nad(p) reservoir for nad(p)-dependent enzyme-based sensors

Abstract

The present disclosure provides an analyte sensor for detecting an analyte comprising one or more NAD(P)-dependent enzymes and an internal supply of NAD(P). The present disclosure also provides methods of using such analyte sensors to detect one or more analytes present in a biological sample of a subject, as well as methods of manufacturing the analyte sensors.

Description

[0001] Cross Reference to Related Applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 125,846, filed December 15, 2020, the contents of which are incorporated by reference in their entirety. TECHNICAL FIELD

[0003] The subject matter described herein relates to analyte sensors comprising NAD(P) reservoirs and methods of use thereof. BACKGROUND

[0004] Detection of various analytes within an individual can sometimes be critical to monitoring their health condition, as deviations from normal analyte levels can be indicative of a physiological condition. For example, monitoring glucose levels can enable a person with diabetes to take appropriate corrective measures, including taking medication or consuming a particular food or beverage product, to avoid serious physiological harm. Other analytes can be required to monitor other physiological conditions. In some cases, more than one analyte can be required to monitor a single or multiple physiological conditions, particularly if a person has a comorbid condition that causes two or more analytes to become simultaneously dysregulated in combination with each other.

[0005] Analyte monitoring in an individual can be performed periodically or continuously over a period of time. Periodic analyte monitoring can be performed by drawing samples of a bodily fluid, such as blood or urine, at set time intervals and performing ex vivo analysis. Periodic ex vivo analyte monitoring can be sufficient to determine the physiological condition of many individuals. However, in some cases, ex vivo analyte monitoring can be inconvenient or painful. Moreover, if an analyte measurement is not obtained at the appropriate time, the lost data cannot be recovered.

[0006] Continuous analyte monitoring can be performed using one or more sensors implanted at least partially within the tissue of an individual, such as intradermally, subcutaneously, or intravenously, so that analysis can be performed in vivo. Implanted sensors can collect analyte data on demand, on a set schedule, or continuously, depending on the specific health needs of the individual and / or previously measured analyte levels. Monitoring analytes using in vivo implanted sensors can be a more desirable approach for individuals with severe analyte dysregulation and / or rapidly fluctuating analyte levels, but it can also be beneficial for other individuals. Because implanted analyte sensors will often remain within the tissue of an individual for an extended period of time, it is highly desirable that such analyte sensors be made from stable materials that exhibit a high degree of biocompatibility.

[0007] However, implantable sensors suffer from short lifetimes or reduced sensitivity. For example, many implantable sensors use enzymes to continuously monitor in vivo analyte levels, where the activity of many of the enzymes relies on coenzymes. 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 often necessary for the activity of enzymes such as dehydrogenases found in implantable sensors. The amount of NAD or NADP available to the enzymes present in an implantable sensor can affect the sensitivity of the sensor to accurately monitor in vivo analyte levels. In certain instances, the amount of exogenous NAD or NADP present can be insufficient to support sensor operation, or even if sufficient exogenous amounts are present, such molecules are too large to readily diffuse to the sensor region where enzymes that rely on NAD or NADP are retained, which can result in reduced sensitivity. Thus, there is a need in the art for sensors that maintain sensitivity over longer periods of time. SUMMARY

[0008] The purposes and advantages of the disclosed subject matter will be set forth in part in the description which follows, and in part will be obvious from the description or can be learned by practice of the disclosed subject matter. The purposes and advantages of the disclosed subject matter will be realized and attained by means of the elements and combinations particularly pointed out in the written description and claims and appended drawings.

[0009] To achieve these and other advantages and in accordance with the purpose of the disclosed subject matter, as embodied and broadly described, the disclosed subject matter provides an analyte sensor comprising an internal supply of NAD(P). For example, and not by way of limitation, the analyte sensor of the present disclosure comprises an internal supply of NAD(P), a permeable polymer covering the internal supply of NAD(P), at least a first working electrode disposed on a surface of the permeable polymer, an analyte-responsive active area disposed on a surface of the first working electrode, and, optionally, a permeable analyte mass transport limiting membrane covering at least the analyte-responsive area.

[0010] In certain embodiments, the analyte is selected from the group consisting of glucose, ketones, alcohols, 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.

[0011] In certain embodiments, the first working electrode is a permeable working electrode. In certain embodiments, the permeable working electrode comprises carbon nanotubes.

[0012] In certain embodiments, the analyte-responsive active area 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 within the analyte-responsive active area is a beta-hydroxybutyrate dehydrogenase. In certain embodiments, the NAD(P)-dependent enzyme present within the analyte-responsive active area is a glucose dehydrogenase. In certain embodiments, the NAD(P)-dependent enzyme present within the analyte-responsive active area is a lactate dehydrogenase. In certain embodiments, the NAD(P)-dependent enzyme present within the analyte-responsive active area is an alcohol dehydrogenase. In certain embodiments, the NAD(P)-dependent enzyme present within the analyte-responsive active area is a diaphorase.

[0013] In certain embodiments, the permeable polymer comprises a poly(propylene glycol)-based polymer. In certain embodiments, the permeable polymer comprises a poly(propylene glycol) methacrylate and / or a 2-hydroxyethyl methacrylate.

[0014] In certain embodiments, the analyte-responsive active area further comprises a diaphorase. In certain embodiments, the analyte-responsive active area further comprises a redox mediator.

[0015] In certain embodiments, the analyte sensor of the present disclosure further comprises a second working electrode and a second active area disposed on a surface of the second working electrode and responsive to a second analyte different from the first analyte. In certain embodiments, the second active area comprises 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.

[0016] The present disclosure also provides methods for monitoring an analyte in vivo. In certain embodiments, the method can comprise providing an analyte sensor comprising (a) an internal supply of NAD(P), (b) a permeable polymer covering the internal supply of NAD(P), (c) at least a first working electrode disposed on a surface of the permeable polymer, wherein the first working electrode is a permeable working electrode, (d) an analyte-responsive active area disposed on a surface of the first working electrode, wherein the analyte-responsive active area comprises an NAD(P)-dependent enzyme, and (e) a mass transport limiting membrane permeable to the analyte covering at least the analyte-responsive area. In certain embodiments, the method further comprises applying an electrical potential to the first working electrode, obtaining a first signal at or above the redox potential of the first active area, wherein the first signal is proportional to a concentration of the first analyte in a fluid contacting the first active area, and correlating the first signal to the concentration of the first analyte in the fluid.

[0017] In certain embodiments, the analyte sensor used in the disclosed methods can also include a second working electrode and a second active area disposed on a surface of the second working electrode and responsive to a second analyte different from the first analyte. In 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 DESCRIPTION OF DRAWINGS

[0018] The following drawings are included to illustrate certain aspects of the present disclosure, and should not be considered exclusive embodiments. The disclosed subject matter can be modified, changed, combined, and equivalents substituted for elements thereof without departing from the scope of the present disclosure.

[0019] FIG. 1A is a system overview of sensor applicators, reader devices, monitoring systems, networks, and remote systems.

[0020] FIG. 1B is a diagram illustrating an operating environment for an example analyte monitoring system used with the technology described herein.

[0021] FIG. 2A is a block diagram depicting an example embodiment of a reader device.

[0022] FIG. 2B is a block diagram illustrating an example data receiving device for communicating with a sensor, in accordance with example embodiments of the disclosed subject matter.

[0023] FIG. 2C and 2D is a block diagram depicting an example embodiment of a sensor control device.

[0024] FIG. 2E is a block diagram illustrating an example analyte sensor, in accordance with example embodiments of the disclosed subject matter.

[0025] FIG. 3A is a proximal perspective view depicting an example embodiment of a user preparing a tray for assembly.

[0026] FIG. 3B is a side view depicting an example embodiment of a user preparing an applicator device for assembly.

[0027] FIG. 3C is a proximal perspective view depicting an example embodiment of a user inserting an applicator device into a tray during assembly.

[0028] FIG. 3D is a proximal perspective view depicting an example embodiment of a user removing an applicator device from a tray during assembly.

[0029] FIG. 3Eis a proximal perspective view depicting an example embodiment of a patient applying a sensor with an applicator device.

[0030] FIG. 3F is a proximal perspective view depicting an example embodiment of a patient with an applied sensor and a used applicator device.

[0031] FIG. 4A is a side view depicting an example embodiment of an applicator device connected with a cap.

[0032] FIG. 4B is a side perspective view depicting an example embodiment of a decoupled applicator device and cap.

[0033] FIG. 4C is a perspective view depicting an example embodiment of a distal end of an applicator device and an electronics housing.

[0034] FIG. 4D is a top perspective view of an example applicator device according to the disclosed subject matter.

[0035] FIG. 4E is a bottom perspective view of the applicator device of FIG. 4D

[0036] FIG. 4F is an exploded view of the applicator device of FIG. 4D

[0037] FIG. 4G is a side cross-sectional view of the applicator device of FIG. 4D

[0038] FIG. 5 is a proximal perspective view depicting an example embodiment of a tray coupled with a sterilization cover.

[0039] FIG. 6A is a proximal perspective cross-sectional view depicting an example embodiment of a tray with a sensor delivery component.

[0040] FIG. 6B is a proximal perspective view depicting a sensor delivery component.

[0041] FIG. 7A and 7B are an isometric exploded top view and a bottom view, respectively, of an example sensor control device.

[0042] FIGS. 8A-8C are assembly and cross-sectional views of an on-body device including an integrated connector for a sensor assembly.

[0043] FIG. 9A and 9B are a sensor applicator FIG. 1A FIG. 2C ​​​​side view and cross-sectional side view of an exemplary embodiment of a sensor applicator coupled to a sensor control device.

[0044] FIG. 10A and 10B are an isometric view and a side view, respectively, of another example sensor control device.

[0045] FIG. 11A-11C are progressive cross-sectional side views showing assembly of a sensor applicator to FIG. 10A-10B a sensor control device of

[0046] FIG. 12A-12C are progressive cross-sectional side views showing assembly and disassembly of an exemplary embodiment of a sensor applicator of a sensor control device of FIG. 10A-10B

[0047] FIG. 13A-13F illustrates a cross-sectional view depicting an example embodiment of an applicator during a deployment phase.

[0048] FIG. 14 is a plot depicting an example of in-vitro sensitivity of an analyte sensor.

[0049] FIG. 15 is a plot illustrating example operational states of a sensor according to exemplary embodiments of the disclosed subject matter.

[0050] FIG. 16 is a plot illustrating example operations and data flow for wireless programming of a sensor according to the disclosed subject matter.

[0051] FIG. 17 is a plot illustrating example data flow for secure exchange of data between two devices according to the disclosed subject matter.

[0052] FIG. 18A-18C shows a cross-sectional view of an analyte sensor including a single active area.

[0053] FIG. 19A-19C shows a cross-sectional view of an analyte sensor including two active areas.

[0054] FIG. 20 shows a cross-sectional view of an analyte sensor including two active areas.

[0055] FIG. 21A-21C shows a perspective view of an analyte sensor including two active areas deployed on separate working electrodes.

[0056] FIG. 22 provides a cross-sectional view of an example sensor including an NAD(P) reservoir for controlled NAD(P) release.

[0057] ​FIG. 23A A schematic of an exemplary analyte sensor including an NAD reservoir is provided.

[0058] FIG. 23B A cross-sectional view of an exemplary analyte sensor not including an NAD reservoir for use as a control is provided.

[0059] FIG. 24 A stability profile plot of ketone detection over time using an analyte sensor including an NAD reservoir (as shown in FIG. 23B FIG. 23A DETAILED DESCRIPTION

[0060] The present disclosure is directed to analyte sensors including one or more active regions that include a nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP) dependent enzyme (collectively referred to herein as “NAD(P) dependent enzyme”). In particular, the analyte sensors of the present disclosure include an internal reservoir of the cofactor NAD and / or NADP (collectively referred to herein as “NAD(P)”) for the NAD(P) dependent enzyme.

[0061] The use of an internal reservoir of NAD(P) within an analyte sensor can overcome some of the limitations associated with analyte sensors including NAD(P) dependent enzymes. For example, the amount of exogenous NAD(P) present in the environment surrounding the sensor can be insufficient in quantity to support operation of the analyte sensor, which can result in reduced sensitivity of the sensor. Further, even if sufficient exogenous NAD(P) is present in the environment surrounding the analyte sensor, the molecular size of NAD(P) can 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., active region) of the analyte sensor.

[0062] The analyte sensors provided by the present disclosure include an internal supply of NAD(P) that can release NAD(P) over an extended period of time to allow for monitoring of an analyte in vivo. In certain embodiments, the NAD(P) internal supply (also referred to herein as an “NAD(P) reservoir”) can be coated with or distributed through a permeation layer (e.g., a polymeric permeation layer) that controls diffusion of NAD(P) from the NAD(P) reservoir to maintain a sufficient concentration of NAD(P) for the sensing chemistry during use of the analyte sensor.

[0063] The present disclosure also provides methods of detecting an analyte using the disclosed sensors and methods of manufacturing the disclosed analyte sensors.

[0064] ​​For clarity, but without being limiting, the detailed description of the presently disclosed subject matter is divided into the following subsections:

[0065] I. Definitions;

[0066] II. Analyte Sensors;

[0067] 1. General Structure of Analyte Sensor Systems;

[0068] 2. NAD(P)-Reservoirs;

[0069] 3. Enzymes;

[0070] 4. Redox Mediators;

[0071] 5. Polymer Backbones;

[0072] 6. Mass Transport Limiting Membranes;

[0073] 7. Interference Domains; and

[0074] 8. Manufacture;

[0075] III. Analyte Monitoring.

[0076] I. Definitions

[0077] The terms used in this specification generally have their ordinary meanings in the context of the present disclosure and in the specific context of each term as used in the specification. Certain terms are discussed below or elsewhere in the specification, to provide additional guidance to the practitioner in describing the compositions and methods of the present disclosure and how to make and use them.

[0078] As used herein, the use of the word "a" or "an" when used in conjunction with the term "comprising" can mean "one," but it is also consistent with the meaning of "one or more."

[0079] As used herein, the terms "comprising," "including," "containing," "having," "may," and variations thereof, are to be construed as open-ended transitional phrases, terms or words that do not preclude the additional acts or structures. The present disclosure also contemplates other embodiments "comprising," "consisting of," and "consisting essentially of," the embodiments or elements presented herein, whether explicitly set forth or not.

[0080] The term“about” or“approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend 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 standard deviations, per the practice in the art. Alternatively, “about” can mean ranges approximately 20%, preferably approximately 10%, more preferably approximately 5%, and still more preferably approximately 1% of a given value. Alternatively, especially with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold and more preferably within 2-fold of a value.

[0081] As used herein, an“analyte sensor” or“sensor” can refer to any device capable of receiving sensor information from a user, including by way of illustration and not limitation, a body temperature sensor, a blood pressure sensor, a pulse or heart rate sensor, a glucose level sensor, an analyte sensor, a body activity sensor, a body movement sensor, or any other sensor for collecting physical or biological information. Analytes measured by an analyte sensor can include, for example, but are not limited to, glucose, ketones, lactate, oxygen, hemoglobin A1C, albumin, alcohol, alkaline phosphatase, alanine transaminase, aspartate transaminase, bilirubin, blood urea nitrogen, calcium, carbon dioxide, chloride, creatinine, hematocrit, lactic acid, magnesium, oxygen, pH, phosphorus, potassium, sodium, total protein, uric acid, etc.

[0082] As used herein, the term“biological fluid” 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, etc. In certain embodiments, the biological fluid is dermal fluid or interstitial fluid. In certain embodiments, the biological fluid is interstitial fluid.

[0083] As used herein, the term“electrolysis” refers to the electrooxidation or electroreduction of a compound directly at an electrode or via one or more electron transfer agents (e.g., a redox mediator or enzyme).

[0084] As used interchangeably herein, the terms“enzyme composition” and“sensing chemistry” refer to a composition including one or more enzymes for detecting and / or measuring an analyte. In certain non-limiting embodiments, an enzyme composition can include one or more enzymes, polymers, redox mediators, and / or cross-linking agents.

[0085] As used herein, the term“homogeneous membrane” refers to a membrane including a single type of membrane polymer.

[0086] As used herein, the term“multi-component membrane” refers to a membrane including two or more types of membrane polymers.

[0087] As used herein, the term "NAD(P)" refers to the co-factors NAD (and its reduced form NADH) or NADP (and its reduced form NADPH) or derivatives thereof.

[0088] As used herein, the term "NAD(P)-dependent enzyme" refers to an enzyme that uses NAD (and its reduced form NADH) or NADP (and its reduced form NADPH) as a coenzyme in an oxidation-reduction reaction.

[0089] As used herein, the term "permeable electrode" refers to an electrode composed of a material that allows molecules (e.g., NAD(P)) to pass through the electrode.

[0090] As used herein, the term "polyvinylpyridine-based polymer" refers to a polymer or copolymer that includes polyvinylpyridine (e.g., poly(2-vinylpyridine) or poly(4-vinylpyridine)) or derivatives thereof.

[0091] As used herein, the term "redox mediator" refers to an electron transfer agent used to carry electrons between an enzyme of an analyte or analyte reduction or analyte oxidation and an electrode, either directly or via one or more additional electron transfer agents. In certain embodiments, a redox mediator that includes a polymeric backbone can also be referred to as a "redox polymer."

[0092] As used herein, the term "reference electrode" can refer to either 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 a counter electrode and a counter electrode that also functions as a reference electrode.

[0093] II. Analyte Sensors

[0094] Before describing the present subject matter in detail, it is to be understood that the disclosure is not limited to the specific embodiments described herein because such can 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 only be limited by the appended claims.

[0095] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present disclosure is not entitled to antedate such publications by virtue of prior disclosure. Further, the dates of publication provided can be different from the dates that can be disclosed in the applications as actual publication dates can vary depending on jurisdiction. Accordingly, no admission is made that any publication cited in this disclosure is prior art.

[0096] Generally, embodiments of the present disclosure include systems, devices, and methods for analyte sensor insertion applicators for use with in vivo analyte monitoring systems. The applicator can be provided to a user in a sterile package with an electronics housing containing a sensor control device. According to some embodiments, a structure separate from the applicator, such as a container, can also be provided to the user as a sterile package containing a sensor module and a sharp module. The user can couple the sensor module to the electronics housing and can couple the sharp to the applicator through an assembly process involving inserting the applicator into the container in a particular manner. In other embodiments, the applicator, sensor control device, sensor module, and sharp module can be provided in a single package. The applicator can be used to position the sensor control device on a human body with the sensor in contact with the wearer's bodily fluids. Embodiments provided herein are improvements to reduce the likelihood of the sensor being improperly inserted or damaged, or causing an adverse physiological response. Other improvements and advantages are also provided. Various configurations of these devices will be described in detail by way of example only.

[0097] Further, many embodiments include in vivo analyte sensors structured to have at least a portion of the sensor positioned or positionable within a user's body to obtain information about at least one analyte of the body. However, it should be noted that embodiments disclosed herein can be used with in vivo analyte monitoring systems incorporating in vitro capabilities as well as purely in vitro or ex vivo analyte monitoring systems, including completely non-invasive systems.

[0098] Further, for each embodiment of the methods disclosed herein, systems and devices capable of performing each of those embodiments are covered within the scope of the present disclosure. For example, embodiments of sensor control devices are disclosed and these devices can have one or more sensors, analyte monitoring 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 any and all method steps or facilitate performance of any and all method steps. These sensor control device embodiments can be used and are capable of being used to implement those steps performed by the sensor control devices according to any and all methods described herein.

[0099] Further, the systems and methods presented herein can be used for operation of sensors used in analyte monitoring systems, such as but not limited to health, fitness, diet, research, information, or any purpose involving analyte sensing over time. As used herein, "analyte sensor" or "sensor" can refer to any device capable of receiving sensor information from a user, including but not limited to, body temperature sensors, blood pressure sensors, pulse or heart rate sensors, glucose level sensors, analyte sensors, physical activity sensors, body movement sensors, or any other sensor for collecting physical or biological information for illustrative purposes. In certain embodiments, the analyte sensors of the present disclosure can also measure analytes including, but not limited to, glucose, ketones, lactate, oxygen, hemoglobin A1C, albumin, alcohol, alkaline phosphatase, alanine transaminase, aspartate transaminase, bilirubin, blood urea nitrogen, calcium, carbon dioxide, chloride, creatinine, hematocrit, lactic acid, magnesium, oxygen, pH, phosphorus, potassium, sodium, total protein, uric acid, and the like.

[0100] As mentioned, a number of embodiments of systems, devices, and methods are described herein that provide for improved assembly and use of skin sensor insertion devices for use with in vivo analyte monitoring systems. In particular, several embodiments of the present disclosure are designed to improve the sensor insertion process with respect to in vivo analyte monitoring systems, and in particular, prevent premature retraction of the insertion tip during the sensor insertion process. For example, some embodiments include skin sensor insertion mechanisms with increased fire speed and delayed tip retraction. In other embodiments, the tip retraction mechanism can be motion-actuated such that the tip does not retract until the user pulls the applicator away from the skin. Thus, these embodiments can reduce the likelihood of premature withdrawal of the insertion tip during the sensor insertion process; reduce the likelihood of improper sensor insertion; and reduce the likelihood of damage to the sensor during the sensor insertion process, to name a few advantages. Several embodiments of the present disclosure also provide improved insertion tip modules to address the small scale of skin sensors and the relatively shallow insertion path that exists in the dermal layer of a subject. Further, several embodiments of the present disclosure are designed to prevent unwanted axial and / or rotational movement of the applicator components during sensor insertion. As such, these embodiments can reduce the likelihood of an unstable positioned skin sensor, irritation of the insertion site, damage to surrounding tissue, and capillary breakage leading to contamination of the dermal fluid with blood, to name a few advantages. Further, to mitigate inaccurate sensor readings that can result from trauma at the insertion site, several embodiments of the present disclosure can reduce the depth of penetration of the needle relative to the tip of the sensor during insertion.

[0101] Before these aspects of the embodiments are described in detail, however, it is first desired to describe examples of devices that can exist within, for example, in vivo analyte monitoring systems, as well as examples of their operation, all of which can be used with the embodiments described herein.

[0102] There are various types of in vivo analyte monitoring systems. For example, a "continuous analyte monitoring" system (or "continuous glucose monitoring" system) can transmit data from a sensor control device to a reader device continuously without prompting, e.g., automatically according to a schedule. As another example, a "flash" analyte monitoring system (or "flash" glucose monitoring system or simply "flash" system) can transmit data from a sensor control device in response to a scan or request for data by a reader device, such as using near field communication (NFC) or radio frequency identification (RFID) protocols. In vivo analyte monitoring systems can also operate without the need for finger stick calibration.

[0103] In vivo analyte monitoring systems can be distinguished from "in vitro" systems that contact a biological sample outside the body (or "ex vivo"), which typically include a meter device with a port for receiving an analyte test strip carrying a user's bodily fluid, which can be analyzed to determine the user's blood glucose level.

[0104] An in vivo monitoring system can include a sensor that, when positioned in vivo, is in contact with a user's bodily fluid and senses analyte levels contained therein. The sensor can be part of a sensor control device that resides on the user's body and contains electronics and a power source that enable and control analyte sensing. The sensor control device and variations thereof can also be referred to as a "sensor control unit," an "on-body electronics" device or unit, an "on-body" device or unit, or a "sensor data communication" device or unit, to name a few.

[0105] An in vivo monitoring system can also include a device that receives sensed analyte data from the sensor control device and processes and / or displays the detected analyte data to the user in any number of forms. This device and variations thereof can be referred to as a "handheld reader device," a "reader device" (or simply "reader"), a "handheld electronics device" (or simply "handheld device"), a "portable data processing" device or unit, a "data receiver," a "receiver" device or unit (or simply "receiver"), or a "remote" device or unit, to name a few. Other devices, such as personal computers, have also been used with or incorporated into in vivo and in vitro monitoring systems.

[0106] 1. General Structure of an Analyte Sensor System

[0107] A. Exemplary In Vivo Analyte Monitoring System

[0108] FIG. 1Ais a conceptual diagram depicting 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 where the sensor 104 is maintained in place by an adhesive patch 105 for a period of time. The sensor control device 102 is further described in FIG. 2B and 2C and can communicate with the reader device 120 via a communication path or link 1140 using wired or wireless, one-way or two-way, and encrypted or non-encrypted technologies. Example wireless protocols include Bluetooth, Bluetooth Low Energy (BLE, BTLE, Bluetooth Smart, etc.), Near Field Communication (NFC), etc. The user can monitor the application in the memory installed on the reader device 120 using the display 122 and input 121, and can recharge the device battery using the power port 123. More details about the reader device 120 are set forth below with respect to FIG. 2A According to certain embodiments, the reader device 120 can constitute an output medium for viewing analyte concentrations and alarms or notifications determined by the sensor 104 or processor associated therewith, as well as allowing one or more user inputs. The reader device 120 can be a multi-functional smart phone or a dedicated electronic reader. While only one reader device 120 is shown, there can be multiple reader devices 120 in certain instances.

[0109] Reader device 120 can communicate with local computer system 170 via communication path 141, which can also be wired or wireless, one-way or two-way, encrypted or non-encrypted. Local computer system 170 can include one or more of a laptop computer, a desktop computer, a tablet computer, a phablet, a smartphone, a set-top box, a video game console, a remote terminal, or other computing device, and wireless communication can include any of a number of applicable wireless networking protocols, including Bluetooth, Bluetooth Low Energy (BTLE), Wi-Fi, or others. Local computer system 170 can communicate with network 190 via communication path 143, similar to how reader device 120 can communicate with network 190 via communication path 142, as previously described, by wired or wireless technology. Network 190 can be any of a number of networks, such as private and public, local area or wide area networks, and the like. Trusted computer system 180 can include a server and can provide authentication services and protected data storage and can communicate with network 190 via communication path 144 by wired or wireless technology. According to certain embodiments, local computer system 170 and / or trusted computer system 180 can be accessible by individuals other than the primary user who are interested in the user's analyte levels. Reader device 120 can include display 122 and optional input 121. According to certain embodiments, display 122 can include a touch screen interface.

[0110] Sensor control device 102 includes a sensor housing, which can house circuitry and a power source for operating sensor 104. Optionally, the power source and / or active circuitry can be omitted. A processor (not shown) can be communicatively coupled to sensor 104, the processor being physically located within sensor housing or reader device 120. According to certain embodiments, sensor 104 protrudes from an underside of sensor housing and extends through adhesive patch 105, which is adapted to adhere sensor housing to a tissue surface, such as skin.

[0111] FIG. 1BAn operating environment for an analyte monitoring system 100a capable of implementing the techniques described herein is illustrated. The analyte monitoring system 100a can include a system of components designed to provide monitoring of a parameter, such as an analyte level, of a human or animal body or can provide other operations based on the configuration of the various components. As embodied herein, the system can include a low power analyte sensor 110, or simply a "sensor" worn by a user or attached to a body for which information is collected. As embodied herein, the analyte sensor 110 can be a sealed, disposable device with a predetermined effective useful life (e.g., 1 day, 14 days, 30 days, etc.). The sensor 110 can be applied to the skin of a user's body and remain adhered during the sensor life or can be designed to be selectively removed and remain functional upon reapplication. The low power analyte monitoring system 100a can also include a data receiving device 129 or a multi-purpose data receiving device 130 configured to facilitate retrieval and delivery of data, including analyte data, from the analyte sensor 110 as described herein.

[0112] As embodied herein, the analyte monitoring system 100a can include software or firmware libraries or applications provided to third parties, for example via a remote application server 1150 or application storefront server 160, and incorporated into a multi-purpose data receiving device 130, such as a mobile phone, tablet, personal computing device, or other similar computing device capable of communicating with the analyte sensor 110 over a communication link. The multi-purpose hardware can also include embedded devices, including but not limited to an insulin pump or insulin pen, with embedded libraries configured to communicate with the analyte sensor 110. While the illustrated embodiment of the analyte monitoring system 100a includes only one of each illustrated device, the present disclosure contemplates the analyte monitoring system 100a incorporating multiple of each component interacting throughout the system. For example, and without limitation, as embodied herein, the data receiving device 129 and / or the multi-purpose data receiving device 130 can include multiple of each device. As embodied herein, multiple multi-purpose data receiving devices 130 can communicate directly with the sensor 110 as described herein. Additionally or alternatively, the multi-purpose data receiving device 130 can communicate with an auxiliary multi-purpose data receiving device 130 to provide analyte data, or a visualization or analysis of the data, for auxiliary display to a user or other authorized party.

[0113] FIG. 1AThe sensor 104 is adapted to be at least partially inserted into tissue of interest, such as into the dermis or subcutaneous layer of the skin. The sensor 104 can include a sensor tail of sufficient length for insertion to a desired depth in a given tissue. The sensor tail can include at least one working electrode. In certain configurations, the sensor tail can include an active area for detecting an analyte, e.g., including one or more NAD(P)-dependent enzymes. A counter electrode can be present in combination with the at least one working electrode. Particular electrode configurations on the sensor tail are described in greater detail below.

[0114] One or more mass transport limiting membranes can cover the active area, as described in further detail below.

[0115] The active area can be configured for detecting a particular analyte described herein. For example, but not by way of limitation, the analyte can include glucose, ketones, lactate, oxygen, hemoglobin A1C, albumin, alcohol, alkaline phosphatase, alanine transaminase, aspartate transaminase, 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, the analyte detected using the disclosed analyte sensor includes alcohol, ketones, creatinine, glucose, and lactate. In certain embodiments, the active area can be configured for detecting two or more analytes described herein. In certain embodiments, the active area of the presently disclosed sensor is configured to detect ketones. In certain embodiments, the active area of the presently disclosed sensor is configured to detect glucose. In certain embodiments, the active area of the presently disclosed sensor is configured to detect lactate. In certain embodiments, the active area of the presently disclosed sensor is configured to detect creatinine. In certain embodiments, the active area of the presently disclosed sensor is configured to detect alcohol, e.g., ethanol.

[0116] In certain embodiments of the present disclosure, one or more analytes can be monitored in any biological fluid of interest, such as dermal fluid, interstitial fluid, blood plasma, blood, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, amniotic fluid, etc. In certain particular embodiments, the analyte sensors of the present disclosure can be adapted to assay dermal fluid or interstitial fluid to determine in vivo concentrations of one or more analytes. In certain embodiments, the biological fluid is interstitial fluid.

[0117] A guide can be temporarily present to facilitate introduction of the sensor 104 into the tissue. In certain illustrative embodiments, the guide can comprise a needle or similar pointed tip. Other types of guides, such as sheaths or blades, can be present in alternative embodiments, as will be readily appreciated by those skilled in the art. More particularly, a needle or other guide can temporarily reside near the sensor 104 prior to tissue insertion and then be withdrawn. When present, the needle or other guide can facilitate insertion of the sensor 104 into the tissue by opening an entry path for the sensor 104 to follow. For example, but not by way of limitation, according to one or more embodiments, a needle can facilitate penetration of the epidermis as a passageway to the dermis to allow implantation of the sensor 104 to occur. After opening the passageway, the needle or other guide can be withdrawn so that the pointed tip does not pose a hazard. In certain embodiments, a suitable needle can be solid or hollow in cross-section, beveled or non-beveled, and / or be circular or non-circular in cross-section. In more particular non-limiting embodiments, a suitable needle can have a cross-sectional diameter and / or tip design comparable to an acupuncture needle, which can have a cross-sectional diameter of about 250 microns. However, a suitable needle can have a larger or smaller cross-sectional diameter if certain particular applications so require.

[0118] In certain embodiments, the tip of the needle (when present) can be angled above the end of the sensor 104 so that the needle first pierces the tissue and opens a passageway for the sensor 104. In certain embodiments, the sensor 104 can reside within the lumen or channel of the needle, which similarly opens a passageway for the sensor 104. In either case, after facilitating sensor insertion, the needle is then withdrawn.

[0119] B. Exemplary Reader Device

[0120] FIG. 2A is a block diagram depicting an example embodiment of a reader device configured as a smart phone. Here, the reader device 120 can include a display 122, an input 121, and a processing core 206 including a communication processor 222a coupled with a memory 223 and an application processor 224 coupled with a memory 225. Separate memory 230 can also be included, as well as an RF transceiver 228 with an antenna 229, and a power source 226 with a power management module 238. A multi-function transceiver 232 can also be included, which can communicate with an antenna 234 via Wi-Fi, NFC, Bluetooth, BTLE, and GPS. These components are electrically and communicatively connected in a manner that results in a functioning device, as will be appreciated by one of skill in the art.

[0121] C. Exemplary Data Receiver Device Architecture

[0122] For purposes of illustration and not limitation, reference is made to the following figures with the understanding that the figures are not necessarily drawn to scale and that they are merely intended to aid in the description of the exemplary embodiments. FIG. 2BAn exemplary embodiment of the data receiving device 129 used in conjunction with the disclosed subjects is shown. The data receiving device 129 and the associated multipurpose data receiving device 130 include components closely related to the discussion of the analyte sensor 110 and its operation, and may include additional components. In certain embodiments, the data receiving device 129 and the multipurpose data receiving device 130 may be or include components supplied by third parties, and are not necessarily limited to including devices manufactured by the same manufacturer as the sensor 110.

[0123] like FIG. 2B As shown, the data receiving device 129 includes an ASIC 4000, which includes a microcontroller 4010, a memory 4020, and a storage device 4030 and is communicatively coupled to a communication module 4040. Power to the components of the data receiving device 129 may be delivered by a power module 4050, which, as implemented herein, may include a rechargeable battery. The data receiving device 129 may also include a display 4070 for facilitating viewing of analyte data received from the analyte sensor 110 or other devices (e.g., user equipment 140 or remote application server 1150). The data receiving device 129 may include separate user interface components (e.g., physical buttons, a light sensor, a microphone, etc.).

[0124] Communication module 4040 may include BLE module 4041 and NFC module 4042. Data receiving device 129 may be configured to wirelessly couple with analyte sensor 110 and send commands to and receive data from analyte sensor 110. As implemented herein, data receiving device 129 may be configured to operate relative to analyte sensor 110 as an NFC scanner and BLE endpoint via a specific module of communication module 4040 (e.g., BLE module 4041 or NFC module 4042). For example, data receiving device 129 may use a first module of communication module 4040 to send commands to analyte sensor 110 (e.g., an activation command for the sensor's data broadcast mode; a pairing command for identifying data receiving device 129) and use a second module of communication module 4040 to receive and transmit data to analyte sensor 110. Data receiving device 129 may be configured to communicate with user equipment 140 via Universal Serial Bus (USB) module 4045 of communication module 4040.

[0125] As another example, the communication module 4040 can include, for example, a cellular radio module 4044. The cellular radio module 4044 can include one or more radio transceivers for communicating using wide-area cellular networks, including but not limited to third generation (3G), fourth generation (4G), and fifth generation (5G) networks. Additionally, the communication module 4040 of the data receiving device 129 can include a Wi-Fi radio module 4043 for communicating using a wireless local area network according to one or more of the IEEE 802.11 standards (e.g., 802.11a, 802.11b, 802.11g, 802.11n (a.k.a. Wi-Fi 4), 802.11ac (a.k.a. Wi-Fi 5), 802.11ax (a.k.a. Wi-Fi 6)). Using the cellular radio module 4044 or the Wi-Fi radio module 4043, the data receiving device 129 can communicate with the remote application server 1150 to receive analyte data or provide updates or inputs received from a user (e.g., through one or more user interfaces). Although not shown, the communication module 5040 of the analyte sensor 110 can similarly include a cellular radio module or a Wi-Fi radio module.

[0126] As embodied herein, the on-board storage 4030 of the data receiving device 129 can store analyte data received from the analyte sensor 110. Additionally, the data receiving device 129, the multi-purpose data receiving device 130, or the user device 140 can be configured to communicate with the remote application server 1150 via a wide-area network. As embodied herein, the analyte sensor 110 can provide data to the data receiving device 129 or the multi-purpose data receiving device 130. The data receiving device 129 can transmit the data to the user device 140. The user device 140 (or the multi-purpose data receiving device 130) in turn can transmit the data to the remote application server 1150 for processing and analysis.

[0127] As embodied herein, the data receiving device 129 can also 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 129 can be configured to operate in coordination with the analyte sensor 110 and based on analyte data received from the analyte sensor 110. As an example, where the analyte sensor 110 is a glucose sensor, the data receiving device 129 can be or include an insulin pump or an insulin injection pen. In coordination, a compatible multi-purpose data receiving device 130 can adjust a user’s insulin dosage based on glucose values received from the analyte sensor.

[0128] D. Exemplary Sensor Control Device

[0129] FIG. 2C and2D is a block diagram depicting an example embodiment of a sensor control device 102 having an analyte sensor 104 and sensor electronics 160, including analyte monitoring circuitry, which can have most of the processing capability for rendering final result data suitable for display to a user. In FIG. 2C In the embodiment of FIG. 1, a single semiconductor chip 161 is described, which can be a custom application specific integrated circuit (ASIC). Shown within the ASIC 161 are certain high-level functional units, including an analog front end (AFE) 162, power management (or control) circuitry 164, a processor 166, and communication circuitry 168 (which can be implemented as a transmitter, receiver, transceiver, passive circuit, or other manner according to a communication protocol). In this embodiment, both the AFE 162 and the processor 166 are used as analyte monitoring circuitry, but in other embodiments either circuit can perform the analyte monitoring function. The processor 166 can include one or more processors, microprocessors, controllers, and / or microcontrollers, each of which can be a discrete chip or distributed among multiple different chips (and portions thereof).

[0130] Memory 163 is also included within the ASIC 161 and can be shared by the various functional units present within the ASIC 161, or can be distributed among two or more of them. The memory 163 can also be a separate chip. The memory 163 can be volatile and / or non-volatile memory. In this embodiment, the ASIC 161 is coupled with a power source 170, which can be a coin cell battery or the like. The AFE 162 interfaces with the in vivo analyte sensor 104 and receives measurement data therefrom, and outputs the data in digital form to the processor 166, which in turn processes the data to obtain final result glucose discrete values and trend values, among others. This data can then be provided to the communication circuitry 168 for transmission to the reader device 120 (not shown) through an antenna 171, for example, where a resident software application requires minimal further processing to display the data.

[0131] FIG. 2D Similar to FIG. 2CBut instead includes two discrete semiconductor chips 162 and 174, which can be packaged together or separately. Here, the AFE 162 resides on the ASIC 161. The processor 166 is integrated on chip 174 with power management circuitry 164 and communication circuitry 168. The AFE 162 includes memory 163 and chip 174 includes memory 165, which can be isolated or distributed among them. In one example embodiment, the AFE 162 is combined on one chip with the power management circuitry 164 and the processor 166, while the communication circuitry 168 is on a separate chip. In another example embodiment, the AFE 162 and the communication circuitry 168 are both on one chip, and the processor 166 and the power management circuitry 164 are on another chip. It should be noted that other chip combinations are possible, including three or more chips, each responsible for separate functions described, or sharing one or more functions for fail-safe redundancy.

[0132] For the purposes of illustration and not limitation, reference is made to an exemplary embodiment of an analyte sensor 110 used with the disclosed subject matter as shown in FIG. 2E FIG. 1. FIG. 2E A block diagram illustrating an exemplary analyte sensor 110 according to exemplary embodiments compatible with the security architecture and communication scheme described herein.

[0133] As embodied herein, the analyte sensor 110 can include an application specific integrated circuit ("ASIC") 5000 communicatively coupled with a communication module 5040. The ASIC 5000 can include a microcontroller 2210, on-board memory 5020, and storage memory 2230. The storage memory 2230 can store data for the authentication and encryption security architecture. The storage memory 2230 can store programming instructions for the sensor 110. As embodied herein, certain communication chips can be embedded in the ASIC 5000 (e.g., NFC transceiver 5025). The ASIC 5000 can receive power from a power module 5050, such as an on-board battery, or from an NFC pulse. The storage memory 2230 of the ASIC 5000 can be programmed to include information, such as an identifier of the sensor 110 for identification and tracking purposes. The storage memory 2230 can also be programmed with configuration or calibration parameters for use by the sensor 110 and its various components. The storage memory 2230 can include rewritable or one-time programmable (OTP) memory. The storage memory 2230 can be updated using the techniques described herein to extend the usefulness of the sensor 110.

[0134] As embodied herein, the communication module 5040 of the sensor 110 can be or include one or more modules to support communication of the analyte sensor 110 with other devices of the analyte monitoring system 100. By way of example only and without limitation, an example communication module 5040 can include a Bluetooth Low Energy ("BLE") module 5041. As used throughout this disclosure, Bluetooth Low Energy ("BLE") refers to a short-range communication protocol optimized so that end users can easily pair Bluetooth devices. The communication module 5040 can transmit and receive data and commands via interaction with a similar-capability communication module of a data receiving device 129 or user device 140. The communication module 5040 can include additional or alternative chipsets for similar short-range communication schemes, such as a personal area network according to IEEE 802.15 protocols, IEEE 802.11 protocols, infrared communication (infrared light) according to Infrared Data Association standards, etc.

[0135] To perform its functions, the sensor 110 can also include suitable sensing hardware 5060 appropriate to its functions. As embodied herein, the sensing hardware 5060 can include an analyte sensor positioned transcutaneously or subcutaneously in contact with a bodily fluid of a subject. The analyte sensor can generate sensor data containing values corresponding to one or more analyte levels within the bodily fluid.

[0136] E. Example Assembly Process for Sensor Control Device

[0137] The components of the sensor control device 102 can be acquired by a user in a number of packaged forms, requiring the user to perform final assembly before delivery to an appropriate user location. FIG. 3A-3D An example embodiment of the assembly process for the sensor control device 102 by a user is depicted, including preparation of separate components prior to coupling the components in order to prepare the sensor for delivery. FIG. 3E-3F An example embodiment of the delivery of the sensor control device 102 to an appropriate user location by selecting an appropriate delivery location and applying the sensor control device 102 to the location is depicted.

[0138] FIG. 3A is a proximal perspective view depicting an example embodiment of a user preparing a container 810, here configured as a tray (but other packaging can be used) for the assembly process. The user can accomplish this preparation by removing a lid 812 from the container 810 to expose a platform 808, for example by peeling an unadhered portion of the lid 812 from the container 810 such that an adhered portion of the lid 812 is removed. Removal of the lid 812 can be appropriate in various embodiments so long as the platform 808 is sufficiently exposed within the container 810. The lid 812 can then be set aside.

[0139] FIG. 3Bis a side view depicting an example embodiment of a user preparing a patch device 150 for assembly. The patch device 150 can be provided in a sterile package sealed by a cap 708. Preparation of the patch device 150 can include decoupling the housing 702 from the cap 708 to expose the sheath 704 FIG. 3C ). This can be accomplished by unscrewing (or otherwise detaching) the cap 708 from the housing 702. The cap 708 can then be set aside.

[0140] FIG. 3C is a proximal perspective view depicting an example embodiment of a user inserting the patch device 150 into the receptacle 810 during assembly. Initially, after aligning the housing orientation feature 1302 (or slot or groove) and the tray orientation feature 924 (abutment or detent), the user can insert the sheath 704 into the platform 808 within the receptacle 810. Inserting the sheath 704 into the platform 808 temporarily unlocks the sheath 704 relative to the housing 702 and also temporarily unlocks the platform 808 relative to the receptacle 810. At this stage, removing the patch device 150 from the receptacle 810 will result in the same state as before initially inserting the patch device 150 into the receptacle 810 (i.e., the process can be reversed or aborted at this point and then repeated without consequence).

[0141] The sheath 704 can be maintained in position within the platform 808 relative to the housing 702 while the housing 702 is advanced distally, coupling with the platform 808 to advance the platform 808 distally relative to the receptacle 810. This step unlocks and unfolds the platform 808 within the receptacle 810. The sheath 704 can contact and disengage locking features (not shown) within the receptacle 810 that unlock the sheath 704 relative to the housing 702 and prevent the sheath 704 from moving (relatively) while the housing 702 continues to advance the platform 808 distally. At the end of the housing 702 and platform 808 advancement, the sheath 704 is permanently unlocked relative to the housing 702. At the end of the housing 702 distal advancement, prongs and sensors (not shown) within the receptacle 810 can couple with electronics housing (not shown) within the housing 702. Operation and interaction of the patch device 150 and receptacle 810 are further described below.

[0142] FIG. 3D is a proximal perspective view depicting an example embodiment of a user removing the patch device 150 from the receptacle 810 during assembly. The user can remove the patch 150 from the receptacle 810 by advancing the housing 702 proximally relative to the receptacle 810 or other motion with the same end result of detaching the patch 150 and receptacle 810. The patch device 150 is removed with the sensor control device 102 (not shown) fully assembled (prongs, sensors, electronics) and positioned for delivery.

[0143] FIG. 3Eis a proximal perspective view depicting an example embodiment of a patient applying the sensor control device 102 to a target area of skin (e.g., on the abdomen or other suitable location) using the applicator device 150. Advancing the housing 702 causes the sheath 704 within the housing 702 to retract distally and apply the sensor to the target location such that the adhesive layer on the bottom side of the sensor control device 102 adheres to the skin. When the housing 702 is fully advanced, the prongs automatically retract while the sensor (not shown) remains in place to measure analyte levels.

[0144] FIG. 3F is a proximal perspective view depicting an example embodiment of a patient with the sensor control device 102 in the applied position. The user can then remove the applicator 150 from the application site.

[0145] In comparison to prior art systems, with respect to FIG. 3A-3F and analyte monitoring systems 100 described elsewhere herein can reduce or eliminate the chance of accidental breakage, permanent deformation, or incorrect assembly of the applicator components. Because the applicator housing 702 directly engages the platform 808 when the sheath 704 is unlocked, rather than indirectly engaging via the sheath 704, the relative angle between the sheath 704 and the housing 702 does not cause breakage or permanent deformation of the arms or other components. The likelihood of generating relatively high forces during assembly, such as in conventional devices, will be reduced, which in turn reduces the likelihood of unsuccessful assembly by the user.

[0146] F. Example Sensor Applicator Device

[0147] FIG. 4A is a side view depicting an example embodiment of the applicator device 150 coupled with the screw cap 708. This is an example of how the applicator 150 is shipped to and received by a user prior to assembly with a sensor by the user. FIG. 4B is a side perspective view depicting the applicator 150 and cap 708 after decoupling. FIG. 4C is a perspective view depicting an example embodiment of the distal end of the applicator device 150 with the electronics housing 706 and adhesive patch 105 removed from the position in which they would remain within the sensor carrier 710 of the sheath 704 when the cap 708 is in place.

[0148] Reference is made to the accompanying drawings that form a part of this patent FIG. 4D -G, the applicator device 20150 can be provided to the user as a single integrated assembly. FIG. 4D and 4E provide a top perspective view and a bottom perspective view, respectively, of the applicator device 20150, FIG. 4F provides an exploded view of the applicator device 20150, while FIG. 4GA side cross-sectional view is provided. The perspective view illustrates how the applicator device 20150 is shipped to a user and how it is received by the user. The exploded view and cross-sectional view illustrate the components of the applicator device 20150. The applicator device 20150 can include a housing 20702, a grommet 20701, a sheath 20704, a sharp carrier 201102, a spring 205612, a sensor carrier 20710 (also referred to as a "disc carrier"), a sharp hub 205014, a sensor control device (also referred to as a "disc") 20102, a patch 20105, a desiccant 20502, a cap 20708, a serial tag 20709, and a tamper-evident feature 20712. When received by the user, only the housing 20702, the cap 20708, the tamper-evident feature 20712, and the tag 20709 are visible. The tamper-evident feature 20712 can be, for example, a sticker coupled to each of the housing 20702 and the cap 20708, and the tamper-evident feature 20712 can be, for example, irreparably damaged as a result of decoupling the housing 20702 from the cap 20708, thereby indicating to the user that the housing 20702 and the cap 20708 have been previously separated. These features will be described in more detail below.

[0149] G. Exemplary Tray and Sensor Module Assembly

[0150] FIG. 5 is a proximal perspective view depicting an exemplary embodiment of a container 810, to which a lid 812 is removably coupled, which can represent how the package is shipped to a user and how it is received by the user prior to assembly.

[0151] FIG. 6A is a proximal perspective cross-sectional view depicting the sensor delivery components within the container 810. The platform 808 is slidably coupled within the container 810. The desiccant 502 is stationary relative to the container 810. The sensor module 504 is mounted within the container 810.

[0152] FIG. 6B is a proximal perspective view depicting the sensor module 504 in more detail. Here, the retaining arm extension 1834 of the platform 808 releasably secures the sensor module 504 in place. The module 2200 is coupled with the connector 2300, the sharp module 2500, and the sensor (not shown) so that they can be removed together as the sensor module 504 during assembly.

[0153] H. Exemplary Applicator and Sensor Control Device for One-Piece Architecture

[0154] Briefly referring again to FIG. 1A and 3A- 3G, for two-piece architecture systems, the sensor tray 202 and the sensor applicator 150 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 discrete, sealed packages allow the sensor tray 202 and the sensor applicator 150 to be sterilized in separate sterilization processes that are unique to the contents of each package and otherwise incompatible with the contents of the other. More specifically, the sensor tray 202 including the plug assembly 207, including the sensor 110 and the prong 220, can be sterilized using a radiation sterilization such as electron beam (or "e-beam") irradiation. Suitable radiation sterilization processes include, but are not limited to, electron beam (e-beam) radiation, gamma ray radiation, X-ray radiation, or any combination thereof. However, radiation sterilization can damage the electronic components disposed within the electronic housing of the sensor control device 102. Thus, if the sensor applicator 150 containing the electronic housing of the sensor control device 102 needs to be sterilized, it can be sterilized via another method such as a gaseous chemical sterilization using, for example, ethylene oxide. However, gaseous chemical sterilization can damage the enzymes or other chemical and biological substances included in the sensor 110. Due to this sterilization incompatibility, the sensor tray 202 and the sensor applicator 150 are typically sterilized in separate sterilization processes and subsequently packaged separately, which requires the user to ultimately assemble the components for use.

[0155] FIG. 7A and 7B are an exploded top view and a bottom view, respectively, of a sensor control device 3702 according to one or more embodiments. The housing 3706 and the mount 3708 operate as opposing clamshell halves that enclose or otherwise substantially encapsulate the various electronic components of the sensor control device 3702. As shown, the sensor control device 3702 can include a printed circuit board assembly (PCBA) 3802 that includes a printed circuit board (PCB) 3804 having a plurality of electronic modules 3806 coupled thereto. Example electronic modules 3806 include, but are not limited to, resistors, transistors, capacitors, inductors, diodes, and switches. Existing sensor control devices typically stack the PCB components on only one side of the PCB. In contrast, the PCB components 3806 in the sensor control device 3702 can be dispersed across the surface area (i.e., the top surface and the bottom surface) on both sides of the PCB 3804.

[0156] In addition to the electronics module 3806, the PCBA 3802 can also include a data processing unit 3808 mounted to the PCB 3804. The data processing unit 3808 can include, for example, an application specific integrated circuit (ASIC) configured to implement one or more functions or routines associated with the operation of the sensor control device 3702. More specifically, the data processing unit 3808 can be configured to perform data processing functions, where such functions can include, but are not limited to, filtering and encoding of data signals, where each data signal corresponds to a sampled analyte level of a user. The data processing unit 3808 can also include or otherwise be in communication with an antenna for communicating with the reader device 120 FIG. 1A ) over a wireless link.

[0157] A battery hole 3810 can be defined in the PCB 3804 that can accommodate and mount a battery 3812 configured to power the sensor control device 3702. Axial battery contacts 3814a and radial battery contacts 3814b can be coupled to the PCB 3804 and extend into the battery hole 3810 to facilitate the transmission of power from the battery 3812 to the PCB 3804. As the names suggest, the axial battery contacts 3814a can be configured to provide axial contacts for the battery 3812, while the radial battery contacts 3814b can provide radial contacts for the battery 3812. Positioning the battery 3812 within the battery hole 3810 with the axial battery contacts 3814a, radial battery contacts 3814b helps to reduce the height H of the sensor control device 3702, which allows the PCB 3804 to be centrally located and its components to be spread out on both sides (i.e., the top and bottom surfaces). This also helps to facilitate the provision of the chamfer 3718 on the electronics housing 3704.

[0158] The sensor 3716 can be centrally positioned relative to the PCB 3804 and include a tail 3816, a flag 3818, and a neck 3820 interconnecting the tail 3816 and the flag 3818. The tail 3816 can be configured to extend through the central hole 3720 of the mount 3708 for transcutaneous reception under the skin of a user. Also, the tail 3816 can have an enzyme or other chemical thereon to help facilitate analyte monitoring.

[0159] The flag 3818 can include a generally planar surface having one or more sensor contacts 3822 FIG. 7B illustrated as three) disposed thereon. The sensor contacts 3822 can be configured to be in electrical communication with a corresponding one or more circuit contacts 3824 FIG. 7AThe three (shown in the diagram) are aligned and engaged. In some embodiments, one or more sensor contacts 3822 may comprise carbon-impregnated polymer printed or otherwise digitally applied to the flag 3818. Existing sensor control devices typically include connectors made of silicone rubber that encapsulate one or more compatible carbon-impregnated polymer modules as conductive contacts between the sensor and the PCB. In contrast, the currently disclosed one or more sensor contacts 3822 provide a direct connection between the sensor 3716 and the PCB 3804, eliminating the need for prior art connectors and advantageously reducing the height H. Moreover, eliminating the compatible carbon-impregnated polymer module eliminates significant circuit resistance, thus improving circuit conductivity.

[0160] The sensor control device 3702 may also include a compliance member 3826, which may be arranged to insert into the inner surface of the flag 3818 and the housing 3706. More specifically, when the housing 3706 and the mounting 3708 are assembled together, the compliance member 3826 may be configured to provide a passive bias load against the flag 3818, which forces one or more sensor contacts 3822 to continuously engage with one or more corresponding circuit system contacts 3824. In the illustrated embodiment, the compliance member 3826 is an elastomeric O-ring, but may alternatively include any other type of biasing device or mechanism, such as a compression spring, without departing from the scope of this disclosure.

[0161] Sensor control device 3702 may also include one or more electromagnetic shields, shown as a first shield 3828a and a second shield. Housing 3706 may provide or otherwise define a first clock socket 3830a. FIG. 7B ) and second clock socket 3830b ( FIG. 7B ), and mounting 3708 can provide or otherwise define the first timing column 3832a ( FIG. 7A ) and the second timing column 3832b ( FIG. 7A Match the first and second clock sockets 3830a and b with the first and second timing columns 3832a and b respectively, and align the housing 3706 correctly with the mounting piece 3708.

[0162] For details, please refer to the following: FIG. 7A The inner surface of the mounting member 3708 may provide or otherwise define a plurality of recesses or depressions configured to accommodate various components of the sensor control device 3702 when the housing 3706 mates with the mounting member 3708. For example, the inner surface of the mounting member 3708 may define a battery positioner 3834 configured to accommodate a portion of the battery 3812 when the sensor control device 3702 is assembled. Adjacent contact recesses 3836 may be configured to accommodate a portion of an axial battery contact 3814a.

[0163] Moreover, a plurality of module pockets 3838 can be defined in the inner surface of the mount 3708 to accommodate various electronic modules 3806 arranged on the bottom of the PCB 3804. In addition, a shield locator 3840 can be defined in the inner surface of the mount 3708 to accommodate at least a portion of the second shield 3828b when assembling the sensor control device 3702. The battery locator 3834, the contact pocket 3836, the module pockets 3838, and the shield locator 3840 all extend a short distance into the inner surface of the mount 3708, and thus, the overall height H of the sensor control device 3702 can be reduced as compared to existing sensor control devices. The module pockets 3838 can also help minimize the diameter of the PCB 3804 by allowing PCB components to be arranged on both sides (i.e., the top and bottom surfaces).

[0164] Still referring to FIG. 7A The mount 3708 can also include a plurality of carrier grip features 3842 (two shown) defined about the outer periphery of the mount 3708. The carrier grip features 3842 are axially offset from the bottom 3844 of the mount 3708, where transfer adhesive (not shown) can be applied during assembly. In contrast to existing sensor control devices that typically include conical carrier grip features that intersect the bottom of the mount, the presently disclosed carrier grip features 3842 are offset from the plane in which the transfer adhesive is applied (i.e., the bottom 3844). This proves advantageous in helping to ensure that the delivery system does not inadvertently stick to the transfer adhesive during assembly. Moreover, the presently disclosed carrier grip features 3842 eliminate the need for a scalloped transfer adhesive, which simplifies the manufacture of the transfer adhesive and eliminates the need to accurately time the transfer adhesive with respect to the mount 3708. This also increases the bonding area, thereby increasing the bond strength.

[0165] Referring to FIG. 7B The bottom 3844 of the mount 3708 can provide or otherwise define a plurality of grooves 3846 that can be defined at or near the outer periphery of the mount 3708 and spaced equidistant from one another. Transfer adhesive (not shown) can be coupled to the bottom 3844 and the grooves 3846 can be configured to help transport (transfer) moisture away from the sensor control device 3702 and toward the periphery of the mount 3708 during use. In some embodiments, the pitch of the grooves 3846 can be inserted between the module pockets 3838 (defined on the opposing sides (inner surfaces) of the mount 3708) to help minimize the diameter of the PCB 3804. FIG. 7A). As will be appreciated, the positions of the alternating grooves 3846 and the module pockets 3838 ensure that the opposing features on either side of the mount 3708 do not extend into one another. This can help to maximize the material of the mount 3708, thereby helping to maintain a minimum height H of the sensor control device 3702. The module pockets 3838 can also significantly reduce mold draft and improve the flatness of the bottom 3844 to which the transfer adhesive is bonded.

[0166] Still referring to FIG. 7B , the inner surface of the housing 3706 can also provide or otherwise define a plurality of pockets or recesses that are configured to house various components of the sensor control device 3702 when the housing 3706 is mated with the mount 3708. For example, the inner surface of the housing 3706 can define opposing battery locators 3848 that can be arranged opposite the battery locators 3834( FIG. 7A ) of the mount 3708 and configured to house a portion of the battery 3812 when the sensor control device 3702 is assembled. The opposing battery locators 3848 extend a small distance into the inner surface of the housing 3706, which helps to reduce the overall height H of the sensor control device 3702.

[0167] A prong and sensor locator 3852 can also be provided by or otherwise defined on the inner surface of the housing 3706. The prong and sensor locator 3852 can be configured to receive a portion of the prong (not shown) and the sensor 3716. Moreover, the prong and sensor locator 3852 can be configured to align and / or mate with a corresponding prong and sensor locator 2054( FIG. 7A ) provided on the inner surface of the mount 3708.

[0168] According to embodiments of the present disclosure, an alternative sensor assembly / electronics assembly connection method is illustrated in FIGS. 8A-8C. As shown, the sensor assembly 14702 includes a sensor 14704, a connector bracket 14706, and a prong 14708. Notably, a recess or socket 14710 can be defined in the bottom of the mounting base of the electronics assembly 14712 and provides a location where the sensor assembly 14702 can be received and coupled to the electronics assembly 14712, thereby fully assembling the sensor control device. The profile of the sensor assembly 14702 can match or be shaped in a complementary manner to the socket 14710, which includes an elastomeric seal member 14714 (including a conductive material coupled to the circuit board and aligned with the electrical contacts of the sensor 14704). Thus, when the sensor assembly 14702 is snap-fitted or otherwise adhered to the electronics assembly 14712 by driving the sensor assembly 14702 into the integrally formed recess 14710 in the electronics assembly 14712, the on-body device 14714 depicted in FIG. 8C is formed. This embodiment provides an integrated connector for the sensor assembly 14702 within the electronics assembly 14712.

[0169] Additional information regarding sensor assemblies is provided in U.S. Publication No. 2013 / 0150691 and U.S. Publication No. 2021 / 0204841, each of which is incorporated herein by reference in its entirety.

[0170] 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 one-piece architecture sensor control devices. The one-piece architecture allows the sensor applicator 150 and the sensor control device 102 to be shipped to the user in a single, sealed package that does not require any end-user assembly steps. More specifically, the user only needs to open one package and then deliver the sensor control device 102 to the target monitoring location. The one-piece system architecture described herein can prove advantageous in eliminating parts, various manufacturing process steps, and user assembly steps. As a result, packaging and waste are reduced, and the potential for user error or system contamination is reduced.

[0171] FIG. 9A and 9B are side and cross-sectional side views, respectively, of an exemplary embodiment of the sensor applicator 150 with the applicator cap 210 coupled thereto. More specifically, FIG. 9A depicts how the sensor applicator 150 can be shipped to the user and how it can be received by the user, and FIG. 9BA sensor control device 4402 disposed within the sensor applicator 150 is depicted. As such, the fully assembled sensor control device 4402 can have been assembled and installed within the sensor applicator 150 prior to delivery to the user, thus removing any additional assembly steps that would otherwise have to be performed by the user.

[0172] The fully assembled sensor control device 4402 can be loaded into the sensor applicator 150, and the applicator cap 210 can be subsequently coupled to the sensor applicator 150. In some embodiments, the applicator cap 210 can be threaded to the housing 208 and include a tamper-evident ring 4702. Upon rotation (e.g., unscrewing) of the applicator cap 210 relative to the housing 208, the tamper-evident ring 4702 can shear and thereby release the applicator cap 210 from the sensor applicator 150.

[0173] According to the present disclosure, when loaded in the sensor applicator 150, the sensor control device 4402 can be subjected to a gaseous chemical sterilization 4704 configured to sterilize the electronics housing 4404 and any other exposed portions of the sensor control device 4402. To accomplish this, a chemical can be injected into a sterilization chamber 4706 defined collectively by the sensor applicator 150 and the interconnecting cap 210. In some applications, the chemical can be injected into the sterilization chamber 4706 via one or more vents 4708 defined in the applicator cap 210 at its proximal end 610. Example chemicals that can be used for the 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 steam.

[0174] Because the distal end portion and the sharp 4412 of the sensor 4410 are sealed within the sensor cap 4416, the chemicals used during the gaseous chemical sterilization do not interact with enzymes, chemicals, and biological agents provided on the tail 4524 and other sensor components, such as membrane coatings that regulate analyte influx.

[0175] Once a desired level of sterility assurance is achieved within the sterilization chamber 4706, the gaseous solution can be removed and the sterilization chamber 4706 can be aerated. Aeration can be accomplished by a series of vacuums and subsequent circulation of a gas (e.g., nitrogen) or filtered air through the sterilization chamber 4706. Once the sterilization chamber 4706 is properly aerated, the vents 4708 can be closed with a seal 4712 (shown in dashed lines).

[0176] In some embodiments, the seal 4712 can include two or more layers of different materials. The first layer can be made of a synthetic material (e.g., flash-spun high-density polyethylene fibers), such as that available from very durable and puncture-resistant and allows for vapor permeation.​ The layer can be applied prior to the gaseous chemical sterilization process, and after the gaseous chemical sterilization process, the layer can be sealed (e.g., heat sealed) with a layer of foil or other vapor and moisture resistant material to prevent contaminants and moisture from entering the sterilization chamber 4706. In other embodiments, the seal 4712 can include only a single protective layer applied to the applicator cap 210. In such embodiments, the single layer can be vapor permeable for the sterilization process, but can also be capable of preventing moisture and other harmful elements once the sterilization process is complete. The layer can be applied prior to the gaseous chemical sterilization process, and after the gaseous chemical sterilization process, the layer can be sealed (e.g., heat sealed) with a layer of foil or other vapor and moisture resistant material to prevent contaminants and moisture from entering the sterilization chamber 4706. In other embodiments, the seal 4712 can include only a single protective layer applied to the applicator cap 210. In such embodiments, the single layer can be vapor permeable for the sterilization process, but can also be capable of preventing moisture and other harmful elements once the sterilization process is complete.

[0177] With the seal 4712 in place, the applicator cap 210 provides a barrier against external contamination, thereby maintaining a sterile environment for the assembled sensor control device 4402 until the user removes (unclips) the applicator cap 210. The applicator cap 210 can also form a dust-free environment during shipping and storage to prevent the adhesive patch 4714 from becoming dirty.

[0178] FIG. 10A and 10B are an isometric view and a side view, respectively, of another example sensor control device 5002 according to one or more embodiments of the present disclosure. The sensor control device 5002 can be similar in some respects to the sensor control device 102 of FIG. 1A , and therefore can be best understood with reference thereto. Also, the sensor control device 5002 can be substituted for the sensor control device 102 of FIG. 1A , and therefore can be used in conjunction with the sensor applicator 150 of FIG. 1A , which can deliver the sensor control device 5002 to a target monitoring location on a user’s skin.

[0179] However, unlike the sensor control device 102 of FIG. 1A , the sensor control device 5002 can include a one-piece system architecture that does not require the user to open multiple packages and finally assemble the sensor control device 5002 prior to application. Rather, upon receipt by the user, the sensor control device 5002 can already be fully assembled and properly positioned within the sensor applicator 150 FIG. 1A . To use the sensor control device 5002, the user need only open one barrier (e.g., the applicator cap 708 of FIG. 3B ) and then quickly deliver the sensor control device 5002 to the target monitoring location for use.

[0180] As shown, the sensor control device 5002 includes an electronics housing 5004, which is generally disc-shaped and can have a circular cross-section. However, in other embodiments, the electronics housing 5004 can assume other cross-sectional shapes, such as oval or polygonal, without departing from the scope of the present disclosure. The electronics housing 5004 can be structured to house or otherwise contain various electronic components for operating the sensor control device 5002. In at least one embodiment, an adhesive patch (not shown) can be disposed at the bottom of the electronics housing 5004. The adhesive patch can be similar to the adhesive patch 105 of FIG. 1, and thus can help adhere the sensor control device 5002 to a user’s skin for use. FIG. 1A

[0181] As shown, the sensor control device 5002 includes an electronics housing 5004, which includes a shell 5006 and a mount 5008 that mates with the shell 5006. The shell 5006 can be secured to the mount 5008 in a variety of ways, such as a snap-fit engagement, an interference fit, a sonic weld, 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 such that a sealed interface is generated therebetween.

[0182] The sensor control device 5002 can also include a sensor 5010 (partially visible) and a sharp 5012 (partially visible) for helping to transcutaneously deliver the sensor 5010 under a user’s skin during application of the sensor control device 5002. As shown, corresponding portions of the sensor 5010 and the sharp 5012 extend distally from the bottom of the electronics housing 5004 (e.g., the mount 5008). The sharp 5012 can include a sharp hub 5014 structured to secure and carry the sharp 5012. As best shown in FIG. 5B, the sharp hub 5014 can include or otherwise define a mating member 5016. To couple the sharp 5012 to the sensor control device 5002, the sharp 5012 can be axially advanced through the electronics device housing 5004 until the sharp hub 5014 engages an upper surface of the shell 5006 and the mating member 5016 extends distally from the bottom of the mount 5008. As the sharp 5012 penetrates the electronics housing 5004, an exposed portion of the sensor 5010 can be housed within a hollow or recessed (arcuate) portion of the sharp 5012. The remaining portion of the sensor 5010 is disposed within the interior of the electronics housing 5004. FIG. 10B The sensor control device 5002 can also include a sensor cap 5018, which can be disposed over the sensor 5010 and the sharp 5012. The sensor cap 5018 can be structured to protect the sensor 5010 and the sharp 5012 from damage and / or contamination. In some cases, the sensor cap 5018 can be structured to be removed prior to use of the sensor control device 5002.

[0183] FIG. 10A-10B ​​The sensor cap 5016 is shown in a disassembled or detached manner from the electronic housing 5004. The sensor cap 5016 can be removably coupled to the sensor control device 5002 (e.g., the electronic housing 5004) at or near the bottom of the mounting 5008. The sensor cap 5018 helps provide a sealed barrier surrounding and protecting the exposed portions of the sensor 5010 and the tip 5012 from gaseous chemical sterilization. As shown, the sensor cap 5018 may include a generally cylindrical body having a first end 5020a and a second end 5020b opposite to the first end 5020a. The first end 5020a may be open to provide an entrance to an inner chamber 5022 defined within the body. In contrast, the second end 5020b may be closed and may provide or otherwise define an engagement feature 5024. As described herein, the engagement feature 5024 helps to fit the sensor cap 5018 to a sensor applicator (e.g., Figure 1 and...). FIG. 3A -3G sensor applicator 150) cap (e.g., FIG. 3B The applicator cap 708) can also help remove the sensor cap 5018 from the sensor control device 5002 when removing the cap from the sensor applicator.

[0184] Sensor cap 5018 may be removably coupled to or near the bottom of mounting member 5008 and electronic housing 5004. More specifically, sensor cap 5018 may be removably coupled to mating member 5016 extending distally from the bottom of mounting member 5008. In at least one embodiment, for example, mating member 5016 may define an assembly of external threads 5026a. FIG. 10B ), which can be combined with the set of internal threads 5026b defined by sensor cap 5018 ( FIG. 10A In some embodiments, the external thread 5026a and internal thread 5026b may include a flat thread design (e.g., without helical curvature), which may prove advantageous when molding parts. Alternatively, the external and internal threads 5026a, b may include a helical thread engagement. Thus, the sensor cap 5018 may be threaded to the sensor control device 5002 at the mating member 5016 of the pointed hub 5014. In other embodiments, the sensor cap 5018 may be removably coupled to the mating member 5016 by other types of engagement, including but not limited to interference fits or friction fits, or to fragile components or materials that can be broken with minimal separation force (e.g., axial force or rotational force).

[0185] In some embodiments, the sensor cap 5018 can comprise a monolithic (single) structure extending between the first end 5020a and the second end 5020b. However, in other embodiments, the sensor cap 5018 can comprise two or more components. In the illustrated embodiment, for example, the sensor cap 5018 can comprise a seal ring 5028 at the first end 5020a and a desiccant cap 5030 disposed at the second end 5020b. The seal ring 5028 can be configured to help seal the inner chamber 5022, as described in greater detail below. In at least one embodiment, the seal ring 5028 can comprise an elastomeric O-ring. The desiccant cap 5030 can house or include a desiccant to help maintain a preferred humidity level within the inner chamber 5022. The desiccant cap 5030 can also define or otherwise provide the engagement feature 5024 of the sensor cap 5018.

[0186] FIG. 11A-11C is a progressive cross-sectional side view showing assembly of the sensor applicator 150 with the sensor control device 5002, in accordance with one or more embodiments. Once the sensor control device 5002 is fully assembled, it can be loaded into the sensor applicator 150. Referring to FIG. 11A , the prong hub 5014 can include or otherwise define a hub snap detent 5302 configured to help couple the sensor control device 5002 to the sensor applicator 150. More specifically, the sensor control device 5002 can be advanced into the interior of the sensor applicator 150 and the hub snap detent 5302 can be positioned for receipt by a corresponding arm 5304 of the prong carrier 5306 within the sensor applicator 150.

[0187] In FIG. 11B , the sensor control device 5002 is shown as being received by the prong carrier 5306, thus secured within the sensor applicator 150. Once the sensor control device 5002 is loaded into the sensor applicator 150, the applicator cap 210 can be coupled to the sensor applicator 150. In some embodiments, the applicator cap 210 and the housing 208 can have opposing, mateable sets of threads 5308 such that the applicator cap 210 can be screwed onto the housing 208 in a clockwise (or counterclockwise) direction, thereby securing the applicator cap 210 to the sensor applicator 150.

[0188] As shown, the sheath 212 is also positioned within the sensor applicator 150, and the sensor applicator 150 can include a sheath locking mechanism 5310 configured to ensure that the sheath 212 does not prematurely collapse during an impact event. In the illustrated embodiment, the sheath locking mechanism 5310 can include a threaded engagement between the applicator cap 210 and the sheath 212. More specifically, one or more internal threads 5312a can be defined or otherwise provided on an inner surface of the applicator cap 210, and one or more external threads 5312b can be defined or otherwise provided on the sheath 212. The internal and external threads 5312a, b can be configured to threadably mate when the applicator cap 210 is threaded onto the sensor applicator 150 at the threads 5308. The internal and external threads 5312a, b can have the same pitch as the threads 5308, enabling the applicator cap 210 to be screwed onto the housing 208.

[0189] In FIG. 11C the applicator cap 210 is shown fully threaded (coupled) to the housing 208. As shown, the applicator cap 210 can also provide or otherwise define a cap post 5314 centrally located within the interior of the applicator cap 210 and extending proximally from a bottom thereof. The cap post 5314 can be configured to receive at least a portion of the sensor cap 5018 when the applicator cap 210 is threaded onto the housing 208.

[0190] With the sensor control device 5002 loaded within the sensor applicator 150 and the applicator cap 210 properly secured, the sensor control device 5002 can then be subjected to a gaseous chemical sterilization configured to sterilize the electronics housing 5004 and any other exposed portions of the sensor control device 5002. Since the distal portion of the sensor 5010 and the prong 5012 are sealed within the sensor cap 5018, the chemicals used during the gaseous chemical sterilization cannot interact with enzymes, chemicals, and biological substances provided on the tail 5104 and other sensor components, such as membrane coatings that regulate analyte influx.

[0191] FIG. 12A-12C is a progressive cross-sectional side view according to one or more additional embodiments showing assembly and disassembly of an alternative embodiment of the sensor applicator 150 with the sensor control device 5002. The fully assembled sensor control device 5002 can be loaded into the sensor applicator 150 by coupling the hub snap detent 5302 into the arm 5304 of the prong carrier 5306 positioned within the sensor applicator 150, as generally described above.

[0192] In the illustrated embodiment, the sheath arm 5604 of the sheath 212 can be configured to interact with a first stop 5702a and a second stop 5702b defined inside the housing 208. The first stop 5702a can alternatively be referred to as a “lock” stop, and the second stop 5702b can alternatively be referred to as a “fire” stop. When the sensor control device 5002 is initially installed in the sensor applicator 150, the sheath arm 5604 can be received within the first stop 5702a. As described below, the sheath 212 can be actuated to move the sheath arm 5604 to the second stop 5702b, which places the sensor applicator 150 in a fire position.

[0193] In FIG. 12B which the applicator cap 210 is aligned with and advanced toward the housing 208 such that the sheath 212 is received within the applicator cap 210. Instead of rotating the applicator cap 210 relative to the housing 208, threads of the applicator cap 210 can be engaged onto corresponding threads of the housing 208 to couple the applicator cap 210 to the housing 208. An axial cutout or slot 5703 (one shown) defined in the applicator cap 210 can allow a portion of the applicator cap 210 proximate its threads to flex outward to snapably engage with threads of the housing 208. When the applicator cap 210 is snapped to the housing 208, the sensor cap 5018 can correspondingly snap into the cap post 5314.

[0194] Similar to FIG. 11A-11C the embodiment of FIG. 6, the sensor applicator 150 can include a sheath lock mechanism configured to ensure that the sheath 212 does not prematurely collapse during a shock event. In the illustrated embodiment, the sheath lock mechanism includes one or more ribs 5704 (one shown) defined near a base of the sheath 212 and configured to interact with one or more ribs 5706 (two shown), and includes a shoulder 5708 defined near a base of the applicator cap 210. The ribs 5704 can be configured to interlock between the ribs 5706 and the shoulder 5708 upon attachment of the applicator cap 210 to the housing 208. More specifically, once the applicator cap 210 is snapped onto the housing 208, the applicator cap 210 can be rotated (e.g., clockwise), which positions the ribs 5704 of the sheath 212 between the ribs 5706 and the shoulder 5708 of the applicator cap 210, thereby “locking” the applicator cap 210 into place until the user reversely rotates the applicator cap 210 to remove the applicator cap 210 for use. The engagement of the ribs 5704 between the ribs 5706 and the shoulder 5708 of the applicator cap 210 can also prevent the sheath 212 from prematurely collapsing.

[0195] In FIG. 12C which the applicator cap 210 is removed from the housing 208. Similar to FIG. 21A-21CAs with the embodiment of FIG. 6, the applicator cap 210 can be removed by reverse-rotating the applicator cap 210, which correspondingly rotates the cap post 5314 in the same direction and causes the sensor cap 5018 to unclamp from the mating member 5016, as generally described above. Moreover, detaching the sensor cap 5018 from the sensor control device 5002 exposes the sensor 5010 and the distal portion of the prong 5012.

[0196] When the applicator cap 210 is unscrewed from the housing 208, the rib 5704 defined on the sheath 212 can slidingly engage the top of the rib 5706 defined on the applicator cap 210. The top of the rib 5706 can provide a corresponding ramped surface that causes the sheath 212 to displace upward as the applicator cap 210 is rotated, and moving the sheath 212 upward causes the sheath arms 5604 to flex out of engagement with the first detent 5702a to be received within the second detent 5702b. When the sheath 212 moves to the second detent 5702b, the radial shoulder 5614 moves out of radial engagement with the carrier arm(s) 5608, which allows the passive spring force of the spring 5612 to push the prong carrier 5306 upward and force the carrier arm(s) 5608 out of engagement with the groove(s) 5610. As the prong carrier 5306 moves upward within the housing 208, the mating member 5016 can correspondingly retract until it becomes flush, substantially flush, or half-flush with the bottom of the sensor control device 5002. At this point, the sensor applicator 150 is in the primed position. Thus, in this embodiment, removing the applicator cap 210 correspondingly causes the mating member 5016 to retract.

[0197] I. Exemplary priming mechanisms for one-piece and two-piece applicators

[0198] FIG. 13A-13F FIGS. 19-22 illustrate example details of an embodiment of a "priming" applicator 216 to apply a sensor control device 222 to a subject and including an internal device mechanism that safely retracts the prong 1030 into the used applicator 216. Collectively, these figures represent an example sequence of driving the prong 1030 (supporting a sensor coupled to the sensor control device 222) into the skin of a user, withdrawing the prong while leaving the sensor in effective contact with the interstitial fluid of the user, and adhering the sensor control device to the skin of the user with adhesive. Those skilled in the art can refer to the same to understand modifications of this activity for use with alternative applicator assembly embodiments and components. Moreover, the applicator 216 can be a sensor applicator having a one-piece architecture or a two-piece architecture as disclosed herein.

[0199] Turning now to FIG. 13AThe sensor 1102 is supported within the prong 1030 just above the user's skin 1104. Tracks 1106 (optionally three of them) of the upper guide 1108 can be provided to control the motion of the applicator 216 relative to the sheath 318. The sheath 318 is held by a stopper feature 1110 within the applicator 216 such that an appropriate downward force along the longitudinal axis of the applicator 216 will cause the resistance provided by the stopper feature 1110 to be overcome such that the prong 1030 and sensor control device 222 can be translated along the longitudinal axis into (and onto) the user's skin 1104. Further, the carrier arm 1112 of the sensor carrier 1022 engages the prong retraction assembly 1024 to hold the prong 1030 in position relative to the sensor control device 222.

[0200] In FIG. 13B , the user force is applied to overcome or exceed the stop feature 1110 and the sheath 318 collapses into the housing 314 driving the sensor control device 222 (with associated parts) to translate down the longitudinal axis as indicated by arrow L. The inner diameter of the upper guide 1108 of the sheath 318 limits the position of the carrier arm 1112 throughout the stroke of the sensor / prong insertion process. The retention of the stop surface 1114 of the carrier arm 1112 against the complementary surface 1116 of the prong retraction component 1024 maintains the position of the member with the reset spring 1118 fully energized. According to embodiments, rather than using a user force to drive the sensor control device 222 to translate down the longitudinal axis as indicated by arrow L, the housing 314 can 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 drive the sensor control device 222.

[0201] In FIG. 13C , the sensor 1102 and prong 1030 have reached full insertion depth. In doing so, the carrier arm 1112 clears the inner diameter of the upper guide 1108. The compression force of the coil reset spring 1118 then drives the angled stop surface 1114 radially outward, releasing the force to drive the prong carrier 1102 of the prong retraction assembly 1024 to pull the (slotted or otherwise configured) prong 1030 out of the user and away from the sensor 1102 as indicated by arrow R in FIG. 13D .

[0202] As shown in FIG. 13E , with the prong 1030 fully retracted, the upper guide portion 1108 of the sheath 318 is provided with a final locking feature 1120. As shown in FIG. 13F , the used applicator assembly 216 is removed from the insertion position leaving the sensor control device 222, and the prong 1030 safely secured within the applicator component 216. The used applicator component 216 can now be disposed of.

[0203] When the applicator sensor controls device 222, the operation of applicator 216 is designed to provide the user with the sensation that the insertion and retraction of tip 1030 are automatically performed by the internal mechanism of applicator 216. In other words, the invention avoids the user experiencing the sensation that they are manually inserting tip 1030 into their skin. Therefore, once the user applies sufficient force to overcome the resistance from the stop feature of applicator 216, the final action of applicator 216 is perceived as an automatic response to the applicator being "triggered". Although all the driving force is provided by the user and no additional bias / driving means are used to insert tip 1030, the user is not aware that they are providing additional force to drive tip 1030 to pierce their skin. As above FIG. 13C As detailed in the description, the retraction of the tip 1030 is automatically completed by the spiral return spring 1118 of the applicator 216.

[0204] Regarding any applicator embodiments and any components thereof described herein, including but not limited to tips, tip modules, and sensor module embodiments, those skilled in the art will understand that the dimensions of these embodiments are designed and configured for use with a sensor configured to sense analyte levels in bodily fluids in the epidermis, dermis, or subcutaneous tissue of a subject. In some embodiments, for example, the tip and distal portion of the analyte sensor disclosed herein may be dimensionally set and configured to be positioned at a specific distal depth (i.e., the furthest point of penetration in the tissue or layer of the subject's body, e.g., in the epidermis, dermis, or subcutaneous tissue). Regarding some applicator embodiments, those skilled in the art will recognize that certain embodiments of the tip may be dimensionally set and configured to be positioned at different distal depths in the subject's body relative to the final distal depth of the analyte sensor. In some embodiments, for example, the tip may be positioned at a first distal depth in the subject's epidermis before retraction, while the distal portion of the analyte sensor may be positioned at a second distal depth in the subject's dermis. In other embodiments, the tip may be positioned at a first distal depth in the subject's dermis before retraction, while the distal portion of the analyte sensor may be positioned at a second distal depth in the subject's subcutaneous tissue. In other embodiments, the tip may be positioned at the first distal depth before retraction and the analyte sensor may be positioned at the second distal depth, wherein both the first and second distal depths are located in the same layer or tissue of the subject's body.

[0205] Further, with respect to any of the applicator embodiments described herein, those skilled in the art will appreciate that the analyte sensor, as well as one or more structural components coupled thereto, including but not limited to one or more spring mechanisms, can be disposed within the applicator in an off-center position relative to one or more axes of the applicator. In some applicator embodiments, for example, the analyte sensor and spring mechanism can be disposed on a first side of the applicator in a first off-center position relative to an axis of the applicator, and the sensor electronics can be disposed on a second side of the applicator in an off-center position relative to the axis of the applicator. In other applicator embodiments, the analyte sensor, spring mechanism, and sensor electronics can be disposed on the same side in off-center positions relative to the axis of the applicator. Those skilled in the art will recognize other arrangements and configurations in which any or all of the analyte sensor, spring mechanism, sensor electronics, and other components of the applicator are disposed in centered or off-center positions relative to one or more axes of the applicator are possible and well within the scope of the present disclosure.

[0206] Additional details of suitable devices, systems, methods, components, and operations thereof, along with related features, are set forth in International Publication No. WO 2018 / 136898 to Rao et al., International Publication No. WO 2019 / 236850 to Thomas et al., International Publication No. WO 2019 / 236859 to Thomas et al., International Publication No. WO 2019 / 236876 to Thomas et al., and U.S. Patent Publication No. 2020 / 0196919, filed June 6, 2019, each of which is incorporated by reference herein 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 by reference herein in their entirety for all purposes. Further details regarding embodiments of sharp modules, sharps, their components, and variations thereof, are described in U.S. Patent Publication No. 2014 / 0171771, which is incorporated by reference herein in its entirety for all purposes.

[0207] J. Exemplary methods of calibrating analyte sensors

[0208] Biochemical sensors can be described by one or more sensing characteristics. One common sensing characteristic is referred to as the sensitivity of the biochemical sensor, which is a measure of the responsiveness of the sensor to the concentration of the chemical or component it is designed to detect. For electrochemical sensors, this response can be in the form of electrical current (amperometric) or electrical charge (coulometric). For 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 a variety of factors, including whether the sensor is in an in vitro or in vivo state.

[0209] FIG. 14 is a plot depicting 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 a regression (e.g., linear or non-linear) 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 according to the equation y = mx + b, where y is the electrical output current of the sensor, x is the analyte level (or concentration), m is the slope of the sensitivity and b is the intercept of the sensitivity, where the intercept generally corresponds to a background signal (e.g., noise). For sensors with a linear or substantially linear response, the analyte level corresponding to a given current can be determined from the slope and intercept of the sensitivity. Sensors with non-linear sensitivities require additional information to determine the analyte level resulting from the output current of the sensor, and those of ordinary skill in the art are familiar with ways to model non-linear sensitivities. In certain embodiments of in vivo sensors, the in vitro sensitivity can be the same as the in vivo sensitivity, but in other embodiments, an in vitro sensitivity is translated to an in vivo sensitivity using a transfer (or conversion) function appropriate for the intended in vivo use of the sensor.

[0210] Calibration is a technique to improve or maintain accuracy by adjusting the measured output of a sensor to reduce differences from the expected output of the sensor. One or more parameters describing the sensing characteristics of the sensor, such as its sensitivity, are established for use in the calibration adjustment.

[0211] Certain in vivo analyte monitoring systems require calibration, either through user interaction or in an automated manner by the system itself, after the sensor is implanted in the user or patient. For example, when user interaction is required, the user performs an ex vivo measurement (e.g., a blood glucose (BG) measurement using a fingertip and ex vivo test strips) and enters it into the system while the analyte sensor is implanted. The system then compares the ex vivo measurement to the in vivo signal and uses the difference to determine an estimate of the in vivo sensitivity of the sensor. The in vivo sensitivity can then be used in an algorithmic process to convert data collected with the sensor into values indicative of the user’s analyte level. This process and other processes that require user operation to perform calibration are referred to as “user calibration.” User calibration can be required by the system due to sensor sensitivity instability, such that the sensitivity drifts or changes over time. Thus, multiple user calibrations can be required (e.g., according to a periodic (e.g., daily) schedule, a variable schedule, or as needed) to maintain accuracy. While embodiments described herein can incorporate some degree of user calibration for certain implementations, generally this is not preferred as it requires the user to perform a painful or otherwise burdensome BG measurement and can introduce user error.

[0212] Some in vivo analyte monitoring systems can periodically adjust calibration parameters through automated measurements by the system itself of characteristics of the sensor (e.g., processing circuitry executing software). Repeated adjustment of the sensitivity of the sensor based on variables measured by the system (rather than the user) is generally referred to as “system” (or automated) calibration, and can be performed with or without user calibration, such as an early BG measurement. As with repeated user calibration, drift in the sensor sensitivity over time generally requires repeated system calibration. Thus, while embodiments described herein can be used with some degree of automated system calibration, preferably the sensitivity of the sensor is relatively stable over time such that post-implant calibration is not required.

[0213] Some in vivo analyte monitoring systems operate with sensors that are factory calibrated. Factory calibration refers to the determination or estimation of one or more calibration parameters prior to distribution to a user or healthcare professional (HCP). The calibration parameters can 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 manufacture sensors in groups or batches, referred to as production lots, manufacturing stage lots, or simply lots. A single lot can include thousands of sensors.

[0214] The sensors can include calibration codes or parameters that can be derived or determined during one or more sensor manufacturing processes and encoded or programmed in the data processing device of the analyte monitoring system as part of the manufacturing process or provided on the sensor itself, e.g., as a bar code, laser tag, RFID tag, or other machine-readable information provided on the sensor. If the codes are provided to the receiver (or other data processing device), user calibration of the sensor during in vivo use can be avoided or the frequency of in vivo calibration during sensor wear can be reduced. In embodiments where the calibration codes or parameters are provided on the sensor itself, the calibration codes or parameters can be automatically transmitted or provided to the data processing device in the analyte monitoring system prior to or at the start of sensor use.

[0215] Some in vivo analyte monitoring systems operate with sensors that can be one or more of factory calibrated, system calibrated, and / or user calibrated. For example, a sensor can be provided with a calibration code or parameter that can allow for factory calibration. If the information is provided to the receiver (e.g., entered by the user), the sensor can operate as a factory calibrated sensor. If the information is not provided to the receiver, the sensor can operate as a user calibrated sensor and / or a system calibrated sensor.

[0216] In another aspect, programming or executable instructions can be provided or stored in the data processing device and / or receiver / controller unit of the analyte monitoring system to provide a time-varying adjustment algorithm to an in vivo sensor during use. For example, based on retrospective statistical analysis of in vivo used analyte sensors and corresponding glucose level feedback, a time-based predetermined or analytical curve or database can be generated and configured to provide additional adjustments to one or more in vivo sensor parameters to compensate for potential sensor drift in stability profile or other factors.

[0217] According to the disclosed subject matter, an analyte monitoring system can be configured to compensate or adjust sensor sensitivity based on a sensor drift curve. The time-varying parameter β(t) can be defined or determined based on analysis of sensor behavior during in vivo use, and a time-varying drift curve can be determined. In certain aspects, the compensation or adjustment to sensor sensitivity can be programmed in the receiver unit, controller, or data processor of the analyte monitoring system, such that the compensation or adjustment, or both, can be performed automatically and / or iteratively as sensor data is received from the analyte sensor. According to the disclosed subject matter, the adjustment or compensation algorithm can be initiated or performed by the user (rather than spontaneously initiated or performed), such that the adjustment or compensation of the analyte sensor sensitivity profile is performed upon user initiation or activation of a corresponding function or routine, or upon user entry of a sensor calibration code.

[0218] According to the disclosed subject matter, each sensor in a batch of sensors (in some cases not including sample sensors for in vitro testing) can be non-destructively inspected to determine or measure its characteristics, such as the membrane thickness at one or more points of the sensor, and other characteristics, including physical characteristics, such as the surface area / volume of the active area, can be measured or determined. Such measurements or determinations can be performed in an automated fashion using, for example, an optical scanner or other suitable measurement device or system, and the determined sensor characteristics of each sensor in the batch of sensors are compared to the corresponding average values based on the sampled sensors for possible correction of the calibration parameters or codes assigned to each sensor. For example, for a calibration parameter defined as the sensor sensitivity, which is approximately inversely proportional to the membrane thickness, so that, for example, if the measured membrane thickness of a sensor is approximately 4% greater than the average of the sampled sensors from the same batch of sensors as that sensor, then in one embodiment the sensitivity assigned to that sensor is the average sensitivity of the sampled sensors divided by 1.04. Likewise, since sensitivity is approximately directly proportional to the active area of the sensor, if the measured active area of a sensor is approximately 3% less than the average active area of the sampled sensors from the same batch of sensors, then the sensitivity assigned to that sensor is the average value sensitivity multiplied by 0.97. By making multiple successive adjustments to each inspection or measurement of the sensors, the assigned sensitivity can be determined from the average sensitivity of the sampled sensors. In certain embodiments, the inspection or measurement of each sensor can additionally include a measurement of the membrane consistency or texture in addition to the membrane thickness and / or the surface area or volume of the active sensing area.

[0219] Additional information regarding sensor calibration is provided in U.S. Publication No. 2010 / 00230285 and U.S. Publication No. 2019 / 0274598, each of which is incorporated by reference herein in its entirety.

[0220] K. Exemplary Bluetooth Communication Protocol

[0221] The storage memory 2230 of the sensor 110 can include software blocks related to the communication protocols of the communication module. For example, the storage memory 2230 can include a BLE service software block having functionality that provides an interface to make the BLE module 5041 available to the computing hardware of the sensor 110. These software functionalities can include a BLE logical interface and an interface resolver. The BLE services provided by the communication module 5040 can include a generic access profile service, a generic attribute service, a generic access service, a device information service, a data transfer service, and a security service. The data transfer service can be the primary service for transferring data such as sensor control data, sensor status data, analyte measurement data (historical and current), and event log data. The sensor status data can include error data, current active time, and software status. The analyte measurement data can include information such as current and historical raw measurements, current and historical values after processing using appropriate algorithms or models, predictions and trends of measurement levels, comparisons of other values to patient-specific average values, calls to action as determined by algorithms or models, and other similar types of data.

[0222] According to aspects of the disclosed subject matter, and as embodied herein, the sensor 110 can be configured to concurrently communicate with multiple devices by adapting the characteristics of the communication protocols or media supported by the hardware and radio transceivers of the sensor 110. As an example, the BLE module 5041 of the communication module 5040 can be provisioned with software or firmware to enable multiple concurrent connections between the sensor 110 as a central device and other devices as peripheral devices, or as a peripheral device with another device as a central device.

[0223] The characteristics of a connection and a subsequent communication session between two devices using a communication protocol such as BLE are characterized by a similar physical channel operating between the two devices (e.g., the sensor 110 and the data receiving device 129). The physical channel can include a single channel or a series of channels, including, for example, but not limited to, a negotiated series of channels using a common clock and channel or frequency hopping sequence. The communication session can use a similar amount of available communication spectrum, and multiple such communication sessions can exist in proximity. In certain embodiments, each set of devices in a communication session uses a different physical channel or series of channels to manage interference of devices of the same proximity.

[0224] For purposes of illustration and not limitation, reference is made to an exemplary embodiment of a process for a sensor-receiver connection for use with the disclosed subject matter. Initially, the sensor 110 repeatedly advertises its connection information to its environment in a search for a data receiving device 129. The sensor 110 can repeat the advertisement periodically until a connection is established. The data receiving device 129 detects the advertisement packet and scans and filters for a sensor 110 to connect with using the data provided in the advertisement packet. Next, the data receiving device 129 sends a scan request command and the sensor 110 responds with a scan response packet providing additional details. Then, the data receiving device 129 sends a connection request using the Bluetooth device address associated with the data receiving device 129. The data receiving device 129 can also continuously request a connection with the sensor 110 having the specific Bluetooth device address. The devices then establish an initial connection allowing them to begin exchanging data. The devices begin the process of initializing a data exchange service and performing a mutual authentication procedure.

[0225] During the first connection between the sensor 110 and the data receiving device 129, the data receiving device 129 can initialize a service, feature, and attribute discovery process. The data receiving device 129 can evaluate these features of the sensor 110 and store them for use during subsequent connections. Next, the devices enable notification of a custom security service for mutual authentication of the sensor 110 and the data receiving device 129. The mutual authentication process can proceed automatically, requiring no user interaction. After successfully completing the mutual authentication process, the sensor 110 sends a connection parameter update to request that the data receiving device 129 use connection parameter settings preferred by the sensor 110 and configured to maximize longevity.

[0226] The data receiving device 129 then performs 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 129 sends a request to initiate a backfill process. The request can specify a range of records defined based on, for example, measurement values, timestamps, or the like as appropriate. The sensor 110 responds with the requested data until all previously unsent data in the memory of the sensor 110 is delivered to the data receiving device 129. The sensor 110 can respond to a backfill request from the data receiving device 129 that all data has been sent. Once the backfill is complete, the data receiving device 129 can notify the sensor 110 that it is ready to receive periodic measurement readings. The sensor 110 can send readings across multiple notification results on a repeated basis. As embodied herein, multiple notifications can be redundant notifications to ensure that data is properly transmitted. Alternatively, multiple notifications can comprise a single payload.

[0227] For purposes of illustration and not limitation, reference is made to an exemplary embodiment of the process of sending a shutdown command to sensor 110. If sensor 110 is in, for example, an error state, a plug-in failure state, or a sensor expired state, a shutdown operation is performed. If sensor 110 is not in those states, sensor 110 can log the command and perform the shutdown when sensor 110 transitions to an error state or a sensor expired state. Data receiving device 129 sends a properly formatted shutdown command to sensor 110. If sensor 110 is actively processing another command, sensor 110 will respond with a standard error response indicating that sensor 110 is busy. Otherwise, sensor 110 sends a response upon receiving the command. In addition, sensor 110 sends a success notification through sensor control features to confirm that sensor 110 has received the command. Sensor 110 registers the shutdown command. At the next appropriate opportunity (e.g., depending on the current sensor state, as described herein), sensor 110 will shut down.

[0228] L. Exemplary Sensor States and Activation

[0229] For purposes of illustration and not limitation, reference is made to an exemplary embodiment of the process of sending a shutdown command to sensor 110. If sensor 110 is in, for example, an error state, a plug-in failure state, or a sensor expired state, a shutdown operation is performed. If sensor 110 is not in those states, sensor 110 can log the command and perform the shutdown when sensor 110 transitions to an error state or a sensor expired state. Data receiving device 129 sends a properly formatted shutdown command to sensor 110. If sensor 110 is actively processing another command, sensor 110 will respond with a standard error response indicating that sensor 110 is busy. Otherwise, sensor 110 sends a response upon receiving the command. In addition, sensor 110 sends a success notification through sensor control features to confirm that sensor 110 has received the command. Sensor 110 registers the shutdown command. At the next appropriate opportunity (e.g., depending on the current sensor state, as described herein), sensor 110 will shut down. FIG. 15

[0230] ​Upon entering state 6025, sensor 110 can store information related to the device authenticated to communicate with the sensor as set during activation, or initialize algorithms related to performing and interpreting measurements from sensing hardware 5060. Sensor 110 can also initialize a life cycle timer, responsible for maintaining an active count of the operational time of sensor 110 and begin communicating with the authenticated device to transmit recorded data. While in the insertion detection state 6025, the sensor can enter state 6030, in which sensor 110 checks whether the operational time equals a predetermined threshold. This operational time threshold can correspond to a timeout function used to determine whether insertion has been successful. If the operational time has reached the threshold, then sensor 110 proceeds to state 6035, in which sensor 110 checks whether the average data read is greater than a threshold amount corresponding to an expected amount of data read used to trigger a successful insertion detection. If the amount of data read is below the threshold in state 6035, then the sensor proceeds to state 6040, corresponding to a failed insertion. If the amount of data read satisfies the threshold, then the sensor proceeds to active pairing state 6055.

[0231] Active pairing state 6055 of sensor 110 reflects a state when sensor 110 is operating normally by recording measurements, processing measurements, and reporting them appropriately. While in active pairing state 6055, sensor 110 transmits measurement results or attempts to establish a connection with receiving device 129. Sensor 110 also increments the operational time. Once sensor 110 reaches a predetermined threshold operational time (e.g., once the operational time reaches a predetermined threshold), sensor 110 transitions to active expiration state 6065. Active expiration state 6065 of sensor 110 reflects a state when sensor 110 has operated for its maximum predetermined amount of time.

[0232] While in active expiration state 6065, sensor 110 can generally perform operations related to ending operation and ensure that collected measurements have been securely transmitted to a receiving device as needed. For example, while in active expiration state 6065, sensor 110 can transmit collected data, and if no connection is available, can increase efforts to find an authenticated device nearby and establish a connection with it. While in active expiration state 6065, sensor 110 can receive a shutdown command in state 6070. If no shutdown command is received, sensor 110 can also check whether the operational time has exceeded a final operational threshold in state 6075. The final operational threshold can be based on the battery life of sensor 110. Normal termination state 6080 corresponds to a final operation of sensor 110 and final shut down of sensor 110.

[0233] Prior to activation of the sensor, the ASIC 5000 is in a low power storage mode state. For example, when an incoming RF field (e.g., an NFC field) drives the power supply voltage of the ASIC 5000 above a reset threshold, the activation process can begin, which causes the sensor 110 to enter a wake-up state. When in the wake-up state, the ASIC 5000 enters an activation sequence state. The ASIC 5000 then wakes up the communication module 5040. The communication module 5040 is initialized, triggering a power-on self-test. The power-on self-test can include the ASIC 5000 communicating with the communication module 5040 using prescribed read and write data sequences to verify that the memory and one-time programmable memory are undamaged.

[0234] When the ASIC 5000 first enters the measurement mode, an insertion detection sequence is performed to verify that the sensor 110 has been properly installed on the patient's body before a proper measurement can be taken. First, the sensor 110 interprets a command to activate the measurement configuration process, causing the ASIC 5000 to enter a measurement command mode. The sensor 110 then temporarily enters a measurement life cycle state to run a number of consecutive measurements to test whether the insertion has been successful. The communication module 5040 or the ASIC 5000 evaluates the measurement results to determine whether the insertion was successful. When the insertion is deemed successful, the sensor 110 enters a measurement state in which the sensor 110 begins taking periodic measurements using the sensing hardware 5060. If the sensor 110 determines that the insertion was not successful, then the sensor 110 is triggered to enter an insertion failure mode in which the ASIC 5000 is commanded back to the storage mode while the communication module 5040 disables itself.

[0235] M. Exemplary Wireless Updates

[0236] FIG. 1B An example operating environment for providing wireless ("OTA") updates for use with the techniques described herein is further illustrated. An operator of the analyte monitoring system 100 can bundle updates for the data receiving device 129 or the sensor 110 into updates for an application executing on the multi-purpose data receiving device 130. Using available communication channels between the data receiving device 129, the multi-purpose data receiving device 130, and the sensor 110, the multi-purpose data receiving device 130 can receive regular updates for the data receiving device 129 or the sensor 110 and initiate installation of the updates on the data receiving device 129 or the sensor 110. The multi-purpose data receiving device 130 acts as an installation or update platform for the data receiving device 129 or the sensor 110 because an application that enables the multi-purpose data receiving device 130 to communicate with the analyte sensor 110, the data receiving device 129, and / or the remote application server 1150 can update software or firmware on the data receiving device 129 or the sensor 110 without wide area network capabilities.

[0237] As embodied herein, the remote application server 1150, operated by the manufacturer of the analyte sensor 110 and / or the operator of the analyte monitoring system 100, can provide software and firmware updates to the devices of the analyte monitoring system 100. In particular embodiments, the remote application server 1150 can provide updated software and firmware to the user device 140 or directly to the multi-purpose data receiving device. As embodied herein, the remote application server 1150 can also provide application software updates to the application storefront server 160 using an interface provided by the application storefront. The multi-purpose data receiving device 130 can periodically contact the application storefront server 160 to download and install updates.

[0238] After the multi-purpose data receiving device 130 downloads an application update including a firmware or software update for the data receiving device 129 or sensor 110, the data receiving device 129 or sensor 110 establishes a connection with the multi-purpose data receiving device 130. The multi-purpose data receiving device 130 determines that the firmware or software update is available for the data receiving device 129 or sensor 110. The multi-purpose data receiving device 130 can prepare the software or firmware update for delivery to the data receiving device 129 or sensor 110. As an example, the multi-purpose data receiving device 130 can compress or segment the data associated with the software or firmware update, can encrypt or decrypt the firmware or software update, or can perform an integrity check of the firmware or software update. The multi-purpose data receiving device 130 sends the data for the firmware or software update to the data receiving device 129 or sensor 110. The multi-purpose data receiving device 130 can also send a command to the data receiving device 129 or sensor 110 to initiate the update. Additionally or alternatively, the multi-purpose data receiving device 130 can provide a notification to the user of the multi-purpose data receiving device 130 and include instructions to facilitate the update, such as instructions to keep the data receiving device 129 and multi-purpose data receiving device 130 connected to a power source and in close proximity until the update is complete.

[0239] The data receiving device 129 or sensor 110 receives the data for the update and the command to initiate the update from the multi-purpose data receiving device 130. The data receiving device 129 can then install the firmware or software update. To install the update, the data receiving device 129 or sensor 110 can place itself or reboot into a so-called “safe” mode with limited operational capabilities. Once the update is complete, the data receiving device 129 or sensor 110 re-enters or resets to a standard operational mode. The data receiving device 129 or sensor 110 can perform one or more self-tests to determine that the firmware or software update has been successfully installed. The multi-purpose data receiving device 130 can receive a notification of the successful update. The multi-purpose data receiving device 130 can then report the confirmation of the successful update to the remote application server 1150.

[0240] In particular embodiments, the storage memory 2230 of the sensor 110 comprises one-time programmable (OTP) memory. The term OTP memory can refer to memory that includes access restrictions and security to facilitate writing to a particular address or segment in the memory a predetermined number of times. The storage memory 2230 can be pre-arranged into a plurality of pre-allocated memory blocks or containers. The containers are pre-allocated as fixed sizes. If the storage memory 2230 is one-time programming memory, the containers can be considered in a non-programmable state. Additional containers that have not yet been written to can be placed in a programmable or writable state. Containerizing the storage memory 2230 in this way can improve the transportability of code and data to be written to the storage memory 2230. Updating software of a device (e.g., a sensor device described herein) stored in OTP memory can be performed by replacing code in a particular container previously written to with updated code written to a new container or multiple new containers, rather than replacing the entire code in the memory. In a second embodiment, the memory is not pre-arranged. Instead, the space allocated for data is dynamically allocated or determined as needed. Incremental updates can be issued as containers of different sizes can be defined where updates are expected.

[0241] FIG. 16 is a diagram illustrating example operations and data flow for wireless (OTA) programming of the storage memory 2230 in the sensor 110 and use of the memory after OTA programming in the execution of the sensor device 110 in accordance with the disclosed subject matter. In FIG. 5 In the example OTA programming 500 shown in FIG. 11, a request is sent from an external device (e.g., the multi-purpose data receiving device 130) to initiate OTA programming (or reprogramming). At 511, the communication module 5040 of the sensor device 110 receives the OTA programming command. The communication module 5040 sends the OTA programming command to the microcontroller 2210 of the sensor device 110.

[0242] At 531, after receiving the OTA programming command, the microcontroller 2210 validates the OTA programming command. The microcontroller 2210 can determine, for example, whether the OTA programming command is signed with an appropriate digital signature token. Upon determining that the OTA programming command is valid, the microcontroller 2210 can set the sensor device to an OTA programming mode. At 532, the microcontroller 2210 can validate the OTA programming data. At 533, the microcontroller 2210 can reset the sensor device 110 to reinitialize the sensor device 110 to a programming state. Once the sensor device 110 has transitioned to the OTA programming state, the microcontroller 2210 can begin writing data to the re-writable memory 540 (e.g., memory 5020) of the sensor device and to the OTP memory 550 (e.g., memory 2230) of the sensor device at 534 and 535, respectively. The data written by the microcontroller 2210 can be based on the validated OTA programming data. The microcontroller 2210 can write data such that one or more programming blocks or regions of the OTP memory 550 are marked as invalid or inaccessible. Data written to the free or unused portions of the OTP memory can be used to replace the invalid or inaccessible programming blocks of the OTP memory 550. After the microcontroller 2210 writes data to the respective memories at 534 and 535, the microcontroller 2210 can perform one or more software integrity checks to ensure that no errors were introduced to the programming blocks during the writing process. Once the microcontroller 2210 is able to determine that the data has been written without error, the microcontroller 2210 can resume standard operation of the sensor device.

[0243] In the execution mode, at 536, the microcontroller 2210 can retrieve a programming manifest or profile from the re-writable memory 540. The programming manifest or profile can include a list of valid software programming blocks and can include guidelines for program execution of the sensor 110. By following the programming manifest or profile, the microcontroller 2210 can determine which memory blocks of the OTP memory 550 are suitable for execution and avoid executing outdated or invalid programming blocks or references to outdated data. At 537, the microcontroller 2210 can selectively retrieve memory blocks from the OTP memory 550. At 538, the microcontroller 2210 can use the retrieved memory blocks by executing the stored program code or using variables stored in memory.

[0244] N. Exemplary Security and Other Architectural Features

[0245] As embodied herein, the first layer of security for communications between the analyte sensor 110 and other devices can be established based on security protocols specified by and integrated in the communication protocols used for the communications. Another layer of security can be based on the communication protocol requiring the communication devices to be in close proximity. Further, certain packets and / or certain data included in packets can be encrypted while other packets and / or data in packets are encrypted or not encrypted. Additionally or alternatively, application layer encryption can be used with one or more block ciphers or stream ciphers to establish mutual authentication and communication encryption with other devices in the analyte monitoring system 100.

[0246] The ASIC 5000 of the analyte sensor 110 can be configured to dynamically generate authentication and encryption keys using data held within the storage memory 2230. The storage memory 2230 can also be preprogrammed with a set of valid authentication and encryption keys for use with a particular class of devices. The ASIC 5000 can also be configured to perform an authentication process with other devices using received data and apply the generated keys to sensitive data prior to transmission of the sensitive data. The generated keys can be unique to the analyte sensor 110, unique to a pair of devices, unique to a communication session between the analyte sensor 110 and other devices, unique to a message sent during a communication session, or unique to a block of data contained in a message.

[0247] Both the sensor 110 and the data receiving device 129 can ensure authorization of the other party in a communication session, e.g., to issue commands or receive data. In particular embodiments, identity authentication can be performed through two features. First, the party asserting its identity provides a valid certificate signed by the manufacturer of the device or the operator of the analyte monitoring system 100. Second, authentication can be enforced through the use of public and private keys established by the devices of the analyte monitoring system 100 or established by the operator of the analyte monitoring system 100 and a shared secret derived therefrom. To confirm the identity of the other party, the party can provide proof that the party has control of its private key.

[0248] The manufacturer of the analyte sensor 110, the data receiving device 129, or the provider of the application of the multi-purpose data receiving device 130 can provide the information and programming needed for the devices to securely communicate through secure programming and updates. For example, the manufacturer can provide information that can be used to generate encryption keys for each device, including a protected root key for the analyte sensor 110 and, optionally, the data receiving device 129, which can be used in conjunction with device-specific information and operational data (e.g., entropy-based random values) to generate encryption values specific to the device, session, or data transmission as needed.

[0249] The analyte data associated with a user is at least partially sensitive data because this information can be used for a variety of purposes, including health monitoring and drug dosage decisions. In addition to user data, the analyte monitoring system 100 can enforce security to prevent reverse engineering by outside parties. The communication connection can be encrypted using a device-unique or session-unique encryption key. The encrypted or unencrypted communication between any two devices can be verified with a built-in transmission integrity check in the communication. The operation of the analyte sensor 110 can be protected from tampering by limiting access to the read and write functions of the memory 5020 through the communication interface. The sensor can be configured to grant access only to known or "trusted" devices, which are provided in a "whitelist," or to devices that can provide a predetermined code associated with the manufacturer or other certified user. The whitelist can represent an exclusive range, i.e., no connection identifiers other than those included in the whitelist are used, or a preferential range, in which the whitelist is searched first, but other devices can still be used. The sensor 110 can also reject and shut down connection requests if the requester cannot complete the login process through the communication interface within a predetermined time period, e.g., within four seconds. These features prevent certain denial-of-service attacks, particularly denial-of-service attacks against the BLE interface.

[0250] As embodied herein, the analyte monitoring system 100 can employ periodic key rotation to further reduce the likelihood of key compromise and exploitation. The key rotation strategy employed by the analyte monitoring system 100 can be designed to support backward compatibility for field-deployed or distributed devices. As an example, the analyte monitoring system 100 can employ keys that are designed to be compatible with multiple generations of keys used by upstream devices (e.g., field devices or devices that cannot practically be provided with updates).

[0251] For the purpose of illustration and not limitation, with reference to the example embodiment of the message sequence chart 600, the analyte monitoring system 100 can be configured to employ a key rotation strategy that is designed to be compatible with multiple generations of keys used by upstream devices (e.g., field devices or devices that cannot practically be provided with updates). FIG. 17The disclosed subject matter shown in the middle is used together and demonstrates an example data exchange between a pair of devices, specifically sensor 110 and data receiving device 129. Data receiving device 129 can be a data receiving device 129 or a multi-purpose data receiving device 130 as embodied herein. At step 605, data receiving device 129 can transmit a sensor activation command 605 to sensor 110, for example via a short range communication protocol. Prior to step 605, sensor 110 can be in a primarily dormant state, conserving its battery until full activation is required. After activation during step 610, sensor 110 can collect data or perform other operations appropriate to the sensing hardware 5060 of sensor 110. At step 615, data receiving device 129 can initiate an authentication request command 615. In response to authentication request command 615, both sensor 110 and data receiving device 129 can engage in a mutual authentication process 620. Mutual authentication process 620 can involve the transfer of data, including challenge parameters that allow sensor 110 and data receiving device 129 to ensure that the other device is sufficiently able to adhere to the agreed upon security framework described herein. Mutual authentication can be based on a mechanism by which two or more entities authenticate each other, with or without an online trusted third party to verify the establishment of a challenge-response authentication key. Mutual authentication can be performed using two-pass, three-pass, four-pass, or five-pass authentication or similar versions thereof.

[0252] After a successful mutual authentication process 620, at step 625, sensor 110 can provide a sensor secret 625 to data receiving device 129. The sensor secret can contain a value unique to the sensor and derived from a random value generated during manufacturing. The sensor secret can be encrypted prior to or during transmission to prevent third parties from accessing the secret. The sensor secret 625 can be encrypted by one or more keys generated by or in response to mutual authentication process 620. At step 630, data receiving device 129 can derive a sensor-specific encryption key from the sensor secret. The sensor-specific encryption key can also be session-specific. Thus, the sensor-specific encryption key can be determined by each device without the need for transmission between sensor 110 or data receiving device 129. At step 635, sensor 110 can encrypt data for inclusion in a payload. At step 640, sensor 110 can transmit the encrypted payload 640 to data receiving device 129 using a communication link established between the appropriate communication models of sensor 110 and data receiving device 129. At step 645, data receiving device 129 can decrypt the payload using the sensor-specific encryption key derived during step 630. After step 645, sensor 110 can deliver additional (including newly collected) data and data receiving device 129 can process the received data as appropriate.

[0253] As discussed herein, the sensor 110 can be a device with limited processing capabilities, battery supply, and storage. The encryption techniques used by the sensor 110 (e.g., selection of a cryptographic algorithm or implementation of an algorithm) can be selected based at least in part on these limitations. The data receiving device 129 can be a more powerful device with fewer such limitations. Accordingly, the data receiving device 129 can employ more complex, computationally intensive encryption techniques, such as cryptographic algorithms and implementations.

[0254] O. Exemplary Payload / Communication Frequency

[0255] The analyte sensor 110 can be configured to alter its discoverability behavior to attempt to increase the probability that a receiving device receives an appropriate data packet and / or to provide a confirmation signal or otherwise reduce limitations that can result in an inability to receive a confirmation signal. Altering the discoverability behavior of the analyte sensor 110 can include, for example and without limitation, altering the frequency with which connection data is included in a data packet, altering the frequency with which data packets are generally transmitted, lengthening or shortening a broadcast window for data packets, altering the amount of time the analyte sensor 110 listens for a confirmation or scan signal after a broadcast, including a directional transmission to one or more devices that have previously communicated with the analyte sensor 110 (e.g., through one or more attempted transmissions) and / or one or more devices on a white list, altering a transmission power associated with the communication module when broadcasting a data packet (e.g., increasing the range of the broadcast or reducing the energy expended and lengthening the life of the analyte sensor's battery), altering the rate at which data packets are prepared and broadcast, or a combination of one or more other alterations. Additionally or alternatively, a receiving device can similarly adjust parameters related to the listening behavior of the device to increase the likelihood of receiving a data packet including connection data.

[0256] As implemented herein, the analyte sensor 110 can be configured to broadcast data packets using two types of windows. The first window refers to the rate at which the analyte sensor 110 is configured to operate the communication hardware. The second window refers to the rate at which the analyte sensor 110 is configured to actively transmit data packets (e.g., broadcast). As an example, the first window can indicate that the analyte sensor 110 operates the communication hardware to send and / or receive data packets (including connection data) during the first 2 seconds of every 60 second period. The second window can indicate that, during each 2 second window, the analyte sensor 110 transmits one data packet every 60 milliseconds. For the remainder of the 2 second window, the analyte sensor 110 is scanning. The analyte sensor 110 can lengthen or shorten either window to modify the discoverability behavior of the analyte sensor 110.

[0257] In particular embodiments, the discoverability behavior of an analyte sensor can be stored in a discoverability profile and can be altered based on one or more factors, such as the status of the analyte sensor 110 and / or by applying rules based on the status of the analyte sensor 110. For example, when the battery level of the analyte sensor 110 is below a certain amount, a rule can 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 can be adjusted based on the ambient temperature, the temperature of the analyte sensor 110, or the temperature of certain components of the communication hardware of the analyte sensor 110. In addition to modifying transmission power, other parameters associated with the transmission capabilities or processes of the communication hardware of the analyte sensor 110 can be modified, including but not limited to transmission rate, frequency, and timing. As another example, when the analyte data indicates that the subject is experiencing or is about to experience a negative health event, a rule can cause the analyte sensor 110 to increase its discoverability to alert receiving devices of the negative health event.

[0258] P. Exemplary sensor sensitivity initialization / adjustment features

[0259] As embodied herein, certain calibration features for the sensing hardware 5060 of the analyte sensor 110 can be adjusted based on external or interval environmental features, as well as to compensate for degradation of the sensing hardware 5060 during extended periods of disuse (e.g., a “shelf life” prior to use). Calibration features of the sensing hardware 5060 can be adjusted autonomously by the sensor 110 (e.g., by operation of the ASIC 5000 to modify memory 5020 or store features in memory 2230) or can be adjusted by other devices of the analyte monitoring system 100.

[0260] As an example, the sensor sensitivity of the sensing hardware 5060 can be adjusted based on external temperature data or time since manufacture. When monitoring the external temperature during storage of the sensor, the disclosed subject matter can adaptively change compensation for sensor sensitivity over time as the device experiences changing storage conditions. For purposes of illustration and not limitation, the adaptive sensitivity adjustment can be performed in an "active" storage mode in which the analyte sensor 110 periodically wakes up to measure temperature. These features can conserve the battery of the analyte device and extend the life of the analyte sensor. At each temperature measurement, the analyte sensor 110 can calculate a sensitivity adjustment for that time period based on the measured temperature. The temperature-weighted adjustments can then be accumulated during the active storage mode to calculate a total sensor sensitivity adjustment value at the end of the active storage mode (e.g., at insertion). Similarly, at insertion, the sensor 110 can determine the time difference between manufacture of the sensor 110 (which can be written to the storage memory 2230 of the ASIC 5000) or the sensing hardware 5060 and modify the sensor sensitivity or other calibration features according to one or more known decay rates or formulas.

[0261] Further, for purposes of illustration and not limitation, as embodied herein, the sensor sensitivity adjustment can take into account other sensor conditions, such as sensor drift. The sensor sensitivity adjustment can be hard-coded into the sensor 110 during manufacture, for example, based on an estimate of how much an average sensor will drift in the case of sensor drift. The sensor 110 can use a calibration function with a time-varying function of sensor offset and gain, which can account for drift during sensor wear. Thus, the sensor 110 can utilize a device-dependent function that describes the drift of the sensor 110 over time to transform the interstitial current into a function of interstitial glucose, which can represent the sensor sensitivity, and can be specific to the device, in combination with a baseline of the glucose profile. Such a function that takes into account sensor sensitivity and drift can improve the accuracy of the sensor 110 during wear, and without the need for user calibration.

[0262] Q. Example model-based analyte measurement

[0263] The sensor 110 detects raw measurements from the sensing hardware 5060. On-sensor processing can be performed, such as by one or more models trained to interpret the raw measurements. The models can be machine-learned models trained off-device to detect, predict, or interpret the raw measurements to detect, predict, or interpret the level of one or more analytes. Other trained models can operate on the output of the machine-learned models trained to interact with the raw measurements. As an example, a model can 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 can include the start or completion of a physical activity, a meal, the application of a medical or pharmaceutical, an urgent health event, and other events of similar nature.

[0264] The models can be provided to the sensor 110, the data receiving device 129, or the multi-purpose data receiving device 130 during manufacturing or during a firmware or software update. The models can be periodically refined based on data received from the sensor 110 and the data receiving devices of individual users or multiple users, such as by the manufacturer of the sensor 110 or the operator of the analyte monitoring system 100. In certain embodiments, the sensor 110 includes sufficient computing components to assist in further training or refining the machine-learned models, such as based on the unique characteristics of the user to which the sensor 110 is attached. The machine-learned models can include, for example and without limitation, models trained using or encompassing decision tree analysis, gradient boosting, ada boosting, artificial neural networks or variants thereof, linear discriminant analysis, nearest neighbor analysis, support vector machines, supervised or unsupervised classification, etc. In addition to the machine-learned models, the models can also include algorithm- or rule-based models. Once data is received from the sensor 110 (or other downstream devices), the model-based processing can be performed by other devices, including the data receiving device 129 or the multi-purpose data receiving device 130.

[0265] R. Exemplary Alert Features

[0266] Data transmitted between the sensor 110 and the data receiving device 129 can include raw or processed measurement values. Data transmitted between the sensor 110 and the data receiving device 129 can also include alerts or notifications for display to a user. The data receiving device 129 can display or otherwise communicate notifications to a user based on raw or processed measurement values, or can display alerts upon receipt from the sensor 110. Alerts that can be triggered for display to a user include alerts based on direct analyte values (e.g., a one-time reading exceeding or failing to meet a threshold value), analyte value trends (e.g., an average reading over a period of time exceeding or failing to meet a threshold value; a slope), analyte value predictions (e.g., an algorithmic calculation based on analyte values exceeding or failing to meet a threshold value), sensor alerts (e.g., detection of a suspected malfunction), communication alerts (e.g., no communication between the sensor 110 and the data receiving device 129 for a threshold period of time; an unknown device attempting or failing to initiate a communication session with the sensor 110), reminders (e.g., a reminder to charge the data receiving device 129; a reminder to take medication or perform other activities), and other alerts of similar nature. For purposes of illustration and not limitation, as embodied herein, alert parameters described herein can be user-configurable or can be fixed during manufacture, or a combination of user-settable and non-user-settable parameters.

[0267] S. Exemplary electrode configurations

[0268] Sensor configurations having a single active area configured for detection of a corresponding single analyte can employ a two-electrode or three-electrode detection motif, as described further herein with reference to FIG. 18A-18C Sensor configurations having two different active areas for detection of separate analytes, either on separate working electrodes or on the same working electrode, are described separately below with reference to FIG. 19A-21C Sensor configurations having multiple working electrodes can be particularly advantageous for incorporating two different active areas within the same sensor tail, as the signal contribution from each active area can be more readily determined.

[0269] When a single working electrode is present in an analyte sensor, a three-electrode sensor configuration can include a working electrode, a counter electrode, and a reference electrode. A related two-electrode sensor configuration can include a working electrode and a second electrode, where the second electrode can function as both a counter electrode and a reference electrode (i.e., counter / reference electrode). The various electrodes can be at least partially stacked (layered) with one another and / or laterally spaced apart from one another on a sensor tail. Suitable sensor configurations can be substantially planar in shape or substantially cylindrical in shape, or any other suitable shape. In any of the sensor configurations disclosed herein, the various electrodes can be electrically isolated from one another by a dielectric material or similar insulator.

[0270] An analyte sensor having multiple working electrodes can similarly include at least one additional electrode. When there is one additional electrode, the one additional electrode can function as a counter electrode / reference electrode for each of the multiple working electrodes. When there are two additional electrodes, one of the additional electrodes can function as a counter electrode for each of the multiple working electrodes, and the other of the additional electrodes can function as a reference electrode for each of the multiple working electrodes.

[0271] FIG. 18A A diagram illustrating an illustrative two-electrode analyte sensor configuration is shown, which is suitable for the disclosure herein. As shown, analyte sensor 200 includes a substrate 30212 disposed between working electrode 214 and counter / reference electrode 30216. Alternatively, working electrode 214 and counter / reference electrode 30216 can be located on the same side of substrate 30212 with a dielectric material interposed therebetween (configuration not shown). Active area 218 is disposed as at least one layer on at least a portion of working electrode 214. As discussed further herein, active area 218 can include multiple spots or a single spot configured for detecting an analyte.

[0272] Still referring to FIG. 18A , film 220 at least coats active area 218. In certain embodiments, film 220 can also coat some or all of working electrode 214 and / or counter / reference electrode 30216, or the entire analyte sensor 200. One or both faces of analyte sensor 200 can be coated with film 220. Film 220 can include one or more polymeric film materials having the ability to limit the flow of analyte to active area 218 (i.e., film 220 is a mass transport limiting film that is permeable to the analyte of interest to some extent). In accordance with the disclosure herein, film 220 can be crosslinked with a branched crosslinker in certain particular sensor configurations. The composition and thickness of film 220 can vary to facilitate the desired flow of analyte to active area 218, providing the desired signal strength and stability. Analyte sensor 200 can be operable for determining an analyte by any of coulometric, amperometric, voltammetric, or potentiometric electrochemical detection techniques.

[0273] FIG. 18B and 18C A diagram illustrating an illustrative three-electrode analyte sensor configuration is shown, which is also suitable for the disclosure herein. The three-electrode analyte sensor configuration can be similar to the two-electrode analyte sensor configuration described above, except for the inclusion of additional electrode 217 in analyte sensors 201 and 202 FIG. 18B and 18C ) above. FIG. 18AThe configuration shown for the analyte sensor 200 is illustrated below. With the additional electrode 217, the anti / reference electrode 30216 can then be used as either an anti electrode or a reference electrode, and the additional electrode 217 performs other electrode functions not otherwise described. The working electrode 214 continues to perform its original function. The additional electrode 217 can be deployed on the working electrode 214 or electrode 30216, with a dielectric material separating the two. For example, but not as a limitation, such as... FIG. 18B As depicted, dielectric layers 219a, 219b, and 219c separate electrodes 214, 30216, and 217 from each other and provide electrical isolation. Alternatively, at least one of electrodes 214, 30216, and 217 may be located on opposite surfaces of substrate 30212, as shown. FIG. 18C As shown in the diagram. Therefore, in some embodiments, electrode 214 (working electrode) and electrode 30216 (counter electrode) may be located on opposite surfaces of substrate 30212, and electrode 217 (reference electrode) may be located on one of electrodes 214 or 30216 and spaced apart from it by a dielectric material. A reference material layer 30230 (e.g., Ag / AgCl) may be present on electrode 217, and the location of the reference material layer 30230 is not limited to... FIG. 18B and 18C The location depicted in the text. (And) FIG. 18A Similar to sensor 200 shown, the active region 218 in analyte sensors 201 and 202 may include multiple points or a single point. Furthermore, analyte sensors 201 and 202 are operable for determining analytes using any of coulometric, amperometric, voltammetric, or potentiochemical detection techniques.

[0274] Similar to analyte sensor 200, membrane 220 can also cover the active regions 218 and other sensor components in analyte sensors 201 and 202, thereby serving as a mass transport limiting membrane. In some embodiments, additional electrode 217 can be covered by membrane 220. Although FIG. 18B and 18C Electrodes 214, 30216, and 217 are depicted as being covered by membrane 220, but it should be recognized that in some embodiments only the working electrode 214 is covered. Furthermore, the thickness of the membrane 220 at each electrode 214, 30216, and 217 may be the same or different. (Configuration with a dual-electrode analyte sensor) FIG. 18A Like ) in FIG. 18B and 18C In the sensor configuration, one or two surfaces of the analyte sensors 201 and 202 can be covered by the membrane 220, or the entire analyte sensors 201 and 202 can be covered. Therefore, FIG. 18B and 18C The three-electrode sensor configuration shown should be understood as not limiting the embodiments disclosed herein, and alternative electrode and / or layer configurations are still within the scope of this disclosure.

[0275] FIG. 19A An illustrative configuration of sensor 203 is shown, which has a single working electrode with two distinct active regions deployed thereon. In addition to the presence of 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 from each other on the surface of the working electrode 214), FIG. 19A Similar to FIG. 19A Active regions 218a and 218b may include multiple points or a single point configured to detect each analyte. The composition of membrane 220 may vary at 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 some embodiments, either or both of active regions 218a and 218b may be configured to detect analytes using an NAD(P)-dependent enzyme. In some embodiments, either or both of active regions 218a and 218b may be configured to detect analytes using an NAD(P)-dependent enzyme, for example, by detecting ketones using an enzyme system comprising NADH oxidase and β-hydroxybutyrate dehydrogenase. In some embodiments, only one active region of 218a and 218b is configured to detect analytes using an NAD(P)-dependent enzyme. In some embodiments, the other active regions are configured not to detect a second analyte using an NAD(P)-dependent enzyme.

[0276] FIG. 19B and 19C Cross-sectional views of illustrative three-electrode sensor configurations of sensors 204 and 205 are shown, each sensor having a single working electrode with a first active region 218a and a second active region 218b deployed thereon. FIG. 19B and 19C In other aspects and FIG. 18B and 18C Similar, and can be better understood through reference. With FIG. 19A Similarly, the composition of membrane 220 may vary or be the same in active regions 218a and 218b.

[0277] refer to FIG. 20-21CAn illustrative sensor configuration having multiple working electrodes, particularly two working electrodes, is described in greater detail. While the following description is primarily directed to a sensor configuration having two working electrodes, it should be recognized that more than two working electrodes can be incorporated by extending the disclosure herein. In addition to a first analyte and a second analyte, additional working electrodes can be used to impart additional sensing capabilities to the analyte sensor, e.g., for detecting a third and / or fourth analyte.

[0278] FIG. 20 A cross-sectional view of an illustrative analyte sensor configuration having two working electrodes, a reference electrode, and a counter electrode is shown, which is suitable for the disclosure herein. As shown, the analyte sensor 300 includes working electrodes 304 and 306 disposed on opposite faces of a 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. A counter electrode 320 is electrically isolated from working electrode 304 by a dielectric layer 322, and a reference electrode 321 is electrically isolated from working electrode 306 by a dielectric layer 323. Outer dielectric layers 330 and 332 are positioned over the reference electrode 321 and counter electrode 320, respectively. According to various embodiments, a membrane 340 can at least coat the active areas 310a and 310b, and other components of the analyte sensor 300 or the entire analyte sensor 300 are optionally also coated with a first membrane portion 340a and / or a second membrane portion 340b. Again, the membrane 340 can be continuous but differentiated within the first membrane portion 340a and the second membrane portion 340b (i.e., over the active areas 310a and 310b) so as to provide different permeability values to differentially regulate analyte flux at each location. For example, different membrane formulations can be sprayed and / or printed onto the opposite faces of the analyte sensor 300. Dip coating techniques can also be suitable, particularly for depositing at least a portion of a bilayer membrane over one of the active areas 310a and 310b. Thus, according to particular embodiments of the disclosure, one of the first membrane portion 340a and the second membrane portion 340b can include a bilayer membrane and the other of the first membrane portion 340a and the second membrane portion 340b can include a single membrane polymer. As with the analyte sensors 200, 201, and 202, the analyte sensor 300 can be operable for electrochemical detection of ketones (and / or a second analyte) by any of coulometric, amperometric, voltammetric, or potentiometric analysis. In certain embodiments, the analyte sensor can include more than one membrane 340, e.g., two or more membranes. For example, but not by way of limitation, the analyte sensor can include a membrane coating one or more active areas (e.g., 310a and 310a), and an additional membrane coating the entire sensor, as FIG. 20In certain embodiments, either or both of active areas 310a and 310b can be configured to detect an analyte using NAD(P)-dependent enzymes, e.g., by detecting ketones using an enzyme system including NADH oxidase and beta-hydroxybutyrate dehydrogenase or beta-hydroxybutyrate dehydrogenase and diaphorase. In certain embodiments, only one of 310a and 310b is configured to detect an analyte using NAD(P)-dependent enzymes, e.g., by detecting ketones using an enzyme system including NADH oxidase and beta-hydroxybutyrate dehydrogenase or beta-hydroxybutyrate dehydrogenase and diaphorase. In certain embodiments, the other active area is configured to detect a second analyte, e.g., that does not use NAD(P)-dependent enzymes for detection.

[0279] An alternative sensor configuration having multiple working electrodes and configured differently than that shown in FIG. 20 An alternative sensor configuration having multiple working electrodes and configured differently than that shown in FIG. 20 may be characterized by a counter / reference electrode rather than separate counter and reference electrodes 320, 321, and / or by different film arrangements than those explicitly depicted. For example, but not by way of limitation, the positioning of counter electrode 320 and reference electrode 321 can be reversed from that depicted in FIG. 20 In addition, working electrodes 304 and 306 need not reside on opposite faces of substrate 302 in the manner shown in

[0280] While a suitable sensor configuration can feature substantially planar electrodes, it should be appreciated that sensor configurations featuring non-planar electrodes can be advantageous and particularly suitable for the disclosure herein. In particular, substantially cylindrical electrodes deployed concentrically with respect to one another can facilitate deposition of mass transport limiting membranes, as described below. In particular, concentric working electrodes spaced apart along the length of a sensor tail can facilitate membrane deposition by successive dip coating operations in a manner similar to that described above for substantially planar sensor configurations. FIG. 21A-21C A perspective view of an analyte sensor is shown having two working electrodes deployed concentrically with respect to one another. It should be appreciated that sensor configurations having concentric electrode deployment but lacking a second working electrode are also possible in the present disclosure.

[0281] FIG. 21AA perspective view showing an illustrative sensor configuration in which the plurality of electrodes are substantially cylindrical and disposed concentrically with respect to one another about a central substrate is shown. As shown, the analyte sensor 400 includes a central substrate 402 about which all of the electrodes and dielectric layers are disposed concentrically with respect to one another. In particular, a working electrode 410 is disposed on a surface of the central substrate 402, and a dielectric layer 412 is disposed on a portion of the working electrode 410 distal from the sensor tip 404. A working electrode 420 is disposed on the dielectric layer 412, and a dielectric layer 422 is disposed on a portion of the working electrode 420 distal from the sensor tip 404. A counter electrode 430 is disposed on the dielectric layer 422, and a dielectric layer 432 is disposed on a portion of the counter electrode 430 distal from the sensor tip 404. A reference electrode 440 is disposed on the dielectric layer 432, and a dielectric layer 442 is disposed on a portion of the reference electrode 440 distal from the sensor tip 404. Thus, the exposed surfaces of the working electrode 410, the working electrode 420, the counter electrode 430, and the reference electrode 440 are spaced apart from one another along the longitudinal axis B of the analyte sensor 400.

[0282] Still referring to FIG. 21A , first and second active regions 414a and 414b responsive to different analytes or the same analyte are disposed on the exposed surfaces of the working electrodes 410 and 420, respectively, allowing contact with a fluid for sensing. In certain embodiments, either or both of the active regions 414a and 414b can be configured to detect an analyte using an NAD(P)-dependent enzyme. In certain embodiments, either or both of the active regions 414a and 414b can be configured to detect a ketone, for example, by using an enzyme system including NADH oxidase and beta-hydroxybutyrate dehydrogenase. In certain embodiments, only one of the active regions 414a and 414b is configured to detect a ketone, for example, by using an enzyme system including NADH oxidase and beta-hydroxybutyrate dehydrogenase. In certain embodiments, the other active region is configured to detect a second analyte. In certain embodiments, either or both of the active regions 414a and 414b can be configured to detect an analyte using an NAD(P)-dependent enzyme, for example, by using an enzyme system including NADH oxidase and beta-hydroxybutyrate dehydrogenase or beta-hydroxybutyrate dehydrogenase and diaphorase to detect a ketone. In certain embodiments, only one of the active regions 414a and 414b is configured to detect an analyte using an NAD(P)-dependent enzyme, for example, by using an enzyme system including NADH oxidase and beta-hydroxybutyrate dehydrogenase or beta-hydroxybutyrate dehydrogenase and diaphorase to detect a ketone. In certain embodiments, the other active region is configured to detect a second analyte, for example, without using an NAD(P)-dependent enzyme. Although the active regions 414a and 414b are shown as being disposed on the working electrodes 410 and 420, respectively, in other embodiments, the active regions 414a and 414b can be disposed on other electrodes, such as the counter electrode 430 or the reference electrode 440. FIG. 21AThe active areas are depicted as three discrete points, but it should be recognized that there can be fewer or more than three points in alternative sensor configurations, including a continuous layer of active area.

[0283] In FIG. 21A the sensor 400 is partially coated with a film 450 over the working electrodes 410 and 420 and the active areas 414a and 414b disposed thereon. FIG. 21B An alternative sensor configuration is shown in which the sensor 401 is entirely coated with a film 450. The composition of the film 450 at the active areas 414a and 414b can be the same or different. For example, the film 450 can include a bilayer coating active area 414a and be a homogenous film coating active area 414b. In certain embodiments, one or more films are deposited on the exposed electroactive surface of the working electrode, such as a platinum surface, including an interference domain and a mass transport limiting film. For example, but not by way of limitation, an interference domain can be disposed on the working electrode, an active area can be disposed on the interference domain, and a mass transport limiting film can be disposed on the active area.

[0284] It should also be recognized that, FIG. 21A and 21B the positioning of the various electrodes can be different than explicitly depicted. For example, the positions of the counter electrode 430 and the reference electrode 440 can be reversed from the configuration depicted in FIG. 21A and 21B . Similarly, the positions of the working electrodes 410 and 420 are not limited to the positions explicitly depicted in FIG. 21A and 21B . FIG. 21C An alternative sensor configuration is shown in which the configuration shown in FIG. 21B is shown with the sensor 405 containing the counter electrode 430 and the reference electrode 440 located closer to the sensor tip 404 and the working electrodes 410 and 420 located further from the sensor tip 404. The sensor configuration with the working electrodes 410 and 420 located further from the sensor tip 404 can be advantageous by providing a greater surface area for deposition of the active areas 414a and 414b (the five discrete sensing points exemplarily shown in FIG. 21C ) thereby facilitating increased signal strength in some cases. Similarly, the central substrate 402 can be omitted in any of the concentric sensor configurations disclosed herein, with the innermost electrode instead supporting the layers subsequently deposited.

[0285] In certain embodiments, one or more electrodes of the analyte sensors described herein are wire electrodes, such as permeable wire electrodes. In certain embodiments, the sensor tail includes a working electrode and a reference electrode that is helically wrapped around the working electrode. In certain embodiments, an insulator is disposed between the working electrode and the reference electrode. In certain embodiments, portions of the electrodes are exposed to allow one or more enzymes to react with an analyte on the electrodes. In certain embodiments, each electrode is formed from a thin wire having a diameter of about 0.001 inch or less to about 0.010 inch or more. In certain embodiments, the working electrode has a diameter of about 0.001 inch or less to about 0.010 inch or more, such as about 0.002 inch to about 0.008 inch, more preferably about 0.004 inch to about 0.005 inch. In certain embodiments, the electrodes are formed from a plated insulator, a plated wire, or a bulk conductive material. In certain embodiments, the working electrode includes a wire formed from a conductive material, such as platinum, platinum-iridium, palladium, graphite, gold, carbon, conductive polymers, alloys, and the like. In certain embodiments, the conductive material is a permeable conductive material. In certain embodiments, the electrodes can be formed by a variety of manufacturing techniques (e.g., bulk metal machining, metal deposition onto a substrate, etc.), the electrodes can be formed from a plated wire (e.g., platinum over steel wire) or a bulk metal (e.g., platinum wire). In certain embodiments, the electrodes are formed from a tantalum wire that is covered with platinum.

[0286] In certain embodiments, the reference electrode, which can be used alone as a reference electrode 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 wrapped on or around the working electrode. In certain embodiments, the reference electrode is helically wrapped around the working electrode. In certain embodiments, the assembly of wires can be coated with an insulating material or adhered together to provide an insulating attachment.

[0287] In certain embodiments, additional electrodes can be included in the sensor tail. For example, and without limitation, 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 can be juxtaposed with a reference electrode disposed therearound (e.g., helically wrapped around the two or more working electrodes). In certain embodiments, the two or more working electrodes can extend parallel to one another. In certain embodiments, the reference electrode is coiled around the working electrodes and extends toward the distal end (i.e., the in vivo end) of the sensor tail. In certain embodiments, the reference electrode extends (e.g., helically) to the exposed region of the working electrodes.

[0288] In certain embodiments, one or more working electrodes are helically wound around the reference electrode. In certain embodiments that provide two or more working electrodes, the working electrodes can form a double helix, triple helix, quadruple helix, etc. configuration along the length of the sensor tail (e.g., surrounding the reference electrode, an insulating 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.

[0289] In certain embodiments, the working electrode comprises a tube with the reference electrode disposed or coiled inside, including an insulator therebetween. Alternatively, the reference electrode comprises a tube with the working electrode disposed or coiled inside, including an insulator therebetween. In certain embodiments, a polymeric (e.g., insulating) rod is provided, with one or more electrodes (e.g., one or more electrode layers) disposed thereon (e.g., by electroplating). In certain embodiments, a metal (e.g., steel or tantalum) rod or wire is provided, coated with an insulating material (described herein), with one or more working electrodes and a reference electrode disposed thereon. For example, but not by way of limitation, the present disclosure provides a sensor, e.g., a sensor tail, comprising one or more tantalum wires, with platinum disposed on a portion of the one or more tantalum wires to serve as a working electrode. In certain embodiments, the platinum-coated tantalum wire is covered with an insulating material, with the insulating material partially covered with a silver / silver chloride composition to serve as a reference electrode and / or a counter electrode.

[0290] In certain embodiments where an insulator is disposed on the working electrode (e.g., on the platinum surface of the electrode), a portion of the insulator can be stripped or otherwise removed to expose an electroactive surface of the working electrode. For example, but not by way of limitation, a portion of the insulator can be removed by hand, excimer laser, chemical etching, laser ablation, sandblasting, etc. Alternatively, a portion of the electrode can be masked from the insulator prior to deposition of the insulator to maintain an exposed electroactive surface area. In certain embodiments, the length of the portion of the insulator that is stripped and / or removed can be about 0.1 mm (about 0.004 inch) or less to about 2 mm (about 0.078 inch) or more, e.g., from about 0.5 mm (about 0.02 inch) to about 0.75 mm (0.03 inch) in length. In certain embodiments, the insulator is a non-conductive polymer. In certain embodiments, the insulator comprises parylene, a fluorinated polymer, polyethylene terephthalate, polyvinylpyrrolidone, polyurethane, polyimide, and other non-conductive polymers. In certain embodiments, a glass or ceramic material can also be used in the insulating layer. In certain embodiments, the insulator comprises parylene. In certain embodiments, the insulator comprises polyurethane. In certain embodiments, the insulator comprises polyurethane and polyvinylpyrrolidone.

[0291] Several portions of the sensor are further described below.

[0292] 2. NAD(P) reservoir

[0293] The present disclosure provides analyte sensors that can include an internal supply of a co-factor. For example, but not by way of limitation, analyte sensors provided by the present disclosure can include an internal supply of a co-factor that allows for controlled release of the co-factor over an extended period of time.

[0294] In certain embodiments, the co-factor internal supply can be coated with or distributed within a permeable layer that controls diffusion of the co-factor from the co-factor supply to maintain sufficient concentrations of the co-factor in the active area (e.g., sensing chemistry layer) during use of the analyte sensor. The exact nature, size, and configuration of the co-factor reservoir present within the analyte sensor can vary depending 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 the analyte is being detected.

[0295] In certain embodiments, the co-factor is NAD or NADP (both referred to herein collectively as "NAD(P)"). In certain embodiments, the NAD(P) is a derivative of NAD(P). Non-limiting examples of NAD(P) derivatives are disclosed in WO 2007 / 012494 and WO 1998 / 033936, the contents of each of which are incorporated herein in their entirety. In certain embodiments, analyte sensors provided by the present disclosure can include an internal supply of NAD(P) that allows for controlled release of NAD(P) or a derivative thereof over an extended period of time. In certain embodiments, the NAD(P) internal supply can be coated with or distributed within a permeable layer that controls diffusion of the NAD(P) from the NAD(P) supply to maintain sufficient concentrations of NAD(P) in the active area (e.g., sensing chemistry layer) during use of the analyte sensor including one or more NAD(P)-dependent enzymes.

[0296] FIG. 22 and 23A Non-limiting embodiments of analyte sensors including NAD(P) reservoirs are provided in FIG. 22 and 23A In certain embodiments, the NAD(P) reservoir can be disposed on the substrate 30212. In certain embodiments, the NAD(P) reservoir can be deposited between dielectric materials (e.g., two dielectric layers) as shown in FIG. 23A

[0297] In certain embodiments, the deposited NAD(P) can be covered with a permeable layer. As shown in FIG. 22 and 23A ​As shown in FIGS. 1-3, the NAD(P) reservoir is at least partially coated with a permeable layer. For example, and without limitation, 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) reservoir is coated with a permeable layer. In certain embodiments, the NAD(P) reservoir is completely coated 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 NAD(P) release kinetics (e.g., the rate of release of NAD(P) from the internal supply).

[0298] Alternatively or additionally, NAD(P) can be present within the permeable layer. For example, and without limitation, NAD(P) can be mixed directly into the permeable layer, such as a polymer permeable layer, rather than added as a separate layer coated by the permeable layer. In certain embodiments, an analyte sensor of the present disclosure can include a permeable layer including NAD(P) disposed on the substrate 30212. In certain embodiments, an analyte sensor of the present disclosure can include a NAD(P) reservoir disposed on the substrate 30212, which is covered with a permeable layer including a separate supply of NAD(P).

[0299] In certain embodiments, the analyte sensor further includes a permeable working electrode, such as 214, 30216, and / or 217. In certain embodiments, as shown in FIGS. 1-3, the permeable working electrode is disposed on the permeable layer. FIG. 22 and 23A In certain embodiments, at least one active area (which contains a sensing chemical) 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 certain embodiments, the active area is disposed on the working electrode as described herein. FIG. 22 and 23A In the non-limiting exemplary embodiments shown in FIGS. 1-3, NAD(P) diffuses through the permeable layer, such as a polymer layer, and the permeable working electrode to contact the active area to maintain sufficient NAD(P) concentration in the active area over time.

[0300] In certain embodiments, the amount of NAD(P) present within the NAD(P) reservoir can vary depending on the duration of use of the analyte sensor. For example, and not by way of limitation, NAD(P) can be present in the NAD(P) reservoir in an amount of about 0.1 pg to about 1,000 pg. In certain embodiments, about 0.1 pg to about 900 pg, about 0.1 pg to about 800 pg, about 0.1 pg to about 700 pg, about 0.1 pg to about 600 pg, about 0.1 pg to about 500 pg, about 0.1 pg to about 400 pg, about 0.1 pg to about 300 pg, about 0.1 pg to about 200 pg, about 0.1 pg to about 100 pg, about 0.1 pg to about 90 pg, about 0.1 pg to about 80 pg, about 0.1 pg to about 70 pg, about 0.1 pg to about 60 pg, about 0.1 pg to about 50 pg, about 0.1 pg to about 40 pg, about 0.1 pg to about 30 pg, about 0.1 pg to about 20 pg, about 0.1 pg to about 10 pg, about 0.1 pg to about 9 pg, about 0.1 pg to about 8 pg, about 0.1 pg to about 7 pg, about 0.1 pg to about 6 pg, about 0.1 pg to about 5 pg, about 0.1 pg to about 4 pg, about 0.1 pg to about 3 pg, about 0.1 pg to about 2 pg, about 0.1 pg to about 1 pg, about 0.1 pg to about 0.9 pg, about 0.1 pg to about 0.8 pg, about 0.1 pg to about 0.7 pg, about 0.1 pg to about 0.6 pg, about 0.1 pg to about 0.5 pg, about 0.1 pg to about 0.4 pg, about 0.1 pg to about 0.3 pg, about 0.1 pg to about 0.2 pg, about 0.2 pg to about 1,000 pg, about 0.3 pg to about 1,000 pg, about 0.4 pg to about 1,000 pg, about 0.5 pg to about 1,000 pg, about 0.6 pg to about 1,000 pg, about 0.7 pg to about 1,000 pg, about 0.8 pg to about 1,000 pg, about 0.9 pg to about 1,000 pg, about 1 pg to about 1,000 pg, about 2 pg to about 1,000 pg, about 3 pg to about 1,000 pg, about 4 pg to about 1,000 pg, about 5 pg to about 1,000 pg, about 6 pg to about 1,000 pg, about 7 pg to about 1,000 pg, about 8 pg to about 1,000 pg, about 9 pg to about 1,000 pg, about 10 pg to about 1,000 pg, about 11 pg to about 1,000 pg, about 12 pg to about 1,000 pg, about 13 pg to about 1,000 pg, about 14 pg to about 1,000 pg, about 15 pg to about 1,000 pg, about 16 pg to about 1,000 pg about 1,000 pg, about 17 pg to about 1,000 pg, about 18 pg to about 1,000 pg, about 19 pg to about 1,000 pg, about 20 pg to about 1,000 pg, from about 30 pg to about 1,000 pg, about 40 pg to about 1,000 pg, about 50 pg to about 1,000 pg, about 60 pg to about 1,000 pg, about 70 pg to about 1,000 pg, about 80 pg to about 1,000 pg, about 90 pg to about 1,000 pg, about 100 pg to about 1,000 pg, about 200 pg to about 1,000 pg, about 300 pg to about 1,000 pg, about 400 pg to about 1,000 pg, about 500 pg to about 1,000 pg, about 600 pg to about 1,000 pg, about 700 pg to about 1,000 pg, about 800 pg to about 1,000 pg, about 900 pg to about 1,000 pg, about 0.1 pg to about 100 pg, about 1 pg to about 100 pg, about 1 pg to about 90 pg, about 1 pg to about 80 pg, about 1 pg to about 70 pg, about 1 pg to about 60 pg, about 1 pg to about 50 pg, about 1 pg to about 40 pg, about 1 pg to about 30 pg, about 1 pg to about 20 pg, about 1 pg to about 15 pg, about 1 pg to about 10 pg, or about 5 pg to about 15 pg of NAD(P) can be present in the NAD(P) reservoir. In certain embodiments, NAD(P) can be present in the NAD(P) reservoir in an amount of about 0.1 pg to about 100 pg.

[0301] In certain embodiments, the amount of NAD(P) present in the NAD(P) reservoir varies depending on the lifetime of the analyte sensor. For example, but not by way of limitation, the amount of NAD(P) in the NAD(P) reservoir allows the analyte sensor to detect an 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) reservoir allows the analyte sensor to detect an 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) reservoir allows the analyte sensor to detect an analyte using an NAD(P)-dependent enzyme for more than about two weeks, more than about three weeks, more than about four weeks, more than about five weeks, more than about six weeks, more than about seven weeks, or more than about eight weeks.

[0302] In certain embodiments, the permeable layer can include a polymer. In certain embodiments, the permeable polymer layer can include a diffusion controlling polymer. In certain embodiments, the permeable polymer layer can include any polymer that allows for controlled diffusion of a co-factor. In certain embodiments, the permeable polymer layer can include any polymer that allows for controlled diffusion of its NAD(P).

[0303] In certain embodiments, the permeable polymer layer can include hyaluronic acid (HA), poly(ethylene glycol) (PEG), phosphorylcholine-based polymers, and other hydrophilic polymers with comparable hydrophilicity to HA, PEG, or phosphorylcholine, ethylene-vinyl alcohol copolymer, polyhydroxyalkanoate, poly(hydroxyvalerate), poly(hydroxyvalerate), poly(caprolactone), poly(lactide-co-glycolide), poly(hydroxybutyrate), poly(hydroxybutyrate-co-valerate), polydioxanone, polyorthoester, polyanhydride, poly(glycolic acid), poly(D,L-lactic acid) (DLPLA), poly(ortho ester), poly(glycolic acid-co-trimethylene carbonate), polyphosphoester, polyphosphoester urethane, poly(amino acid), cyanoacrylate, poly(trimethylene carbonate), poly(imino carbonates), polyurethane, copoly(ether-esters) (e.g., PEO / PLA), polyalkylene oxalates, polyphosphazenes, biomolecules such as fibrin, fibrinogen, cellulose, starch, and collagen, polyurethanes, silicones, polyesters, polyolefins, polyisobutylene, and ethylene-alphaolefin copolymers, acrylic polymers and copolymers, halogen-containing vinyl polymers and copolymers, poly(amide ester)s (PEA), poly(caprolactone) (PCL), poly(hexafluoropropylene) (HFP), poly(ethylene vinyl alcohol) (EVAL), polyvinyl ethers such as poly(vinyl methyl ether), poly(vinylidene fluoride) (PVDF), poly(vinylidene halides) such as poly(vinylidene chloride), polyacrylonitrile, polyvinyl ketones, poly(vinyl aromatics) such as polystyrene, polyvinyl esters such as polyvinyl acetate, copolymers of vinyl monomers with each other and with other olefins, olefinic copolymers such as ethylene-methyl methacrylate copolymers, acrylonitrile-styrene copolymers, ABS resins, and ethylene-vinyl acetate copolymers, polyamides such as nylon 66 and polycaprolactam, alkyd resins, polycarbonates, polyformaldehyde, polyimides, polyethers, epoxy resins, polyurethanes, rayon, rayon-triacetate, cellulose acetate, cellulose butyrate, cellulose acetate butyrate, cellophane, nitrocellulose, propylene cellulose, cellulose ethers, and carboxymethyl cellulose. In certain embodiments, a suitable polymer is a copolymer comprising poly(ethylene terephthalate) and poly(butylene terephthalate) (PEGT / PBT) segments.

[0304] Other non-limiting examples of polymers that can be present in the permeable layer include polycarboxylic acids, cellulosic polymers, gelatin, polyvinylpyrrolidone, cross-linked polyvinylpyrrolidone, poly anhydrides including maleic anhydride polymers, polyvinyl alcohol, polyvinyl arenes such as copolymer monomers of polystyrene with other vinyls 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 including polyethylene terephthalate, polyacrylamide, polyethersulfone, polyolefins including polypropylene, polyethylene, and high molecular weight polyethylene, halogenated polyolefins including polytetrafluoroethylene, natural and synthetic rubbers including polyisoprene, polybutadiene, polyisobutylene, and copolymer polymers thereof with other vinyl monomers such as polyorthoesters, proteins, polypeptides, siloxanes, polylactic acid, polyglycolic acid, polyhydroxybutyrate valerate, and blends and copolymers thereof, as well as other biodegradable, bioabsorbable, and bio-stable polymers and copolymers. In certain embodiments, suitable polymers include polyacrylic acid and copolymers of polylactic acid with polycaprolactone.

[0305] In certain embodiments, the permeable layer can include a polyether-based polymer. In certain embodiments, the permeable layer can include poly(ethylene glycol). In certain embodiments, the permeable layer can include a poly(ethylene glycol)-based polymer. In certain embodiments, the permeable layer can include poly(propylene glycol). In certain embodiments, the permeable layer can include a poly(propylene glycol)-based polymer. In certain embodiments, the permeable layer can include poly(propylene glycol) methacrylate (POMA). In certain embodiments, the permeable layer can include 2-hydroxyethyl methacrylate (HEMA). In certain embodiments, the permeable layer can include a mixture of POMA and HEMA. In certain embodiments, the permeable layer can include a mixture of POMA and HEMA to generate a permeable polymer. For example, but not by way of limitation, the permeable layer can include a ratio of POMA to HEMA of about 10: 1 to about 1 : 10, such as 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. 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 include about 20% to about 80% by weight POMA, such as about 30% to about 70% or about 40% to about 60% by weight. In certain embodiments, the permeable layer can include about 40% to about 60% by weight POMA. In certain embodiments, the permeable layer can include about 20% to about 80% by weight HEMA, such as about 30% to about 70% or about 40% to about 60% by weight. In certain embodiments, the permeable layer can include about 40% to about 60% by weight HEMA.

[0306] In certain embodiments, the permeable polymer is a hydrogel. In certain embodiments, the permeable polymer, e.g., a hydrogel for use in the present disclosure, is capable of absorbing water in an amount of about 30% to about 95% of its weight, e.g., about 30% to about 70% or about 40% to about 60%. In certain embodiments, the permeable polymer, e.g., a hydrogel, is capable of absorbing water in an amount of at least about 30% of its weight. In certain embodiments, the permeable polymer, e.g., a hydrogel, is capable of absorbing water in an amount of at least about 40% of its weight. In certain embodiments, the permeable polymer, e.g., a hydrogel, is capable of absorbing water in an amount of at least about 50% of its weight. In certain embodiments, the permeable polymer, e.g., a hydrogel, is capable of absorbing water in an amount of at least about 60% of its weight. In certain embodiments, the permeable polymer, e.g., a hydrogel, is capable of absorbing water in an amount of at least about 70% of its weight. In certain embodiments, the permeable polymer, e.g., a hydrogel, is capable of absorbing water in an amount of about 30% to about 60% of its weight.

[0307] In certain embodiments, the NAD(P) reservoir has a thickness, e.g., a dry thickness, ranging from about 0.1 pm to about 1,000 pm, e.g., about 1 pm to about 500 pm, about 10 pm to about 100 pm, or about 10 pm to about 100 pm. In certain embodiments, the NAD(P) reservoir can have a thickness of about 0.1 pm to about 10 pm, e.g., about 0.5 pm to about 10 pm, about 1 pm to about 10 pm, about 1 pm to about 5 pm, or about 0.1 pm to about 5 pm.

[0308] In certain embodiments, the permeable electrode can include any material that is permeable to NAD(P). In certain embodiments, the permeable electrode can include any electrically conductive material that is permeable to NAD(P), e.g., an electrically conductive ink or polymer. For example, but not by way of limitation, the permeable electrode can include carbon, silver, amorphous carbon, graphite, graphene, glassy carbon, platinum-coated carbon, gold, platinum, and / or palladium. In certain embodiments, the permeable electrode can include a carbon material. In certain embodiments, the permeable electrode can include a carbon material that includes an additive, such as, but not limited to, silver, amorphous carbon, graphite, graphene, glassy carbon, platinum-coated carbon, gold, platinum, and / or palladium. In certain embodiments, the permeable electrode can consist at least in part of carbon nanotubes. In certain embodiments, the permeable electrode can include an electrically conductive polymer, e.g., poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS). In certain embodiments, the electrically conductive material can be present in a polymer, e.g., a polymeric carrier. In certain embodiments, the electrode includes a polymer containing an electrically conductive material and / or electrically conductive particles.

[0309] 3. Enzyme

[0310] 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 AlC, 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 analyte to be detected using the NAD(P)-dependent enzyme is glucose, lactate, ketones, creatinine, alcohol, e.g., ethanol, and the like. In certain embodiments, the active region can include multiple enzymes, e.g., an enzyme system, that collectively respond to the analyte.

[0311] In certain embodiments, the active region of the presently disclosed analyte sensors includes at least one NAD(P)-dependent enzyme. In certain embodiments, the active region of the presently disclosed analyte sensors includes two or more NAD(P)-dependent enzymes. In certain embodiments, the analyte sensors of the present disclosure include two active sites, each active site including at least one NAD(P)-dependent enzyme. Alternatively, in certain embodiments, the analyte sensors of the present disclosure include two active sites, where only one of the active sites includes a 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.

[0312] In certain embodiments, the analyte sensors of the present disclosure include one or more internal supplies of NAD(P) for the NAD(P)-dependent enzymes contained in one or more active sites of the analyte sensor. For example, but not by way of limitation, a reservoir of NAD(P) can be disposed under each electrode configured to detect an analyte. Alternatively, a reservoir of NAD(P) can be disposed under only one electrode configured to detect an analyte.

[0313] In certain embodiments, the active site can include 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), a-hydroxybutyric acid dehydrogenase, sorbitol dehydrogenase, L-amino acid dehydrogenase (EC 1.4.1.5), diaphorase (EC 1.8.1.4), and combinations thereof.

[0314] In certain embodiments, the NAD(P)-dependent dehydrogenase can include diaphorase, glucose dehydrogenase, alcohol dehydrogenase, lactate dehydrogenase, and b-hydroxybutyric acid dehydrogenase. In certain embodiments, the enzyme system can include two or more NAD(P)-dependent dehydrogenases, such as a first NAD(P)-dependent dehydrogenase and diaphorase. For example, but not by way of limitation, the NAD(P)-dependent dehydrogenase can convert an analyte and oxidized nicotinamide adenine dinucleotide (NAD + ) to oxidized analyte and reduced nicotinamide adenine dinucleotide (NADH), respectively. The enzyme cofactors NAD + and NADH help facilitate the synergistic enzymatic reactions disclosed herein. NADH can then be reduced under diaphorase mediation, in the process of which the transferred electrons provide the basis for analyte detection at the working electrode.

[0315] In certain embodiments, the analyte sensors of the present disclosure can include a glucose-responsive active area, a ketone-responsive active area, a lactate-responsive active area, a creatinine-responsive active area, an alcohol-responsive active area, or combinations thereof. In certain embodiments, the glucose-responsive active area can include one or more NAD(P)-dependent enzymes for detecting glucose. In certain embodiments, the ketone-responsive active area can include one or more NAD(P)-dependent enzymes for detecting ketones. In certain embodiments, the lactate-responsive active area can include one or more NAD(P)-dependent enzymes for detecting lactate. In certain embodiments, the creatinine-responsive active area can include one or more NAD(P)-dependent enzymes for detecting creatinine. In certain embodiments, the alcohol-responsive active area can include one or more NAD(P)-dependent enzymes for detecting alcohol. In certain embodiments, the active area can include an enzyme system comprising two or more enzymes that collectively respond to an analyte. For example, but not by way of limitation, the ketone-responsive active area can include an enzyme system containing at least one NAD(P)-dependent enzyme.

[0316] 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 a glucose dehydrogenase. For example, but not by way of limitation, an analyte sensor of the present disclosure for detecting glucose can include an NAD(P) reservoir and an active region that includes a glucose dehydrogenase.

[0317] 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, but not by way of limitation, an analyte sensor of the present disclosure for detecting alcohols can include an NAD(P) reservoir and an active region that includes an alcohol dehydrogenase.

[0318] 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 a beta-hydroxybutyrate dehydrogenase. For example, but not by way of limitation, an analyte sensor of the present disclosure for detecting ketones can include an NAD(P) reservoir and an active region that includes an enzyme system that includes a beta-hydroxybutyrate dehydrogenase.

[0319] 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, but not by way of limitation, the lactate-responsive active site can include a lactate dehydrogenase. In certain embodiments, an analyte sensor of the present disclosure for detecting lactate can include an NAD(P) reservoir and an active region that includes a lactate dehydrogenase.

[0320] In certain embodiments, an analyte sensor disclosed herein can include at least one active site that includes one or more NAD(P)-dependent enzymes for detecting an analyte, as disclosed herein. Alternatively, an analyte sensor disclosed herein can include two or more active sites, each active site containing one or more enzymes, e.g., where at least one active site includes one or more NAD(P)-dependent enzymes. For example, but not by way of limitation, an analyte sensor of the present disclosure can include a first active region that includes a first enzyme (or enzyme system) for detecting a first analyte and a second active site (or second enzyme system) that includes a second enzyme for detecting a second analyte, where at least the first active region or the second active region includes an NAD(P)-dependent enzyme.

[0321] In certain embodiments, the analyte-responsive active area can include about 10% to about 80% by weight, such as about 15% to about 75%, about 20% to about 70%, about 25% to about 65%, or about 30% to about 60% by weight of one or more enzymes disclosed herein (e.g., one or more NAD(P)-dependent enzymes). In certain embodiments, the analyte-responsive active area can include about 10% to about 80% by weight, such as about 15% to about 75%, about 20% to about 70%, about 25% to about 65%, about 30% to about 60%, about 20% to about 60%, or about 20% to about 50% by weight of one or more enzymes disclosed herein (e.g., one or more NAD(P)-dependent enzymes). In certain embodiments, the analyte-responsive active area can include about 10% to about 80% by weight, such as about 15% to about 75%, about 20% to about 70%, about 25% to about 65%, or about 30% to about 60% by weight of one or more enzymes disclosed herein (e.g., one or more NAD(P)-dependent enzymes). In certain embodiments, the analyte-responsive active area can include about 10% to about 80% by weight, such as about 15% to about 75%, about 20% to about 70%, about 25% to about 65%, or about 30% to about 60% by weight of one or more enzymes disclosed herein (e.g., one or more NAD(P)-dependent enzymes).

[0322] In certain embodiments, the analyte-responsive active region can further include a stabilizing agent, e.g., for stabilizing an enzyme. For example, and not by way of limitation, the stabilizing agent can be an albumin, e.g., a serum albumin. Non-limiting examples of serum albumins include bovine serum albumin and human serum albumin. In certain embodiments, the stabilizing agent is human serum albumin. In certain embodiments, the stabilizing agent is bovine serum albumin. In certain embodiments, the stabilizing agent can be catalase. In certain embodiments, the analyte-responsive active region can include a ratio of stabilizing agent present in the analyte-responsive active region to one or more enzymes (e.g., NAD(P)-dependent enzymes) 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 :30 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 include a ratio of stabilizing agent present in the analyte-responsive active region to one or more enzymes (e.g., NAD(P)-dependent enzymes) of about 2: 1 to about 1 :2. In certain embodiments, the analyte-responsive active region can include a ratio of stabilizing agent 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 :30 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 include a ratio of stabilizing agent 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 include about 10% to about 50% by weight, e.g., about 15% to about 45%, about 20% to about 40%, about 20% to about 35%, or about 20% to about 30%, of a stabilizing agent. In certain embodiments, the analyte-responsive active region can include about 15% to about 35% by weight of a stabilizing agent.

[0323] In certain embodiments, in addition to the presence of a NAD(P) reservoir, the analyte-responsive active area can also include a co-factor for one or more enzymes present in the analyte-responsive active area. In certain embodiments, the co-factor is NAD(P). In certain embodiments, the co-factor is a co-factor other than NAD(P). In certain embodiments, the analyte-responsive active area can include a ratio of co-factor to enzyme of about 40: 1 to about 1 :40, such as 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 area can include a ratio of co-factor to enzyme of about 2: 1 to about 1 :2. In certain embodiments, the analyte-responsive active area can include about 10% to about 50% by weight, such as about 15% to about 45% by weight, about 20% to about 40% by weight, about 20% to about 35% by weight, about 20% to about 30% by weight, of the co-factor. In certain embodiments, the analyte-responsive active area can include about 15% to about 35% by weight of the co-factor. In certain embodiments, the co-factor, e.g., NAD(P), can be physically retained within the analyte-responsive active area. For example, and not by way of limitation, a membrane that coats the analyte-responsive active area can help retain the co-factor in the analyte-responsive active area while still allowing the analyte to sufficiently diffuse inward to allow for detection thereof.

[0324] In certain embodiments, an analyte sensor of the present disclosure can include a sensor tail including at least one NAD(P) reservoir, at least one working electrode (e.g., a permeable electrode), and an analyte-responsive active area disposed on a surface of the working electrode, wherein the analyte-responsive active area includes at least one NAD(P)-dependent enzyme. In certain embodiments, an analyte sensor of the present disclosure can include a sensor tail including a substrate, at least one NAD(P) reservoir disposed on a surface of the substrate, at least one working electrode (e.g., a permeable electrode), and an analyte-responsive active area disposed on a surface of the working electrode, wherein the analyte-responsive active area includes at least one NAD(P)-dependent enzyme. In certain embodiments, the NAD(P)-dependent enzyme is an NAD(P)-dependent dehydrogenase. For example, but not by way of limitation, a sensor of the present disclosure can include a sensor tail including at least one NAD(P) reservoir, a permeable layer disposed on the NAD(P) reservoir, at least one permeable working electrode, and an analyte-responsive active area disposed on a surface of the permeable working electrode, wherein the analyte-responsive active area includes an enzyme system including an NAD(P)-dependent dehydrogenase.

[0325] In certain embodiments, a sensor of the present disclosure can include a sensor tail including at least one NAD(P) reservoir, a permeable layer disposed on top of the NAD(P) reservoir, at least one permeable working electrode, and a ketone-responsive active area disposed on a surface of the permeable working electrode, wherein the ketone-responsive active area includes an enzyme system including an NAD(P)-dependent dehydrogenase, e.g., a beta-hydroxybutyrate dehydrogenase. In certain embodiments, the enzyme system further includes diaphorase.

[0326] In certain embodiments, an analyte sensor of the present disclosure can include a second active area, e.g., for detecting an analyte different from the analyte detected by the first active area. In certain embodiments, the second active area is disposed on the same working electrode or a second working electrode as the first active area. In certain embodiments, the second active area is a glucose-responsive active area, a lactate-responsive active area, a creatinine-responsive active area, or an alcohol-responsive active area.

[0327] In certain embodiments, a second active area of an analyte sensor of the present disclosure can include one or more enzymes for detecting glucose. For example, but not by way of limitation, an analyte sensor of the present disclosure can include an active area (e.g., a second active area) including one or more enzymes for detecting glucose, e.g., disposed on a second working electrode. In certain embodiments, an analyte sensor can include an active site including a glucose oxidase and / or a glucose dehydrogenase for detecting glucose.

[0328] In certain embodiments, the second active area can include one or more enzymes for detecting lactate. For example, and not by way of limitation, the analyte sensors of the present disclosure can include an active area (e.g., a second active area) comprising one or more enzymes, e.g., an enzyme system, for detecting lactate, e.g., disposed on a second working electrode. In certain embodiments, the analyte sensors can include active sites containing lactate dehydrogenase and / or lactate oxidase.

[0329] In certain embodiments, the second enzyme-responsive active area, e.g., present on a second working electrode of an analyte sensor of the present disclosure, can include one or more enzymes for detecting alcohol. For example, and not by way of limitation, the analyte sensors of the present disclosure can include an active area (e.g., a second active area) comprising one or more enzymes, e.g., an enzyme system, for detecting alcohol, e.g., disposed on a second working electrode. In certain embodiments, the analyte sensors can include active sites containing alcohol dehydrogenase.

[0330] In certain embodiments, the second enzyme-responsive active area, e.g., present on a second working electrode of an analyte sensor of the present disclosure, can include one or more enzymes for detecting creatinine. For example, and not by way of limitation, the analyte sensors of the present disclosure can include an active area (e.g., a second active area) comprising one or more enzymes, e.g., an enzyme system, for detecting creatinine, e.g., disposed on a second working electrode. In certain embodiments, the analyte sensors can include active sites containing amidohydrolase, creatinine amidohydrolase, and / or creatine oxidase.

[0331] In certain embodiments, an analyte sensor can include two working electrodes, e.g., a first active area disposed on a first working electrode (e.g., a permeable electrode) and a second active area disposed on a second working electrode. For example, but not by way of limitation, an analyte sensor disclosed herein can have the features of at least one NAD(P) reservoir, a first analyte-responsive active area disposed on a first working electrode, and a second analyte-responsive active area disposed on the surface of a different working electrode (e.g., a second working electrode), wherein at least one analyte-responsive active area includes an NAD(P)-dependent enzyme. In certain embodiments, the second analyte-responsive active area can be configured to detect a different analyte or the same analyte as detected by the first analyte-responsive active area. In certain embodiments, such an analyte sensor can include a sensor tail having at least one NAD(P) reservoir, a first working electrode and a second working electrode, a first analyte-responsive active area disposed on the surface of the first working electrode, and a second analyte-responsive active area disposed on the surface of the second working electrode, wherein at least one analyte-responsive active area includes an NAD(P)-dependent enzyme and at least one working electrode is permeable. For example, but not by way of limitation, an analyte-responsive active area including an NAD(P)-dependent enzyme is disposed on a permeable working electrode.

[0332] In certain embodiments, when a sensor is configured to detect two or more analytes using two working electrodes, detection of each analyte can include applying a potential to each working electrode separately, such that a separate signal is obtained from each analyte. The signal obtained from each analyte can then be related to analyte concentration by using a calibration curve or function, or by employing a lookup table. In certain particular embodiments, the relating of the analyte signal to analyte concentration can be performed by using a processor.

[0333] In certain other analyte sensor configurations, the first active area and the second active area can be disposed on a single working electrode. For example, but not by way of limitation, the analyte sensors disclosed herein can have at least one NAD(P) reservoir, a first analyte-responsive active area and a second analyte-responsive active area disposed on the surface of a single permeable working electrode, where at least one of the analyte-responsive active areas includes a NAD(P)-dependent enzyme. In certain embodiments, a first signal can be obtained from the first active area, e.g., at a low potential, and a second signal can be obtained at a higher potential that includes signal contributions from both active areas. Subtracting the first signal from the second signal can then allow determination of the signal contribution by the second analyte. The signal contribution from each analyte can then be associated with analyte concentration in a similar manner as described for sensor configurations having multiple working electrodes. In certain embodiments, when a ketone-responsive active area and a second active area configured to detect a different analyte (e.g., a glucose-responsive active area) are arranged in this manner on a single working electrode, one of the active areas can be configured such that it can be interrogated separately to facilitate detection of each analyte. For example, the ketone-responsive active area or the glucose-responsive active area can generate a signal independently of the other active area.

[0334] It should also be recognized that the sensitivity (output current) of an analyte sensor to each analyte can be altered by varying the footprint (area or size) of the active areas, the area ratio of the active areas relative to each other, the properties, thickness, and / or composition of the mass transport limiting membranes that overcoat the active areas. Variations in these parameters can be readily made by one of ordinary skill in the art once the benefit of the disclosure herein is granted.

[0335] 4. Oxidation-Reduction Mediators

[0336] In certain embodiments, the analyte sensors disclosed herein can include an electron transfer agent. For example, but not by way of limitation, one or more active sites of an analyte sensor can include an electron transfer agent. In certain embodiments, an 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 an electron transfer agent in an active area can depend on the enzyme or enzyme system used to detect the analyte and / or the composition of the working electrode. Alternatively, an analyte sensor can include two active sites, where both active sites include an electron transfer agent.

[0337] Suitable electron transfer agents can facilitate the transfer of electrons to an adjacent working electrode after the analyte undergoes an enzymatic oxidation-reduction reaction within the corresponding active area, thereby generating a current indicative of the presence of that particular analyte. The amount of current generated is proportional to the amount of analyte present. In certain embodiments, suitable electron transfer agents can include ions, complexes, or molecules (e.g., quinones) that are electroreducible and electrooxidizable with an oxidation-reduction potential that is several hundred millivolts more positive or more negative than the oxidation-reduction potential of a standard saturated calomel electrode (SCE). In certain embodiments, the oxidation-reduction mediator can include osmium complexes and other transition metal complexes such as those described in U.S. Patent Nos. 6,134,461 and 6,605,200, the disclosures of which are incorporated herein by reference in their entireties. Additional examples of suitable oxidation-reduction mediators include those described in U.S. Patent Nos. 6,736,957, 7,501,053, and 7,754,093, the disclosures of each of which are also incorporated herein by reference in their entireties. Other examples of suitable oxidation-reduction mediators include metal compounds or complexes of ruthenium, osmium, iron (e.g., polyvinyl ferrocene or hexacyanoferrate) or cobalt, including, for example, metallocene compounds thereof. Suitable ligands for the metal complexes can also include, for example, bidentate or higher dentate 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 the metal complex, for example, osmium complexes, to achieve a fully coordinated sphere.

[0338] In certain embodiments, the electron transfer agents disclosed herein can include suitable groups to facilitate covalent bonding to a polymer (also referred to herein as a polymer backbone) within the active area, as further discussed below. For example, and without limitation, the electron transfer agents for use in the present disclosure can include polymer-bound electron transfer agents. Suitable, non-limiting examples of polymer-bound electron transfer agents include those described in U.S. Patent Nos. 8,444,834, 8,268,143, and 6,605,201, which are incorporated herein by reference in their entireties. In certain embodiments, the electron transfer agent is a bidentate osmium complex bound to a polymer described herein, for example, a polymer backbone described in Section 5 below. In certain embodiments, the polymer-bound electron transfer agents illustrated in FIG. 3 of U.S. Patent No. 8,444,834 can be used in the sensors of the present disclosure.

[0339] In certain embodiments of the present disclosure, an analyte sensor can include at least one NAD(P) reservoir, at least one permeable layer disposed over the NAD(P) reservoir, at least one working electrode (e.g., a permeable electrode), and at least one analyte-responsive active area disposed on a surface of the working electrode, wherein the analyte-responsive active area includes 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 area includes an enzyme system that includes diaphorase, an NAD(P)-dependent dehydrogenase, e.g., beta-hydroxybutyrate dehydrogenase, and a redox mediator, e.g., an osmium complex.

[0340] 5. Polymer backbone

[0341] In certain embodiments, one or more active sites for facilitating analyte detection can include a polymer to which an enzyme and / or a redox mediator is covalently bonded. Any suitable polymer backbone can be present in the active area to facilitate detection of an analyte by covalent bonding of an enzyme and / or a redox mediator thereto. Non-limiting examples of suitable polymers within the active area include polyvinylpyridines, e.g., poly(4-vinylpyridine) and / or poly(2-vinylpyridine), and polyvinylimidazoles, e.g., poly(N-vinylimidazole) and poly(l-vinylimidazole) or copolymers thereof, e.g., where the quaternized pyridine groups serve as a point of attachment for a redox mediator or enzyme thereto. Illustrative copolymers that can be suitable for inclusion in the active area include those containing monomeric units such as styrene, acrylamide, methacrylamide, or acrylonitrile. In certain embodiments, polymers that can be present in the active area include polyurethanes or copolymers thereof, and / or polyvinylpyrrolidone. In certain embodiments, polymers that can be present in the active area include, but are not limited to, those described in U.S. Patent 6,605,200, which is incorporated by reference herein in its entirety, e.g., poly(acrylic acid), styrene / maleic anhydride copolymer, methyl vinyl ether / maleic anhydride copolymer (GANTREZ polymers), poly(vinylbenzyl chloride), poly(allylamine), polylysine, poly(4-vinylpyridine) quaternized with carboxypentyl, and poly(4-sodium styrene sulfonate). In certain embodiments where the analyte sensor includes two active sites, the polymer within each active area can be the same or different.

[0342] In certain embodiments, the polymer is a polyvinylpyridine or a copolymer thereof. In certain embodiments, the polymer is a copolymer of vinylpyridine and styrene.

[0343] In certain embodiments, when an enzyme system having multiple enzymes is present in a given active area, all of the multiple enzymes can be covalently bonded to the polymer. In certain other embodiments, only a subset of the multiple enzymes is covalently bonded to the polymer. For example, but not by way of limitation, one or more enzymes within the enzyme system can be covalently bonded to the polymer, and at least one enzyme can be non-covalently bonded to the polymer, such that the non-covalently bonded enzyme is physically retained in the polymer. In certain embodiments, the NAD(P)-dependent enzymes can be covalently bonded to the polymer. Alternatively, the NAD(P)-dependent enzymes can be non-covalently bonded to the polymer. In certain embodiments, the NAD(P)-dependent dehydrogenase and the diaphorase can be covalently bonded to the polymer within the active area of the disclosed analyte sensor. In certain embodiments, the NAD(P)-dependent dehydrogenase can be covalently bonded to the polymer and the diaphorase can be non-covalently bonded to the polymer. Alternatively, the diaphorase can be covalently bonded to the polymer and the NAD(P)-dependent dehydrogenase can be non-covalently bonded to the polymer.

[0344] In certain embodiments, when a stabilizing agent is present in the active area, one or more enzymes within the area can be covalently bonded to the stabilizing agent. For example, but not by way of limitation, one or more enzymes (e.g., one or more NAD(P)-dependent enzymes) within the enzyme system can be covalently bonded to a stabilizing agent (e.g., albumin) present in the active area.

[0345] In certain specific embodiments, covalent bonding of one or more enzymes and / or redox mediators to the polymer and / or stabilizing agent in a given active area can occur through cross-linking introduced by a suitable cross-linking agent. In certain embodiments, cross-linking 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 cross-linking agents can include one or more cross-linkable functionalities, such as, but not limited to, vinyl, alkoxy, acetoxy, alkenoxy, oximato, amino, hydroxyl, cyano, halogen, acrylate, epoxy, and isocyanate groups. In certain embodiments, the cross-linking agent comprises one or more, two or more, three or more, or four or more epoxy groups. For example, but not by way of limitation, cross-linking agents for use in the present disclosure can include mono-, di-, tri-, and tetra-ethylene oxide. In certain embodiments, cross-linking agents for reaction with free amino groups in the enzymes (e.g., with free side chain amines in lysine) can include cross-linking agents such as, for example, polyethylene glycol dibutyl ether, polypropylene glycol dimethyl ether, polyalkylene glycol allyl methyl ether, polyethylene glycol diglycidyl ether (PEGDGE) or other polyepoxides, cyanuric chloride, N-hydroxysuccinimide, imidoesters, epichlorohydrin, or derivatives thereof. In certain embodiments, the cross-linking agent is PEGDGE, for example, having an average molecular weight (Mn) of about 200 to 1,000. n), for example, about 400. In certain embodiments, the crosslinking agent is PEGDGE 400. In certain embodiments, the crosslinking agent can be glutaraldehyde. Suitable crosslinking agents for reacting with free carboxylic acid groups in the enzyme can include, for example, carbodiimides. In certain embodiments, the crosslinking agent 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.

[0346] 6. Mass transport limiting membrane

[0347] In certain embodiments, the analyte sensors disclosed herein further include an analyte permeable membrane that at least coats the active area, e.g., the first active area and / or the second active area.

[0348] In certain embodiments, the analyte sensors disclosed herein further include an analyte permeable membrane that coats at least one active area, e.g., the first active area and / or the second active area. In certain embodiments, the membrane coats each active area of the analyte sensor. Alternatively, a first membrane coats one of the active areas and a second membrane covers the second active area. Alternatively, a first membrane coats one of the active areas and a second membrane coats both the first and second active areas.

[0349] In certain embodiments, the membrane coating the analyte-responsive active area can function as a mass transport limiting membrane and / or improve biocompatibility. The mass transport limiting membrane can act as a diffusion limiting barrier to reduce the mass transport rate of an analyte (e.g., glucose, alcohol, ketone, lactate, or beta-hydroxybutyrate) when the sensor is in use. For example, but not by way of limitation, limiting the entry of an analyte (e.g., a ketone) into the analyte-responsive active area using a mass transport limiting membrane can help avoid sensor overload (saturation), thereby improving detection performance and accuracy. In certain embodiments, the mass transport limiting layer limits the flux of analyte to the electrode in the electrochemical sensor such that the sensor responds linearly over a wide range of analyte concentrations.

[0350] In certain embodiments, the mass transport limiting membrane can be homogenous and can be a single component (comprising a single membrane polymer). Alternatively, the mass transport limiting membrane can be a multi-component (comprising two or more different membrane polymers). In certain embodiments, the mass transport limiting membrane can comprise two or more layers, e.g., a bilayer or a trilayer membrane. In certain embodiments, each layer can comprise a different polymer or the same polymer at a different concentration or thickness. In certain embodiments, the first analyte-responsive active area can be covered by a multi-layer membrane, e.g., a bilayer membrane, and the second analyte-responsive active area can be covered by a single layer membrane. In certain embodiments, the first analyte-responsive active area can be covered by a multi-layer membrane, e.g., a bilayer membrane, and the second analyte-responsive active area can be covered by a multi-layer membrane, e.g., a bilayer membrane. In certain embodiments, the first analyte-responsive active area can be covered by a single layer membrane and the second analyte-responsive active area can be covered by a multi-layer membrane, e.g., a bilayer membrane covered by a single layer membrane. In certain embodiments, the first analyte-responsive active area can be covered by a single layer membrane and the second analyte-responsive active area can be covered by a single layer membrane.

[0351] In certain embodiments, the mass transport limiting membrane can comprise a cross-linked polymer comprising 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., in Formula 2b), the disclosure of which is incorporated by reference herein in its entirety.

[0352] In certain embodiments, the mass transport limiting membrane can comprise a polyvinylpyridine (e.g., poly(4-vinylpyridine) or poly(4-vinylpyridine)), a polyvinylimidazole, a polyvinylpyridine copolymer (e.g., a copolymer of vinylpyridine and styrene), a polyacrylate, a polyurethane, a polyether polyurethane, a silicone, a polytetrafluoroethylene, a polyethylene-tetrafluoroethylene copolymer, a polyolefin, a polyester, a polycarbonate, a biostable polytetrafluoroethylene, a homo-, co- or ter-polymer of polyurethane, a polypropylene, a polyvinyl chloride, a polyvinylidene fluoride, a polybutylene terephthalate, a polymethyl methacrylate, a polyether ether ketone, a cellulose polymer, a polysulfone, and block copolymers thereof, including, e.g., di-, tri-, alternating, random, and graft copolymers or chemically related materials, and the like.

[0353] In certain embodiments, films (e.g., single component films) used in the present disclosure can include polyvinylpyridine (e.g., poly(4-vinylpyridine) and / or poly(2-vinylpyridine)). In certain embodiments, films (e.g., single component films) used in the present disclosure can include poly(4-vinylpyridine). In certain embodiments, films (e.g., single component films) used in the present disclosure can include a copolymer of vinylpyridine and styrene. In certain embodiments, the film can comprise a polyvinylpyridine-co-styrene copolymer. For example, but not by way of limitation, polyvinylpyridine-co-styrene copolymers used in the present disclosure can include polyvinylpyridine-co-styrene copolymers in which a portion of the pyridine nitrogen atoms are functionalized with non-crosslinked polyethylene glycol tail and a portion of the pyridine nitrogen atoms are functionalized with alkyl sulfonic acid groups. In certain embodiments, the derivatized polyvinylpyridine-co-styrene copolymer used as the film polymer can be the 10Q5 polymer described in U.S. Patent No. 8,761,857, the contents of which are incorporated by reference in their entirety. In certain embodiments, the polyvinylpyridine-based polymer has a molecular weight of about 50 Da to about 500 kDa.

[0354] In certain embodiments, the film can include a polymer such as, but not limited to, poly(styrene co-maleic anhydride), dodecylamine, and poly(propylene glycol)-block-poly(ethylene glycol)-block-poly(propylene glycol) (2-aminopropyl ether) crosslinked with 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.

[0355] In certain embodiments, the membrane comprises a polyurethane membrane comprising a hydrophilic region and a hydrophobic region. In certain embodiments, the hydrophobic polymer component is a polyurethane, a polyurethane urea, or a poly(ether-polyurethane-urea). In certain embodiments, the polyurethane is a polymer produced by condensation reaction of a diisocyanate and a bifunctional hydroxyl-containing material. In certain embodiments, the polyurethane urea is a polymer produced by condensation reaction of a diisocyanate and a bifunctional amine-containing material. In certain embodiments, the diisocyanate used herein includes aliphatic diisocyanates, for example, diisocyanates containing about 4 to about 8 methylene units, or diisocyanates containing alicyclic moieties. Other non-limiting examples of polymers that can be used to generate the membranes of the sensors of the present disclosure include vinyl polymers, polyethers, polyesters, polyamides, inorganic polymers (e.g., polysiloxanes and polycarbosiloxanes), natural polymers (e.g., cellulose and protein-based materials), and mixtures (e.g., mixtures or layered structures) or combinations thereof. In certain embodiments, the hydrophilic polymer component is a polyethylene oxide and / or a polyethylene glycol. In certain embodiments, the hydrophilic polymer component is a polyurethane copolymer. For example, but not by way of limitation, the hydrophobic-hydrophilic copolymer component used in the present disclosure is a polyurethane polymer that includes about 10% to about 50% (e.g., 20%) of a hydrophilic polyethylene oxide.

[0356] In certain embodiments, the membrane comprises a silicone polymer / hydrophobic- hydrophilic polymer blend. In certain embodiments, the hydrophobic-hydrophilic polymer used 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 copolymers can be substituted with hydroxyl substituents.

[0357] In certain embodiments, a hydrophilic or hydrophobic modifier can be used to "tune" the permeability of the resulting membrane to a target analyte. In certain embodiments, a hydrophilic modifier such as a poly(ethylene glycol), a hydroxyl or polyhydroxyl modifier, and the like, or any combination thereof, can be used to enhance the biocompatibility of the polymer or the resulting membrane.

[0358] In certain embodiments where multiple active areas are present, a mass transport limiting membrane can coat each active area, including the option for varying composition according to different active areas, which can be achieved through sequential dip coating operations to produce a bilayer membrane portion on the working electrode positioned closer to the sensor tip.

[0359] In certain embodiments where multiple active areas are present, separate mass transport limiting membranes can coat each active area. For example, but not by way of limitation, a mass transport limiting membrane can be disposed on a first active area (e.g., a ketone-responsive active area), and a separate second mass transport limiting membrane can coat a second active area (e.g., a glucose-responsive active area). In certain embodiments, the two mass transport limiting membranes are spatially separated and do not overlap one another. In certain embodiments, the first mass transport limiting membrane does not overlap the second mass transport limiting membrane and the second mass transport limiting membrane does not overlap the first mass transport limiting membrane. In certain embodiments, the first mass transport limiting membrane includes a different polymer than the second mass transport limiting membrane. Alternatively, the first mass transport limiting membrane includes the same polymer as the second mass transport limiting membrane. In certain embodiments, the first mass transport limiting membrane includes the same polymer as the second mass transport limiting membrane but includes a different crosslinker.

[0360] In certain embodiments, the composition of the mass transport limiting membrane disposed on an analyte sensor having two active areas can be the same or different, where a mass transport limiting membrane coats each active area. For example, but not by way of limitation, a portion of the mass transport limiting membrane coating a ketone-responsive active area can be multi-component and / or a portion of the mass transport limiting membrane coating a glucose-responsive active area can be single-component. Alternatively, a portion of the mass transport limiting membrane coating a ketone-responsive active area can be single-component and / or a portion of the mass transport limiting membrane coating a glucose-responsive active area can be multi-component.

[0361] In certain embodiments, a glucose-responsive active area can be coated with a membrane including a polyurethane, a polyurethane urea, or a poly(ether- urethane-urea). In certain embodiments, a glucose-responsive active area can be coated with a membrane including a polyurethane. In certain embodiments of the present disclosure, a ketone-responsive active area and a second analyte-responsive region (e.g., a glucose-responsive active area) can be coated with a membrane including a poly(ethylene-co-vinylpyridine-co-styrene) copolymer.

[0362] In certain embodiments, a membrane (e.g., a single-component membrane) can include a poly(ethylene-co-vinylpyridine). In certain embodiments, a membrane (e.g., a single-component membrane) can include a copolymer of a vinylpyridine and a styrene (or derivative thereof).

[0363] In some embodiments, the multicomponent membrane may 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 the solution onto a working electrode (e.g., impregnation). In some embodiments of this disclosure, a first analyte-responsive region (e.g., a ketone-responsive region) may be coated with a multicomponent membrane comprising polyvinylpyridine and a polyvinylpyridine-copolystyrene copolymer, either as a bilayer membrane or as a homogeneous mixture, and a second analyte-responsive region (e.g., a glucose-responsive region) may be coated with a membrane comprising a polyvinylpyridine-copolystyrene copolymer.

[0364] The styrene content of suitable copolymers of vinylpyridine and styrene ranges from about 0.01% to about 50% molar percentage, or about 0.05% to about 45% molar percentage, or about 0.1% to about 40% molar percentage, or about 0.5% to about 35% molar percentage, or about 1% to about 30% molar percentage, or about 2% to about 25% molar percentage, or about 5% to about 20% molar percentage. Substituted styrene may be used similarly and in similar amounts. The molecular weight of suitable copolymers of vinylpyridine and styrene may be 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 more, or about 40 kDa or more, or about 50 kDa or more, or about 75 kDa or more, or about 90 kDa or more, or about 100 kDa or more. In non-limiting examples, suitable copolymers of vinylpyridine and styrene have molecular weights 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.

[0365] Polydimethylsiloxane (PDMS) can be incorporated into any mass transport restriction film disclosed herein.

[0366] In some embodiments, the analyte sensor described herein may include a sensor tail that includes at least an NAD(P) reservoir, a permeable polymer covering the NAD(P) reservoir, a first permeable working electrode, a first active region deployed on the surface of the first working electrode, and a mass transport restriction membrane of a first analyte permeable to at least cover the first active region.

[0367] In certain embodiments, the first active area includes at least one NAD(P)-dependent enzyme responsive to the first analyte (optionally covalently bonded to a first polymer present within the active area). For example, but not by way of limitation, an analyte sensor described herein can include a sensor tail including at least an NAD(P) reservoir, a permeable polymer overcoating the NAD(P) reservoir, a first working electrode, an analyte-responsive active area including at least one NAD(P)-dependent enzyme disposed on a surface of the first working electrode, and a mass transport limiting membrane permeable to overcoat the analyte-responsive active area.

[0368] In certain embodiments, the first active area includes a first polymer and an enzyme responsive to a first analyte (e.g., glucose), such as an NAD(P)-dependent enzyme, optionally covalently bonded to the first polymer. For example, but not by way of limitation, an analyte sensor described herein can include a sensor tail including at least an NAD(P) reservoir, a first working electrode, a glucose-responsive active area including glucose dehydrogenase (optionally covalently bonded to a first polymer) disposed on a surface of the first working electrode, and a mass transport limiting membrane permeable to overcoat the glucose-responsive active area.

[0369] In certain embodiments, the first active area includes a first polymer and an enzyme system responsive to a first analyte (e.g., a ketone), the enzyme system including at least one enzyme, such as an NAD-dependent enzyme, optionally covalently bonded to the first polymer. For example, but not by way of limitation, an analyte sensor described herein can include a sensor tail including at least an NAD(P) reservoir, a first working electrode, a ketone-responsive active area including an enzyme system (the enzyme system including beta-hydroxybutyrate dehydrogenase and heart muscle yellow enzyme, one or both of which enzymes are covalently bonded to a polymer) disposed on a surface of the first working electrode, and a mass transport limiting membrane permeable to overcoat the ketone-responsive active area.

[0370] In some embodiments, when the first and second active regions configured for determining different analytes are deployed on separate working electrodes, the mass transport restriction membrane can have different permeability values ​​for the first and second analytes. For example, but not limited to, the mass transport restriction membrane covering at least one active region can include a mixture of a first membrane polymer and a second membrane polymer, or a bilayer of the first and second membrane polymers. A homogenized membrane can be used to cover uncoated active regions with a mixture or a bilayer, wherein the homogenized membrane comprises only one of the first or second membrane polymers. Advantageously, the architecture of the analyte sensor disclosed herein readily allows a continuous membrane having a homogenized membrane portion to be deployed on the first active region of the analyte sensor and a multi-component membrane portion to be deployed on the second active region of the analyte sensor, thereby concurrently utilizing the permeability value of each analyte to provide improved sensitivity and detection accuracy. In a particular embodiment, continuous membrane deposition can be performed via a sequential dip-coating operation.

[0371] In some embodiments, when multiple active regions are present, the mass transport restriction membrane may cover each active region. In some embodiments, the mass transport restriction layer is a membrane composed of a crosslinked polymer containing heterocyclic nitrogen groups, such as a polymer of polyvinylpyridine and polyvinylimidazole. Embodiments also include membranes made of polyurethane or polyether urethane or chemically related materials, or membranes made of silicone resins, etc. In some embodiments, the mass transport restriction membrane may include a membrane polymer, such as a polyvinylpyridine or polyvinylimidazole homopolymer or copolymer, which may be further crosslinked with a suitable crosslinking agent. In certain specific embodiments, the membrane polymer may include a copolymer of vinylpyridine and styrene.

[0372] In some embodiments, the mass transport restriction membrane may comprise a membrane polymer crosslinked with a crosslinking agent disclosed herein and in Section 5 above. In some embodiments where two mass transport restriction membranes are present (e.g., a first mass transport restriction membrane and a second mass transport restriction membrane), each membrane may be crosslinked with a different crosslinking agent. For example, but not as a limitation, the crosslinking agent may produce a membrane that restricts the diffusion of certain compounds (e.g., analytes within the membrane) more or less, for example, by influencing the size of the pores within the membrane. For example, but not as a limitation, in a sensor configured to detect ketones and glucose, the mass transport restriction membrane covering the ketone response region may have a pore size that restricts the diffusion of compounds larger than ketones (e.g., glucose) through the membrane.

[0373] In certain embodiments, crosslinking agents used in the present disclosure can include polyepoxide, carbodiimide, cyanuric chloride, triglycidyl glycerol, N-hydroxysuccinimide, imido ester, epichlorohydrin, or a derivatized variant thereof. In certain embodiments, the film polymer encapsulating one or more active regions can be crosslinked with a branched crosslinking agent, for example, which can reduce the amount of extractables available from a mass transport limiting film. Non-limiting examples of branched crosslinking agents include branched glycidyl ether crosslinking agents, for example, including branched glycidyl ether crosslinking agents including two or three or more crosslinkable groups. In certain embodiments, a branched crosslinking agent can include two or more crosslinkable groups, such as polyethylene glycol diglycidyl ether. In certain embodiments, a branched crosslinking agent can include three or more crosslinkable groups, such as polyethylene glycol tetraglycidyl ether. In certain embodiments, a mass transport limiting film can include polyvinylpyridine or a copolymer of vinylpyridine and styrene crosslinked with a branched glycidyl ether crosslinking agent including two or three crosslinkable groups, such as polyethylene glycol tetraglycidyl ether or polyethylene glycol diglycidyl ether. In certain embodiments, the epoxy groups of a polyepoxide (e.g., polyethylene glycol tetraglycidyl ether or polyethylene glycol diglycidyl ether) can form covalent bonds with pyridine or imidazole through epoxide ring opening, resulting in hydroxylalkyl bridged crosslinking agents to the main body of the film polymer to the heterocycle.

[0374] In certain embodiments, the crosslinking agent is polyethylene glycol diglycidyl ether (PEGDGE). In certain embodiments, PEGDGE used to facilitate crosslinking (e.g., intermolecular crosslinking) between two or more film 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 5,000 g / mol. The number of ethylene glycol repeat units in each arm of PEGDGE can be the same or different and can generally vary within a given sample to provide an average molecular weight. In certain embodiments, PEGDGE used in the present disclosure has an average molecular weight (Mn) of about 200 to 1,000 (e.g., about 400). n ) In certain embodiments, the crosslinking agent is PEGDGE 400.

[0375] In certain embodiments, polyethylene glycol tetraglycidyl ether for facilitating 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 with 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 1,000 g / mol to about 5,000 g / mol. The number of ethylene glycol repeat units in each arm of the polyethylene glycol tetraglycidyl ether can be the same or different and can generally vary within a given sample to provide an average molecular weight. In certain embodiments, the mass transport limiting membrane can be deposited directly onto the active area.

[0376] In certain embodiments, the thickness, e.g., dry thickness, of the mass transport limiting membrane ranges from about 0.1 pm to about 1,000 pm, e.g., from about 1 pm to about 500 pm, from about 10 pm to about 100 pm, or from about 10 pm to about 100 pm. In certain embodiments, the thickness of the mass transport limiting membrane can be from about 0.1 pm to about 10 pm, e.g., from about 0.5 pm to about 10 pm, from about 1 pm to about 10 pm, from about 1 pm to about 5 pm, or from about 0.1 pm to about 5 pm. In certain embodiments, the sensor can be immersed in the mass transport limiting membrane solution more than once. For example, and not by way of limitation, the sensor (or working electrode) of the present disclosure can be immersed in the interferent domain solution at least twice, at least three times, at least four times, or at least five times to achieve the desired interferent domain thickness.

[0377] 7. Interferent domain

[0378] In certain embodiments, the sensor, e.g., sensor tail, of the present disclosure can also include an interferent domain. In certain embodiments, the interferent domain can include a polymer domain that limits the flow of one or more interferents to the surface of, e.g., the working electrode. In certain embodiments, the interferent domain can function as a molecular sieve that allows passage of analytes and other substances to be measured by the working electrode while blocking passage of other substances, such as interferents. In certain embodiments, the interferents can affect the signal obtained at the working electrode. Non-limiting examples of interferents include acetaminophen, ascorbate, ascorbic acid, bilirubin, cholesterol, creatinine, dopamine, ephedrine, ibuprofen, levodopa, methyldopa, salicylate, tetracycline, tolazamide, tolbutamide, triglycerides, urea, and uric acid.

[0379] In certain embodiments, the interference domain is positioned between the working electrode and one or more active areas, e.g., ketone-responsive active areas. 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 void size. In certain embodiments, the interference domain is formed from one or more cellulose derivatives. Non-limiting examples of cellulose derivatives include polymers such as cellulose acetate, cellulose acetate butyrate, 2-hydroxyethyl cellulose, cellulose acetate phthalate, cellulose acetate propionate, cellulose acetate trimellitate, and the like.

[0380] In certain embodiments, the interference domain is part of the mass transport limiting membrane rather than a separate membrane.

[0381] In certain embodiments, the interference domain includes a hydrophobic film that is non-swellable and limits diffusion of high molecular weight species. For example, but not by way of limitation, the interference domain can permeate relatively low molecular weight species (such as hydrogen peroxide) while limiting passage of higher molecular weight species (such as ketones, glucose, acetaminophen, and / or ascorbic acid).

[0382] In certain embodiments, the interference domain can be deposited directly onto the working electrode, e.g., onto the surface of a permeable working electrode. In certain embodiments, the thickness (e.g., dry thickness) of the interference domain ranges from about 0.1 pm to about 1,000 pm, e.g., from about 1 pm to about 500 pm, from about 10 pm to about 100 pm, or from about 10 pm to about 100 pm. In certain embodiments, the interference domain can have a thickness of from about 0.1 pm to about 10 pm, e.g., from about 0.5 pm to about 10 pm, from about 1 pm to about 10 pm, from about 1 pm to about 5 pm, or from about 0.1 pm to about 5 pm. In certain embodiments, the sensor can be immersed in the interference domain solution more than once. For example, but not by way of limitation, a sensor (or working electrode) of the present disclosure can be immersed in the interference domain solution at least twice, at least three times, at least four times, or at least five times to achieve a desired thickness of the interference domain.

[0383] 8. Manufacturing

[0384] The present disclosure also provides methods for manufacturing the presently disclosed analyte sensors including one or more active areas, one or more NAD(P) reservoirs, and one or more working electrodes.

[0385] In certain embodiments, the method includes depositing a composition containing NAD(P) on a substrate to generate an NAD(P) reservoir. For example, but not by way of limitation, the composition can be NAD and / or NADP, depending on the enzymes present in the active area. In certain embodiments, the method can also include adding a permeable layer on top of the NAD(P) reservoir. In certain embodiments, the permeable layer can include a polymer that controls the release of NAD(P) from the reservoir. Alternatively, a composition including a polymer and NAD(P) can be deposited onto a substrate to generate an NAD(P) reservoir. In certain embodiments, the polymer of the NAD(P) reservoir is curable, for example, UV curable.

[0386] In certain embodiments, the method can also include creating a permeable working electrode, for example, a carbon nanotube electrode, on the permeable layer.

[0387] In certain embodiments, the method can also include depositing an enzyme composition including one or more NAD(P)-dependent enzymes (for example, NAD(P)-dependent dehydrogenases) on the working electrode. In certain embodiments, the enzyme composition can include one or more additional enzymes (for example, diaphorase), a crosslinking agent (for example, polyethylene glycol diglycidyl ether), a polymer, and / or a redox mediator. In certain embodiments, the enzyme composition can be deposited on the surface of the working electrode as one large patch covering the desired portion of the working electrode, or in the form of an array of multiple enzyme compositions, for example, spaced apart from one another, to generate one or more active areas for detecting one or more analytes. In certain embodiments, the method can also include curing the enzyme composition.

[0388] In certain embodiments, the NAD(P), permeable polymer, permeable working electrode, and enzyme composition can be prepared as a solution that dries or cures upon deposition to solidify. Thus, in certain embodiments, all layers can be deposited in an automated fashion using small volume liquid handling or similar techniques used for high throughput sensor manufacturing.

[0389] In certain embodiments, the method can also include adding a film composition on top of the cured enzyme composition and / or around the entire sensor. In certain embodiments, the film composition can include a polymer (for example, polyvinylpyridine), and / or a crosslinking agent (for example, polyethylene glycol diglycidyl ether). In certain embodiments, the method can include curing the film polymer composition.

[0390] Generally, the thickness of the film is controlled by the concentration of the film solution, by the number of droplets of the film solution applied, by the number of times the sensor is dipped or sprayed with the film solution, by the volume of the film solution sprayed on the sensor, and the like, and by any combination of these factors. In certain embodiments, the films described herein can have a thickness in the range of, for example, about 0.1 micrometers (pm) to about 1,000 pm, about 1 pm to about 500 pm, about 10 pm to about 100 pm, or about 10 pm to about 100 pm. In certain embodiments, the sensor can be dipped in the film solution more than once. For example, and without limitation, the sensor (or working electrode) of the present disclosure can be dipped in the film solution at least twice, at least three times, at least four times, or at least five times to achieve the desired film thickness.

[0391] In certain embodiments, the film can cover one or more active areas, and in certain embodiments, the active areas can have a thickness of about 0.1 pm to about 10 pm, for example, about 0.5 pm to about 10 pm, about 1 pm to about 10 pm, about 1 pm to about 5 pm, or about 0.1 pm to about 5 pm. In certain embodiments, a series of droplets can be applied sequentially on top to achieve the desired thickness of the active area and / or film without significantly increasing the diameter of the applied droplets (i.e., maintaining the desired diameter or range thereof). In certain embodiments, each individual droplet can be applied and then allowed to cool or dry, followed by one or more additional droplets. For example, and without limitation, at least one drop, at least two drops, at least three drops, at least four drops, or at least five drops are added sequentially on top to achieve the desired thickness of the active area.

[0392] III. Analyte Monitoring

[0393] The present disclosure also provides methods of detecting an analyte in vivo using the analyte sensors disclosed herein. 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 by way of limitation, the present disclosure provides methods for detecting one or more analytes using one or more NAD(P)-dependent enzymes, including glucose, ketones, lactate, oxygen, hemoglobin A1C, albumin, alcohol, alkaline phosphatase, alanine transaminase, aspartate transaminase, bilirubin, blood urea nitrogen, calcium, carbon dioxide, chloride, creatinine, hematocrit, lactate, magnesium, oxygen, pH, phosphorus, potassium, sodium, total protein, and / or uric acid. 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 by way of limitation, the present disclosure provides methods for detecting one or more ketones. In certain embodiments, the present disclosure provides methods for detecting glucose. In certain embodiments, the present disclosure provides methods for detecting creatinine. In certain embodiments, the present disclosure provides methods for detecting lactate. In certain embodiments, the present disclosure provides methods for detecting alcohol.

[0394] In certain embodiments, the present disclosure provides methods for monitoring in vivo levels of an analyte over time using an analyte sensor comprising an NAD(P) reservoir and one or more NAD(P)-dependent enzymes, such as an NAD(P)-dependent dehydrogenase. Generally, monitoring in vivo concentrations of an analyte in a subject's bodily fluid includes inserting an in vivo analyte sensor disclosed herein at least partially beneath the surface of the skin, contacting the fluid to be monitored (interstitial, blood, dermis, 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, forwarded, and / or otherwise processed. A variety of methods can be employed to determine the concentration of an analyte (e.g., glucose, alcohol, ketones, and / or lactate) with the current sensor. In certain embodiments, monitoring the concentration of an analyte using the sensor signal can be performed by coulometry, amperometry, voltammetry, potentiometry, or any other convenient electrochemical detection technique.

[0395] In some embodiments, a method for detecting an analyte includes: (i) providing an analyte sensor including: (a) an internal supply of NAD(P); (b) a permeable polymer covering the internal supply of NAD(P); (c) at least a first working electrode deployed on the surface of the permeable polymer, wherein the first working electrode is a permeable working electrode; (d) an analyte-responsive active region deployed on the surface of the first working electrode, wherein the analyte-responsive active region includes an NAD(P)-dependent enzyme; and (e) a mass transport restriction membrane permeable to the analyte, at least covering the analyte-responsive region; (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, the first signal being proportional to the concentration of a first analyte in a fluid contacting the analyte-responsive active region; and (iv) correlating the first signal with the concentration of the first analyte in the fluid.

[0396] In some embodiments, a method for detecting one or more ketones includes: (i) providing an analyte sensor including: (a) an internal supply of NAD(P); (b) a permeable polymer covering the internal supply of NAD(P); (c) at least a first working electrode deployed on the surface of the permeable polymer, wherein the first working electrode is a permeable working electrode; (d) a ketone-responsive active region deployed on the surface of the first working electrode, wherein the analyte-responsive active region includes β-hydroxybutyrate dehydrogenase and myocardial flavin enzyme; and (e) a ketone-permeable mass transport restriction membrane covering at least the analyte-responsive region; (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 with the concentration of the ketone in the fluid.

[0397] In certain embodiments, the methods of the present disclosure can further comprise detecting a second analyte by providing an analyte sensor comprising a second active area and / or exposing an analyte sensor comprising a second active area to a fluid comprising a first analyte and a second analyte. In certain embodiments, the analyte sensor for use in the method of detecting a first analyte and a second analyte can further comprise a second working electrode; a second active area disposed on a surface of the second working electrode and responsive to a second analyte different from the first analyte, wherein the second active area comprises a second polymer, at least one enzyme responsive to the second analyte covalently bonded to the second polymer and, optionally, a redox mediator covalently bonded to the second polymer; wherein a portion (e.g., a second portion) of the mass transport limiting membrane encases the second active area. Alternatively, the second active site can be covered by a second mass transport limiting membrane that is separate and / or different from the mass transport limiting membrane that encases the ketone-responsive active area. In certain embodiments, the at least one enzyme responsive to the second analyte comprises an enzyme system comprising a plurality of enzymes collectively responsive to the second analyte.

[0398] In certain embodiments, the method further comprises attaching an electronics unit to the skin of the patient, coupling the conductive contacts of the electronics unit to the contacts of the sensor, using the electronics unit to collect data about the analyte level from signals generated by the sensor, relaying 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 comprises an application related to the analyte being monitored. In certain embodiments, the analyte information is relayed by an RFID protocol, such as Bluetooth, etc.

[0399] In certain embodiments, the analyte sensor can be positioned within a user for automatic analyte sensing, e.g., continuously or periodically. In certain embodiments, the level of the analyte can be monitored over a period of seconds to minutes, hours, days, weeks, or months. In certain embodiments, the methods disclosed herein can be used to predict future levels of the analyte, such as but not limited to the current analyte level at time zero, and the rate or amount of change in the analyte concentration, based on the information obtained.

[0400] IV. Exemplary Embodiments

[0401] A. In certain non-limiting embodiments, the presently disclosed subject matter provides an analyte sensor comprising:

[0402] (i) an internal supply of NAD(P);

[0403] (ii) a permeable polymer encasing the internal supply of NAD(P);

[0404] (iii) at least a first working electrode disposed on a surface of the permeable polymer, wherein the first working electrode is a permeable working electrode;

[0405] (iv) an analyte-responsive active area disposed on a surface of the first working electrode, wherein the analyte-responsive active area comprises an NAD(P)-dependent enzyme; and

[0406] (v) a mass transport limiting membrane permeable to the analyte covering at least the analyte-responsive area.

[0407] Al. The analyte sensor of A, wherein the NAD(P)-dependent enzyme is an NAD(P)- dependent dehydrogenase.

[0408] A2. The analyte sensor of A or Al, wherein the permeable working electrode comprises carbon nanotubes.

[0409] A3. The analyte sensor of any one of A-A2, wherein the permeable polymer comprises poly(propylene glycol) methacrylate and 2-hydroxyethyl methacrylate.

[0410] A4. The analyte sensor of any one of A-A3, wherein the analyte is selected from the group consisting of glucose, a ketone, an alcohol, creatinine, lactate, and combinations thereof.

[0411] A5. The analyte sensor of A4, wherein the analyte is glucose.

[0412] A6. The analyte sensor of A4, wherein the analyte is lactate.

[0413] A7. The analyte sensor of A4, wherein the analyte is an alcohol.

[0414] A8. The analyte sensor of A4, wherein the analyte is a ketone.

[0415] A9. The analyte sensor of A5, wherein the NAD(P)-dependent enzyme is a glucose dehydrogenase.

[0416] A10. The analyte sensor of A6, wherein the NAD(P)-dependent enzyme is a lactate dehydrogenase.

[0417] Al l. The analyte sensor of A7, wherein the NAD(P)-dependent enzyme is an alcohol dehydrogenase.

[0418] A12. The analyte sensor of A8, wherein the NAD(P)-dependent enzyme is a beta-hydroxybutyrate dehydrogenase.

[0419] A13. The analyte sensor of any one of A-A12, wherein the analyte-responsive active area further comprises diaphorase.

[0420] A14. The analyte sensor of any of A-A13, wherein the analyte-responsive active area further comprises a redox mediator.

[0421] A15. The analyte sensor of any of A-A14, wherein the analyte-responsive active area further comprises a stabilizer.

[0422] A16. The analyte sensor of A15, wherein the stabilizer comprises albumin.

[0423] A17. The analyte sensor of any of A-A16, wherein the analyte-responsive active area further comprises a cross-linking agent.

[0424] A18. The analyte sensor of any of A-A17, wherein the internal supply of NAD(P) comprises about 1 pg to about 1,000 pg of NAD(P).

[0425] A19. The analyte sensor of any 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., vinylpyridine and styrene), polyacrylate, polyurethane, polyether polyurethane, silicone, or a combination thereof.

[0426] A20. The analyte sensor of any of A-A19, further comprising:

[0427] (vi) a second working electrode; and

[0428] (vii) a second active area disposed on a surface of the second working electrode and responsive to a second analyte different from the first analyte, wherein the second active area comprises at least one enzyme responsive to the second analyte;

[0429] wherein the second portion of the mass transport limiting membrane covers the second active area.

[0430] B. In certain non-limiting embodiments, the presently disclosed subject matter provides a method of detecting an analyte, comprising:

[0431] (i) providing an analyte sensor comprising:

[0432] (a) an internal supply of NAD(P);

[0433] (b) a permeable polymer covering the internal supply of NAD(P);

[0434] (c) at least a first working electrode disposed on a surface of the permeable polymer, wherein the first working electrode is a permeable working electrode;

[0435] (d) an analyte-responsive active area disposed on a surface of the first working electrode, wherein the analyte-responsive active area comprises an NAD(P)-dependent enzyme; and

[0436] (e) a mass transport limiting membrane permeable to the analyte covering at least the analyte-responsive active area;

[0437]

[0438] (ii) applying a potential to the first working electrode;

[0439] (iii) obtaining a first signal at or above a redox potential of the first active area, the first signal being proportional to a concentration of a first analyte in a fluid contacting the first active area; and

[0440] (iv) correlating the first signal to the concentration of the first analyte in the fluid.

[0441] B1. The method of B, wherein the NAD(P)-dependent enzyme is an NAD(P)- dependent dehydrogenase.

[0442] B2. The method of B or B1, wherein the permeable working electrode comprises carbon nanotubes.

[0443] B3. The method of any of B-B2, wherein the permeable polymer comprises poly(propylene glycol) methacrylate and 2-hydroxyethyl methacrylate.

[0444] B4. The method of any of B-B3, wherein the analyte is selected from the group consisting of glucose, a ketone, an alcohol, lactate, and combinations thereof.

[0445] B5. The method of B4, wherein the analyte is glucose.

[0446] B6. The method of B4, wherein the analyte is lactate.

[0447] B7. The method of B4, wherein the analyte is an alcohol.

[0448] B8. The method of B4, wherein the analyte is a ketone.

[0449] B9. The method of B5, wherein the NAD(P)-dependent enzyme is glucose dehydrogenase.

[0450] B10. The method of B6, wherein the NAD(P)-dependent enzyme is lactate dehydrogenase.

[0451] B11. The method of B7, wherein the NAD(P)-dependent enzyme is alcohol dehydrogenase.

[0452] B12. The method of B8, wherein the NAD(P)-dependent enzyme is beta-hydroxybutyrate dehydrogenase.

[0453] ​B13. The method of any of B-B12, wherein the analyte-responsive active area further comprises diaphorase.

[0454] B14. The method of any of B-B13, wherein the analyte-responsive active area further comprises a redox mediator.

[0455] B15. The method of any of B-B14, wherein the analyte-responsive active area further comprises a stabilizer.

[0456] B16. The method of B15, wherein the stabilizer comprises albumin.

[0457] B17. The method of any of B-B16, wherein the analyte-responsive active area further comprises a cross-linking agent.

[0458] B18. The method of any of B-B17, wherein the internal supply of NAD(P) comprises about 1 pg to about 1,000 pg of NAD(P).

[0459] B19. The method of any of B-B18, wherein the mass transport limiting membrane comprises polyvinylpyridine (e.g., poly(4-vinylpyridine) or poly(4-vinylpyridine)), polyvinylimidazole, polyvinylpyridine copolymers (e.g., copolymers of vinylpyridine and styrene), polyacrylate, polyurethane, polyether polyurethane, silicone, or combinations thereof.

[0460] B20. The method of any of B-B14, wherein the analyte sensor further comprises:

[0461] (f) a second working electrode; and

[0462] (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, wherein the second active area comprises at least one enzyme responsive to the second analyte;

[0463] wherein the second portion of the mass transport limiting membrane covers the second active area.

[0464] Examples

[0465] The presently disclosed subject matter will be better understood by reference to the following examples, which are offered by way of illustration of the presently disclosed subject matter and not by way of limitation.

[0466] Example 1: Preparation of a Polymer-Controlled NAD Release System

[0467] This example provides a method of manufacturing a sensor having an NAD reservoir as shown in FIG. 23A Example 1: Preparation of a Polymer-Controlled NAD Release System

[0468] An analyte sensor was prepared by depositing various solutions. An NAD solution was first deposited on a thin plastic substrate (support layer) and allowed to dry, leaving solid NAD. Subsequently, a polymer solution consisting of a mixture of poly(propylene glycol) methacrylate (POMA) and 2-hydroxyethyl methacrylate (HEMA) was deposited on the solid NAD and polymerized by UV exposure. A carbon nanotube solution was then deposited and allowed to dry, forming a permeable electrode. A ketone sensing enzyme composition including an enzyme system containing diaphorase and beta-hydroxybutyrate dehydrogenase was then deposited onto the permeable electrode. Finally, the electrode was sectioned and dip-coated in a membrane solution including polyvinylpyridine and polystyrene copolymer and a cross-linking agent to form an outer membrane. A control sensor was similarly manufactured, except that there was no NAD deposited on the thin plastic substrate FIG. 23B

[0469] The sensors and controls were then evaluated for response with 2 mM beta-hydroxybutyrate, which was used as a surrogate for ketones present in vivo. As shown in FIG. 24 Without being bound by theory, it is believed that the outer membrane of the sensor can allow leaching of NAD from the sensing layer, resulting in a decrease in ketone response over time, as NAD is required to facilitate the flow of electrons from the analyte to the electrode. As shown in this example, the use of an NAD reservoir can overcome such limitations by allowing for the continuous release of NAD from the reservoir to maintain sufficient NAD concentration for use by the NAD-dependent enzymes in the sensing layer.

[0470] ***

[0471] 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 herein without departing from the spirit and scope of the disclosed subject matter. Moreover, the scope of the present application is not intended to be limited to the particular embodiments of the process, machine, manufacture, composition of matter, means, methods and steps described in the specification. As one of ordinary skill in the art will readily appreciate from the disclosure of the presently disclosed subject matter, processes, machines, manufacture, compositions of matter, methods, or steps, presently existing or later to be developed, that perform substantially the same function or achieve substantially the same result as the corresponding embodiments described herein can be utilized. Accordingly, the appended claims are intended to include within their scope such processes, machines, manufacture, compositions of matter, methods, or steps.

[0472] ​Throughout this application various patents, patent applications, publications, product descriptions, protocols and sequence accession numbers have been referenced. The disclosures of the above publications in their entireties are hereby incorporated by reference for all purposes.

Claims

1. An analyte sensor, comprising: (i) Internal supply of NAD(P); (ii) Permeable polymers that cover the internal supply of NAD(P); (iii) At least a first working electrode is deployed on the surface of a permeable polymer, wherein the first working electrode is a permeable working electrode; (iv) An analyte-responsive active region, deployed on the surface of the first working electrode, wherein the analyte-responsive active region includes an NAD(P)-dependent enzyme. as well as (v) A mass transport restriction membrane permeable to the analyte, covering at least the analyte response-active region.

2. The analyte sensor of claim 1, wherein the NAD(P)-dependent enzyme is an NAD(P)-dependent dehydrogenase.

3. The analyte sensor of claim 1, wherein the permeable working electrode comprises carbon nanotubes.

4. The analyte sensor of claim 1, wherein the permeable polymer comprises a polyether-based polymer.

5. The analyte sensor of claim 1, wherein the analyte is selected from glucose, ketones, alcohols, lactates, and combinations thereof.

6. The analyte sensor of claim 5, wherein the NAD(P)-dependent enzyme is glucose dehydrogenase, lactate dehydrogenase, alcohol dehydrogenase, or β-hydroxybutyrate dehydrogenase.

7. The analyte sensor of claim 6, wherein the analyte-responsive active region further comprises myocardial flavin.

8. The analyte sensor of claim 1, wherein the analyte-responsive region further comprises a redox mediator.

9. The analyte sensor as claimed in claim 1, further comprising: (vi) Second working electrode; as well as (vii) A second active region, deployed on the surface of the second working electrode and responsive to a second analyte different from the first analyte, wherein the second active region includes at least one enzyme responsive to the second analyte; The second part of the mass transport restriction membrane covers the second active region.

10. A method for detecting an analyte, the method comprising: (i) Provide an analyte sensor, including: (a) Internal supply of NAD(P); (b) Permeable polymers that cover the internal supply of NAD(P); (c) At least a first working electrode is deployed on the surface of a permeable polymer, wherein the first working electrode is a permeable working electrode; (d) An analyte-responsive active region, deployed on the surface of the first working electrode, wherein the analyte-responsive active region includes an NAD(P)-dependent enzyme; and (e) A mass transport restriction membrane permeable to the analyte, covering at least the analyte-responsive region; (ii) Apply a potential to the first working electrode; (iii) Obtaining a first signal at or above the redox potential of the first active region, the first signal being proportional to the concentration of a first analyte in the fluid contacting the first active region; and (iv) Correlate the first signal with the concentration of the first analyte in the fluid.

11. The method of claim 10, wherein the NAD(P)-dependent enzyme is an NAD(P)-dependent dehydrogenase.

12. The method of claim 10, wherein the permeable working electrode comprises carbon nanotubes.

13. The method of claim 10, wherein the permeable polymer comprises a polyether-based polymer.

14. The method of claim 10, wherein the analyte is selected from glucose, ketones, alcohols, lactates, and combinations thereof.

15. The method of claim 14, wherein the NAD(P)-dependent enzyme is glucose dehydrogenase, lactate dehydrogenase, alcohol dehydrogenase, or β-hydroxybutyrate dehydrogenase.

16. The method of claim 15, wherein the analyte-responsive active region further comprises myocardial flavin.

17. The method of claim 10, wherein the analyte responsive region further comprises a redox mediator.

18. The method of claim 10, wherein the analyte sensor further comprises: (f) Second working electrode; as well as (g) A second active region, deployed on the surface of the second working electrode and responsive to a second analyte different from the first analyte, wherein the second active region includes at least one enzyme responsive to the second analyte; The second part of the mass transport restriction membrane covers the second active region.

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