Analyte sensors for sensing ketones and methods of using the same

Abstract

To provide an analyte sensor for use in detecting ketones.SOLUTION: In certain embodiments, a ketones-responsive active site of a presently disclosed analyte sensor includes an enzyme system comprising β-hydroxybutyrate dehydrogenase and NADH oxidase disposed on a surface of a platinum working electrode. The present disclosure further provides methods for detecting ketones using the disclosed analyte sensors.SELECTED DRAWING: Figure 1A

Description

Technical Field

[0001] The subject matter described herein relates to an analyte sensor for detecting ketones and methods of using the same.

Background Art

[0002] The detection of various analytes in an individual can be important for monitoring the health of the individual, as deviations from normal analyte levels can sometimes be indicative of a physiological condition. For example, monitoring ketone levels can enable a person suffering from diabetes to receive appropriate corrective measures to avoid serious physiological distress due to ketoacidosis. Other analytes may also be desirable for monitoring other physiological conditions. In some cases, it may be desirable to monitor two or more analytes to monitor multiple physiological conditions, particularly if the individual is suffering from a coexisting condition that results in a simultaneous dysregulation of two or more analytes, especially when combined with each other.

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

[0004] Many analytes represent attractive targets for physiological analysis on the premise that suitable detection chemistries can be identified. For this purpose, enzyme-based amperometric sensors configured to continuously assay glucose in vivo have been developed and refined over recent years to assist in monitoring the health of diabetic individuals. Other analytes that are commonly prone to concurrent dysregulation along with glucose in diabetic individuals include, for example, lactate, oxygen, A1c, ketones, and others. Apart from glucose dysregulation, it may also be desirable to monitor these and other analytes. Analyte sensors configured to detect analytes other than glucose in vivo are known, but currently are not very well refined. For example, low sensitivity to analytes present in low abundance can be a particular problem. Accordingly, there is a need in the art for improved sensors for detecting ketones in vivo. SUMMARY OF THE INVENTION

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

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

[0007] In certain embodiments, the ketone-responsive active region further includes a stabilizer and / or a crosslinking agent. In certain embodiments, one or more of the enzymes in the enzyme system comprising β-hydroxybutyrate dehydrogenase and NADH oxidase are covalently bound to a stabilizer in the ketone-responsive active region. In certain embodiments, one or more of the enzymes in the enzyme system comprising β-hydroxybutyrate dehydrogenase and NADH oxidase are covalently bound to a polymer in the ketone-responsive active region. In certain embodiments, the mass transfer limiting membrane comprises polyvinylpyridine, polyvinylimidazole, a polyvinylpyridine copolymer, polyacrylate, polyurethane, polyetherurethane, or a combination thereof. In certain embodiments, the mass transfer limiting membrane comprises polyvinylpyridine. Alternatively or additionally, the mass transfer limiting membrane comprises a copolymer of vinylpyridine and styrene.

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

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

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

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

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

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

[0014] In certain embodiments, the test substance sensors of the present disclosure are for use in a subject in need thereof. For example, without limitation, the subject may be a subject with diabetes. In certain embodiments, the subject may be on a ketogenic diet. In certain embodiments, the subject is in a state of ketosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The following figures are included to illustrate certain aspects of the present disclosure and should not be considered exclusive embodiments. The disclosed subject matter is capable of considerable modification, alteration, combination, and equivalents in form and function without departing from the scope of the present disclosure.

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DETAILED DESCRIPTION OF THE INVENTION

[0016] The present disclosure generally describes an analyte sensor employing one or more enzymes for the detection of an analyte. For example, but not limited to, the present disclosure provides an analyte sensor employing a number of enzymes for the detection of an analyte, such as one or ketones. In certain embodiments, the present disclosure further provides an analyte sensor employing a number of enzymes for the detection of two different analytes, such as ketones and a second analyte, such as glucose. Depending on the configuration of the sensor, the analyte sensor of the present disclosure can be configured to detect one analyte or a number of analytes simultaneously or substantially simultaneously. The present disclosure further provides a method for detecting one or more analytes using the disclosed analyte sensor.

[0017] Glucose-responsive analytes sensors have been well studied and are still an area of development to help diabetic individuals better manage their health. Despite the frequent dysregulation of analytes in diabetic individuals, sensor chemistries suitable for detecting ketones and other analytes that are generally dysregulated are significantly lagging behind the more developed glucose detection chemistries. The present disclosure alleviates this deficiency by providing a sensor chemistry suitable for detecting ketones with good response stability over a wide range of ketone concentrations, in particular a detection chemistry that utilizes an enzyme system comprising at least two enzymes that can act in concert to facilitate the detection of ketones. As used herein, the term "in concert" means a coupled enzyme reaction in which the product of a first enzyme reaction becomes the substrate of a second enzyme reaction, and the second enzyme reaction serves as a basis for measuring the concentration of the substrate (e.g., analyte) that reacted during the first enzyme reaction. In certain embodiments, the product and / or substrate of the reaction may be the reduced and / or oxidized form of a cofactor or coenzyme of the enzymes of the enzyme system, such as NAD or NADP. Although defined with respect to two coupled enzyme reactions, it should be recognized that in some cases three or more coupled enzyme reactions may also occur. For example, the product of a first enzyme reaction may become the substrate of a second enzyme reaction, the product of the second enzyme reaction may become the substrate of a third enzyme reaction, and the third enzyme reaction may serve as a basis for measuring the concentration of the substrate (e.g., analyte) that reacted during the first enzyme reaction. A discussion of enzyme systems suitable for detecting ketones according to the present disclosure is presented below.

[0018] For clarity, but not by way of limitation, the detailed description of the subject matter disclosed herein is divided into the following subsections. I. Definitions; and II. Analyte Sensors; 1. General Structure of Analyte Sensor Systems; 2. Enzymes; 3. Redox Mediators; 4. Polymer Backbones; 5. Mass Transfer Limiting Membranes; 6. Interference domain; and 7. Manufacture; III. Method of use; and IV. Exemplary embodiments I. Definitions The terms used herein generally have their ordinary meaning in the art, within the context of the present disclosure and in the particular context in which each term is used. To provide further guidance to the practitioner in the description of the compositions and methods of the present disclosure and the methods of making and using them, certain terms are discussed below or elsewhere in this specification.

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

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

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

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

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

[0024] As used herein, the term "electrolysis" means the direct electrolytic oxidation or reduction of a compound at an electrode or via one or more electron transfer agents (redox mediators or enzymes).

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

[0026] As used herein, the term "polyvinylpyridine-based polymer" means a polymer or copolymer comprising polyvinylpyridine (e.g., poly(2-vinylpyridine) or poly(4-vinylpyridine)) or derivatives thereof.

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

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

[0029] II. Analyte Sensor Before describing the subject matter in detail, it should be understood that the present disclosure is not limited to the specific embodiments described and may, of course, vary. It should also be understood that the terms used herein are for the purpose of describing only the specific embodiments and are not intended to be limiting, since the scope of the present disclosure is limited only by the appended claims.

[0030] The publications discussed in this specification merely provide disclosure prior to the filing date of the present application. Nothing in this specification should be construed as an admission that the present disclosure is not entitled to antedate such publications by virtue of prior disclosure. Further, the provided publication dates may be different from the actual publication dates, which may need to be independently verified.

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

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

[0033] Furthermore, for each one of the embodiments of the methods disclosed herein, systems and devices capable of performing each of these embodiments are encompassed 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), a power source, communication circuitry, a transmitter, a receiver, a processor, and / or a controller (e.g., for executing instructions) capable of performing or facilitating the execution of any method step. These embodiments of sensor control devices can be used to perform the steps executed by the sensor control device from any of the methods described herein and can be capable of being used.

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

[0035] As described above, some embodiments of systems, devices, and methods are described herein that provide improved assembly and use of a skin sensor insertion device for use with an in vivo analyte monitoring system. In particular, some embodiments of the present disclosure are designed to improve a sensor insertion method for an in vivo analyte monitoring system and, in particular, to prevent premature retraction of the insertion tip during the sensor insertion process. For example, some embodiments include a skin sensor insertion mechanism with an increased firing speed and a slower tip retraction. In other embodiments, the tip retraction mechanism can be made to operate by actuation, such that the tip does not retract until the user withdraws the applicator from the skin. As a result, these embodiments can, among other advantages, reduce the likelihood that the insertion tip will be prematurely retracted 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. Some embodiments of the present disclosure also provide an improved insertion tip module that accounts for small-scale skin sensors and relatively shallow insertion paths present in the target skin layer. Additionally, some embodiments of the present disclosure are designed to prevent unwanted axial and / or rotational movement of the applicator components during sensor insertion. Thus, these embodiments can, among other advantages, reduce the instability of the placed skin sensor, irritation at the insertion site, damage to surrounding tissue, and the likelihood of capillary disruption resulting in contamination of the skin fluid with blood. Further, to reduce inaccurate sensor readings that can occur due to injury at the insertion site, some embodiments of the present disclosure can reduce the penetration of the needle to the end depth of the sensor chip during insertion.

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

[0037] There are various types of in vivo analyte monitoring systems. For example, a "continuous analyte monitoring" system (or "continuous glucose monitoring" system) can automatically transmit data from a sensor control device to a reader device continuously, for example according to a schedule, without instruction. 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, for example by means of a near field communication (NFC) or radio frequency identification (RFID) protocol. An in vivo analyte monitoring system can also operate without the need for fingerstick calibration.

[0038] An in vivo analyte monitoring system can be distinguished from an "in vitro" system that contacts a biological sample outside the body (i.e., "ex vivo") and generally includes a measuring device having a port for receiving an analyte test strip that transports a body fluid that can be analyzed to determine a user's blood glucose level.

[0039] An in vivo monitoring system can include a sensor that contacts a user's body fluid while disposed in vivo and detects the level of an analyte contained therein. The sensor can be part of a sensor control device that is present on the user's body and includes electronic equipment and a power source that enables and controls the detection of the analyte. The sensor control device and variations thereof can also be referred to as, among other things, 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.

[0040] An in vivo monitoring system may also include a device that receives the detected analyte data from the sensor control device and processes and / or presents the detected analyte data to the user in any number of forms. This device and its variations may be referred to, among other things, as a "handheld reader device", a "reader device" (or simply a "reader"), a "handheld electronic device" (or simply a "handheld"), a "portable data processing" device or unit, a "data receiver", a "receiver" device or unit (or simply a "receiver"), or a "remote" device or unit. Other devices such as personal computers are also utilized with or incorporated into in vivo and in vitro monitoring systems.

[0041] 1. General Structure of Analyte Sensor System A. Exemplary In Vivo Analyte Monitoring System Before describing the analyte sensors and their components of the present disclosure in more detail, a brief overview of the configuration of a suitable in vivo analyte sensor and the sensor system employing the analyte sensor is provided so that the embodiments of the present disclosure can be better understood.

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

[0043] FIG. 1B shows the operating environment of a test substance monitoring system 100a that can implement the technology described herein. The test substance monitoring system 100a includes a system of components designed to provide monitoring of parameters such as test substance levels in a human or animal body, or can provide other operations based on various component configurations. As implemented herein, the system can include a low-power test substance sensor 110 or simply a "sensor" that is worn by the user or attached to the body from which information is collected. As implemented herein, the test substance sensor 110 can be a sealed disposable device having a predetermined active service life (e.g., 1 day, 14 days, 30 days, etc.). The sensor 110 can be attached to the skin of the user's body and can be designed to remain adhered throughout the life of the sensor or to be selectively removed and retain functionality when reattached. The low-power test substance monitoring system 100a can further include a data reader device 120 or a multi-purpose data receiving device 130 configured as described herein to facilitate the retrieval and delivery of data including test substance data from the test substance sensor 110.

[0044] As embodied herein, the test substance monitoring system 100a may be provided to a third party via, for example, a remote application server 150 or an application storefront server 160, and may be incorporated into a multi-purpose hardware device 130 such as a mobile phone, a tablet, a personal computing device, or other similar computing devices capable of communicating with the test substance sensor 110 via a communication link. The multi-purpose hardware may further include an embedded device including, but not limited to, an insulin pump or an insulin pen having an embedded library configured to communicate with the test substance sensor 110. The illustrated embodiment of the test substance monitoring system 100a includes only one of each of the illustrated devices, but the present disclosure contemplates that the test substance monitoring system 100a incorporates a number of each of the respective components that interact throughout the system. For example, without limitation, as embodied herein, the data reader device 120 and / or the multi-purpose data receiving device 130 may include a number of each. As embodied herein, the multiple data receiving device 130 may communicate directly with the sensor 110 described herein. Further or alternatively, the data receiving device 130 may communicate with a secondary data receiving device 130 to provide test substance data or visualization or analysis of data for secondary display to a user or other authorized party.

[0045] FIG. 1C shows a schematic diagram of an exemplary detection system into which the analyte sensor of the present disclosure can be incorporated. As shown, the detection system 100 includes a sensor control device 102 and a reader device 120 configured to communicate with each other via a local communication path or link 140, which may be wired or wireless, may be unidirectional or bidirectional, and may or may not be encrypted. According to certain embodiments, the reader device 120 may constitute an output medium for visually recognizing the analyte concentration and warnings or cautions determined by the sensor 104 or an associated processor, and for enabling one or more user inputs. The reader device 120 may be a multi-purpose smartphone or a dedicated electronic reader device. Although only one reader device 120 is shown, in certain cases, a plurality of reader devices 120 may be present. The reader device 120 may communicate with a remote terminal 90170 and / or a reliable computer system 90180 via communication paths / links 90141 and / or 90142, which may be wired or wireless, may be unidirectional or bidirectional, and may or may not be encrypted. The reader device 120 may also or instead communicate with a network 150 (e.g., a mobile phone network, the Internet, or a cloud server) via a communication path / link 151. The network 150 may be further communicatively connected to the remote terminal 90170 via a communication path / link 152 and / or to the reliable computer system 90180 via a communication path / link 153. Alternatively, the sensor 104 may communicate directly with the remote terminal 90170 and / or the reliable computer system 90180 without the presence of an intervening reader device 120. For example, but not limited to, according to certain embodiments described in U.S. Patent Application Publication No. 2011 / 0213225, which is hereby incorporated by reference in its entirety, the sensor 104 may communicate with the remote terminal 90170 and / or the reliable computer system 90180 via a direct communication link to the network 150.For each communication path or link, near field communication (NFC), radio frequency identification (RFID), Bluetooth® or Bluetooth® low energy protocol, WiFi, or any other suitable electronic communication protocol can be used. According to certain embodiments, the remote terminal 90170 and / or the trusted computer system 90180 can be accessed by individuals other than the primary user who is interested in the user's analyte level. The reader device 120 may include a display unit 122 and an optional input component 121. According to certain embodiments, the display unit 122 may include a touch screen interface.

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

[0047] The sensor 104 is adapted to be at least partially inserted into a target tissue such as within a skin layer or a subcutaneous layer of the skin. The sensor 104 may include a sensor tail of sufficient length for insertion to a desired depth in a given tissue. The sensor tail may include at least one working electrode. In a particular configuration, the sensor tail may include an active region for detecting an analyte. A counter electrode may be present in combination with the at least one working electrode. A particular electrode configuration on the sensor tail is described in more detail below.

[0048] The active region may be configured to detect a specific analyte. In certain embodiments, the active region of the sensors disclosed herein is configured to detect ketones. In certain embodiments, the active region may be configured to detect two or more analytes. In certain embodiments, additional analytes to be detected using the sensors of the present disclosure include any analyte that is dysregulated along with ketones. For example, without limitation, the analyte sensors of the present disclosure can detect ketones and additional analytes such as creatinine, oxygen, glucose, and / or lactate. In certain embodiments, the additional analyte is glucose.

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

[0050] Still referring to FIG. 1C, sensor 104 can automatically send data to reader device 120. For example, without limitation, data on the concentration of the analyte (i.e., the concentration of glucose and / or ketones) can be communicated automatically and periodically, such as at a certain frequency, when the data is obtained or after a certain period of time has elapsed, and the data is stored in memory until transmission (e.g., every minute, every 5 minutes, or other predetermined period). In other specific embodiments, sensor 104 may communicate with reader device 120 in a non-automatic manner, not following a set schedule. For example, without limitation, data may be communicated from sensor 104 using RFID technology when the electronics of the sensor enter the communication range of reader device 120. The data may remain stored in the memory of sensor 104 until it is communicated to reader device 120. That is, the user does not need to always maintain close proximity to reader device 120 and instead can upload data when convenient. In other specific embodiments, a combination of automatic and non-automatic data transfer may be implemented. For example, without limitation, data transfer may continue automatically until reader device 120 is no longer within the communication range of sensor 104.

[0051] To facilitate introduction of the sensor 104 into tissue, an introducer may be present temporarily. In certain exemplary embodiments, the introducer may include a needle or similar tip. As will be readily appreciated by those skilled in the art, other types of introducers such as sheaths or blades may be present in alternative embodiments. More specifically, a needle or other introducer may be present temporarily proximal to the sensor 104 prior to insertion into tissue and then withdrawn later. While present, the needle or other introducer can facilitate insertion of the sensor 104 into tissue by opening an access path for the sensor 104 to follow. For example, without limitation, according to one or more embodiments, the needle can facilitate penetration of the epidermis as an access path to the dermis to enable implantation of the sensor 104. After opening the access path, the needle or other introducer can be withdrawn so as not to represent damage by the tip. In certain embodiments, suitable needles can be solid or hollow, beveled or non-beveled, and / or have a circular or non-circular cross-section. In more specific embodiments, suitable needles can be similar to acupuncture needles in cross-sectional diameter and / or tip design, and these can have a cross-sectional diameter of about 250 microns (250 μm). However, suitable needles can have a larger or smaller cross-sectional diameter if required for a particular application.

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

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

[0054] C. Architecture of an Exemplary Data Receiving Device For purposes of illustration and not limitation, reference is made to an exemplary embodiment of a data receiving device 120 for use in accordance with the disclosed subject matter shown in FIG. 2B. The data receiving device 120 and related multipurpose data receiving device 130 include components that are closely related to the consideration of the analyte sensor 110, and its operation and additional components may be included. In certain embodiments, the data receiving device 120 and multipurpose data receiving device 130 may be or include components provided by a third party and are not necessarily limited to including devices made by the same manufacturer as the sensor 110.

[0055] As shown in FIG. 2B, the data receiving device 120 includes a microcontroller 4010, a memory 4020, and a storage 4030, and includes an ASIC 4000 communicatively connected to a communication module 4040. Power for the components of the data receiving device 120 can be delivered by a power module 4050, which may include a rechargeable battery as embodied herein. The data receiving device 120 may further include a display unit 4070 to facilitate verification of the test substance data received from the test substance sensor 110 or other devices (e.g., the user device 140 or the remote application server 150). The data receiving device 120 may include separate user interface components (e.g., physical keys, optical sensors, microphones, etc.).

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

[0057] As another example, the communication module 4040 may include, for example, a cellular radio module 4044. The cellular radio module 4044 may include one or more radio transceivers for communicating using broadband cellular networks including, but not limited to, third generation (3G), fourth generation (4G), and fifth generation (5G) networks. Further, the communication module 4040 of the data receiving device 120 may 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 (also referred to as Wi-Fi 4), 802.11ac (also referred to as Wi-Fi 5), 802.11ax (also referred to as Wi-Fi 6)). Using the cellular radio module 4044 or the Wi-Fi radio module 4043, the data receiving device 120 may communicate with the remote application server 150 to receive the analyte data or provide updates or inputs received from the user (e.g., via one or more user interfaces). Although not shown, the communication module 5040 of the analyte sensor 120 may similarly include a cellular radio module or a Wi-Fi radio module.

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

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

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

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

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

[0063] By way of example and not limitation, reference is made to an exemplary embodiment of the analyte sensor 110 for use by the disclosed subject matter shown in FIG. 2E. FIG. 2E shows a block schematic of an example of an analyte sensor 110 according to an exemplary embodiment that is compliant with the security architecture and communication scheme described herein.

[0064] As embodied herein, the analyte sensor 110 may include an application specific integrated circuit ( "ASIC") 5000 communicatively coupled to a communication module 5040. The ASIC 5000 may include a microcontroller core 5010, on-board memory 5020, and storage memory 5030. The storage memory 5030 can store data used in the authentication and encryption security architecture. The storage memory 5030 can store programming instructions for the sensor 110. As embodied herein, a particular communication chipset may be embedded within 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 NFC pulse. The storage memory 5030 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 5030 can also be programmed with configuration or calibration parameters for use by the sensor 110 and its various components. The storage memory 5030 may include rewritable or one-time programmable (OTP) memory. The storage memory 5030 can be updated using the techniques described herein to extend the usefulness of the sensor 110.

[0065] As embodied herein, the communication module 5040 of the sensor 100 may be or include one or more modules that assist the analyte sensor 110 in communicating with other devices of the analyte monitoring system 100. By way of example only and not limitation, an example of the communication module 5040 may include a Bluetooth® Low Energy (“BLE”) module 5041. As used throughout this disclosure, Bluetooth® Low Energy (“BLE”) means a short-range communication protocol optimized to simplify the pairing of Bluetooth® devices for the end user. The communication module 5040 can transmit and receive data and commands via interaction with a similarly capable communication module of the data receiving device 120 or the user device 140. The communication module 5040 may include additional or alternative chip sets for use with a personal area network according to the IEEE802.15 protocol, the IEEE802.11 protocol, infrared communication according to the Infrared Data Association standard (IrDA), and other similar short-range communication schemes.

[0066] To perform its functionality, the sensor 100 may further include suitable sensing hardware 5060 suitable for its function. As embodied herein, the sensing hardware 5060 may include an analyte sensor disposed transcutaneously or subcutaneously in contact with the bodily fluid of interest. The analyte sensor can generate sensor data that includes values corresponding to the levels of one or more analytes in the bodily fluid.

[0067] E. Exemplary Assembly Process for the Sensor Control Device The components of the sensor control device 102 can be obtained by the user in multiple packaging that requires the user to perform the final assembly before delivery to the appropriate user location. FIGS. 3A - 3D show examples of embodiments of the assembly process of the sensor control device 102 by the user, including preparing separate components before connecting the components to prepare the sensor for delivery. FIGS. 3E - 3F show examples of embodiments of delivering the sensor control device 102 to the appropriate user location by selecting the appropriate delivery location and fitting the device 102 to that location.

[0068] FIG. 3A is a proximal perspective view showing an example of an embodiment of a user preparing a container 810 configured as a tray herein (although other packaging can also be used) for the assembly process. The user can achieve this preparation, for example, by removing the lid 812 from the tray 810 such that the non - adhesive portion of the lid 812 is peeled from the tray 810 and the adhesive portion of the lid 812 is removed, exposing the platform 808. As long as the platform 808 is appropriately exposed within the tray 810, the removal of the lid 812 can be appropriate in various embodiments. Next, the lid 812 may be placed sideways.

[0069] FIG. 3B is a side view showing an example of an embodiment of a user preparing an applicator device 150 for assembly. The applicator device 150 may be provided in a sterile package sealed by a cap 708. The preparation of the applicator device 150 may include removing the housing 702 from the cap 708 to expose the sheath 704 (FIG. 3C). This can be achieved by turning the cap 708 off the housing 702 (or removing it in other ways). Next, the cap 708 may be placed sideways.

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

[0071] While the housing 702 is advancing distally, the sheath 704 is maintained in position within the platform 808 relative to the housing 702 and is coupled to the platform 808 to advance the platform 808 distally relative to the tray 810. This process unlocks and folds the platform 808 within the tray 810. A fixed feature (not shown) within the tray 810 contacts and disengages the sheath 704 to prevent the sheath 704 from (relatively) moving while the sheath 704 is unlocked with respect to the housing 702 and the housing 702 continues to advance the platform 808 distally. When the advancement of the housing 702 and the platform 808 is complete, the sheath 704 is permanently unlocked with respect to the housing 702. At the end of the distal advancement of the housing 702, a tip and sensor (not shown) within the tray 810 can be coupled to an electronics housing (not shown) within the housing 702. The operation and interaction of the applicator device 150 and the tray 810 are further described below.

[0072] Figure 3D is a proximal perspective view showing an example of an embodiment in which the user removes the applicator device 150 from the tray 810 during assembly. The user can remove the applicator 150 from the tray 810 by advancing the housing 702 in the proximal direction relative to the tray 810 or by other operations having the same final effect as the disconnection of the connection between the applicator 150 and the tray 810. The applicator device 150 is removed with the sensor control device 102 (not shown) fully assembled therein (tip, sensor, electronics) and arranged for delivery.

[0073] Figure 3E is a proximal perspective view showing an example of an embodiment in which the patient applies the sensor control device 102 to a target area of the skin, such as the abdomen or other suitable location, using the applicator device 150. By advancing the housing 702 in the distal direction, the sheath 704 is folded within the housing 702 and the sensor is applied to the target location, as a result of which the adhesive layer on the bottom side of the sensor control device 102 adheres to the skin. While the tip is automatically retracted when the housing 702 is fully advanced, the sensor (not shown) remains in position to measure the analyte level.

[0074] Figure 3F is a proximal perspective view showing an example of an embodiment of the patient with the sensor control device 102 attached in place. Next, the user can remove the applicator 150 from the site where it was attached.

[0075] The system 100 described with respect to FIGS. 3A - 3F and elsewhere in this specification can reduce or eliminate the chance of accidental breakage, permanent deformation, or improper assembly of the applicator components as compared to prior art systems. Instead of an indirect engagement via the sheath 704, the applicator housing 702 engages directly with the platform 808 while the sheath 704 unlocks, so the relative angle between the sheath 704 and the housing 702 does not result in breakage or permanent deformation of the arm or other components. The relatively large forces (such as in conventional devices) that may be applied during assembly are reduced, thereby reducing the chance that the user's assembly will be unsuccessful.

[0076] F. Exemplary Sensor Applicator Device FIG. 4A is a side view showing an example of an embodiment of an applicator device 150 coupled to a screw cap 708. This is an example of how the applicator 150 is shipped and received by the user before being assembled with the sensor by the user. FIG. 4B is a side perspective view showing the applicator 150 and the cap 708 after the connection has been removed. FIG. 4C is a perspective view showing an example of an embodiment of the distal end of the applicator device 150 with the electronic device housing 706 and the adhesive patch 105 removed from the position where they were held within the sensor carrier 710 of the sheath 704 when the cap 708 was in a predetermined position.

[0077] For purposes of illustration and not limitation, referring to FIGS. 4D - G, the applicator device 20150 can be provided to the user as a single integrated assembly. FIGS. 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, and FIG. 4G provides a side cross - sectional view. The perspective views show how the applicator 20150 is shipped and received by the user. The exploded view and cross - sectional view show the components of the applicator device 20150. The applicator device 20150 may include a housing 20702, a gasket 20701, a sheath 20704, a tip carrier 201102, a spring 205612, a sensor carrier 20710 (also referred to as a "pack carrier"), a tip hub 205014, a sensor control device (also referred to as a "pack") 20102, an adhesive patch 20105, a desiccant 20502, a cap 20708, a serial label 20709, and a tamper evidence form 20712. When received by the user, only the housing 20702, the cap 20708, the tamper evidence form 20712, and the label 20709 are visible. The tamper evidence form 20712 may be, for example, a sticker connected to each of the housing 20702 and the cap 20708, and the tamper evidence form 20712 is irreparably damaged, for example, by removing the connection between the housing 20702 and the cap 20708, thereby indicating to the user that the connection between the housing 20702 and the cap 20708 has been previously removed. These forms are described in more detail below.

[0078] G. Exemplary Tray and Sensor Module Assembly FIG. 5 is a proximal perspective view showing an example of an embodiment of a tray 810 to which a sterilization lid 812 is removably connected, which can show how the package is shipped and received by the user prior to assembly.

[0079] FIG. 6A is a proximal perspective cross-sectional view showing the sensor delivery components within tray 810. Platform 808 is slidably coupled within tray 810. Desiccant 502 is fixed relative to tray 810. Sensor module 504 is provided within tray 810.

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

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

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

[0083] In addition to the electronic modules 3806, the PCBA 3802 may include a data processing unit 3808 attached to the PCB 3804. The data processing unit 3808 may comprise, for example, an application specific integrated circuit (ASIC) configured to execute one or more functions or routines associated with the operation of the sensor control device 3702. More specifically, the data processing unit 3808 may be configured to perform data processing functions such as, but not limited to, filtering and encoding data signals each corresponding to a user-sampled analyte level. The data processing unit 3808 may also include or otherwise communicate with an antenna for communicating with the reader device 106 (FIG. 1A).

[0084] The battery patch 3810 may be sized to receive and seat a battery 3812 defined within the PCB 3804 and configured to supply power to the sensor control device 3702. Axial battery contacts 3814a and radial battery contacts 3814b may extend into the battery patch 3810 to couple to the PCB 3804 to facilitate delivery of power from the battery 3812 to the PCB 3804. As their names suggest, the axial battery contacts 3814a may be configured to provide axial contacts to the battery 3812 and the radial battery contacts 3814b may provide radial contacts to the battery 3812. Positioning the battery 3812 within the battery patch 3810 having the battery contacts 3814a and b helps to lower the height H of the sensor control device 3702, thereby allowing the PCB 3804 to be centered and its components to be dispersed on both sides (i.e., the top and bottom surfaces). This also helps to facilitate mounting the chamber 3718 on top of the electronic device housing 3704.

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

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

[0087] Sensor control device 3702 may further include a compliant member 3826, which may be disposed to intervene between flag 3818 and the inner surface of shell 3706. More specifically, when shell 3706 and mount 3708 are assembled together, compliant member 3826 may be configured to provide a passive biasing load to flag 3818 that forces the sensor contact(s) 3822 to continuously engage corresponding circuit contact(s) 3824. In the illustrated embodiment, compliant member 3826 is an elastomeric O-ring, but alternatively may comprise any other type of biasing device or mechanism, such as a compression spring, without departing from the scope of the present disclosure.

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

[0089] Specifically referring to FIG. 7A, the inner surface of the mount 3708 may include a plurality of pockets or recesses configured to accommodate various components of the sensor control device 3702 when the shell 3706 is paired with the mount 3708 or may be defined in other ways. For example, the inner surface of the mount 3708 may define a battery locator 3834 configured to accommodate a portion of the battery 3812 when the sensor control device 3702 is assembled. The adjacent contact pocket 3836 may be configured to accommodate a portion of the axial contact 3814a.

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

[0091] Referring further to FIG. 7A, mount 3708 may further include a plurality of carrier grip formations 3842 (two shown) defined near the outer periphery of mount 3708. The carrier grip formations 3842 are axially offset from the bottom 3844 of mount 3708, and a transfer adhesive may be applied thereto during assembly. In contrast to conventional sensor control devices that include conical carrier grip formations that generally intersect the bottom of the mount, the carrier grip formations 3842 of the present disclosure are offset from the plane (i.e., bottom 3844) to which the transfer adhesive is applied. This may be advantageous in helping to ensure that the delivery system does not inadvertently adhere to the transfer adhesive during assembly. Further, the carrier grip formations 3842 of the present disclosure eliminate the need for a wavy transfer adhesive, which simplifies the manufacture of the transfer adhesive and eliminates the need to accurately register the transfer adhesive to mount 3708. This also increases the adhesive area and thus the adhesive strength.

[0092] Referring to FIG. 7B, the bottom 3844 of the mount 3708 may comprise a plurality of grooves 3846 or be defined in some other way, which are defined on or near the outer periphery of the mount 3708 and are equidistant from each other. A transfer adhesive (not shown) may be coupled to the bottom 3844, and the grooves 3846 may be configured to help carry (transfer) moisture away from the sensor control device 3702 and around the mount 3708 during use. In some embodiments, the spacing of the grooves 3846 may sandwich the module pocket 3838 (FIG. 7A) defined on the opposite (inner) side of the mount 3708. As will be appreciated, by alternating the positions of the grooves 3846 and the module pocket 3838, it is ensured that the opposing features on either side of the mount 3708 do not extend into each other. This helps to maximize the amount of material used for the mount 3708 and may serve to maintain the minimum height H of the sensor control device 3702. The module pocket 3838 may also significantly reduce the mold sink and improve the flatness of the bottom 3844 to which the transfer adhesive adheres.

[0093] Further, referring to FIG. 7B, the inner surface of the shell 3706 may comprise a plurality of pockets or recesses configured to accommodate various components of the sensor control device 3702 when the shell 3706 is paired with the mount 3708 or be defined in some other way. For example, the inner surface of the shell 3706 may define opposing battery locators 3848 that are positioned to face the battery locator 3834 (FIG. 7A) of the mount 3708 and are configured to accommodate a portion of the battery 3812 when the sensor control device 3702 is assembled. The opposing battery locators 3848 extend a short distance into the inner surface of the shell 3706, thereby serving to reduce the overall height H of the sensor control device 3702.

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

[0095] Alternative sensor assembly / electronic device assembly connection approaches are shown in FIGS. 8A - 8C according to embodiments of the present disclosure. As shown, the sensor assembly 14702 includes a sensor 14704, a connector support 14706, and a tip 14708. In particular, a recess or receptacle 14710 is defined at the bottom of the mount of the electronic device assembly 14712, and the sensor assembly 14702 is received and coupled to the electronic device assembly 14712, thereby providing a location where the sensor control device can be fully assembled. The profile of the sensor assembly 14702 may be shaped to match or be complementary to the receptacle 14710, which includes an elastomeric sealing member 14714 (including a conductive material connected to the circuit board and aligned with the electrical contacts of the sensor 14704). Thus, by driving the sensor assembly 14702 into the integrally formed recess 14710 of the electronic device assembly 14712, an on - body device 14714 as shown in FIG. 8C is formed when the sensor assembly 14702 snap - fits or is otherwise adhered to the electronic device assembly 14712. This embodiment provides an integrated connector for the sensor assembly 14702 within the electronic device assembly 14712.

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

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

[0098] Figures 9A and 9B are respectively a side view and a side cross-sectional view of an example embodiment of a sensor applicator 102 with an applicator cap 210 connected thereto. More specifically, Figure 9A shows how the sensor applicator 102 is shipped and received by the user, and Figure 9B shows the sensor control device 4402 disposed within the sensor applicator 102. Accordingly, the fully assembled sensor control device 4402 may already be assembled and attached within the sensor applicator 102 before being delivered to the user, thereby eliminating any additional assembly steps that would otherwise have to be performed by the user.

[0099] The fully assembled sensor control device 4402 may be attached to the sensor applicator 102, and subsequently the applicator cap 210 may be connected to the sensor applicator 102. In some embodiments, the applicator cap 210 may be screwed onto the housing 208 and may include a tampering 4702. When the applicator cap 210 is rotated (e.g., unscrewed) relative to the housing 208, the tampering 4702 may shear, thereby enabling the applicator cap 210 to be released from the sensor applicator 102.

[0100] According to the present disclosure, while attached to the sensor applicator 102, the sensor control device 4402 can receive gaseous chemical sterilization 4704 configured to sterilize the electronic device housing 4404 and any other exposed portions of the sensor control device 4402. To achieve this, chemicals may be injected into the sterilization chamber 4706 cooperatively defined by the sensor applicator 102 and the interconnected cap 210. In some embodiments, chemicals may be injected into the sterilization chamber 4706 through one or more vents 4708 defined at its proximal end 610 by the applicator cap 210. Examples of chemicals that can be used for gaseous chemical sterilization 4704 include, but are not limited to, ethylene oxide, vaporized hydrogen peroxide, nitrogen oxides (such as nitrous oxide, nitrogen dioxide, etc.), and steam.

[0101] Since the distal portions of the sensor 4410 and the tip 4412 are sealed within the sensor cap 4416, the chemicals used during the gaseous chemical sterilization process do not interact with the enzymes, chemicals, and biologic agents provided in the tail 4524 and other sensor components such as the membrane coating that regulates the influx of, for example, the analyte.

[0102] Once the desired sterilization assurance level is achieved within the sterilization chamber 4706, the gaseous solution may be removed and the sterilization chamber 4706 may be vented. Venting may be achieved by a series of vacuums through the sterilization chamber 4706 followed by circulation of a gas (such as nitrogen) or filtered air. Once the sterilization chamber 4706 is properly vented, the vent 4708 may be closed by a seal 4712 (shown in dashed lines).

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

[0104] Once seal 4712 is placed, 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 (unscrews) applicator cap 210. Applicator cap 210 can also create a dust-free environment that prevents adhesive patch 4714 from becoming contaminated during shipping and storage.

[0105] FIGS. 10A and 10B are, respectively, an isometric view and a side view of another example of a sensor control device 5002 according to one or more embodiments of the present disclosure. Sensor control device 5002 may be similar to sensor control device 102 of FIG. 1A in some respects and may thus be best understood by reference thereto. Additionally, sensor control device 5002 may replace sensor control device 102 of FIG. 1A and may thus be used in conjunction with sensor applicator 102 of FIG. 1A that can deliver sensor control device 5002 to a target monitoring location on a user's skin.

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

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

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

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

[0110] The sensor control device 5002 may further include a sensor cap 5018 shown disassembled or separated from the electronic device housing 5004 of FIGS. 10A - 10B. The sensor cap 5016 may be removably coupled to the sensor control device 5002 (e.g., the electronic device housing 5004) at or near the bottom of the mount 5008. The sensor cap 5018 can serve to surround the exposed portions of the sensor 5010 and the tip 5012 and provide a sealed barrier that protects them from gaseous chemical sterilization. As shown, the sensor cap 5018 may comprise a generally cylindrical body having a first end 5020a and a second end 5020b opposite the first end 5020a. The first end 5020a may be open to provide access to an internal chamber 5022 defined within the body. In contrast, the second end 5020b may be closed and may comprise an engaging formation 5024 or be defined in some other way. As described herein, the engaging formation 5024 may serve to mate the sensor cap 5018 with a cap (e.g., the applicator cap 708 of FIG. 3B) of a sensor applicator (e.g., the sensor applicator 150 of FIGS. 1 and 3A - 3G) and may serve to remove the sensor cap 5018 from the sensor control device 5002 when removing the cap from the sensor applicator.

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

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

[0113] Figures 11A - 11C are sequential side cross - sectional views showing an assembly of the sensor applicator 102 with the sensor control device 5002 according to one or more embodiments. Once the sensor control device 5002 is fully assembled, the sensor control device can be mounted within the sensor applicator 102. Referring to FIG. 11A, the tip hub 5014 may include hub snap poles 5302 configured to assist in coupling the sensor control device 5002 to the sensor applicator 102 or may be defined in other ways. More specifically, the sensor control device 5002 may be advanced into the interior of the sensor applicator 102, and the hub snap poles 5302 may be received by corresponding arms 5304 of the tip carrier 5306 disposed within the sensor applicator 102.

[0114] In FIG. 11B, it is shown that the sensor control device 5002 is received by the tip carrier 5306 and thus fixed within the sensor applicator 102. Once the sensor control device 5002 is mounted within the sensor applicator 102, the applicator cap 210 may be coupled to the sensor applicator 102. In some embodiments, the applicator cap 210 and the housing 208 may have opposing mating sets of threads 5308 that allow the applicator cap 210 to be screwed into the housing 208 in a clockwise (or counter - clockwise) direction, thereby securing the applicator cap 210 to the sensor applicator 102.

[0115] As shown, the sheath 212 may also be disposed within the sensor applicator 102, and the sensor applicator 102 may include a sheath fixation mechanism 5310 configured to ensure that the sheath 212 is not prematurely folded during impact occurrence. In the illustrated embodiment, the sheath fixation mechanism 5310 may comprise a threaded engagement between the applicator cap 210 and the sheath 212. More specifically, one or more female threads 5312a may be defined or otherwise provided on the inner surface of the applicator cap 210, and one or more male threads 5312b may be defined or otherwise provided on the sheath 212. The female or male threads 5312a, b may be configured such that the applicator cap 210 threads onto the sensor applicator 102 at the thread 5308 and engages by threading. The female and male threads 5312a, b may have the same thread pitch as the thread 5308 that allows the applicator cap 210 to be screwed into the housing 208.

[0116] In FIG. 11C, the applicator cap 210 is shown as being fully screwed (connected) into the housing 208. As shown, the applicator cap 210 may further comprise or otherwise define a cap post 5314 located at the center inside the applicator cap 210 and extending proximally from its bottom. The cap post 5314 may be configured such that the applicator cap 210 is screwed into the housing 208 and receives at least a portion of the sensor cap 5018.

[0117] The sensor control device 5002 is mounted within the sensor applicator 102, and when the applicator cap 210 is properly secured, the sensor control device 5002 can receive gaseous chemical sterilization configured to sterilize the electronic device housing 5004 and any other exposed portions of the sensor control device 5002. Since the distal portions of the sensor 5010 and tip 5012 are sealed within the sensor cap 5018, the chemicals used during the gaseous chemical sterilization process cannot interact with the enzymes, chemicals, and biologic agents provided at the tail 5104 and other sensor components such as the membrane coating that regulates the influx of the analyte.

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

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

[0120] In FIG. 12B, the applicator cap 210 is aligned with the housing 208 and advanced toward the housing 208, whereby the sheath 212 is received within the applicator cap 210. Instead of rotating the applicator cap 210 relative to the housing 208, the threads of the applicator cap 210 may be snap - fit to the corresponding threads of the housing 208 to couple the applicator cap 210 to the housing 208. A defined axial cut or slot 5703 (one shown) in the applicator cap 210 may allow a portion of the applicator cap 210 near its threads to bend outward and be snap - fit to engage the threads of the housing 208. When the applicator cap 210 is snap - fit to the housing 208, the sensor cap 5018 may correspondingly be snap - fit to the cap post 5314.

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

[0122] In FIG. 12C, the applicator cap 210 is removed from the housing 208. Similar to the embodiments of FIGS. 21A - 21C, generally as described above, the applicator cap 210 can be removed by rotating the applicator cap 210 counter - clockwise, whereby the corresponding cap post 5314 rotates in the same direction and the screw engagement between the sensor cap 5018 and the fitting member 5016 is disengaged. Further, by removing the sensor cap 5018 from the sensor control device 5002, the distal portions of the sensor 5010 and the tip 5012 are exposed.

[0123] When the screwing engagement between the applicator cap 210 and the housing 208 is disengaged, the rib 5704 defined on the sheath 212 can engage with the upper part of the rib 5706 defined on the applicator cap 210 while sliding. The upper part of the rib 5706 may provide a corresponding inclined surface that causes the applicator cap 210 to rotate and results in upward movement of the sheath 212. Moving the sheath 212 upward disengages it from the engagement with the first detent 5702a so that the sheath arm 5604 is deflected and received within the second detent 5702b. When the sheath 212 moves to the second detent 5702b, the radial shoulder 5614 moves in a direction to disengage from the radial engagement with the carrier arm(s) 5608, thereby enabling the passive spring force of the spring 5612 to push the tip carrier 5306 upward and disengage the carrier arm(s) 5608 from the engagement with the groove(s) 5610. When the tip carrier 5306 moves upward within the housing 208, the fitting member 5016 can correspondingly retract until it is flush with, substantially flush with, or quasi-flush with the bottom of the sensor control device 5002. At this point, the sensor applicator 102 is in the firing position. Thus, in this embodiment, removing the applicator cap 210 correspondingly retracts the fitting member 5016.

[0124] I. Exemplary Firing Mechanisms for One-Piece and Two-Piece Applicators Figures 13A - 13F show detailed examples of an internal device mechanism that "fires" applicator 216 to apply sensor control device 222 to a user and safely retracts tip 1030 into the used applicator 216. Collectively, these drawings depict a sequence of driving tip 1030 (which supports a sensor coupled to sensor control device 222) into the user's skin, leaving the sensor in operable contact with the user's interstitial fluid and retracting the tip, and adhering the sensor control device to the user's skin with an adhesive. Modifications of such operations for use with alternative applicator assembly embodiments and components can be understood by those skilled in the art with reference to the same. Further, applicator 216 may be a sensor applicator having a one-piece architecture or a two-piece architecture as disclosed herein.

[0125] Referring now to FIG. 13A, sensor 1102 is supported within tip 1030, just above user's skin 1104. To control the movement of applicator 216 relative to sheath 318, rails 1106 (optionally, three of them) of upper guide section 1108 may be provided. Sheath 318 is held by a detent formation 1110 within applicator 216, and an appropriate downward force along the longitudinal axis of applicator 216 overcomes the resistance force provided by detent mechanism 1110, allowing tip 1030 and sensor control device 222 to translate into (and out of) user's skin 1104 along the longitudinal axis. Further, catch arm 1112 of sensor carrier 1022 engages tip retraction assembly 1024 to maintain tip 1030 in position relative to sensor control device 222.

[0126] In FIG. 13B, a user's force is applied such that it overcomes or exceeds the detent form 1110, and the sheath 318 is folded within the housing 314, translating the sensor control device 222 (along with its attendant components) downward along the longitudinal axis as indicated by arrow L. The inner diameter of the upper guide section 1108 of the sheath 318 constrains the position of the carrier arm 1112 throughout the full stroke of the sensor / tip insertion process. The retention of the stop surface 1114 of the carrier arm 1112 against the complementary surface 1116 of the tip retraction assembly 1024 maintains the position of the member along with the fully biased telescoping spring 1118. According to an embodiment, rather than employing a user's force to translate the sensor control device 222 downward along the longitudinal axis as indicated by arrow L, a drive spring (e.g., but not limited to, a coil spring) may be actuated to drive a button (e.g., but not limited to, a push button) that drives the sensor control device 222 and is included in the housing 314.

[0127] In FIG. 13C, the sensor 1102 and the tip 1030 have reached the depth at which they are fully inserted. Thereby, the carrier arm 1112 moves away from the inner diameter of the upper guide section 1108. Next, the compressive force of the coil compression spring 1118 drives the angled stop surface 1114 radially outward, releasing force to drive the tip carrier 1102 of the tip retraction assembly 1024, pulling the tip 1030 (configured with slots or otherwise) away from the user and away from the sensor 1102 as indicated by arrow R in FIG. 13D.

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

[0129] The operation of the applicator 216 when applying the sensor control device 222 is designed to give the user the sense that both the insertion and retraction of the tip 1030 are automatically performed by the internal mechanism of the applicator 216. In other words, according to the present invention, the user is spared the experience of feeling that they are manually driving the tip 1030 into their skin. That is, if the user applies sufficient force to overcome the resistance of the applicator 216's detent form, the resulting movement of the applicator 216 is perceived as an automatic response to the "induced" applicator. Despite the fact that all of the driving force is provided by the user and no additional biasing / driving means are used to insert the tip 1030, the user does not perceive that they are supplying additional force to drive the tip 1030 to pierce the skin. As described above with reference to FIG. 13C, the retraction of the tip 1030 is automated by the coil expansion and contraction spring 1118 of the applicator 216.

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

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

[0132] Further details of suitable devices, systems, methods, components, and their operations, along with related features, are described in International Publication Nos. WO2018 / 136898 by Rao et al., WO2019 / 236850 by Thomas et al., WO2019 / 236859 by Thomas et al., WO2019 / 236876 by Thomas et al., and U.S. Patent Publication No. 2020 / 0196919 filed on Jun. 6, 2019, each of which is hereby incorporated by reference in its entirety into the present specification. Further details regarding embodiments of applicators, their components, and their variations are described in U.S. Patent Publication Nos. 2013 / 0150691, 2016 / 0331283, and 2018 / 0235520, all of which are hereby incorporated by reference in their entirety into the present specification for all purposes. Further details regarding embodiments of tip modules, tips, their components, and their variations are described in U.S. Patent Publication No. 2014 / 0171771, which is hereby incorporated by reference in its entirety into the present specification for all purposes.

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

[0134] FIG. 14 is a graph showing the in vitro sensitivity of a current-measuring substance sensor. The in vitro sensitivity can be obtained by testing the sensor in vitro at various substance concentrations and performing regression (e.g., linear or non-linear) or other curve fitting on the obtained data. In this example, the sensitivity of the substance sensor is linear or substantially linear and can be modeled by the equation y = mx + b. Here, y is the electrical output current of the sensor, x is the level (or concentration) of the substance, m is the slope of the sensitivity, and b is the intercept of the sensitivity, and the intercept generally corresponds to the background signal (e.g., noise). For sensors with a linear or substantially linear response, the substance level corresponding to a given current can be determined from the slope and intercept of the sensitivity. Sensors with non-linear sensitivity require additional information to determine the substance level resulting from the output current of the sensor, and those skilled in the art are familiar with methods for modeling non-linear sensitivity. In certain embodiments of in vivo sensors, the in vitro sensitivity may be the same as the in vivo sensitivity, but in other embodiments, a transfer (or conversion) function is used to replace the in vitro sensitivity with the in vivo sensitivity applicable to the intended in vivo use of the sensor.

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

[0136] In certain in vivo analyte monitoring systems, after the sensor is implanted in the user or patient, it is necessary to perform calibration either by user intervention or in an automated manner by the system itself. For example, when user intervention is required, the user performs an in vitro measurement (e.g., a blood glucose (BG) measurement using a fingerstick and an in vitro test strip) while the analyte sensor is implanted and inputs this into the system. The system then compares the in vitro measurement value to the in vivo signal and uses the difference to determine an estimated value of the in vivo sensitivity of the sensor. The in vivo sensitivity can then be used in an algorithmic process to convert the data collected by the sensor into a value indicative of the user's analyte level. This and other processes that require user action to perform calibration are referred to as "user calibration." The system may require user calibration due to the instability of the sensor's sensitivity, where the sensitivity drifts or changes over time. Thus, multiple user calibrations (e.g., on a periodic (e.g., daily) schedule, a variable schedule, or an as-needed schedule) may be necessary to maintain accuracy. While the embodiments described herein may incorporate some degree of user calibration for certain implementations, this is generally not preferred as it can be painful for the user or require inconvenient BG measurements to be performed, which can lead to user error.

[0137] Some in vivo analyte monitoring systems can periodically adjust calibration parameters by using an automated measurement of sensor characteristics (e.g., software executed by a processing circuit) created by the system itself. Repeated adjustment of sensor sensitivity based on variables measured by the system (not the user) is generally referred to as “system” (or automatic) calibration and can be performed with or without user calibration, such as early BG measurement. Similar to repeated user calibration, repeated system calibration is generally required due to drift in sensor sensitivity over time. Accordingly, the embodiments described herein can be used with some degree of automated system calibration, but preferably the sensor sensitivity is relatively stable over time and thus post-implant calibration is not necessary.

[0138] Some in vivo analyte monitoring systems operate using factory-calibrated sensors. Factory calibration means determining or estimating one or more calibration parameters prior to distribution to the user or healthcare provider (HCP). The calibration parameters can be determined by the sensor manufacturer (or, if two components are different, the manufacturer of the other components of the sensor control device). Many in vivo sensor manufacturing processes assemble sensors in groups or batches referred to as manufacturing lots, manufacturing stage lots, or simply lots. A single lot can contain thousands of sensors.

[0139] The sensor may include calibration codes or parameters that are induced or determined during one or more sensor manufacturing processes, encoded or programmed into the data processing device of the analyte monitoring system as part of the manufacturing process, or provided to the sensor itself as machine-readable other information such as barcodes, laser tags, RFID tags, or the like provided with the sensor. When the code is provided to the receiver (or other data processing device), user calibration during in vivo use of the sensor can be eliminated, or the frequency of in vivo calibration while the sensor is worn can be reduced. In embodiments where the calibration code or parameters are provided to the sensor itself before or at the start of use of the sensor, the calibration code or parameters can be automatically transmitted or provided to the data processing device of the analyte monitoring system.

[0140] Some in vivo analyte monitoring systems operate using sensors that can be one or more of factory calibration, system calibration, and / or user calibration. For example, the sensor can be provided with calibration codes or parameters that enable factory calibration. When information is provided to the receiver (e.g., input 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 calibration sensor and / or a system calibration sensor.

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

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

[0143] According to the disclosed subject matter, each sensor in a sensor lot (in some embodiments excluding sample sensors used in in vitro testing) can be non-destructively inspected to determine or measure its characteristics, such as the thickness of the membrane at one or more points of the sensor, and other characteristics including physical characteristics such as the surface area / volume of the active region can be measured or determined. Such measurements or determinations can be performed in an automated manner using, for example, an optical scanner or other suitable measuring device or system, and the sensor characteristics determined for each sensor in the sensor lot are compared to corresponding average values based on a sample sensor for possible correction of the calibration parameters or codes assigned to each sensor. For example, for a calibration parameter defined as sensor sensitivity, the sensitivity is approximately inversely proportional to the thickness of the membrane. Thus, for example, for a sensor having a measured membrane thickness that is approximately 4% thicker than the average membrane thickness of sensors sampled from the same sensor lot as a certain sensor, in one embodiment the sensitivity assigned to that sensor is the average sensitivity determined from the sampled sensors divided by 1.04. Similarly, since the sensitivity is approximately proportional to the active area of the sensor, for a sensor having a measured active area that is approximately 3% smaller than the average active region of sensors sampled from the same sensor lot, the sensitivity assigned to that sensor is the average sensitivity multiplied by 0.97. The assigned sensitivity can be determined from the average sensitivity from the sampled sensors by a plurality of successive adjustments for each inspection or measurement of the sensor. In certain embodiments, the inspection or measurement of each sensor can further include a measurement of the uniformity or structure of the membrane in addition to the thickness of the membrane and / or the surface area or volume of the active sensing region.

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

[0145] K. Exemplary Bluetooth® Communication Protocol The storage memory 5030 of the sensor 110 may include software blocks related to the communication protocol of the communication module. For example, the storage memory 5030 may include a BLE service software block having a function of providing an interface that makes the BLE module 5041 available to the computing hardware of the sensor 110. These software functions may include a BLE logical interface and an interface parser. The BLE services provided by the communication module 5040 may include a generic access profile service, a generic attribute service, a generic access service, a device information service, a data transmission service, and a security service. The data transmission service may be a primary service used to transmit data such as sensor control data, sensor status data, analyte measurement data (past and present), and event log data. The sensor status data may include error data, current active time, and software status. The analyte measurement data may include information such as current and past raw measurement values, current and past values after being processed using appropriate algorithms or models, prediction and trend of measurement levels, comparison of other values with patient-specific average values, action requests determined by algorithms or models, and other similar types of data.

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

[0147] The connection between two devices using a communication protocol such as BLE and the subsequent communication session can be characterized by a similar physical channel operating between the two devices (e.g., sensor 110 and data receiving device 120). The physical channel can include a single channel or a series of channels, and can include, for example, but not limited to, using a common clock and a series of agreed channels determined by a channel or frequency hopping sequence. The communication session can use a similar amount of available communication spectrum, and a number of 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 between devices that are also in proximity.

[0148] For purposes of illustration and not limitation, reference is made to an exemplary embodiment of a process for connection of a sensor to a receiver for use by the disclosed subject matter. First, sensor 110 repeatedly announces its connection information in its vicinity during discovery of data receiving device 120. Sensor 110 can repeatedly announce periodically until a connection is established. Data receiving device 120 detects the advertising packet and performs scanning and filtering to connect via the data provided by sensor 120 in the advertising packet. Next, data receiving device 120 transmits a scan request command, and sensor 110 responds with a scan response packet providing additional details. Next, data receiving device 120 transmits a connection request using the Bluetooth® device address associated with data receiving device 120. Data receiving device 120 can also continuously request to establish a connection to sensor 110 having a specific Bluetooth® device address. Next, the devices establish an initial connection to enable data exchange. The devices initiate a process of initializing the data exchange service and perform a mutual authentication process.

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

[0150] Next, the data receiving device 120 executes sensor control processing to backfill historical data, current data, event logs, and factory data. As an example, for each type of data, the data receiving device 120 sends a request to start a backfill process. The request may specify a range of records defined based on, for example, measurement values, timestamps, etc., as required. 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 120. The sensor 110 may respond to the backfill request from the data receiving device 120 that all data has already been sent. When the backfill is complete, the data receiving device 120 may notify the sensor 110 that it is ready to receive periodic measurement values. The sensor 110 may send measurement values over multiple notification results on a repeating basis. As embodied herein, the multiple notifications may be redundant notifications to ensure that the data is correctly transmitted. Alternatively, the multiple notifications may constitute a single payload.

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

[0152] L. Exemplary Sensor States and Operations For purposes of illustration and not limitation, reference is made to an exemplary embodiment of a high-level depiction of a state machine 6000 of actions that may be performed by sensor 110 shown in FIG. 15. After initialization, the sensor enters a state 6005 related to the manufacture of sensor 110. In the manufacture state 6005, sensor 110 can be configured for operation, for example, storage memory 5030 can be written. At various times while in state 6005, sensor 110 checks for received commands to proceed to a save state 6015. When entering the save state 6015, the sensor executes a software integrity check. While in the save state 6015, the sensor may also receive an operation request command before proceeding to an insertion detection state 6025.

[0153] When entering state 6025, sensor 110 can save information about the authenticated device for communicating with the sensors set during operation, or can initialize an algorithm for performing and interpreting measurements from the detection hardware 5060. Sensor 110 can also initialize a life cycle timer involved in maintaining an active count of the operating time of sensor 110 and start communication with the authenticated device for transmitting the recorded data. While in the insertion detection state 6025, the sensor can enter state 6030, where sensor 110 checks whether the operating time is equal to a predetermined threshold. This operating time threshold can correspond to a timeout function for determining whether the insertion was successful. If the operating time reaches the threshold, sensor 110 proceeds to state 6035, where sensor 110 checks whether the average data read amount is greater than a threshold corresponding to the expected data read amount for inducing the detection of successful insertion. If the data read amount is lower than the threshold while in state 6035, the sensor proceeds to state 6040 corresponding to failed insertion. If the data read amount meets the threshold, the sensor proceeds to the active pairing state 6055.

[0154] The active pairing state 6055 of sensor 110 indicates the state while sensor 110 is operating normally by recording measurement values, processing the measurement values, and reporting them as necessary. While in the active pairing state 6055, sensor 110 attempts to transmit the measurement results or establish a connection with the receiving device 120. Sensor 110 also increases the operating time. When sensor 110 reaches a predetermined operating time threshold (for example, when the operating time reaches the predetermined threshold), sensor 110 transitions to the active expiration state 6065. The active expiration state 6065 of sensor 110 indicates the state while sensor 110 has been operating for its maximum predetermined time.

[0155] While in the expired active state 6065, the sensor 110 may generally perform operations related to the end of operation and ensure that the collected measurements are safely transmitted to the receiving device as needed. For example, while in the expired active state 6065, the sensor 110 can transmit the collected data and, if the connection is not executable, increase attempts to find and establish a connection with a nearby authenticated device. While in the expired active state 6065, the sensor 110 may receive a shutdown command in state 6070. If the shutdown command is not received, the sensor 110 may also check in state 6075 whether the operating time has exceeded a final operation threshold. The final operation threshold may be based on the battery life of the sensor 110. The normal end state 6080 corresponds to the final operation of the sensor 110 and ultimately shuts down the sensor 110.

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

[0157] When the ASIC 5000 enters the measurement mode for the first time, an insertion detection sequence is executed to verify that the sensor 110 is properly placed on the patient's body before appropriate measurements can be made. First, the sensor 110 interprets the command to activate the measurement setting process and causes the ASIC 5000 to enter the measurement command mode. Next, the sensor 110 temporarily enters the measurement life cycle state and performs several consecutive measurements to check whether the insertion was successful. The communication module 5040 or the ASIC 5000 evaluates the measurement results to determine the success of the insertion. If the insertion is considered successful, the sensor 110 enters the measurement state and the sensor 110 begins to perform periodic measurements using the detection hardware 5060. If the sensor 110 determines that the insertion was not successful, the sensor 110 is induced into the insertion failure mode and while the communication module 5040 disables itself, the ASIC 5000 is instructed to return to the save mode.

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

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

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

[0161] The data receiving device 120 or the sensor 110 receives data for update and a command to start the update from the multi-purpose data receiving device 130. Next, the data receiving device 120 may install a firmware or software update. To install the update, the data receiving device 120 or the sensor 110 can put itself into a so-called "safe" mode with restricted operating functions or restart. When the update is complete, the data receiving device 120 or the sensor 110 re-enters the standard operating mode or is reset. The data receiving device 120 or the sensor 110 may perform one or more self-tests to determine that the firmware or software update has been successfully installed. The multi-purpose data receiving device 130 may receive a notification of update success. Next, the multi-purpose data receiving device 130 may report the confirmation of update success to the remote application server 150.

[0162] In certain embodiments, the storage memory 5030 of the sensor 110 includes one-time programmable (OTP) memory. The term OTP memory may refer to a memory that includes access restrictions and security to facilitate a predetermined number of writes to specific addresses or segments within the memory. The memory 5030 may be pre-arranged into a plurality of pre-assigned memory blocks or containers. The containers are pre-assigned a fixed size. If the storage memory 5030 is one-time programmable memory, the containers may be considered in a non-programmable state. Additional containers that have not yet been written to can be made programmable or writable. By containerizing the storage memory 5030 in this way, the transportability of the code and data to be written to the storage memory 5030 can be improved. Updating the software of a device (e.g., the sensor device described herein) stored in the OTP memory can be performed by replacing only the code within one or more specific previously written containers with updated code written in one or more new containers, rather than replacing the entire code within 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. Since containers of various sizes that are expected to be updated can be defined, incremental updates can be issued.

[0163] FIG. 16 is a schematic diagram showing exemplary operations and data flows for over-the-air (OTA) programming of the storage memory 5030 within the sensor device 100 and use of the memory after OTA programming in the execution of the process by the sensor device 110, according to the disclosed subject matter. In the example of OTA programming 500 shown in FIG. 5, a request is transmitted from an external device (e.g., 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 transmits the OTA programming command to the microcontroller 5010 of the sensor device 110.

[0164] At 531, after receiving the OTA programming command, the microcontroller 5010 verifies the OTA programming command. The microcontroller 5010 can determine, for example, whether the OTA programming command is signed with a proper digital signature token. If it is determined that the OTA programming command is valid, the microcontroller 5010 can set the sensor device to the OTA programming mode. At 532, the microcontroller 5010 can verify the OTA programming data. At 533, the microcontroller 5010 can reset the sensor device 110 to re-initialize the sensor device 110 to the programming state. When the sensor device 110 transitions to the OTA programming state, the microcontroller 5010 starts writing data to the rewritable memory 540 (e.g., memory 5020) of the sensor device at 534 and can start writing data to the OTP memory 550 (e.g., storage memory 5030) of the sensor device at 535. The data written by the microcontroller 5010 can be based on the verified OTA programming data. The microcontroller 5010 can write data and mark one or more programming blocks or regions of the OTP memory 550 as invalid or inaccessible. The data written to the empty or unused portion of the OTP memory can be used to replace the invalidated or inaccessible programming blocks of the OTP memory 550. After the microcontroller 5010 writes data to each memory at 534 and 535, the microcontroller 5010 can perform one or more software integrity checks to ensure that no error was introduced into the programming block during the writing process. If the microcontroller 5010 can determine that the data was written without error, the microcontroller 5010 can resume the standard operation of the sensor device.

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

[0166] N. Exemplary Security and Other Architectural Features As embodied herein, a first layer of security for communication between the analyte sensor 110 and other devices can be specified by the communication protocol used for communication and established based on a security protocol integrated into that communication protocol. Another layer of security can be based on a communication protocol that requires proximity of the communication devices. Additionally, certain packets and / or certain data contained within a packet can be encrypted, while other packets and / or data within a packet can be encrypted in a different manner 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 within the analyte monitoring system 100.

[0167] The ASIC 5000 of the analyte sensor 110 can be configured to dynamically generate authentication and encryption keys using the data held in the storage memory 5030. The storage memory 5030 can also be pre-programmed with a set of valid authentication and encryption keys for use with a particular class of devices. The ASIC 5000 can be further configured to perform an authentication process with other devices using the received data and to apply the generated keys to the confidential data before transmitting the confidential 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 transmitted during the communication session, or unique to a block of data included in the message.

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

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

[0170] The test substance data associated with the user is at least partially confidential data, because this information can be used for various purposes including health monitoring and drug administration decisions. In addition to user data, the test substance monitoring system 100 can perform security enhancements against implementation by external parties for reverse engineering. The communication connection can be encrypted using device-specific or session-specific encryption keys. Encrypted or unencrypted communication between any two devices can be verified using a transmission integrity check incorporated into the communication. The operation of the test substance sensor 110 can be protected from tampering by restricting access to the read and write functions to the memory 5020 via the communication interface. The sensor can be configured to allow access only to known or "trusted" devices provided within a "white list", or only to devices that can provide a predetermined code associated with a user authenticated by the manufacturer or other means. The white list can represent an exclusive range meaning that connection identifiers other than those included in the white list are not used, or a preferred range where the white list is searched first but other devices can still be used. The sensor 110 can further reject the connection request and shut down if the requester cannot complete the login process via the communication interface within a predetermined time (e.g., within 4 seconds). These features protect against specific service disruption attacks, particularly service disruption attacks against the BLE interface.

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

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

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

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

[0175] O. Exemplary Payload / Communication Frequency The analyte sensor 110 may be configured to attempt to change its discoverability behavior to increase the probability that the receiving device receives an appropriate data packet and / or the establishment of providing a response signal, or to otherwise reduce limitations that may prevent the response signal from being received. Changing the discoverability behavior of the analyte sensor 110 may include, for example, but is not limited to, changing the frequency with which connection data is included in data packets, changing the frequency with which data packets are generally transmitted, extending or shortening the broadcast window of data packets, changing the time after broadcast that the analyte sensor 110 accepts response or scan signals, including direct transmissions to one or more devices previously communicated with by the analyte sensor 110 (e.g., via one or more attempted transmissions) and / or one or more devices on a whitelist, changing the transmission power associated with the communication module when broadcasting data packets (e.g., to increase the range of the broadcast, reduce the energy consumed, and extend the life of the analyte sensor's battery), changing the speed at which data packets are prepared and broadcast, or including a combination of one or more other changes. Additionally or alternatively, the receiving device may similarly adjust parameters regarding the listening behavior of the device to increase the likelihood of receiving data packets containing connection data.

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

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

[0178] P. Exemplary Sensor Sensitivity Initialization / Adjustment Mechanism As embodied herein, a calibration mechanism for the detection hardware 5060 of the analyte sensor 110 can be adjusted based on external or ambient environmental characteristics and to compensate for attenuation of the detection hardware 5060 during periods when it is not in use (e.g., the “storage time” before use). The calibration mechanism of the detection hardware 5060 can be adjusted autonomously by the sensor 110 (e.g., by the operation of the ASIC 5000 to change characteristics within the memory 5020 or storage 5030), or can be adjusted by other devices of the analyte monitoring system 100.

[0179] As an example, the sensor sensitivity of the detection hardware 5060 can be adjusted based on external temperature data or time since manufacture. When the external temperature is monitored during storage of the sensor, the disclosed subject matter can adaptively change the compensation for sensor sensitivity over time as the device experiences changes in storage conditions. For purposes of illustration and not limitation, in an “active” storage mode where the analyte sensor 110 periodically wakes up to measure temperature, an adaptive sensitivity adjustment can be performed. These mechanisms 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 period based on the measured temperature. Next, the temperature-weighted adjustment values can be accumulated over the active storage mode period to calculate a total sensor sensitivity adjustment value at the end of the active storage mode (e.g., at insertion). Similarly, at insertion, the sensor 110 can determine the time difference between the manufacture of the sensor 110 (which can be written to the storage 5030 of the ASIC 5000) or the detection hardware 5060 and change the sensor sensitivity or other calibration mechanism according to one or more known attenuation rates or equations.

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

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

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

[0183] R. Exemplary alarm mechanism The data transmitted between the sensor 110 and the data receiving device 120 may include raw measurement values or processed measurement values. The data transmitted between the sensor 110 and the data receiving device 120 may further include an alarm or notification for display to the user. The data receiving device 120 can display a notification to the user based on the raw measurement value or the processed measurement value, or convey it in another way, or can display an alarm when received from the sensor 110. Alarms that can be triggered for display to the user include direct analyte values (e.g., a single reading that exceeds a threshold or does not meet a threshold), analyte value trends (e.g., average readings, gradients over a set period that exceed a threshold or do not meet a threshold), analyte value predictions (e.g., algorithm calculations based on analyte values that exceed a threshold or do not meet a threshold), sensor alerts (e.g., detected suspected malfunctions), communication alerts (e.g., no communication between the sensor 110 and the data receiving device 120 over a threshold period, an unknown device attempting to start or failing to start a communication session with the sensor 110), reminders (e.g., a reminder to charge the data receiving device 120, a reminder to take medication, or perform other activities), and alarms based on other alerts of a similar nature. By way of example and not limitation, as embodied herein, the alarm parameters described herein may be configurable by the user, may be fixed during manufacture, or may be a combination of user-configurable and non-user-configurable parameters.

[0184] S. Exemplary Electrode Configuration A sensor configuration characterized by a single active region configured for the detection of a corresponding single analyte may use a two - electrode or three - electrode detection motif, as further described herein with reference to FIGS. 18A - 18C. A sensor configuration characterized by two different active regions for the detection of different analytes, either on separate working electrodes or on the same working electrode, will be described individually later with reference to FIGS. 19A - 21C. A sensor configuration having multiple working electrodes can be particularly advantageous for incorporating two different active regions within the same sensor tail, since the signal contributions from each active region can be more readily determined.

[0185] When a single working electrode is present within 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 includes a working electrode and a second electrode, where the second electrode can function as both a counter electrode and a reference electrode (i.e., a counter / reference electrode). The various electrodes may be at least partially stacked on top of each other (layered) and / or laterally spaced from each other on the sensor tail. A suitable sensor configuration can be substantially flat or substantially cylindrical in shape. In any of the sensor configurations disclosed herein, the various electrodes can be electrically insulated from each other by a dielectric material or a similar insulator.

[0186] An analyte sensor characterized by multiple working electrodes may similarly include at least one additional electrode. When one additional electrode is present, the one additional electrode can function as a counter / reference electrode for each of the multiple working electrodes. When two additional electrodes are present, one of the additional electrodes may function as a counter electrode for each of the multiple working electrodes, and the other of the additional electrodes may function as a reference electrode for each of the multiple working electrodes.

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

[0188] Referring further to FIG. 18A, a membrane 220 covers at least the active region 218. In certain embodiments, the membrane 220 can cover a portion or all of the working electrode 214 and / or the counter / reference electrode 30216, or the entire analyte sensor 200. One or both sides of the analyte sensor 200 can be covered by the membrane 220. The membrane 220 can include one or more polymeric membrane materials having the ability to restrict the analyte flux to the active region 218 (i.e., the membrane 220 is a mass transfer - limiting membrane having some permeability to the analyte of interest (e.g., ketones)). According to the disclosure herein, as further described below, the membrane 220 can be cross - linked by a branching cross - linker in certain sensor configurations. For example, but not limited to, the membrane 220 is cross - linked by a branched glycidyl ether. The composition and thickness of the membrane 220 can vary to promote the desired analyte (e.g., ketones) flux to the active region 218, thereby providing the desired signal intensity and stability. The analyte sensor 200 can be operable to assay the analyte by any of the electrochemical detection techniques of coulometry, amperometry, voltammetry, or potentiometry.

[0189] In certain embodiments, one or more membranes including an interference domain and a mass transport limiting membrane are deposited on the exposed electroactive surface of the working electrode, such as a platinum surface. For example, without limitation, the interference domain may be disposed on the working electrode, the active region may be disposed on the interference domain, and the mass transport limiting membrane may be disposed on the active region.

[0190] Figures 18B and 18C show schematic diagrams of exemplary three - electrode analyte sensors, which are also compatible for use in the disclosure herein. The three - electrode analyte sensor configuration can be similar to that shown as analyte sensor 200 in Figure 18A, except that it includes additional electrodes 217 within analyte sensors 201 and 202 (Figures 18B and 18C). With the additional electrodes 217, the counter / reference electrode 30216 may then function either as a counter electrode or a reference electrode, and the additional electrode 217 serves other electrode functions not otherwise described. The working electrode 214 continues to perform its original function. The additional electrode 217 can be disposed on either the working electrode 214 or the electrode 30216, with a separation layer of dielectric material in between. For example, but not limited to, as shown in Figure 18B, dielectric layers 219a, 219b, and 219c separate the electrodes 214, 30216, and 217 from each other and provide electrical insulation. Alternatively, as shown in Figure 18C, at least one of the electrodes 214, 30216, and 217 may be positioned on the opposite side of the substrate 30212. Thus, in certain embodiments, the electrode 214 (working electrode) and the electrode 30216 (counter electrode) may be disposed on opposite sides of the substrate 30212, and the electrode 217 (reference electrode) is disposed on one of the electrode 214 or the electrode 30216 and is spaced therefrom by a dielectric material. A reference material layer 30230 (e.g., Ag / AgCl) may be present on the electrode 217, and the position of the reference material layer 30230 is not limited to the positions shown in Figures 18B and 18C. Similar to the sensor 200 shown in Figure 18A, the active regions 218 (for detection of ketones) in analyte sensors 201 and 202 can include multiple spots or a single spot. In addition, analyte sensors 201 and 202 can be operable to assay an analyte by any of the electrochemical detection techniques of coulometry, amperometry, voltammetry, or potentiometry.

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

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

[0193] Figures 19B and 19C show cross-sectional views of exemplary three-electrode sensor configurations for sensors 204 and 205, respectively, each featuring a single working electrode with a first active region 218a and a second active region 218b disposed thereon. Figures 19B and 19C are similar to Figures 18B and 18C in other respects and can be better understood by referring to them. Similar to Figure 19A, the composition of the membrane 220 may vary in the active regions 218a and 218b or may be compositionally the same.

[0194] An exemplary sensor configuration having a plurality of working electrodes, specifically two working electrodes, will be described in more detail with reference to FIGS. 4 to 21C. The following description mainly targets a sensor configuration having two working electrodes, but it should be understood that more than two working electrodes can be incorporated by the extension of the disclosure herein. In addition to the first analyte and the second analyte, additional working electrodes can be used to endow the analyte sensor with additional sensing capabilities for detecting, for example, third and / or fourth analytes.

[0195] Figure 4 shows a cross-sectional view of an exemplary analyte sensor configuration having two working electrodes, a reference electrode, and a counter electrode that is adapted for use in the disclosure of this specification. As shown, the analyte sensor 300 includes working electrodes 304 and 306 disposed on opposite surfaces of a substrate 302. A first active region 310a is disposed on the surface of working electrode 304, and a second active region 310b is disposed on the surface of working electrode 306. The counter electrode 320 is electrically insulated from the working electrode 304 by a dielectric layer 322, and the reference electrode 321 is electrically insulated from the working electrode 306 by a dielectric layer 323. Outer dielectric layers 330 and 332 are disposed on the reference electrode 321 and the counter electrode 320, respectively. The membrane 340 may, according to various embodiments, cover at least the active regions 310a and 310b, and optionally, other components of the analyte sensor 300 or the entire analyte sensor 300 may also be covered by a first membrane portion 340a and / or a second membrane portion 340b. Further, the membrane 340 may be continuous, but may vary compositionally between the first membrane portion 340a and the second membrane portion 340b (i.e., over the active regions 310a and 310b) to allow for different permeability values to separately regulate the analyte flux at each location. For example, different membrane formulations may be sprayed and / or printed on opposite surfaces of the analyte sensor 300. Dip coating techniques may also be suitable, particularly for depositing at least a portion of a bilayer membrane over one of the active regions 310a and 310b. Thus, according to certain embodiments of the present disclosure, one of the first membrane portion 340a and the second membrane portion 340b may comprise a bilayer membrane, and the other of the first membrane portion 340a and the second membrane portion 340b may comprise a single membrane polymer. Similar to the analyte sensors 200, 201, and 202, the analyte sensor 300 may be operable to assay ketones (and / or a second analyte) by any of the electrochemical detection techniques of coulometry, amperometry, voltammetry, or potentiometry. In certain embodiments, the analyte sensor may include more than one membrane 340, for example, two or more membranes.For example, without limitation, the analyte sensor may include one or more active regions, such as the films coating 310a and 310a, and a further film coating the entire sensor as shown in FIG. 20. In certain embodiments, either or both of the active regions 310a and 310b may be configured to detect ketones by using, for example, an enzyme system comprising NADH oxidase and β-hydroxybutyrate dehydrogenase. In certain embodiments, only one of the active regions 310a and 310b is configured to detect ketones by using, for example, an enzyme system comprising NADH oxidase and β-hydroxybutyrate dehydrogenase. In certain embodiments, another active region is configured to detect a second analyte. In certain embodiments, the second analyte is glucose.

[0196] An alternative sensor configuration having a plurality of working electrodes and different from the configuration shown in FIG. 20 may feature a counter / reference electrode instead of separate counter and reference electrodes 320, 321, and / or may feature a different arrangement of layers and / or films than that explicitly shown. For example, without limitation, the positions of the counter electrode 320 and the reference electrode 321 may be opposite to those shown in FIG. 20. Further, the working electrodes 304 and 306 do not necessarily have to be present on the opposite side of the substrate 302 as in the positions shown in FIG. 20.

[0197] Suitable sensor configurations can be characterized by electrodes that are substantially planar as a characteristic, but sensor configurations characterized by non-planar electrodes may also be advantageous and it should be recognized that they may be particularly suitable for use in the disclosure herein. In particular, substantially cylindrical electrodes arranged concentrically with each other can facilitate the deposition of a mass transfer limiting membrane, as described below. In particular, concentric working electrodes spaced along the length of the sensor tail can facilitate film deposition by a continuous dip coating operation in a manner similar to that described above for substantially planar sensor configurations. FIGS. 21A-21C show perspective views of an analyte sensor characterized by two working electrodes arranged concentrically with each other. It should be recognized that sensor configurations having a concentric electrode arrangement but no second working electrode are also possible in the present disclosure.

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

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

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

[0201] It should be further understood that the positioning of the various electrodes in FIGS. 21A and 21B may be different from that explicitly shown. For example, the positions of the counter electrode 430 and the reference electrode 440 may be reversed from the configurations shown in FIGS. 21A and 21B. Similarly, the positions of the working electrodes 410 and 420 are not limited to the positions explicitly shown in FIGS. 21A and 21B. FIG. 21C shows an alternative to the sensor configuration shown in FIG. 21B, where the sensor 405 includes a counter electrode 430 and a reference electrode 440 positioned more proximally with respect to the sensor tip 404, and working electrodes 410 and 420 positioned more distally with respect to the sensor tip 404. A sensor configuration in which the working electrodes 410 and 420 are positioned more distally with respect to the sensor tip 404 provides a larger surface area for the deposition of the active regions 414a and 414b (exemplarily showing five separate detection spots in FIG. 21C), thereby potentially being advantageous by facilitating an increase in signal strength in some cases. Similarly, the central substrate 402 can be omitted in any of the concentric sensor configurations disclosed herein, in which case the innermost electrode can instead support the subsequently deposited layers.

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

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

[0204] In certain embodiments, additional electrodes may be included in the sensor tail. For example, without limitation, a three - electrode system (working electrode, reference electrode, and counter electrode) and / or additional working electrodes (e.g., an electrode for detecting a second analyte). In certain embodiments where the sensor comprises two working electrodes, the two working electrodes may be juxtaposed and a reference electrode may be disposed around them (e.g., wound helically around two or more working electrodes). In certain embodiments, two or more working electrodes may extend parallel to each other. In certain embodiments, the reference electrode is wound around the working electrodes and extends towards the distal end (i.e., the in - vivo end) of the sensor tail. In certain embodiments, the reference electrode extends (e.g., helically) into the exposed area of the working electrodes.

[0205] In certain embodiments, one or more working electrodes are wound helically around the reference electrode. In certain embodiments where two or more working electrodes are provided, the working electrodes may be formed in a double, triple, quadruple, etc. helical configuration (e.g., surrounding the reference electrode, an insulated rod, or other support structure) along the length of the sensor tail. In certain embodiments, the electrodes, e.g., two or more working electrodes, are formed coaxially. For example, without limitation, all the electrodes share the same central axis.

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

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

[0208] Some components of the analyte sensor and the film of the present disclosure are further described below. 2. Enzyme The active region of the analyte sensor of the present disclosure can be configured to detect one or more analytes. In certain embodiments, the analyte sensor of the present disclosure can include two or more active regions, and each active region is configured to detect the same analyte or different analytes. Non-limiting examples of analytes that can be detected using the disclosed analyte sensor include ketones, glucose, oxygen, creatinine, alcohol, such as ethanol, and lactate. In certain embodiments, the analyte is one or more ketones.

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

[0210] In certain embodiments, the analyte sensor of the present disclosure includes at least one active region configured to detect ketones. A particular enzyme system that can be used to detect ketones is described in FIG. 22. In the shown enzyme reaction, β-hydroxybutyrate acts as a surrogate for ketones formed in vivo. As shown in FIG. 22, a pair of concerted enzymes can be used to detect ketones by the disclosure herein. For example, without limitation, the pair of concerted enzymes can include dehydrogenase and oxidase. In certain embodiments, the dehydrogenase is β-hydroxybutyrate dehydrogenase. In certain embodiments, the oxidase is NADH oxidase. The enzyme cofactors NAD+ and NADH are disclosed herein and assist in facilitating the concerted enzyme reaction shown in FIG. 22. When the ketone-responsive active region includes this pair of concerted enzymes, β-hydroxybutyrate dehydrogenase (HBDH) catalyzes the reaction between β-hydroxybutyrate and oxidized nicotinamide adenine dinucleotide (NAD +) can be converted into acetoacetate and reduced nicotinamide adenine dinucleotide (NADH), respectively. Next, NADH oxidase (NADHOx) can catalyze the reaction between molecular oxygen and NADH to produce hydrogen peroxide and NAD⁺. Next, hydrogen peroxide can be catalytically oxidized at the anode, i.e., the working electrode, according to the following equation.

[0211] H2O2→2H + +O2+2e - [1] The electrons transferred during this reaction provide the basis for the detection of ketones at the working electrode and eliminate the need for a redox mediator. Next, the obtained electrochemical signal can be correlated with the amount of ketones initially present in the sample.

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

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

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

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

[0216] In certain embodiments, the ketone-responsive active region can comprise one or more enzymes of an enzyme system at 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, for example β-hydroxybutyrate dehydrogenase and / or NADH oxidase. In certain embodiments, the ketone-responsive active region can comprise both enzymes of an enzyme system at 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, for example β-hydroxybutyrate dehydrogenase and NADH oxidase. In certain embodiments, the ketone-responsive active region can comprise β-hydroxybutyrate dehydrogenase at 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. In certain embodiments, the ketone-responsive active region can comprise NADH oxidase at 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.

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

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

[0219] In certain embodiments, the ketone-responsive active region may further comprise a cofactor (or a derivative thereof) for the enzymes of the enzyme systems disclosed herein. Non-limiting examples of cofactors include NADH or NADPH or derivatives thereof. In certain embodiments, the cofactor is NADH or a derivative thereof. In certain embodiments, the ketone-responsive active region may comprise a ratio of cofactor to NADH oxidase of from about 40:1 to about 1:40, such as from about 35:1 to about 1:35, from about 30:1 to about 1:30, from about 25:1 to about 1:25, from about 20:1 to about 1:20, from about 15:1 to about 1:15, from about 10:1 to about 1:10, from about 9:1 to about 1:9, from about 8:1 to about 1:8, from about 7:1 to about 1:7, from about 6:1 to about 1:6, from about 5:1 to about 1:5, from about 4:1 to about 1:4, from about 3:1 to about 1:3, from about 2:1 to about 1:2, or about 1:1. In certain embodiments, the ketone-responsive active region may comprise a ratio of cofactor to NADH oxidase of from about 2:1 to about 1:2. In certain embodiments, the ketone-responsive active region may comprise a ratio of cofactor to β-hydroxybutyrate dehydrogenase of from about 40:1 to about 1:40, such as from about 35:1 to about 1:35, from about 30:1 to about 1:30, from about 25:1 to about 1:25, from about 20:1 to about 1:20, from about 15:1 to about 1:15, from about 10:1 to about 1:10, from about 9:1 to about 1:9, from about 8:1 to about 1:8, from about 7:1 to about 1:7, from about 6:1 to about 1:6, from about 5:1 to about 1:5, from about 4:1 to about 1:4, from about 3:1 to about 1:3, from about 2:1 to about 1:2, or about 1:1. In certain embodiments, the ketone-responsive active region may comprise a ratio of cofactor to β-hydroxybutyrate dehydrogenase of from about 2:1 to about 1:2. In certain embodiments, the ketone-responsive active region may comprise from about 10% to about 50% by weight, such as from about 15% to about 45% by weight, from about 20% to about 40% by weight, from about 20% to about 35% by weight, from about 20% to about 30% by weight of the cofactor. In certain embodiments, the ketone-responsive active region may comprise from about 15% to about 35% by weight of the cofactor. In certain embodiments, the cofactor, such as NADH, may be physically retained within the ketone-responsive active region. For example, without limitation, a membrane coating the ketone-responsive active region can allow sufficient inward diffusion of ketones to enable detection of ketones while helping to retain the cofactor within the ketone-responsive active region.

[0220] In certain embodiments, the ketone-responsive active region is disposed on a portion of the working electrode. For example, but not limited to, the ketone-responsive active region is disposed on a portion of the working electrode in a spot pattern, such as two or more spots on the working electrode. In certain embodiments, the ketone-responsive active region is disposed on a portion of the working electrode in a slot-shaped pattern. In certain embodiments, the ketone-responsive active region is disposed over the entire length of the working electrode or in a continuous pattern on the working electrode. In certain embodiments, the ketone-responsive active region is about 0.01 mm 2 to about 2.0 mm 2 , for example about 0.1 mm 2 to about 1.0 mm 2 , or about 0.2 mm 2 to about 0.5 mm 2 in area.

[0221] In certain embodiments, the analyte sensor of the present disclosure may include a second active region for detecting an analyte different from ketones on, for example, the same working electrode as the ketone-responsive active region or on a second working electrode. In certain embodiments, the second active region is a glucose-responsive active region, a lactate-responsive active region, a creatinine-responsive active region, or an alcohol-responsive active region.

[0222] In certain embodiments, the second active region of the analyte sensor of the present disclosure may include one or more enzymes for detecting glucose. For example, but not limited to, the analyte sensor of the present disclosure may include an active region (e.g., the second active region) having one or more enzymes (e.g., an enzyme system) for detecting glucose disposed, for example, on the second working electrode. In certain embodiments, the analyte sensor may include an active site comprising glucose oxidase and / or glucose dehydrogenase for detecting glucose. In certain embodiments, glucose may be detected using glucose oxidase present in the second active region that produces H2O2 as a product. H2O2 reacts with the electrochemically reactive surface of the second working electrode, such as a platinum surface, to generate a detectable current.

[0223] In certain embodiments, the second active region may include one or more enzymes for detecting lactate. For example, but not limited to, the analyte sensor of the present disclosure may include an active region (e.g., the second active region) having one or more enzymes (e.g., an enzyme system) for detecting lactate disposed, for example, on the second working electrode. In certain embodiments, the analyte sensor may include an active site comprising lactate dehydrogenase and / or lactate oxidase.

[0224] In certain embodiments, the second enzyme-responsive active region, such as on the second working electrode of the analyte sensor of the present disclosure, may include one or more enzymes for detecting alcohol. For example, but not limited to, the analyte sensor of the present disclosure may include an active region (e.g., the second active region) having one or more enzymes (e.g., an enzyme system) for detecting alcohol disposed, for example, on the second working electrode. In certain embodiments, the analyte sensor may include an active site comprising alcohol dehydrogenase.

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

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

[0227] In the configuration of certain other analyte sensors, the first active region and the second active region can be disposed on a single working electrode. The first signal can be obtained, for example, at a low potential from the first active region, and the second signal, which includes the contributions of the signals from both active regions, can be obtained at a high potential. Next, by subtracting the first signal from the second signal, it becomes possible to determine the signal contribution resulting from the second analyte. Next, the signal contribution from each analyte can be correlated with the analyte concentration in a manner similar to that described for the sensor configuration having a plurality of working electrodes. In a particular embodiment, when a ketone-responsive active region and a second active region configured to detect a different analyte, such as a glucose-responsive active region, are disposed on a single working electrode in this manner, one of the active regions can be configured to respond separately so as to facilitate the detection of each analyte. For example, the ketone-responsive active region or the glucose-responsive active region can generate a signal independently of the other active regions.

[0228] It should also be understood that the sensitivity (output current) of the analyte sensor directed to each analyte can vary by changing the coverage (area or size) of the active region, the area ratio of the active regions relative to each other, the identity, the thickness and / or composition of the mass transfer limiting film covering the active region. Given the benefit of the disclosure herein, changes in these parameters can be readily implemented by those skilled in the art.

[0229] 3. Redox mediator In a particular embodiment, the analyte sensor disclosed herein can include an electron transfer agent. As discussed above, due to the composition of the ketone-responsive active region, such as the enzyme composition and the composition of the working electrode, in any of the exemplary sensor configurations disclosed herein, the electron transfer agent is not included in the ketone-responsive active region. For example, without limitation, a ketone-responsive active region comprising an enzyme system including β-hydroxybutyrate dehydrogenase and NADH oxidase does not include an electron transfer agent, such as a redox mediator, such as an osmium redox mediator.

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

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

[0232] In certain embodiments, suitable electron transfer agents can include electroreducible and electrooxidizable ions, complexes, or molecules (e.g., quinones) having a redox potential that is several hundred millivolts higher or lower than the redox potential of the standard calomel electrode (SCE). In certain embodiments, redox mediators can include osmium complexes and other transition metal complexes (e.g., as described in U.S. Pat. Nos. 6,134,461 and 6,605,200, which are hereby incorporated by reference in their entirety). Further examples of suitable redox mediators include those described in U.S. Pat. Nos. 6,736,957, 7,501,053, and 7,754,093, the disclosures of each of which are also hereby incorporated by reference in their entirety. Examples of other suitable redox mediators can include metal compounds or complexes of ruthenium, osmium, iron (e.g., polyvinylferrocene or hexacyanoferrate), or cobalt (e.g., including their metallocene compounds). Suitable ligands for metal complexes can also include polydentate ligands such as, for example, bipyridine, biimidazole, phenanthroline, or pyridyl(imidazole). Other suitable polydentate ligands can include, for example, amino acids, oxalic acid, acetylacetone, diaminoalkanes, or o-diaminoarenes. Any combination of monodentate, bidentate, tridentate, tetradentate, or higher denticity ligands can be present in the metal complex to achieve a complete coordination sphere.

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

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

[0235] In certain embodiments, the polymer is polyvinylpyridine or its copolymer. In certain embodiments, the polymer is a copolymer of vinylpyridine and styrene. In certain embodiments, when an enzyme system comprising a plurality of enzymes including NADH oxidase is present in a given active region, all of the plurality of enzymes may be covalently bound to a polymer. In certain other embodiments, only some of the plurality of enzymes are covalently bound to the polymer. For example, without limitation, one or more enzymes in the enzyme system may be covalently bound to the polymer and at least one enzyme may be non-covalently bound to the polymer, whereby the non-covalently bound enzyme may be physically retained within the polymer. In certain embodiments, β-hydroxybutyrate dehydrogenase and NADH oxidase may be covalently bound to a polymer within the ketone-responsive active region of the disclosed analyte sensor. In certain embodiments, β-hydroxybutyrate dehydrogenase may be covalently bound to the polymer and NADH oxidase may be non-covalently bound to the polymer. Alternatively, NADH oxidase may be covalently bound to the polymer and β-hydroxybutyrate dehydrogenase may be non-covalently bound to the polymer. In certain embodiments, NAD + may be covalently bound to the polymer. In certain embodiments, NAD+ is not covalently bound to the polymer. NAD + In certain embodiments where it is not covalently bound to the polymer, NAD + may be physically retained within the ketone-responsive active region. In certain embodiments, the membrane coating the ketone-responsive active region can help retain NAD + within the ketone-responsive active region while still allowing sufficient inward diffusion of ketones to enable detection of ketones. Suitable membrane polymers for coating the ketone-responsive active region are further discussed herein.

[0236] Alternatively or additionally, the active region, e.g., the ketone-responsive active region, does not contain polyvinylpyridine or a copolymer of vinylpyridine and styrene. In certain embodiments, one or more enzymes present in the active region can be immobilized in the active region by use of a crosslinking agent, as described herein, in the presence of a stabilizer. For example, without limitation, one or more enzymes in the ketone-responsive active region of the present disclosure, such as NADH oxidase and / or β-hydroxybutyrate dehydrogenase, can be immobilized in the active region by using a crosslinking agent, such as polyethylene glycol diglycidyl ether. In certain embodiments, one or more enzymes in the ketone-responsive active region, such as NADH oxidase and / or β-hydroxybutyrate dehydrogenase, can covalently bind to a stabilizer in the active region by using a crosslinking agent. In certain embodiments, the stabilizer is serum albumin, such as bovine serum albumin (BSA).

[0237] In certain embodiments, the covalent attachment of one or more enzymes and / or redox mediators to a polymer and / or stabilizer in a given active region can occur via crosslinks introduced by a suitable crosslinking agent. Suitable crosslinking agents can include, but are not limited to, one or more crosslinkable functional groups such as vinyl, alkoxy, acetoxy, enoxy, oxime, amino, hydroxyl, cyano, halo, acrylate, epoxide, and isocyanate groups. In certain embodiments, the crosslinking agent comprises one or more, two or more, three or more, or four or more epoxide groups. For example, without limitation, crosslinking agents for use with the present disclosure can include mono-, di-, tri-, and tetra-ethylene oxide. In certain embodiments, crosslinking agents for reaction with free amino groups in the enzyme (e.g., with free side chain amines in lysine) can include crosslinking agents such as polyethylene glycol dibutyl ethers, polypropylene glycol dimethyl ethers, polyalkylene glycol allyl methyl ethers, polyethylene glycol diglycidyl ether (PEGDGE), or other polyepoxides, cyanuric chloride, N-hydroxysuccinimide, imido esters, epichlorohydrin, or derivatized variants thereof. In certain embodiments, the crosslinking agent is PEGDGE having an average molecular weight (M n ) of, for example, about 200 to 1000, for example about 400. In certain embodiments, the crosslinking agent is PEGDGE400. In certain embodiments, the crosslinking agent can be glutaraldehyde. Suitable crosslinking agents for reaction with free carboxylic acid groups in the enzyme can include, for example, carbodiimides. In certain embodiments, the crosslinking of the enzyme to the polymer or stabilizer is generally intermolecular.

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

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

[0240] In certain embodiments, the film coating the analyte-responsive active region can function as a mass transfer limiting film and / or to improve biocompatibility. The mass transfer limiting film can act as a diffusion barrier to reduce the rate of mass transfer of the analyte. For example, but not limited to, by restricting access of the analyte, such as a ketone, to the analyte-responsive active region by the mass transfer limiting film, it can help avoid sensor overload (saturation), thereby improving detection performance and accuracy.

[0241] In certain embodiments, the mass transfer limiting film can be uniform and can be a single component (including a single film polymer). Alternatively, the mass transfer limiting film can be multi-component (including two or more different film polymers). In certain embodiments, the mass transfer limiting film can include two or more layers, such as a bilayer or a trilayer film. In certain embodiments, each layer can have different concentrations or thicknesses and can comprise different polymers or the same polymer. In certain embodiments, the ketone-responsive active region can be covered by a multilayer film, such as a bilayer film, and the second analyte-responsive active region can be covered by a single film. In certain embodiments, the ketone-responsive active region can be covered by a multilayer film, such as a bilayer film, and the second analyte-responsive active region can be covered by a multilayer film, such as a bilayer film.

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

[0243] In certain embodiments, a membrane for use in the present disclosure, such as a single-component membrane, may include polyvinylpyridine (e.g., poly(4-vinylpyridine) and / or poly(2-vinylpyridine)). In certain embodiments, a membrane for use in the present disclosure, such as a single-component membrane, may include poly(4-vinylpyridine). In certain embodiments, a membrane for use in the present disclosure, such as a single-component membrane, may include a copolymer of vinylpyridine and styrene. In certain embodiments, the membrane may comprise a polyvinylpyridine-co-styrene copolymer. For example, without limitation, a polyvinylpyridine-co-styrene copolymer for use in the present disclosure may include a polyvinylpyridine-co-styrene copolymer functionalized with a polyethylene glycol tail in which some of the pyridine nitrogen atoms are not crosslinked and some of the pyridine nitrogen atoms are functionalized with an alkylsulfonic acid group. In certain embodiments, the derivatized polyvinylpyridine-co-styrene copolymer for use as a membrane polymer may be the 10Q5 polymer described in U.S. Patent No. 8,761,857, the contents of which are incorporated herein by reference in their entirety. In certain embodiments, the polyvinylpyridine-based polymer has a molecular weight of from about 50 Da to about 500 kDa.

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

[0245] In certain embodiments, the membrane comprises a polyurethane membrane having both hydrophilic and hydrophobic regions. In certain embodiments, the hydrophobic polymer component is polyurethane, polyurethane urea, or poly(ether-urethane-urea). In certain embodiments, the polyurethane is a polymer produced by a condensation reaction of a diisocyanate and a bifunctional hydroxyl-containing material. In certain embodiments, the polyurethane urea is a polymer produced by a condensation reaction of a diisocyanate and a bifunctional amine-containing material. In certain embodiments, the diisocyanate for use herein includes, for example, an aliphatic diisocyanate containing from about 4 to about 8 methylene units, or a diisocyanate containing an alicyclic moiety. Further non-limiting examples of polymers that can be used for the production of the membranes of the sensors of the present disclosure include vinyl polymers, polyethers, polyesters, polyamides, inorganic polymers (such as polysiloxanes and polycarbosiloxanes), natural polymers (such as cellulose-based and protein-based materials), and mixtures (such as mixed or laminated structures), or combinations thereof. In certain embodiments, the hydrophilic polymer component is polyethylene oxide and / or polyethylene glycol. For example, but not limited to, the hydrophobic-hydrophilic copolymer component for use in the present disclosure is a polyurethane polymer comprising from about 10% to about 50%, such as about 20%, hydrophilic polyethylene oxide.

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

[0247] In certain embodiments, a hydrophilic or hydrophobic modifier can be used to "fine-tune" the permeability of the resulting membrane to a target analyte, such as ketones. In certain embodiments, hydrophilic modifiers such as polyethylene glycol, hydroxyl or polyhydroxyl modifiers, and any combination thereof can be used to improve the biocompatibility of the polymer or the resulting membrane.

[0248] In certain embodiments where there are multiple active regions, the mass transfer limiting membrane can cover each active region, including the selection of compositional changes in different active regions, which can be achieved via a continuous dip coating operation that produces a bilayer membrane portion at the working electrode disposed closer to the sensor tip.

[0249] In certain embodiments where there are multiple active regions, separate mass transfer limiting membranes may cover each active region. For example, without limitation, a mass transfer limiting membrane may be disposed over a first active region, such as a ketone-responsive active region, and a separate second mass transfer limiting membrane may cover a second active region, such as a glucose-responsive active region. In certain embodiments, the two mass transfer limiting membranes are spatially separated and do not overlap with each other. In certain embodiments, the first mass transfer limiting membrane does not overlap with the second mass transfer limiting membrane, and the second mass transfer limiting membrane does not overlap with the first mass transfer limiting membrane. In certain embodiments, the first mass transfer limiting membrane comprises a different polymer than the second mass transfer limiting membrane. Alternatively, the first mass transfer limiting membrane comprises the same polymer as the second mass transfer limiting membrane. In certain embodiments, the first mass transfer limiting membrane comprises the same polymer as the second mass transfer limiting membrane but a different cross-linking agent.

[0250] In certain embodiments, the composition of the mass transfer limiting membrane disposed on an analyte sensor having two active regions may be the same or different when the mass transfer limiting membrane covers each active region. For example, without limitation, the portion of the mass transfer limiting membrane covering the ketone-responsive active region may be multi-component, and / or the portion of the mass transfer limiting membrane covering a second analyte-responsive region, such as a glucose-responsive active region, may be single-component. Alternatively, the portion of the mass transfer limiting membrane covering the ketone-responsive active region may be single-component, and / or the portion of the mass transfer limiting membrane covering a second analyte-responsive region, such as a glucose-responsive active region, may be multi-component. In certain embodiments, the mass transfer limiting membrane covering the ketone-responsive active region may be single-component, and the mass transfer limiting membrane covering a second analyte-responsive region, such as a glucose-responsive active region, may also be single-component. In certain embodiments, the mass transfer limiting membrane covering the ketone-responsive active region comprises a different polymer than the mass transfer limiting membrane covering a second analyte-responsive region, such as a glucose-responsive active region.

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

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

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

[0254] In certain other embodiments, the membrane polymer that coats one or more active regions can be crosslinked using the crosslinking agents disclosed herein and in Section 4 above. In certain embodiments where there are two mass transfer limiting membranes, e.g., a first mass transfer limiting membrane and a second mass transfer limiting membrane, each membrane can be crosslinked to a different crosslinking agent. For example, without limitation, the crosslinking agent can result in a membrane that more restricts the diffusion of a particular compound, e.g., an analyte in the membrane, or less restricts the diffusion of a particular compound, e.g., by affecting the size of the pores in the membrane. For example, without limitation, in a sensor configured to detect ketones and glucose, the mass transfer limiting membrane that coats the ketone-responsive region can have a pore size that restricts the diffusion of compounds larger than ketones, e.g., glucose, through the membrane.

[0255] In certain embodiments, crosslinking agents for use in the present disclosure can include polyepoxides, carbodiimides, cyanuric chloride, triglycidyl glycerol, N-hydroxysuccinimide, imido esters, epichlorohydrin, or derivatized variants thereof. In certain embodiments, the membrane polymer that coats one or more active regions can be crosslinked to a branched crosslinking agent that can, for example, reduce the amount of extract obtained from a mass transfer limiting membrane. Non-limiting examples of branched crosslinking agents include, for example, branched glycidyl ether crosslinking agents that include a branched glycidyl ether crosslinking agent having two, three, or more crosslinkable groups. In certain embodiments, the branched crosslinking agent can include two or more crosslinkable groups such as polyethylene glycol diglycidyl ether. In certain embodiments, the branched crosslinking agent can include three or more crosslinkable groups such as polyethylene glycol tetraglycidyl ether. In certain embodiments, the mass transfer limiting membrane can include polyvinyl pyridine or a copolymer of vinyl pyridine and styrene crosslinked to a branched glycidyl ether crosslinking agent having two or three crosslinkable groups such as polyethylene glycol tetraglycidyl ether or polyethylene glycol diglycidyl ether. In certain embodiments, an epoxy group such as polyethylene glycol tetraglycidyl ether or polyethylene glycol diglycidyl ether, can form a covalent bond with pyridine or imidazole by ring opening of the epoxy ring, resulting in a hydroxyalkyl group that crosslinks the body of the crosslinking agent and the heterocycle of the membrane polymer.

[0256] In certain embodiments, the crosslinking agent is polyethylene glycol diglycidyl ether (PEGDGE). In certain embodiments, the PEGDGE used to facilitate crosslinking (e.g., intermolecular crosslinking) between two or more membrane polymer backbones can exhibit a wide range of suitable molecular weights. In certain embodiments, the molecular weight of PEGDGE can range from about 100 g / mol to about 5000 g / mol. The number of ethylene glycol repeating units in each arm of PEGDGE may be the same or different and generally can vary over a range within a given sample for which an average molecular weight is obtained. In certain embodiments, the PEGDGE for use in the present disclosure has an average molecular weight (M n ) of about 200 - 1000, such as about 400. In certain embodiments, the crosslinking agent is PEGDGE400.

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

[0258] In certain embodiments, polydimethylsiloxane (PDMS) can be incorporated into any of the mass transfer limiting membranes disclosed herein. In certain embodiments, the analyte sensor described herein can include a sensor tail having at least a first working electrode, a first active region disposed on the surface of the first working electrode, and a mass transfer limiting membrane that at least covers the first active region and is permeable to the first analyte. In certain embodiments, the first active region includes at least one enzyme (optionally covalently bound to a first polymer and / or stabilizer) and an enzyme system that responds to the first analyte, such as an enzyme system that responds to ketones. For example, without limitation, the analyte sensor described herein can include a sensor tail having at least a first working platinum electrode, a ketone-responsive active region that includes an enzyme system having β-hydroxybutyrate dehydrogenase and NADH oxidase disposed on the surface of the first working electrode (where one or both of the enzymes are optionally covalently bound to a polymer and / or stabilizer), and a mass transfer limiting membrane that covers the ketone-responsive active region and is permeable to ketones. In certain embodiments, the mass transfer limiting membrane includes a membrane polymer crosslinked with a branched glycidyl ether crosslinking agent having two or more crosslinkable groups, such as polyethylene glycol diglycidyl ether or polyethylene glycol tetraglycidyl ether.

[0259] In certain embodiments, the analyte sensor of the present disclosure includes a sensor tail portion having at least a first working electrode and a second working electrode spaced apart from each other along the length of the sensor tail. In certain embodiments, a first active region is disposed on the surface of the first working electrode, a second active region is disposed on the surface of the second working electrode, and the first active region and the second active region respond to different analytes. For example, but not by way of limitation, the first active region is a ketone-responsive active region. In certain embodiments, the second active region may respond to glucose. In certain embodiments, a mass transfer limiting film covers the first active region and the second active region, and the mass transfer limiting film includes a bilayer film portion covering the first active region and a uniform film portion covering the second active region. In certain embodiments, each layer of the bilayer film portion comprises a different film polymer. In certain embodiments, the bottom layer of the bilayer portion over the first active region comprises a different film polymer than the uniform film portion over the second active region, such as a polyvinylpyridine polymer.

[0260] In certain embodiments, when the first active region and the second active region configured to assay different analytes are disposed on different working electrodes, the mass transfer limiting film may have different permeability values for the first analyte and the second analyte. For example, but not by way of limitation, the mass transfer limiting film covering at least one of the active regions may include a mixture of a first film polymer and a second film polymer, or a bilayer of the first film polymer and the second film polymer. A uniform film may cover the active region not covered by the mixture or bilayer, and the uniform film includes only one of the first film polymer or the second film polymer. Advantageously, the architecture of the analyte sensor disclosed herein enables a continuous film having a uniform film portion to be disposed over the first active region of the analyte sensor and a multi-component film portion to be disposed over the second active region of the analyte sensor, thereby simultaneously equalizing the permeability values for each analyte and improving sensitivity and detection accuracy. In certain embodiments, continuous film deposition can be performed by a continuous dip coating operation.

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

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

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

[0264] In certain embodiments, the interference domain can be deposited directly on the working electrode, for example, on the platinum surface of the working electrode. In certain embodiments, the interference domain has a thickness in the range of about 0.1 μm to about 1000 μm, such as about 1 μm to about 500 μm, about 10 μm to about 100 μm, or about 10 μm to about 100 μm, for example, a dry thickness. In certain embodiments, the interference domain can have a thickness of about 0.1 μm to about 10 μm, such as about 0.5 μm to about 10 μm, about 1 μm to about 10 μm, about 1 μm to about 5 μm, or about 0.1 μm to about 5 μm. In certain embodiments, the sensor can be immersed in the solution of the interference domain two or more times. For example, but not limited to, the sensor (or working electrode) of the present disclosure can be immersed in the solution of the interference domain at least 2 times, at least 3 times, at least 4 times, or at least 5 times to obtain the desired thickness of the interference domain.

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

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

[0267] In certain embodiments, the method may further include adding a membrane composition on an enzyme composition, such as a cured enzyme composition. In certain embodiments, the membrane composition may include a polymer, such as polyvinyl pyridine and / or a crosslinking agent, such as polyethylene glycol diglycidyl ether. In certain embodiments, the membrane is applied onto the working electrode on the enzyme composition by dip coating (or similar techniques), spray coating, painting, inkjet printing, roller coating, etc. In certain embodiments, the method may include curing the membrane polymer composition.

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

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

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

[0271] Generally, the thickness of the film is controlled by the concentration of the film solution, the number of droplets of the film solution applied, the number of times the sensor is immersed in the film solution or the film solution is sprayed onto the sensor, the volume of the film solution sprayed onto the sensor, etc., and any combination of these factors. In certain embodiments, the films described herein can have a thickness in the range of about 0.1 micrometers (μm) to about 1000 μm, such as about 1 μm to about 500 μm, about 10 μm to about 100 μm, or about 10 μm to about 100 μm. In certain embodiments, the sensor can be immersed in the film solution more than twice. For example, but not limited to, the sensors (or working electrodes) of the present disclosure can be immersed in the film solution at least twice, at least three times, at least four times, or at least five times to obtain a desired film thickness.

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

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

[0274] In certain embodiments, the present disclosure provides a method for detecting ketone levels in a subject in need thereof. In certain embodiments, the present disclosure provides a method for detecting in vivo ketone levels in a subject. In certain embodiments, the present disclosure provides a method for detecting ketone levels in interstitial fluid in a subject. In certain embodiments, the present disclosure provides a method for detecting ketone levels in a diabetic subject. In certain embodiments, the present disclosure provides a method for detecting ketone levels in a subject on a ketogenic diet. In certain embodiments, the present disclosure provides a method for detecting ketone levels in a subject in a state of ketosis or for detecting ketone levels in a subject maintaining a state of ketosis. In certain embodiments, the analyte sensor of the present disclosure can be used to ensure that a subject adheres to a ketogenic diet. For example, but not limited to, the analyte sensor of the present disclosure can be used to measure the level of ketones in a sample and convey to adjust or modify the diet of the subject to maintain ketosis. In certain embodiments, the present disclosure provides a method for detecting ketone levels in a subject at risk of developing ketoacidosis. In certain embodiments, the present disclosure provides a method for detecting ketone levels in a subject at risk of developing diabetic ketoacidosis. In certain embodiments, the sensor of the present disclosure can be used to monitor and / or prevent diabetic ketoacidosis. For example, but not limited to, the sensor of the present disclosure includes a sensing chemical for detecting ketones and glucose to monitor and / or prevent diabetic ketoacidosis in a subject, such as a diabetic subject. Alternatively or additionally, the sensor of the present disclosure can be used in combination with a glucose sensor to monitor and / or prevent diabetic ketoacidosis. In certain embodiments, the sensor of the present disclosure can be used for the purpose of monitoring the level of ketones in a subject, for example, monitoring compliance with a ketogenic diet, maintaining a state of ketosis, and / or monitoring and / or preventing diabetic ketoacidosis.

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

[0276] In certain embodiments, the method of the present disclosure comprises: (i) exposing an analyte sensor comprising: (a) a sensor tail portion comprising at least a first working electrode, (b) a ketone-responsive active region disposed on the surface of the first working electrode and comprising, for example, an enzyme system comprising β-hydroxybutyrate dehydrogenase and NADH oxidase that responds to ketones and optionally a first polymer and that responds to ketones, and (c) a mass transfer limiting membrane that covers the ketone-responsive active region and is permeable to ketones, to a fluid comprising ketones; (ii) applying a potential, for example a low potential, to the first working electrode; (iii) obtaining a first signal that is above the redox potential of the ketone-responsive active region and that is proportional to the concentration of ketones in the fluid; and (iv) correlating the first signal with the concentration of ketones in the fluid. In certain embodiments, the potential applied to the first working electrode is a potential at which NADH is not oxidized. In certain embodiments, the potential applied to the first working electrode is a potential at which NADH is not reversibly oxidized. In certain embodiments, the potential applied to the first working electrode is from about +0.2 V to about +0.5 V relative to an Ag / AgCl reference. In certain embodiments, the potential applied to the first working electrode is from about +0.3 V to about +0.4 V, for example about +0.35 V, relative to an Ag / AgCl reference.

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

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

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

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

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

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

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

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

[0285] The analyte sensor according to A8 or A9, wherein the mass transfer limiting membrane comprises polyvinylpyridine. The analyte sensor according to any one of A to A10, wherein the sensor tail is configured for insertion into tissue.

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

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

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

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

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

[0291] The analyte sensor according to any one of A to A16, further comprising (iv) a second working electrode, and (v) a second active region disposed on the surface of the second working electrode and responsive to a second analyte different from ketones, the second active region comprising at least one enzyme responsive to the second analyte.

[0292] The analyte sensor according to A17, wherein the second part of the mass transfer limiting membrane covers the second active region. The analyte sensor according to A17, wherein the second mass transfer limiting membrane covers the second active region.

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

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

[0295] The analyte sensor according to any one of A to A23, which is implanted in a subject having diabetes. The analyte sensor according to any one of A to A24, which is implanted in a subject who is undergoing or at risk of undergoing ketoacidosis.

[0296] The analyte sensor according to any one of A to A24, which is implanted in a subject who is consuming a ketogenic diet. The analyte sensor according to any one of A to A24, which is implanted in a subject who is in a state of ketosis or needs to maintain a state of ketosis.

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

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

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

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

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

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

[0303] B7. The method according to B6, wherein the crosslinking agent is polyethylene glycol diglycidyl ether. B8. The method according to any one of B to B7, wherein the mass transfer limiting membrane comprises polyvinyl pyridine, polyvinyl imidazole, a copolymer of vinyl pyridine and styrene, or a combination thereof.

[0304] Method according to B8, wherein the mass transfer limiting membrane comprises polyvinylpyridine. Method according to B8, wherein the mass transfer limiting membrane comprises polyurethane. Method according to any one of B - B10, wherein the ketone-responsive active region further comprises a polymer.

[0305] Method according to B11, wherein the polymer comprises polyurethane. Method according to any one of B - B12, wherein the sensor tail is configured for insertion into tissue.

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

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

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

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

[0310] The method according to any one of B to B18, wherein the analyte sensor further comprises (d) a second working electrode, and (e) a second active region disposed on the surface of the second working electrode and responsive to a second analyte different from ketones.

[0311] The method according to B19, wherein the second portion of the mass transfer limiting membrane covers the second active region. The method according to B19, wherein the second mass transfer limiting membrane covers the second active region.

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

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

[0314] The method according to any one of B to B25, wherein the analyte sensor is implanted in a subject suffering from or at risk of developing ketoacidosis. The method according to any one of B to B26, wherein the analyte sensor is implanted in a subject consuming a ketogenic diet.

[0315] The method according to any one of B to B27, wherein the analyte sensor is implanted in a subject in a state of ketosis or in need of maintaining a state of ketosis. The method according to any one of B to B28, wherein hydrogen peroxide generated by the reaction of the enzyme system and ketones in the ketone-responsive active region is detected at the working electrode.

[0316] The analyte sensor according to any one of A to A30 for use in detecting ketones in a subject in need of an analyte sensor. Subject having diabetes, a test substance sensor for use of C.

[0317] Subject undergoing or at risk of undergoing ketoacidosis, a test substance sensor for use of C. Subject consuming a ketogenic diet, a test substance sensor for use of C.

[0318] Subject in a state of ketosis or in need of maintaining a state of ketosis, a test substance sensor for use of C. Examples The subject matter of this disclosure is provided as an illustration of the subject matter disclosed herein and is not limiting, but will be better understood by reference to the following examples.

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

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

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

Table 1

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

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

[0324] Although the subject matter and its advantages disclosed herein have been described in detail, it is to be understood that various changes, substitutions, and modifications can be made herein without departing from the technical idea and scope of the disclosed subject matter. Further, the scope of this application is not intended to be limited to the particular embodiments of the processes, machines, manufactures, and compositions of matter, methods, and processes described herein. Those skilled in the art will readily appreciate that presently existing or later developed processes, machines, manufactures, compositions of matter, methods, or steps that perform substantially the same function or achieve substantially the same result as the corresponding embodiments described herein can be utilized in accordance with the disclosed subject matter. Accordingly, the appended claims are intended to include within their scope such processes, machines, manufactures, compositions of matter, methods, or steps.

[0325] Various patents, patent applications, publications, product descriptions, protocols, and sequence accession numbers are cited through this application, and those inventions are hereby incorporated by reference in their entirety for all purposes.

Claims

1. 1. An analyte sensor for detecting ketones in vivo, comprising: (i) a first working electrode comprising a conductive material that oxidizes hydrogen peroxide to produce a signal that correlates to the concentration of ketones; (ii) a ketone-responsive active region disposed on the surface of the first working electrode, the ketone-responsive active region comprising an enzyme system for producing hydrogen peroxide in the presence of a ketone, the enzyme system comprising β-hydroxybutyrate dehydrogenase, nicotinamide adenine dinucleotide (NAD), and NADH oxidase; (iii) a mass transport limiting membrane adapted to allow inward diffusion of ketones and retain NAD, the mass transport limiting membrane being disposed over the ketone-responsive active region; the ketone-responsive active region responds to ketones at a potential of +0.2 V to +0.5 V versus an Ag / AgCl reference; The analyte sensor, wherein a distal portion of the analyte sensor is configured to be inserted into the skin of a subject to detect ketones in vivo.

2. A test substance sensor as described in claim 1, wherein the ketone-responsive active region does not contain an electron transfer agent.

3. A test substance sensor as described in claim 1 or 2, wherein the ketone-responsive active region does not contain superoxide dismutase.

4. A test substance sensor described in any one of claims 1 to 3, wherein the ketone-responsive active region further comprises a stabilizer for stabilizing the enzyme system.

5. A test substance sensor described in any one of claims 1 to 4, wherein the mass transfer limiting membrane comprises polyvinylpyridine, polyvinylimidazole, polyvinylpyridine copolymer, polyacrylate, polyurethane, polyether urethane, or a combination thereof.

6. A test substance sensor as described in claim 5, wherein the mass transfer limiting membrane comprises polyvinylpyridine.

7. A test substance sensor described in any one of claims 1 to 6, wherein the conductive material is selected from the group consisting of platinum, platinum-iridium, palladium, graphite, gold, carbon, conductive polymers, and alloys thereof.

8. A test substance sensor described in any one of claims 1 to 7, wherein the conductive material comprises platinum.

9. A test substance sensor described in any one of claims 1 to 8, wherein the ketone-responsive active region further comprises a first polymer.

10. The test substance sensor of claim 9, wherein one or both of the β-hydroxybutyrate dehydrogenase and the NADH oxidase are covalently bonded to the first polymer. (iv) a second working electrode; and 11. The analyte sensor of claim 1, further comprising: (v) a second active region disposed on a surface of the second working electrode and responsive to a second analyte other than ketones, the second active region comprising at least one enzyme responsive to the second analyte.

12. A test substance sensor as described in claim 11, further comprising a second mass transport limiting membrane covering the second active region.

13. A test substance sensor as described in claim 11 or 12, wherein the second test substance is glucose.

14. The test substance sensor of claim 13, wherein the at least one enzyme in the second active region is glucose oxidase or glucose dehydrogenase.

15. A test substance sensor described in any one of claims 11 to 14, wherein the second active region is provided with an electron transfer agent.

16. (i) a sensor electronic device; (ii) A sensor control device comprising: a test substance sensor according to any one of claims 1 to 15, the test substance sensor obtaining a signal correlating with the concentration of ketones and transmitting the signal to the sensor electronics.

17. A sensor control device as described in Claim 16, wherein the sensor electronics are configured to correlate the signal with the concentration of ketones. (i) a test substance sensor according to any one of claims 1 to 15; (ii) a ketone detection system comprising: (a) sensor electronics configured to correlate a signal obtained by the test substance sensor to a ketone concentration; and (b) transmit the ketone concentration to a reader device for display.

19. A method for controlling the operation of a test substance sensor according to any one of claims 1 to 10, wherein the test substance sensor is introduced into tissue, and the control method comprises: (i) applying a potential to the first working electrode; (ii) obtaining a signal proportional to the concentration of ketones in the fluid at or above the redox potential of the ketone-responsive active region; (iii) correlating the signal with a concentration of ketones in the fluid.

20. The control method described in claim 19, wherein the fluid is interstitial fluid.

21. A method for controlling the operation of a test substance sensor according to any one of claims 11 to 14, wherein the test substance sensor is introduced into tissue, and the control method comprises: (i) applying a potential to the first working electrode; (ii) applying a potential to the second working electrode; (iii) obtaining a signal proportional to the concentration of ketones in the fluid at or above the redox potential of the ketone-responsive active region; (iv) obtaining a second signal at or above the redox potential of the second active region, the second signal being proportional to the concentration of a second analyte in the fluid; (v) correlating the signal with the concentration of ketones in the fluid; (vi) correlating the second signal with a concentration of a second analyte in the fluid.

22. The control method described in claim 21, wherein the fluid is interstitial fluid.

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