication process with the other device using the received data and apply a generated key to the sensitive data before transmitting the sensitive data. The generated key may be specific to the analyte sensor 110, specific to a pair of devices, specific to a communication session between the analyte sensor 110 and the other device, specific to a message sent during the communication session, or specific to a block of data contained within a message.

[0168] Both the sensor 110 and the data receiving device 120 may ensure the authentication of the other party in a communication session, for example to issue commands or receive data. In certain embodiments, identity authentication may be performed through two mechanisms. First, the party asserting its identity provides a valid certificate signed by the device manufacturer or the operator of the analyte monitoring system 100. Second, authentication may be performed through the use of public and private keys established by the device of the analyte monitoring system 100 or by the operator of the analyte monitoring system 100, and a shared secret key derived therefrom. To verify the identity of the other party, a party may provide proof that the party has control of the private key.

[0169] The manufacturer of the analyte sensor 110, data receiving device 120, or a provider of an application for the general-purpose data receiving device 130 may provide the information and programming necessary for the devices to communicate securely through secure programming and updates. For example, the manufacturer may provide information that can be used to generate encryption keys for each device, including a secure root key for the analyte sensor 110 and optionally the data receiving device 120, which may be used in combination with device specific information and operational data (e.g., entropy-based random values) to generate encryption values ​​specific to the device, session, or data transmission, as needed.

[0170] The analyte data associated with the user is at least partially sensitive data, since this information may be used for a variety of purposes, including health monitoring and medication administration decisions. In addition to the user data, the analyte monitoring system 100 may implement security enhancements against reverse engineering by external parties. The communication connection may be encrypted using a device-specific or session-specific encryption key. Encrypted or unencrypted communication between any two devices may be verified using a transmission integrity check built into the communication. The operation of the analyte sensor 110 may be protected from tampering by restricting access to read and write functions to the memory 5020 via the communication interface. The sensor may be configured to allow access only to known or "trusted" devices provided in a "white list" or to devices that can provide a predefined code associated with the manufacturer or an otherwise authenticated user. The white list may represent an exclusive scope, meaning that no connection identifiers other than those included in the white list are used, or a preferred scope where the white list is searched first but still other devices may be used. The sensor 110 may further reject and shut down a connection request if the requester fails to complete the login process over the communication interface within a predetermined time (e.g., within 4 seconds). These properties protect against certain denial of service attacks, particularly those against BLE interfaces.

[0171] As embodied herein, analyte monitoring system 100 may use periodic key rotation to further reduce the likelihood of key leakage and misuse. The key rotation strategy employed by analyte monitoring system 100 may be designed to support backward compatibility of field-deployed or distributed devices. As an example, analyte monitoring system 100 may employ keys for downstream devices (e.g., devices that are in the field or cannot feasibly provide updates) that are designed to be compatible with multiple generations of keys used by upstream devices.

[0172] For purposes of illustration and not limitation, reference is made to an exemplary embodiment of a message sequence diagram 600 for use with the disclosed subject matter shown in FIG. 17, illustrating an example of data exchange between a pair of devices, specifically a sensor 110 and a data receiving device 120. The data receiving device 120 may be a data receiving device 120 or a general-purpose data receiving device 130 as embodied herein. In step 605, the data receiving device 120 may transmit a sensor activation command 605 to the sensor 110, for example via a short-range communication protocol. The sensor 110 may be primarily dormant prior to step 605, conserving its battery until full activation is required. After activation, during step 610, the sensor 110 may collect data or perform other operations as appropriate for the sensing hardware 5060 of the sensor 110. In step 615, the data receiving device 120 may initiate an authentication request command 615. In response to the authentication request command 615, both the sensor 110 and the data sink 120 may engage in a mutual authentication process 620. The mutual authentication process 620 may involve the transfer of data, including challenge parameters that allow the sensor 110 and the data sink 120 to ensure that the other device is capable of fully complying with the agreed upon security framework described herein. Mutual authentication may be based on mechanisms for authenticating two or more entities to each other, with or without an online trusted third party, to verify the establishment of a secret key via a challenge response. Mutual authentication may be performed using two-pass, three-pass, four-pass or five-pass authentication, or similar versions thereof.

[0173] Following a successful mutual authentication process 620, in step 625, the sensor 110 may provide the data receiving device 120 with a sensor secret 625. The sensor secret may include a sensor-specific value and may be derived from a random value generated during manufacturing. The sensor secret may be encrypted before or during transmission to prevent a third party from accessing the secret. The sensor secret 625 may be encrypted via one or more of the keys generated by or in response to the mutual authentication process 620. In step 630, the data receiving device 120 may derive a sensor-specific encryption key from the sensor secret. The sensor-specific encryption key may further be session-specific. Thus, the sensor-specific encryption key may be determined by each device without being transmitted between the sensor 110 or the data receiving device 120. In step 635, the sensor 110 may encrypt the data contained in the payload. In step 640, using the communication link established between the appropriate communication model of the sensor 110 and the data receiving device 120, the sensor 110 may transmit the encrypted payload 640 to the data receiving device 120. In step 645, the data sink 120 may decrypt the payload using the sensor-specific encryption key derived during step 630. Following step 645, the sensor 110 may deliver additional (including newly collected) data, and the data sink 120 may process the received data appropriately.

[0174] As described herein, the sensor 110 may be a device with limited processing power, battery supply, and storage. The encryption techniques (e.g., selection of cryptographic algorithms or implementations of algorithms) used by the sensor 110 may be selected based at least in part on these limitations. The data receiving device 120 may be a more powerful device with fewer limitations of this nature. Thus, the data receiving device 120 may employ more sophisticated and computationally intensive encryption techniques, such as cryptographic algorithms and implementations.

[0175] O. Exemplary Payloads / Communication Frequencies The analyte sensor 110 may be configured to modify its discoverability behavior to attempt to increase the probability that a receiving device will receive a proper data packet and / or provide a response signal, or to otherwise reduce limitations that may prevent a response signal from being received. Modifying the discoverability behavior of the analyte sensor 110 may include, for example, without limitation, modifying the frequency at which connection data is included in a data packet, modifying the frequency at which data packets are generally transmitted, lengthening or shortening the broadcast window of a data packet, modifying the time at which the analyte sensor 110 will accept a response or scan signal after broadcasting, including direct transmissions to one or more devices that previously communicated with the analyte sensor 110 (e.g., via one or more attempted transmissions) and / or one or more devices on a whitelist, modifying the transmit power associated with the communications module when broadcasting a data packet (e.g., to increase the range of a broadcast or reduce energy consumed and extend the battery life of the analyte sensor), modifying the rate at which data packets are prepared and broadcast, or a combination of one or more other modifications. Additionally or alternatively, the receiving device may similarly adjust parameters related to the device's listening behavior in order to increase the likelihood of receiving data packets containing the connection data.

[0176] As embodied herein, the analyte sensor 110 may be configured to broadcast data packets using two types of windows. The first window indicates the rate at which the analyte sensor 110 is configured to operate its communications hardware. The second window indicates the rate at which the analyte sensor 110 is configured to actively transmit (e.g., broadcast) data packets. As an example, the first window may indicate that the analyte sensor 110 operates its communications hardware to transmit and / or receive data packets (including connection data) during the first 2 seconds of each 60 second period. The second window may indicate that during each 2 second window, the analyte sensor 110 transmits a data packet every 60 milliseconds. The remaining time during the 2 second window, the analyte sensor 110 is scanning. The analyte sensor 110 may lengthen or shorten either window to change the discoverability behavior of the analyte sensor 110.

[0177] In certain embodiments, the discoverability behavior of the analyte sensor may be stored in a discoverability profile and may be altered based on one or more factors, such as the state of the analyte sensor 110, and / or by applying rules based on the state of the analyte sensor 110. For example, when the battery level of the analyte sensor 110 falls below a certain amount, a rule may cause the analyte sensor 110 to reduce the power consumed by the broadcast process. As another example, configuration settings associated with broadcasting or otherwise transmitting packets may be adjusted based on the ambient temperature, the temperature of the analyte sensor 110, or the temperature of a particular part of the communication hardware of the analyte sensor 110. In addition to altering the transmission power, other parameters associated with the transmission capabilities or processes of the communication hardware of the analyte sensor 110 may be altered, including, but not limited to, transmission rate, frequency, and timing. As another example, when the analyte data indicates that a subject is experiencing or is about to experience a negative health event, a rule may cause the analyte sensor 110 to increase its discoverability to alert a receiving device of the negative health event.

[0178] P. Exemplary Sensor Sensitivity Initialization / Adjustment Mechanism As embodied herein, certain calibration mechanisms for the sensing hardware 5060 of the analyte sensor 110 may be adjusted based on external or interval environmental characteristics and to compensate for decay of the sensing hardware 5060 during periods of non-use (e.g., "storage time" before use). The calibration mechanisms of the sensing hardware 5060 may be adjusted autonomously by the sensor 110 (e.g., by operation of the ASIC 5000 to modify characteristics in the memory 5020 or storage 5030) or may be adjusted by other devices in the analyte monitoring system 100.

[0179] As an example, the sensor sensitivity of the sensing hardware 5060 may be adjusted based on external temperature data or time since manufacture. When external temperature is monitored during sensor storage, the disclosed subject matter may adaptively change compensation to the sensor sensitivity over time as the device experiences changing storage conditions. By way of example and not limitation, adaptive sensitivity adjustments may be performed in an "active" storage mode in which the analyte sensor 110 periodically wakes up to measure temperature. These mechanisms may conserve the battery of the analyte device and extend the life of the analyte sensor. At each temperature measurement, the analyte sensor 110 may calculate a sensitivity adjustment for that period based on the measured temperature. The temperature-weighted adjustments may then be accumulated over the active storage mode period to calculate a total sensor sensitivity adjustment at the end of the active storage mode (e.g., upon insertion). Similarly, upon insertion, the sensor 110 may determine the time difference between manufacture of the sensor 110 (which may be written to the storage 5030 of the ASIC 5000) or the sensing hardware 5060 and alter the sensor sensitivity or other calibration mechanisms according to one or more known attenuation rates or formulas.

[0180] Additionally, for purposes of illustration and not limitation, as embodied herein, the sensor sensitivity adjustment may account for other sensor conditions, such as sensor drift. The sensor sensitivity adjustment may be hard-coded into the sensor 110 during manufacturing, for example, based on an estimate of how much the average sensor drifts in the case of sensor drift. The sensor 110 may use a calibration function having time-varying functions for sensor offset and gain, which may account for drift over the sensor's wear period. Thus, the sensor 110 may utilize a device-dependent function that describes the sensor's 110 drift over time, and a function used to convert interstitial current to interstitial glucose, which may indicate sensor sensitivity and may be device-specific in combination with a baseline glucose profile. Such a function to account for sensor sensitivity and drift may improve the accuracy of the sensor 110 over the wear period without user calibration.

[0181] Q. Exemplary Model-Based Analyte Measurements The sensor 110 detects raw measurements from the sensing hardware 5060. On-sensor processing may be performed, such as by one or more models trained to interpret the raw measurements. The models may be machine learning models trained off-device to detect, predict, or interpret the raw measurements to detect, predict, or interpret the levels of one or more analytes. Additional trained models may operate on the output of the machine learning models trained to interact with the raw measurements. As an example, the models may be used to detect, predict, or recommend an event based on the raw measurements and the type of analyte(s) detected by the sensing hardware 5060. The events may include the initiation or completion of a physical activity, a meal, the application of a medical procedure or medication, an emergency health event, and other events of a similar nature.

[0182] The model may be provided to the sensor 110, data receiving device 120, or general-purpose data receiving device 130 during manufacturing or during a firmware or software update. The model may be periodically refined, such as by the manufacturer of the sensor 110 or an operator of the analyte monitoring system 100, based on data received from the sensor 110 and data receiving device of an individual user or multiple users collectively. In certain embodiments, the sensor 110 includes sufficient computational components to support further training or refinement of the machine learning model, such as based on unique characteristics of the user to which the sensor 110 is attached. The machine learning model may include models trained using or incorporating, by way of example and not limitation, decision tree analysis, gradient boosting, ada boosting, artificial neural networks or variations thereof, linear discriminant analysis, nearest neighbor analysis, support vector machines, supervised or unsupervised classification, and the like. The model may also include algorithmic or rule-based models in addition to machine learning models. Model-based processing may be performed by other devices, including the data receiving device 120 or general-purpose data receiving device 130, upon receiving data from the sensor 110 (or other downstream devices).

[0183] R. Exemplary Alarm Mechanism The data transmitted between the sensor 110 and the data sink 120 may include raw measurements or processed measurements. The data transmitted between the sensor 110 and the data sink 120 may further include alarms or notifications for display to a user. The data sink 120 may display or otherwise convey notifications to a user based on the raw measurements or processed measurements, or may display alarms as received from the sensor 110. Alarms that may be triggered for display to a user include alarms based on direct analyte values ​​(e.g., a single reading that exceeds or does not meet a threshold), analyte value trends (e.g., average readings over a set period of time that exceed or do not meet a threshold, slope), analyte value predictions (e.g., algorithmic calculations based on analyte values ​​that exceed or do not meet a threshold), sensor alerts (e.g., a suspected malfunction detected), communication alerts (e.g., no communication between the sensor 110 and the data receiving device 120 for a threshold period of time, an unknown device attempting to initiate or failing to initiate a communication session with the sensor 110), reminders (e.g., a reminder to charge the data receiving device 120, take medication, or perform other activities), and other alerts of a similar nature. For purposes of example and not limitation, as embodied herein, the alarm parameters described herein may be configurable by the user, or may be fixed during manufacture, or may be a combination of user-configurable and non-user-configurable parameters.

[0184] S. Exemplary Electrode Configurations Sensor configurations featuring a single active area configured for detection of a single corresponding analyte may use a two-electrode or three-electrode detection motif, as described further herein with reference to Figures 18A-18C. Sensor configurations featuring two different active areas for detection of separate analytes, either on separate working electrodes or on the same working electrode, are described separately below with reference to Figures 19A-21C. Sensor configurations having multiple working electrodes may be particularly advantageous in incorporating two different active areas within the same sensor tail, since the signal contribution from each active area may be more easily determined.

[0185] When a single working electrode is present in the analyte sensor, a three-electrode sensor configuration may include a working electrode, a counter electrode, and a reference electrode. A related two-electrode sensor configuration includes 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). The various electrodes may be at least partially stacked (layered) on one another and / or spaced laterally from one another on the sensor tail. Suitable sensor configurations may be substantially planar or substantially cylindrical in shape. In any of the sensor configurations disclosed herein, the various electrodes may be electrically insulated from one another by a dielectric material or similar insulator.

[0186] Analyte sensors featuring multiple working electrodes may also include 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.

[0187] 18A shows a schematic diagram of an exemplary two-electrode analyte sensor configuration suitable for use in the present disclosure. As shown, the analyte sensor 200 includes a substrate 30212 disposed between a working electrode 214 and a counter / reference electrode 30216. Alternatively, the working electrode 214 and the counter / reference electrode 30216 may be disposed on the same side of the substrate 30212 with a dielectric material between them (configuration not shown). The active area 218 is disposed as at least one layer on at least a portion of the working electrode 214. The active area 218 may include multiple spots or a single spot configured for detection of an analyte (e.g., ketones), as discussed further herein. In certain embodiments, the active area 218 includes an enzyme system comprising NADH oxidase and β-hydroxybutyrate dehydrogenase.

[0188] 18A, membrane 220 covers at least active area 218. In certain embodiments, membrane 220 may cover some or all of working electrode 214 and / or counter / reference electrode 30216, or the entire analyte sensor 200. One or both sides of analyte sensor 200 may be covered with membrane 220. Membrane 220 may include one or more polymeric membrane materials capable of limiting analyte flux to active area 218 (i.e., membrane 220 is a mass transport limiting membrane having some permeability to the analyte of interest (e.g., ketones)). In accordance with the disclosure herein, membrane 220 may be crosslinked by a branched crosslinker in certain sensor configurations, as described further below. For example, but not limited to, membrane 220 is crosslinked by a branched glycidyl ether. The composition and thickness of membrane 220 may be varied to facilitate desired analyte (e.g., ketones) flux to active area 218, thereby providing desired signal strength and stability. Analyte sensor 200 may be operable to assay the analyte by any of the following electrochemical detection techniques: coulometry, amperometry, voltammetry, or potentiometry.

[0189] In certain embodiments, one or more membranes, including an interference domain and a mass transport limiting membrane, are deposited on an exposed electroactive surface of a working electrode, such as a platinum surface. For example, but not limited to, an interference domain can be disposed on the working electrode, an active region can be disposed on the interference domain, and a mass transport limiting membrane can be disposed on the active region.

[0190] 18B and 18C show schematic diagrams of exemplary three-electrode analyte sensor configurations, which are also suitable for use in the present disclosure. The three-electrode analyte sensor configuration may be similar to that shown as analyte sensor 200 in FIG. 18A, except for the inclusion of an additional electrode 217 in analyte sensors 201 and 202 (FIGS. 18B and 18C). With the additional electrode 217, counter / reference electrode 30216 may then function as either a counter electrode or a reference electrode, and additional electrode 217 performs other electrode functions not otherwise described. Working electrode 214 continues to perform its original function. Additional electrode 217 may be disposed on either working electrode 214 or electrode 30216, with a separating layer of dielectric material therebetween. For example, but not by way of limitation, as shown in FIG. 18B, dielectric layers 219a, 219b, and 219c separate electrodes 214, 30216, and 217 from one another and provide electrical insulation. Alternatively, at least one of the electrodes 214, 30216, and 217 may be located on the opposite side of the substrate 30212, as shown in FIG. 18C. Thus, in certain embodiments, the electrode 214 (working electrode) and the electrode 30216 (counter electrode) may be disposed on the opposite side of the substrate 30212, and the electrode 217 (reference electrode) is disposed on one of the electrodes 214 or 30216 and separated therefrom by a dielectric material. A reference material layer 30230 (e.g., Ag / AgCl) may be present on the electrode 217, and the location of the reference material layer 30230 is not limited to the location shown in FIG. 18B and FIG. 18C. Similar to the sensor 200 shown in FIG. 18A, the active area 218 (for detection of ketones) in the analyte sensors 201 and 202 may include multiple spots or a single spot. Additionally, analyte sensors 201 and 202 may be operable to assay the analyte by any of the following electrochemical detection techniques: coulometry, amperometry, voltammetry, or potentiometry.

[0191] Similar to analyte sensor 200, membrane 220 may also cover active area 218 and other sensor components in analyte sensors 201 and 202, thereby functioning as a mass transport limiting membrane. In certain embodiments, additional electrode 217 may be covered by membrane 220. Although FIGS. 18B and 18C show electrodes 214, 30216, and 217 as being covered by membrane 220, it should be appreciated that in certain embodiments, only working electrode 214 is covered. Furthermore, the thickness of membrane 220 on each of electrodes 214, 30216, and 217 may be the same or different. As in the two-electrode analyte sensor configuration (FIG. 18A), one or both faces of analyte sensors 201 and 202 may be covered by membrane 220 in the sensor configuration of FIGS. 18B and 18C, or the entire analyte sensors 201 and 202 may be covered. Thus, the three-electrode sensor configuration shown in Figures 18B and 18C should be understood as not limiting the embodiments disclosed herein, and alternative electrode and / or layer configurations remain within the scope of the present disclosure.

[0192] FIG. 19A shows an exemplary configuration of a sensor 203 having a single working electrode with two different active regions disposed thereon. FIG. 19A is similar to FIG. 18A, except that there are two active regions on the working electrode 214, a first active region 218a and a second active region 218b, which are responsive to different analytes and are laterally spaced apart from each other on the surface of the working electrode 214. The active regions 218a and 218b may include multiple spots or a single spot configured for detection of each analyte. The composition of the membrane 220 may vary in the active regions 218a and 218b or may be compositionally the same. The first active region 218a and the second active region 218b may be configured to detect their corresponding analytes at different working electrode potentials, as discussed further below. In certain embodiments, either or both of active areas 218a and 218b may be configured to detect ketones, for example, by using an enzyme system including NADH oxidase and β-hydroxybutyrate dehydrogenase. In certain embodiments, only one of active areas 218a and 218b is configured to detect ketones, for example, by using an enzyme system including NADH oxidase and β-hydroxybutyrate dehydrogenase. In certain embodiments, the other active area is configured to detect a second analyte, for example, lactate, glucose, creatinine, and / or oxygen. In certain embodiments, the second analyte is glucose.

[0193] Figures 19B and 19C show cross-sectional views of exemplary three-electrode sensor configurations for sensors 204 and 205, respectively, each featuring a single working electrode having a first active area 218a and a second active area 218b disposed thereon. Figures 19B and 19C are otherwise similar to Figures 18B and 18C and may be better understood by reference thereto. As with Figure 19A, the composition of membrane 220 may vary in active areas 218a and 218b or may be compositionally the same.

[0194] Exemplary sensor configurations having multiple working electrodes, specifically two working electrodes, are described in further detail with reference to Figures 4-21C. The following description is primarily directed to sensor configurations having two working electrodes, but it should be understood that extensions of the disclosure herein may incorporate more than two working electrodes. Additional working electrodes may be used to provide the analyte sensor with further sensing capabilities for detection of not only the first and second analytes, but also, for example, a third and / or fourth analyte.

[0195] 4 illustrates a cross-sectional view of an exemplary analyte sensor configuration having two working electrodes, a reference electrode, and a counter electrode suitable for use in the present disclosure. As shown, the analyte sensor 300 includes working electrodes 304 and 306 disposed on opposite sides of a substrate 302. A first active area 310a is disposed on a surface of the working electrode 304, and a second active area 310b is disposed on a surface of the working electrode 306. The counter electrode 320 is electrically insulated from the working electrode 304 by a dielectric layer 322, and the reference electrode 321 is electrically insulated from the working electrode 306 by a dielectric layer 323. Outer dielectric layers 330 and 332 are disposed on the reference electrode 321 and the counter electrode 320, respectively. The membrane 340 may cover at least the active areas 310a and 310b according to various embodiments, and optionally other components of the analyte sensor 300 or the entire analyte sensor 300 are similarly covered by the first membrane portion 340a and / or the second membrane portion 340b. Furthermore, the membrane 340 may be continuous but compositionally varied within the first membrane portion 340a and the second membrane portion 340b (i.e., over the active areas 310a and 310b) to allow different permeability values ​​for separately adjusting the analyte flux at each location. For example, different membrane formulations may be sprayed and / or printed on opposite sides of the analyte sensor 300. Dip-coating techniques may also be suitable, particularly for depositing at least a portion of the bilayer membrane over one of the active areas 310a and 310b. Thus, according to certain embodiments of the present disclosure, one of the first membrane portion 340a and the second membrane portion 340b may comprise a bilayer membrane, and the other of the first membrane portion 340a and the second membrane portion 340b may comprise a single membrane polymer. Similar to analyte sensors 200, 201, and 202, analyte sensor 300 may be operable to assay ketones (and / or a second analyte) by any of a coulometric, amperometric, voltammetric, or potentiometric electrochemical detection techniques. In certain embodiments, the analyte sensor may include more than one membrane 340, e.g., two or more membranes.For example, without limitation, an analyte sensor may include one or more active areas, such as 310a and a membrane covering 310a, and an additional membrane covering the entire sensor as shown in FIG. 20. In certain embodiments, either or both of active areas 310a and 310b may be configured to detect ketones, for example, by using an enzyme system including NADH oxidase and β-hydroxybutyrate dehydrogenase. In certain embodiments, only one of active areas 310a and 310b is configured to detect ketones, for example, by using an enzyme system including NADH oxidase and β-hydroxybutyrate dehydrogenase. In certain embodiments, the other active area is configured to detect a second analyte. In certain embodiments, the second analyte is glucose.

[0196] Alternative sensor configurations having multiple working electrodes and that differ from the configuration shown in Figure 20 may feature counter / reference electrodes in place of separate counter and reference electrodes 320, 321, and / or different layer and / or film arrangements than those explicitly shown. For example, but not by way of limitation, the locations of counter electrode 320 and reference electrode 321 may be opposite those shown in Figure 20. Additionally, working electrodes 304 and 306 need not necessarily be on opposite sides of substrate 302 as in the location shown in Figure 20.

[0197] Although preferred sensor configurations may feature electrodes that are substantially planar in nature, it should be appreciated that sensor configurations featuring non-planar electrodes may be advantageous and may be particularly suitable for use in the present disclosure. In particular, substantially cylindrical electrodes arranged concentrically with respect to one another may facilitate deposition of a mass transport limiting film, as described below. In particular, concentric working electrodes spaced apart along the length of the sensor tail may facilitate film deposition by successive dip coating operations in a similar manner as described above for the substantially planar sensor configuration. Figures 21A-21C show perspective views of an analyte sensor featuring two working electrodes arranged concentrically with respect to one another. It should be appreciated that sensor configurations having a concentric electrode arrangement but no second working electrode are also possible in the present disclosure.

[0198] 21A shows a perspective view of an exemplary sensor configuration in which multiple electrodes are substantially cylindrical and arranged concentrically with respect to one another with respect to a central substrate. As shown, the analyte sensor 400 includes a central substrate 402 around which all of the electrodes and dielectric layers are arranged concentrically with respect to one another. In particular, a working electrode 410 is disposed on a surface of the central substrate 402, with a dielectric layer 412 disposed over a portion of the working electrode 410 distal to the sensor tip 404. A working electrode 420 is disposed over the dielectric layer 412, with a dielectric layer 422 disposed over a portion of the working electrode 420 distal to the sensor tip 404. A counter electrode 430 is disposed over the dielectric layer 422, with a dielectric layer 432 disposed over a portion of the counter electrode 430 distal to the sensor tip 404. A reference electrode 440 is disposed over the dielectric layer 432, with a dielectric layer 442 disposed over a portion of the reference electrode 440 distal to the sensor tip 404. Thus, the exposed surfaces of working electrode 410 , working electrode 420 , counter electrode 430 , and reference electrode 440 are spaced apart from one another along the longitudinal axis B of analyte sensor 400 .

[0199] 21A, a first active area 414a and a second active area 414b associated with different or the same analyte are disposed on the exposed surfaces of the working electrodes 410 and 420, respectively, thereby allowing contact with a fluid for sensing to occur. In certain embodiments, either or both of the active areas 414a and 414b may be configured to detect ketones, for example, by using an enzyme system comprising NADH oxidase and β-hydroxybutyrate dehydrogenase. In certain embodiments, only one of the active areas 414a and 414b is configured to detect ketones, for example, by using an enzyme system comprising NADH oxidase and β-hydroxybutyrate dehydrogenase. In certain embodiments, the other active area is configured to detect a second analyte. In certain embodiments, the second analyte is glucose. Although the active areas 414a and 414b are shown as three separate spots in FIG. 21A, it should be understood that there may be fewer or more than three spots comprising a continuous layer of active areas in alternative sensor configurations.

[0200] In Fig. 21A, the sensor 400 is partially covered by a membrane 450 over the working electrodes 410 and 420 and the active areas 414a and 414b disposed thereon. Fig. 21B illustrates an alternative sensor configuration in which substantially all of the sensor 401 is covered by a membrane 450. The membrane 450 may be the same or compositionally different in the active areas 414a and 414b. For example, the membrane 450 may include a bilayer membrane covering the active area 414a or may be a uniform membrane covering the active area 414b.

[0201] It should be further understood that the positioning of the various electrodes in Figures 21A and 21B may differ from that explicitly shown. For example, the positions of the counter electrode 430 and the reference electrode 440 may be reversed from the configuration shown in Figures 21A and 21B. Similarly, the positions of the working electrodes 410 and 420 are not limited to those explicitly shown in Figures 21A and 21B. Figure 21C illustrates an alternative sensor configuration to that shown in Figure 21B, where the sensor 405 includes the counter electrode 430 and the reference electrode 440 located more proximally relative to the sensor tip 404, and the working electrodes 410 and 420 located more distally relative to the sensor tip 404. A sensor configuration in which the working electrodes 410 and 420 are located more distally relative to the sensor tip 404 may be advantageous by providing a larger surface area for deposition of the active regions 414a and 414b (five separate sensing spots are illustratively shown in Figure 21C), thereby facilitating increased signal strength in some cases. Similarly, the center substrate 402 may be omitted in any of the concentric sensor configurations disclosed herein, in which case the innermost electrode may instead support subsequently deposited layers.

[0202] In certain embodiments, the electrodes are wire electrodes. In certain embodiments, the sensor tail comprises a working electrode and a reference electrode spirally wound around the working electrode. In certain embodiments, an insulator is disposed between the working electrode and the reference electrode. In certain embodiments, a portion of the electrode is exposed to allow reaction of one or more enzymes with the analyte on the electrode, as described below. In certain embodiments, each electrode is formed from a thin wire having a diameter of about 25.4 micrometers (0.001 inches) or less to about 254 micrometers (0.010 inches) or more. In certain embodiments, the working electrode has a diameter of about 25.4 micrometers (0.001 inches) or less to about 254 micrometers (0.010 inches) or more, such as about 50.8 micrometers (0.002 inches) to about 203 micrometers (0.008 inches), or about 102 micrometers (0.004 inches) to about 127 micrometers (0.005 inches). In certain embodiments, the electrodes are formed from plated insulators, plated wires, or bulk conductive materials. In certain embodiments, the working electrode comprises a wire formed from, for example, platinum, platinum-iridium, palladium, graphite, gold, carbon, conductive polymers, alloys, or other conductive materials. In certain embodiments, the electrodes can be formed by a variety of manufacturing techniques (e.g., bulk metal processing, deposition of metal on a substrate, etc.), and the electrodes can be formed from plated wires (e.g., platinum on steel wire) or bulk metals (e.g., platinum wire). In certain embodiments, the electrodes are formed from tantalum wire coated with platinum.

[0203] In certain embodiments, the reference electrode, which may function as a reference electrode alone or as a dual reference and counter electrode, is formed from silver, silver / silver chloride, etc. In certain embodiments, the reference electrode is juxtaposed and / or twisted with or around the working electrode. In certain embodiments, the reference electrode is spirally wrapped around the working electrode. In certain embodiments, the assembly of wires may be coated or glued with an insulating material to provide an insulating attachment.

[0204] In certain embodiments, additional electrodes may be included in the sensor tail. For example, but not limited to, a three-electrode system (working, reference, and counter electrodes) and / or additional working electrodes (e.g., an electrode for detecting a second analyte). In certain embodiments where the sensor comprises two working electrodes, the two working electrodes may be juxtaposed and the reference electrode may be disposed therearound (e.g., spirally wrapped around the two or more working electrodes). In certain embodiments, the two or more working electrodes may extend parallel to one another. In certain embodiments, the reference electrode is wrapped around the working electrodes and extends toward the distal end (i.e., the in vivo end) of the sensor tail. In certain embodiments, the reference electrode extends (e.g., spirally) into the exposed area of ​​the working electrodes.

[0205] In certain embodiments, one or more working electrodes are spirally wound around the reference electrode. In certain embodiments where more than one working electrode is provided, the working electrodes may be formed in a double, triple, quadruple, etc. spiral configuration (e.g., surrounding a reference electrode, an insulated rod, or other supporting structure) along the length of the sensor tail. In certain embodiments, the electrodes, e.g., two or more working electrodes, are formed coaxially. For example, but not by way of limitation, the electrodes all share the same central axis.

[0206] In certain embodiments, the working electrode comprises a tube with an insulator therebetween and within which the reference electrode is disposed or wound. Alternatively, the reference electrode comprises a tube with an insulator therebetween and within which the working electrode is disposed or wound. In certain embodiments, a polymeric (e.g., insulating) rod is provided, and one or more electrodes (e.g., one or more electrode layers) are disposed thereon (e.g., by electroplating). In certain embodiments, a metal (e.g., steel or tantalum) rod or wire is provided that is coated with an insulating material (as described herein) and has one or more working and reference electrodes disposed thereon. For example, but not by way of limitation, the present disclosure provides a sensor, e.g., a sensor tail, that comprises one or more tantalum wires, with platinum disposed on a portion of the one or more tantalum wires to function as the working electrode. In certain embodiments, the platinum-coated tantalum wire is covered with an insulating material, which is partially covered with a silver / silver chloride composition that functions as the reference and / or counter electrode.

[0207] In certain embodiments where an insulator is disposed on the working electrode (e.g., on the platinum surface of the electrode), a portion of the insulator can be stripped or otherwise removed to expose the electroactive surface of the working electrode. For example, but not limited to, a portion of the insulator can be removed by hand, excimer laser, chemical etching, laser ablation, grit blasting, and the like. Alternatively, a portion of the electrode can be masked prior to deposition of the insulator to maintain an exposed electroactive surface area. In certain embodiments, the stripped and / or removed portion of the insulator can be about 0.1 mm (about 0.004 inches) or less to about 2 mm (about 0.078 inches) or more in length, such as about 0.5 mm (about 0.02 inches) to about 0.75 mm (about 0.03 inches) in length. In certain embodiments, the insulator is a non-conductive polymer. In certain embodiments, the insulator comprises parylene, fluorinated polymers, polyethylene terephthalate, polyvinylpyrrolidone, polyurethane, polyimide, and other non-conductive polymers. In certain embodiments, glass or ceramic materials can also be used for the insulator layer. In certain embodiments, the insulator comprises parylene. In certain embodiments, the insulator comprises polyurethane. In certain embodiments, the insulator comprises polyurethane and polyvinylpyrrolidone.

[0208] Some components of the active region and membrane of the analyte sensor of the present disclosure are further described below. 2. Enzymes The active area of ​​the disclosed analyte sensor may be configured to detect one or more analytes. In certain embodiments, the disclosed analyte sensor may include two or more active areas, each active area configured to detect the same analyte or a different analyte. Non-limiting examples of analytes that may be detected using the disclosed analyte sensors include ketones, glucose, oxygen, creatinine, alcohol, e.g., ethanol, and lactate. In certain embodiments, the analyte is one or more ketones.

[0209] In certain embodiments, the analyte sensor of the present disclosure may include a ketone-responsive active region, a glucose-responsive active region, a lactate-responsive active region, a creatinine-responsive active region, an alcohol-responsive active region, or a combination thereof. In certain embodiments, the ketone-responsive active region may include one or more enzymes for detecting ketones. In certain embodiments, the glucose-responsive active region may include one or more enzymes for detecting glucose. In certain embodiments, the lactate-responsive active region may include one or more enzymes for detecting lactate. In certain embodiments, the creatinine-responsive active region may include one or more enzymes for detecting creatinine. In certain embodiments, the alcohol-responsive active region may include one or more enzymes for detecting alcohol. In certain embodiments, the active region may include an enzyme system comprising two or more enzymes that collectively respond to the analyte.

[0210] In certain embodiments, the analyte sensor of the present disclosure includes at least one active area configured to detect ketones. A particular enzyme system that can be used to detect ketones is described in FIG. 22. In the enzymatic reaction shown, β-hydroxybutyrate serves as a surrogate for ketones formed in vivo. As shown in FIG. 22, a concerted enzyme pair can be used to detect ketones according to the disclosure herein. For example, but not limited to, the concerted enzyme pair can include a dehydrogenase and an oxidase. In certain embodiments, the dehydrogenase is β-hydroxybutyrate dehydrogenase. In certain embodiments, the oxidase is NADH oxidase. The enzyme cofactors NAD+ and NADH help facilitate the concerted enzymatic reaction disclosed herein and shown in FIG. 22. When the ketone-responsive active site contains this pair of concerted enzymes, β-hydroxybutyrate dehydrogenase (HBDH) converts β-hydroxybutyrate and oxidized nicotinamide adenine dinucleotide (NAD +) to acetoacetate and reduced nicotinamide adenine dinucleotide (NADH), respectively. NADH oxidase (NADHOx) can then catalyze the reaction of molecular oxygen with NADH to produce hydrogen peroxide and NAD+. Hydrogen peroxide can then be catalytically oxidized at the anode, or working electrode, according to the following equation:

[0211] H 2 O 2 →2H + +O 2 +2e - [1] The electrons transferred during this reaction provide the basis for detection of the ketones at the working electrode, eliminating the need for a redox mediator. The resulting electrochemical signal can then be correlated to the amount of ketones initially present in the sample.

[0212] In certain embodiments, the working electrode comprises a metal capable of oxidizing hydrogen peroxide. In certain embodiments, the working electrode may comprise platinum, a platinum alloy, carbon, or a combination thereof. In certain embodiments, the working electrode is a platinum electrode. In certain embodiments, the working electrode may include a platinum-carbon mixture.

[0213] The ketone-sensing enzyme system comprising β-hydroxybutyrate dehydrogenase and NADH oxidase offers several advantages over previously disclosed enzyme systems. For example, the enzyme system eliminates the need for the use of electron transfer agents, such as redox mediators, such as osmium complexes. In addition, lower potentials can be used to prevent irreversible oxidation of NADH. For example, previously disclosed systems require application of a potential of about +0.7 V versus an Ag / AgCl reference. At this potential, NADH is also irreversibly oxidized, which is undesirable. As disclosed herein, a potential of less than about +0.7 V versus an Ag / AgCl reference, such as less than about +0.6 V, less than about +0.5 V, or less than about +0.4 V versus an Ag / AgCl reference, is applied to the sensor (e.g., working electrode or enzyme system) of the present disclosure. In certain embodiments, the potential applied to the enzyme system of the present disclosure is about +0.2 V to about +0.5 V relative to an Ag / AgCl reference, e.g., about +0.3 V to about +0.4 V relative to an Ag / AgCl reference. In certain embodiments, the potential applied to the enzyme system of the present disclosure is about +0.35 V relative to an Ag / AgCl reference. At this potential, NADH is not oxidized, e.g., is not irreversibly oxidized.

[0214] In certain embodiments, an analyte sensor of the present disclosure may include at least one working electrode and a sensor tail including a ketone-responsive active area (e.g., active area 218 or 310a) disposed on a surface of the working electrode, the ketone-responsive active area including an enzyme system including a dehydrogenase and an oxidase. In certain embodiments, the enzyme system includes β-hydroxybutyrate dehydrogenase and NADH oxidase. In certain embodiments, the enzyme system consists essentially of β-hydroxybutyrate dehydrogenase and NADH oxidase. In certain embodiments, the enzyme system consists essentially of β-hydroxybutyrate dehydrogenase and NADH oxidase. In certain embodiments, the enzyme system does not include superoxide dismutase.

[0215] In certain embodiments, the ketone-responsive active region can comprise a ratio of β-hydroxybutyrate dehydrogenase to NADH oxidase of about 40:1 to about 1:40, e.g., about 35:1 to about 1:35, about 30:1 to about 1:30, about 25:1 to about 1:25, about 20:1 to about 1:20, about 15:1 to about 1:15, about 10:1 to about 1:10, about 9:1 to about 1:9, about 8:1 to about 1:8, about 7:1 to about 1:7, about 6:1 to about 1:6, about 5:1 to about 1:5, about 4:1 to about 1:4, about 3:1 to about 1:3, about 2:1 to about 1:2, about 2:1, or about 1:1. In certain embodiments, the ketone-responsive activity region may comprise a ratio of β-hydroxybutyrate dehydrogenase to NADH oxidase of about 5:1 to about 1:5. In certain embodiments, the ketone-responsive activity region may comprise a ratio of β-hydroxybutyrate dehydrogenase to NADH oxidase of about 3:1 to about 1:3. In certain embodiments, the ketone-responsive activity region may comprise a ratio of β-hydroxybutyrate dehydrogenase to NADH oxidase of about 2:1 to about 1:2. In certain embodiments, the ketone-responsive activity region may comprise a ratio of β-hydroxybutyrate dehydrogenase to NADH oxidase of about 2:1.

[0216] In certain embodiments, the ketone-responsive active region may comprise about 10% to about 80% by weight, e.g., about 15% to about 75% by weight, about 20% to about 70% by weight, about 25% to about 65% by weight, or about 30% to about 60% by weight of one or more enzymes of the enzyme system, e.g., β-hydroxybutyrate dehydrogenase and / or NADH oxidase. In certain embodiments, the ketone-responsive active region may comprise about 10% to about 80% by weight, e.g., about 15% to about 75% by weight, about 20% to about 70% by weight, about 25% to about 65% by weight, about 30% to about 60% by weight, about 20% to about 60% by weight, or about 20% to about 50% by weight of both enzymes of the enzyme system, e.g., β-hydroxybutyrate dehydrogenase and NADH oxidase. In certain embodiments, the ketone-responsive active region may comprise about 10% to about 80% by weight of β-hydroxybutyrate dehydrogenase, for example, about 15% to about 75% by weight, about 20% to about 70% by weight, about 25% to about 65% by weight, or about 30% to about 60% by weight of NADH oxidase.

[0217] In certain embodiments, the ketone-responsive active region may comprise about 15% to about 35% by weight of one enzyme of the enzyme system, such as β-hydroxybutyrate dehydrogenase and / or NADH oxidase. In certain embodiments, the ketone-responsive active region may comprise about 10% to about 50% by weight, such as about 15% to about 45%, about 20% to about 40%, about 20% to about 35%, or about 20% to about 30% by weight of β-hydroxybutyrate dehydrogenase. In certain embodiments, the ketone-responsive active region may comprise about 15% to about 35% by weight of β-hydroxybutyrate dehydrogenase. In certain embodiments, the ketone-responsive active region may comprise about 10% to about 50% by weight of NADH oxidase, for example, about 15% to about 45%, about 20% to about 40%, about 20% to about 35%, or about 20% to about 30% by weight. In certain embodiments, the ketone-responsive active region may comprise about 15% to about 35% by weight of NADH oxidase.

[0218] In certain embodiments, the ketone-responsive active region may further comprise a stabilizer, for example, to stabilize the enzyme. For example, but not limited to, the stabilizer may be albumin, such as serum albumin. Non-limiting examples of serum albumin include bovine serum albumin and human serum albumin. In certain embodiments, the stabilizer is human serum albumin. In certain embodiments, the stabilizer is bovine serum albumin. In certain embodiments, the stabilizer may be catalase. In certain embodiments, the ketone-responsive active region can include a ratio of stabilizer to enzymes of the enzyme system, such as NADH oxidase and β-hydroxybutyrate dehydrogenase, of about 40:1 to about 1:40, e.g., about 35:1 to about 1:35, about 30:1 to about 1:30, about 25:1 to about 1:25, about 20:1 to about 1:20, about 15:1 to about 1:15, about 10:1 to about 1:10, about 9:1 to about 1:9, about 8:1 to about 1:8, about 7:1 to about 1:7, about 6:1 to about 1:6, about 5:1 to about 1:5, about 4:1 to about 1:4, about 3:1 to about 1:3, about 2:1 to about 1:2, or about 1:1. In certain embodiments, the ketone-responsive active region can include a ratio of stabilizer to enzymes of the enzyme system, such as NADH oxidase and β-hydroxybutyrate dehydrogenase, of about 2:1 to about 1:2. In certain embodiments, the ketone-responsive active region may comprise a ratio of stabilizer to NADH oxidase of about 40:1 to about 1:40, e.g., about 35:1 to about 1:35, about 30:1 to about 1:30, about 25:1 to about 1:25, about 20:1 to about 1:20, about 15:1 to about 1:15, about 10:1 to about 1:10, about 9:1 to about 1:9, about 8:1 to about 1:8, about 7:1 to about 1:7, about 6:1 to about 1:6, about 5:1 to about 1:5, about 4:1 to about 1:4, about 3:1 to about 1:3, about 2:1 to about 1:2, or about 1:1. In certain embodiments, the ketone-responsive active region may comprise a ratio of stabilizer to NADH oxidase of about 2:1 to about 1:2.In certain embodiments, the ketone-responsive active region may comprise a ratio of stabilizer to β-hydroxybutyrate dehydrogenase of about 40:1 to about 1:40, e.g., about 35:1 to about 1:35, about 30:1 to about 1:30, about 25:1 to about 1:25, about 20:1 to about 1:20, about 15:1 to about 1:15, about 10:1 to about 1:10, about 9:1 to about 1:9, about 8:1 to about 1:8, about 7:1 to about 1:7, about 6:1 to about 1:6, about 5:1 to about 1:5, about 4:1 to about 1:4, about 3:1 to about 1:3, about 2:1 to about 1:2, or about 1:1. In certain embodiments, the ketone-responsive active region may comprise a ratio of stabilizer to β-hydroxybutyrate dehydrogenase of about 2:1 to about 1:2. In certain embodiments, the ketone-responsive active region may comprise about 10% to about 50% by weight of the stabilizer, e.g., about 15% to about 45%, about 20% to about 40%, about 20% to about 35%, or about 20% to about 30%. In certain embodiments, the ketone-responsive active region may comprise about 15% to about 35% by weight of the stabilizer.

[0219] In certain embodiments, the ketone-responsive active region may further comprise a cofactor (or a derivative thereof) for an enzyme of the enzyme system disclosed herein. Non-limiting examples of cofactors include NADH or NADPH or derivatives thereof. In certain embodiments, the cofactor is NADH or a derivative thereof. In certain embodiments, the ketone-responsive active region may comprise a ratio of cofactor to NADH oxidase of about 40:1 to about 1:40, e.g., about 35:1 to about 1:35, about 30:1 to about 1:30, about 25:1 to about 1:25, about 20:1 to about 1:20, about 15:1 to about 1:15, about 10:1 to about 1:10, about 9:1 to about 1:9, about 8:1 to about 1:8, about 7:1 to about 1:7, about 6:1 to about 1:6, about 5:1 to about 1:5, about 4:1 to about 1:4, about 3:1 to about 1:3, about 2:1 to about 1:2, or about 1:1. In certain embodiments, the ketone-responsive active region may comprise a ratio of cofactor to NADH oxidase of about 2:1 to about 1:2. In certain embodiments, the ketone-responsive active region may comprise a ratio of cofactor to β-hydroxybutyrate dehydrogenase of about 40:1 to about 1:40, e.g., about 35:1 to about 1:35, about 30:1 to about 1:30, about 25:1 to about 1:25, about 20:1 to about 1:20, about 15:1 to about 1:15, about 10:1 to about 1:10, about 9:1 to about 1:9, about 8:1 to about 1:8, about 7:1 to about 1:7, about 6:1 to about 1:6, about 5:1 to about 1:5, about 4:1 to about 1:4, about 3:1 to about 1:3, about 2:1 to about 1:2, or about 1:1. In certain embodiments, the ketone-responsive active region may comprise a ratio of cofactor to β-hydroxybutyrate dehydrogenase of about 2:1 to about 1:2. In certain embodiments, the ketone-responsive active region may contain about 10% to about 50% by weight of the cofactor, for example, about 15% to about 45%, about 20% to about 40%, about 20% to about 35%, or about 20% to about 30% by weight. In certain embodiments, the ketone-responsive active region may contain about 15% to about 35% by weight of the cofactor. In certain embodiments, the cofactor, for example, NADH, may be physically retained in the ketone-responsive active region. For example, but not limited to, a membrane covering the ketone-responsive active region may help retain the cofactor in the ketone-responsive active region while allowing sufficient inward diffusion of ketones to allow detection of ketones.

[0220] In certain embodiments, the ketones-responsive active area is disposed on a portion of the working electrode. For example, without limitation, the ketones-responsive active area is disposed on the portion of the working electrode in a spot pattern, e.g., two or more spots on the working electrode. In certain embodiments, the ketones-responsive active area is disposed on the portion of the working electrode in a slot-like pattern. In certain embodiments, the ketones-responsive active area is disposed over the entire length of the working electrode or in a continuous pattern on the working electrode. In certain embodiments, the ketones-responsive active area is disposed within a range of about 0.01 mm. 2 ~ approx. 2.0 mm 2 , for example, about 0.1 mm 2 ~ approx. 1.0 mm 2 , or about 0.2 mm 2 ~about 0.5mm 2 It has an area of.

[0221] In certain embodiments, the analyte sensors of the present disclosure may include a second active region for detecting an analyte other than ketones, e.g., on the same working electrode as the ketone-responsive active region or on a second working electrode, hi certain embodiments, the second active region is a glucose-responsive active region, a lactate-responsive active region, a creatinine-responsive active region, or an alcohol-responsive active region.

[0222] In certain embodiments, the second active region of the analyte sensor of the present disclosure may include one or more enzymes for detecting glucose. For example, without limitation, the analyte sensor of the present disclosure may include an active region (e.g., a second active region) comprising one or more enzymes for detecting glucose, e.g., disposed on a second working electrode. In certain embodiments, the analyte sensor may include an active site comprising glucose oxidase and / or glucose dehydrogenase for detecting glucose. In certain embodiments, glucose may be reacted with H as a product. 2 O 2 The glucose oxidase present in the second active region can be used to detect the glucose oxidase that produces H. 2 O2 reacts with an electrochemically reactive surface, such as a platinum surface, of a second working electrode to generate a detectable current.

[0223] In certain embodiments, the second active region may include one or more enzymes for detecting lactate. For example, without limitation, an analyte sensor of the present disclosure may include an active region (e.g., a second active region) comprising one or more enzymes (e.g., an enzyme system) for detecting lactate, e.g., disposed on a second working electrode. In certain embodiments, the analyte sensor may include an active site comprising lactate dehydrogenase and / or lactate oxidase.

[0224] In certain embodiments, a second enzyme-responsive active region present, for example, on a second working electrode of an analyte sensor of the present disclosure may include one or more enzymes for detecting alcohol. For example, without limitation, an analyte sensor of the present disclosure may include an active region (e.g., a second active region) that includes one or more enzymes (e.g., an enzyme system) for detecting alcohol, for example, disposed on a second working electrode. In certain embodiments, an analyte sensor may include an active site that includes alcohol dehydrogenase.

[0225] In certain embodiments, a second enzyme-responsive active region present, for example, on a second working electrode of an analyte sensor of the present disclosure may include one or more enzymes for detecting creatinine. For example, without limitation, an analyte sensor of the present disclosure may include an active region (e.g., a second active region) comprising one or more enzymes (e.g., an enzyme system) for detecting creatinine, for example, disposed on a second working electrode. In certain embodiments, an analyte sensor may include an active site comprising amidohydrolase, creatine amidinohydrolase, and / or sarcosine oxidase.

[0226] In certain embodiments, an analyte sensor may include two working electrodes, e.g., a first active area disposed on a first working electrode and a second active area disposed on a second working electrode. In certain embodiments, the first active area and the second active area are configured to detect different analytes. In certain embodiments, the first active area is configured to detect ketones. In certain embodiments, the second active area is configured to detect an analyte different from ketones, e.g., glucose, creatinine, lactate, and / or alcohol. For example, without limitation, an analyte sensor disclosed herein may feature a ketones-responsive active area on a surface of a first working electrode and a second active area, e.g., a glucose-responsive active area, configured to detect a different analyte on a surface of a different working electrode, e.g., a second working electrode. In certain embodiments, such an analyte sensor may include a sensor tail having at least a first working electrode and a second working electrode, a ketone-responsive active region disposed on the surface of the first working electrode, and a glucose-responsive active region comprising a glucose-responsive enzyme disposed on the surface of the second working electrode. For example, without limitation, when the sensor is configured to detect more than one analyte, detection of each analyte may include applying a potential to each working electrode individually, thereby resulting in a separate signal from each analyte. The signal obtained from each analyte may then be correlated to an analyte concentration by use of a calibration curve or function or by employing a look-up table. In certain embodiments, the correlation of the analyte signal to the analyte concentration may be performed by use of a processor.

[0227] In certain other analyte sensor configurations, the first active area and the second active area may be disposed on a single working electrode. A first signal may be obtained from the first active area, for example at a low potential, and a second signal, including signal contributions from both active areas, may be obtained at a high potential. Subtracting the first signal from the second signal then allows the signal contribution resulting from the second analyte to be determined. The signal contribution from each analyte may then be correlated with the analyte concentration in a manner similar to that described for the sensor configurations having multiple working electrodes. In certain embodiments, when a ketone-responsive active area and a second active area configured to detect a different analyte, for example a glucose-responsive active area, are disposed on a single working electrode in this manner, one of the active areas may be configured to be able to respond separately to facilitate detection of each analyte. For example, the ketone-responsive active area or the glucose-responsive active area may generate a signal independently of the other active area.

[0228] It should also be understood that the sensitivity (output current) of the analyte sensor directed to each analyte may be varied by varying the coverage (area or size) of the active regions, the area ratio of the active regions to one another, the identity, the thickness and / or composition of the mass transport limiting membrane covering the active regions. Variations in these parameters can be readily implemented by one of ordinary skill in the art given the benefit of the disclosure herein.

[0229] 3. Redox mediators In certain embodiments, the analyte sensor disclosed herein may include an electron transfer agent. As discussed above, the composition of the ketone-responsive active region in any of the exemplary sensor configurations disclosed herein, such as the enzyme composition and the working electrode composition, does not include an electron transfer agent in the ketone-responsive active region. For example, but not limited to, a ketone-responsive active region comprising an enzyme system including β-hydroxybutyrate dehydrogenase and NADH oxidase does not include an electron transfer agent, such as a redox mediator, such as an osmium redox mediator.

[0230] In certain embodiments, an active region configured to detect another analyte, such as glucose, present in an analyte sensor of the present disclosure may include an electron transfer agent. For example, without limitation, an analyte sensor of the present disclosure may include a sensor tail having at least a first working electrode and a second working electrode, a ketone-responsive active region comprising β-hydroxybutyrate dehydrogenase and NADH oxidase disposed on the surface of the first working electrode, and a glucose-responsive active region comprising a glucose-responsive enzyme and an electron transfer agent disposed on the surface of the second working electrode. In certain embodiments, the first working electrode comprises platinum, and the ketone-responsive active region does not comprise an electron transfer agent. In certain embodiments, the ketone-responsive active region generates hydrogen peroxide in the presence of ketones, which is directly oxidized at the surface of the working electrode comprising platinum without the need for an electron transfer agent to produce a detectable current, e.g., that correlates with the concentration of ketones in the sample. Alternatively or additionally, in certain embodiments, the second working electrode comprises platinum, and the glucose-responsive active region disposed on the second working electrode does not comprise an electron transfer agent. In certain embodiments, the glucose-responsive active region generates hydrogen peroxide in the presence of glucose, which is directly oxidized at the surface of a platinum-containing working electrode without the need for an electron transfer agent to produce a detectable current that correlates, for example, with the concentration of glucose in the sample.

[0231] A suitable electron transfer agent can facilitate the transfer of electrons to an adjacent working electrode after the analyte undergoes an enzymatic redox reaction within the corresponding active area, thereby generating a current indicative of the presence of a particular analyte, the amount of current generated being proportional to the amount of analyte present.

[0232] In certain embodiments, suitable electron transfer agents may include electroreducible and electrooxidizable ions, complexes, or molecules (e.g., quinones) that have redox potentials several hundred millivolts higher or lower than the redox potential of a standard calomel electrode (SCE). In certain embodiments, redox mediators may include osmium complexes and other transition metal complexes, such as those described in U.S. Pat. Nos. 6,134,461 and 6,605,200, which are incorporated herein by reference in their entireties. Further examples of suitable redox mediators include those described in U.S. Pat. Nos. 6,736,957, 7,501,053, and 7,754,093, the disclosures of each of which are also incorporated herein by reference in their entireties. Other examples of suitable redox mediators include metal compounds or complexes of ruthenium, osmium, iron (e.g., polyvinylferrocene or hexacyanoferrate), or cobalt, including, for example, metallocene compounds thereof. Suitable ligands for metal complexes may also include bidentate or higher 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 dentate ligands may be present in the metal complex to achieve a complete coordination sphere.

[0233] In certain embodiments, the electron transfer agents disclosed herein may be provided with suitable functional groups to facilitate covalent attachment to a polymer (referred to herein as a polymer backbone) in the active region, as discussed further below. For example, without limitation, the electron transfer agents for use in the present disclosure may include polymer-bound electron transfer agents. Non-limiting examples of suitable polymer-bound electron transfer agents include those described in U.S. Pat. Nos. 8,444,834, 8,268,143, and 6,605,201, the disclosures of which are incorporated herein by reference in their entireties. In certain embodiments, the electron transfer agent is a bidentate osmium complex bound to a polymer described herein, such as a polymer backbone described in Section 4 below. In certain embodiments, the polymer-bound electron transfer agents shown in FIG. 3 of U.S. Pat. No. 8,444,834 may be used in the sensors of the present disclosure.

[0234] 4. Polymer backbone In certain embodiments, one or more active sites for facilitating detection of an analyte may include a polymer to which an enzyme and / or a redox mediator is covalently attached. Any suitable polymer backbone may be present in the active region to facilitate detection of an analyte via covalent attachment of an enzyme and / or a redox mediator thereto. Non-limiting examples of suitable polymers in the active region include polyvinylpyridines, such as poly(4-vinylpyridine) and / or poly(2-vinylpyridine), and polyvinylimidazoles, such as poly(N-vinylimidazole) and poly(1-vinylimidazole), or copolymers thereof, for example, where quaternized pyridine groups serve as attachment points for a redox mediator or enzyme thereto. Exemplary copolymers that may be suitable for inclusion in the active region include those that include monomer units such as, for example, styrene, acrylamide, methacrylamide, or acrylonitrile. In certain embodiments, polymers that may be present in the active region include polyurethane or copolymers thereof and / or polyvinylpyrrolidone. In certain embodiments, polymers that may be present in the active region include, but are not limited to, those described in U.S. Patent No. 6,605,200 (hereby incorporated by reference in its entirety), such as poly(acrylic acid), styrene / maleic anhydride copolymer, methyl vinyl ether / maleic anhydride copolymer (GANTREZ polymer), poly(vinylbenzyl chloride), poly(allylamine), polylysine, poly(4-vinylpyridine) quaternized with carboxypentyl groups, and poly(sodium 4-styrenesulfonate). In certain embodiments where the analyte sensor includes two active sites, the polymers in each active region may be the same or different.

[0235] In certain embodiments, the polymer is polyvinylpyridine or a copolymer thereof, hi certain embodiments, the polymer is a copolymer of vinylpyridine and styrene. In certain embodiments, when an enzyme system including multiple enzymes, including NADH oxidase, is present in a given active area, all of the multiple enzymes may be covalently bound to the polymer. In certain other embodiments, only a portion of the multiple enzymes are covalently bound to the polymer. For example, but not limited to, one or more enzymes in the enzyme system may be covalently bound to the polymer and at least one enzyme may be non-covalently bound to the polymer, thereby physically retaining the non-covalently bound enzyme within the polymer. In certain embodiments, β-hydroxybutyrate dehydrogenase and NADH oxidase may be covalently bound to the polymer in the ketone-responsive active area of ​​the disclosed analyte sensor. In certain embodiments, β-hydroxybutyrate dehydrogenase may be covalently bound to the polymer and NADH oxidase may be non-covalently bound to the polymer. Alternatively, NADH oxidase may be covalently bound to the polymer and β-hydroxybutyrate dehydrogenase may be non-covalently bound to the polymer. In certain embodiments ... + may be covalently attached to the polymer. In certain embodiments, NAD+ is not covalently attached to the polymer. + In certain embodiments, where NAD is not covalently attached to a polymer, + can be physically retained within the ketone-responsive active region. In certain embodiments, the membrane covering the ketone-responsive active region retains NAD within the ketone-responsive active region while still allowing sufficient inward diffusion of ketones to permit detection of ketones. + Suitable membrane polymers for coating the ketone-responsive active regions are discussed further herein.

[0236] Alternatively or additionally, the active region, e.g., the ketones-responsive active region, does not include polyvinylpyridine or a copolymer of vinylpyridine and styrene. In certain embodiments, one or more enzymes present in the active region can be immobilized in the active region by the use of a cross-linking agent, as described herein, in the presence of a stabilizer. For example, but not limited to, one or more enzymes in the ketones-responsive active region of the present disclosure, e.g., NADH oxidase and / or β-hydroxybutyrate dehydrogenase, can be immobilized in the active region by the use of a cross-linking agent, e.g., polyethylene glycol diglycidyl ether. In certain embodiments, one or more enzymes in the ketones-responsive active region, e.g., NADH oxidase and / or β-hydroxybutyrate dehydrogenase, can be covalently attached to a stabilizer in the active region by the use of a cross-linking agent. In certain embodiments, the stabilizer is serum albumin, e.g., bovine serum albumin (BSA).

[0237] In certain embodiments, the covalent attachment of one or more enzymes and / or redox mediators to the polymer and / or stabilizer in a given active area may occur through crosslinking introduced by a suitable crosslinking agent. Suitable crosslinking agents may include one or more crosslinkable functional groups, such as, but not limited to, vinyl, alkoxy, acetoxy, enoxy, oxime, amino, hydroxyl, cyano, halo, acrylate, epoxide, and isocyanate groups. In certain embodiments, the crosslinking agent comprises one or more, two or more, three or more, or four or more epoxide groups. For example, but not limited to, crosslinking agents for use in the present disclosure may include mono-, di-, tri-, and tetra-ethylene oxide. In certain embodiments, crosslinkers for reaction with free amino groups in an enzyme (e.g., with the free side chain amine in lysine) can include crosslinkers such as, for example, polyethylene glycol dibutyl ethers, polypropylene glycol dimethyl ethers, polyalkylene glycol allyl methyl ethers, polyethylene glycol diglycidyl ether (PEGDGE), or other polyepoxides, cyanuric chloride, N-hydroxysuccinimide, imidoesters, epichlorohydrin, or derivatized variants thereof. In certain embodiments, the crosslinker has an average molecular weight (M) of, for example, about 200 to 1000, e.g., about 400. n ) is PEGDGE. In certain embodiments, the crosslinker is PEGDGE400. In certain embodiments, the crosslinker can be glutaraldehyde. Crosslinkers suitable for reaction with free carboxylic acid groups in the enzyme can include, for example, carbodiimides. In certain embodiments, the crosslinking of the enzyme to the polymer or stabilizer is generally intermolecular.

[0238] In certain embodiments, the ketone-responsive active region can comprise a ratio of crosslinker to one or more or both enzymes of the enzyme system, e.g., NADH oxidase and / or β-hydroxybutyrate dehydrogenase, of about 40:1 to about 1:40, e.g., about 35:1 to about 1:35, about 30:1 to about 1:30, about 25:1 to about 1:25, about 20:1 to about 1:20, about 15:1 to about 1:15, about 10:1 to about 1:10, about 9:1 to about 1:9, about 8:1 to about 1:8, about 7:1 to about 1:7, about 6:1 to about 1:6, about 5:1 to about 1:5, about 4:1 to about 1:4, about 3:1 to about 1:3, about 2:1 to about 1:2, or about 1:1. In certain embodiments, the ketones-responsive active region may comprise a ratio of about 5:1 to about 1:5 of crosslinker to one or more or both enzymes of the enzyme system, such as NADH oxidase and / or β-hydroxybutyrate dehydrogenase. In certain embodiments, the ketones-responsive active region may comprise a ratio of about 3:1 to about 1:3 of crosslinker to one or more or both enzymes of the enzyme system, such as NADH oxidase and / or β-hydroxybutyrate dehydrogenase. In certain embodiments, the ketones-responsive active region may comprise a ratio of about 2:1 to about 1:2 of crosslinker to one or more or both enzymes of the enzyme system, such as NADH oxidase and / or β-hydroxybutyrate dehydrogenase. In certain embodiments, the ketones-responsive active region may comprise about 5% to about 50% crosslinker by weight. In certain embodiments, the ketones-responsive active region may comprise about 5% to about 20% crosslinker by weight, such as about 10% to about 15% crosslinker by weight.

[0239] 5. Mass-transfer limiting membrane In certain embodiments, the analyte sensors disclosed herein further comprise a membrane that is permeable to the analyte and that covers at least an active area, such as the first active area and / or the second active area.

[0240] In certain embodiments, the membrane covering the analyte-responsive active area may function as a mass transport limiting membrane and / or to improve biocompatibility. The mass transport limiting membrane may act as a diffusion limiting barrier to reduce the rate of mass transport of the analyte. For example, but not by way of limitation, limiting the access of the analyte, such as a ketone, to the analyte-responsive active area by the mass transport limiting membrane may help to avoid overloading (saturation) of the sensor, thereby improving detection performance and accuracy.

[0241] In certain embodiments, the mass transport limiting membrane may be homogeneous and may be single component (comprising a single membrane polymer). Alternatively, the mass transport limiting membrane may be multi-component (comprising two or more different membrane polymers). In certain embodiments, the mass transport limiting membrane may include two or more layers, for example, a bi-layer or tri-layer membrane. In certain embodiments, each layer may comprise a different polymer or the same polymer, but at a different concentration or thickness. In certain embodiments, the ketone-responsive active region may be covered with a multi-layer membrane, for example, a bi-layer membrane, and the second analyte-responsive active region may be covered with a single membrane. In certain embodiments, the ketone-responsive active region may be covered with a multi-layer membrane, for example, a bi-layer membrane, and the second analyte-responsive active region may be covered with a multi-layer membrane, for example, a bi-layer membrane.

[0242] In certain embodiments, the mass transport limiting membrane can include a crosslinked polymer that includes a heterocyclic nitrogen group. In certain embodiments, the mass transport limiting membrane can include a polyvinylpyridine-based polymer. Non-limiting examples of polyvinylpyridine-based polymers are disclosed in U.S. Patent Publication No. 2003 / 0042137 (e.g., Formula 2b), the contents of which are incorporated herein by reference in their entirety. In certain embodiments, the mass transport limiting membrane can comprise polyvinylpyridine (e.g., poly(4-vinylpyridine) or poly(4-vinylpyridine), polyvinylimidazole, polyvinylpyridine copolymers (e.g., copolymers of vinylpyridine and styrene), polyacrylates, polyurethanes, polyetherurethanes, silicones, polytetrafluoroethylene, polyethylene-co-tetrafluoroethylene, polyolefins, polyesters, polycarbonates, biostable polytetrafluoroethylene, homopolymers, copolymers, or terpolymers of polyurethanes, polypropylenes, polyvinyl chloride, polyvinylidene difluoride, polybutylene terephthalate, polymethyl methacrylate, polyether ether ketones, cellulose polymers, polysulfones, and block copolymers thereof, including, for example, diblock, triblock, alternating, random, and graft copolymers, or other chemically related materials.

[0243] In certain embodiments, membranes, e.g., single component membranes, for use in the present disclosure may include polyvinylpyridine (e.g., poly(4-vinylpyridine) and / or poly(2-vinylpyridine). In certain embodiments, membranes, e.g., single component membranes, for use in the present disclosure may include poly(4-vinylpyridine). In certain embodiments, membranes, e.g., single component membranes, for use in the present disclosure may include a copolymer of vinylpyridine and styrene. In certain embodiments, the membrane may comprise a polyvinylpyridine-co-styrene copolymer. For example, but not limited to, polyvinylpyridine for use in the present disclosure The polyvinylpyridine-co-styrene copolymer may include a polyvinylpyridine-co-styrene copolymer in which a portion of the pyridine nitrogen atoms are functionalized with non-crosslinked polyethylene glycol tails and a portion of the pyridine nitrogen atoms are functionalized with alkylsulfonic acid groups. In certain embodiments, the derivatized polyvinylpyridine-co-styrene copolymer for use as a membrane polymer may be the 10Q5 polymer described in U.S. Patent No. 8,761,857, the contents of which are incorporated herein by reference in their entirety. In certain embodiments, the polyvinylpyridine-based polymer has a molecular weight of about 50 Da to about 500 kDa.

[0244] In certain embodiments, the membrane may comprise a polymer such as, but not limited to, poly(styrene-co-maleic anhydride), poly(propylene glycol)-block-poly(ethylene glycol)-block-poly(propylene glycol) crosslinked with dodecylamine and poly(propylene glycol)-block-poly(ethylene glycol)-block-poly(propylene glycol) bis(2-aminopropyl ether); poly(N-isopropylacrylamide); copolymers of poly(ethylene oxide) and poly(propylene oxide); or combinations thereof.

[0245] In certain embodiments, the membrane comprises a polyurethane membrane with both hydrophilic and hydrophobic regions. In certain embodiments, the hydrophobic polymer component is a polyurethane, a polyurethaneurea, or a poly(ether-urethane-urea). In certain embodiments, the polyurethane is a polymer produced by the condensation reaction of a diisocyanate with a difunctional hydroxyl-containing material. In certain embodiments, the polyurethaneurea is a polymer produced by the condensation reaction of a diisocyanate with a difunctional amine-containing material. In certain embodiments, the diisocyanate for use herein comprises an aliphatic diisocyanate containing, for example, about 4 to about 8 methylene units, or a diisocyanate containing an alicyclic moiety. Further non-limiting examples of polymers that may be used for the production of the membrane of the sensor of the present disclosure include vinyl polymers, polyethers, polyesters, polyamides, inorganic polymers (e.g., polysiloxanes and polycarbosiloxanes), natural polymers (e.g., cellulosic and protein-based materials), and mixtures (e.g., mixed or layered structures), or combinations thereof. In certain embodiments, the hydrophilic polymer component is polyethylene oxide and / or polyethylene glycol. For example, and without limitation, a hydrophobic-hydrophilic copolymer component for use in the present disclosure is a polyurethane polymer comprising about 10% to about 50%, e.g., about 20%, hydrophilic polyethylene oxide.

[0246] In certain embodiments, the membrane comprises a silicone polymer / hydrophobic-hydrophilic polymer blend. In certain embodiments, the hydrophobic-hydrophilic polymer for use in the blend can be any suitable hydrophobic-hydrophilic polymer, such as, but not limited to, polyethers such as polyvinylpyrrolidone, polyhydroxyethyl methacrylate, polyvinyl alcohol, polyacrylic acid, polyethylene glycol or polypropylene oxide, and copolymers thereof, including, for example, diblock, triblock, alternating, random, comb, star, dendritic, and graft copolymers. In certain embodiments, the hydrophobic-hydrophilic polymer is a copolymer of poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO). Non-limiting examples of copolymers of PEO and PPO include PEO-PPO diblock copolymers, PPO-PEO-PPO triblock copolymers, PEO-PPO-PEO triblock copolymers, alternating block copolymers of PEO-PPO, random copolymers of ethylene oxide and propylene oxide, and blends thereof. In certain embodiments, the copolymers may be substituted with hydroxy substituents.

[0247] In certain embodiments, hydrophilic or hydrophobic modifiers can be used to "tweak" the permeability of the resulting membrane to analytes of interest, such as ketones. In certain embodiments, hydrophilic modifiers such as polyethylene glycol, hydroxyl or polyhydroxyl modifiers, and any combination thereof, can be used to improve the biocompatibility of the polymer or the resulting membrane.

[0248] In certain embodiments where multiple active regions are present, a mass transport limiting membrane can cover each active region, including the option of varying composition in the different active regions, which can be accomplished through successive dip coating operations that create a bilayer membrane portion at the working electrode that is positioned more closely to the sensor tip.

[0249] In certain embodiments where there are multiple active regions, a separate mass transport limiting membrane may cover each active region. For example, but not by way of limitation, a mass transport limiting membrane may be disposed over a first active region, such as a ketone-responsive active region, and a separate second mass transport limiting membrane may cover a second active region, such as a glucose-responsive active region. In certain embodiments, the two mass transport limiting membranes are spatially separated and do not overlap with each other. In certain embodiments, the first mass transport limiting membrane does not overlap with the second mass transport limiting membrane, and the second mass transport limiting membrane does not overlap with the first mass transport limiting membrane. In certain embodiments, the first mass transport limiting membrane comprises a different polymer than the second mass transport limiting membrane. Instead, the first mass transport limiting membrane comprises the same polymer as the second mass transport limiting membrane. In certain embodiments, the first mass transport limiting membrane comprises the same polymer as the second mass transport limiting membrane, but with a different crosslinker.

[0250] In certain embodiments, the composition of a mass transport limiting membrane disposed on an analyte sensor having two active areas may be the same or different when a mass transport limiting membrane covers each active area. For example, but not by way of limitation, the portion of the mass transport limiting membrane covering the ketone-responsive active area may be multi-component and / or the portion of the mass transport limiting membrane covering the second analyte responsive area, e.g., the glucose-responsive active area, may be single-component. Alternatively, the portion of the mass transport limiting membrane covering the ketone-responsive active area may be single-component and / or the portion of the mass transport limiting membrane covering the second analyte responsive area, e.g., the glucose-responsive active area, may be multi-component. In certain embodiments, the mass transport limiting membrane covering the ketone-responsive active area may be single-component and the mass transport limiting membrane covering the second analyte responsive area, e.g., the glucose-responsive active area, may be single-component. In certain embodiments, the mass transport limiting membrane covering the ketone-responsive active region comprises a different polymer than the mass transport limiting membrane covering a second analyte-responsive region, such as a glucose-responsive active region.

[0251] In certain embodiments of the present disclosure, the ketone-responsive active area may be coated with a single-component membrane comprising polyvinylpyridine (e.g., poly(4-vinylpyridine)) and the second analyte-responsive area, e.g., the glucose-responsive active area, may be coated with a membrane comprising polyvinylpyridine-co-styrene copolymer. In certain embodiments, the membrane covering the second analyte-responsive area, e.g., the glucose-responsive active area, may be coated with a membrane covering the ketone-responsive active area, e.g., to create a bilayer membrane over the ketone-responsive active area. In certain embodiments, the glucose-responsive active area may be coated with a membrane comprising polyurethane, polyurethane urea, or poly(ether-urethane-urea). In certain embodiments, the glucose-responsive active area may be coated with a membrane comprising polyurethane.

[0252] In certain embodiments of the present disclosure, the ketone-responsive active region and the second analyte-responsive region, e.g., the glucose-responsive active region, can be coated with a membrane comprising polyvinylpyridine-co-styrene copolymer. In certain embodiments, the multi-component membrane can exist as a bilayer membrane or as a homogeneous mixture of two or more membrane polymers. The homogeneous mixture can be deposited by mixing two or more membrane polymers in a solution and then depositing the solution on the working electrode. In certain embodiments of the present disclosure, the ketone-responsive active region can be coated with a multi-component membrane comprising polyvinylpyridine and polyvinylpyridine-co-styrene copolymer (or derivatives thereof) as a bilayer membrane or homogeneous mixture, and the second analyte-responsive region, e.g., the glucose-responsive active region, can be coated with a membrane comprising polyvinylpyridine-co-styrene copolymer (or derivatives thereof). In certain embodiments of the present disclosure, the ketone-responsive active region may be coated in a multi-component membrane comprising polyvinylpyridine and polyvinylpyridine-co-styrene copolymer (or a derivative thereof) as a bilayer membrane, and a second analyte-responsive region, e.g., a glucose-responsive active region, may be coated in a single-component membrane comprising polyvinylpyridine-co-styrene copolymer (or a derivative thereof).

[0253] Suitable copolymers of vinylpyridine and styrene (polyvinylpyridine-co-styrene copolymers) may have a styrene content ranging from about 0.01% to about 50% mole percent, or from about 0.05% to about 45% mole percent, or from about 0.1% to about 40% mole percent, or from about 0.5% to about 35% mole percent, or from about 1% to about 30% mole percent, or from about 2% to about 25% mole percent, or from about 5% to about 20% mole percent. Substituted styrenes may be used in similar amounts as well. Suitable copolymers of vinylpyridine and styrene may have a molecular weight of 5 kDa or more, or about 10 kDa or more, or about 15 kDa or more, or about 20 kDa or more, or about 25 kDa or more, or about 30 kDa or more, or about 40 kDa or more, or about 50 kDa or more, or about 75 kDa or more, or about 90 kDa or more, or about 100 kDa or more. In non-limiting examples, suitable copolymers of vinylpyridine and styrene may have a molecular weight in the range of about 5 kDa to about 150 kDa, or about 10 kDa to about 125 kDa, or about 15 kDa to about 100 kDa, or about 20 kDa to about 80 kDa, or about 25 kDa to about 75 kDa, or about 30 kDa to about 60 kDa.

[0254] In certain other embodiments, the membrane polymer covering one or more active regions may be crosslinked using a crosslinking agent as disclosed herein and in section 4 above. In certain embodiments where there are two mass transport limiting membranes, e.g., a first mass transport limiting membrane and a second mass transport limiting membrane, each membrane may be crosslinked to a different crosslinking agent. For example, but not by way of limitation, a crosslinking agent may result in a membrane that is more restrictive of the diffusion of a particular compound, e.g., an analyte, through the membrane, or less restrictive of the diffusion of a particular compound, e.g., by affecting the size of the pores in the membrane. For example, but not by way of limitation, in a sensor configured to detect ketones and glucose, the mass transport limiting membrane covering the ketones-responsive region may have a pore size that limits the diffusion of compounds larger than ketones, e.g., glucose, through the membrane.

[0255] In certain embodiments, crosslinkers for use in the present disclosure may include polyepoxides, carbodiimides, cyanuric chloride, triglycidyl glycerol, N-hydroxysuccinimide, imide esters, epichlorohydrin, or derivatized variants thereof. In certain embodiments, the membrane polymers covering one or more active regions may be crosslinked to a branched crosslinker that may, for example, reduce the amount of extractables obtained from the mass transport limiting membrane. Non-limiting examples of branched crosslinkers include branched glycidyl ether crosslinkers, including branched glycidyl ether crosslinkers that include two, three, or more crosslinkable groups. In certain embodiments, the branched crosslinker may include two or more crosslinkable groups, such as polyethylene glycol diglycidyl ether. In certain embodiments, the branched crosslinker may include three or more crosslinkable groups, such as polyethylene glycol tetraglycidyl ether. In certain embodiments, the mass transport limiting membrane may include polyvinylpyridine or a copolymer of vinylpyridine and styrene crosslinked to a branched glycidyl ether crosslinker that includes two or three crosslinkable groups, such as polyethylene glycol tetraglycidyl ether or polyethylene glycol diglycidyl ether. In certain embodiments, the epoxide group of a polyepoxide, for example, polyethylene glycol tetraglycidyl ether or polyethylene glycol diglycidyl ether, can form a covalent bond with a pyridine or imidazole by opening the epoxide ring to provide a hydroxyalkyl group that bridges the body of the crosslinker with the heterocycle of the membrane polymer.

[0256] In certain embodiments, the crosslinker is polyethylene glycol diglycidyl ether (PEGDGE). In certain embodiments, the PEGDGE used to facilitate crosslinking (e.g., intermolecular crosslinking) between two or more membrane polymer backbones may exhibit a wide range of suitable molecular weights. In certain embodiments, the molecular weight of the PEGDGE may range from about 100 g / mol to about 5000 g / mol. The number of ethylene glycol repeat units in each arm of the PEGDGE may be the same or different, and generally may vary over a range within a given sample from which the average molecular weight is obtained. In certain embodiments, the PEGDGE for use in the present disclosure has an average molecular weight (M n In certain embodiments, the crosslinker is PEGDGE400.

[0257] In certain embodiments, the polyethylene glycol tetraglycidyl ether used to promote cross-linking (e.g., intermolecular cross-linking) between two or more membrane polymer backbones may exhibit a wide range of suitable molecular weights. Up to four polymer backbones may be cross-linked by a single molecule of polyethylene glycol tetraglycidyl ether cross-linker. In certain embodiments, the molecular weight of the polyethylene glycol tetraglycidyl ether may range from about 1000 g / mol to about 5000 g / mol. The number of ethylene glycol repeat units in each arm of the polyethylene glycol tetraglycidyl ether may be the same or different, and generally may vary over a range within a given sample for which the average molecular weight is obtained.

[0258] In certain embodiments, polydimethylsiloxane (PDMS) may be incorporated into any of the mass transport limiting membranes disclosed herein. In certain embodiments, the analyte sensors described herein may comprise a sensor tail comprising at least a first working electrode, a first active area disposed on a surface of the first working electrode, and a mass transport limiting membrane covering at least the first active area and permeable to the first analyte. In certain embodiments, the first active area comprises an enzyme system comprising at least one enzyme (optionally covalently bound to a first polymer and / or stabilizer) and responsive to the first analyte, e.g., ketones responsive. For example, without limitation, the analyte sensors described herein may comprise a sensor tail comprising at least a first working platinum electrode, a ketones-responsive active area comprising an enzyme system comprising β-hydroxybutyrate dehydrogenase and NADH oxidase (wherein one or both enzymes are optionally covalently bound to a polymer and / or stabilizer) disposed on a surface of the first working electrode, and a mass transport limiting membrane covering the ketones-responsive active area and permeable to ketones. In certain embodiments, the mass transport limiting membrane comprises a membrane polymer crosslinked to a branched glycidyl ether crosslinker comprising two or more crosslinkable groups, such as polyethylene glycol diglycidyl ether or polyethylene glycol tetraglycidyl ether.

[0259] In certain embodiments, the analyte sensor of the present disclosure includes a sensor tail comprising at least a first working electrode and a second working electrode spaced apart from one another along the length of the sensor tail. In certain embodiments, a first active area is disposed on a surface of the first working electrode and a second active area is disposed on a surface of the second working electrode, and the first active area and the second active area are responsive to different analytes. For example, and without limitation, the first active area is a ketones-responsive active area. In certain embodiments, the second active area may be responsive to glucose. In certain embodiments, a mass transport limiting membrane covers the first active area and the second active area, the mass transport limiting membrane comprising a bilayer membrane portion covering the first active area and a uniform membrane portion covering the second active area. In certain embodiments, each layer of the bilayer membrane portion comprises a different membrane polymer. In certain embodiments, the bottom layer of the bilayer portion over the first active area comprises a different membrane polymer, e.g., a polyvinylpyridine polymer, than the uniform membrane portion over the second active area.

[0260] In certain embodiments, when a first active area and a second active area configured to assay different analytes are placed on separate working electrodes, the mass transport limiting membrane may have different permeability values ​​for the first analyte and the second analyte. For example, but not by way of limitation, the mass transport limiting membrane covering at least one of the active areas may include a mixture of a first membrane polymer and a second membrane polymer, or a bilayer of a first membrane polymer and a second membrane polymer. A homogeneous membrane may cover the active areas not covered by the mixture or bilayer, the homogeneous membrane including only one of the first membrane polymer or the second membrane polymer. Advantageously, the architecture of the analyte sensor disclosed herein allows for a continuous membrane having a homogeneous membrane portion to be placed on the first active area of ​​the analyte sensor, and a multi-component membrane portion to be placed on the second active area of ​​the analyte sensor, whereby the permeability values ​​for each analyte are simultaneously equalized, improving sensitivity and detection accuracy. In certain embodiments, successive film depositions can be accomplished by successive dip coating operations.

[0261] 6. Interference Domains In certain embodiments, the sensor of the present disclosure, e.g., the sensor tail, may further comprise an interference domain. In certain embodiments, the interference domain may include, for example, a polymer domain that restricts the flow of one or more interferents to the surface of the working electrode. In certain embodiments, the interference domain may function as a molecular sieve that allows the passage of the analyte and other substances measured by the working electrode while preventing the passage of other substances, such as interferents. In certain embodiments, the interferents may affect the signal obtained at the working electrode. Non-limiting examples of interferents include acetaminophen, ascorbate, ascorbic acid, bilirubin, cholesterol, creatinine, dopamine, ephedrine, ibuprofen, L-dopa, methyldopa, salicylate, tetracycline, tolazamide, tolbutamide, triglycerides, urea, and uric acid.

[0262] In certain embodiments, the interference domain is disposed between the working electrode and one or more active regions, such as a ketone-responsive active region. In certain embodiments, non-limiting examples of polymers that can be used in the interference domain include polyurethanes, polymers with pendant ionic groups, and polymers with controlled pore sizes. In certain embodiments, the interference domain is formed from one or more cellulosic derivatives. Non-limiting examples of cellulosic derivatives include polymers such as cellulose acetate, cellulose acetate butyrate, 2-hydroxyethyl cellulose, cellulose acetate phthalate, cellulose acetate propionate, and cellulose acetate trimellitate.

[0263] In certain embodiments, the interference domain comprises a thin, hydrophobic membrane that is non-swelling and limits the diffusion of high molecular weight species, for example, but not by way of limitation, the interference domain is permeable to relatively low molecular weight substances, such as hydrogen peroxide, while restricting the passage of high molecular weight substances, such as ketones, glucose, acetaminophen, and / or ascorbic acid.

[0264] In certain embodiments, the interference domain may be deposited directly onto the working electrode, e.g., onto the platinum surface of the working electrode. In certain embodiments, the interference domain has a thickness, e.g., a dry thickness, in the range of about 0.1 μm to about 1000 μm, e.g., about 1 μm to about 500 μm, about 10 μm to about 100 μm, or about 10 μm to about 100 μm. In certain embodiments, the interference domain may have a thickness of about 0.1 μm to about 10 μm, e.g., about 0.5 μm to about 10 μm, about 1 μm to about 10 μm, about 1 μm to about 5 μm, or about 0.1 μm to about 5 μm. In certain embodiments, the sensor may be immersed in the solution of the interference domain more than once. For example, but not by way of limitation, a sensor (or working electrode) of the present disclosure may be immersed in the solution of the interference domain at least twice, at least three times, at least four times, or at least five times to obtain a desired thickness of the interference domain.

[0265] 7.Manufacturing The present disclosure further provides a method for manufacturing the disclosed analyte sensor comprising one or more active sites. In certain embodiments, the method includes screen printing one or more working electrodes. In certain embodiments, one of the working electrodes is a platinum electrode. In certain embodiments, a conductive material comprising, for example, platinum, of one or more working electrodes is screen printed onto a substrate.

[0266] In certain embodiments, the method may further include adding a composition comprising an enzyme onto the surface of the working electrode to generate active sites on the working electrode. For example, but not limited to, the composition may include β-hydroxybutyrate dehydrogenase and NADH oxidase, for example, in the amounts and / or ratios disclosed herein. In certain embodiments, the composition may further include a cofactor for the enzyme present in the composition, for example, NAD, for example, in the amounts and / or ratios disclosed herein. In certain embodiments, the composition may further include a crosslinker, for example, polyethylene glycol diglycidyl ether, and a stabilizer (e.g., BSA), for example, in the amounts and / or ratios disclosed herein. In certain embodiments, the method may further include curing the enzyme composition.

[0267] In certain embodiments, the method may further include adding a membrane composition onto the enzyme composition, e.g., the cured enzyme composition. In certain embodiments, the membrane composition may include a polymer, e.g., polyvinylpyridine, and / or a cross-linker, e.g., polyethylene glycol diglycidyl ether. In certain embodiments, the membrane is applied to the working electrode above the enzyme composition by dip coating (or similar technique), spray coating, painting, inkjet printing, roller coating, etc. In certain embodiments, the method may include curing the membrane polymer composition.

[0268] In certain embodiments, the analyte sensor may further include a second working electrode, and a method of manufacturing such a sensor may include, for example, depositing a second enzyme composition on a surface of the second working electrode for detecting a second analyte. In certain embodiments, the method may further include curing or drying the second enzyme composition and depositing a second film composition over the second enzyme composition.

[0269] In certain embodiments, a first dip-coating operation deposits a first membrane polymer over a first active area (e.g., a ketone-responsive active area) and a second dip-coating operation deposits a second membrane polymer over both the first active area and the second active area (e.g., a second analyte-responsive active area) to define a bilayer membrane portion over the first active area and a uniform membrane portion over the second active area. In certain embodiments, the first membrane polymer and the second membrane polymer are different from each other. In certain embodiments, the bottom layer of the bilayer membrane portion and the uniform membrane portion comprise the same membrane polymer. In certain embodiments, the top layer of the bilayer membrane portion and the uniform membrane portion comprise the same membrane polymer. In certain embodiments, the bottom layer of the bilayer membrane portion and the uniform membrane portion comprise a different membrane polymer.

[0270] In certain embodiments, the first dip-coating operation deposits a first membrane polymer over both the first active area and the second active area, and the second dip-coating operation deposits a second membrane polymer over the first active area to define a bilayer membrane portion over the first active area. In certain embodiments, the first membrane polymer and the second membrane polymer are different from each other.

[0271] In general, the thickness of the membrane is controlled by the concentration of the membrane solution, the number of droplets of the membrane solution applied, the number of times the sensor is dipped into the membrane solution or sprayed onto the sensor, the volume of the membrane solution sprayed onto the sensor, and the like, and any combination of these factors. In certain embodiments, the membranes described herein may have a thickness ranging from about 0.1 micrometers (μm) to about 1000 μm, such as from about 1 μm to about 500 μm, from about 10 μm to about 100 μm, or from about 10 μm to about 100 μm. In certain embodiments, the sensor may be dipped into the membrane solution more than once. For example, and without limitation, the sensor (or working electrode) of the present disclosure may be dipped into the membrane solution at least twice, at least three times, at least four times, or at least five times to obtain a desired membrane thickness.

[0272] In certain embodiments, the film can cover one or more active regions, and in certain embodiments, the active regions can have a thickness of about 0.1 μm to about 10 μm, such as about 0.5 μm to about 10 μm, about 1 μm to about 10 μm, about 1 μm to about 5 μm, or about 0.1 μm to about 5 μm. In certain embodiments, a series of droplets may be applied on top of each other without substantially increasing the diameter of the droplets applied (i.e., while maintaining the desired diameter or range) to achieve a desired thickness of the active region and / or film. In certain embodiments, each single droplet may be applied and then allowed to cool or dry, followed by application of one or more additional droplets. For example, but not limited to, at least one droplet, at least two droplets, at least three droplets, at least four droplets, or at least five droplets may be added on top of each other to achieve a desired thickness of the active region.

[0273] III.How to use The present disclosure further provides methods of using the analyte sensors disclosed herein. In certain embodiments, the present disclosure provides methods for detecting an analyte. For example, but not limited to, the present disclosure provides methods for detecting one or more analytes including ketones, glucose, alcohol, lactate, and / or creatinine, or combinations thereof. In certain embodiments, the present disclosure provides methods for detecting one or more ketones. In certain embodiments, the present disclosure provides methods for detecting one or more ketones and a second analyte. In certain embodiments, the second analyte may be selected from the group consisting of glucose, alcohol, lactate, and creatinine. In certain embodiments, the second analyte comprises glucose.

[0274] In certain embodiments, the present disclosure provides a method for detecting ketone levels in a subject in need thereof. In certain embodiments, the present disclosure provides a method for detecting ketone levels in vivo in a subject. In certain embodiments, the present disclosure provides a method for detecting ketone levels in interstitial fluid in a subject. In certain embodiments, the present disclosure provides a method for detecting ketone levels in a diabetic subject. In certain embodiments, the present disclosure provides a method for detecting ketone levels in a subject undergoing a ketogenic diet. In certain embodiments, the present disclosure provides a method for detecting ketone levels in a subject in a state of ketosis or maintaining a state of ketosis. In certain embodiments, the analyte sensor of the present disclosure may be used to ensure that a subject is adhering to a ketogenic diet. For example, but not by way of limitation, the analyte sensor of the present disclosure may be used to measure the level of ketones in a sample to inform a subject to adjust or modify their diet to maintain ketosis. In certain embodiments, the present disclosure provides a method for detecting ketone levels in a subject at risk of developing ketoacidosis. In certain embodiments, the present disclosure provides a method for detecting ketone levels in a subject at risk of developing diabetic ketoacidosis. In certain embodiments, the sensor of the present disclosure can be used to monitor and / or prevent diabetic ketoacidosis. For example, but not limited to, the sensor of the present disclosure includes sensing chemistry for detecting ketones and glucose to monitor and / or prevent diabetic ketoacidosis in a subject, such as a diabetic subject. Alternatively or additionally, the sensor of the present disclosure can be used in combination with a glucose sensor to monitor and / or prevent diabetic ketoacidosis. In certain embodiments, the sensor of the present disclosure can be used in applications to monitor the level of ketones in a subject, such as to monitor compliance with a ketogenic diet, to maintain a state of ketosis, and / or to monitor and / or prevent diabetic ketoacidosis.

[0275] In certain embodiments, a method for detecting ketones includes (i) providing an analyte sensor including (a) a sensor tail including at least a first working electrode, (b) a ketone-responsive active region disposed on a surface of the first working electrode and responsive to ketones, e.g., at a low potential, the ketone-responsive active region including an enzyme system including β-hydroxybutyrate dehydrogenase and NADH oxidase responsive to ketones, and optionally a first polymer, and (c) a mass transport limiting membrane covering the ketone-responsive active region and permeable to ketones, (ii) applying a potential, e.g., a low potential, to the first working electrode, (iii) obtaining a first signal that is equal to or greater than the redox potential of the ketone-responsive active region and proportional to the concentration of ketones in a fluid in contact with the ketone-responsive active region, and (iv) correlating the first signal with the concentration of ketones in the fluid. In certain embodiments, the potential applied to the first working electrode is a potential at which NADH is not oxidized. In certain embodiments, the potential applied to the first working electrode is a potential at which NADH is not reversibly oxidized. In certain embodiments, the potential applied to the first working electrode is about +0.2 V to about +0.5 V relative to an Ag / AgCl reference. In certain embodiments, the potential applied to the first working electrode is about +0.3 V to about +0.4 V relative to an Ag / AgCl reference, for example, about +0.35 V relative to an Ag / AgCl reference.

[0276] In certain embodiments, the disclosed method may include (i) exposing an analyte sensor including (a) a sensor tail including at least a first working electrode, (b) a ketone-responsive active region disposed on a surface of the first working electrode and responsive to ketones, e.g., at a low potential, the ketone-responsive active region including an enzyme system including β-hydroxybutyrate dehydrogenase and NADH oxidase responsive to ketones, and optionally a first polymer, and (c) a mass transport limiting membrane covering the ketone-responsive active region and permeable to ketones, to a fluid including ketones, (ii) applying a potential, e.g., a low potential, to the first working electrode, (iii) obtaining a first signal that is equal to or greater than the redox potential of the ketone-responsive active region and proportional to the concentration of ketones in the fluid, and (iv) correlating the first signal with the concentration of ketones in the fluid. In certain embodiments, the potential applied to the first working electrode is a potential at which NADH is not oxidized. In certain embodiments, the potential applied to the first working electrode is a potential at which NADH is not reversibly oxidized. In certain embodiments, the potential applied to the first working electrode is about +0.2 V to about +0.5 V relative to an Ag / AgCl reference. In certain embodiments, the potential applied to the first working electrode is about +0.3 V to about +0.4 V relative to an Ag / AgCl reference, for example, about +0.35 V relative to an Ag / AgCl reference.

[0277] In certain embodiments, the method of the present disclosure may further include detecting a second analyte by providing an analyte sensor including a second active region and / or by exposing the analyte sensor including the second active region to a fluid comprising ketones and a second analyte, such as glucose. In certain embodiments, an analyte sensor for use in a method for detecting ketones and a second analyte may further include a second working electrode and a second active region disposed on a surface of the second working electrode and responsive to a second analyte different from the first analyte, the second active region including a second polymer, at least one enzyme responsive to the second analyte covalently bound to the second polymer, and optionally a redox mediator covalently bound to the second polymer, and a portion, such as a second portion, of the mass transport limiting membrane covering the second active region. Alternatively, the second active site may be covered by a second mass transport limiting membrane separate and / or different from the mass transport limiting membrane covering the ketones-responsive active region. In certain embodiments, the at least one enzyme responsive to the second analyte comprises an enzyme system comprising a plurality of enzymes collectively responsive to the second analyte, hi certain embodiments, the second analyte comprises glucose.

[0278] In certain embodiments, the membrane polymer comprises polyvinylpyridine or polyvinylimidazole. In certain embodiments, the membrane polymer comprises a copolymer of vinylpyridine and styrene. In certain embodiments, the mass transport limiting membrane of the analyte sensor comprises a membrane polymer crosslinked with a branched crosslinker comprising two or more or three or more crosslinkable groups. In certain embodiments, the branched crosslinker comprises polyethylene glycol diglycidyl ether. In certain embodiments, the branched crosslinker comprises polyethylene glycol tetraglycidyl ether.

[0279] IV. Illustrative Embodiments A. In certain non-limiting embodiments, the presently disclosed subject matter provides an analyte sensor comprising: (i) a sensor tail comprising at least a first working electrode; (ii) a ketone-responsive active region disposed on a surface of the first working electrode and comprising an enzyme system comprising β-hydroxybutyrate dehydrogenase and NADH oxidase; and (iii) a mass transport limiting membrane covering at least a portion of the ketone-responsive active region, the mass transport limiting membrane being permeable to ketones.

[0280] A1. The analyte sensor according to A, wherein the ketone-responsive active region does not contain an electron transfer agent. A2. The test substance sensor according to A or A1, wherein the ketone-responsive active region does not contain superoxide dismutase.

[0281] A3. The analyte sensor according to any one of A to A2, wherein the working electrode comprises platinum. A4. The analyte sensor according to any one of A to A2, wherein the ketone-responsive active region further comprises a stabilizer.

[0282] A5. The analyte sensor according to A4, wherein the stabilizer is serum albumin. A6. The analyte sensor according to any one of A to A5, wherein the ketone-responsive active region further comprises a crosslinker.

[0283] A7. The analyte sensor according to A6, wherein the cross-linking agent is polyethylene glycol diglycidyl ether. A8. The analyte sensor according to any one of A to A7, wherein the mass transport limiting membrane comprises polyvinylpyridine, polyvinylimidazole, polyvinylpyridine copolymer, polyacrylate, polyurethane, polyether urethane, or a combination thereof.

[0284] A9. The analyte sensor of any one of A-A7, wherein the mass transport limiting membrane comprises polyvinylpyridine, polyvinylimidazole, a copolymer of vinylpyridine and styrene, or a combination thereof.

[0285] A10. The analyte sensor of A8 or A9, wherein the mass transport limiting membrane comprises polyvinylpyridine. A11. The test substance sensor of any one of A-A10, wherein the sensor tail is configured for insertion into tissue.

[0286] A12. The test substance sensor according to any one of A to A11, wherein a ratio of β-hydroxybutyrate dehydrogenase to NADH oxidase present in the ketone-responsive active region is about 5:1 to about 1:5.

[0287] A13. The test substance sensor according to any one of A to A12, wherein the ratio of β-hydroxybutyrate dehydrogenase to NADH oxidase present in the ketone-responsive active region is about 2:1 to about 1:2.

[0288] A14. The test substance sensor according to any one of A to A13, wherein β-hydroxybutyrate dehydrogenase and NADH oxidase are present in the ketone-responsive active region in an amount of about 10% to about 80% by weight of the ketone-responsive active region.

[0289] A15. The analyte sensor according to any one of A to A14, wherein the ketone-responsive active region responds to ketones at a potential of about +0.2 V to about +0.5 V versus an Ag / AgCl reference.

[0290] A16. The analyte sensor according to any one of A to A15, wherein the ketone-responsive active region responds to ketones at a potential of about +0.3 V to about +0.4 V versus an Ag / AgCl reference.

[0291] A17. A test substance sensor described in any one of A to A16, further comprising: (iv) a second working electrode; and (v) a second active area disposed on the surface of the second working electrode and responsive to a second test substance other than ketones, the second active area having at least one enzyme responsive to the second test substance.

[0292] A18. The analyte sensor of A17, wherein a second portion of the mass transport limiting membrane covers the second active area. A19. The analyte sensor according to A17, wherein a second mass transport limiting membrane covers the second active area.

[0293] A20. The analyte sensor according to A17, wherein a second mass transport limiting membrane covers the second active area and the first active area. A21. The analyte sensor of any one of A17 to A20, wherein the second analyte comprises glucose, lactate, creatinine, or alcohol.

[0294] A22. The analyte sensor of A21, wherein the second analyte comprises glucose. A23. A test substance sensor according to any one of A to A22, configured to detect ketones in interstitial fluid from a subject.

[0295] A24. A test substance sensor according to any one of A to A23, which is implanted in a subject having diabetes. A25. A test substance sensor according to any one of A to A24, which is implanted in a subject experiencing or at risk of experiencing ketoacidosis.

[0296] A26. A test substance sensor according to any one of A to A24, which is implanted in a subject taking a ketogenic diet. A27. A test substance sensor according to any one of A to A24, which is implanted in a subject who is in a state of ketosis or who needs to maintain a state of ketosis.

[0297] A28. The analyte sensor according to any one of A to A27, wherein hydrogen peroxide produced by a reaction between the enzyme system in the ketone-responsive active region and a ketone is detected at the working electrode.

[0298] A29. The analyte sensor according to any one of A to A28, wherein the ketone-responsive active region further comprises a polymer. A30. The analyte sensor of any one of A-A29, wherein the polymer comprises polyurethane.

[0299] B. In certain non-limiting embodiments, the presently disclosed subject matter provides a method for detecting ketones, the method including: (i) providing an analyte sensor comprising: (a) a sensor tail comprising at least a first working electrode; (b) a ketone-responsive active region disposed on a surface of the first working electrode and comprising an enzyme system comprising β-hydroxybutyrate dehydrogenase and NADH oxidase; and (c) a mass transport limiting membrane covering at least a portion of the ketone-responsive active region and permeable to ketones; (ii) applying a potential to the first working electrode; (iii) obtaining a first signal that is equal to or greater than the redox potential of the ketone-responsive active region and proportional to a concentration of ketones in a fluid in contact with the ketone-responsive active region; and (iv) correlating the first signal to the concentration of ketones in the fluid.

[0300] B1. The method according to B, wherein the ketone-responsive active region does not contain an electron transfer agent. B2. The method according to B or B1, wherein the ketone-responsive active region does not include superoxide dismutase.

[0301] B3. The method of B-B2, wherein the working electrode comprises platinum. B4. The method of any one of B-B3, wherein the ketone-responsive active region further comprises a stabilizer.

[0302] B5. The method of B4, wherein the stabilizer is serum albumin. B6. The method according to any one of B-B5, wherein the ketone-responsive active region further comprises a cross-linker.

[0303] B7. The method of B6, wherein the cross-linking agent is polyethylene glycol diglycidyl ether. B8. The method of any one of B-B7, wherein the mass transport limiting membrane comprises polyvinylpyridine, polyvinylimidazole, a copolymer of vinylpyridine and styrene, or a combination thereof.

[0304] B9. The method of B8, wherein the mass transport limiting membrane comprises polyvinylpyridine. B10. The method of B8, wherein the mass transport limiting membrane comprises polyurethane. B11. The method of any one of B-B10, wherein the ketone-responsive active region further comprises a polymer.

[0305] B12. The method of B11, wherein the polymer comprises a polyurethane. B13. The method of any one of B-B12, wherein the sensor tail is configured for insertion into tissue.

[0306] B14. The method according to any one of B to B13, wherein the ratio of β-hydroxybutyrate dehydrogenase to NADH oxidase present in the ketone-responsive activity region is from about 5:1 to about 1:5.

[0307] B15. The method according to any one of B to B14, wherein the ratio of β-hydroxybutyrate dehydrogenase to NADH oxidase present in the ketone-responsive activity region is from about 2:1 to about 1:2.

[0308] B16. The method according to any one of B to B15, wherein β-hydroxybutyrate dehydrogenase and NADH oxidase are present in the ketone-responsive active region in an amount of about 10% to about 80% by weight of the ketone-responsive active region.

[0309] B17. The method of any one of B-B16, wherein the ketone-responsive active region responds to ketones at a potential of about +0.2 V to about +0.5 V versus an Ag / AgCl reference. B18. The method of any one of B-B17, wherein the ketone-responsive active region responds to ketones at a potential of about +0.3 V to about +0.4 V versus an Ag / AgCl reference.

[0310] B19. The method of any one of B-B18, wherein the analyte sensor further comprises (d) a second working electrode, and (e) a second active area disposed on a surface of the second working electrode and responsive to a second analyte other than ketones.

[0311] B20. The method of B19, wherein a second portion of the mass transport limiting membrane covers a second active area. B21. The method of B19, wherein a second mass transport limiting membrane covers the second active area.

[0312] B22. The method of any one of B19-B21, wherein the second analyte comprises glucose, lactate, creatinine, or alcohol. B23. The method of B22, wherein the second test substance is glucose.

[0313] B24. The method of any one of B-B23, wherein the fluid is interstitial fluid from the subject. B25. The method of any one of B-B24, wherein the analyte sensor is implanted in a subject having diabetes.

[0314] B26. The method of any one of B-B25, wherein the analyte sensor is implanted in a subject experiencing or at risk of experiencing ketoacidosis. B27. The method of any one of B-B26, wherein the test substance sensor is implanted in a subject consuming a ketogenic diet.

[0315] B28. The method of any one of B-B27, wherein the analyte sensor is implanted in a subject who is in a state of ketosis or who needs to maintain a state of ketosis. B29. The method according to any one of B to B28, wherein hydrogen peroxide produced by the reaction of the enzyme system in the ketone-responsive active region with a ketone is detected at a working electrode.

[0316] C. An analyte sensor according to any one of A-A30 for use in detecting ketones in a subject in need thereof. C1. Analyte sensor for use in C, wherein the subject has diabetes.

[0317] C2. An analyte sensor for use in C, wherein a subject is experiencing or at risk of experiencing ketoacidosis. C3. Analyte sensor for use in C, where the subject is on a ketogenic diet.

[0318] C4. An analyte sensor for use in C, where a subject is in a state of ketosis or needs to maintain a state of ketosis. Working Example The presently disclosed subject matter will be better understood by reference to the following examples, which are provided by way of illustration and not limitation of the presently disclosed subject matter.

[0319] Example 1: Selection of electrode potential This example provides a process for selecting an electrode potential for an analyte sensor having a ketone-responsive active region as disclosed herein.

[0320] The oxidation characteristics of hydrogen peroxide and NADH in phosphate buffered saline buffer (PBS) were determined using a platinum (Pt) electrode. The solution was maintained at a temperature of 33° C. Linear scan voltammetry was performed using a CH Instrument CHI1030B potentiostat and the results were recorded versus an Ag / AgCl reference electrode. The results are shown in FIG. 23. As shown in FIG. 23, the oxidation potential of NADH is approximately +0.6 V versus the Ag / AgCl reference. Furthermore, FIG. 23 shows that at a potential of +0.35 V versus the Ag / AgCl reference, the oxidation of hydrogen peroxide plateaus and there is very little oxidation of NADH. This potential was selected as the operating potential of the ketone sensor described herein and used in Example 2.

[0321] Example 2: Ketone Sensor This example provides a sensor for detecting β-hydroxybutyrate, which is used as a surrogate for ketones in vivo. For this example, the enzyme system of FIG. 22 was used to facilitate the detection of ketones. In particular, an enzyme system including NADH oxidase (NADHOx) and hydroxybutyrate dehydrogenase (HBDH) was used to detect β-hydroxybutyrate. The chemical composition of the sensor is shown in Table 1. The components were 10 mM 2-(N-morpholino)ethanesulfonic acid (MES) buffer, pH 5.5. [Table 1]

[0322] A Pt electrode was used as the sensor electrode. The formulation in Table 1 was deposited on the Pt electrode. A control sensor was also fabricated using the same sensing chemistry formulation in Table 1, except that NADHOx was not included in the formulation. The sensor was cured overnight. Following the curing process, the sensor was immersed in a mixture of polyvinylpyridine (PVP) and polyethylene glycol diglycidyl ether 400 (PEGDGE400). The sensor was then cured again overnight. A beaker test was then performed in 100 mM PBS buffer at 33°C. The sensor current was recorded at +0.35 V versus an Ag / AgCl reference electrode using a CH Instrument CHI1030B potentiostat.

[0323] FIG. 24 shows the current response for the four NADHOx sensors and the control. As shown, the current increased over the course of several minutes after exposure to a new concentration of β-hydroxybutyrate, and then stabilized. It is also shown that this effect is not observed in the control sensor, indicating that the production of hydrogen peroxide is proceeding via the proposed sensing mechanism. FIG. 25 provides an exemplary plot of the current response versus the concentration of β-hydroxybutyrate for each of the NADHOx sensors and the control.

[0324] Although the subject matter disclosed herein and its advantages have been described in detail, it should be understood that various changes, substitutions, and alterations can be made therein without departing from the spirit and scope of the disclosed subject matter. Moreover, the scope of the present application is not intended to be limited to the particular embodiments of the processes, machines, manufacture, and compositions of matter, methods, and processes described herein. As one skilled in the art will readily appreciate from the disclosed subject matter of the presently disclosed subject matter, any now existing or later developed process, machine, manufacture, composition of matter, method, or step that performs substantially the same function or achieves substantially the same results as the corresponding embodiments described herein may be utilized in accordance with the subject matter disclosed herein. Accordingly, it is intended that the appended claims include within their scope such processes, machines, manufacture, compositions of matter, methods, or steps.

[0325] Various patents, patent applications, publications, product descriptions, protocols, and sequence accession numbers are cited throughout this application, the inventions of which are hereby incorporated by reference in their entireties for all purposes.

Claims

1. A test substance sensor for detecting ketones in vivo, comprising: (i) at least a first working electrode comprising a conductive material that oxidizes hydrogen peroxide; (ii) a ketone-responsive active area disposed on a surface of the first working electrode, the ketone-responsive active area comprising an enzyme system for producing hydrogen peroxide in the presence of a ketone, the enzyme system comprising β-hydroxybutyrate dehydrogenase and NADH oxidase, the ketone-responsive active area being free of superoxide dismutase; and (iii) a mass transport limiting membrane that covers at least a portion of the ketone-responsive active region and is permeable to ketones; the ketone-responsive active region responds to ketones at potentials between +0.2 V and +0.5 V versus an Ag / AgCl reference; The analyte sensor, wherein a distal portion of the analyte sensor is configured to be inserted into the skin of a subject to detect ketones in vivo.

2. 2. The analyte sensor of claim 1, wherein the ketone-responsive active region does not include an electron transfer agent.

3. 3. The analyte sensor according to claim 1, wherein the ketone-responsive active region further comprises a stabilizer for stabilizing the enzyme system.

4. 4. The analyte sensor of claim 1, wherein the mass transport limiting membrane comprises polyvinylpyridine, polyvinylimidazole, polyvinylpyridine copolymer, polyacrylate, polyurethane, polyether urethane, or a combination thereof.

5. 5. The analyte sensor of claim 4, wherein the mass transport limiting membrane comprises polyvinylpyridine.

6. (iv) a second working electrode; and 6. The analyte sensor of claim 1, further comprising: (v) a second active area disposed on a surface of the second working electrode and responsive to a second analyte other than ketones, the second active area comprising at least one enzyme responsive to the second analyte.

7. A test substance sensor as described in claim 6, further comprising a second mass transport limiting membrane covering the second active region.

8. The analyte sensor of claim 6 or 7, wherein the second analyte comprises glucose.

9. 2. A method of controlling an operation of a test substance sensor according to claim 1, wherein the test substance sensor is introduced into tissue, the method comprising: (i) applying a potential to the first working electrode; (ii) obtaining a first signal proportional to a concentration of ketones in a fluid in contact with the ketone-responsive active region at or above the redox potential of the ketone-responsive active region; and (iii) correlating the first signal with a concentration of ketones in the fluid.

10. The method of claim 9 , wherein the ketone-responsive active region does not contain an electron transfer agent.

11. 11. The method of claim 9 or 10, wherein the conductive material comprises platinum.

12. The control method according to claim 9 , wherein the ketone-responsive active region further comprises a stabilizer for stabilizing the enzyme system.

13. 13. The method of claim 9, wherein the mass transport limiting membrane comprises polyvinylpyridine, polyvinylimidazole, polyvinylpyridine copolymer, polyacrylate, polyurethane, polyether urethane, or a combination thereof.

14. 14. The method of claim 13, wherein the mass transport limiting membrane comprises polyvinylpyridine.

15. The analyte sensor comprises: A second working electrode; and a second active area disposed on a surface of the second working electrode and responsive to a second analyte different from ketones; The method of claim 9 , further comprising: a second mass transport limiting membrane covering the second active region.

16. A test substance sensor as described in claim 7, wherein the second mass transport limiting membrane also covers the ketones-responsive active region.

17. A test substance sensor as described in any one of claims 1-8, 16, wherein the conductive material comprises platinum.

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