NAD (p) depots for NAD (p) - dependent enzyme-based sensors

Abstract

To provide an NAD (P) depot for an NAD (P) - dependent enzyme-based sensor.SOLUTION: The present disclosure provides an analyte sensor comprising one or more NAD (P) - dependent enzymes and an internal source of NAD (P) for the detection of an analyte. The present disclosure further provides methods of using such analyte sensors to detect one or more analytes present in a biological sample of a subject and methods of making the analyte sensors.SELECTED DRAWING: Figure 1A

Description

[Technical Field]

[0001] The subject matter described herein relates to analyte sensors comprising an NAD(P) depot and methods of use thereof. [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 glucose levels may allow people suffering from diabetes to take appropriate corrective measures, including administering medication or consuming certain food or beverage products, to avoid serious physiological problems. Other analytes may also be desirable for monitoring other physiological conditions. In certain instances, it may be desirable to monitor two or more analytes to monitor one or more physiological conditions, especially when a person suffers from a co-occurring condition in which two or more analytes are combined with each other and simultaneously cause dysregulation.

[0003] Analyte monitoring in an individual may be performed periodically or continuously over a period of time. Periodic analyte monitoring can be performed by taking samples of bodily fluids, such as blood or urine, at set intervals and analyzing them ex vivo. Periodic ex vivo analyte monitoring may be sufficient to measure the physiological status of many individuals. However, ex vivo analyte monitoring can be inconvenient or painful in some cases. Furthermore, if analyte measurements are not obtained at the appropriate time, there is no way to recover lost data.

[0004] Continuous analyte monitoring can be performed using one or more sensors that remain at least partially implanted within an individual's tissue, such as cutaneously, subcutaneously, or intravenously, so that analysis can be performed in vivo. The implanted sensors can collect analyte data on demand, on a set schedule, or continuously, depending on the individual's specific health needs and / or previously measured analyte levels. Analyte monitoring with in vivo implanted sensors may be a more desirable approach for individuals with severe analyte dysregulation and / or rapidly fluctuating analyte levels, but may be beneficial for other individuals as well. Because implanted analyte sensors often remain within an individual's tissue 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.

[0005] However, implantable sensors can suffer from short lifespans or reduced sensitivity. For example, many implantable sensors use enzymes for continuous in vivo monitoring of analyte levels, and many of these enzymes depend on coenzymes for activity. For example, nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP) are two of the most important coenzymes found in living cells and are frequently required for the activity of enzymes, such as dehydrogenases, found in implantable sensors. The amount of NAD or NADP available for use by enzymes present in an implantable sensor can affect the sensor's sensitivity for accurately monitoring analyte levels in vivo. Under certain circumstances, exogenous NAD or NADP may not be present in sufficient amounts to support sensor operation, or even if sufficient exogenous amounts are present, such molecules may be too large to easily diffuse into regions of the sensor that harbor NAD- or NADP-dependent enzymes, which can result in reduced sensitivity. Therefore, there is a need in the art for sensors that retain their sensitivity for longer periods of time. Summary of the Invention

[0006] The objects and advantages of the disclosed subject matter will be set forth in and become apparent from the following description and may 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 accompanying drawings.

[0007] To achieve these and other advantages, and in accordance with the objectives of the disclosed subject matter, as embodied and broadly described, the disclosed subject matter provides an analyte sensor that includes an internal source of NAD(P). For example, but not by way of limitation, the disclosed analyte sensor includes an internal source of NAD(P), a permeable polymer covering the internal source of NAD(P), at least a first working electrode disposed on a surface of the permeable polymer, an analyte-responsive active region disposed on a surface of the first working electrode, and optionally a mass transport-limiting membrane covering at least the analyte-responsive region and permeable to the analyte.

[0008] In certain embodiments, the analyte is selected from the group consisting of glucose, a ketone, an alcohol, lactate, and combinations thereof. In certain embodiments, the analyte is glucose. In certain embodiments, the analyte is a ketone. In certain embodiments, the analyte is lactate. In certain embodiments, the analyte is an alcohol, such as ethanol.

[0009] In certain embodiments, the first working electrode is a permeable working electrode. In certain embodiments, the permeable working electrode comprises carbon nanotubes. In certain embodiments, the analyte-responsive active region comprises an NAD(P)-dependent enzyme. In certain embodiments, the NAD(P)-dependent enzyme is an NAD(P)-dependent dehydrogenase. In certain embodiments, the NAD(P)-dependent enzyme present in the analyte-responsive active region is β-hydroxybutyrate dehydrogenase. In certain embodiments, the NAD(P)-dependent enzyme present in the analyte-responsive active region is glucose dehydrogenase. In certain embodiments, the NAD(P)-dependent enzyme present in the analyte-responsive active region is lactate dehydrogenase. In certain embodiments, the NAD(P)-dependent enzyme present in the analyte-responsive active region is alcohol dehydrogenase. In certain embodiments, the NAD(P)-dependent enzyme present in the analyte-responsive active region is diaphorase.

[0010] In certain embodiments, the permeable polymer comprises a poly(propylene glycol)-based polymer. In certain embodiments, the permeable polymer comprises poly(propylene glycol) methacrylate and / or 2-hydroxyethyl methacrylate.

[0011] In certain embodiments, the analyte-responsive active region further comprises diaphorase.In certain embodiments, the analyte-responsive active region further comprises a redox mediator. In certain embodiments, the analyte sensor of the present disclosure further includes a second working electrode and a second active area disposed on the second working electrode and responsive to a second analyte different from the first analyte. 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.

[0012] The present disclosure further provides methods for monitoring an analyte in vivo. In certain embodiments, the method may include providing an analyte sensor comprising: (a) an internal source of NAD(P); (b) a permeable polymer coating the internal source of NAD(P); (c) at least a first working electrode disposed on the surface of the permeable polymer and being a permeable working electrode; (d) an analyte-responsive active region disposed on the surface of the first working electrode and comprising an NAD(P)-dependent enzyme; and (e) a mass transport-limiting membrane coating at least the analyte-responsive region and permeable to the analyte. In certain embodiments, the method further includes applying a potential to the first working electrode; obtaining a first signal at or above the redox potential of the first active region that is proportional to the concentration of the first analyte in a fluid contacting the first active region; and correlating the first signal to the concentration of the first analyte in the fluid.

[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 the second working electrode and responsive to a second analyte different from the first analyte, hi certain embodiments, the second active area includes at least one enzyme responsive to the second analyte, and the second portion of the mass transport limiting membrane covers the second active area. [Brief explanation of the drawings]

[0014] The following figures are included to illustrate certain aspects of the present disclosure and should not be considered exclusive embodiments. The disclosed subject matter is capable of numerous modifications, variations, 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 2A]1 is a 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 block diagram illustrating an example embodiment of a sensor control device. [Figure 2D] 1 is a 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] 10A-10C are proximal perspective views illustrating an example embodiment of a user preparing a tray for assembly. [Figure 3B] 10A-10C are side views illustrating an example embodiment of a user preparing an applicator device for assembly. [Figure 3C] 10A-10C are proximal perspective views illustrating an example embodiment of a user inserting an applicator device into a tray during assembly. [Figure 3D] 10A-10C 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 and 1B are side views illustrating 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 released. FIG. [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] FIG. 4E is a side cross-sectional view of the applicator device of FIG. 4D. [Figure 5] FIG. 10 is a proximal perspective view showing an example embodiment of a tray with an associated sterilization lid. [Figure 6A] 10A-10C are proximal perspective cross-sectional views illustrating an example embodiment of a tray with a sensor delivery component. [Figure 6B] FIG. 13 is a proximal perspective view showing the sensor delivery component. [Figure 7A] FIG. 1 illustrates an isometric exploded top view of an exemplary sensor control device. [Figure 7B] FIG. 2 is an isometric exploded bottom view of an exemplary sensor control device. [Figure 8A] 1A and 1B are assembly and cross-sectional views of an on-body device including an integrated connector for sensor assembly. [Figure 8B] 1A and 1B are assembly and cross-sectional views of an on-body device including an integrated connector for sensor assembly. [Figure 8C] 1A and 1B 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. 10 is an isometric view of another example sensor control device. [Figure 10B] FIG. 10 is a side view of another example of a sensor control device. [Figure 11A] 10A-10B are side cross-sectional views illustrating the assembly of a sensor applicator having the sensor control device of FIGS. [Figure 11B] 10A-10B are side cross-sectional views illustrating the assembly of a sensor applicator having the sensor control device of FIGS. [Figure 11C] 10A-10B are side cross-sectional views illustrating the assembly of a sensor applicator having the sensor control device of FIGS. [Figure 12A] 10A-10B are side cross-sectional views illustrating the assembly and disassembly of an example embodiment of a sensor applicator having the sensor control device of FIGS. [Figure 12B] 10A-10B are side cross-sectional views illustrating the assembly and disassembly of an example embodiment of a sensor applicator having the sensor control device of FIGS. [Figure 12C] 10A-10B are side cross-sectional views illustrating the assembly and disassembly of an example embodiment of a sensor applicator having the sensor control device of FIGS. [Figure 13A] 1A-1C show cross-sectional views illustrating an example embodiment of an applicator during a placement stage. [Figure 13B] 1A-1C show cross-sectional views illustrating an example embodiment of an applicator during a placement stage. [Figure 13C] 1A-1C show cross-sectional views illustrating an example embodiment of an applicator during a placement stage. [Figure 13D] 1A-1C show cross-sectional views illustrating an example embodiment of an applicator during a placement stage. [Figure 13E] 1A-1C show cross-sectional views illustrating an example embodiment of an applicator during a placement stage. [Figure 13F] 1A-1C 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 an example of an operational state of a sensor according to an exemplary embodiment of the disclosed subject matter. [Figure 16] 1 is a diagram illustrating an example of the 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 secure 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 containing a single active area. [Figure 18B] 1 shows a schematic cross-sectional view of an analyte sensor containing a single active area. [Figure 18C]1 shows a schematic cross-sectional view of an analyte sensor containing a single active area. [Figure 19A] 1 shows a schematic cross-sectional view of an analyte sensor containing two active regions. [Figure 19B] 1 shows a schematic cross-sectional view of an analyte sensor containing two active regions. [Figure 19C] 1 shows a schematic cross-sectional view of an analyte sensor containing two active regions. [Figure 20] 1 shows a schematic cross-sectional view of an analyte sensor containing two active regions. [Figure 21A] FIG. 1 shows a perspective view of an analyte sensor including two active areas disposed on separate working electrodes. [Figure 21B] FIG. 1 shows a perspective view of an analyte sensor including two active areas disposed on separate working electrodes. [Figure 21C] FIG. 1 shows a perspective view of an analyte sensor including two active areas disposed on separate working electrodes. [Figure 22] 1 provides a cross-sectional schematic of an exemplary sensor including a NAD(P) depot for controlled NAD(P) release. [Figure 23A] 1 provides a schematic diagram of an exemplary analyte sensor including a NAD depot. [Figure 23B] 1 provides a cross-sectional view of an exemplary analyte sensor excluding the NAD depot for use as a control. [Figure 24] 23A provides a stability profile of ketone detection over time by an analyte sensor with an NAD depot (shown in FIG. 23A) compared to an analyte sensor without an NAD depot (shown in FIG. 23B). DETAILED DESCRIPTION OF THE INVENTION

[0015] The present disclosure is directed to analyte sensors that include one or more active regions that include nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP)-dependent enzymes (collectively referred to herein as "NAD(P)-dependent enzymes"). In particular, the analyte sensors of the present disclosure include an internal reservoir of the cofactors NAD and / or NADP (collectively referred to herein as "NAD(P)") for the NAD(P)-dependent enzymes.

[0016] The use of an internal reservoir of NAD(P) within the analyte sensor may overcome some of the limitations associated with analyte sensors that contain NAD(P)-dependent enzymes. For example, the amount of exogenous NAD(P) present in the environment surrounding the sensor may not be sufficient to support the operation of the analyte sensor, potentially resulting in reduced sensor sensitivity. Additionally, even if sufficient exogenous NAD(P) is present in the environment surrounding the analyte sensor, the molecular size of NAD(P) may prevent the molecule from diffusing through the surrounding sensor membrane to reach one or more NAD(P)-dependent enzymes present in the sensing chemistry layer, e.g., the active region, of the analyte sensor.

[0017] The present disclosure provides analyte sensors that include an internal source of NAD(P) that can release NAD(P) over an extended period of time to enable in vivo analyte monitoring. In certain embodiments, the internal NAD(P) source (also referred to herein as an "NAD(P) depot") can be coated with or dispersed within a permeable layer (e.g., a polymeric permeable layer) that controls the diffusion of NAD(P) from the NAD(P) depot to maintain a sufficient concentration of NAD(P) for the sensing chemistry during use of the analyte sensor.

[0018] The present disclosure further provides methods of detecting analytes using the disclosed sensors, and methods of manufacturing the disclosed analyte sensors. For purposes of clarity, but not limitation, the detailed description of the subject matter of this disclosure is divided into the following subsections.

[0019] I. Definition; II. Analyte sensor; 1. General structure of the analyte sensor system; 2.NAD(P) Depot; 3. Enzymes; 4. Redox mediators; 5. Polymer backbone; 6. Mass transfer limiting membrane; 7. Interference domain; and 8. Manufacturing; III. Test substance monitoring.

[0020] 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 herein to provide further guidance to the practitioner in describing the compositions and methods of the present disclosure and how to make and use them.

[0021] 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 also consistent with the meaning of "one or more," "at least one," and "one or more."

[0022] 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 explicitly stated or not.

[0023] The term "about" or "approximately" means within an acceptable error range for a particular value as determined by a person skilled in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 3 or more than 3 standard deviations, according to the practice in the art. Alternatively, "about" can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and even more preferably up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, this term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold of a value.

[0024] As used herein, "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 the like.

[0025] The term "biological fluid," as used herein, refers to any bodily fluid or bodily fluid derivative 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, and the like. In certain embodiments, the biological fluid is dermal fluid or interstitial fluid. In certain embodiments, the biological fluid is interstitial fluid.

[0026] The term "electrolysis," as used herein, refers to the electro-oxidation or electro-reduction of a compound either directly at an electrode or via one or more electron transfer agents (e.g., redox mediators or enzymes).

[0027] The terms "enzyme composition" and "sensing chemical," used interchangeably herein, refer to a composition comprising one or more enzymes for detecting and / or measuring an analyte. In certain non-limiting embodiments, the enzyme composition can comprise one or more enzymes, a polymer, a redox mediator, and / or a cross-linking agent.

[0028] As used herein, the term "homogeneous membrane" refers to a membrane comprising a single type of membrane polymer. As used herein, the term "multi-component membrane" refers to a membrane comprising two or more types of membrane polymers.

[0029] As used herein, the term "NAD(P)" refers to the cofactor NAD (and its reduced form NADH) or NADP (and its reduced form NADPH) or derivatives thereof. As used herein, the term "NAD(P)-dependent enzyme" refers to an enzyme that utilizes NAD (and its reduced form NADH) or NADP (and its reduced form NADPH) as a coenzyme in an oxidation-reduction reaction.

[0030] As used herein, the term "permeable electrode" refers to an electrode that is constructed from a material that allows molecules (e.g., NAD(P)) to pass through the material of the electrode. 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.

[0031] As used herein, the term "redox mediator" refers to an electron transfer agent that transfers electrons between an analyte or analyte reductase or analyte oxidase and an electrode, either directly or via one or more additional electron transfer agents. In certain embodiments, a redox mediator comprising a polymer backbone may also be referred to as a "redox polymer."

[0032] 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, as used herein, the term "counter electrode" can refer to both a counter electrode and a counter electrode that also functions as a reference electrode.

[0033] 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.

[0034] The publications discussed herein are provided solely 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 publication by virtue of prior disclosure. Further, the publication dates provided may be different from the actual publication dates, which may need to be independently confirmed.

[0035] 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 may be provided to a user in a sterile package with the electronics housing of the sensor control unit housed therein. According to some embodiments, a structure separate from the applicator, such as a container, may also be provided to a user in a sterile package with the sensor module and tip module contained therein. The 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 may 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 of the sensor being improperly inserted or damaged, or eliciting an adverse physiological response. Other improvements and advantages are also provided. Various configurations of these devices are described in detail by way of example embodiments only.

[0036] 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 may be used with in vivo analyte monitoring systems that incorporate in vitro capabilities, as well as solely in vitro or ex vivo analyte monitoring systems, including systems that are completely non-invasive.

[0037] Furthermore, for any and all embodiments of the methods disclosed herein, systems and devices capable of performing each of those 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 performance of any method step. These sensor control device embodiments may be used, or may be capable of being used, to perform steps performed by the sensor control device from any method described herein.

[0038] 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 involving analyte detection over time. As used herein, "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. 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.

[0039] 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 particularly 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 may be actuated by motion so that the tip is not retracted until the user pulls the applicator away from the skin. As a result, these embodiments may 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 sensor damage 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 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 components during sensor insertion. These embodiments can therefore reduce the likelihood of capillary disruption, which can lead to 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 inaccurate sensor readings that may be caused by trauma at the insertion site, some embodiments of the present disclosure can reduce the depth of needle end penetration relative to the sensor tip during insertion.

[0040] However, before describing these aspects of the embodiments in detail, it is desirable to first 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.

[0041] There are various types of in-vivo analyte monitoring systems. A "continuous analyte monitoring" system (or "continuous glucose monitoring" system), for example, may transmit data from a sensor controller to a reader device continuously and 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, may transmit data from the 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 may also operate without the need for fingerstick calibration.

[0042] In vivo analyte monitoring systems can be distinguished from "in vitro" systems that 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.

[0043] An in-vivo monitoring system may include a sensor that contacts a user's bodily fluids while positioned in vivo and detects the analyte level 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 analyte detection. 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 communications" devices or units, to name a few.

[0044] 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. These devices and variations thereof may be referred to as "handheld reader devices," "reader devices" (or simply "readers"), "handheld electronic devices" (or simply "handhelds"), "portable data processing" devices or units, "data receivers," "receiver" devices or units (or simply "receivers"), or "remote" devices or units, to name a few. Other devices, such as personal computers, are also utilized with or incorporated into in-vivo and in-vitro monitoring systems.

[0045] 1. General structure of the test substance sensor system A. Exemplary In Vivo Analyte Monitoring Systems 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. Here, the sensor applicator 150 can be used to deliver the sensor control device 102 to a monitoring location on a user's skin, with the sensor 104 maintained in place for a period of time by an adhesive patch 105. The sensor control device 102 can communicate with the reader device 120 via a communication path or link 140, further described in FIGS. 2B and 2C, using wired or wireless, unidirectional or bidirectional, and encrypted or unencrypted 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 with respect to FIG. 2A below. The reader device 120, according to certain embodiments, may provide an output medium for visually indicating 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.

[0046] The reader device 120 can communicate with a local computer system 170 via communication path 141, which can be wired or wireless, unidirectional or bidirectional, and encrypted or unencrypted. The local computer system 170 can include one or more of a laptop, desktop, tablet, phablet, smartphone, set-top box, video game console, remote terminal, 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, similar to how the reader device 120 can communicate with the network 190 via communication path 142, via the wired or wireless technologies already mentioned. The network 190 can be any of a private network, a public network, a local area network, a wide area network, or some other network. The trusted computer system 180 may include a server, provide authentication services and secure data storage, and communicate with the network 190 via communication path 144 using wired or wireless technology. According to certain embodiments, the local computer system 170 and / or the trusted computer system 180 may be accessible by individuals other than the primary user who have an interest 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 touchscreen interface.

[0047] The sensor control device 102 includes a sensor housing that can 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 or the reader device 120. According to certain embodiments, the sensor 104 protrudes from the underside of the sensor housing and extends through an adhesive layer 105 adapted to adhere the sensor housing to a tissue surface, such as skin.

[0048] FIG. 1B illustrates the operating environment of an analyte monitoring system 100a that can embody the technology described herein. The analyte monitoring system 100a can include a system of components designed to provide monitoring of a parameter, such as an analyte level, in a human or animal body, or provide other operations based on the configuration of various components. As embodied herein, the system can include a low-power analyte sensor 110, or simply a “sensor,” that is 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 to remain functional when selectively removed and reattached. The low-power analyte monitoring system 100a may further include a data reader device 120 or a multipurpose data receiving device 130 configured as described herein to facilitate retrieval and delivery of data, including analyte data, from the analyte sensor 110.

[0049] As embodied herein, analyte monitoring system 100a may include software or firmware libraries or applications provided to a third party, for example, via remote application server 150 or application storefront server 160, and embedded in a multipurpose hardware device 130, such as a mobile phone, tablet, personal computing device, or other similar computing device capable of communicating with analyte sensor 110 via a communications link. Multipurpose hardware may further include embedded devices, including, but not limited to, insulin pumps or insulin pens, having embedded libraries configured to communicate with analyte sensor 110. While the illustrated embodiment of analyte monitoring system 100a includes only one of each of the illustrated devices, the present disclosure contemplates that analyte monitoring system 100a may incorporate multiple of each of the respective components interacting throughout the system. For example, but not limited to, as embodied herein, data reader device 120 and / or multipurpose 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 data visualization or analysis for secondary display to a user or other authorized party.

[0050] The sensor 104 of FIG. 1A is adapted to be at least partially inserted into a tissue of interest, such as into the dermal layer or 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 region containing, for example, one or more NAD(P)-dependent enzymes to detect an analyte. A counter electrode may be present in combination with the at least one working electrode. Specific electrode configurations on the sensor tail are described in more detail below.

[0051] One or more mass transport limiting membranes may cover the active area, as described in more detail below. The active region can be configured to detect specific analytes described herein. For example, without limitation, analytes can include 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, etc. In certain embodiments, analytes for detection using the disclosed analyte sensors include alcohol, ketones, creatinine, glucose, and lactate. In certain embodiments, the active region can be configured to detect two or more analytes described herein. In certain embodiments, the active region of a sensor of the present disclosure is configured to detect ketones. In certain embodiments, the active region of a sensor of the present disclosure is configured to detect glucose. In certain embodiments, the active region of a sensor of the present disclosure is configured to detect lactate. In certain embodiments, the active area of ​​the sensor of the present disclosure is configured to detect creatinine. In certain embodiments, the active area of ​​the sensor of the present disclosure is configured to detect alcohol, such as ethanol.

[0052] 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.

[0053] 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 pointed tip. As will be readily recognized by one of ordinary skill in the art, other types of introducers, such as a sheath or blade, may be present in alternative embodiments. More specifically, the needle or other introducer may be temporarily present near the sensor 104 prior to tissue insertion and then withdrawn. While present, the needle or other introducer may facilitate the insertion of the sensor 104 into the tissue by opening an access path for the sensor 104 to follow. For example, without limitation, according to one or more embodiments, the needle may facilitate penetration of the epidermis as an access path to the dermis to allow implantation of the sensor 104 to occur. After opening the access path, the needle or other introducer may be withdrawn so that it does not present a sharps hazard. 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 non-limiting embodiments, suitable needles may be similar in cross-sectional diameter and / or tip design to acupuncture needles, which may have a cross-sectional diameter of about 250 microns (250 μm). However, suitable needles may have larger or smaller cross-sectional diameters if needed for a particular application.

[0054] In certain embodiments, the tip of the needle (while present) may be angled beyond the end of the sensor 104, so 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, so that the needle similarly opens an access path for the sensor 104. In either case, the needle may be subsequently withdrawn after facilitating sensor insertion.

[0055] B. Exemplary Reader Device 2A is a block diagram illustrating an example embodiment of a reader device configured as a smartphone. Here, reader device 120 may include display 122, input component 121, and processing core 206 including communication processor 222 connected to memory 223 and application processor 224 connected to memory 225. Separate memory 230, RF transceiver 228 with antenna 229, and power supply 226 with power management module 238 may also be included. A multi-function transceiver 232 capable of communicating with antenna 234 via Wi-Fi, NFC, Bluetooth, BTLE, and GPS may further be included. As will be understood by those skilled in the art, these components are electrically and communicatively connected in a manner to create a functional device.

[0056] C. Exemplary Data Receiving Device Architecture For purposes of illustration 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 multi-purpose data receiving device 130 include components germane to the discussion of the analyte sensor 110 and its operation, and additional components may be included. In certain embodiments, the data receiving device 120 and multi-purpose data receiving device 130 may be or include components provided by third parties, and are not necessarily limited to including devices made by the same manufacturer as the sensor 110.

[0057] 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 may 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.).

[0058] The communications module 4040 may include a BLE module 4041 and an NFC module 4042. The data receiving device 120 may be configured to wirelessly connect to the analyte sensor 110, send commands to the analyte sensor 110, 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 particular module of the communications module 4040 (e.g., the BLE module 4042 or the NFC module 4043) with respect to the analyte sensor 110 described herein. For example, the data receiving device 120 may use a first module of the communications module 4040 to issue commands (e.g., an activation command for the sensor's data broadcast mode, a pairing command for identifying the data receiving device 120) to the analyte sensor 110 and use a second module of the communications module 4040 to receive data from and send data to the analyte sensor 110. The data receiving device 120 may be configured to communicate with the user device 140 via a universal serial bus (USB) module 4045 of the communication module 4040 .

[0059] 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 known as Wi-Fi 4), 802.11ac (also known as Wi-Fi 5), and 802.11ax (also known as Wi-Fi 6)). 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 communication module 5040 of the analyte sensor 120 may similarly include a cellular radio module or a Wi-Fi radio module.

[0060] As embodied herein, the onboard storage 4030 of the data receiving device 120 may store 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 may be configured to communicate with a remote application server 150 over a wide area network. As embodied herein, the analyte sensor 110 may provide data to the data receiving device 120 or the multi-purpose data receiving device 130. The data receiving device 120 may transmit the data to the user computing device 140. The user computing device 140 (or the multi-purpose data receiving device 130) may then transmit the data to the remote application server 150 for processing and analysis.

[0061] 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 particular embodiments, the data receiving device 120 may be configured to interface with the analyte sensor 110 and act based on 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.

[0062] 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), which may have most of the processing power for rendering final 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 communications circuit 168 (which may be implemented as a transmitter, receiver, transceiver, passive circuitry, or in other ways according to a communications 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 (and portions of) multiple different chips.

[0063] Memory 163 is also included in ASIC 161 and may be shared by the various functional units present in ASIC 161 or distributed among two or more of them. Memory 163 may also be a separate chip. Memory 163 may be volatile and / or non-volatile memory. In this embodiment, ASIC 161 is connected to a power source 170, which may be a button cell battery or the like. AFE 162 interfaces with and receives measurement data from in-vivo analyte sensor 104 and outputs the data in digital form to 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 communication circuitry 168 for transmission via antenna 171 to reader device 120 (not shown), where minimal further processing by a resident software application is required to display the data, for example.

[0064] 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 on ASIC 161. Processor 166 is integrated with power management circuitry 164 and communications 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 exemplary embodiment, AFE 162 is combined with power management circuitry 164 and processor 166 on one chip, and communications circuitry 168 is on a separate chip. In another exemplary embodiment, both AFE 162 and communications circuitry 168 are on one chip, and processor 166 and power management circuitry 164 are on another chip. Note that other chip combinations are possible, including three or more chips, each performing the distinct functions described or sharing one or more functions for fail-safe redundancy.

[0065] For purposes of illustration and not limitation, reference is made to an exemplary embodiment of an analyte sensor 110 for use in accordance with the disclosed subject matter shown in Figure 2E. Figure 2E shows a block diagram of an example analyte sensor 110 in accordance with an exemplary embodiment that is compatible with the security architecture and communication schemes described herein.

[0066] As embodied herein, the analyte sensor 110 may include an application specific integrated circuit ("ASIC") 5000 communicatively coupled to a communications 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 the authentication and encryption security architecture. The storage memory 5030 may store programming instructions for the sensor 110. As embodied herein, a specific communications chipset may be incorporated into the ASIC 5000 (e.g., an NFC transceiver 5025). The ASIC 5000 may receive power from a power module 5050, such as an on-board battery, or from NFC pulses. The storage memory 5030 of the ASIC 5000 may be programmed to include information such as an identifier for 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 rewritable or one-time programming (OTP) memory that can be updated using techniques described herein to extend the usefulness of the sensor 110.

[0067] As embodied herein, the communications 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 in the analyte monitoring system 100. By way of example only and not limitation, an example communications 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 communications protocol optimized to simplify pairing of Bluetooth® devices for end users. The communications module 5040 can send and receive data and commands via interaction with a similarly capable communications module in the data receiving device 120 or user device 140. The communications module 5040 may include additional or alternative chipsets for use with personal area networks according to the IEEE 802.15 protocol, IEEE 802.11 protocol, infrared communications according to the Infrared Data Association standard (IrDA), or other similar short-range communications schemes.

[0068] To perform its functionality, sensor 100 may further include suitable sensing hardware 5060 appropriate for that function. As embodied herein, 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 the level of one or more analytes in the bodily fluid.

[0069] E. Exemplary Assembly Process for a Sensor Control Unit The components of the sensor control device 102 may be acquired by the user in multiple packages that require final assembly by the user before delivery to the appropriate user location. Figures 3A-3D show an example embodiment of a user assembly process for the sensor control device 102, including preparation of the separate components before joining the components to prepare the sensor for delivery. Figures 3E-3F show an example embodiment of delivery of the sensor control device 102 to the appropriate user location by selecting the appropriate delivery location and applying the device 102 to that location.

[0070] 3A is a proximal perspective view showing an example embodiment of a user preparing a container 810, here configured as a tray (although other packaging may be used), for the assembly process. The user may accomplish this preparation by, for example, removing the lid 812 from the tray 810 by peeling the non-adhesive portion of the lid 812 from the tray 810 so that the adhesive portion of the lid 812 is removed, 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.

[0071] 3B is a side view illustrating an example embodiment of a user preparing 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.

[0072] 3C is a proximal perspective view showing an example embodiment of a user inserting 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 from housing 702, and platform 808 from 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).

[0073] While the housing 702 is advanced distally, the sheath 704 is maintained in position within the platform 808 relative to the housing 702 and couples with the platform 808, advancing the platform 808 distally relative to the tray 810. This process unlocks the platform 808 and folds it into the tray 810. The sheath 704 contacts and disengages a locking feature (not shown) in the tray 810 that prevents the sheath 704 from moving (relatively) while the housing 702 continues to advance the platform 808 distally. When the advancement of the housing 702 and platform 808 is complete, the sheath 704 is permanently unlocked from the housing 702. A tip and sensor (not shown) in the tray 810 may couple with an electronics housing (not shown) in the housing 702 at the end of distal advancement of the housing 702. The operation and interaction of applicator device 150 and tray 810 is further described below.

[0074] 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 uncoupling 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.

[0075] 3E is a proximal perspective view illustrating an example embodiment in which a patient uses applicator device 150 to apply sensor control unit 102 to a target area of ​​skin, such as the abdomen or other suitable location. Distal advancement of housing 702 collapses sheath 704 within housing 702, and the sensor is applied to the target location so that the adhesive layer on the bottom side of sensor control unit 102 adheres to the skin. When housing 702 is fully advanced, the tip is automatically retracted, while the sensor (not shown) is left in place to measure the analyte level.

[0076] 3F is a proximal perspective view of an example patient embodiment with the sensor control unit 102 in the application position. The user can then remove the applicator 150 from the application site.

[0077] 3A-3F and elsewhere herein, system 100 may reduce or eliminate the possibility of accidental breakage, permanent deformation, or incorrect assembly of applicator components compared to prior art systems. Because applicator housing 702 directly engages platform 808 while sheath 704 unlocks, rather than indirect engagement through sheath 704, the relative angle between sheath 704 and housing 702 does not result in breakage or permanent deformation of arms or other components. The likelihood of relatively large forces (as in conventional devices) during assembly is reduced, which in turn reduces the likelihood of user assembly failure.

[0078] F. Exemplary Sensor Applicator Device Figure 4A is a side view of an example embodiment of applicator device 150 coupled with screw cap 708. This is an example of how applicator 150 may be shipped and received by a user before assembly with a sensor by the user. Figure 4B is a side perspective view of applicator 150 and cap 708 after the coupling has been removed. Figure 4C is a perspective view of an example embodiment of the distal end of applicator device 150 with electronics housing 706 and adhesive patch 105 removed from their positions in sensor carrier 710 of sheath 704 while cap 708 is in place.

[0079] 4D-4G, for purposes of illustration and not limitation, the applicator device 20150 may 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 view illustrates how the applicator 20150 is shipped and received by a user. The exploded and cross-sectional views illustrate 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. Tamper evidence feature 20712 may be, for example, a sticker coupled to each of housing 20702 and cap 20708, such that tamper evidence feature 20712 can be irreparably damaged by, for example, disconnecting housing 20702 from cap 20708, thereby indicating to a user that housing 20702 has previously been disconnected from cap 20708. These features are described in more detail below.

[0080] G. Exemplary Tray and Sensor Module Assembly 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.

[0081] 6A is a proximal perspective cross-sectional view showing the sensor delivery components within the tray 810. The platform 808 is slidably coupled within the tray 810. The desiccant 502 is fixed relative to the tray 810. The sensor module 504 is mounted within the tray 810.

[0082] 6B is a proximal perspective view showing the sensor module 504 in more 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, the tip module 2500, and the sensor (not shown) so that they can be removed together as the sensor module 504 during assembly.

[0083] H. Exemplary Applicator and Sensor Controller for One-Piece Architecture 1A and 3A-3G, in 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 separately 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 sensors 110 and tips 220, can 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. Therefore, if the sensor applicator 102, including the electronics housing of the sensor control device 102, needs to be sterilized, it can be sterilized by another method, such as gas chemical sterilization using ethylene oxide. However, gas chemical sterilization may damage enzymes or other chemical and biological materials contained in the sensors 110. Because of this sterilization incompatibility, the sensor tray 202 and the sensor applicator 102 are typically sterilized in separate sterilization processes and then packaged separately, which requires the user to perform final assembly of the parts for use.

[0084] 7A and 7B are top and bottom exploded views, respectively, of a sensor control device 3702 in accordance with one or more embodiments. The shell 3706 and mount 3708 operate as opposing clamshell halves that contain or otherwise substantially encapsulate the 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 that includes a printed circuit board (PCB) 3804 to which multiple 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 across both surface areas (i.e., the top and bottom) of the PCB 3804.

[0085] In addition to the electronics module 3806, the PCBA 3802 may also 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 control device 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 user's sampled analyte level. The data processing unit 3808 may also include an antenna for communicating with or otherwise communicating with the reader device 106 (FIG. 1A).

[0086] 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 extend into the battery aperture 3810 to couple to the PCB 3804 and facilitate the 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 for the battery 3812, while the radial battery contact 3814b may provide a radial contact for the battery 3812. Locating the battery 3812 within the battery aperture 3810 with the battery contacts 3814a,b helps to reduce the height H of the sensor controller 3702, which allows the PCB 3804 to be centrally located and its components to be distributed on both sides (i.e., top and bottom). This also helps make it easier to mount the chamfer 3718 onto the electronics housing 3704 .

[0087] The sensor 3716 may be centrally disposed relative 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 that is received transcutaneously 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.

[0088] 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 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 serve 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, thereby eliminating the need for a prior art connector and advantageously reducing the height H. Furthermore, by eliminating the compliant carbon-impregnated polymer modules, circuit resistance is significantly eliminated, thus improving circuit conductivity.

[0089] The sensor control device 3702 may further include a compliant member 3826, which may be disposed so as to be interposed between the flag 3818 and the 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.

[0090] 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). Pairing the first and second clocking receptacles 3830a,b with the first and second clocking posts 3832a,b, respectively, properly aligns the shell 3706 with the mount 3708.

[0091] 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 to 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.

[0092] Additionally, a plurality 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 within the inner surface of the mount 3708, such that the overall height H of the sensor control device 3702 may be reduced compared to conventional sensor control devices. The module pocket 3838 may also help minimize the diameter of the PCB 3804 by allowing PCB components to be disposed on both sides (i.e., the top and bottom).

[0093] 7A , the mount 3708 may further include a plurality of carrier grip features 3842 (two shown) defined around the circumference of the mount 3708. The carrier grip features 3842 are axially offset from a bottom 3844 of the mount 3708, where a transfer adhesive (not shown) can be applied during assembly. In contrast to conventional sensor control devices that generally include a conical carrier grip feature that intersects 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. Furthermore, 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 relative to the mount 3708. This also increases the bonding area and, therefore, the bond strength.

[0094] 7B , the bottom 3844 of the mount 3708 may include or otherwise define a plurality of grooves 3846, which may be defined at or near the periphery of the mount 3708 and spaced equidistant 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 (migrate) moisture away from the sensor control unit 3702 toward the periphery 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 positions 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 material usage for the mount 3708 and may help maintain a minimum height H of the sensor control unit 3702. The module pocket 3838 may also significantly reduce mold sink and improve the flatness of the bottom 3844 where the transfer adhesive adheres.

[0095] 7B , the inner surface of the shell 3706 may also include or otherwise define a plurality of pockets or recesses configured to accommodate various components of the sensor control unit 3702 when the shell 3706 is mated to 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 when the sensor control unit 3702 is assembled and configured to accommodate a portion of the battery 3812. The opposing battery locator 3848 extends a short distance on the inner surface of the shell 3706, thereby helping to reduce the overall height H of the sensor control unit 3702.

[0096] A tip and sensor locator 3852 may also be provided or otherwise defined on the interior surface of the shell 3706. The tip and sensor locator 3852 may be configured to receive both a tip (not shown) and a portion of the sensor 3716. Furthermore, 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 the interior surface of the mount 3708.

[0097] 8A-8C show an alternative sensor assembly / electronics assembly connection approach 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 in the bottom of the mount of the electronics assembly 14712 to provide a location where the sensor assembly 14702 may 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 the circuit board and including a conductive material aligned with the electrical contacts of the sensor 14704). 8C is formed when the sensor assembly 14702 is snap-fit ​​or otherwise adhered to the electronics assembly 14712 by driving the sensor assembly 14702 into the integrally formed recess 14710 of the electronics assembly 14712. This embodiment provides an integrated connector for the sensor assembly 14702 in the electronics assembly 14712.

[0098] 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.

[0099] According to embodiments of the present disclosure, the sensor control device 102 can be modified to provide a one-piece architecture that can be subjected to sterilization techniques specifically designed for the one-piece architecture sensor control device. The one-piece architecture allows the sensor applicator 150 and sensor control device 102 to be shipped to a user in a single, sealed package that does not require any final user assembly steps. Rather, the user simply opens one package and then delivers the sensor control device 102 to the target monitoring location. The one-piece system architecture described herein can prove advantageous in eliminating component parts, various manufacturing process steps, and user assembly steps. As a result, packaging material and waste are reduced, and the possibility of user error or contamination of the system is mitigated.

[0100] Figures 9A and 9B are a side view and a side cross-sectional view, 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, the fully assembled sensor control unit 4402 may already be assembled and installed within the sensor applicator 102 before delivery to the user, thereby eliminating any additional assembly steps that the user would otherwise have to perform.

[0101] The fully assembled sensor control unit 4402 may be installed within the sensor applicator 102, and the applicator cap 210 may then be coupled to the sensor applicator 102. In some embodiments, the applicator cap 210 may be threaded onto the housing 208 and may include a tamper ring 4702. When the applicator cap 210 is rotated (e.g., unscrewed) relative to the housing 208, the tamper ring 4702 may shear, thereby releasing the applicator cap 210 from the sensor applicator 102.

[0102] According to the present disclosure, while installed within the sensor applicator 102, the sensor control unit 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 unit 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 applications, chemicals may be injected into the sterilization chamber 4706 via one or more vents 4708 defined in the proximal end 610 of the applicator cap 210. Examples of chemicals that can be used for gaseous chemical sterilization 4704 include, but are not limited to, ethylene oxide, vaporized hydrogen peroxide, nitrogen oxides (e.g., nitrous oxide, nitrogen dioxide, etc.), and water vapor.

[0103] The sensor 4410 and distal portion of tip 4412 are sealed within sensor cap 4416 so that the chemicals used during the gas chemical sterilization process do not interact with the enzymes, chemicals, and biologicals provided on other sensor components such as tail 4524 and membrane coatings that regulate analyte influx.

[0104] Once the desired level of sterility assurance has been achieved within the sterilization chamber 4706, the gaseous solution may be removed and the sterilization chamber 4706 may be vented. Venting may be achieved by a series of vacuums followed by the 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 with a seal 4712 (shown in dotted lines).

[0105] In some embodiments, the seal 4712 may comprise two or more layers of different materials. A first layer may be made from a synthetic material (e.g., flash-spun high-density polyethylene fiber) such as Tyvek® available from DuPont®. Tyvek® is highly durable and puncture-resistant while allowing vapor transmission. A 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 the 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, the single layer may be gas-permeable for the sterilization process but may also be able to protect from moisture and other harmful elements once the sterilization process is complete.

[0106] 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 during shipping and storage that prevents the adhesive patch 4714 from becoming contaminated.

[0107] 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 therefore may be best understood by reference thereto. Furthermore, the sensor control device 5002 may replace the sensor control device 102 of FIG. 1A and therefore 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 a user's skin.

[0108] 1A, the sensor control device 5002 may comprise a one-piece system architecture that does not require the user to open multiple packages and ultimately assemble the sensor control device 5002 prior to application. Rather, upon receipt by the 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, the user only needs to open one barrier (e.g., applicator cap 708 of FIG. 3B) before quickly delivering the sensor control device 5002 to the target monitoring location for use.

[0109] As shown, the sensor control unit 5002 includes an electronics housing 5004 that is generally disc-shaped and may 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 adhesive patch 105 of FIG. 1A and, therefore, may serve to adhere the sensor control unit 5002 to a user's skin for use.

[0110] 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 cases, the shell 5006 can be secured to the mount 5008 so that a sealed interface is created therebetween.

[0111] The sensor control device 5002 may further include a sensor 5010 (partially visible) and a tip 5012 (partially visible) that are used to facilitate transcutaneous delivery of the sensor 5010 under the user's skin 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 that is 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.

[0112] The sensor control unit 5002 may further include a sensor cap 5018, which is shown exploded or separated from the electronics housing 5004 in FIGS. 10A-10B . The sensor cap 5018 may be removably coupled to the sensor control unit 5002 (e.g., the electronics housing 5004) at or near the bottom of the mount 5008. The sensor cap 5018 may serve to provide a sealed barrier surrounding the exposed portions of the sensor 5010 and tip 5012, protecting them from gaseous chemical sterilization. As shown, the sensor cap 5018 may comprise a generally cylindrical body having a first end 5020 a and a second end 5020 b opposite the first end 5020 a. The first end 5020 a may be open to provide access to an interior chamber 5022 defined within the body. In contrast, the second end 5020 b may be closed and may include or otherwise define an engagement feature 5024. As described herein, the engagement feature 5024 may help to fit the sensor cap 5018 to 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 help to remove the sensor cap 5018 from the sensor control device 5002 when the cap is removed from the sensor applicator.

[0113] 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 extending distally from the bottom of the mount 5008. In at least one embodiment, for example, the mating member 5016 may define a set of external threads 5026a (FIG. 10B) that are matable with a set of internal threads 5026b (FIG. 10A) defined by the sensor cap 5018. In some embodiments, the external and internal threads 5026a,b may comprise a flat thread design (e.g., lack of a helical curve), which may prove advantageous in molding the part. Alternatively, the external and internal threads 5026a,b may comprise a helical thread engagement. Thus, the sensor cap 5018 may be threadedly 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 other types of engagement, including, but not limited to, an interference fit or a friction fit, or a frangible member or material that can be broken with minimal separation force (e.g., axial or rotational force).

[0114] In some embodiments, the sensor cap 5018 may comprise a monolithic (single) structure extending between the first and second ends 5020 a, b. However, in other embodiments, the sensor cap 5018 may comprise two or more components. In the illustrated embodiment, for example, the sensor cap 5018 may include a sealing ring 5028 disposed at the first end 5020 a and a desiccant cap 5030 disposed at the second end 5020 b. The sealing ring 5028 may be configured to help seal the internal chamber 5022, as described in more detail below. In at least one embodiment, the sealing 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 internal chamber 5022. The desiccant cap 5030 may also define or otherwise comprise the engagement feature 5024 of the sensor cap 5018.

[0115] 11A-11C are step-by-step cross-sectional side views illustrating the assembly of the sensor applicator 102 with a sensor control device 5002, according to one or more embodiments. Once the sensor control device 5002 is fully assembled, it may be installed within the sensor applicator 102. Referring to FIG. 11A, the tip hub 5014 may include or otherwise define a hub snap pole 5302 configured to help couple 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.

[0116] 11B, the sensor control device 5002 is shown received by the tip carrier 5306 and thus secured within the sensor applicator 102. Once the sensor control device 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, interlocking threads 5308 that allow the applicator cap 210 to be threaded onto the housing 208 in a clockwise (or counterclockwise) direction, thereby securing the applicator cap 210 to the sensor applicator 102.

[0117] As shown, the sheath 212 is also disposed within the sensor applicator 102, which may include a sheath locking mechanism 5310 configured to ensure that the sheath 212 does not prematurely collapse during an impact event. In the embodiment shown, the sheath locking 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 to threadably engage and allow the applicator cap 210 to threadably engage 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 threaded onto the housing 208 .

[0118] 11C , applicator cap 210 is shown as being fully threaded (coupled) to housing 208. As shown, applicator cap 210 further includes, or may otherwise define, a cap post 5314 centrally located within and extending proximally from the 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.

[0119] 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 can undergo a gas chemical sterilization process 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 biologicals provided on the tail 5104 and the membrane coating that regulates the influx of the analyte.

[0120] 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 device 5002, according to one or more further embodiments. As generally described above, the fully assembled sensor control device 5002 may be attached to the sensor applicator 102 by coupling the hub snap pole 5302 into the arm 5304 of the tip carrier 5306 disposed within the sensor applicator 102.

[0121] 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 the fired position.

[0122] 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 on applicator cap 210 may snap onto corresponding threads on 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 outward and snap into engagement with threads on housing 208. Once applicator cap 210 is snapped onto housing 208, sensor cap 5018 may correspondingly snap onto cap post 5314.

[0123] 11A-11C , the sensor applicator 102 may include a sheath fixation mechanism configured to ensure that the sheath 212 does not prematurely collapse during an impact event. In the illustrated embodiment, the sheath fixation mechanism includes one or more ribs 5704 (one shown) defined near the base of the sheath 212 and configured to interact with one or more ribs 5706 (two shown), and a shoulder 5708 defined near the 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, once applicator cap 210 is snapped onto housing 208, applicator cap 210 may be rotated (e.g., clockwise), which positions rib 5704 of sheath 212 between rib 5706 and shoulder 5708 of applicator cap 210, thereby "locking" applicator cap 210 in place until a user counter-rotates applicator cap 210 and removes applicator cap 210 for use. The engagement of rib 5704 with rib 5706 and shoulder 5708 of applicator cap 210 may also prevent sheath 212 from prematurely collapsing.

[0124] 12C, the applicator cap 210 is removed from the housing 208. Similar to the embodiment of FIGS. 21A-21C, the applicator cap 210 may be removed by counter-rotating the applicator cap 210, which correspondingly rotates the cap post 5314 in the same direction, unscrewing the sensor cap 5018 from the fitting 5016, generally as described above. Additionally, removing the sensor cap 5018 from the sensor control device 5002 exposes the sensor 5010 and a distal portion of the tip 5012.

[0125] 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 that, when applicator cap 210 is rotated, causes upward movement of sheath 212, which deflects sheath arm 5604 out of engagement with first detent 5702a so as 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, allowing the passive spring force of the spring 5612 to urge the tip carrier 5306 upward, disengaging the carrier arms 5608 from engagement with the grooves 5610. As the tip carrier 5306 moves upward within the housing 208, the engaging 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 engaging member 5016.

[0126] I. Exemplary Firing Mechanisms for One-Piece and Two-Piece Applicators 13A-13F show detailed example embodiments of the internal device mechanisms that "fire" the applicator 216 to apply the sensor control unit 222 to a user and safely retract 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 a user's skin, retracting the tip leaving the sensor in operable 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 can be understood by those skilled in the art with reference thereto. Additionally, the applicator 216, as disclosed herein, may be a sensor applicator having a one-piece or two-piece architecture.

[0127] 13A , the sensor 1102 is supported within 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 retained by detent features 1110 within the applicator 216, and 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.

[0128] 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 unit 222 (along with accompanying 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, along with the fully biased return spring 1118, maintains the member's position. According to an embodiment, rather than employing user force to translate the sensor control unit 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 unit 222.

[0129] In Figure 13C, the sensor 1102 and tip 1030 have reached their fully inserted depth, causing the carrier arm 1112 to clear the inner diameter of the upper guide section 1108. The compressive 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, pulling 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.

[0130] With tip 1030 fully retracted, as shown in Figure 13E, upper guide section 1108 of sheath 318 is secured by final securement feature 1120. As shown in Figure 13F, the used applicator assembly 216 is removed from the insertion site, leaving behind sensor control device 222, with tip 1030 safely secured inside applicator assembly 216. The used applicator assembly 216 is now ready for disposal.

[0131] 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 applicator's 216 internal mechanisms. 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 applicator's 216 detent features, 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 they are supplying additional force to drive the tip 1030 and pierce the skin. As described above in FIG. 13C , retraction of the tip 1030 is automated by the applicator's 216 coil return spring 1118.

[0132] With respect to any of the applicator embodiments described herein and any of its components, including, but not limited to, embodiments of the tip, tip module, and sensor module, those skilled in the art will understand that the embodiments can be sized and configured for use with a sensor configured to detect analyte levels in bodily fluids within the epidermis, dermis, or subcutaneous tissue of a subject. In some embodiments, for example, the tip and distal portion of an analyte sensor disclosed herein can both be sized and configured to be positioned at a particular distal depth (i.e., the furthest point of penetration within a tissue or layer of a subject's body, e.g., the epidermis, dermis, or subcutaneous tissue). With respect to some applicator embodiments, those skilled in the art will understand that particular embodiments of the tip can be sized and configured to be positioned at a different distal depth within a subject's body relative to the final distal depth of the analyte sensor. In some embodiments, for example, the tip can be positioned at a first distal depth within the subject's epidermis, while the distal portion of the analyte sensor can be positioned at a second distal depth within the subject's dermis prior to retraction. In other embodiments, the tip may be positioned at a first distal depth within the subject's dermis prior to retraction, while the distal portion of the analyte sensor may be positioned at a second distal depth within the subject's subcutaneous tissue. In yet other embodiments, the tip may be positioned 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.

[0133] Additionally, with respect to any of the applicator embodiments described herein, one skilled in the art will understand that the analyte sensor and one or more structural components coupled to the analyte sensor, including, but not limited to, one or more spring mechanisms, may be positioned within the applicator in an eccentric position 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 position relative to the applicator axis on a first side of the applicator, and the sensor electronics may be positioned in a second eccentric position 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 position relative to the applicator axis on the same side. One skilled in the art will understand 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 central or eccentric positions relative to one or more axes of the applicator are possible and fully within the scope of the present disclosure.

[0134] Further details of suitable devices, systems, methods, components, and their operation, along with relevant 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 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, tip, their components, and variations thereof are described in U.S. Patent Publication No. 2014 / 0171771, which is incorporated herein by reference in its entirety for all purposes.

[0135] J. Exemplary Methods for Calibrating Analyte Sensors Biochemical sensors can be described by one or more sensing properties. A common sensing property is referred to as the sensitivity of a biochemical sensor, which is a measure of the sensor's responsiveness to the concentration of the chemical or composition it is designed to detect. In electrochemical sensors, this response can be in the form of current (amperometric) or charge (coulometric). In other types of sensors, the response can 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, including whether the sensor is in vitro or in vivo.

[0136] FIG. 14 is a graph showing the in vitro sensitivity of an amperometric analyte sensor. In vitro sensitivity can be obtained by testing the sensor in vitro at various analyte concentrations and then 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 by 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 sensitivity slope, and b is the sensitivity intercept, with the intercept generally corresponding to 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 sensitivity slope and intercept. Sensors with nonlinear sensitivity require additional information to determine the analyte level resulting from the sensor's output current, and those skilled in the art are familiar with methods for modeling 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 convert the in vitro sensitivity to an in vivo sensitivity applicable to the sensor's intended in vivo use.

[0137] Calibration is a technique for improving or maintaining accuracy by adjusting the measured output of a sensor to reduce the difference from the sensor's expected output. One or more parameters describing the sensing characteristics of the sensor, such as its sensitivity, are established for use in calibration adjustments.

[0138] Certain 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 manner. For example, when user intervention is required, the user performs an in vitro measurement (e.g., a blood glucose (BG) measurement using a fingerstick 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 with the in vivo signal and uses the difference to determine an estimate of the sensor's in vivo sensitivity. The in vivo sensitivity may then be used in an algorithmic process to convert data collected by the sensor into a value indicative of the user's analyte level. This and other processes requiring user action to perform calibration are referred to as "user calibration." Systems may require user calibration due to instability in sensor sensitivity, such as sensitivity drifting or changing over time. Thus, multiple user calibrations (e.g., on a periodic (e.g., daily) schedule, a variable schedule, or as needed) may be required to maintain accuracy. Although the embodiments described herein may incorporate some degree of user calibration for particular implementations, this is generally not preferred as it requires the user to perform painful or otherwise cumbersome BG measurements and can introduce user error.

[0139] 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 commonly referred to as "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 typically necessitated by drift in sensor sensitivity over time. Thus, while the embodiments described herein can be used with some degree of automatic system calibration, preferably the sensor sensitivity is relatively stable over time so that post-implant calibration is not required.

[0140] 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 before distribution to users or healthcare professionals (HCPs). The calibration parameters may be determined by the sensor manufacturer (or the manufacturer of other components of the sensor control device, if the two entities are different). Many in vivo sensor manufacturing processes produce sensors in groups or batches called production lots, production-stage lots, or simply lots. A single lot may contain thousands of sensors.

[0141] 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. If 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 during sensor wear may be reduced. In embodiments in which the calibration codes or parameters are provided on the sensor itself, the calibration codes or parameters may be automatically transmitted or provided to the data processing device in the analyte monitoring system prior to or upon initiation of sensor use.

[0142] 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.

[0143] In further aspects, programming or executable instructions may be provided or stored in the data processing device and / or receiver / controller unit of the analyte monitoring system to provide time-varying adjustment algorithms for the in-vivo sensor during use. For example, based on retrospective statistical analysis of analyte sensors used in vivo and corresponding glucose level feedback, a time-based predetermined or analytical curve or database may be generated and 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.

[0144] In accordance with the disclosed subject matter, an analyte monitoring system can 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 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 performed or executed upon user initiation or activation of a corresponding function or routine or when the user enters a sensor calibration code.

[0145] According to the disclosed subject matter, each sensor in a sensor lot (in some examples, not including sample sensors used for in vitro testing) can be non-destructively inspected to determine or measure its characteristics, such as film thickness at one or more points on the sensor, and other characteristics, including physical characteristics such as surface area / volume of the active area, can be measured or determined. Such measurements or determinations may be performed in an automated manner, for example, using an optical scanner or other suitable measurement device or system, and the determined sensor characteristics of each sensor in the sensor lot are compared to corresponding average values ​​based on sample sensors for possible correction of calibration parameters or codes assigned to each sensor. For example, for a calibration parameter defined as sensor sensitivity, sensitivity is approximately inversely proportional to film thickness; thus, for example, for a sensor having a measured film thickness that is about 4% greater than the average film thickness of sensors sampled from the same sensor lot as the sensor, in one embodiment the sensitivity assigned to that sensor 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 for a sensor with a measured active area that is approximately 3% smaller than the average active area of ​​sensors sampled from the same sensor lot, the sensitivity assigned to 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 test or measurement of the sensor. In certain embodiments, the test or measurement of each sensor may further include measurement of the film uniformity or structure in addition to the film thickness and / or surface area or volume of the active sensing area.

[0146] 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 herein in its entirety.

[0147] 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 that makes 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. 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. Sensor status data may include error data, current active time, and software state. 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, comparisons of other values ​​to patient-specific averages, calls to action determined by algorithms or models, and other similar types of data.

[0148] In accordance with aspects of the disclosed subject matter, as embodied herein, 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 sensor 110. As an example, BLE module 5041 of communication module 5040 may comprise software or firmware to enable multiple simultaneous connections between sensor 110 as a central device and other devices as peripheral devices, or sensor 110 as a peripheral device when another device is the central device.

[0149] 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, but not limited to, 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 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 similarly nearby devices.

[0150] For purposes of illustration and not limitation, reference will be made to an exemplary embodiment of a process for sensor-receiver connection 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 can 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 specific Bluetooth® device address. The device then establishes the initial connection so that data exchange can begin. The device begins the process of initializing the data exchange service and performing a mutual authentication process.

[0151] During the first connection between the sensor 110 and the data receiving device 120, the data receiving device 120 may initialize a service, feature, and attribute discovery process. The data receiving device 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 receiving device 120. The mutual authentication process can be automated and does not require user intervention. After successful completion of the mutual authentication process, the sensor 110 sends a connection parameter update requesting the data receiving device 120 to use connection parameter settings that are preferred for the sensor 110 and configured to maximize longevity.

[0152] The data receiving device 120 then executes a sensor control process to backfill historical data, current data, event logs, and factory data. As an example, for each type of data, the data receiving device 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 untransmitted data in the sensor 110's memory is delivered to the data receiving device 120. The sensor 110 may respond to a backfill request from the data receiving device 120 that all data has already been transmitted. Once backfilling is complete, the data receiving device 120 may notify the sensor 110 that it is ready to receive periodic measurements. The sensor 110 may transmit measurements over multiple notifications on a repeating basis. As embodied herein, the multiple notifications may be redundant notifications to ensure that the data is transmitted correctly. Alternatively, the multiple notifications may comprise a single payload.

[0153] For purposes of illustration and not limitation, reference will be 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 one of these states, the sensor 110 may record the command and execute the shutdown when the sensor 110 transitions to the error state or the sensor expired state. The data sink 120 sends an appropriately 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, to acknowledge that the sensor 110 received the command, the sensor 110 sends a success notification via the sensor control property. 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.

[0154] L. Exemplary Sensor States and Operation 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 performed 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. At various times while in state 6005, the sensor 110 checks for a received command to proceed to the 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 detect state 6025.

[0155] Upon entering state 6025, the sensor 110 may store information about authorized devices to communicate with the configured sensor during operation or initialize algorithms related to performing and interpreting measurements from the sensing hardware 5060. The sensor 110 may also initialize a lifecycle timer responsible for maintaining an active count of the sensor's 110 operation time and begin communicating with authorized devices to transmit recorded data. While in insertion detection state 6025, the sensor may enter state 6030, in which the sensor 110 checks whether the operation time equals a predetermined threshold. This operation time threshold may correspond to a timeout function for determining whether the insertion is successful. If the operation time threshold is reached, the sensor 110 proceeds to state 6035, in which 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.

[0156] The active pair state 6055 of the sensor 110 indicates the 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 transmits measurements or attempts to establish a connection with the receiving device 120. The sensor 110 also increases its operating time. When the sensor 110 reaches a predetermined operating time threshold (e.g., when the operating time reaches a predetermined threshold), the sensor 110 transitions to the active expired state 6065. The active expired state 6065 of the sensor 110 indicates the state during which the sensor 110 has been operating for its maximum predetermined time.

[0157] While in the active expired state 6065, the sensor 110 may perform operations generally related to terminating operation and ensuring that collected measurements are securely transmitted to receiving devices as needed. 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 terminal operation threshold. The terminal operation threshold may be based on the battery life of the sensor 110. The normal termination state 6080 corresponds to the final operation of the sensor 110 and ultimately shuts down the sensor 110.

[0158] Before the sensor is activated, the ASIC 5000 is in a low-power storage mode. 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, causing 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 activates 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.

[0159] When the ASIC 5000 first enters measurement mode, an insertion detection sequence is executed to verify that the sensor 110 is properly placed on the patient's body before proper measurements can be taken. First, the sensor 110 interprets commands to activate the measurement configuration process, causing the ASIC 5000 to enter measurement command mode. Next, the sensor 110 temporarily enters a measurement lifecycle state, performing several consecutive measurements to check for successful insertion. The communications module 5040 or the ASIC 5000 evaluates the measurement results to determine successful insertion. If insertion is deemed successful, the sensor 110 enters a measurement state, where the sensor 110 begins taking periodic measurements using the sensing hardware 5060. If the sensor 110 determines that insertion was not successful, the sensor 110 is triggered into insertion failure mode, and the ASIC 5000 is instructed to return to storage mode, while the communications module 5040 disables itself.

[0160] M. Exemplary Over-the-Air Update 1B further illustrates an example operating environment for providing over-the-air ("OTA") updates for use with the techniques described herein. An operator of the analyte monitoring system 100 may bundle updates for the data receiving device 120 or sensor 110 into updates for an application running on the multipurpose data receiving device 130. Using available communication channels between the data receiving device 120, the multipurpose data receiving device 130, and the sensor 110, the multipurpose data receiving device 130 may receive periodic updates for the data receiving device 120 or sensor 110 and initiate installation of the updates on the data receiving device 120 or sensor 110. Applications that enable the multipurpose data receiving device 130 to communicate with the analyte sensor 110, the data receiving device 120, and / or the remote application server 150 can update software or firmware on the data receiving device 120 or sensor 110 without wide area network capability, so the multipurpose data receiving device 130 serves as an installation or update platform for the data receiving device 120 or sensor 110.

[0161] 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 particular embodiments, the remote application server 150 may provide updated software and firmware to the user device 140 or directly to the multipurpose 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 multipurpose data receiving device 130 may periodically contact the application storefront server 160 to download and install updates.

[0162] After the multipurpose data receiving device 130 downloads an application update including a 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, encrypt or decrypt the firmware or software update, or perform an integrity check on 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.

[0163] The data receiving device 120 or the sensor 110 receives the data for the update and a command to initiate 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 place itself in a so-called "safe" mode with limited operational 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 notification of a successful update. The multipurpose data receiving device 130 may then report confirmation of the successful update to the remote application server 150.

[0164] In certain embodiments, the storage memory 5030 of the sensor 110 includes one-time programmable (OTP) memory. The term OTP memory may refer to memory that includes access restrictions and security to facilitate writing to specific addresses or segments within the memory a predetermined number of times. The memory 5030 may be pre-configured into multiple pre-allocated memory blocks or containers. The containers are pre-allocated to a fixed size. When the storage memory 5030 is 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 software of a device (e.g., a sensor device described herein) stored in OTP memory may be performed by replacing only the code in one or more specific previously written containers with updated code written to one or more new containers, rather than replacing the entire code in the memory. In a second embodiment, the memory is not pre-configured. 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.

[0165] 16 is a schematic diagram illustrating example operations and data flows for over-the-air (OTA) programming of storage memory 5030 in sensor device 100 and use of the memory after OTA programming in execution of a process by 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 communications module 5040 of sensor device 110 receives the OTA programming command. The communications module 5040 transmits the OTA programming command to the microcontroller 5010 of sensor device 110.

[0166] 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 an appropriate digital signature token. Upon determining that the OTA programming command is valid, the microcontroller 5010 may place the sensor device in 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 re-initialize the sensor device 110 to the 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 writes 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 determines that the data was written without error, the microcontroller 5010 may resume normal operation of the sensor device.

[0167] At 536, in 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 in the OTP memory 550 are appropriate to execute and can avoid executing outdated or invalidated programming blocks or referencing outdated 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.

[0168] N. Exemplary Security and Other Architectural Features As embodied herein, a first layer of security for communications between the 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. Furthermore, certain packets and / or certain data contained within packets may be encrypted, while other packets and / or data within packets may be 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 the analyte monitoring system 100.

[0169] The ASIC 5000 of the analyte sensor 110 may be configured to dynamically generate authentication and encryption keys using data maintained 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 the message.

[0170] 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 in the analyte monitoring system 100 or by the operator of the analyte monitoring system 100, and a shared secret key derived from them. To verify the identity of another party, a party may provide proof that they have control of the private key.

[0171] The manufacturer of the analyte sensor 110, the data receiving device 120, or the provider of the application for the multi-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 can be used in combination with device-specific information and operational data (e.g., entropy-based random values) to generate encryption values ​​unique to the device, session, or data transmission, as needed.

[0172] The analyte data associated with a user is, at least in part, sensitive data because this information can be used for a variety of purposes, including health monitoring and medication administration decisions. In addition to user data, the analyte monitoring system 100 may implement security enhancements against reverse engineering by external parties. Communication connections may be encrypted using device-specific or session-specific encryption keys. Encrypted or unencrypted communications between any two devices may be verified using transmission integrity checks built into the communications. 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 "whitelist," or to devices that can provide a predetermined code associated with the manufacturer or otherwise authenticated user. The whitelist may indicate an exclusive range, meaning that no connection identifiers other than those included in the whitelist may be used, or a preferred range, in which the whitelist is searched first but other devices may still be used. The sensor 110 may further reject a connection request and shut down if the requester fails to complete the login process over the communication interface within a predetermined time (e.g., within 4 seconds). These features protect against certain denial-of-service attacks, particularly those against BLE interfaces.

[0173] As embodied herein, analyte monitoring system 100 may use periodic key rotation to further reduce the likelihood of key compromise and misuse. The key rotation strategy employed by analyte monitoring system 100 may be designed to support backward compatibility for fielded or distributed devices. As an example, analyte monitoring system 100 may employ keys for downstream devices (e.g., devices in the field or unable to feasibly provide updates) that are designed to be compatible with multiple generations of keys used by upstream devices.

[0174] 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 send 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 receiving device 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 receiving device 120 to ensure that the other device is capable of sufficient compliance with the agreed-upon security framework described herein. Mutual authentication may be based on a mechanism 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.

[0175] Following a successful mutual authentication process 620, in step 625, the sensor 110 may provide a sensor secret 625 to the data receiving device 120. 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 third parties 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 included in the payload. In step 640, using the appropriate communication model of the sensor 110 and the communication link established between 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 receiving device 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 receiving device 120 may process the received data appropriately.

[0176] As described herein, the sensor 110 may be a device with limited processing power, battery power, and storage. The encryption techniques (e.g., selection of cryptographic algorithms or algorithm implementations) 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.

[0177] O. Exemplary Payloads / Communication Frequencies The analyte sensor 110 may be configured to modify its discoverability behavior to increase the probability that a receiving device will receive an appropriate data packet and / or provide a response signal, or to otherwise attempt to 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 data packets, modifying the frequency at which data packets are generally transmitted, lengthening or shortening the broadcast window of data packets, modifying the time after broadcast at which the analyte sensor 110 will accept a response or scan signal, 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 the 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 also adjust parameters related to the device's listening behavior to increase the likelihood of receiving data packets containing connection data.

[0178] 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 two seconds of each 60-second period. The second window may indicate that the analyte sensor 110 transmits a data packet every 60 milliseconds during each two-second window. The remainder of the time during the two-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.

[0179] In certain embodiments, the discoverability behavior of the analyte sensor can be stored in a discoverability profile and can be modified 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 component of the analyte sensor's 110 communications hardware. In addition to modifying transmission power, other parameters related to the transmission capabilities or processes of the analyte sensor's 110 communications hardware can be modified, including, but not limited to, transmission rate, frequency, and timing. As another example, when 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 in order to alert receiving devices of the negative health event.

[0180] 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 alter characteristics in memory 5020 or storage 5030) or may be adjusted by other devices in the analyte monitoring system 100.

[0181] 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 for sensor sensitivity over time as the device experiences changing storage conditions. By way of example and not limitation, adaptive sensitivity adjustment 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 adjustment value may then be accumulated over the active storage mode period to calculate a total sensor sensitivity adjustment value at the end of the active storage mode (e.g., upon insertion). Similarly, upon insertion, the sensor 110 may determine the time difference between the manufacture of the sensor 110 (which can 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.

[0182] 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. For example, in the case of sensor drift, the sensor sensitivity adjustment may be hard-coded into the sensor 110 during manufacturing based on an estimate of how much the average sensor will drift. The sensor 110 may use a calibration function with time-varying functions for sensor offset and gain, which may account for drift over sensor wear. Thus, the sensor 110 may utilize a device-dependent function that describes the sensor 110's drift over time, and utilize a function used to convert interstitial current to interstitial glucose, which may indicate sensor sensitivity and may be device-specific combined with a baseline glucose profile. Such a function to account for sensor sensitivity and drift may improve the accuracy of the sensor 110 over wear without user calibration.

[0183] 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, a model 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. Events may include the initiation or completion of physical activity, a meal, the application of a medical procedure or medication, an emergency health event, and other events of a similar nature.

[0184] The model may be provided to the sensor 110, data receiving device 120, or multipurpose data receiving device 130 during manufacturing or during a firmware or software update. The model may be periodically refined, for example, by the sensor 110 manufacturer or the 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. Machine learning models may include, by way of example and not limitation, models trained using or incorporating decision tree analysis, gradient boosting, ADA boosting, artificial neural networks or variants thereof, linear discriminant analysis, nearest neighbor analysis, support vector machines, supervised or unsupervised classification, etc. Models 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 multipurpose data receiving device 130, upon receiving data from the sensor 110 (or other downstream devices).

[0185] R. Exemplary Alarm Mechanism The data transmitted between the sensor 110 and the data receiving device 120 may include raw measurements or processed measurements. The data transmitted between the sensor 110 and the data receiving device 120 may further include alarms or notifications for display to a user. The data receiving device 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 detected suspected malfunction), 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. By way of example and not limitation, as embodied herein, the alarm parameters described herein may be configurable by the user, may be fixed during manufacturing, or may be a combination of user-configurable and non-user-configurable parameters.

[0186] S. Exemplary Electrode Configurations Sensor configurations featuring a single active region configured for the detection of a single corresponding analyte may use two-electrode or three-electrode detection motifs, as further described herein with reference to Figures 18A-18C. Sensor configurations featuring two different active regions for the detection of different analytes, either on separate working electrodes or on the same working electrode, are described separately below with reference to Figures 19A-21C. Sensor configurations with multiple working electrodes may be particularly advantageous for incorporating two different active regions within the same sensor tail, since the signal contribution from each active region can be more easily determined.

[0187] 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 may include a working electrode and a second electrode, which 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, or any other suitable 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.

[0188] An analyte sensor 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 additional electrode may function as a reference electrode for each of the multiple working electrodes.

[0189] 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 can be disposed on the same side of the substrate 30212 with a dielectric material between them (configuration not shown). An active area 218 is disposed as at least one layer over at least a portion of the working electrode 214. The active area 218 can include multiple spots or a single spot configured for analyte detection, as discussed further herein.

[0190] 18A , a membrane 220 covers at least the active area 218. In certain embodiments, the membrane 220 may cover part or all of the working electrode 214 and / or the counter / reference electrode 30216, or the entire analyte sensor 200. One or both sides of the analyte sensor 200 may be covered by the membrane 220. The membrane 220 may include one or more polymeric membrane materials capable of limiting analyte flux to the active area 218 (i.e., the membrane 220 is a mass transport limiting membrane having some permeability to the analyte of interest). In accordance with the disclosure herein, the membrane 220 may be crosslinked with a branched crosslinker in certain sensor configurations. The composition and thickness of the membrane 220 may be varied to promote the desired analyte flux to the active area 218, thereby providing the 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.

[0191] 18B and 18C show schematic diagrams of exemplary three-electrode analyte sensor configurations, also suitable for use in the present disclosure. The three-electrode analyte sensor configuration can be similar to that shown as analyte sensor 200 in FIG. 18A , except for the inclusion of an additional electrode 217 within 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, with the additional electrode 217 performing other electrode functions not otherwise described. Working electrode 214 continues to perform its original function. The additional electrode 217 can be disposed on either working electrode 214 or electrode 30216, with a separating layer of dielectric material sandwiched therebetween. For example, but not by way of limitation, as shown in FIG. 18B , dielectric layers 219 a, 219 b, and 219 c separate electrodes 214, 30216, and 217 from one another and provide electrical insulation. Alternatively, as shown in FIG. 18C , at least one of electrodes 214, 30216, and 217 may be located on the opposite side of substrate 30212. Thus, in certain embodiments, electrode 214 (working electrode) and electrode 30216 (counter electrode) may be disposed on the opposite side of substrate 30212, and electrode 217 (reference electrode) may be disposed on one of electrode 214 or electrode 30216 and separated therefrom by a dielectric material. A reference material layer 30230 (e.g., Ag / AgCl) may be present on electrode 217, and the location of reference material layer 30230 is not limited to the location shown in FIGS. 18B and 18C . Similar to sensor 200 shown in FIG. 18A , active area 218 in 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.

[0192] 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. While 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 sides 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. Therefore, 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.

[0193] FIG. 19A shows an exemplary configuration of a sensor 203 having a single working electrode with two distinct active regions disposed thereon. FIG. 19A is similar to FIG. 19A except that there are two active regions on the working electrode 214: a first active region 218a and a second active region 218b, which respond 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 identical. 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 the active regions 218a and 218b may be configured to detect the analyte using an NAD(P)-dependent enzyme. In certain embodiments, either or both of active regions 218a and 218b may be configured to detect an analyte using an NAD(P)-dependent enzyme, e.g., ketones, by using an enzyme system comprising NADH oxidase and β-hydroxybutyrate dehydrogenase. In certain embodiments, only one active region of 218a and 218b is configured to detect an analyte using an NAD(P)-dependent enzyme. In certain embodiments, the other active region is configured to detect a second analyte without using an NAD(P)-dependent enzyme.

[0194] 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.

[0195] Exemplary sensor configurations having multiple working electrodes, specifically two working electrodes, are described in further detail with reference to Figures 20 through 21C. While the following description is primarily directed to sensor configurations having two working electrodes, 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 example, for detecting a third and / or fourth analyte in addition to the first and second analytes.

[0196] 20 shows 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, analyte sensor 300 includes working electrodes 304 and 306 disposed on opposite sides of substrate 302. A first active area 310a is disposed on the surface of working electrode 304, and a second active area 310b is disposed on the surface of working electrode 306. Counter electrode 320 is electrically insulated from working electrode 304 by dielectric layer 322, and reference electrode 321 is electrically insulated from working electrode 306 by dielectric layer 323. Outer dielectric layers 330 and 332 are disposed on reference electrode 321 and counter electrode 320, respectively. According to various embodiments, membrane 340 may cover at least active areas 310a and 310b, with other components of analyte sensor 300 or the entire analyte sensor 300 optionally similarly covered by first membrane portion 340a and / or second membrane portion 340b. Additionally, membrane 340 may be continuous but compositionally varied within first membrane portion 340a and second membrane portion 340b (i.e., over active areas 310a and 310b) to enable different permeability values ​​for separately adjusting analyte flux at each location. For example, different membrane formulations may be sprayed and / or printed on opposite sides of analyte sensor 300. Dip-coating techniques may also be suitable, particularly for depositing at least a portion of a bilayer membrane over one of 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 the following electrochemical detection techniques: coulometry, amperometry, voltammetry, or potentiometry. 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 can 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 can be configured to detect an analyte using an NAD(P)-dependent enzyme, e.g., ketones, by using an enzyme system comprising NADH oxidase and β-hydroxybutyrate dehydrogenase, or β-hydroxybutyrate dehydrogenase and diaphorase. In certain embodiments, only one of active areas 310a and 310b is configured to detect an analyte using an NAD(P)-dependent enzyme, e.g., ketones, by using an enzyme system comprising NADH oxidase and β-hydroxybutyrate dehydrogenase, or β-hydroxybutyrate dehydrogenase and diaphorase. In certain embodiments, the other active area is configured to detect a second analyte, e.g., an analyte not detected using an NAD(P)-dependent enzyme.

[0197] Alternative sensor configurations having multiple working electrodes that differ from the configuration shown in Figure 20 may feature a counter / reference electrode in place of separate counter and reference electrodes 320, 321, and / or may feature layer and / or film arrangements that differ from those explicitly shown. For example, without limitation, the locations of counter electrode 320 and reference electrode 321 may be opposite those shown in Figure 20. Furthermore, working electrodes 304 and 306 need not necessarily be on opposite sides of substrate 302 as in the location shown in Figure 20.

[0198] While preferred sensor configurations can feature electrodes that are substantially planar in nature, it should be appreciated that sensor configurations featuring non-planar electrodes may be advantageous and 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 manner similar to that described above for substantially planar sensor configurations. Figures 21A-21C show perspective views of analyte sensors 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.

[0199] 21A shows a perspective view of an exemplary sensor configuration in which multiple electrodes are substantially cylindrical and concentrically arranged relative to a center substrate. As shown, the analyte sensor 400 includes a center substrate 402 around which all of the electrodes and dielectric layers are concentrically arranged relative to one another. In particular, a working electrode 410 is disposed on the surface of the center substrate 402, with a dielectric layer 412 disposed on a portion of the working electrode 410 distal to the sensor tip 404. A working electrode 420 is disposed on the dielectric layer 412, with a dielectric layer 422 disposed on a portion of the working electrode 420 distal to the sensor tip 404. A counter electrode 430 is disposed on the dielectric layer 422, with a dielectric layer 432 disposed on a portion of the counter electrode 430 distal to the sensor tip 404. A reference electrode 440 is disposed on the dielectric layer 432, with a dielectric layer 442 disposed on 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 longitudinal axis B of analyte sensor 400 .

[0200] 21A , first and second active areas 414a and 414b associated with different or the same analyte are disposed on the exposed surfaces of working electrodes 410 and 420, respectively, allowing for fluid contact for sensing. In certain embodiments, either or both of active areas 414a and 414b may be configured to detect the analyte using an NAD(P)-dependent enzyme. In certain embodiments, either or both of active areas 414a and 414b 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 414a and 414b 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, either or both of active regions 414a and 414b can be configured to detect an analyte using an NAD(P)-dependent enzyme, e.g., ketones, by using an enzyme system comprising NADH oxidase and β-hydroxybutyrate dehydrogenase, or β-hydroxybutyrate dehydrogenase and diaphorase. In certain embodiments, only one of active regions 414a and 414b is configured to detect an analyte using an NAD(P)-dependent enzyme, e.g., ketones, by using an enzyme system comprising NADH oxidase and β-hydroxybutyrate dehydrogenase, or β-hydroxybutyrate dehydrogenase and diaphorase. In certain embodiments, the other active region is configured to detect a second analyte, e.g., an analyte not detected using an NAD(P)-dependent enzyme. While active regions 414a and 414b are shown as three separate spots in FIG. 21A , it should be understood that alternative sensor configurations may include fewer or more than three spots comprising a continuous layer of active regions.

[0201] In FIG. 21A, the sensor 400 is partially coated with a membrane 450 over the working electrodes 410 and 420 and the active regions 414a and 414b disposed thereon. FIG. 21B illustrates an alternative sensor configuration in which substantially all of the sensor 401 is coated with a membrane 450. The membrane 450 may be the same or compositionally different in the active regions 414a and 414b. For example, the membrane 450 may include a bilayer membrane covering the active region 414a or may be a uniform membrane covering the active region 414b. In certain embodiments, one or more membranes, including an interference domain and a mass transport limiting membrane, are deposited on the exposed electroactive surface of the working electrode, e.g., a platinum surface. For example, without limitation, an interference domain may be disposed over the working electrode, an active region may be disposed over the interference domain, and a mass transport limiting membrane may be disposed over the active region.

[0202] It should be further understood that the positioning of the various electrodes in FIGS. 21A and 21B may differ from that explicitly shown. For example, the positions of the counter electrode 430 and reference electrode 440 may be reversed from the configuration shown in FIGS. 21A and 21B. Similarly, the positions of the working electrodes 410 and 420 are not limited to those explicitly shown in FIGS. 21A and 21B. FIG. 21C illustrates an alternative sensor configuration to that shown in FIG. 21B, in which the sensor 405 includes the counter electrode 430 and 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 FIG. 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.

[0203] In certain embodiments, one or more electrodes of the analyte sensors described herein are wire electrodes, e.g., permeable 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. In certain embodiments, each electrode is formed from a thin wire having a diameter of about 25.4 micrometers (0.001 inch) or less to about 254 micrometers (0.010 inch) or more. In certain embodiments, the working electrode has a diameter of about 25.4 micrometers (0.001 inch) or less to about 254 micrometers (0.010 inch) or more, e.g., about 50.8 micrometers (0.002 inch) to about 203 micrometers (0.008 inch), more preferably about 102 micrometers (0.004 inch) to about 127 micrometers (0.005 inch). 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 a conductive material, such as platinum, platinum-iridium, palladium, graphite, gold, carbon, a conductive polymer, an alloy, or other conductive material. In certain embodiments, the conductive material is a transparent conductive material. In certain embodiments, the electrodes can be formed by various 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 a steel wire) or bulk metals (e.g., platinum wire). In certain embodiments, the electrodes are formed from platinum-coated tantalum wire.

[0204] 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, or the like. In certain embodiments, the reference electrode is juxtaposed and / or twisted with or around the working electrode. In certain embodiments, the reference electrode is spirally wrapped around the working electrode. In certain embodiments, the wire assembly may be coated or glued with an insulating material to provide an insulating attachment.

[0205] In certain embodiments, additional electrodes may be included in the sensor tail. For example, but not limited to, a three-electrode system (working electrode, reference electrode, and counter electrode) and / or an additional working electrode (e.g., an electrode for detecting a second analyte). In certain embodiments in which the sensor includes 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 electrode 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 region of the working electrode.

[0206] In certain embodiments, one or more working electrodes are spirally wound around the reference electrode. In certain embodiments where two or more working electrodes are provided, the working electrodes may be formed in a double, triple, quadruple, etc. spiral configuration along the length of the sensor tail (e.g., surrounding the reference electrode, an insulated rod, or other support structure). 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.

[0207] In certain embodiments, the working electrode comprises a tube with an insulator therebetween and the reference electrode disposed or wound therein. Alternatively, the reference electrode comprises a tube with an insulator therebetween and the working electrode disposed or wound therein. 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, without limitation, the present disclosure provides sensors, e.g., sensor tails, that comprise 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 coated with an insulating material that is partially coated with a silver / silver chloride composition that functions as the reference and / or counter electrode.

[0208] In certain embodiments in which 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, without limitation, a portion of the insulator can be removed by hand, excimer laser, chemical etching, laser ablation, grit blasting, or 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, e.g., 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.

[0209] Some parts of the sensor are further described below. 2. NAD(P) Depot The present disclosure provides analyte sensors that can include an internal source of cofactor. For example, without limitation, the present disclosure provides analyte sensors that can include an internal source of cofactor that allows for controlled release of the cofactor over an extended period of time.

[0210] In certain embodiments, the internal cofactor source may be coated with or distributed within a permeable layer that controls the diffusion of cofactor from the cofactor source to maintain a sufficient concentration of cofactor in the active region, e.g., the sensing chemistry layer, during use of the analyte sensor. The exact nature, size, and configuration of the cofactor depot present within the analyte sensor may vary based on the particular application of the analyte sensor, e.g., which analyte is being detected, the duration of analyte detection, and the conditions under which analyte detection is performed.

[0211] In certain embodiments, the cofactor is NAD or NADP (both of which are collectively referred to herein as "NAD(P)"). In certain embodiments, NAD(P) is a derivative of NAD(P). Non-limiting examples of NAD(P) derivatives are disclosed in International Publication Nos. WO 2007 / 012494 and WO 1998 / 033936, the contents of each of which are herein incorporated by reference in their entirety. In certain embodiments, the present disclosure provides analyte sensors that can include an internal source of NAD(P) that allows for the controlled release of NAD(P) or a derivative thereof over an extended period of time. In certain embodiments, the internal NAD(P) source can be coated with or distributed within a permeable layer that controls the diffusion of NAD(P) from the NAD(P) source to maintain a sufficient concentration of NAD(P) in an active region, e.g., a sensing chemistry layer, comprising one or more NAD(P)-dependent enzymes during use of the analyte sensor.

[0212] Non-limiting embodiments of analyte sensors including a NAD(P) depot are provided in Figures 22 and 23A. For example, without limitation, the NAD(P) can be deposited on a substrate, such as a plastic substrate, as shown in Figures 22 and 23A. In certain embodiments, the NAD(P) depot can be disposed on substrate 30212. In certain embodiments, the NAD(P) depot can be deposited on a dielectric material, such as between two dielectric layers, as shown in Figure 23A.

[0213] In certain embodiments, the deposited NAD(P) can be covered with a permeable layer. As shown in Figures 22 and 23A, the NAD(P) depot is at least partially covered with a permeable layer. For example, but not limited to, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% of the NAD(P) depot is covered with a permeable layer. In certain embodiments, the NAD(P) depot is completely covered with a permeable layer. In certain embodiments, the permeable layer provides sustained NAD(P) release over time. The composition of the permeable layer can vary depending on the desired release kinetics of NAD(P) from an internal source, e.g., the NAD(P) release rate.

[0214] Additionally or alternatively, NAD(P) can be present within the permeable layer. For example, without limitation, NAD(P) can be mixed directly into the permeable layer, e.g., a polymeric permeable layer, rather than being added as a separate layer covered by the permeable layer. In certain embodiments, the analyte sensor of the present disclosure can include a permeable layer comprising NAD(P) disposed on the substrate 30212. In certain embodiments, the analyte sensor of the present disclosure can include an NAD(P) depot disposed on the substrate 30212 covered with a permeable layer containing a separate source of NAD(P).

[0215] In certain embodiments, the analyte sensor further includes a permeable working electrode, e.g., 214, 30, 216, and / or 217. In certain embodiments, the permeable working electrode is disposed on the permeable layer, as shown in Figures 22 and 23A. In certain embodiments, at least one active area (containing the sensing chemistry) is disposed on the working electrode as described herein. In certain embodiments, two or more active areas are disposed on the working electrode as described herein. In the non-limiting exemplary embodiment shown in Figures 22 and 23A, NAD(P) diffuses through the permeable layer (e.g., polymer layer) and the permeable working electrode to contact the active area and maintain a sufficient NAD(P) concentration in the active area over time.

[0216] In certain embodiments, the amount of NAD(P) present in the NAD(P) depot can vary depending on the duration of use of the analyte sensor. For example, without limitation, NAD(P) can be present in the NAD(P) depot from about 0.1 μg to about 1000 μg. In certain embodiments, the NAD(P) depot can contain from about 0.1 μg to about 900 μg, about 0.1 μg to about 800 μg, about 0.1 μg to about 700 μg, about 0.1 μg to about 600 μg, about 0.1 μg to about 500 μg, about 0.1 μg to about 400 μg, about 0.1 μg to about 300 μg, about 0.1 μg to about 200 μg, about 0.1 μg to about 100 μg, about 0.1 μg to about 90 μg, about 0.1 μg to about 80 μg, about 0.1 μg to about 70 μg, about 0.1 μg to about 60 μg, about 0.1 μg to about 50 μg, about 0.1 μg to about 40 μg, about 0.1 μg to about 30 μg, about 0.1 μg to about 20 μg, about 0.1 μg to about 10 μg, about 0.1 μg to about 9 μg, about 0.1 μg to about 8 μg, about 0.1 μg to about 7 μg, about 0.1 μg to about 6 μg, about 0.1 μg to about 5 μg, about 0.1 μg to about 4 μg, about 0.1 μg to about 3 μg, about 0.1 μg to about 2 μg, about 0.1 μg to about 1 μg, about 0.1 μg to about 0.9 μg, about 0.1 μg to about 0.8 μg, about 0.1 μg to about 0.7 μg, about 0.1 μg to about 0.6 μg, about 0.1 μg to about 0.5 μg, about 0.1 μg to about 0.4 μg, about 0.1 μg to about 0.3 μg, about 0.1 μg to about 0.2 μg, about 0.2 μg to about 1000 μg, about 0.3 μg to about 1000 μg, about 0.4 μg to about 1000 μg, about 0.5 μg to about 1000 μg, about 0.6 μg to about 1000 μg, about 0.7 μg to about 1000 μg, about 0.8 μg to about 1000 μg, about 0.9 μg to about 1000 μg, about 1 μg to about 1000 μg, about 2 μg to about 1000 μg, about 3 μg to about 1000 μg, about 4 μg to about 1000 μg, about 5 μg to about 1000 μg, about 6 μg to about 1000 μg, about 7 μg to about 1000 μg, about 8 μg to about 1000 μg, about 9 μg to about 1000 μg, about 10 μg to about 1000 μg, about 11 μg to about 1000 μg, about 12 μg to about 1000 μg, about 13 μg to about 1000 μg, about 14 μg to about 1000 μg, about 15 μg to about 1000 μg, about 16 μg to about 1000 μg, about 17 μg to about 1000 μg, about 18 μg to about 1000 μg, about 19 μg to about 1000 μg, about 20 μg to about 1000 μg, about 30 μg to about 1000 μg, about 40 μg to about 1000 μg, about 50 μg to about 1000 μg, about 60 μg to about 1000 μg , about 70 μg to about 1000 μg, about 80 μg to about 1000 μg, about 90 μg to about 1000 μg, about 100 μg to about 1000 μg, about 200 μg to about 1000 μg, about 300 μg to about 1000 μg, about 400 μg to about 1000 μg, about 500 μg to about 1000 μg, about 600 μg to about 1000 μg, about 700 μg to about 1000 μg, about 800 μg to about 1000 μg, about 900 μg to about 1000 μg NAD(P) may be present at about 1000 μg, about 0.1 μg to about 100 μg, about 1 μg to about 100 μg, about 1 μg to about 90 μg, about 1 μg to about 80 μg, about 1 μg to about 70 μg, about 1 μg to about 60 μg, about 1 μg to about 50 μg, about 1 μg to about 40 μg, about 1 μg to about 30 μg, about 1 μg to about 20 μg, about 1 μg to about 15 μg, about 1 μg to about 10 μg, or about 5 μg to about 15 μg. In certain embodiments, NAD(P) may be present in the NAD(P) depot at about 0.1 μg to about 100 μg.

[0217] In certain embodiments, the amount of NAD(P) present in the NAD(P) depot varies depending on the lifetime of the analyte sensor. For example, without limitation, the amount of NAD(P) in the NAD(P) depot enables the analyte sensor to detect the analyte using an NAD(P)-dependent enzyme for at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 15 days, at least about 16 days, at least about 17 days, at least about 18 days, at least about 19 days, at least about 20 days, at least about 25 days, at least about 30 days, at least about 35 days, or at least about 40 days. In certain embodiments, the amount of NAD(P) in the NAD(P) depot enables the analyte sensor to detect the analyte using an NAD(P)-dependent enzyme for at least about 14 days. In certain embodiments, the amount of NAD(P) in the NAD(P) depot enables the analyte sensor to detect the analyte using an NAD(P)-dependent enzyme for more than about 2 weeks, more than about 3 weeks, more than about 4 weeks, more than about 5 weeks, more than about 6 weeks, more than about 7 weeks, or more than about 8 weeks.

[0218] In certain embodiments, the permeable layer can comprise a polymer. In certain embodiments, the permeable polymer layer can comprise a diffusion-controlling polymer. In certain embodiments, the permeable polymer layer can comprise any polymer that allows for controlled diffusion of the cofactor. In certain embodiments, the permeable polymer layer can comprise any polymer that allows for controlled diffusion of the cofactor NAD(P).

[0219] In certain embodiments, the permeable polymer layer may be hyaluronic acid (HA), poly(ethylene glycol) (PEG), phosphorylcholine-based polymers and other hydrophilic polymers with hydrophilicity comparable to that of HA, PEG, or phosphorylcholine, ethylene vinyl alcohol copolymers, polyhydroxyalkanoates, poly(hydroxyvalerate), polycaprolactone, poly(lactide-co-glycolide), poly(hydroxybutyrate), poly(hydroxybutyrate-co-valerate), polydioxanone, polyorthoesters, polyanhydrides, poly(glycolic acid), poly(D,L-lactic acid) (DLPLA), poly(orthoesters), poly(glycolic acid-co-trimethylene carbonate), polyphosphoesters, polyphosphoesterurethanes, poly(amino acids), cyanoacrylates, poly(trimethylene carbonate), poly(iminocarbonates), polyurethanes, copoly(ether-esters) (e.g., PEO / PLA), polyalkylene oxalates, polyphosphazenes, biomolecules (e.g., fibrin, fibrinogen, cellulose, starch, and collagen), polyurethanes, silicones, polyesters, polyolefins, polyisobutylene and ethylene-α-olefin copolymers, acrylic polymers and copolymers, vinyl halide polymers and copolymers, poly(amide esters) (PEA), polycaprolactone (PCL), poly(hexafluoropropylene) (HFP), poly(ethylene vinyl alcohol) (EVAL), polyvinyl ethers ( Examples of suitable polymers include polyvinyl methyl ether, polyvinylidene halides (e.g., polyvinylidene fluoride (PVDF) and polyvinylidene chloride), polyacrylonitrile, polyvinyl ketones, polyvinylaromatics (e.g., polystyrene), polyvinyl esters (e.g., polyvinyl acetate), copolymers of vinyl monomers and olefins (e.g., ethylene-methyl methacrylate copolymer, acrylonitrile-styrene copolymer, ABS resin, and ethylene-vinyl acetate copolymer), polyamides (e.g., nylon 66 and polycaprolactam), alkyd resins, polycarbonates, polyoxymethylene, polyimides, polyethers, epoxy resins, polyurethanes, rayon, rayon-triacetate, cellulose acetate, cellulose butyrate, cellulose acetate butyrate, cellophane, cellulose nitrate, cellulose propionate, cellulose ethers, and carboxymethyl cellulose. In certain embodiments, suitable polymers are copolymers comprising poly(ethylene glycol terephthalate) and poly(butylene terephthalate) (PEGT / PBT) segments.

[0220] Further non-limiting examples of polymers that may be present in the permeable layer include polycarboxylic acids, cellulose polymers, gelatin, polyvinylpyrrolidone, cross-linked polyvinylpyrrolidone, polyanhydrides including maleic anhydride polymers, polyvinyl alcohol, polyvinyl aromatics (e.g., copolymers of polystyrene with other vinyl monomers such as isobutylene, isoprene, and butadiene (e.g., styrene-isobutylene-styrene (SIBS), styrene-isoprene-styrene (SIS) copolymers, styrene-butadiene-styrene (SBS) copolymers)), polyethylene oxide, glycosaminoglycans, polysaccharides, polyesters (polyethylene terephthalates), polyacrylamides, polyethersulfones, polyalkylenes (including polypropylene, polyethylene, and high molecular weight polyethylene), halogenated polyalkylenes (including polytetrafluoroethylene), natural and synthetic rubbers (including polyisoprene, polybutadiene, polyisobutylene, and copolymers thereof with other vinyl monomers (e.g., polyorthoesters)), proteins, polypeptides, siloxane polymers, polylactic acid, polyglycolic acid, polyhydroxybutyrate valerate, and blends and copolymers thereof, and other biodegradable, bioabsorbable, and biostable polymers and copolymers. In certain embodiments, suitable polymers include polyacrylic acid, and copolymers of polylactic acid and polycaprolactone.

[0221] In certain embodiments, the permeable layer can comprise a polyether-based polymer. In certain embodiments, the permeable layer can comprise poly(ethylene glycol). In certain embodiments, the permeable layer can comprise a poly(ethylene glycol)-based polymer. In certain embodiments, the permeable layer can comprise poly(propylene glycol). In certain embodiments, the permeable layer can comprise a poly(propylene glycol)-based polymer. In certain embodiments, the permeable layer can comprise poly(propylene glycol) methacrylate (POMA). In certain embodiments, the permeable layer can comprise 2-hydroxyethyl methacrylate (HEMA). In certain embodiments, the permeable layer can comprise a blend of POMA and HEMA. In certain embodiments, the permeable layer can comprise a blend of POMA and HEMA to produce a permeable polymer. For example, and without limitation, the permeable layer can comprise a ratio of POMA to HEMA of about 10:1 to about 1:10, e.g., 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, or about 2:1 to about 1:2. In certain embodiments, the ratio of POMA to HEMA can be about 2:1 to about 1:2. In certain embodiments, the ratio of POMA to HEMA can be about 1:1. In certain embodiments, the permeable layer can comprise about 20% to about 80% by weight of POMA, e.g., about 30% to about 70% by weight or about 40% to about 60% by weight. In certain embodiments, the permeable layer can be about 40% to about 60% by weight of POMA. In certain embodiments, the permeable layer can be about 20% to about 80% by weight HEMA, e.g., about 30% to about 70% by weight or about 40% to about 60% by weight HEMA. In certain embodiments, the permeable layer can be about 40% to about 60% by weight HEMA.

[0222] In certain embodiments, the permeable polymer is a hydrogel. In certain embodiments, a permeable polymer for use in the present disclosure, e.g., a hydrogel, can absorb about 30% to about 95%, e.g., about 30% to about 70%, or about 40% to about 60%, of its weight in water. In certain embodiments, a permeable polymer, e.g., a hydrogel, can absorb at least about 30% of its weight in water. In certain embodiments, a permeable polymer, e.g., a hydrogel, can absorb at least about 40% of its weight in water. In certain embodiments, a permeable polymer, e.g., a hydrogel, can absorb at least about 50% of its weight in water. In certain embodiments, a permeable polymer, e.g., a hydrogel, can absorb at least about 60% of its weight in water. In certain embodiments, a permeable polymer, e.g., a hydrogel, can absorb at least about 70% of its weight in water. In certain embodiments, a permeable polymer, e.g., a hydrogel, can absorb about 30% to about 60% of its weight in water.

[0223] In certain embodiments, the NAD(P) depot has a thickness, e.g., a dry thickness, ranging from about 0.1 μm to about 1000 μm, e.g., 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 NAD(P) depot can have a thickness of about 0.1 μm to about 10 μm, e.g., from about 0.5 μm to about 10 μm, from about 1 μm to about 10 μm, from about 1 μm to about 5 μm, or from about 0.1 μm to about 5 μm.

[0224] In certain embodiments, the permeable electrode can comprise any material that is permeable to NAD(P). In certain embodiments, the permeable electrode can comprise any conductive material that is permeable to NAD(P), such as a conductive ink or polymer. For example, without limitation, the permeable electrode can comprise carbon, silver, amorphous carbon, graphite, graphene, glassy carbon, platinized carbon, gold, platinum, and / or palladium. In certain embodiments, the permeable electrode can comprise a carbon material. In certain embodiments, the permeable electrode can comprise a carbon material containing an additive, such as, but not limited to, silver, amorphous carbon, graphite, graphene, glassy carbon, platinized carbon, gold, platinum, and / or palladium. In certain embodiments, the permeable electrode can be at least partially composed of carbon nanotubes. In certain embodiments, the permeable electrode can comprise a conductive polymer, such as poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS). In certain embodiments, the conductive material can be present in a polymer, such as a polymeric carrier. In certain embodiments, the electrodes comprise a polymer comprising a conductive material and / or conductive particles.

[0225] 3. Enzymes The sensors of the present disclosure include one or more enzymes for detecting one or more analytes in at least one active region. Enzymes suitable for use in the sensors of the present disclosure include, but are not limited to, any NAD(P)-dependent enzyme. For example, NAD(P)-dependent enzymes for use in the present disclosure can be used to detect 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. In certain embodiments, the analytes detected using NAD(P)-dependent enzymes include glucose, lactate, ketones, creatinine, alcohol, such as ethanol, and the like. In certain embodiments, the active region can include multiple enzymes, e.g., enzyme systems, that collectively respond to the analyte.

[0226] In certain embodiments, the active region of an analyte sensor of the present disclosure comprises at least one NAD(P)-dependent enzyme. In certain embodiments, the active region of an analyte sensor of the present disclosure comprises two or more NAD(P)-dependent enzymes. In certain embodiments, an analyte sensor of the present disclosure comprises two active sites, each comprising at least one NAD(P)-dependent enzyme. Alternatively, in certain embodiments, an analyte sensor of the present disclosure comprises two active sites, with only one active site comprising an NAD(P)-dependent enzyme. Non-limiting examples of NAD(P)-dependent enzymes are disclosed in Vidal et al., Biochimica et Biophysica Acta-Proteins and Proteomics 1866(2):327-347 (2018) (see Tables 1-2), the contents of which are incorporated herein by reference.

[0227] In certain embodiments, an analyte sensor of the present disclosure includes one or more internal sources of NAD(P) for an NAD(P)-dependent enzyme contained in one or more active sites of the analyte sensor. For example, without limitation, an NAD(P) depot can be disposed beneath each electrode configured to detect the analyte. Alternatively, an NAD(P) depot can be disposed beneath only one electrode configured to detect the analyte.

[0228] In certain embodiments, the active site may comprise an NAD(P)-dependent dehydrogenase. Non-limiting examples of NAD(P)-dependent dehydrogenases include glucose dehydrogenase (EC 1.1.1.47), lactate dehydrogenase (EC 1.1.1.27 and EC 1.1.1.28), malate dehydrogenase (EC 1.1.1.37), glycerol dehydrogenase (EC 1.1.1.6), alcohol dehydrogenase (EC 1.1.1.1), α-hydroxybutyrate dehydrogenase, sorbitol dehydrogenase, amino acid dehydrogenases such as L-amino acid dehydrogenase (EC 1.4.1.5), diaphorase (EC 1.8.1.4), and combinations thereof.

[0229] In certain embodiments, the NAD(P)-dependent dehydrogenase can include diaphorase, glucose dehydrogenase, alcohol dehydrogenase, lactate dehydrogenase, and β-hydroxybutyrate dehydrogenase. In certain embodiments, the enzyme system can include two or more NAD(P)-dependent dehydrogenases, e.g., a first NAD(P)-dependent dehydrogenase and diaphorase. For example, but not limited to, the NAD(P)-dependent dehydrogenase can react with the test substance and oxidized nicotinamide adenine dinucleotide (NAD + ) can be converted to the oxidized analyte and reduced nicotinamide adenine dinucleotide (NADH), respectively. +and NADH help drive the concerted enzymatic reactions disclosed herein. NADH can then undergo diaphorase-mediated reduction, and the electrons transferred during this process provide the basis for analyte detection at the working electrode.

[0230] In certain embodiments, the analyte sensors of the present disclosure may include a glucose-responsive active region, a ketone-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 glucose-responsive active region may include one or more NAD(P)-dependent enzymes for detecting glucose. In certain embodiments, the ketone-responsive active region may include one or more NAD(P)-dependent enzymes for detecting ketones. In certain embodiments, the lactate-responsive active region may include one or more NAD(P)-dependent enzymes for detecting lactate. In certain embodiments, the creatinine-responsive active region may include one or more NAD(P)-dependent enzymes for detecting creatinine. In certain embodiments, the alcohol-responsive active region may include one or more NAD(P)-dependent 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. For example, but not by way of limitation, the ketone-responsive active region can include an enzyme system comprising at least one NAD(P)-dependent enzyme.

[0231] In certain embodiments, the active site can be a glucose-responsive active site that includes at least one NAD(P)-dependent enzyme for detecting glucose. In certain embodiments, the glucose-responsive active site can include glucose dehydrogenase. For example, without limitation, an analyte sensor of the present disclosure for detecting glucose can include an NAD(P) depot and an active region comprising glucose dehydrogenase.

[0232] In certain embodiments, the active site can be an alcohol-responsive active site that includes at least one NAD(P)-dependent enzyme for detecting one or more alcohols. In certain embodiments, the alcohol-responsive active site can include an alcohol dehydrogenase. For example, without limitation, an analyte sensor of the present disclosure for detecting alcohol can include an NAD(P) depot and an active region comprising an alcohol dehydrogenase.

[0233] In certain embodiments, the active site can be a ketone-responsive active site that includes at least one NAD(P)-dependent enzyme for detecting one or more ketones. In certain embodiments, the ketone-responsive active site can include β-hydroxybutyrate dehydrogenase. For example, without limitation, an analyte sensor of the present disclosure for detecting ketones can include an NAD(P) depot and an active region with an enzyme system that includes β-hydroxybutyrate dehydrogenase.

[0234] In certain embodiments, the active site can be a lactate-responsive active site that includes at least one NAD(P)-dependent enzyme for detecting lactate. For example, without limitation, the lactate-responsive active site can include lactate dehydrogenase. In certain embodiments, an analyte sensor of the present disclosure for detecting lactate can include an NAD(P) depot and an active region comprising lactate dehydrogenase.

[0235] In certain embodiments, the analyte sensors disclosed herein can include at least one active site comprising one or more NAD(P)-dependent enzymes as disclosed herein for detecting an analyte. Alternatively, the analyte sensors disclosed herein can include two or more active sites, each containing one or more enzymes, e.g., at least one of the active sites comprises one or more NAD(P)-dependent enzymes. For example, without limitation, the analyte sensors of the present disclosure can include a first active region comprising a first enzyme (or enzyme system) for use in detecting a first analyte and a second active region comprising a second enzyme (or second enzyme system) for detecting a second analyte, wherein at least the first active region or the second active region comprises an NAD(P)-dependent enzyme.

[0236] In certain embodiments, the analyte-responsive active region can 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 (e.g., one or more NAD(P)-dependent enzymes) disclosed herein. In certain embodiments, the analyte-responsive active region can 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 one or more enzymes (e.g., one or more NAD(P)-dependent enzymes) disclosed herein. In certain embodiments, the analyte-responsive active region can 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 (e.g., one or more NAD(P)-dependent enzymes) disclosed herein. In certain embodiments, the analyte-responsive active region can 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 (e.g., one or more NAD(P)-dependent enzymes) disclosed herein.

[0237] In certain embodiments, the analyte-responsive active region can further comprise a stabilizer, for example, to stabilize the enzyme. For example, without limitation, the stabilizer can 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 can be catalase. In certain embodiments, the analyte-responsive active region may comprise a ratio of stabilizer to one or more enzymes present in the analyte-responsive active region (e.g., an NAD(P)-dependent enzyme) 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 analyte-responsive active region can comprise a ratio of stabilizer to one or more enzymes present in the analyte-responsive active region (e.g., NAD(P)-dependent enzymes) of about 2:1 to about 1:2. In certain embodiments, the test substance-responsive active region may comprise a ratio of stabilizer to NAD(P)-dependent enzyme (e.g., NAD(P)-dependent 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 analyte-responsive active region can comprise a ratio of stabilizer to NAD(P)-dependent enzyme (e.g., NAD(P)-dependent dehydrogenase) of about 2:1 to about 1:2. In certain embodiments, the analyte-responsive active region can comprise about 10% to about 50% by weight of stabilizer, for example, about 15% to about 45% by weight, about 20% to about 40% by weight, about 20% to about 35% by weight, or about 20% to about 30% by weight.In certain embodiments, the analyte-responsive active region can include about 15% to about 35% by weight of the stabilizer.

[0238] In certain embodiments, in addition to the presence of an NAD(P) depot, the analyte-responsive active region can further include a cofactor for one or more enzymes present in the analyte-responsive active region. In certain embodiments, the cofactor is NAD(P). In certain embodiments, the cofactor is a cofactor different from NAD(P). In certain embodiments, the analyte-responsive active region may comprise a cofactor to enzyme ratio 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 analyte-responsive active region may comprise a cofactor to enzyme ratio of about 2:1 to about 1:2. In certain embodiments, the analyte-responsive active region can comprise about 10% to about 50% by weight of the cofactor, e.g., about 15% to about 45% by weight, about 20% to about 40% by weight, about 20% to about 35% by weight, or about 20% to about 30% by weight. In certain embodiments, the analyte-responsive active region can comprise about 15% to about 35% by weight of the cofactor. In certain embodiments, the cofactor, e.g., NAD(P), can be physically retained within the analyte-responsive active region. For example, but not by way of limitation, a membrane covering the analyte-responsive active region can help retain the cofactor within the analyte-responsive active region while allowing sufficient inward diffusion of the analyte to enable detection of the analyte.

[0239] In certain embodiments, an analyte sensor of the present disclosure can include a sensor tail comprising at least one NAD(P) depot, at least one working electrode, e.g., a permeable electrode, and an analyte-responsive active region disposed on the surface of the working electrode, wherein the analyte-responsive active region comprises at least one NAD(P)-dependent enzyme. In certain embodiments, an analyte sensor of the present disclosure can include a sensor tail comprising a substrate, at least one NAD(P) depot disposed on the surface of the substrate, at least one working electrode, e.g., a permeable electrode, and an analyte-responsive active region disposed on the surface of the working electrode, wherein the analyte-responsive active region comprises at least one NAD(P)-dependent enzyme. In certain embodiments, the NAD(P)-dependent enzyme is an NAD(P)-dependent dehydrogenase. For example, without limitation, a sensor of the present disclosure can include a sensor tail comprising at least one NAD(P) depot, a permeable layer disposed on the NAD(P) depot, at least one permeable working electrode, and an analyte-responsive active region disposed on a surface of the permeable working electrode, wherein the analyte-responsive active region includes an enzyme system comprising an NAD(P)-dependent dehydrogenase.

[0240] In certain embodiments, a sensor of the present disclosure can include a sensor tail comprising at least one NAD(P) depot, a permeable layer disposed on top of the NAD(P) depot, at least one permeable working electrode, and a ketone-responsive active region disposed on a surface of the permeable working electrode, wherein the ketone-responsive active region comprises an enzyme system comprising an NAD(P)-dependent dehydrogenase, e.g., β-hydroxybutyrate dehydrogenase. In certain embodiments, the enzyme system further comprises diaphorase.

[0241] In certain embodiments, an analyte sensor of the present disclosure can include a second active region for detecting an analyte different from the analyte detected by the first active region, for example. In certain embodiments, the second active region is disposed on the same working electrode as the first active region or on a second working electrode. In 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.

[0242] In certain embodiments, the second active region of the analyte sensor of the present disclosure can include one or more enzymes for detecting glucose. For example, without limitation, the analyte sensor of the present disclosure can include an active region (e.g., second active region) comprising one or more enzymes for detecting glucose, disposed, for example, on a second working electrode. In certain embodiments, the analyte sensor can include an active site comprising glucose oxidase and / or glucose dehydrogenase for detecting glucose.

[0243] In certain embodiments, the second active region can include one or more enzymes for detecting lactate. For example, without limitation, an analyte sensor of the present disclosure can include an active region (e.g., second active region) comprising one or more enzymes (e.g., enzyme systems) for detecting lactate, disposed, for example, on a second working electrode. In certain embodiments, the analyte sensor can include an active site comprising lactate dehydrogenase and / or lactate oxidase.

[0244] In certain embodiments, a second enzyme-responsive active region present on, for example, a second working electrode of an analyte sensor of the present disclosure can include one or more enzymes for detecting alcohol. For example, without limitation, an analyte sensor of the present disclosure can include an active region (e.g., a second active region) comprising one or more enzymes (e.g., an enzyme system) for detecting alcohol, disposed on, for example, a second working electrode. In certain embodiments, an analyte sensor can include an active site comprising alcohol dehydrogenase.

[0245] In certain embodiments, a second enzyme-responsive active region present on, e.g., the second working electrode of an analyte sensor of the present disclosure can include one or more enzymes for detecting creatinine. For example, without limitation, an analyte sensor of the present disclosure can include an active region (e.g., a second active region) disposed on, e.g., the second working electrode, that includes one or more enzymes (e.g., an enzyme system) for detecting creatinine. In certain embodiments, the analyte sensor can include an active site that includes amidohydrolase, creatine amidinohydrolase, and / or sarcosine oxidase.

[0246] In certain embodiments, an analyte sensor can include two working electrodes, e.g., a first active region disposed on a first working electrode (e.g., a permeable electrode) and a second active region disposed on a second working electrode. For example, without limitation, an analyte sensor disclosed herein can feature at least one NAD(P) depot, a first analyte-responsive active region disposed on the first working electrode, and a second analyte-responsive active region disposed on a different working electrode, e.g., the second working electrode, where at least one of the analyte-responsive active regions comprises an NAD(P)-dependent enzyme. In certain embodiments, the second analyte-responsive active region can be configured to detect a different analyte or the same analyte detected by the first analyte-responsive active region. In certain embodiments, such an analyte sensor can include a sensor tail having at least one NAD(P) depot, a first working electrode, a second working electrode, a first analyte-responsive active region disposed on the surface of the first working electrode, and a second analyte-responsive active region disposed on the surface of the second working electrode, wherein at least one of the analyte-responsive active regions includes an NAD(P)-dependent enzyme, and at least one of the working electrodes is permeable. For example, but not limited to, an analyte-responsive active region including an NAD(P)-dependent enzyme is disposed on the permeable working electrode.

[0247] In certain embodiments, when a sensor is configured to detect two or more analytes using two working electrodes, detection of each analyte can include separately applying a potential to each working electrode so that a separate signal is obtained from each analyte. The signal obtained from each analyte can then be correlated to an analyte concentration through the use of a calibration curve or function or by employing a look-up table. In certain embodiments, the correlation between the analyte signal and the analyte concentration can be performed through the use of a processor.

[0248] In certain other analyte sensor configurations, the first and second active regions can be disposed on a single working electrode. For example, without limitation, the analyte sensors disclosed herein can feature at least one NAD(P) depot, a first analyte-responsive active region, and a second analyte-responsive active region disposed on the surface of a single permeable working electrode, with at least one of the analyte-responsive active regions comprising an NAD(P)-dependent enzyme. In certain embodiments, a first signal can be obtained from the first active region, e.g., at a low potential, and a second signal including signal contributions from both active regions can 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 can then be correlated with analyte concentration in a manner similar to that described for sensor configurations with multiple working electrodes. In certain embodiments, when a ketone-responsive active region and a second active region configured to detect a different analyte, such as a glucose-responsive active region, are disposed on a single working electrode in this manner, one of the active regions can be configured to respond separately to facilitate detection of each analyte. For example, either the ketone-responsive active region or the glucose-responsive active region can generate a signal independently of the other active region.

[0249] It should also be understood that the sensitivity (output current) of the analyte sensor directed to each analyte can be varied by varying the coverage (area or size) of the active regions, the area ratio of the active regions to each other, the identity, and the thickness and / or composition of the mass transport limiting membrane covering the active regions. Variations of these parameters can be readily implemented by one of ordinary skill in the art given the benefit of the disclosure herein.

[0250] 4. Redox mediators In certain embodiments, the analyte sensors disclosed herein can include an electron transfer agent. For example, without limitation, one or more active sites of the analyte sensor can include an electron transfer agent. In certain embodiments, the analyte sensor can include one active site that includes an electron transfer agent and a second active site that does not include an electron transfer agent. In certain embodiments, the presence of the electron transfer agent in the active site can depend on the composition of the enzyme or enzyme system and / or working electrode used to detect the analyte. Alternatively, the analyte sensor can include two active sites, both of which include an electron transfer agent.

[0251] 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 is proportional to the amount of analyte present. In certain embodiments, suitable electron transfer agents can include electroreducible and electrooxidizable ions, complexes, or molecules (e.g., quinones) with redox potentials several hundred millivolts higher or lower than the redox potential of a standard calomel electrode (SCE). In certain embodiments, redox mediators can include osmium complexes and other transition metal complexes (e.g., as 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. Examples of other suitable redox mediators include metal compounds or complexes (including, for example, metallocene compounds) of ruthenium, osmium, iron (e.g., polyvinylferrocene or hexacyanoferrate), or cobalt. Suitable ligands for metal complexes can also include bidentate or higher ligands, such as, for example, bipyridine, biimidazole, phenanthroline, or pyridyl (imidazole). Other suitable bidentate ligands can include, for example, amino acids, oxalic acid, acetylacetone, diaminoalkanes, or o-diaminoarenes. Any combination of monodentate, bidentate, tridentate, tetradentate, or higher dentate ligands can be present in a metal complex (e.g., an osmium complex) to achieve a complete coordination sphere.

[0252] In certain embodiments, the electron transfer agents disclosed herein may comprise functional groups suitable for facilitating covalent attachment to a polymer (referred to herein as the polymer backbone) in the active region, as discussed further below. For example, without limitation, 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 attached to a polymer described herein, such as the polymer backbone described in Section 5 below. In certain embodiments, the polymer-bound electron transfer agent shown in Figure 3 of U.S. Pat. No. 8,444,834 may be used in the sensors of the present disclosure.

[0253] In certain embodiments of the present disclosure, the analyte sensor can include at least one NAD(P) depot, at least one permeable layer disposed on the NAD(P) depot, at least one working electrode, e.g., a permeable electrode, and at least one analyte-responsive active region disposed on the surface of the working electrode, the analyte-responsive active region comprising at least one NAD(P)-dependent enzyme and at least one redox mediator, e.g., an osmium complex. In certain embodiments, the analyte-responsive active region comprises an enzyme system comprising diaphorase, an NAD(P)-dependent dehydrogenase, e.g., β-hydroxybutyrate dehydrogenase, and a redox mediator, e.g., an osmium complex.

[0254] 5. Polymer backbone In certain embodiments, one or more active sites for facilitating analyte detection may comprise 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 analyte detection via covalent attachment of an enzyme and / or a redox mediator thereto. Non-limiting examples of suitable polymers in the active region include polyvinylpyridines, e.g., poly(4-vinylpyridine) and / or poly(2-vinylpyridine), and polyvinylimidazoles, e.g., poly(N-vinylimidazole) and poly(1-vinylimidazole), or copolymers thereof, where, for example, quaternized pyridine groups serve as attachment points for the redox mediator or enzyme. Exemplary copolymers that may be suitable for inclusion in the active region include those containing monomer units such as 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 (incorporated herein 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 in which the analyte sensor includes two active sites, the polymers in each active region can be the same or different.

[0255] In certain embodiments, the polymer is polyvinylpyridine or a copolymer thereof. In certain embodiments, the polymer is a copolymer of vinylpyridine and styrene. In certain embodiments, when an enzyme system comprising multiple enzymes 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 may be covalently bound to the polymer. For example, without limitation, 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, the NAD(P)-dependent enzyme may be covalently bound to the polymer. Alternatively, the NAD(P)-dependent enzyme may be non-covalently bound to the polymer. In certain embodiments, the NAD(P)-dependent dehydrogenase and diaphorase may be covalently bound to the polymer within the active area of ​​the disclosed analyte sensor. In certain embodiments, the NAD(P)-dependent dehydrogenase may be covalently bound to the polymer, and the diaphorase may be non-covalently bound to the polymer. Alternatively, the diaphorase may be covalently bound to the polymer, and the NAD(P)-dependent dehydrogenase may be non-covalently bound to the polymer.

[0256] In certain embodiments, when a stabilizer is present in an active region, one or more enzymes in the region can be covalently bound to the stabilizer. For example, without limitation, one or more enzymes in an enzyme system, such as one or more NAD(P)-dependent enzymes, can be covalently bound to a stabilizer, such as albumin, present in the active region.

[0257] In certain embodiments, covalent bonding of one or more enzymes and / or redox mediators to the polymer and / or stabilizer in a given active area can occur through crosslinking introduced by a suitable crosslinking agent. In certain embodiments, crosslinking of the polymer to one or more enzymes and / or redox mediators can reduce the occurrence of delamination of the enzyme composition from the electrode. Suitable crosslinking agents can 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 can include mono-, di-, tri-, and tetra-ethylene oxide. In certain embodiments, crosslinkers for reaction with free amino groups in enzymes (e.g., with free side chain amines in lysines) can include crosslinkers such as 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), e.g., of 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 crosslinker is polyethylene glycol diglycidyl ether. In certain embodiments, the crosslinking of the enzyme to the polymer is generally intermolecular. In certain embodiments, the crosslinking of the enzyme to the polymer is generally intramolecular.

[0258] 6. Mass transfer limiting membrane In certain embodiments, the analyte sensors disclosed herein further include a membrane that is permeable to the analyte and that covers at least the active area, e.g., the first active area and / or the second active area.

[0259] In certain embodiments, the analyte sensors disclosed herein further include a membrane that is permeable to the analyte and that covers at least one active area, e.g., the first active area and / or the second active area. In certain embodiments, a membrane covers each of the active areas of the analyte sensor. Alternatively, the first membrane covers one of the active areas and the second membrane covers the second active area. Alternatively, the first membrane covers one of the active areas and the second membrane covers both the first and second active areas.

[0260] In certain embodiments, the membrane covering the analyte-responsive active region 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 mass transfer rate of the analyte (e.g., glucose, alcohol, ketone, lactate, or β-hydroxybutyrate) during use of the sensor. For example, but not by way of limitation, limiting the access of the analyte, such as a ketone, to the analyte-responsive active region with a mass transport limiting membrane may help avoid sensor overload (saturation), thereby improving detection performance and accuracy. In certain embodiments, the mass transport limiting layer limits the flux of the analyte to the electrodes in the electrochemical sensor, resulting in a linear sensor response over a wide range of analyte concentrations.

[0261] In certain embodiments, the mass transport limiting membrane may be homogeneous and may be a single component (comprising a single membrane polymer). Alternatively, the mass transport limiting membrane may be multicomponent (comprising two or more different membrane polymers). In certain embodiments, the mass transport limiting membrane may include two or more layers, e.g., a bilayer or trilayer 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 first analyte-responsive active region may be covered by a multilayer membrane, e.g., a bilayer membrane, and the second analyte-responsive active region may be covered by a single membrane. In certain embodiments, the first analyte-responsive active region may be covered by a multilayer membrane, e.g., a bilayer membrane, and the second analyte-responsive active region may be covered by a multilayer membrane, e.g., a bilayer membrane. In certain embodiments, the first analyte-responsive active region may be covered by a single membrane and the second analyte-responsive active region may be covered by a multilayer membrane, e.g., a bilayer membrane may be covered by a single membrane. In certain embodiments, the first analyte-responsive active area may be covered by a single membrane, and the second analyte-responsive active area may be covered by a single membrane.

[0262] In certain embodiments, the mass transport limiting membrane can comprise a crosslinked polymer containing a heterocyclic nitrogen group. In certain embodiments, the mass transport limiting membrane can comprise 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.

[0263] 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), polyacrylate, polyurethane, polyetherurethane, silicone, polytetrafluoroethylene, polyethylene-co-tetrafluoroethylene, polyolefin, polyester, polycarbonate, biostable polytetrafluoroethylene, polyurethane homopolymers, copolymers, or terpolymers, polypropylene, polyvinyl chloride, polyvinylidene difluoride, polybutylene terephthalate, polymethyl methacrylate, polyether ether ketone, cellulose polymers, polysulfone, and block copolymers thereof, including, for example, diblock, triblock, alternating, random, and graft copolymers, or other chemically related materials.

[0264] In certain embodiments, membranes, e.g., single-component membranes, for use in the present disclosure may comprise 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 comprise poly(4-vinylpyridine). In certain embodiments, membranes, e.g., single-component membranes, for use in the present disclosure may comprise 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- The 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.

[0265] 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)(2-aminopropyl ether) 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.

[0266] In certain embodiments, the membrane comprises a polyurethane membrane containing both hydrophilic and hydrophobic regions. In certain embodiments, the hydrophobic polymer component is a polyurethane, polyurethaneurea, or poly(ether-urethane-urea). In certain embodiments, polyurethane is a polymer produced by the condensation reaction of a diisocyanate with a difunctional hydroxyl-containing material. In certain embodiments, polyurethaneurea is a polymer produced by the condensation reaction of a diisocyanate with a difunctional amine-containing material. In certain embodiments, diisocyanates for use herein include aliphatic diisocyanates containing, for example, about 4 to about 8 methylene units, or diisocyanates containing alicyclic moieties. Further non-limiting examples of polymers that can be used to produce membranes for sensors 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. In certain embodiments, the hydrophilic polymer component is a polyurethane copolymer. 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., 20%, hydrophilic polyethylene oxide.

[0267] 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, polyvinylpyrrolidone, polyhydroxyethyl methacrylate, polyvinyl alcohol, polyacrylic acid, polyethers such as 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 PEO and PPO copolymers include PEO-PPO diblock copolymers, PPO-PEO-PPO triblock copolymers, PEO-PPO-PEO triblock copolymers, alternating block copolymers of PEO-PPO, random copolymers of ethylene oxide and propylene oxide, and blends thereof. In certain embodiments, the copolymer may be substituted with hydroxy substituents.

[0268] In certain embodiments, hydrophilic or hydrophobic modifiers can be used to "fine tune" the permeability of the resulting membrane to an analyte of interest. In certain embodiments, hydrophilic modifiers such as poly(ethylene) glycol, hydroxyl or polyhydroxyl modifiers, and any combination thereof, can be used to improve the biocompatibility of the polymer or the resulting membrane.

[0269] 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 different active regions, which can be achieved through successive dip-coating operations to create a bilayer membrane portion at the working electrode located more closely to the sensor tip.

[0270] In certain embodiments where multiple active regions are present, a separate mass transport limiting membrane can cover each active region. For example, without limitation, a mass transport limiting membrane can be disposed over a first active region, e.g., a ketone-responsive active region, and a separate second mass transport limiting membrane can cover a second active region, e.g., a glucose-responsive active region. In certain embodiments, the two mass transport limiting membranes are spatially separated and do not overlap. 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. Alternatively, 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.

[0271] In certain embodiments, the composition of a mass transport limiting membrane disposed on an analyte sensor having two active regions can be the same or different when a mass transport limiting membrane covers each active region. For example, without limitation, the portion of the mass transport limiting membrane covering the ketone-responsive active region can be multi-component and / or the portion of the mass transport limiting membrane covering the glucose-responsive active region can be single-component. Alternatively, the portion of the mass transport limiting membrane covering the ketone-responsive active region can be single-component and / or the portion of the mass transport limiting membrane covering the glucose-responsive active region can be multi-component.

[0272] In certain embodiments, the glucose-responsive active region can be coated with a membrane comprising polyurethane, polyurethaneurea, or poly(ether-urethane-urea). In certain embodiments, the glucose-responsive active region can be coated with a membrane comprising polyurethane. In certain embodiments of the present disclosure, the ketone-responsive active region and a second analyte-responsive region, e.g., a glucose-responsive active region, can be coated with a membrane comprising polyvinylpyridine-co-styrene copolymer.

[0273] In certain embodiments, the membrane, e.g., the single-component membrane, can include polyvinylpyridine. In certain embodiments, the membrane, e.g., the single-component membrane, can include a copolymer of vinylpyridine and styrene (or a derivative thereof).

[0274] In certain embodiments, the multi-component membrane can exist as a bilayer membrane or as a homogeneous mixture of two or more membrane polymers. A homogeneous mixture can be deposited by mixing two or more membrane polymers in a solution and then depositing (e.g., dipping) this solution onto the working electrode. In certain embodiments of the present disclosure, a first analyte-responsive active region, e.g., a ketone-responsive active region, can be coated with a multi-component membrane comprising polyvinylpyridine and polyvinylpyridine-co-styrene copolymer, either as a bilayer membrane or a homogeneous mixture, and a second analyte-responsive active region, e.g., a glucose-responsive active region, can be coated with a membrane comprising polyvinylpyridine-co-styrene copolymer.

[0275] Suitable copolymers of vinylpyridine and styrene 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 also be used in similar amounts. Suitable copolymers of vinylpyridine and styrene may have a molecular weight of 5 kDa or greater, or about 10 kDa or greater, or about 15 kDa or greater, or about 20 kDa or greater, or about 25 kDa or greater, or about 30 kDa or greater, or about 40 kDa or greater, or about 50 kDa or greater, or about 75 kDa or greater, or about 90 kDa or greater, or about 100 kDa or greater. In non-limiting examples, suitable copolymers of vinylpyridine and styrene can have a molecular weight ranging from about 5 kDa to about 150 kDa, or from about 10 kDa to about 125 kDa, or from about 15 kDa to about 100 kDa, or from about 20 kDa to about 80 kDa, or from about 25 kDa to about 75 kDa, or from about 30 kDa to about 60 kDa.

[0276] Polydimethylsiloxane (PDMS) can be incorporated into any of the mass transport limiting membranes disclosed herein. In certain embodiments, the analyte sensors described herein can comprise a sensor tail comprising at least an NAD(P) depot, a permeable polymer covering the NAD(P) depot, a first permeable working electrode, a first active area disposed on a surface of the first working electrode, and a mass transport limiting membrane permeable to the first analyte covering at least the first active area.

[0277] In certain embodiments, the first active region comprises at least one NAD(P)-dependent enzyme (optionally covalently bound to a first polymer present in the active region) responsive to a first analyte. For example, without limitation, an analyte sensor described herein can comprise a sensor tail comprising at least an NAD(P) depot, a permeable polymer covering the NAD(P) depot, a first working electrode, an analyte-responsive active region disposed on a surface of the first working electrode and comprising at least one NAD(P)-dependent enzyme, and a mass transport limiting membrane covering the analyte-responsive active region and permeable to the analyte.

[0278] In certain embodiments, the first active region comprises a first polymer and, optionally, an enzyme covalently bound to the first polymer that is responsive to a first analyte, e.g., glucose, e.g., an NAD(P)-dependent enzyme. For example, without limitation, an analyte sensor described herein can comprise at least an NAD(P) depot, a first working electrode, a glucose-responsive active region disposed on a surface of the first working electrode and comprising glucose dehydrogenase (optionally covalently bound to the first polymer), and a sensor tail comprising a mass transport limiting membrane covering the glucose-responsive active region and permeable to glucose.

[0279] In certain embodiments, the first active region comprises a first polymer and, optionally, at least one enzyme, e.g., an NAD-dependent enzyme, covalently bound to the first polymer, and an enzyme system responsive to a first analyte, e.g., ketones. For example, without limitation, an analyte sensor described herein can comprise at least an NAD(P) depot, a first working electrode, a ketone-responsive active region disposed on a surface of the first working electrode and comprising an enzyme system comprising β-hydroxybutyrate dehydrogenase and diaphorase, one or both enzymes covalently bound to the polymer, and a mass transport limiting membrane covering the ketone-responsive active region and permeable to ketones.

[0280] In certain embodiments, when a first active region and a second active region configured to assay different analytes are disposed on separate working electrodes, the mass transport limiting membrane can have different permeability values ​​for the first and second analytes. For example, without limitation, the mass transport limiting membrane covering at least one of the active regions can 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 can cover active regions not covered by the mixture or bilayer, where the homogeneous membrane includes only one of the first membrane polymer or the second membrane polymer. Advantageously, the architecture of the analyte sensor disclosed herein allows a continuous membrane having a homogeneous membrane portion to be disposed on the first active region of the analyte sensor, and a multi-component membrane portion to be disposed on the second active region of the analyte sensor, thereby simultaneously equalizing the permeability values ​​for each analyte, improving sensitivity and detection accuracy. In certain embodiments, successive film depositions can be achieved by successive dip coating operations.

[0281] In certain embodiments, when multiple active areas are present, a mass transport limiting membrane can cover each active area. In certain embodiments, the mass transport limiting layer is a membrane composed of a crosslinked polymer containing heterocyclic nitrogen groups, such as polymers of polyvinylpyridine and polyvinylimidazole. Embodiments also include membranes made from polyurethane, polyetherurethane, or chemically related materials, or membranes made from silicone, etc. In certain embodiments, the mass transport limiting membrane can include a membrane polymer, such as a polyvinylpyridine or polyvinylimidazole homopolymer or copolymer, which can be further crosslinked with an appropriate crosslinking agent. In certain embodiments, the membrane polymer can include a copolymer of vinylpyridine and styrene.

[0282] In certain embodiments, the mass transport limiting membrane can comprise a membrane polymer crosslinked with a crosslinker as disclosed herein and in Section 5 above. In certain embodiments where two mass transport limiting membranes are present, e.g., a first mass transport limiting membrane and a second mass transport limiting membrane, each membrane can be crosslinked to a different crosslinker. For example, but not by way of limitation, a crosslinker can 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 ketone-responsive region can have a pore size that limits the diffusion of compounds larger than ketones, e.g., glucose, through the membrane.

[0283] In certain embodiments, crosslinkers for use in the present disclosure may include polyepoxides, carbodiimides, cyanuric chloride, triglycidylglycerol, N-hydroxysuccinimide, imide esters, epichlorohydrin, or derivatized variants thereof. In certain embodiments, membrane polymers covering one or more active regions may be crosslinked to a branched crosslinker, which can, 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 containing two, three, or more crosslinkable groups. In certain embodiments, the branched crosslinker may contain two or more crosslinkable groups, such as polyethylene glycol diglycidyl ether. In certain embodiments, the branched crosslinker may contain 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 containing 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, such as polyethylene glycol tetraglycidyl ether or polyethylene glycol diglycidyl ether, can form a covalent bond with pyridine or imidazole by opening the epoxide ring, resulting in a hydroxyalkyl group that bridges the body of the crosslinker with the heterocycle of the membrane polymer.

[0284] In certain embodiments, the crosslinker is polyethylene glycol diglycidyl ether (PEGDGE). In certain embodiments, PEGDGE used to promote crosslinking (e.g., intermolecular crosslinking) between two or more membrane polymer backbones can exhibit a wide range of suitable molecular weights. In certain embodiments, the molecular weight of PEGDGE can range from about 100 g / mol to about 5000 g / mol. The number of ethylene glycol repeat units in each arm of PEGDGE can be the same or different, and generally can vary over a range within a given sample from which the average molecular weight is obtained. In certain embodiments, PEGDGE for use in the present disclosure has an average molecular weight (M) of about 200 to 1000, e.g., about 400. n In certain embodiments, the crosslinker is PEGDGE400.

[0285] In certain embodiments, the polyethylene glycol tetraglycidyl ether used to promote crosslinking (e.g., intermolecular crosslinking) between two or more membrane polymer backbones can exhibit a wide range of suitable molecular weights. Up to four polymer backbones can be crosslinked by a single molecule of polyethylene glycol tetraglycidyl ether crosslinker. In certain embodiments, the molecular weight of the polyethylene glycol tetraglycidyl ether can 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 can be the same or different, and generally, the average molecular weight can vary over a range within a given sample. In certain embodiments, the mass transport limiting membrane can be deposited directly on the active region.

[0286] In certain embodiments, the mass transport limiting membrane 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 mass transport limiting membrane can 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 can be immersed in the mass transport limiting membrane solution more than once. For example, without limitation, a sensor (or working electrode) of the present disclosure can be immersed in the interference domain solution at least two times, at least three times, at least four times, or at least five times to achieve the desired interference domain thickness.

[0287] 7. Interference Domains In certain embodiments, a sensor of the present disclosure, e.g., a 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, allowing 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, 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.

[0288] 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.

[0289] In certain embodiments, the interference domain is part of the mass transport limiting membrane and is not a separate membrane. In certain embodiments, the interference domain comprises a thin, hydrophobic membrane that is non-swelling and restricts 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.

[0290] In certain embodiments, the interference domain can be deposited directly onto the working electrode, e.g., on the surface of a permeable 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 can 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 can be immersed in the interference domain solution more than once. For example, without limitation, a sensor (or working electrode) of the present disclosure can be immersed in the interference domain solution at least twice, at least three times, at least four times, or at least five times to achieve the desired interference domain thickness.

[0291] 8.Manufacturing The present disclosure further provides methods for manufacturing an analyte sensor of the present disclosure that includes one or more active regions, one or more NAD(P) depots, and one or more working electrodes.

[0292] In certain embodiments, the method includes depositing a composition containing NAD(P) onto a substrate to generate an NAD(P) depot. For example, without limitation, the composition may be NAD and / or NADP, depending on the enzyme present in the active region. In certain embodiments, the method can further include adding a permeable layer on top of the NAD(P) depot. In certain embodiments, the permeable layer can include a polymer that controls the release of NAD(P) from the depot. Alternatively, a composition comprising a polymer and NAD(P) can be deposited onto a substrate to generate the NAD(P) depot. In certain embodiments, the polymer of the NAD(P) depot is curable, e.g., UV-curable.

[0293] In certain embodiments, the method can further include forming a permeable working electrode, eg, a carbon nanotube electrode, on the permeable layer. In certain embodiments, the method may further include depositing an enzyme composition comprising one or more NAD(P)-dependent enzymes, e.g., NAD(P)-dependent dehydrogenases, on the working electrode. In certain embodiments, the enzyme composition may include one or more additional enzymes (e.g., diaphorase), a cross-linker (e.g., polyethylene glycol diglycidyl ether), a polymer, and / or a redox mediator. In certain embodiments, the enzyme composition may be deposited on the surface of the working electrode as one large application covering a desired portion of the working electrode or in the form of an array of multiple enzyme compositions (e.g., spaced apart from one another) to create one or more active areas for detecting one or more analytes. In certain embodiments, the method may further include curing the enzyme composition.

[0294] In certain embodiments, the NAD(P), permeable polymer, permeable working electrode, and enzyme composition can be prepared as solutions that dry or cure to solidify after deposition. Thus, in certain embodiments, all layers can be deposited in an automated manner using low-volume liquid handling or similar techniques for high-throughput sensor fabrication.

[0295] In certain embodiments, the method can further include adding a film composition over the cured enzyme composition and / or around the entire sensor. In certain embodiments, the film composition can include a polymer, such as polyvinylpyridine, and / or a cross-linker, such as polyethylene glycol diglycidyl ether. In certain embodiments, the method can include curing the film polymer composition.

[0296] Generally, the thickness of the film is controlled by the concentration of the film solution, the number of droplets of film solution applied, the number of times the sensor is dipped into or sprayed with the film solution, the volume of film solution sprayed onto the sensor, and any combination of these factors. In certain embodiments, the films described herein can have a thickness ranging from about 0.1 micrometers (μm) to about 1000 μm, e.g., 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 can be dipped into the film solution more than once. For example, without limitation, a sensor (or working electrode) of the present disclosure can be dipped into the film solution at least two times, at least three times, at least four times, or at least five times to achieve a desired film thickness.

[0297] 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, 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, to achieve a desired thickness of the active region and / or film, a series of droplets can be applied on top of each other without substantially increasing the diameter of the applied droplets (i.e., while maintaining the desired diameter or range). In certain embodiments, each single droplet can be applied and then allowed to cool or dry, followed by the 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 can be applied on top of each other to achieve a desired thickness of the active region.

[0298] III. Test substance monitoring The present disclosure further provides methods of using the analyte sensors disclosed herein to detect analytes in vivo. In certain embodiments, the present disclosure provides methods for detecting one or more analytes, e.g., one analyte or two analytes. For example, but not limited to, the present disclosure provides methods for detecting one or more analytes including 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, and / or uric acid using one or more NAD(P)-dependent enzymes. In certain embodiments, the analyte can be ketones, alcohol, glucose, and / or lactate using one or more NAD(P)-dependent enzymes. For example, but not limited to, the present disclosure provides methods for detecting one or more ketones. In certain embodiments, the present disclosure provides a method for detecting glucose. In certain embodiments, the present disclosure provides a method for detecting creatinine. In certain embodiments, the present disclosure provides a method for detecting lactate. In certain embodiments, the present disclosure provides a method for detecting alcohol.

[0299] In certain embodiments, the present disclosure provides methods for monitoring in vivo analyte levels over time using an analyte sensor comprising an NAD(P) depot and one or more NAD(P)-dependent enzymes, such as NAD(P)-dependent dehydrogenases. Generally, monitoring the in vivo concentration of an analyte in a subject's bodily fluid involves at least partially inserting an in vivo analyte sensor disclosed herein beneath the skin surface, contacting the fluid to be monitored (e.g., interstitial fluid, blood, dermal fluid, etc.) with the inserted sensor, and generating a sensor signal at the working electrode. The presence and / or concentration of the analyte detected by the analyte sensor can be displayed, stored, transferred, and / or otherwise processed. Various approaches can be employed to determine the concentration of an analyte (e.g., glucose, alcohol, ketones, and / or lactate) using a subject's sensor. In certain embodiments, monitoring the concentration of the analyte using the sensor signal can be done by coulometry, amperometry, voltammetry, potentiometry, or any other convenient electrochemical detection technique.

[0300] In certain embodiments, a method for detecting an analyte includes: (i) providing an analyte sensor including: (a) an internal source of NAD(P); (b) a permeable polymer coating the internal source of NAD(P); (c) at least a first working electrode disposed on a surface of the permeable polymer and being a permeable working electrode; (d) an analyte-responsive active region disposed on a surface of the first working electrode and comprising an NAD(P)-dependent enzyme; and (e) a mass transport limiting membrane coating at least the analyte-responsive active region and permeable to the analyte; (ii) applying a potential to the first working electrode; (iii) obtaining a first signal at or above the redox potential of the analyte-responsive active region that is proportional to the concentration of the analyte in a fluid contacting the analyte-responsive active region; and (iv) correlating the first signal to the concentration of the analyte in the fluid.

[0301] In certain embodiments, a method for detecting one or more ketones includes: (i) providing an analyte sensor including: (a) an internal source of NAD(P); (b) a permeable polymer coating the internal source of NAD(P); (c) at least a first working electrode disposed on a surface of the permeable polymer and being a permeable working electrode; (d) a ketone-responsive active region disposed on a surface of the first working electrode and comprising β-hydroxybutyrate dehydrogenase and diaphorase; and (e) a mass transport limiting membrane coating at least the analyte-responsive region and permeable to ketones; (ii) applying a potential to the first working electrode; (iii) obtaining a first signal at or above the redox potential of the ketone-responsive active region, the first signal being proportional to the concentration of the analyte in a fluid contacting the analyte-responsive active region; and (iv) correlating the first signal to the concentration of ketones in the fluid.

[0302] In certain embodiments, the methods 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 the first analyte and the second analyte. In certain embodiments, an analyte sensor for use in a method for detecting a first analyte 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 comprising 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, e.g., the 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 that is separate and / or different from the mass transport limiting membrane that covers the ketone-responsive active region. In certain embodiments, the at least one enzyme responsive to the second analyte comprises an enzyme system comprising multiple enzymes that collectively respond to the second analyte.

[0303] In certain embodiments, the method further includes attaching an electronics unit to the patient's skin, coupling conductive contacts of the electronics unit to contacts of the sensor, collecting data related to the analyte level from a signal generated by the sensor using the electronics unit, and transferring the collected data from the electronics unit to a receiver unit, e.g., by RF. In certain embodiments, the receiver unit is a mobile phone. In certain embodiments, the mobile phone includes an application related to the analyte being monitored. In certain embodiments, the analyte information is transferred via an RFID protocol such as Bluetooth®.

[0304] In certain embodiments, an analyte sensor may be positioned within a user for automated analyte detection, e.g., continuously or periodically. In certain embodiments, analyte levels may be monitored over periods ranging from seconds to minutes, hours, days, weeks, or months. In certain embodiments, the methods disclosed herein may be used to predict future levels of an analyte based on acquired information, such as, but not limited to, the current analyte level at time 0 and the rate of change of the analyte concentration or amount.

[0305] IV. Illustrative Embodiments A. In certain non-limiting embodiments, the disclosed subject matter provides an analyte sensor comprising: (a) an internal source of NAD(P); (b) a permeable polymer coating the internal source of NAD(P); (c) at least a first working electrode disposed on a surface of the permeable polymer, the first working electrode being a permeable working electrode; (d) an analyte-responsive active region disposed on a surface of the first working electrode, the analyte-responsive active region comprising an NAD(P)-dependent enzyme; and (e) a mass transport limiting membrane coating at least the analyte-responsive region and permeable to the analyte.

[0306] A1. The analyte sensor according to A, wherein the NAD(P)-dependent enzyme is an NAD(P)-dependent dehydrogenase. A2. The analyte sensor of A or A1, wherein the permeable working electrode comprises carbon nanotubes.

[0307] A3. The analyte sensor of any one of A-A2, wherein the permeable polymer comprises poly(propylene glycol) methacrylate and 2-hydroxyethyl methacrylate.

[0308] A4. The analyte sensor of any one of A-A3, wherein the analyte is selected from the group consisting of glucose, ketone, alcohol, creatinine, lactate, and combinations thereof.

[0309] A5. The analyte sensor according to A4, wherein the analyte is glucose. A6. The analyte sensor according to A4, wherein the analyte is lactate. A7. The test substance sensor according to A4, wherein the test substance is alcohol.

[0310] A8. The analyte sensor according to A4, wherein the analyte is a ketone. A9. The analyte sensor according to A5, wherein the NAD(P)-dependent enzyme is glucose dehydrogenase.

[0311] A10. The analyte sensor according to A6, wherein the NAD(P)-dependent enzyme is lactate dehydrogenase. A11. The analyte sensor according to A7, wherein the NAD(P)-dependent enzyme is alcohol dehydrogenase.

[0312] A12. The analyte sensor according to A8, wherein the NAD(P)-dependent enzyme is β-hydroxybutyrate dehydrogenase. A13. The analyte sensor of any one of A-A12, wherein the analyte-responsive active region further comprises diaphorase.

[0313] A14. The analyte sensor of any one of A-A13, wherein the analyte-responsive active region further comprises a redox mediator. A15. The analyte sensor of any one of A-A14, wherein the analyte-responsive active region further comprises a stabilizer.

[0314] A16. The analyte sensor of A15, wherein the stabilizer comprises albumin. A17. The analyte sensor of any one of A-A16, wherein the analyte-responsive active region further comprises a cross-linking agent.

[0315] A18. The analyte sensor according to any one of A-A17, wherein the internal source of NAD(P) comprises about 1 μg to about 1000 μg of NAD(P). A19. The analyte sensor of any one of A-A18, wherein the mass transport limiting membrane comprises polyvinylpyridine (e.g., poly(4-vinylpyridine) or poly(4-vinylpyridine)), polyvinylimidazole, polyvinylpyridine copolymer (e.g., a copolymer of vinylpyridine and styrene), polyacrylate, polyurethane, polyetherurethane, silicone, or a combination thereof.

[0316] A20. An analyte sensor described in any one of A-A19, further comprising: (vi) a second working electrode; and (vii) a second active area disposed on the surface of the second working electrode and responsive to a second analyte different from the first analyte, the second active area comprising at least one enzyme responsive to the second analyte, wherein a second portion of the mass transport limiting membrane covers the second active area.

[0317] B. In certain non-limiting embodiments, the disclosed subject matter provides methods for detecting an analyte, comprising: (i) providing an analyte sensor comprising: (a) an internal source of NAD(P); (b) a permeable polymer coating the internal source of NAD(P); (c) at least a first working electrode disposed on a surface of the permeable polymer and being a permeable working electrode; (d) an analyte-responsive active region disposed on a surface of the first working electrode and comprising an NAD(P)-dependent enzyme; and (e) a mass transport limiting membrane coating at least the analyte-responsive region and permeable to the analyte; (ii) applying a potential to the first working electrode; (iii) obtaining a first signal at or above the redox potential of the first active region proportional to the concentration of the first analyte in a fluid contacting the first active region; and (iv) correlating the first signal to the concentration of the first analyte in the fluid.

[0318] B1. The method according to B, wherein the NAD(P)-dependent enzyme is an NAD(P)-dependent dehydrogenase. B2. The method of claim B or B1, wherein the permeable working electrode comprises carbon nanotubes.

[0319] B3. The method of any one of B-B2, wherein the permeable polymer comprises poly(propylene glycol) methacrylate and 2-hydroxyethyl methacrylate. B4. The method of any one of B-B3, wherein the test substance is selected from the group consisting of glucose, ketones, alcohol, lactate, and combinations thereof.

[0320] B5. The method according to B4, wherein the test substance is glucose. B6. The method of B4, wherein the test substance is lactate. B7. The method of B4, wherein the test substance is an alcohol.

[0321] B8. The method of B4, wherein the test substance is a ketone. B9. The method according to B5, wherein the NAD(P)-dependent enzyme is glucose dehydrogenase.

[0322] B10. The method according to B6, wherein the NAD(P)-dependent enzyme is lactate dehydrogenase. B11. The method according to B7, wherein the NAD(P)-dependent enzyme is alcohol dehydrogenase.

[0323] B12. The method according to B8, wherein the NAD(P)-dependent enzyme is β-hydroxybutyrate dehydrogenase. B13. The method of any one of B-B12, wherein the analyte-responsive active region further comprises diaphorase.

[0324] B14. The method of any one of B-B13, wherein the analyte-responsive active region further comprises a redox mediator. B15. The method of any one of B-B14, wherein the analyte-responsive active region further comprises a stabilizer.

[0325] B16. The method of B15, wherein the stabilizer comprises albumin. B17. The method of any one of B-B16, wherein the analyte-responsive active region further comprises a cross-linking agent.

[0326] B18. The method of any one of B-B17, wherein the internal source of NAD(P) comprises about 1 μg to about 1000 μg of NAD(P). B19. The method of any one of B-B18, wherein the mass transport limiting membrane comprises polyvinylpyridine (e.g., poly(4-vinylpyridine) or poly(4-vinylpyridine)), polyvinylimidazole, polyvinylpyridine copolymer (e.g., copolymer of vinylpyridine and styrene), polyacrylate, polyurethane, polyetherurethane, silicone, or a combination thereof.

[0327] B20. The method of any one of B-B14, wherein the analyte sensor further comprises: (f) a second working electrode; and (g) a second active area disposed on a surface of the second working electrode and responsive to a second analyte different from the first analyte, the second active area comprising at least one enzyme responsive to the second analyte, and wherein a second portion of the mass transport limiting membrane covers the second active area.

[0328] Example The presently disclosed subject matter will be better understood by reference to the following examples, which are provided by way of illustration of the presently disclosed subject matter, and not by way of limitation.

[0329] Example 1: Preparation of a polymer-controlled NAD release system. This example provides a method for fabricating a sensor with a NAD depot, as shown in Figure 23A.

[0330] The analyte sensors were prepared by depositing various solutions. First, an NAD solution was deposited on a thin plastic substrate (support layer) and dried to leave solid NAD. A polymer solution consisting of a mixture of poly(propylene glycol) methacrylate (POMA) and 2-hydroxyethyl methacrylate (HEMA) was then deposited on the solid NAD and polymerized by UV irradiation. Next, a carbon nanotube solution was deposited and dried to form a permeable electrode. Next, a ketone-sensing enzyme composition containing an enzyme system comprising diaphorase and β-hydroxybutyrate dehydrogenase was deposited on the permeable electrode. Finally, the electrode was singulated and dip-coated in a membrane solution comprising polyvinylpyridine and polystyrene copolymer and a crosslinker to form an outer membrane. A control sensor was similarly prepared, except that no NAD was deposited on the thin plastic substrate (Figure 23B).

[0331] Next, the responses of the sensor and control were evaluated using 2 mM β-hydroxybutyrate, used as a surrogate for ketones present in vivo. As shown in Figure 24, the control sensor without an NAD depot exhibited a decreasing signal over time as NAD diffused from the sensing layer. However, the sensor with an NAD depot exhibited no decrease in signal over time, likely due to the sustained release of NAD from the depot to maintain sufficient NAD concentrations in the sensing layer. Without being bound by theory, it is believed that because NAD is required to facilitate electron flow from the analyte to the electrode, the outer membrane of the sensor allows NAD to leach out of the sensing layer, resulting in a decrease in ketone response over time. As shown in this example, the use of an NAD depot can overcome such limitations by allowing the sustained release of NAD from the depot to maintain sufficient NAD concentrations for use by NAD-dependent enzymes in the sensing layer.

[0332] While the presently disclosed subject matter 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. Furthermore, the scope of this application is not intended to be limited to the particular embodiments of the processes, machines, manufacture, and compositions of matter, methods, and steps described herein. As those skilled in the art will readily appreciate from the disclosed subject matter herein, any now-existing or later-developed process, machine, manufacture, composition of matter, method, or step can be utilized in accordance with the presently disclosed subject matter that performs substantially the same function or achieves substantially the same results as the corresponding embodiment described herein. Accordingly, the appended claims are intended to include within their scope such processes, machines, manufacture, compositions of matter, methods, or steps.

[0333] Various patents, patent applications, publications, product descriptions, protocols, and sequence accession numbers are cited throughout this application, the inventions of which are incorporated herein by reference in their entireties for all purposes.

Claims

1. (i) a NAD(P) depot; (ii) a permeable polymer coating the NAD(P) depot; (iii) a first working electrode; and (iv) an analyte-responsive active region disposed on the surface of the first working electrode, the active region comprising at least one NAD(P)-dependent enzyme; (v) an analyte sensor comprising a sensor tail comprising a mass transport limiting membrane permeable to the analyte and covering the analyte-responsive active region;

2. A test substance sensor as described in claim 1, comprising a counter electrode and a reference electrode, or comprising a second electrode that can function as a counter electrode and a reference electrode.

3. A test substance sensor as described in claim 1, wherein the NAD(P)-dependent enzyme is an NAD(P)-dependent dehydrogenase.

4. A test substance sensor as described in claim 1, wherein the first working electrode is permeable, and optionally, the permeable first working electrode comprises a carbon nanotube.

5. A test substance sensor as described in claim 1, wherein the permeable polymer controls the diffusion of NAD(P) from the NAD(P) depot so as to maintain a sufficient concentration of NAD(P) in the active region during use of the test substance sensor.

6. A test substance sensor as described in claim 1, wherein the permeable polymer includes a polyether-based polymer, and optionally, the permeable polymer includes a poly(ethylene glycol)-based polymer and / or a poly(propylene glycol)-based polymer.

7. The test substance sensor of claim 1, wherein the permeable polymer comprises a mixture of poly(propylene glycol) methacrylate (POMA) and 2-hydroxyethyl methacrylate (HEMA).

8. A test substance sensor as described in claim 1, wherein the amount of NAD(P) present in the NAD(P) depot is 0.1 μg to 1000 μg.

9. The test substance sensor described in claim 1, wherein the test substance is selected from the group consisting of glucose, ketones, alcohol, lactate, and combinations thereof.

10. The test substance sensor according to claim 9, wherein the NAD(P)-dependent enzyme is glucose dehydrogenase, lactate dehydrogenase, alcohol dehydrogenase, or β-hydroxybutyrate dehydrogenase.

11. The test substance sensor of claim 10, wherein the test substance responsive active region further comprises diaphorase.

12. The test substance sensor of claim 1, wherein the test substance responsive active region further comprises a redox mediator. (vi) a second working electrode; and (vii) a second active area disposed on the surface of the second working electrode and responsive to a second analyte different from the analyte, the second active area including at least one enzyme responsive to the second analyte; 10. The analyte sensor of claim 1, wherein a second portion of the mass transport limiting membrane covers the second active area.

14. A test substance sensor as described in claim 1, wherein the test substance sensor is adapted to be inserted at least partially into a target tissue, and optionally adapted to be inserted at least partially into the dermis or subcutaneous layer. (i) preparing a test substance sensor according to any one of claims 1 to 14; (ii) applying a potential to the first working electrode; (iii) obtaining a first signal equal to or greater than the redox potential of the analyte-responsive active region, the first signal being proportional to the concentration of a first analyte in a fluid contacting the analyte-responsive active region; (iv) correlating the first signal to the concentration of the first analyte in the fluid. A method comprising:

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