Sensor for detecting ketones and method for 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 a test substance sensor for detecting ketones and a method for using the same. [Background technology]

[0002] The detection of various test substances within an individual can be important for monitoring their health, as deviations from normal test substance levels can indicate physiological conditions. For example, monitoring ketone levels can enable individuals with diabetes to receive appropriate corrective measures to avoid significant physiological distress caused by ketoacidosis. Other test substances may also be desirable for monitoring other physiological conditions. In some cases, monitoring two or more test substances may be desirable to monitor multiple physiological conditions, especially if the individual suffers from a comorbidity that results in simultaneous dysregulation of two or more test substances in combination.

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

[0004] Many test substances represent an attractive target for physiological analysis, provided that suitable detection chemicals can be identified. For this purpose, enzyme-based current measurement sensors configured to continuously assay glucose in vivo have been developed and improved over recent years to aid in monitoring the health of individuals with diabetes. Other test substances that commonly undergo simultaneous dysregulation with glucose in individuals with diabetes include, for example, lactate, oxygen, A1c, ketones, and others. Monitoring these and other test substances separately from glucose dysregulation may also be desirable. While test substance sensors configured to detect non-glucose test substances in vivo are known, they have not been significantly improved. For example, low sensitivity to test substances in low abundance can be a particular problem. Therefore, improved sensors for detecting ketones in vivo are needed in this art. [Overview of the project]

[0005] The purpose and merits of the disclosed subject matter are described and made clear by the following description and learned through the implementation of the disclosed subject matter. Further merits of the disclosed subject matter may be realized and obtained by the apparatus specifically indicated in the written specification and claims, and also by the accompanying drawings.

[0006] To achieve these and other advantages, and in accordance with the objectives of the disclosed subject matter which are embodied and broadly described, the disclosed subject matter includes a test substance sensor comprising a sensor tail including at least a first working electrode, a ketone-responsive active region disposed on the 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 contain an electron transfer agent. In certain embodiments, the ketone-responsive active region does not contain superoxide dismutase.

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

[0008] In certain embodiments, the test substance sensor of the present disclosure further includes a second working electrode and a second active region disposed on the surface of the second working electrode and responding 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. In certain embodiments, a second portion of a mass transfer limiting membrane covers the second active region. In certain embodiments, the second test substance includes glucose.

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

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

[0011] In certain embodiments, the first working electrode is a platinum electrode. In certain embodiments, the 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 includes a stabilizer and / or a crosslinking agent. In certain embodiments, one or more enzymes in an enzyme system comprising β-hydroxybutyrate dehydrogenase and NADH oxidase are covalently bonded to the stabilizer in the ketone-responsive active region. In certain embodiments, one or more enzymes in an enzyme system comprising β-hydroxybutyrate dehydrogenase and NADH oxidase are covalently bonded to the polymer in the ketone-responsive active region. In certain embodiments, the mass transfer limiting membrane comprises polyvinylpyridine, polyvinylimidazole, polyvinylpyridine copolymer, polyvinylpyrrolidone, polyacrylate, polyurethane, polyether urethane, or copolymers or combinations thereof. In certain embodiments, the mass transfer limiting membrane comprises polyvinylpyridine. Alternatively, or furthermore, 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 approximately +0.2V to approximately +0.5V relative to an Ag / AgCl reference. In certain embodiments, the ketone-responsive active region responds to ketones at a potential of approximately +0.3V to approximately +0.4V relative to an Ag / AgCl reference. In certain embodiments, the ketone-responsive active region responds to ketones at a potential of approximately +0.35V relative to an Ag / AgCl reference.

[0013] In certain embodiments, the test substance sensor for use in the disclosed method may further include a second working electrode and a second active region disposed on the surface of the second working electrode and responding to a second test substance different from ketones. In certain embodiments, the second active region comprises 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 substance sensor of this disclosure is intended for use in subjects requiring it. For example, but not limited to, subjects may be diabetic subjects. In certain embodiments, subjects may be on a ketogenic diet. In certain embodiments, subjects may be in a state of ketosis. [Brief explanation of the drawing]

[0015] The following figures are included to illustrate specific aspects of the disclosure and should not be considered exclusive embodiments. The disclosed subject matter can be substantially modified, altered, combined, and equivalent in form and function without departing from the scope of the disclosure. [Figure 1A] This is a system overview of the sensor applicator, reader device, monitoring system, network, and remote system. [Figure 1B] This is a schematic diagram illustrating the operating environment of a test substance monitoring system as an example of use by the technology described herein. [Figure 1C] A schematic diagram of an exemplary detection system into which the substance sensor of this disclosure may be incorporated is shown. [Figure 2A] This is a schematic block diagram showing an example of an embodiment of the reader device. [Figure 2B] This is a block diagram illustrating an example of a data receiving device for communicating with a sensor according to an exemplary embodiment of the disclosed subject matter. [Figure 2C] This is a schematic block diagram showing an example of an embodiment of a sensor control device. [Figure 2D]It is a block schematic diagram showing an example of an embodiment of a sensor control device. [Figure 2E] It is a block schematic diagram showing an example of a test substance sensor according to an exemplary embodiment of the disclosed subject matter. [Figure 3A] It is a proximal perspective view showing an example of an embodiment of a user preparing a tray for assembly. [Figure 3B] It is a side view showing an example of an embodiment of a user preparing an applicator device for assembly. [Figure 3C] It is a proximal perspective view showing an example of an embodiment of a user inserting an applicator device into a tray during assembly. [Figure 3D] It is a proximal perspective view showing an example of an embodiment of a user removing an applicator device from a tray during assembly. [Figure 3E] It is a proximal perspective view showing an example of an embodiment of a patient applying a sensor using an applicator device. [Figure 3F] It is a proximal perspective view showing an example of an embodiment of a patient having an applied sensor and a used applicator device. [Figure 4A] It is a side view showing an example of an embodiment of an applicator device connected to a cap. [Figure 4B] It is a side perspective view showing an example of an embodiment of a separated applicator device and a cap. [Figure 4C] It is a perspective view showing an example of an embodiment of a distal end of an applicator device and an electronic device housing. [Figure 4D] It is a top perspective view of an exemplary applicator device according to the disclosed subject matter. [Figure 4E] It is a bottom perspective view of the applicator device of FIG. 4D. [Figure 4F] It is an exploded view of the applicator device of FIG. 4D. [Figure 4G] It is a side cross-sectional view of the applicator device of FIG. 4D. [Figure 5] It is a proximal perspective view showing an example of an embodiment of a tray with a sterilization lid connected. [Figure 6A]This is a proximal perspective cross-sectional view showing an example of an embodiment of a tray having sensor delivery components. [Figure 6B] This is a proximal perspective view showing the sensor delivery component. [Figure 7A] This is an isometric top view of an exemplary sensor control device. [Figure 7B] This is an isometrically resolved bottom view of an exemplary sensor control device. [Figure 8A] These are assembly diagrams and cross-sectional views of an on-body device including an integrated connector for sensor assembly. [Figure 8B] These are assembly diagrams and cross-sectional views of an on-body device including an integrated connector for sensor assembly. [Figure 8C] These are assembly diagrams and cross-sectional views of an on-body device including an integrated connector for sensor assembly. [Figure 9A] This is a side view of an example of an embodiment of the sensor applicator shown in Figure 1A, with the caps shown in Figure 2C connected. [Figure 9B] This is a side cross-sectional view of an example of an embodiment of the sensor applicator shown in Figure 1A, with the caps shown in Figure 2C connected. [Figure 10A] This is an isometric view of another example of a sensor control device. [Figure 10B] This is a side view of another example of a sensor control device. [Figure 11A] Figures 10A to 10B are stepwise side cross-sectional views showing the assembly of a sensor applicator having a sensor control device. [Figure 11B] Figures 10A to 10B are stepwise side cross-sectional views showing the assembly of a sensor applicator having a sensor control device. [Figure 11C] Figures 10A to 10B are stepwise side cross-sectional views showing the assembly of a sensor applicator having a sensor control device. [Figure 12A] Figures 10A to 10B are stepwise side cross-sectional views showing the assembly and disassembly of an example embodiment of a sensor applicator having a sensor control device. [Figure 12B] Figures 10A to 10B are stepwise side cross-sectional views showing the assembly and disassembly of an example embodiment of a sensor applicator having a sensor control device. [Figure 12C] Figures 10A to 10B are stepwise side cross-sectional views showing the assembly and disassembly of an example embodiment of a sensor applicator having a sensor control device. [Figure 13A] A cross-sectional view is shown illustrating an example of an applicator embodiment during the deployment phase. [Figure 13B] A cross-sectional view is shown illustrating an example of an applicator embodiment during the deployment phase. [Figure 13C] A cross-sectional view is shown illustrating an example of an applicator embodiment during the deployment phase. [Figure 13D] A cross-sectional view is shown illustrating an example of an applicator embodiment during the deployment phase. [Figure 13E] A cross-sectional view is shown illustrating an example of an applicator embodiment during the deployment phase. [Figure 13F] A cross-sectional view is shown illustrating an example of an applicator embodiment during the deployment phase. [Figure 14] This graph shows an example of the in vitro sensitivity of a test substance sensor. [Figure 15] This is a schematic diagram showing an example of the operating state of a sensor according to an exemplary embodiment of the disclosed subject matter. [Figure 16] This is a schematic diagram illustrating an example of the operation and data flow of over-the-air programming of a sensor based on the disclosed subject matter. [Figure 17] This is a schematic diagram illustrating an example of data flow for reliable data exchange between two devices based on the disclosed subject matter. [Figure 18A] A schematic cross-sectional view of a test substance sensor containing a single active region is shown. [Figure 18B] A schematic cross-sectional view of a test substance sensor containing a single active region is shown. [Figure 18C] A schematic cross-sectional view of a test substance sensor containing a single active region is shown. [Figure 19A] A schematic cross-sectional view of the test substance sensor containing two active regions is shown. [Figure 19B] A schematic cross-sectional view of the test substance sensor containing two active regions is shown. [Figure 19C] A schematic cross-sectional view of the test substance sensor containing two active regions is shown. [Figure 20] A schematic cross-sectional view of the test substance sensor containing two active regions is shown. [Figure 21A] A perspective view of the test substance sensor, including two active regions on separate working electrodes, is shown. [Figure 21B] A perspective view of the test substance sensor, including two active regions on separate working electrodes, is shown. [Figure 21C] A perspective view of the test substance sensor, including two active regions on separate working electrodes, is shown. [Figure 22] This disclosure shows a schematic diagram of a specific enzyme system that may be used to detect ketones. [Figure 23] The voltammograms of NADH and NADH with hydrogen peroxide (H2O2) are shown. [Figure 24] Four replicate experiments of current responses for electrodes containing NAD+, NADH oxidase, and β-hydroxybutyrate dehydrogenase, compared to a control without NADH oxidase, are shown when exposed to varying concentrations of β-hydroxybutyrate. [Figure 25] Figure 24 shows an exemplary plot of the sensor's current response and the concentration of β-hydroxybutyrate for the electrode shown. [Modes for carrying out the invention]

[0016] This disclosure generally describes a test substance sensor employing one or more enzymes for the detection of a test substance. For example, but not limited to, this disclosure provides a test substance sensor employing multiple enzymes for the detection of a test substance, e.g., one or ketones. In certain embodiments, this disclosure further provides a test substance sensor employing multiple enzymes for the detection of two different test substances, e.g., ketones and a second test substance, e.g., glucose. Depending on the sensor configuration, the test substance sensors of this disclosure may be configured to detect one or multiple test substances simultaneously or substantially simultaneously. This disclosure further provides a method for detecting one or more test substances using the disclosed test substance sensors.

[0017] Glucose-responsive chemistem sensors are well-studied and remain a developing field to help individuals with diabetes better manage their health. Despite the prevalence of dysregulation of chemistems in individuals with diabetes, sensor chemistry suitable for detecting ketones and other chemistems that are generally dysregulated lags significantly behind the more well-developed glucose detection chemistry. This disclosure alleviates this deficiency by providing a sensor chemistry suitable for detecting ketones with good response stability over a wide range of ketone concentrations, particularly a detection chemistry utilizing an enzyme system comprising at least two enzymes capable of acting concertedly to facilitate ketone detection. As used herein, the term “concertedly” means a conjugated enzyme reaction in which the product of a first enzymatic reaction becomes a substrate for a second enzymatic reaction, and the second enzymatic reaction serves as a basis for measuring the concentration of the substrate (e.g., chemistem) that reacted during the first enzymatic reaction. In certain embodiments, the product and / or substrate of the reaction may be a reduced and / or oxidized form of a cofactor or coenzyme of the enzymes in the enzyme system, such as NAD or NADP. While defined in terms of two coupled enzyme reactions, it should be recognized that in some cases, three or more coupled enzyme reactions may occur. For example, the product of the first enzyme reaction may become the substrate for the second enzyme reaction, the product of the second enzyme reaction may become the substrate for the third enzyme reaction, and the third enzyme reaction may serve as the basis for measuring the concentration of the substrate (e.g., the test substance) that reacted during the first enzyme reaction. A consideration of suitable enzyme systems for detecting ketones according to this disclosure is described below.

[0018] For clarity, and without limitation, the detailed description of the subject matter disclosed herein will be divided into the following sub-sections. I. Definitions; and II. Sensors for the substance being tested; 1. General structure of a test substance sensor system; 2. Enzymes; 3. Redox mediators; 4. Polymer skeleton; 5. Mass transfer limiting membrane; 6. Interference domains; and 7. Manufacturing; III. Method of use; and IV. Exemplary Embodiments I. Definition Terms used herein generally have the ordinary meanings in the art within the context of this disclosure and in the specific context in which each term is used. Specific terms are discussed below or elsewhere in this specification to provide further guidance to practitioners in describing the compositions and methods of this disclosure and the methods for preparing and using them.

[0019] As used herein, the use of the word “one” ("a" or "an") in conjunction with the term “equipped with” in the claims and / or specification may mean “one,” but is not inconsistent with the meanings 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 their variations as used herein are intended to be open-ended transitional phrases, terms, or words that do not preclude additional acts or structures. This disclosure also assumes other embodiments, whether expressly described or not, that “comprise,” “consist of,” and “essentially consist of” the embodiments or elements shown herein.

[0021] The terms “about” or “approximately” mean within an acceptable margin of error of a particular value as determined by those skilled in the art, which in part depends on how the value was measured or determined, i.e., the limits of the measurement system. For example, “about” may mean within 3 or more standard deviations by convention in the art. Alternatively, “about” may mean a range of up to 20%, preferably 10%, more preferably 5%, and even more preferably 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term may mean within one order of magnitude of a value, preferably within 5 times, and more preferably within 2 times.

[0022] As used herein, “test substance sensor” or “sensor” may mean 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 test substance sensor, a physical activity sensor, a movement sensor, or any other sensor for collecting physical or biological information. Test substances measured by a test substance sensor may include, but are 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, pH, phosphorus, potassium, sodium, total protein, uric acid, and others.

[0023] The term "biological fluid," as used herein, means any bodily fluid or derivative of bodily fluid in which the test substance can be measured. Non-limiting examples of biological fluids include skin fluid, interstitial fluid, plasma, blood, lymph, 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 electro-oxidation or electro-reduction of a compound, either directly at an electrode or via one or more electron-transfer agents (redox mediators or enzymes).

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

[0026] As used herein, the term "polyvinylpyridine 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 test substance or test substance reductase or test substance oxidase and an electrode, either directly or via one or more further 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 an electrode that functions as either a reference electrode or both a reference electrode and a counter electrode. Similarly, as used herein, the term "counter electrode" may mean both a counter electrode and a counter electrode that also functions as a reference electrode.

[0029] II. Sensor for the substance being tested Before going into detail about this subject matter, it should be understood that this disclosure is not limited to the specific embodiments described and is therefore of course subject to change. The scope of this disclosure is limited only by the appended claims, and it should also be understood that the terms used herein are intended solely to describe and not to limit specific embodiments.

[0030] The publications discussed herein merely provide disclosures prior to the filing date of this application. Nothing herein should be construed as acknowledging that this disclosure is not prior to those publications by prior disclosures. Furthermore, the publication dates provided may differ from the actual publication dates, which must be independently verified.

[0031] Generally, embodiments of this disclosure include systems, apparatus, and methods for the use of 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 electronic housing of the sensor control device contained therein. According to some embodiments, a separate structure from the applicator, such as a container, can also be provided to the user in a sterile package together with the sensor module and tip module contained therein. The user can connect the sensor module to the electronic 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 and bring the sensor into contact with the wearer's bodily fluids. Embodiments provided herein are improvements that reduce the possibility of the sensor being improperly inserted or damaged or inducing an adverse physiological response. Other improvements and advantages are also provided. Various configurations of these devices are described in detail by embodiments that are merely illustrative.

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

[0033] Furthermore, for each embodiment of the methods disclosed herein, systems and apparatus capable of carrying out each of these embodiments are included within the scope of this disclosure. For example, embodiments of sensor control devices are disclosed, these devices may have one or more sensors, a substance under test monitoring circuit (e.g., an analog circuit), a memory (e.g., for storing instructions), a power supply, a communication circuit, a transmitter, a receiver, a processor, and / or a controller (e.g., for executing instructions), which can carry out or facilitate the carrying out of any method process. These embodiments of sensor control devices can and may be used to carry out a process performed by a sensor control device from any method described herein.

[0034] Furthermore, the systems and methods presented herein can be used for the operation of sensors used in a test substance monitoring system for any purpose, including, but not limited to, wellness, fitness, diet, research, information, or the detection of a test substance over time. Where used herein, “test substance sensor” or “sensor” may mean 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 test substance sensor, a physical activity sensor, a movement sensor, or any other sensor for collecting physical or biological information. The test substance sensors of this disclosure measure ketones. In certain embodiments, the test substance sensor of this disclosure can further measure test 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, pH, phosphorus, potassium, sodium, total protein, uric acid, and the like.

[0035] As described above, several embodiments of systems, apparatus, and methods that provide improved assembly and use of skin sensor insertion devices for use with in vivo test substance monitoring systems are described herein. In particular, several embodiments of the present disclosure are designed to improve sensor insertion methods relating to in vivo test substance monitoring systems, and especially to prevent premature retraction of the insertion tip during the sensor insertion process. For example, some embodiments include a skin sensor insertion mechanism with increased firing velocity and slower tip retraction. In other embodiments, the tip retraction mechanism can be actuated so that the tip does not retract until the user withdraws the applicator from the skin. As a result, these embodiments can reduce the possibility of premature retraction of the insertion tip during the sensor insertion process, reduce the possibility of improper sensor insertion, and reduce the possibility of sensor damage during the sensor insertion process, to name a few advantages. Some embodiments of the present disclosure also provide improved insertion tip modules that are due to small-scale skin sensors and relatively shallow insertion paths present in the target skin layer. Furthermore, some embodiments of this disclosure are designed to prevent undesirable axial and / or rotational movement of the applicator component during sensor insertion. Thus, these embodiments can reduce the possibility of instability of the placed skin sensor, irritation at the insertion site, damage to surrounding tissue, and disruption of capillaries resulting in blood contamination of skin fluids, among other advantages. In addition, to mitigate inaccurate sensor readings that may occur due to injury at the insertion site, some embodiments of this disclosure can reduce the penetration of the needle to the terminal depth of the sensor tip during insertion.

[0036] However, before describing these embodiments in detail, it is desirable to first describe, for example, examples of devices that may be present in an in vivo test substance 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 test substance monitoring systems. For example, a "continuous test substance monitoring" system (or "continuous glucose monitoring" system) can continuously transmit data from a sensor control unit to a reader unit automatically, for example, according to a schedule, without requiring any instruction. A "flash test substance monitoring" system (or "flash glucose monitoring" system or simply a "flash" system) can, as another example, transmit data from a sensor control unit via, for example, near-field communication (NFC) or radio frequency identification (RFID) protocols in response to data scanning or requests by a reader unit. In vivo test substance monitoring systems can also operate without the need for fingerstick calibration.

[0038] In vivo test substance monitoring systems can be distinguished from “in vitro” systems, which typically involve measuring devices that come into contact with biological samples outside the body (i.e., “ex vivo”) and have ports for receiving test substance test strips that carry bodily fluids that can be analyzed to determine the user’s blood glucose level.

[0039] An in vivo monitoring system may include a sensor that comes into contact with the user's bodily fluids while deployed in vivo and detects the level of a test substance contained therein. The sensor may be part of a sensor control unit that is located on the user's body and includes electronic equipment and a power supply that enables and controls the detection of the test substance. Sensor control units and variations thereof may also be referred to, to some extent, as a “sensor control unit,” an “on-body electronics” device or unit, an “on-body” device or unit, or a “sensor data communication” device or unit.

[0040] An in vivo monitoring system may also include a device that receives detected test substance data from a sensor control device and processes and / or displays the detected test substance data to the user in any number of forms. This device and its variations may be referred to, to some extent, as a “handheld reader device,” “reader device” (or simply “reader”), “handheld electronic device” (or simply “handheld”), “portable data processing” device or unit, “data receiver,” “receiver” device or unit (or simply “receiver”), or “remote” device or unit. Other devices, such as personal computers, may also be used with or incorporated into in vivo and in vitro monitoring systems.

[0041] 1. General structure of the test substance sensor system A. Exemplary in vivo test substance monitoring system Before further detailing the substance sensor and its components of this disclosure, a brief overview of a suitable in vivo substance sensor configuration and a sensor system employing the substance sensor is provided to better understand the embodiments of this disclosure.

[0042] Figure 1A is a conceptual schematic diagram illustrating an example of an embodiment of a substance monitoring system 100, which includes a sensor applicator 150, a sensor control device 102, and a reader device 120. Here, the sensor applicator 150 can be used to deliver the sensor control device 102 to a monitoring location on the user's skin, and the sensor 104 is maintained in place for a period of time by an adhesive patch 105. The sensor control device 102, which is further described in Figures 2B and 2C, can communicate with the reader device 120 via a communication path 140 using wired or wireless technology. Examples of wireless protocols include Bluetooth®, Bluetooth® Low Energy (BLE, BTLE, Bluetooth® SMART, etc.), Near Field Communication (NFC), and others. The user can monitor applications installed in the memory on the reader device 120 using the screen 122, and the input 121 and the device battery can be recharged using the power port 123. Further details regarding the reader device 120 are described below with reference to Figure 2A. The reader device 120 can communicate with the local computer system 170 via the communication path 141 using wired or wireless technology. The local computer system 170 includes one or more laptops, desktops, tablets, phablets, smartphones, set-top boxes, video game consoles, or other computing devices, and wireless communication may 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 the communication path 143 using the wired or wireless technology already described, in the same way that the reader device 120 can communicate with the network 190 via the communication path 142. The network 190 may be any of several networks, including private and public networks, local area networks or wide area networks, or other networks.The trusted computer system 180 includes a server that can provide authentication services and guaranteed data storage, and can communicate with the network 190 via a communication path 144 using wired or wireless technology.

[0043] Figure 1B shows the operating environment of a test substance monitoring system 100a that can embody 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 the body of a human or animal, or can provide other operations based on the configuration of various components. As embodied herein, the system may include a low-power test substance sensor 110 or simply a “sensor” that is worn by a user or attached to the body from which information is collected. As embodied herein, the test substance sensor 110 may 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 a user’s body and may remain attached for the duration of the sensor’s service life or may be selectively removed and designed to remain functional when reattached. The low-power test substance monitoring system 100a may further include a data reader device 120 or a multipurpose data receiver 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 include software or firmware libraries or applications that are provided to a third party, for example via a remote application server 150 or an application storefront server 160, and are incorporated into a multipurpose hardware device 130, such as a mobile phone, tablet, personal computing device, or other similar computing device that can communicate with the test substance sensor 110 via a communication link. The multipurpose hardware may further include embedded devices, including but not limited to insulin pumps or insulin pens, which have embedded libraries configured to communicate with the test substance sensor 110. While the illustrated embodiments of the test substance monitoring system 100a include only one of each of the illustrated devices, this disclosure intends that the test substance monitoring system 100a incorporates a number of each respective component that interact as a whole system. For example, as embodied herein, but not limited to, the data reader device 120 and / or multipurpose data receiver device 130 may include a number of each. As embodied herein, the multiplex data receiver 130 can communicate directly with the sensor 110 described herein. Furthermore, or alternatively, the data receiver 130 can communicate with a secondary data receiver 130 to provide the substance data or data visualization or analysis for secondary display to a user or other authorized party.

[0045] Figure 1C shows a schematic diagram of an exemplary detection system into which the substance sensor of this disclosure may 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 be encrypted or not encrypted. The reader device 120 may, according to a particular embodiment, constitute an output medium for visualizing the substance concentration and warnings or alerts determined by the sensor 104 or its associated processor, and for enabling one or more user inputs. The reader device 120 may be a multipurpose smartphone or a dedicated electronic reader device. Although only one reader device 120 is shown, there may be multiple reader devices 120 in particular cases. The reader device 120 may communicate with the remote terminal 90170 and / or the trusted computer system 90180 via communication paths / links 90141 and / or 90142, which may be wired or wireless, unidirectional or bidirectional, and encrypted or unencrypted. The reader device 120 may also or alternatively communicate with a network 150 (e.g., a mobile phone network, the Internet, or a cloud server) via communication path / link 151. The network 150 may further be connected to communicate with the remote terminal 90170 and / or the trusted computer system 90180 via communication path / link 152 and / or communication path / link 153. Alternatively, the sensor 104 may communicate directly with the remote terminal 90170 and / or the trusted computer system 90180 without the presence of the intervening reader device 120. For example, in accordance with certain embodiments described in, but not limited to, U.S. Patent Application Publication No. 2011 / 0213225, which is incorporated herein by reference in its entirety, the sensor 104 may communicate with a remote terminal 90170 and / or a trusted 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, Wi-Fi, or any other suitable electronic communication protocol may be used. According to certain embodiments, the remote terminal 90170 and / or trusted computer system 90180 may be accessible to individuals other than the primary user who are interested in the user's test substance levels. 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 touchscreen interface.

[0046] The sensor control device 102 includes a sensor housing 103, which can house the circuitry and power supply for operating the sensor 104. Optionally, the power supply and / or active circuitry 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 a particular embodiment, the sensor 104 protrudes from the underside of the sensor housing 103 and extends via an adhesive layer 105 adapted to adhere the sensor housing 103 to a tissue surface such as skin.

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

[0048] The active region may be configured to detect a specific test substance. In certain embodiments, the active region of the sensor disclosed herein is configured to detect ketones. In certain embodiments, the active region may be configured to detect two or more test substances. In certain embodiments, further test substances to be detected using the sensor disclosed herein include any abnormally regulated test substance along with ketones. For example, but not limited to, the test substance sensor disclosed herein can detect ketones and further test substances such as creatinine, oxygen, glucose, and / or lactate. In certain embodiments, the further test substance is glucose.

[0049] In certain embodiments of the Disclosure, one or more test substances may be monitored in skin fluid, interstitial fluid, plasma, blood, lymph, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, amniotic fluid, or any other biological fluid for any purpose. In certain embodiments, the test substance sensor of the Disclosure may be adapted to assay skin fluid or interstitial fluid to determine the concentration of one or more test substances in vivo. In certain embodiments, the biological fluid is interstitial fluid.

[0050] Referring again to Figure 1C, the sensor 104 can automatically send data to the reader device 120. For example, but not limited to, data on the concentration of a test substance (i.e., glucose and / or ketone concentrations) can be communicated automatically and periodically, for example, 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 at other predetermined intervals). In other specific embodiments, the sensor 104 may communicate with the reader device 120 in a non-automatic manner, without following a set schedule. For example, but not limited to, data may be communicated from the sensor 104 using RFID technology when the sensor's electronics enter the communication range of the reader device 120. The data may remain stored in the sensor 104's memory until it is communicated to the reader device 120. That is, the user does not need to maintain close proximity to the reader device 120 at all times, but can instead upload data at their convenience. In other specific embodiments, a combination of automatic and non-automatic data transfer may be performed. For example, though not limited to this, data transfer may be automatically continued until the reader device 120 is no longer within the communication range of the sensor 104.

[0051] An introducer may be temporarily present to facilitate the introduction of the sensor 104 into the tissue. In certain exemplary embodiments, the introducer may include a needle or a similar tip. As will be readily apparent to those skilled in the art, other types of introducers, such as a sheath or blade, may be present in alternative embodiments. More specifically, the needle or other introducer may be temporarily present proximal to the sensor 104 before insertion into the tissue and then withdrawn. While present, the needle or other introducer can facilitate the insertion of the sensor 104 into the tissue by opening an access path therefor for the sensor 104. For example, according to one or more embodiments, but not limited to, the needle may facilitate penetration of the epidermis as an access path to the dermis, allowing the implantation of the sensor 104 to be performed. 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, a preferred needle may be solid or hollow, beveled or non-beveled, and / or have a circular or non-circular cross-section. In more specific embodiments, preferred needles may be similar to acupuncture needles in terms of cross-sectional diameter and / or tip design, and may have a cross-sectional diameter of about 250 microns (250 μm). However, preferred needles may have larger or smaller cross-sectional diameters if required for a particular application.

[0052] In certain embodiments, the tip of the needle (while present) may be angled beyond the end of the sensor 104, thereby allowing the needle to initially penetrate the tissue and open an access path for the sensor 104. In certain embodiments, the sensor 104 may be located in the lumen or groove of the needle, allowing the needle to similarly open an access path for the sensor 104. In either case, the needle is subsequently withdrawn after facilitating the insertion of the sensor.

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

[0054] C. Exemplary data receiving device architecture For illustrative purposes only and not limiting, an exemplary embodiment of the data receiver 120 for use in the disclosed subject matter is shown in Figure 2B. The data receiver 120 and the associated multipurpose data receiver 130 include components closely related to the consideration of the substance sensor 110, and may include its operation and further components. In certain embodiments, the data receiver 120 and the multipurpose data receiver 130 may be components provided by a third party or include such components, and are not necessarily limited to including devices manufactured by the same manufacturer as the sensor 110.

[0055] As shown in Figure 2B, the data receiving device 120 includes a microcontroller 4010, a memory 4020, and a storage 4030, and includes an ASIC 4000 which is communicatively connected to a communication module 4040. Power for the components of the data receiving device 120 can be supplied 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 for facilitating confirmation of the substance data received from the substance sensor 110 or other devices (e.g., a user device 140 or a remote application server 150). The data receiving device 120 may include separate user interface components (e.g., a physical key, an optical sensor, a microphone, etc.).

[0056] The communication module 4040 may include a BLE module 4041 and an NFC module 4042. The data receiving device 120 may be wirelessly connected to the substance sensor 110 and configured to send commands to the substance sensor 110 and receive data from it. As embodied herein, the data receiving device 120 may be configured to operate as an NFC scanner and BLE endpoint with respect to the substance sensor 110 described herein via a specific module of the communication module 4040 (e.g., BLE module 4042 or NFC module 4043). For example, the data receiving device 120 may use a first module of the communication module 4040 to issue commands to the 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), and use a second module of the communication module 4040 to receive data from the substance sensor 110 and send data to it. The data receiving device 120 may be configured for communication with the user device 140 via the 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 communication using a broadband cellular network, including but not limited to third-generation (3G), fourth-generation (4G), and fifth-generation (5G) networks. Furthermore, the communication module 4040 of the data receiving device 120 may include a Wi-Fi radio module 4043 for communication using a wireless local area network based on one or more IEEE 802.11 standards (e.g., 802.11a, 802.11b, 802.11g, 802.11n (also known as Wi-Fi 4), 802.11ac (also known as Wi-Fi 5), 802.11ax (also known as Wi-Fi 6)). Using a cellular radio module 4044 or a Wi-Fi radio module 4043, the data receiving device 120 can communicate with a remote application server 150 to receive test substance data or provide updates or inputs received from a user (e.g., via one or more user interfaces). Although not shown, the communication module 5040 of the test substance sensor 120 may also include a cellular radio module or a Wi-Fi radio module.

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

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

[0060] D. Exemplary Sensor Control Device Figures 2C and 2D are block diagrams illustrating an example of an embodiment of a sensor control device 102 having a test substance sensor 104 and sensor electronics 160 (including a test substance monitoring circuit) that may have most of the processing power to prepare the final result data for display to the user. Figure 2C shows a single semiconductor chip 161, which may be a custom application-specific integrated circuit (ASIC). Within the ASIC 161 are shown specific high-level functionality units, 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 test substance monitoring circuit, but in other embodiments, either circuit may perform the test substance monitoring function. The processor 166 may include one or more processors, microprocessors, controllers, and / or microcontrollers, each of which may be a separate chip or distributed among a number of different chips (and some thereof).

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

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

[0063] For illustrative purposes only and not limiting, an exemplary embodiment of the substance sensor 110 for use in the disclosed subject is shown in Figure 2E. Figure 2E shows a block schematic of an example of the substance sensor 110 according to an exemplary embodiment that conforms to the security architecture and communication scheme described herein.

[0064] As embodied herein, the substance under test sensor 110 may include an application-specific integrated circuit ("ASIC") 5000 communicably connected to a communication module 5040. The ASIC 5000 may include a microcontroller core 5010, onboard memory 5020, and storage memory 5030. The storage memory 5030 can store data used in authentication and encryption security architectures. The storage memory 5030 can store programming instructions for the sensor 110. As embodied herein, a specific communication chipset may be embedded in the ASIC 5000 (e.g., an NFC transceiver 5025). The ASIC 5000 can receive power from a power module 5050, e.g., an onboard battery or NFC pulses. The storage memory 5030 of the ASIC 5000 can be programmed to contain information such as an identifier for 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 programming (OTP) memory. The storage memory 5030 may be updated using 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 one or more modules that help the substance sensor 110 communicate with other devices of the substance monitoring system 100. An example of the communication module 5040, but not limited to this, 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 facilitate pairing of Bluetooth® devices for end users. The communication module 5040 can transmit and receive data and commands through interaction with a similarly capable communication module of the data receiving device 120 or user device 140. The communication module 5040 may include additional or alternative chipsets for use in personal area networks by the IEEE 802.15 protocol, the IEEE 802.11 protocol, infrared communication by the Infrared Data Association Standard (IrDA), and other similar short-range communication schemes.

[0066] To perform its function, the sensor 100 may further include suitable detection hardware 5060 appropriate for that function. As embodied herein, the detection hardware 5060 may include a test substance sensor that is placed transcutaneously or subcutaneously in contact with the body fluid of the subject. The test substance sensor can generate sensor data that includes values ​​corresponding to the levels of one or more test substances in the body fluid.

[0067] E. Exemplary assembly process for sensor control device The components of the sensor control device 102 can be obtained by the user in multi-packaging that requires the user to perform final assembly before delivery to the appropriate user location. Figures 3A-3D show examples of embodiments of the user assembly process of the sensor control device 102, which includes preparing separate components before joining them together to prepare the sensor for delivery. Figures 3E-3F show examples of embodiments of delivering the sensor control device 102 to the appropriate user location by selecting an appropriate delivery location and fitting the device 102 into that location.

[0068] Figure 3A is a proximal perspective view showing an example of a user embodiment of preparing a container 810 configured herein as a tray (although other packaging may also be used) for the assembly process. The user can achieve this preparation by, for example, peeling the non-adhesive portion of the lid 812 from the tray 810 to remove the adhesive portion of the lid 812 and exposing the platform 808. Removal of the lid 812 may be appropriate in various embodiments, as long as the platform 808 is adequately exposed within the tray 810. The lid 812 may then be placed to the side.

[0069] Figure 3B is a side view showing an example of an embodiment in which a user prepares the applicator device 150 for assembly. The applicator device 150 may be supplied in a sterile package sealed by a cap 708. Preparation of the applicator device 150 may include removing the housing 702 from the cap 708 to expose the sheath 704 (Figure 3C). This can be achieved by unscrewing (or otherwise removing) the cap 708 from the housing 702. The cap 708 can then be placed on its side.

[0070] Figure 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, the user aligns the housing orientation feature 1302 (or slot or recess) and the tray orientation feature 924 (contact or stopper), and then inserts the sheath 704 into the platform 808 inside the tray 810. By inserting the sheath 704 into the platform 808, the sheath 704 is temporarily unlocked from the housing 702, and the platform 808 is also temporarily unlocked from the tray 810. At this stage, removing the applicator device 150 from the tray 810 would result in the same state as before the initial insertion of the applicator device 150 into the tray 810 (i.e., this process can be reversed or interrupted at this point and then repeated without result).

[0071] As the housing 702 advances distally, the sheath 704 is maintained in position within the platform 808 relative to the housing 702, engaging with the platform 808 and advancing the platform 808 distally relative to the tray 810. This process unlocks and folds the platform 808 within the tray 810. As the sheath 704 is unlocked relative to the housing 702, it comes into contact with a fixed feature (not shown) within the tray 810 that prevents the sheath 704 from moving (relatively) while the housing 702 continues to advance the platform 808 distally, disengaging them. When the advance of the housing 702 and the platform 808 is complete, the sheath 704 is permanently unlocked relative to the housing 702. At the end of the distal advance of the housing 702, the tip and sensor (not shown) within the tray 810 can be engaged with the electronic equipment housing (not shown) within the housing 702. The operation and interaction of the applicator device 150 and the tray 810 will be described further below.

[0072] Figure 3D is a proximal perspective view showing an example of an embodiment in which a 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 proximal to the tray 810 or by other actions that have the same final effect as disengaging the applicator 150 from the tray 810. The applicator device 150 is removed with the sensor control device 102 (not shown) fully assembled therein (tip, sensor, electronics) and positioned for delivery.

[0073] Figure 3E is a proximal perspective view showing an example of an embodiment in which a patient applies the sensor control unit 102 to a target area of ​​skin, such as the abdomen or another suitable location, using the applicator device 150. By advancing the housing 702 distally, the sheath 704 is folded into the housing 702, the sensor is applied to the target location, and as a result, the adhesive layer on the bottom of the sensor control unit 102 adheres to the skin. The tip automatically retracts when the housing 702 is fully advanced, while the sensor (not shown) remains in place to measure the level of the substance being tested.

[0074] Figure 3F is a proximal perspective view showing an example of a patient embodiment where the sensor control device 102 is attached. The user can then remove the applicator 150 from the attached site.

[0075] System 100, as described in Figures 3A-3F and elsewhere in this specification, can reduce or eliminate the chances of accidental breakage, permanent deformation, or improper assembly of applicator components compared to systems of the prior art. Because the applicator housing 702 engages directly with the platform 808 while the sheath 704 is unlocked, rather than through indirect engagement via the sheath 704, 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 possibility of relatively large forces being applied during assembly (e.g., in conventional devices) is reduced, thereby reducing the chances of unsuccessful user assembly.

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

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

[0078] G. Assembly of exemplary tray and sensor module Figure 5 is a proximal perspective view showing an example of an embodiment of a tray 810 with a detachably connected sterile lid 812, which may illustrate how the package is shipped and received by the user before assembly.

[0079] Figure 6A is a proximal perspective cross-sectional view showing the sensor delivery components in tray 810. Platform 808 is slidably connected to tray 810. Desiccant 502 is fixed to tray 810. Sensor module 504 is installed in tray 810.

[0080] Figure 6B is a proximal perspective view showing the sensor module 504 in more detail. Here, the retaining arm extension 1834 of the platform 808 securely fixes the sensor module 504 in place. Module 2200 is connected to the connector 2300, the tip module 2500, and the sensor (not shown), so that they can be removed together as the sensor module 504 during assembly.

[0081] Exemplary Applicator and Sensor Control Device for H-One Piece Architecture Referring again briefly to Figures 1A and 3A-3G, for a two-piece architectural system, the sensor tray 202 and sensor applicator 102 are provided to the user as separate packages, and therefore the user is required to open each package and ultimately assemble the system. In some applications, the separately sealed packages allow the sensor tray 202 and sensor applicator 102 to be sterilized by separate sterilization processes specific to the contents of each package and otherwise incompatible with the contents of the other package. More specifically, the sensor tray 202, including the sensor 110 and the plug assembly 207 including 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 may damage the electrical components located inside the electronic housing of the sensor control device 102. Therefore, if the sensor applicator 102, including the electronic housing of the sensor control device 102, needs to be sterilized, it can be sterilized by another method, such as gaseous chemical sterilization using ethylene oxide. However, gaseous chemical sterilization may damage 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 usually sterilized by separate sterilization processes and then individually packaged, so that the user ultimately needs to assemble the parts for use.

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

[0083] In addition to the electronic module 3806, PCBA3802 may include a data processing unit 3808 mounted on PCB3804. The data processing unit 3808 may comprise an application-specific integrated circuit (ASIC) configured to perform one or more functions or routines associated with the operation of the sensor control device 3702, for example. More specifically, the data processing unit 3808 may be configured to perform data processing functions, which may include, but are not limited to, filtering and encoding of data signals, each corresponding to the user's sampled test substance level. The data processing unit 3808 may also include an antenna for communicating with the reader device 106 (Figure 1A), or may communicate with it in other ways.

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

[0085] Sensor 3716 may be centrally positioned relative to 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 mount 3708, which is received percutaneously under the user's skin. Furthermore, the tail 3816 may have an enzyme or other chemicals contained therein that helps facilitate monitoring of the test substance.

[0086] Flag 3818 may include a substantially flat surface having one or more sensor contacts 3822 (three shown in Figure 7B) positioned thereon. The sensor contacts 3822 may be configured to align with and engage with one or more corresponding circuit contacts 3824 (three shown in Figure 7A) provided on PCB 3804. In some embodiments, the sensor contacts 3822 may comprise a carbon-impregnated polymer printed on or otherwise digitally applied to 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 contacts 3822 of this disclosure provide a direct connection between the sensor 3716 and PCB 3804, thereby eliminating the need for a conventional connector and advantageously reducing the height H. Furthermore, by eliminating the compliant carbon-impregnated polymer modules, circuit resistance is significantly eliminated, and thus the conductivity of the circuit is improved.

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

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

[0089] Specifically with reference to Figure 7A, the inner surface of the mount 3708 may be provided with or otherwise defined by 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. 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. Adjacent contact pockets 3836 may be configured to accommodate a portion of the axial contact 3814a.

[0090] Furthermore, multiple module pockets 3838 may be defined on the inner surface of the mount 3708 to accommodate various electronic modules 3806 located 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 unit 3702 is assembled. The battery locator 3834, contact pocket 3836, module pockets 3838, and shield locator 3840 all extend a short distance on the inner surface of the mount 3708, and as a result, the overall height H of the sensor control unit 3702 can be reduced compared to conventional sensor control units. The module pockets 3838 may 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 Figure 7A, the mount 3708 may further include a plurality of carrier grip features 3842 (two shown) defined near the outer circumference of the mount 3708. The carrier grip features 3842 are axially offset from the bottom 3844 of the mount 3708, where transfer adhesive may be applied during assembly. In contrast to conventional sensor control devices that generally include conical carrier grip features intersecting the bottom of the mount, the carrier grip features 3842 of this 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. Furthermore, the carrier grip features 3842 of this disclosure eliminate the need for corrugated transfer adhesive, which simplifies the manufacture of the transfer adhesive and eliminates the need to accurately record the transfer adhesive to the mount 3708. This also increases the bonding area and therefore the bonding strength.

[0092] Referring to Figure 7B, the bottom 3844 of the mount 3708 may be provided with or otherwise defined by a plurality of grooves 3846, which are defined on or near the outer circumference of the mount 3708 and are equidistant from each other. A transfer adhesive (not shown) may be connected to the bottom 3844, and the grooves 3846 may be configured to help carry (transfer) moisture away from the sensor control device 3702 towards the periphery of the mount 3708 during use. In some embodiments, the spacing of the grooves 3846 may include module pockets 3838 (Figure 7A) defined on the opposite (inner) side of the mount 3708. As recognized, alternating the positions of the grooves 3846 and module pockets 3838 ensures that opposing features on either side of the mount 3708 do not extend toward each other. This helps to maximize the amount of material used for the mount 3708 and may help to maintain the minimum height H of the sensor control device 3702. The module pocket 3838 can also significantly reduce mold sink and improve the flatness of the bottom 3844 to which the transfer adhesive adheres.

[0093] Furthermore, referring to Figure 7B, the inner surface of the shell 3706 may be provided with or otherwise defined by 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. For example, the inner surface of the shell 3706 may define an opposing battery locator 3848 that can be positioned opposite the battery locator 3834 (Figure 7A) of the mount 3708 when the sensor control device 3702 is assembled, and is configured to accommodate a portion of the battery 3812. The opposing battery locator 3848 extends only a short distance into the inner surface of the shell 3706, thereby helping 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 a tip and sensor locator 3852. The tip and sensor locator 3852 may be configured to receive both the tip (not shown) and a portion of the sensor 3716. Furthermore, the tip and sensor locator 3852 may be configured to align with and / or pair with a corresponding tip and sensor locator 2054 (Figure 7A) provided on the inner surface of the mount 3708.

[0095] Embodiments of this disclosure show alternative sensor assembly / electronic equipment assembly connection approaches in Figures 8A–8C. As shown, the sensor assembly 14702 includes a sensor 14704, a connector support 14706, and a tip 14708. In particular, a recess or receptacle 14710 may be defined at the bottom of the mount of the electronic equipment assembly 14712, providing a place for the sensor assembly 14702 to be received and connected to the electronic equipment assembly 14712, thereby providing a place for the sensor control unit to be fully assembled. The profile of the sensor assembly 14702 may be molded to match or complement the receptacle 14710, which includes an elastomer sealing member 14714 (connected to a circuit board and containing conductive material aligned with the electrical contacts of the sensor 14704). Accordingly, the on-body device 14714 shown in Figure 8C is formed when the sensor assembly 14702 is driven into the integrally formed recess 14710 of the electronics assembly 14712, causing the sensor assembly 14702 to snap into place or otherwise bond to the electronics assembly 14712. This embodiment provides an integrated connector for the sensor assembly 14702 within the electronics assembly 14712.

[0096] Further information relating to the sensor assembly is provided in U.S. Publication No. 2013 / 0150691 and U.S. Publication No. 2021 / 0204841, each of which is incorporated herein by reference in whole.

[0097] According to embodiments of this disclosure, the sensor control device 102 may be modified to provide a one-piece architecture that can undergo sterilization techniques specifically designed for one-piece architecture sensor control devices. The one-piece architecture allows the sensor applicator 150 and sensor control device 102 to be shipped to the user in a single sealed package that requires no final user assembly process. Rather, the user only needs to open one package and subsequently deliver the sensor control device 102 to the target monitoring location. The one-piece system architecture described herein may prove advantageous in reducing parts, various manufacturing processes, and user assembly processes. As a result, packaging materials and waste are reduced, and the possibility of user error or contamination of the system is mitigated.

[0098] Figures 9A and 9B are a side view and a side cross-sectional view, respectively, of an example embodiment of the sensor applicator 102 with the applicator cap 210 connected. More specifically, Figure 9A shows how the sensor applicator 102 is shipped to and received by the user, and Figure 9B shows the sensor control unit 4402 located inside the sensor applicator 102. Thus, the fully assembled sensor control unit 4402 may already be assembled and installed inside 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 unit 4402 can be attached to the sensor applicator 102, and subsequently the applicator cap 210 can be connected to the sensor applicator 102. In some embodiments, the applicator cap 210 may be screwed into the housing 208 and may include a tamper ring 4702. When the applicator cap 210 is rotated relative to the housing 208 (e.g., by loosening a screw), the tamper ring 4702 is sheared, thereby releasing the applicator cap 210 from the sensor applicator 102.

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

[0101] Since the distal portions of the sensor 4410 and 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 biological agents provided in the tail 4524, as well as other sensor components such as membrane coatings that regulate the inflow of the test substance.

[0102] Once the desired level of sterilization assurance is achieved in the sterilization chamber 4706, the gaseous solution may be removed and the sterilization chamber 4706 may be ventilated. Ventilation may be achieved by a series of vacuums and subsequent circulation of gas (e.g., nitrogen) or filtered air through the sterilization chamber 4706. Once the sterilization chamber 4706 is properly ventilated, the vent 4708 may be closed by the seal 4712 (indicated by the dotted line).

[0103] In some embodiments, the 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 fiber) such as Tyvek®, available from DuPont®. Tyvek® is highly durable, puncture-resistant, and vapor-permeable. The Tyvek® layer can be applied before the gaseous chemical sterilization process, and following the gaseous chemical sterilization process, a foil or other vapor and moisture-proof material layer may be applied to cover and seal (e.g., heat-seal) the Tyvek® layer to prevent contaminants and moisture from entering the sterilization chamber 4706. In other embodiments, the seal 4712 may comprise only a single protective layer applied to the applicator cap 210. In such embodiments, the single layer may be gas-permeable for the sterilization process, but can also protect against moisture and other harmful elements once the sterilization process is complete.

[0104] Once the seal 4712 is in place, the applicator cap 210 provides a barrier against external contamination, thereby maintaining a sterile environment for the assembled sensor control unit 4402 until the user removes the applicator cap 210 (loosens the screw). The applicator cap 210 can also create a dust-free environment that prevents the adhesive patch 4714 from becoming contaminated during shipping and storage.

[0105] Figures 10A and 10B are isometric and side views, respectively, of another example of the sensor control device 5002 according to one or more embodiments of the present disclosure. The sensor control device 5002 may be similar in some respects to the sensor control device 102 of Figure 1A, and can therefore be best understood by referring to it. Furthermore, the sensor control device 5002 may replace the sensor control device 102 of Figure 1A, and can therefore be used in conjunction with the sensor applicator 102 of Figure 1A, which can deliver the sensor control device 5002 to a target monitoring position on the user's skin.

[0106] However, unlike the sensor control unit 102 in Figure 1A, the sensor control unit 5002 may have a one-piece system architecture that does not require the user to open multiple packages and ultimately assemble the sensor control unit 5002 before application. Rather, when the user receives it, the sensor control unit 5002 may already be fully assembled and properly positioned in the sensor applicator 150 (Figure 1A). To use the sensor control unit 5002, the user only needs to open one barrier (e.g., the applicator cap 708 in Figure 3B) before the sensor control unit 5002 is immediately delivered to the target monitoring location for use.

[0107] As shown, the sensor control device 5002 includes an electronic equipment housing 5004 which is substantially disk-shaped and may have a circular cross-section. However, in other embodiments, the electronic equipment housing 5004 may have other cross-sectional shapes, such as oval or polygonal, without departing from the scope of the present disclosure. The electronic equipment housing 5004 may be configured to house or otherwise include various electrical components used to operate the sensor control device 5002. In at least one embodiment, an adhesive patch (not shown) may be placed on the bottom of the electronic equipment housing 5004. The adhesive patch may be similar to the adhesive patch 105 in Figure 1A and may thus help 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 equipment housing 5004 which includes a shell 5006 and a mount 5008 which can be paired with the shell 5006. The shell 5006 can be fastened to the mount 5008 by a variety of methods such as snap-fit ​​engagement, interlocking fit, ultrasonic welding, one or more mechanical fasteners (e.g., screws), gaskets, adhesives, or any combination thereof. In some examples, the shell 5006 may be fastened to the mount 5008 such that a sealed interface is formed between them.

[0109] The sensor control device 5002 may further include a sensor 5010 (partially visible) and a tip 5012 (partially visible) used to help deliver the sensor 5010 transdermally under the user's skin while the sensor control device 5002 is being applied. As shown, the corresponding portions of the sensor 5010 and tip 5012 extend distally from the bottom of the electronic equipment housing 5004 (e.g., the mount 5008). The tip 5012 may include a tip hub 5014 configured to fix and deliver the tip 5012. As best seen in Figure 10B, the tip hub 5014 may include a mating member 5016 or be otherwise defined. To connect the tip 5012 to the sensor control device 5002, the tip hub 5014 engages with the upper surface of the shell 5006, allowing the tip 5012 to advance axially through the electronic housing 5004 until the mating member 5016 extends distally from the bottom of the mount 5008. Once the tip 5012 penetrates the electronic housing 5004, the exposed portion of the sensor 5010 can be received in the hollow or recessed (arched) portion of the tip 5012. The remaining portion of the sensor 5010 is located inside the electronic housing 5004.

[0110] The sensor control unit 5002 may further include a sensor cap 5018, as shown disassembled or separated from the electronic housing 5004 in Figures 10A-10B. The sensor cap 5016 may be removably coupled to the sensor control unit 5002 (e.g., the electronic housing 5004) at or near the bottom of the mount 5008. The sensor cap 5018 may serve to surround the exposed portions of the sensor 5010 and tip 5012, providing a sealed barrier to protect them from gaseous chemical sterilization. As shown, the sensor cap 5018 may comprise a substantially 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 form 5024 or otherwise defined. As described herein, the engaging feature 5024 may help to pair the sensor cap 5018 with the cap of the sensor applicator (e.g., the sensor applicator 150 in Figures 1 and 3A-3G) (e.g., the applicator cap 708 in Figure 3B), and may help 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 connected to the electronic equipment housing 5004 at or near the bottom of the mount 5008. More specifically, the sensor cap 5018 may be removably connected to a mating member 5016 extending distally from the bottom of the mount 5008. In at least one embodiment, for example, the mating member 5016 may define a set of male threads 5026a (Figure 10B) that can mate with a set of female threads 5026b (Figure 10A) defined by the sensor cap 5018. In some embodiments, the male and female threads 5026a,b may have a flat thread design (e.g., without helical curvature), which may prove advantageous when forming the part. Alternatively, the male and female threads 5026a,b may have helical thread engagement. Thus, the sensor cap 5018 may be screw-connected 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 connected to the mating member 5016 via an interlocking fit, a friction fit, or other types of engagement including, but not limited to, fragile members or materials that can be broken by a minimal separating force (e.g., axial force or rotational force).

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

[0113] Figures 11A–11C are stepwise side cross-sectional views showing an assembly of a sensor applicator 102 with a sensor control device 5002 according to one or more embodiments. Once the sensor control device 5002 is fully assembled, it can be mounted within the sensor applicator 102. Referring to Figure 11A, the tip hub 5014 may include or be otherwise defined a hub snap pole 5302 configured to help connect the sensor control device 5002 to the sensor applicator 102. More specifically, the sensor control device 5002 may be advanced into the sensor applicator 102, and the hub snap pole 5302 may be received by a corresponding arm 5304 of a tip carrier 5306 located inside the sensor applicator 102.

[0114] Figure 11B shows that the sensor control device 5002 is received by the tip carrier 5306 and thus secured inside the sensor applicator 102. Once the sensor control device 5002 is mounted inside the sensor applicator 102, the applicator cap 210 may be connected to the sensor applicator 102. In some embodiments, the applicator cap 210 and the housing 208 may have a pair of opposing, mating threads 5308 that allow the applicator cap 210 to be screwed into the housing 208 in a clockwise (or counterclockwise) direction, thereby securing the applicator cap 210 to the sensor applicator 102.

[0115] As shown, the sheath 212 is also located within the sensor applicator 102, which may include a sheath locking mechanism 5310 configured to ensure that the sheath 212 does not fold prematurely during an impact. In the embodiment shown, the sheath locking mechanism 5310 may provide a screw 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 to screw and engage with the sensor applicator 102 at the threads 5308. The female and male threads 5312a,b may have the same thread pitch as the threads 5308, which allow the applicator cap 210 to be screwed into the housing 208.

[0116] In Figure 11C, the applicator cap 210 is shown fully screwed into (connected to) the housing 208. As shown, the applicator cap 210 may further comprise or otherwise define a cap post 5314 located in the center of the inside of the applicator cap 210 and extending proximal to its bottom. The cap post 5314 may be configured to allow the applicator cap 210 to screw into the housing 208 and to receive at least a portion of the sensor cap 5018.

[0117] Once the sensor control unit 5002 is mounted inside the sensor applicator 102 and the applicator cap 210 is properly secured, the sensor control unit 5002 can undergo gaseous chemical sterilization configured to sterilize the electronic housing 5004 and any other exposed parts of the sensor control unit 5002. Since the distal portions of the sensor 5010 and tip 5012 are sealed inside the sensor cap 5018, the chemicals used during the gaseous chemical sterilization process cannot interact with other sensor components such as enzymes, chemicals, and biologics provided in the tail 5104, and membrane coatings that regulate the inflow of the test substance.

[0118] Figures 12A to 12C are stepwise side cross-sectional views showing the assembly and disassembly of alternative embodiments of the sensor applicator 102 with the sensor control device 5002 according to one or more further embodiments. Generally as described above, the fully assembled sensor control device 5002 may be attached to the sensor applicator 102 by connecting the hub snap pole 5302 into the arm 5304 of the tip carrier 5306 located inside the sensor applicator 102.

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

[0120] In Figure 12B, the applicator cap 210 is aligned with the housing 208 and advances toward the housing 208, thereby receiving the sheath 212 into the applicator cap 210. Instead of rotating the applicator cap 210 relative to the housing 208, the applicator cap 210 may be connected to the housing 208 by snapping the threads of the applicator cap 210 onto the corresponding threads of the housing 208. An axial cut or slot 5703 (one shown) defined in the applicator cap 210 may allow a portion of the applicator cap 210 near its threads to bend outward and snap into place to engage with the threads of the housing 208. Once the applicator cap 210 is snapped to the housing 208, the sensor cap 5018 may be correspondingly snapped to the cap post 5314.

[0121] Similar to the embodiments shown in Figures 11A-11C, the sensor applicator 102 may include a sheath locking mechanism configured to ensure that the sheath 212 does not fold prematurely during impact. In the embodiments shown, the sheath locking mechanism includes one or more ribs 5704 (shown one) 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 ribs 5704 may be configured to interlock between the ribs 5706 and the shoulder 5708 while the applicator cap 210 is attached to the housing 208. More specifically, once the applicator cap 210 is snapped onto the housing 208, the applicator cap 210 rotates (for example, clockwise), thereby positioning the rib 5704 of the sheath 212 between the rib 5706 of the applicator cap 210 and the shoulder 5708, so that the applicator cap 210 is "locked" in place until the user reverses the rotation to remove the applicator cap 210 for use. The engagement of the rib 5704 between the rib 5706 of the applicator cap 210 and the shoulder 5708 can also prevent the sheath 212 from folding prematurely.

[0122] In Figure 12C, the applicator cap 210 is removed from the housing 208. Similar to the embodiments in Figures 21A to 21C, the applicator cap 210 can generally be removed by rotating it in the reverse direction, which in turn causes the cap post 5314 to rotate in the same direction, disengaging the screw connection between the sensor cap 5018 and the fitting member 5016. Furthermore, by removing the sensor cap 5018 from the sensor control device 5002, the distal portion of the sensor 5010 and tip 5012 is exposed.

[0123] When the applicator cap 210 is disengaged from the housing 208, the rib 5704 defined on the sheath 212 may engage with the upper part of the rib 5706 defined on the applicator cap 210 in a sliding manner. The upper part of the rib 5706 may provide a corresponding inclined surface that causes the applicator cap 210 to rotate and the sheath 212 to move upward, which causes the sheath arm 5604 to disengage from the first retaining arm 5702a and flex so that it is received in the second retaining arm 5702b. As the sheath 212 moves to the second stopper 5702b, the radial shoulder 5614 moves in a direction that disengages radially from the carrier arm(s) 5608, thereby allowing the passive spring force of the spring 5612 to push the tip carrier 5306 upward, disengaging the carrier arm(s) 5608 from the groove(s) 5610. As the tip carrier 5306 moves upward within the housing 208, the mating member 5016 can correspondingly retract until it is coplanar, substantially coplanar, or quasi-coplanar with the bottom of the sensor control device 5002. At this point, the sensor applicator 102 is in the firing position. Therefore, in this embodiment, removing the applicator cap 210 causes the mating member 5016 to retract accordingly.

[0124] I. Exemplary firing mechanisms of one-piece and two-piece applicators Figures 13A–13F show detailed examples of an internal device mechanism that “launches” the applicator 216 to apply the sensor control device 222 to a user and safely retracts the tip 1030 into the used applicator 216. Together, these drawings illustrate an example sequence of driving the tip 1030 (supporting the sensor connected to the sensor control device 222) into the user’s skin, retracting the tip leaving the sensor in operably contacting the user’s interstitial fluid, and adhering the sensor control device to the user’s skin with adhesive. Modifications of such operation for use with alternative applicator assembly embodiments and components can be understood by those skilled in the art by reference to the same. Furthermore, the applicator 216 may be a sensor applicator having a one-piece architecture or a two-piece architecture, as disclosed herein.

[0125] Referring here to Figure 13A, the sensor 1102 is supported within the tip 1030, just above the user's skin 1104. Rails 1106 (optionally, three of them) may be provided on the upper guide section 1108 to control the movement of the applicator 216 relative to the sheath 318. The sheath 318 is held in place by the retaining feature 1110 within the applicator 216, and a suitable downward force along the longitudinal axis of the applicator 216 overcomes the resistance force provided by the retaining mechanism 1110, allowing the tip 1030 and the sensor control device 222 to translate along the longitudinal axis into (and above) the user's skin 1104. Furthermore, the catch arm 1112 of the sensor carrier 1022 engages with the tip retraction assembly 1024 to maintain the tip 1030 in position relative to the sensor control device 222.

[0126] In Figure 13B, a user force is applied to overcome or over the retaining feature 1110, causing the sheath 318 to fold within the housing 314, translating the sensor control device 222 (along with its accompanying 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 entire stroke of the sensor / tip insertion process. The retention of the stop surface 1114 of the carrier arm 1112 against the complementary surface 1116 of the tip retraction assembly 1024 maintains the position of the member together with the fully biased telescopic spring 1118. According to one embodiment, rather than employing a user force to translate the sensor control device 222 downward along the longitudinal axis as indicated by arrow L, a button (e.g., a push button) that drives the sensor control device 222 by acting an actuation spring (e.g., a coil spring) may be included in the housing 314.

[0127] In Figure 13C, the sensor 1102 and tip 1030 have reached the depth where they are fully inserted. This causes the carrier arm 1112 to move away from the inner diameter of the upper guide section 1108. Next, the compressive force of the coil expansion spring 1118 drives the angled stop surface 1114 radially outward, and by releasing the force, drives the tip carrier 1102 of the tip retraction assembly 1024, pulling the tip 1030 (which has a slot or is otherwise configured) outward from the user and away from the sensor 1102, as indicated by arrow R in Figure 13D.

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

[0129] The operation of the applicator 216 when the sensor control device 222 is applied is designed to give the user the sensation that both the insertion and retraction of the tip 1030 are performed automatically by the internal mechanism of the applicator 216. In other words, the present invention avoids the user experiencing the sensation of manually driving the tip 1030 into the user's skin. That is, if the user applies sufficient force to overcome the resistance of the applicator 216's retaining element, the resulting movement of the applicator 216 is perceived as an automatic response to the “induced” applicator. Even though all the driving force is provided by the user and no further biasing / driving means are used to insert the tip 1030, the user does not perceive that they are supplying any further force to drive the tip 1030 to puncture the skin. As described above in Figure 13C, the retraction of the tip 1030 is automated by the coil extension spring 1118 of the applicator 216.

[0130] Those skilled in the art will understand that any of the applicator embodiments described herein, and any of their components, including but not limited to embodiments of the tip, tip module, and sensor module, may be sized and configured for use with a sensor configured to detect the level of a test substance in bodily fluids in the epidermis, dermis, or subcutaneous tissue of a subject. In some embodiments, for example, the tip and distal portion of the test substance sensor disclosed herein may both be sized and configured to be located at a specific terminal depth (i.e., the furthest point of penetration in the tissue or layer of the body of the subject, such as the epidermis, dermis, or subcutaneous tissue). With respect to some applicator embodiments, those skilled in the art will recognize that specific embodiments of the tip may be sized and configured to be located at different terminal depths in the body of the subject relative to the final terminal depth of the test substance sensor. In some embodiments, for example, the tip may be located at a first terminal depth in the epidermis of the subject before retraction, while the distal portion of the test substance sensor may be located at a second terminal depth in the dermis of the subject. In other embodiments, the tip may be located at a first distal depth in the dermis of the subject before retraction, while the distal portion of the substance sensor may be located at a second distal depth in the subcutaneous tissue of the subject. In yet another embodiment, the tip may be located at the first distal depth before retraction, and the substance sensor may be located at the second distal depth, where both the first and second distal depths are in the same layer or tissue of the subject's body.

[0131] Furthermore, with respect to any embodiment of the applicator described herein, those skilled in the art will understand that, with regard to any of the embodiments of the applicator described herein, one or more structural components connected to the substance sensor and one or more spring mechanisms, including but not limited to them, may be positioned eccentrically with respect to one or more axes of the applicator within the applicator. In some embodiments of the applicator, for example, the substance sensor and spring mechanism may be positioned at a first eccentric position with respect to the axis of the applicator on a first side of the applicator, and the sensor electronics may be positioned at a second eccentric position with respect to the axis of the applicator on a second side of the applicator. In other embodiments of the applicator, the substance sensor, spring mechanism, and sensor electronics may be positioned eccentrically with respect to the axis of the applicator on the same side. Those skilled in the art will recognize that other arrangements and configurations are possible in which any or all of the substance sensor, spring mechanism, sensor electronics, and other components of the applicator are positioned centrally or eccentrically with respect to one or more axes of the applicator, and that these are entirely within the scope of the present disclosure.

[0132] Further details of preferred apparatus, systems, methods, components, and their operation, along with relevant features, are described in International Publication WO2018 / 136898 by Rao et al., International Publication WO2019 / 236850 by Thomas et al., International Publication WO2019 / 236859 by Thomas et al., International Publication WO2019 / 236876 by Thomas et al., and U.S. Patent Publication 2020 / 0196919, filed on June 6, 2019, each of which is incorporated herein by reference in whole. Further details relating to applicators, their components, and modified embodiments thereof are described in U.S. Patent Publications 2013 / 0150691, 2016 / 0331283, and 2018 / 0235520, all of which are incorporated herein by reference in whole for any purpose. Further details relating to tip modules, tips, their components, and embodiments of variations thereof are described in U.S. Patent Publication 2014 / 0171771, which is incorporated herein by reference in whole for all purposes.

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

[0134] Figure 14 is a graph showing the in vitro sensitivity of a current-measuring substance sensor. In vitro sensitivity can be obtained by in vitro testing the sensor at various substance concentrations and performing regression (e.g., linear or nonlinear) 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, where y is the electrical output current of the sensor, x is the level (or concentration) of the substance, m is the sensitivity gradient, and b is the sensitivity intercept, which 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 sensitivity gradient and intercept. Sensors with nonlinear sensitivity require further information to determine the substance level resulting from the sensor's output current, and those skilled in the art are familiar with methods for modeling nonlinear sensitivity. In certain embodiments of an in vivo sensor, the in vitro sensitivity may be the same as the in vivo sensitivity, but in other embodiments, a transfer (or conversion) function is used to replace the in vitro sensitivity with an in vivo sensitivity applicable to the in vivo application for which the sensor is intended.

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

[0136] In some in vivo test substance monitoring systems, calibration is required either by the user or by the system itself after the sensor has been implanted in the user or patient. For example, when user intervention is required, the user performs an in vitro measurement (e.g., blood glucose (BG) measurement using finger prick and in vitro test strip) while the test substance sensor is implanted and inputs it into the system. The system then compares the in vitro measurement to the in vivo signal and uses the difference to determine an estimate of the sensor's in vivo sensitivity. The in vivo sensitivity can then be used in an algorithmic process that converts the data collected by the sensor into a value indicating the user's test substance level. This and other processes requiring user action to perform calibration are referred to as "user calibration." The system may require user calibration due to the instability of sensor sensitivity, where sensitivity drifts or changes over time. Therefore, multiple user calibrations (e.g., by a regular (e.g., daily), variable, or as needed schedule) may be necessary to maintain accuracy. The embodiments described herein may incorporate some degree of user calibration for specific executions, which is generally undesirable as it may be cumbersome for the user or require the performance of cumbersome BG measurements and may lead to user errors.

[0137] Some in vivo substance monitoring systems can periodically adjust calibration parameters by using automated measurement of sensor characteristics created by the system itself (e.g., processing circuit execution software). The repeated adjustment of sensor sensitivity based on variables measured by the system (not the user) is generally referred to as "system" (or automated) calibration and can be performed with or without user calibration, such as early BG measurement. As with repeated user calibration, repeated system calibration is generally required due to the drift of sensor sensitivity over time. Therefore, 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 therefore post-implantation calibration is not required.

[0138] Some in vivo test substance 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 professional (HCP). Calibration parameters can be determined by the sensor manufacturer (or, if two components differ, by the manufacturer of other components of the sensor control unit). 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 may 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 and coded or programmed into the data processing unit of the substance monitoring system as part of the manufacturing process, or provided to the sensor itself as, for example, a barcode, laser tag, RFID tag, or other machine-readable information provided on the sensor. When the codes are provided to the receiver (or other data processing unit), 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 codes or parameters are provided to the sensor itself before or at the start of sensor use, the calibration codes or parameters may be automatically transmitted or provided to the data processing unit of the substance monitoring system.

[0140] Some in vivo substance monitoring systems operate with sensors that can be factory calibrated, system calibrated, and / or user calibrated. For example, a sensor may be provided with calibration codes or parameters that enable factory calibration. When information is provided to the receiver (e.g., entered by the user), the sensor may operate as a factory calibrated sensor. If no information is provided to the receiver, the sensor may operate as a user-calibrated sensor and / or system-calibrated sensor.

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

[0142] According to the disclosed subject matter, a test substance monitoring system may be configured to compensate for or adjust sensor sensitivity based on a sensor drift profile. A time-varying parameter β(t) may be defined or determined based on an analysis of the sensor behavior during in vivo use, and a time-varying drift profile may be determined. In certain embodiments, compensation or adjustment for sensor sensitivity may be programmed in the receiver unit, controller, or data processor of the test substance monitoring system so that compensation or adjustment, or both, can be performed automatically and / or iteratively when sensor data is received from the test substance sensor. According to the disclosed subject matter, the adjustment or compensation algorithm may be initiated or executed by the user (rather than self-initiated or executed) so that the adjustment or compensation for the test substance sensor sensitivity profile is performed or executed when the user initiates or activates the 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 (excluding, in some embodiments, sample sensors used in in vitro testing) can be non-destructively inspected to determine or measure its characteristics, such as the film thickness at one or more points on the sensor, and other characteristics, including physical properties 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, for example, using 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 the corresponding average value based on the sample sensor for possible correction of the calibration parameter or code assigned to each sensor. For example, for a calibration parameter defined as sensor sensitivity, the sensitivity is approximately inversely proportional to the film thickness, and therefore, for example, for a sensor having a measured film thickness that is approximately 4% thicker than the average film thickness of sensors sampled from the same sensor lot as a given 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 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 area of ​​sensors sampled from the same sensor lot, the sensitivity assigned to that sensor is the average sensitivity multiplied by 0.97. The assigned sensitivity may be determined from the average sensitivity from sampled sensors by multiple sequential adjustments for each inspection or measurement of the sensor. In certain embodiments, the inspection or measurement of each sensor may further include measuring the uniformity or structure of the film in addition to the film thickness and / or the surface area or volume of the activity sensing area.

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

[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 that has the function of providing an interface to make 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 general access profile service, a general attribute service, a general 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, test substance measurement data (past and present), and event log data. Sensor status data may include error data, current active time, and software status. Test substance measurement data may include information such as current and past raw measurements, current and past values ​​after processing using an appropriate algorithm or model, predictions and trends of measurement levels, comparisons of other values ​​with patient-specific mean values, action requests determined by the algorithm or model, and other similar types of data.

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

[0147] Connections and subsequent communication sessions between two devices using a communication protocol such as BLE may be characterized by similar physical channels operating between the two devices (e.g., sensor 110 and data receiving device 120). The physical channels may include a single channel or a set of channels, and may include, for example, the use of a agreed-upon set of channels determined by a common clock and a channel or frequency hopping sequence, but are not limited to these. Communication sessions may utilize a similar amount of available communication spectrum, and numerous such communication sessions may exist in close proximity. In certain embodiments, each set of devices in a communication session may use different physical channels or sets of channels to manage interference from other nearby devices.

[0148] For illustrative purposes only and not limiting, exemplary embodiments of the process for connecting a sensor and a receiver for use in the disclosed subject matter are referred to. First, the sensor 110 repeatedly announces its connection information to its surroundings in search of the data receiver 120. The sensor 110 may repeatedly announce periodically until a connection is established. The data receiver 120 detects advertising packets and scans and filters them for the sensor 120 to connect via the data provided in the advertising packets. Next, the data receiver 120 sends a scan request command, and the sensor 110 responds with a scan response packet providing additional details. Next, the data receiver 120 sends a connection request using the Bluetooth® device address associated with the data receiver 120. The data receiver 120 may also successively request to establish a connection to the sensor 110 having a specific Bluetooth® device address. Next, the device establishes an initial connection and makes it possible to initiate data exchange. The device initiates a process to initialize the data exchange service and performs mutual authentication processing.

[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 store them for use during subsequent connections. The device then enables notification about customized security services used for mutual authentication between 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 requesting the data receiving device 120 to use connection parameter settings that are suitable for the sensor 110 and configured to maximize its lifespan.

[0150] Next, the data receiver 120 performs sensor control processing to backfill historical data, current data, event logs, and factory data. For example, for each type of data, the data receiver 120 sends a request to initiate the backfill process. The request may, if necessary, specify a range of records defined based on, for example, measured values, timestamps, etc. The sensor 110 responds with the requested data until all previously untransmitted data in the sensor 110's memory is delivered to the data receiver 120. The sensor 110 may respond to the backfill request from the data receiver 120 that all data has already been transmitted. Once backfilling is complete, the data receiver 120 may notify the sensor 110 that it is ready to receive periodic measured values. The sensor 110 may transmit measured values ​​across multiple notification results on a repeating basis. As embodied herein, multiple notifications may be redundant notifications to ensure that the data is transmitted correctly. Alternatively, multiple notifications may constitute a single payload.

[0151] For illustrative purposes only, not limiting, an exemplary embodiment of the process of sending a shutdown command to sensor 110 is described below. The shutdown operation is performed, 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 any of these states, sensor 110 may log a command and perform a 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. Furthermore, to indicate that sensor 110 has received the command, sensor 110 sends a success notification via sensor control characteristics. Sensor 110 registers the shutdown command. On the next appropriate occasion (for example, depending on the current sensor state as described herein), sensor 110 shuts down.

[0152] L. Exemplary sensor state and operation For illustrative purposes only, not limiting, we refer to an exemplary embodiment of a high-level depiction of the state machine indicator 6000 of actions that may be performed by the sensor 110 shown in Figure 15. After initialization, the sensor enters a state 6005 related to the manufacture of the sensor 110. In the manufacture state 6005, the sensor 110 can be configured for operation, and for example, the storage memory 5030 can be written to. At various times while in state 6005, the sensor 110 checks for received commands to proceed to the saved state 6015. Upon entering the saved state 6015, the sensor performs a software integrity check. While in the saved state 6015, the sensor may also receive an actuation request command before proceeding to the insertion detection state 6025.

[0153] Upon entering state 6025, the sensor 110 may store information about the device authenticated to communicate with the sensor during operation, or initialize an algorithm for performing and interpreting measurements from the sensing hardware 5060. The sensor 110 may also initialize a lifecycle timer involved in maintaining an active count of the sensor 110's operating time and initiate communication with the authenticated device to transmit recorded data. While in insertion detection state 6025, the sensor may enter state 6030, where the sensor 110 checks whether the operating time is equal to a predetermined threshold. This operating time threshold may correspond to a timeout function for determining whether the insertion was successful. If the operating time reaches the threshold, the sensor 110 proceeds to state 6035, where the sensor 110 checks whether the average data reading is greater than a threshold corresponding to the expected data reading to trigger the detection of successful insertion. If the data reading is lower than the threshold while in state 6035, the sensor proceeds to state 6040, which corresponds to insertion failure. If the data reading meets the threshold, the sensor proceeds to active pair state 6055.

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

[0155] While in the active expired state 6065, the sensor 110 may perform actions generally related to the termination of operation and to ensure that collected measurements have been safely transmitted to a receiving device as needed. For example, while in the active expired state 6065, the sensor 110 may transmit collected data and, if a connection is not feasible, may increase its attempts to find a nearby authenticated device and establish a connection with it. While in the active expired state 6065, the sensor 110 may receive a shutdown command in state 6070. If no shutdown command is received, the sensor 110 may also check in state 6075 whether the operating time has exceeded the final operating threshold. The final operating threshold may be based on the battery life of the sensor 110. The normal termination state 6080 corresponds to the final operation of the sensor 110 and ultimately shuts down the sensor 110.

[0156] Before the sensor is activated, the ASIC 5000 is in a low-power saving 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, thereby initiating the activation process when the sensor 110 enters the wake-up state. While in the wake-up state, the ASIC 5000 enters the activation sequence state. Next, the ASIC 5000 activates the communication module 5040. The communication module 5040 is initialized and triggers a power-on self-test. The power-on self-test may include the ASIC 5000 communicating with the communication module 5040 using a predetermined sequence of reading and writing data to verify that the memory and one-time programmable memory are not corrupted.

[0157] When the ASIC5000 first enters measurement mode, an insertion detection sequence is performed to verify that the sensor 110 is properly positioned on the patient's body before appropriate measurements can be taken. First, the sensor 110 interprets a command to activate the measurement setup process, causing the ASIC5000 to enter measurement command mode. Next, the sensor 110 temporarily enters a measurement lifecycle state and performs several consecutive measurements to check whether the insertion was successful. The communication module 5040 or the ASIC5000 evaluates the measurement results to determine whether the insertion was successful. If the insertion is deemed successful, the sensor 110 enters a measurement state and begins performing periodic measurements using the sensing hardware 5060. If the sensor 110 determines that the insertion was unsuccessful, the sensor 110 is triggered into insertion failure mode, and the ASIC5000 is instructed to return to save mode while the communication module 5040 disables itself.

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

[0159] As embodied herein, a remote application server 150, operated by the manufacturer of the substance sensor 110 and / or the operator of the substance monitoring system 100, may provide software and firmware updates to the apparatus of the substance monitoring system 100. In certain embodiments, the remote application server 150 may provide the updated software and firmware to the user apparatus 140 or directly to the multipurpose data receiver. 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 receiver 130 may periodically contact the application storefront server 160 to download and install the updates.

[0160] After the multipurpose data receiver 130 downloads an application update, including a firmware or software update, for the data receiver 120 or sensor 110, the data receiver 120 or sensor 110 and the multipurpose data receiver 130 establish a connection. The multipurpose data receiver 130 determines that the firmware or software update is available for the data receiver 120 or sensor 110. The multipurpose data receiver 130 may prepare the software or firmware update for distribution to the data receiver 120 or sensor 110. For example, the multipurpose data receiver 130 may compress or split the data associated with the software or firmware update, encrypt or decrypt the firmware or software update, or perform an integrity check on the firmware or software update. The multipurpose data receiver 130 transmits the data for the firmware or software update to the data receiver 120 or sensor 110. The multipurpose data receiver 130 may also send a command to the data receiver 120 or sensor 110 to initiate the update. In addition, or instead, the multipurpose data receiver 130 may provide notifications to its user, including commands to facilitate updates, such as a command to keep the data receiver 120 and the multipurpose data receiver 130 connected to a power source and in close proximity until the update is complete.

[0161] The data receiver 120 or sensor 110 receives data for the update and a command to start the update from the multipurpose data receiver 130. The data receiver 120 may then install the firmware or software update. To install the update, the data receiver 120 or sensor 110 may put itself into a so-called "safe" mode with limited operational functions, or it may restart. Once the update is complete, the data receiver 120 or sensor 110 returns to the standard operating mode or is reset. The data receiver 120 or sensor 110 may perform one or more self-tests to determine that the firmware or software update has been successfully installed. The multipurpose data receiver 130 may receive a notification of update success. The multipurpose data receiver 130 may then 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 memory that includes access restrictions and security to facilitate a predetermined number of writes to a specific address or segment within the memory. The memory 5030 may be pre-configured into a plurality of pre-allocated memory blocks or containers. The containers are pre-allocated to a fixed size. If the storage memory 5030 is one-time programmable memory, the containers may be considered non-programmable. Additional containers that have not yet been written to can be made programmable or writable. Containerizing the storage memory 5030 in this way can improve the transportability of code and data to be written to the storage memory 5030. Updating the software of a device (e.g., the sensor device described herein) stored in OTP memory may be performed by replacing only the code in one or more specific previously written containers with updated code written to one or more new containers, rather than replacing the entire code in the memory. In a second embodiment, the memory is not pre-configured. Instead, the space allocated for the data is dynamically allocated or determined as needed. Since you can define containers of various sizes that are expected to be updated, incremental updates can be issued.

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

[0164] In step 531, after receiving an OTA programming command, the microcontroller 5010 verifies the OTA programming command. The microcontroller 5010 may, for example, determine whether the OTA programming command is signed with an appropriate digital signature token. If it determines that the OTA programming command is valid, the microcontroller 5010 may set the sensor device to OTA programming mode. In step 532, the microcontroller 5010 may verify the OTA programming data. In step 533, the microcontroller 5010 may reset the sensor device 110 and reinitialize the sensor device 110 to the programming state. Once the sensor device 110 has entered the OTA programming state, the microcontroller 5010 may, in step 534, begin writing data to the rewritable memory 540 (e.g., memory 5020) of the sensor device, and in step 535, begin writing data to the OTP memory 550 (e.g., storage memory 5030) of the sensor device. The data written by the microcontroller 5010 is based on the verified OTA programming data. The microcontroller 5010 may write data to mark one or more programming blocks or areas of the OTP memory 550 as invalid or inaccessible. Data written to the free or unused portion of the OTP memory may be used to replace the invalidated or inaccessible programming blocks in the OTP memory 550. After the microcontroller 5010 has written data to the respective memories 534 and 535, the microcontroller 5010 may perform one or more software integrity checks to ensure that no errors were introduced into the programming blocks during the writing process. If the microcontroller 5010 determines that the data has been written without errors, the microcontroller 5010 may resume the standard operation of the sensor device.

[0165] In 536, in execution mode, the microcontroller 5010 can retrieve a programming manifest or profile from the rewritable memory 540. The programming manifest or profile may include a list of valid software programming blocks and may include guidance for program execution for the sensor 110. By following the programming manifest or profile, the microcontroller 5010 can determine which memory block in the OTP memory 550 is appropriate to execute and avoid executing expired or invalid programming blocks or referencing 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 memory or by using variables stored in memory.

[0166] N. Exemplary security and other architectural features As embodied herein, a first layer of security for communication between the substance sensor 110 and other devices is specified by the communication protocol used for communication and may be established based on a security protocol integrated into that communication protocol. Another layer of security may be based on a communication protocol that requires proximity of the communication devices. Furthermore, certain packets and / or certain data contained within packets may be encrypted, while other packets and / or data within packets may be encrypted in other ways or not encrypted at all. In addition or instead, application layer encryption may be used with one or more block ciphers or stream ciphers to establish mutual authentication and communication encryption with other devices in the substance monitoring system 100.

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

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

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

[0170] The test substance data associated with a user is, at least partially, confidential data because this information may be used for a variety of purposes, including health monitoring and drug administration decisions. In addition to user data, the test substance monitoring system 100 may implement enhanced security against reverse engineering by external parties. Communication connections may be encrypted using device-specific or session-specific encryption keys. Encrypted or unencrypted communications between any two devices may be verified using transmit integrity checks built into the communications. The operation of the test substance sensor 110 may be protected from tampering by restricting access to read and write functions to memory 5020 via the communication interface. The sensor may be configured to allow access only to known or "trusted" devices provided in a "whitelist," or only to devices that can provide a predetermined code associated with the manufacturer or a user authenticated by other means. The whitelist may represent an exclusive range meaning that no connection identifiers other than those included in the whitelist will be used, or a preferred range where the whitelist is searched first, but other devices may still be used. The sensor 110 may also reject a 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 characteristics protect against certain denial-of-service attacks, particularly those against the BLE interface.

[0171] As embodied herein, the substance monitoring system 100 may use periodic key rotation to further reduce the possibility of key leakage and misuse. The key rotation strategy employed by the substance monitoring system 100 may be designed to support backward compatibility of field-placed or distributed equipment. As an example, the substance monitoring system 100 may employ keys for downstream equipment (e.g., equipment located in the field or for which updates cannot be made viably available) that are designed to be compatible with multiple generations of keys used by upstream equipment.

[0172] For illustrative purposes only, not limiting, we refer to an exemplary embodiment of message sequence diagram 600 for use with the disclosed subject, shown in Figure 17, which illustrates an example of data exchange between a pair of devices, in particular sensor 110 and data receiver 120. The data receiver 120 may be data receiver 120 or multipurpose data receiver 130, as embodied herein. In step 605, the data receiver 120 may transmit a sensor activation command 605 to sensor 110, for example, via a short-range communication protocol. Sensor 110 may be primarily in a dormant state prior to step 605, conserving its battery until full operation is required. After activation, during step 610, sensor 110 may collect data or perform other operations as appropriate for the sensing hardware 5060 of sensor 110. In step 615, the data receiver 120 may initiate an authentication request command 615. In response to an authentication request command 615, both the sensor 110 and the data receiving device 120 may participate in a mutual authentication process 620. The mutual authentication process 620 may involve data transfer, including a challenge parameter that enables the sensor 110 and the data receiving device 120 to assure that the other device is fully compliant with the agreed security framework described herein. Mutual authentication may be based on a mechanism for authenticating two or more entities to each other, with or without an online trusted third party, in order to verify the establishment of a secret key via a challenge response. Mutual authentication may be performed using two-pass authentication, three-pass authentication, four-pass authentication, or five-pass authentication, or similar versions thereof.

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

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

[0175] O. Exemplary payload / communication frequency The substance sensor 110 may be configured to modify its discoverability behavior in an attempt to increase the probability that a receiving device will receive a suitable data packet and / or provide a response signal, or to otherwise reduce limitations that may prevent the receiving device from receiving a response signal. Modifying the discoverability behavior of the substance sensor 110 may include, but are 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 a broadcast for the substance sensor 110 to accept a response or scan signal, including direct transmissions to one or more devices that have previously communicated with the substance sensor 110 (e.g., via one or more attempted transmissions) and / or one or more devices on a whitelist, changing the transmit power associated with the communication module when broadcasting data packets (e.g., to increase the broadcast range or decrease the energy consumed and extend the battery life of the substance sensor), changing the rate at which data packets are prepared and broadcast, or one or more other combinations of modifications. In addition, or alternatively, the receiving device may similarly adjust parameters relating to the device's listening behavior to increase the likelihood of receiving data packets containing connection data.

[0176] As embodied herein, the substance sensor 110 may be configured to broadcast data packets using two types of windows. The first window represents the rate at which the substance sensor 110 is configured to operate the communication hardware. The second window represents the rate at which the substance sensor 110 is configured to actively transmit (e.g., broadcast) data packets. As an example, the first window may represent the substance sensor 110 operating the communication hardware to transmit and / or receive data packets (including connection data) during the first two seconds of each 60-second window. The second window may represent the substance sensor 110 transmitting data packets every 60 milliseconds during each two-second window. For the remainder of the two-second window, the substance sensor 110 is scanning. The substance sensor 110 may change its discoverability behavior by lengthening or shortening either window.

[0177] In certain embodiments, the discoverability behavior of a substance sensor can be stored in a discoverability profile and can be modified based on one or more factors, such as the state of the substance sensor 110, and / or by applying rules based on the state of the substance sensor 110. For example, when the battery level of the substance sensor 110 falls below a certain amount, a rule may cause the substance 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 substance sensor 110, or the temperature of specific components of the communication hardware of the substance sensor 110. In addition to changing the transmit power, other parameters related to the transmit capability or process of the communication hardware of the substance sensor 110 can be changed, including, but not limited to, transmit speed, frequency, and timing. As yet another example, when substance data indicates that a subject is experiencing or is experiencing a negative health event, a rule may cause the substance sensor 110 to increase its discoverability in order 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 substance sensor 110 may be adjusted based on external or interval environmental characteristics and to compensate for the decay of the detection hardware 5060 during periods of non-use (e.g., "storage time" before use). The calibration mechanism for the detection hardware 5060 may be adjusted autonomously by the sensor 110 (e.g., by the operation of the ASIC 5000 to change characteristics in memory 5020 or storage 5030) or by other devices of the substance monitoring system 100.

[0179] As an example, the sensor sensitivity of the detection hardware 5060 may be adjusted based on external temperature data or time since manufacture. When the external temperature is monitored during sensor storage, the disclosed subject may adaptively change the compensation for sensor sensitivity over time as the device experiences changes in storage conditions. For illustrative purposes only, not limiting, adaptive sensitivity adjustment may be performed in an "active" storage mode in which the substance sensor 110 is periodically activated to measure temperature. These mechanisms may conserve battery power in the substance device and extend the life of the substance sensor. In each temperature measurement, the substance sensor 110 may calculate the sensitivity adjustment for that period based on the measured temperature. The temperature-weighted adjustment values ​​may then be accumulated over the active storage mode period to calculate a total sensor sensitivity adjustment value at the end of the active storage mode (e.g., at insertion). Similarly, upon insertion, the sensor 110 may determine the time difference between the manufacturing of the sensor 110 (which can be written to the storage 5030 of the ASIC 5000) or the sensing hardware 5060, and modify the sensor sensitivity or other calibration mechanism according to one or more known attenuation rates or formulas.

[0180] In addition, for illustrative purposes rather than limiting purposes, as embodied herein, sensor sensitivity adjustments may take into account other sensor conditions, such as sensor drift. Sensor sensitivity adjustments may be hardcoded within the sensor 110 during manufacturing, for example, based on an estimate of how much the average sensor drifts, in the case of sensor drift. The sensor 110 may use a calibration function having time-varying functions for sensor offset and gain, which may account for drift over the sensor's wear period. Thus, the sensor 110 may utilize a device-dependent function that describes the drift of the sensor 110 over time, utilizing a function used to convert interstitial current to interstitial glucose, which may indicate sensor sensitivity and may be device-specific in combination with a baseline of glucose profiles. Such functions for taking into account sensor sensitivity and drift may improve the accuracy of the sensor 110 over its wear period without user calibration.

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

[0182] The model may be provided to the sensor 110, data receiver 120, or multipurpose data receiver 130 during manufacturing or firmware or software updates. The model may be periodically improved by, for example, the manufacturer of the sensor 110 or the operator of the substance monitoring system 100, based on data received from the sensor 110 and data receiver for individual users or multiple users collectively. In certain embodiments, the sensor 110 includes sufficient computational components to support 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 may include, but is not limited to, models trained using or incorporating decision tree analysis, gradient boosting, ADA boosting, artificial neural networks or their variations, linear discriminant analysis, nearest neighbor analysis, support vector machines, supervised or unsupervised classification, etc. The model may also include algorithm or rule-based models in addition to machine learning models. Model-based processing may be performed by other devices, including the data receiver 120 or multipurpose data receiver 130, upon receiving data from the sensor 110 (or other downstream devices).

[0183] R. Exemplary alarm mechanism The data transmitted between the sensor 110 and the data receiving device 120 may include raw or processed measurements. The data transmitted between the sensor 110 and the data receiving device 120 may further include alarms or notifications for display to the user. The data receiving device 120 may display or otherwise communicate notifications to the user based on the raw or processed measurements, or may display alarms when received from the sensor 110. Alarms that may be triggered for display to the user include alarms based on direct test substance values ​​(e.g., a single reading exceeding or falling below a threshold), test substance value trends (e.g., average readings, gradients, over a set period exceeding or falling below a threshold), test substance value predictions (e.g., algorithmic calculations based on test substance values ​​exceeding or falling below a threshold), sensor alerts (e.g., a detected suspicious malfunction), communication alerts (e.g., no communication between sensor 110 and data receiver 120 over a threshold period, an unknown device attempting to initiate or failing to initiate a communication session with sensor 110), reminders (e.g., a reminder to charge data receiver 120, a reminder to take medication, or perform other activities), and other alerts of a similar nature. For illustrative purposes only, not limiting, the alarm parameters described herein, as embodied herein, may be configurable by the user, fixed during manufacturing, or a combination of user-configurable and non-user-configurable parameters.

[0184] S. Exemplary configuration Sensor configurations featuring a single active region configured for the detection of a single corresponding test substance may use a two-electrode or three-electrode detection motif, as further described herein with reference to Figures 18A to 18C. Sensor configurations featuring two different active regions for the detection of a different test substance, either on a different working electrode or on the same working electrode, are described separately later with reference to Figures 19A to 21C. Sensor configurations with multiple working electrodes may be particularly advantageous for incorporating two different active regions within the same sensor tail, as the signal contribution from each active region can be more easily determined.

[0185] When a single working electrode is present within the sensor for the substance being tested, a three-electrode sensor configuration may include a working electrode, a counter electrode, and a reference electrode. A related two-electrode sensor configuration includes a working electrode and a second electrode, in which the second electrode may function as both a counter electrode and a reference electrode (i.e., a counter / reference electrode). The various electrodes may be at least partially stacked (layered) with respect to each other, and / or laterally spaced apart on the sensor tail. A suitable sensor configuration may be substantially flat or substantially cylindrical in shape. In any of the sensor configurations disclosed herein, the various electrodes may be electrically insulated from each other by a dielectric material or similar insulator.

[0186] A sensor for a substance being tested, characterized by multiple working electrodes, may similarly include at least one additional electrode. When one additional electrode is present, it may function as a pair / reference electrode for each of the multiple working electrodes. When two additional electrodes are present, one of the additional electrodes may function as a pair electrode for each of the multiple working electrodes, and the other additional electrode may function as a reference electrode for each of the multiple working electrodes.

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

[0188] Referring further to Figure 18A, the membrane 220 covers at least the active region 218. In certain embodiments, the membrane 220 may cover part or all of the working electrode 214 and / or the pair / reference electrode 30216, or the entire substance sensor 200. One or both sides of the substance sensor 200 may be covered by the membrane 220. The membrane 220 may comprise one or more polymer membrane materials having the ability to restrict the flux of the substance to the active region 218 (i.e., the membrane 220 is a mass transfer limiting membrane with some permeability to the substance of interest (e.g., ketones)). As further described below according to the disclosure herein, the membrane 220 may be crosslinked by a branching crosslinking agent in certain sensor configurations. For example, but not limited to, the membrane 220 may be crosslinked by a branching glycidyl ether. The composition and thickness of the membrane 220 may vary to promote the flux of the desired substance (e.g., ketones) to the active region 218, thereby providing the desired signal intensity and stability. The test substance sensor 200 may be capable of assaying the test substance by any of the following electrochemical detection techniques: coulometry, amperometry, voltammetry, or potentiometry.

[0189] In certain embodiments, one or more films, including interference domains and mass transfer limiting films, are deposited on an exposed electroactive surface of a working electrode, such as a platinum surface. For example, but not limited to, interference domains may be located on the working electrode, an active region on top of the interference domains, and a mass transfer limiting film on top of the active region.

[0190] Figures 18B and 18C show schematic diagrams of exemplary three-electrode substance sensor configurations, which are also suitable for use in the disclosure herein. The three-electrode substance sensor configurations may be similar to those shown as substance sensor 200 in Figure 18A, except that they include an additional electrode 217 within substance sensors 201 and 202 (Figures 18B and 18C). With the additional electrode 217, the pair / reference electrode 30216 may then function as either the pair electrode or the reference electrode, and the additional electrode 217 may perform other electrode functions not otherwise described. The working electrode 214 continues to perform its original function. The additional electrode 217 may be placed on either the working electrode 214 or electrode 30216 with a dielectric material isolation layer in between. For example, but not limited to, as shown in Figure 18B, dielectric layers 219a, 219b and 219c isolate 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 electrodes 214 (working electrode) and 30216 (counter electrode) may be positioned on the opposite side of the substrate 30212, and the electrode 217 (reference electrode) may be positioned on one of the electrodes 214 or 30216 and separated therefrom by a dielectric material. The 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 region 218 (for the detection of ketones) in the test substance sensors 201 and 202 may consist of multiple spots or a single spot. In addition, the test substance sensors 201 and 202 may be capable of assaying the test substance by any of the following electrochemical detection techniques: coulometry, amperometry, voltammetry, or potentiometry.

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

[0192] Figure 19A shows an exemplary configuration of a sensor 203 having a single working electrode on which two different active regions are positioned. Figure 19A is similar to Figure 18A except that there are two active regions on the working electrode 214, namely a first active region 218a and a second active region 218b, which respond to different test substances and are spaced laterally apart from each other on the surface of the working electrode 214. The active regions 218a and 218b may include multiple spots or a single spot configured for the detection of each test substance. The composition of the film 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 test substances at different working electrode potentials, as will be discussed further below. In certain embodiments, either 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 test substance, such as lactate, glucose, creatinine, and / or oxygen. In certain embodiments, the second test substance is glucose.

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

[0194] Exemplary sensor configurations having multiple working electrodes, specifically two working electrodes, are described in further detail with reference to Figures 4 to 21C. The following description primarily concerns sensor configurations having two working electrodes, but it should be understood that more than two working electrodes may be incorporated through the extension of the disclosure herein. Additional working electrodes may be used to give the test substance sensor further detection capability for detecting, for example, a third and / or fourth test substance, in addition to the first and second test substances.

[0195] Figure 4 shows a cross-sectional view of an exemplary substance sensor configuration having two working electrodes, a reference electrode, and a counter electrode, suitable for use in the disclosure herein. As shown, the substance sensor 300 includes working electrodes 304 and 306 located on opposite sides of the substrate 302. A first active region 310a is located on the surface of the working electrode 304, and a second active region 310b is located on the surface of the working electrode 306. The counter electrode 320 is electrically insulated from the working electrode 304 by a dielectric layer 322, and the reference electrode 321 is electrically insulated from the working electrode 306 by a dielectric layer 323. Outer dielectric layers 330 and 332 are located on the reference electrode 321 and the counter electrode 320, respectively. The film 340 may, according to various embodiments, cover at least the active regions 310a and 310b, and other components of the substance sensor 300 or the entire substance sensor 300 may also be optionally covered by the first film portion 340a and / or the second film portion 340b. Furthermore, the film 340 may be continuous but may be compositionally varied within the first film portion 340a and the second film portion 340b (i.e., over the active regions 310a and 310b) to allow different permeability values ​​for independently adjusting the substance flux at each location. For example, different film formulations may be sprayed and / or printed on the opposite side of the substance sensor 300. Dip coating techniques may also be suitable, particularly for depositing at least a portion of the two-layer film on one of the active regions 310a and 310b. Accordingly, according to certain embodiments of this 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 test substance sensors 200, 201, and 202, the test substance sensor 300 may be operable to assay ketones (and / or a second test substance) by any of the electrochemical detection techniques by coulometry, amperometry, voltammetry, or potentiometry. In certain embodiments, the test substance sensor may comprise more than one membrane 340, for example, two or more membranes.For example, but not limited to, the test substance sensor may include one or more active regions, e.g., 310a and a membrane covering 310a, and a further membrane covering the entire sensor as shown in Figure 20. In certain embodiments, either one or both of the active regions 310a and 310b 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 310a and 310b 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 test substance. In certain embodiments, the second test substance is glucose.

[0196] Alternative sensor configurations having multiple working electrodes, different from the configuration shown in Figure 20, may feature different pair / reference electrodes instead of another pair electrode and reference electrode 320, 321, and / or may feature different layer and / or film arrangements than those explicitly shown. For example, but not limited to, the positions of pair electrode 320 and reference electrode 321 may be opposite to those shown in Figure 20. Furthermore, working electrodes 304 and 306 do not necessarily have to be located on the opposite side of the substrate 302 as shown in Figure 20.

[0197] A preferred sensor configuration may feature electrodes that are substantially planar in nature, but a sensor configuration featuring non-planar electrodes may also be advantageous, and may be particularly suitable for use in the disclosure herein. In particular, substantially cylindrical electrodes arranged concentrically with respect to each other can facilitate the deposition of a mass transfer limiting film, as described below. In particular, concentric working electrodes spaced apart along the length of the sensor tail can facilitate film deposition by a continuous dip coating operation in the same manner as described above for a substantially planar sensor configuration. Figures 21A-21C show perspective views of a substance sensor featuring two working electrodes arranged concentrically with respect to each other. It should be noted that a sensor configuration having a concentric electrode arrangement but without a second working electrode is also possible in this disclosure.

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

[0199] Referring further to Figure 21A, a first active region 414a and a second active region 414b, each involved with a different or the same test substance, are positioned on the exposed surfaces of the working electrodes 410 and 420, respectively, thereby allowing contact with the fluid to occur for detection. In certain embodiments, either one or both of the active regions 414a and 414b 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 414a and 414b 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 test substance. In certain embodiments, the second test substance is glucose. While the active regions 414a and 414b are shown as three separate spots in Figure 21A, it should be understood that alternative sensor configurations may have fewer or more spots comprising a continuous layer of active regions.

[0200] In Figure 21A, the sensor 400 is partially covered by a film 450 over the working electrodes 410 and 420 and the active regions 414a and 414b located thereon. Figure 21B shows an alternative sensor configuration in which substantially the entire sensor 401 is covered by the film 450. The film 450 may be the same or compositionally different in the active regions 414a and 414b. For example, the film 450 may include a two-layer film covering the active region 414a, or a uniform film covering the active region 414b.

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

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

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

[0204] In certain embodiments, additional electrodes may be included in the sensor tail. For example, but not limited to, a three-electrode system (working electrode, reference electrode, and counter electrode) and / or an additional working electrode (e.g., an electrode for detecting a second test substance). In certain embodiments where the sensor has two working electrodes, the two working electrodes may be placed side by side, with a reference electrode positioned around them (e.g., spirally wound 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 toward the distal end (i.e., the in vivo end) of the sensor tail. In certain embodiments, the reference electrode extends (e.g., spirally) into the exposed area of ​​the working electrodes.

[0205] In certain embodiments, one or more working electrodes are helically wound around a reference electrode. In certain embodiments where two or more working electrodes are provided, the working electrodes may be formed in a double, triple, quadruple, or other helical configuration along the length of the sensor tail (e.g., surrounding the reference electrode, an insulated rod, or other support structure). In certain embodiments, the electrodes, for example, two or more working electrodes, are formed coaxially. For example, but not limited to, all electrodes share the same central axis.

[0206] In certain embodiments, the working electrode comprises a tube with an insulator between it and a reference electrode arranged or wound therein. Alternatively, the reference electrode comprises a tube with an insulator between it and a working electrode arranged or wound therein. In certain embodiments, a polymer (e.g., insulating) rod is provided, on which one or more electrodes (e.g., one or more electrode layers) are arranged (e.g., by electroplating). In certain embodiments, a metal (e.g., steel or tantalum) rod or wire is provided, coated with an insulating material (as described herein), on which one or more working electrodes and a reference electrode are arranged. For example, but not limited to, the Disclosure provides a sensor comprising one or more tantalum wires, e.g., a sensor tail, on which platinum is arranged on a portion of one or more tantalum wires to function as a working electrode. In certain embodiments, a platinum-coated tantalum wire is covered with an insulating material, and the insulating material 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 placed on the working electrode (e.g., on the platinum surface of the electrode), a portion of the insulator can be peeled off or otherwise removed to expose the electroactive surface of the working electrode. For example, but not limited to, a portion of the insulator may be removed by hand, excimer laser, chemical etching, laser ablation, grit blasting, or other means. Alternatively, a portion of the electrode may be masked before the insulator is deposited in order to maintain the exposed electroactive surface area. In certain embodiments, the peeled and / or removed portion of the insulator may be from about 0.1 mm (about 0.004 inches) or less to about 2 mm (about 0.078 inches) or more in length, for example, 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 includes parylene, fluorinated polymers, polyethylene terephthalate, polyvinylpyrrolidone, polyurethane, polyimide, and other non-conductive polymers. In certain embodiments, glass or ceramic material may 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] The active region and some components of the membrane of the substance sensor described herein are further described below. 2. Enzymes The active regions of the test substance sensors of this disclosure may be configured to detect one or more test substances. In certain embodiments, the test substance sensors of this disclosure may include two or more active regions, each configured to detect the same or different test substances. Non-limiting examples of test substances that can be detected using the disclosed test substance sensors include ketones, glucose, oxygen, creatinine, alcohols such as ethanol, and lactates. In certain embodiments, the test substances are one or more ketones.

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

[0210] In certain embodiments, the test substance sensor of this disclosure includes at least one active region configured to detect ketones. Specific enzyme systems that may be used to detect ketones are shown in Figure 22. In the enzymatic reaction shown, β-hydroxybutyrate acts as a substitute for ketones formed in vivo. As shown in Figure 22, a pair of concerted enzymes may be used to detect ketones by the disclosure herein. For example, but not limited to, a pair of concerted enzymes may include a dehydrogenase and an oxidase. In certain embodiments, the dehydrogenase is β-hydroxybutyrate dehydrogenase. In certain embodiments, the oxidase is NADH oxidase. Enzyme cofactors NAD+ and NADH, disclosed herein, help facilitate the concerted enzymatic reaction shown in Figure 22. When the ketone-responsive active domain contains this pair of concerted enzymes, β-hydroxybutyrate dehydrogenase (HBDH) reacts with β-hydroxybutyrate and oxidized nicotinamide adenine dinucleotide (NAD +These can be converted to acetacetate 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+. Then, hydrogen peroxide can be catalytically oxidized at the anode, i.e., the working electrode, according to the following formula.

[0211] H2O2 → 2H + +O2+2e - [1] The electrons transferred during this reaction provide a basis for detecting ketones at the working electrode, eliminating the need for redox mediators. The resulting electrochemical signal can then 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 may comprise platinum, a platinum alloy, carbon, or a combination thereof. In certain embodiments, the working electrode is a platinum electrode. In certain embodiments, the working electrode may comprise a platinum-carbon mixture.

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

[0214] In certain embodiments, the substance 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, wherein the ketone-responsive active region comprises an enzyme system comprising a dehydrogenase and an oxidase. In certain embodiments, the enzyme system comprises 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 may include ratios of β-hydroxybutyrate dehydrogenase to NADH oxidase of about 40:1 to about 1:40, for example, about 35:1 to about 1:35, about 30:1 to about 1:30, about 25:1 to about 1:25, about 20:1 to about 1:20, about 15:1 to about 1:15, about 10:1 to about 1:10, about 9:1 to about 1:9, about 8:1 to about 1:8, about 7:1 to about 1:7, about 6:1 to about 1:6, about 5:1 to about 1:5, about 4:1 to about 1:4, about 3:1 to about 1:3, about 2:1 to about 1:2, about 2:1 or about 1:1. In certain embodiments, the ketone-responsive active region may include a ratio of β-hydroxybutyrate dehydrogenase to NADH oxidase of about 5:1 to about 1:5. In certain embodiments, the ketone-responsive active region may include a ratio of β-hydroxybutyrate dehydrogenase to NADH oxidase of about 3:1 to about 1:3. In certain embodiments, the ketone-responsive active region may include a ratio of β-hydroxybutyrate dehydrogenase to NADH oxidase of about 2:1 to about 1:2. In certain embodiments, the ketone-responsive active region may include a ratio of β-hydroxybutyrate dehydrogenase to NADH oxidase of about 2:1.

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

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

[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 may be albumin, such as serum albumin. Non-limited examples of serum albumin include bovine serum albumin and human serum albumin. In certain embodiments, the stabilizer is human serum albumin. In certain embodiments, the stabilizer is bovine serum albumin. In certain embodiments, the stabilizer may be catalase. In certain embodiments, the ketone-responsive active region may include ratios of stabilizer to enzymes in an enzyme system, such as NADH oxidase and β-hydroxybutyrate dehydrogenase, of about 40:1 to about 1:40, for example, 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 approximately 2:1 to approximately 1:2 of stabilizer to enzymes in an enzyme system, such as NADH oxidase and β-hydroxybutyrate dehydrogenase. In certain embodiments, the ketone-responsive active region may include stabilizer-to-NADH oxidase ratios of about 40:1 to about 1:40, for example, 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 stabilizer-to-NADH oxidase ratios of about 2:1 to about 1:2.In certain embodiments, the ketone-responsive active region may include a stabilizer-to-β-hydroxybutyrate dehydrogenase ratio of about 40:1 to about 1:40, for example, 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 stabilizer-to-β-hydroxybutyrate dehydrogenase ratio of about 2:1 to about 1:2. In certain embodiments, the ketone-responsive active region may contain about 10% to about 50% by weight of stabilizers, for example, about 15% to about 45%, about 20% to about 40%, about 20% to about 35%, or about 20% to about 30%.

[0219] In certain embodiments, the ketone-responsive active region may further include cofactors (or derivatives 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 include a cofactor-to-NADH oxidase ratio of about 40:1 to about 1:40, for example, 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 cofactor-to-NADH oxidase ratio of about 2:1 to about 1:2. In certain embodiments, the ketone-responsive active region may include a ratio of cofactor to β-hydroxybutyrate dehydrogenase of about 40:1 to about 1:40, for example, 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 cofactor to β-hydroxybutyrate dehydrogenase of about 2:1 to about 1:2. In certain embodiments, the ketone-responsive active region may contain cofactors of about 10% to about 50% by weight, for example, about 15% to about 45%, about 20% to about 40%, about 20% to about 35%, and about 20% to about 30% by weight. In certain embodiments, the ketone-responsive active region may contain cofactors of about 15% to about 35% by weight. In certain embodiments, cofactors, such as NADH, may be physically retained within the ketone-responsive active region. For example, but not limited to, a membrane covering the ketone-responsive active region can help retain cofactors within the ketone-responsive active region while allowing sufficient inward diffusion of ketones to enable detection of ketones.

[0220] In certain embodiments, the ketone-responsive active region is disposed on a portion of the working electrode. For example, without limitation, 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 substance sensor of the present disclosure may include one or more enzymes for detecting glucose. For example, but not limited to, the substance sensor of the present disclosure may include an active region (e.g., a second active region) comprising one or more enzymes for detecting glucose, for example, located on a second working electrode. In certain embodiments, the substance sensor may include an active site comprising glucose oxidase and / or glucose dehydrogenase for detecting glucose. In certain embodiments, glucose can be detected using glucose oxidase present in the second active region, which produces H2O2 as a product. H2O2 reacts with an electrochemically reactive surface of the second working electrode, for example, 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 test substance sensor of this disclosure may include an active region (e.g., a second active region) comprising one or more enzymes (e.g., an enzyme system) for detecting lactate, for example, located on a second working electrode. In certain embodiments, the test substance sensor may include an active site comprising lactate dehydrogenase and / or lactate oxidase.

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

[0225] In certain embodiments, a second enzyme-responsive active region located, for example, on a second working electrode of the test substance sensor of the present disclosure may include one or more enzymes for detecting creatinine. For example, but not limited to, the test substance sensor of the present disclosure may include an active region (e.g., a second active region) located, for example, on a second working electrode, comprising one or more enzymes (e.g., an enzyme system) for detecting creatinine. In certain embodiments, the test substance sensor may include an active site comprising an amide hydrolase, a creatine amidinohydrolase, and / or a sarcosine oxidase.

[0226] In certain embodiments, the test substance sensor may include two working electrodes, for example, a first active region located on a first working electrode and a second active region located on a second working electrode. In certain embodiments, the first and second active regions are configured to detect different test substances. In certain embodiments, the first active region is configured to detect ketones. In certain embodiments, the second active region is configured to detect test substances other than ketones, for example, glucose, creatinine, lactate, and / or alcohol. For example, but not limited to, test substance sensors disclosed herein may feature a ketone-responsive active region on the surface of a first working electrode and a second active region configured to detect different test substances on the surface of a different working electrode, for example, a second working electrode, such as a glucose-responsive active region. In certain embodiments, such a test substance 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 disposed on the surface of the second working electrode, comprising a glucose-responsive enzyme. For example, but not limited to, when the sensor is configured to detect two or more test substances, the detection of each test substance may include applying a potential to each working electrode individually so that a separate signal is obtained from each test substance. The signals obtained from each test substance can then be correlated to the test substance concentration by the use of a calibration curve or function, or by employing a lookup table. In certain embodiments, the correlation between the test substance signal and the test substance concentration may be performed by the use of a processor.

[0227] In certain other test substance sensor configurations, a first active region and a second active region may be located on a single working electrode. The first signal can be obtained from the first active region, for example, at a low potential, and the second signal, which includes signal contributions from both active regions, can be obtained at a high potential. Next, by subtracting the first signal from the second signal, it is possible to determine the signal contribution from the second test substance. Then, the signal contribution from each test substance can be correlated with the test substance concentration in a manner similar to that described for sensor configurations with multiple working electrodes. In certain embodiments, when a ketone-responsive active region and a second active region configured to detect different test substances, such as a glucose-responsive active region, are located on a single working electrode in this manner, one of the active regions may be configured to respond separately to facilitate the detection of each test substance. For example, the ketone-responsive active region or the glucose-responsive active region may generate a signal independently of the other active region.

[0228] It should also be understood that the sensitivity (output current) of a substance sensor directed at each substance can be varied by changing the coverage (area or size) of the active region, the area ratio of the active regions to each other, their identity, and the thickness and / or composition of the mass transfer limiting film covering the active region. If the benefits of the disclosure herein are recognized, these parameter changes can be readily implemented by those skilled in the art.

[0229] 3. Redox mediators In certain embodiments, the test substance sensors disclosed herein may include electron transfer agents. As discussed above, depending on the composition of the ketone-responsive active region in any of the exemplary sensor configurations disclosed herein, such as the enzyme composition and the working electrode composition, electron transfer agents are not included in the ketone-responsive active region. For example, a ketone-responsive active region comprising an enzyme system including β-hydroxybutyrate dehydrogenase and NADH oxidase does not include electron transfer agents, such as redox mediators, such as osmium redox mediators.

[0230] In certain embodiments, an active region configured to detect another substance present in the substance sensor of the present disclosure, such as glucose, may include an electron transfer agent. For example, but not limited to, the substance 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 is made of 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 platinum working electrode without the need for an electron transfer agent to produce a detectable current that correlates, for example, with the concentration of ketones in the sample. Alternatively or further, in certain embodiments, the second working electrode is made of platinum, and the glucose-responsive active region disposed on the second working electrode does not include an electron transfer agent. In certain embodiments, a glucose-responsive active region generates hydrogen peroxide in the presence of glucose, which is directly oxidized on the surface of a platinum-based working electrode without the need for an electron transfer agent, thereby producing a detectable current that correlates, for example, with the concentration of glucose in the sample.

[0231] A suitable electron transfer agent can facilitate the transfer of electrons from the test substance to an adjacent working electrode after it has undergone an enzymatic redox reaction within its corresponding active region, thereby generating a current that indicates the presence of a specific test substance. The amount of current generated is proportional to the amount of test substance present.

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

[0233] In certain embodiments, the electron transfer agents disclosed herein may comprise functional groups suitable for promoting covalent bonding to a polymer (hereinafter referred to as the polymer backbone) within the active region, as will be further discussed below. For example, but not limited to, electron transfer agents for use in this disclosure may include electron transfer agents bonded to a polymer. Non-limiting examples of suitable polymer-bonded electron transfer agents include those described in U.S. Patents 8,444,834, 8,268,143, and 6,605,201, the disclosures of which are incorporated herein by reference in their entirety. In certain embodiments, the electron transfer agent is a bidentate osmium complex bonded to a polymer described herein, for example, a polymer backbone described in Section 4 below. In certain embodiments, the polymer-bonded electron transfer agent shown in Figure 3 of U.S. Patent 8,444,834 may be used in the sensor of this disclosure.

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

[0235] In certain embodiments, the polymer is polyvinylpyridine or a copolymer thereof. 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 furthermore, 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 may be immobilized within the active region by the use of a crosslinking agent, as described herein, in the presence of a stabilizer. For example, but not limited to, one or more enzymes in the ketone-responsive active region of this disclosure, e.g., NADH oxidase and / or β-hydroxybutyrate dehydrogenase, may be immobilized within the active region by using a crosslinking agent, e.g., polyethylene glycol diglycidyl ether. In certain embodiments, one or more enzymes in the ketone-responsive active region, e.g., NADH oxidase and / or β-hydroxybutyrate dehydrogenase, may be covalently bonded to a stabilizer within the active region by using a crosslinking agent. In certain embodiments, the stabilizer is serum albumin, e.g., bovine serum albumin (BSA).

[0237] In certain embodiments, covalent bonding of one or more enzymes and / or redox mediators to a polymer and / or stabilizer in a given active region may occur via crosslinking introduced by a suitable crosslinking agent. Suitable crosslinking agents may 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, but are not limited to, crosslinking agents for use in the present disclosure may include mono-, di-, tri-, and tetra-ethylene oxides. In certain embodiments, the crosslinking agent for the reaction with free amino groups in the enzyme (e.g., with free side-chain amines in lysine) may include, for example, polyethylene glycol dibutyl ethers, polypropylene glycol dimethyl ethers, polyalkylene glycol allyl methyl ethers, polyethylene glycol diglycidyl ether (PEGDGE), or other polyepoxides, cyanuryl chloride, N-hydroxysuccinimide, imide esters, epichlorohydrin, or derivative variants thereof. In certain embodiments, the crosslinking agent may have an average molecular weight (M) of, for example, about 200 to 1000, for example, about 400. n PEGDGE has ) . In certain embodiments, the crosslinking agent is PEGDGE400. In certain embodiments, the crosslinking agent may be glutaraldehyde. Suitable crosslinking agents for reaction with free carboxylic acid groups in the enzyme may include, for example, carbodiimides. In certain embodiments, the crosslinking of the enzyme to the polymer or stabilizer is generally intermolecular.

[0238] In certain embodiments, the ketone-responsive active region can include a ratio of a crosslinker to one or both enzymes of an 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 can include a ratio of a crosslinker to one or both enzymes of an 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 can include a ratio of a crosslinker to one or both enzymes of an 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 can include a ratio of a crosslinker to one or both enzymes of an 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 can include from about 5% to about 50% by weight of a crosslinker. In certain embodiments, the ketone-responsive active region can include from about 5% to about 20% by weight, such as from about 10% to about 15% by weight, of a crosslinker.

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

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

[0241] In certain embodiments, the mass transfer restriction membrane may be homogeneous and single-component (including a single-membrane polymer). Alternatively, the mass transfer restriction membrane may be multi-component (including two or more different membrane polymers). In certain embodiments, the mass transfer restriction membrane may comprise two or more layers, e.g., two or three layers. In certain embodiments, each layer may comprise different polymers or the same polymer at different concentrations or thicknesses. In certain embodiments, the ketone-responsive active region may be covered with a multilayer membrane, e.g., a two-layer membrane, and the second test substance-responsive active region may be covered with a single membrane. In certain embodiments, the ketone-responsive active region may be covered with a multilayer membrane, e.g., a two-layer membrane, and the second test substance-responsive active region may be covered with a multilayer membrane, e.g., a two-layer membrane.

[0242] In certain embodiments, the mass transfer limiting membrane may comprise a crosslinked polymer containing heterocyclic nitrogen groups. In certain embodiments, the mass transfer limiting membrane may comprise a polyvinylpyridine polymer. Non-limiting examples of polyvinylpyridine polymers are disclosed in U.S. Patent Publication 2003 / 0042137 (e.g., formula 2b), the contents of which are incorporated herein by reference in whole. In certain embodiments, the mass transfer limiting membrane may include polyvinylpyridine (e.g., poly(4-vinylpyridine) or poly(4-vinylpyridine), polyvinylimidazole, polyvinylpyridine copolymer (e.g., copolymer of vinylpyridine and styrene), polyacrylate, polyurethane, polyether urethane, silicone, polytetrafluoroethylene, polyethylene-co-tetrafluoroethylene, polyolefin, polyester, polycarbonate, biostable polytetrafluoroethylene, homopolymers, copolymers, or terpolymers of polyurethane, polypropylene, polyvinyl chloride, polyvinylidene difluoride, polybutylene terephthalate, polymethyl methacrylate, polyether ether ketone, cellulose polymer, polysulfone, and their block copolymers, including, for example, diblock, triblock, alternating, random, and graft copolymers, or chemically related materials and others.

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

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

[0245] In certain embodiments, the membrane includes 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, polyurethane is a polymer produced by a condensation reaction between a diisocyanate and a bifunctional hydroxyl-containing material. In certain embodiments, polyurethane urea is a polymer produced by a condensation reaction between a diisocyanate and a bifunctional amine-containing material. In certain embodiments, diisocyanates for use herein include, for example, aliphatic diisocyanates containing about 4 to about 8 methylene units, or diisocyanates containing alicyclic moieties. Further non-limiting examples of polymers that may be used for the formation of sensor membranes of this disclosure include vinyl polymers, polyethers, polyesters, polyamides, inorganic polymers (e.g., polysiloxanes and polycarbosiloxanes), natural polymers (e.g., cellulosic and protein-based materials), and mixtures (e.g., mixed or layered structures), or combinations thereof. In certain embodiments, the hydrophilic polymer component is polyethylene oxide and / or polyethylene glycol. For example, but not limited to, the hydrophobic-hydrophilic copolymer component for use in this disclosure is a polyurethane polymer comprising about 10% to about 50%, for example, about 20%, of hydrophilic polyethylene oxide.

[0246] In certain embodiments, the film comprises a silicone polymer / hydrophobic-hydrophilic polymer blend. In certain embodiments, the hydrophobic-hydrophilic polymer for use in the blend may be any suitable hydrophobic-hydrophilic polymer, but is not limited to polyvinylpyrrolidone, polyhydroxyethyl methacrylate, polyvinyl alcohol, polyacrylic acid, polyethylene glycol, or polyethers such as polyethers such as polyethers such as polyvinylpyrrolidone, polyhydroxyethyl methacrylate, polyvinyl alcohol, polyacrylic acid, or polypropylene oxide, and copolymers thereof, including, for example, 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 PEO and PPO copolymers include PEO-PPO diblock copolymer, PPO-PEO-PPO triblock copolymer, PEO-PPO-PEO triblock copolymer, alternating block copolymer of PEO-PPO, random copolymer of ethylene oxide and propylene oxide, and blends thereof. In certain embodiments, the copolymer may be substituted with a hydroxyl substituent.

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

[0248] In certain embodiments where multiple active regions exist, the mass transfer limiting film can coat each active region, including the selection of changes in composition in different active regions, which can be achieved through a continuous dip coating operation that generates a two-layer film portion at the working electrode, which is positioned more closely at the sensor tip.

[0249] In certain embodiments where multiple active regions exist, separate mass transfer limiting membranes may cover each active region. For example, but not limited to, a mass transfer limiting membrane may be positioned over a first active region, e.g., a ketone-responsive active region, and a separate second mass transfer limiting membrane may cover a second active region, e.g., 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 comprises a different crosslinking agent.

[0250] In certain embodiments, the composition of the mass transfer limiting membranes placed on a test substance sensor having two active regions may be the same or different depending on whether the mass transfer limiting membranes cover each active region. For example, but not limited to, 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 the second test substance-responsive region, such as the 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 the second test substance-responsive region, such as the 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 the second test substance-responsive region, such as the glucose-responsive active region, may be single-component. In certain embodiments, the mass transfer limiting membrane covering the ketone-responsive active region comprises a different polymer from the mass transfer limiting membrane covering the second test substance-responsive region, for example, the glucose-responsive active region.

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

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

[0253] Suitable copolymers of vinylpyridine and styrene (polyvinylpyridine-co-styrene copolymers) may have styrene content ranging from about 0.01% to about 50% mole percent, or about 0.05% to about 45% mole percent, or about 0.1% to about 40% mole percent, or about 0.5% to about 35% mole percent, or about 1% to about 30% mole percent, or about 2% to about 25% mole percent, or about 5% to about 20% mole percent. Substituted styrenes may be used in similar amounts. Suitable copolymers of vinylpyridine and styrene may have molecular weights of 5 kDa or more, or about 10 kDa or more, or about 15 kDa or more, or about 20 kDa or more, or about 25 kDa or more, or about 30 kDa or more, or about 40 kDa or more, or about 50 kDa or more, or about 75 kDa or more, or about 90 kDa or more, or about 100 kDa or more. In non-limiting examples, suitable copolymers of vinylpyridine and styrene may have molecular weights 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, a membrane polymer covering one or more active regions may be crosslinked using crosslinking agents disclosed herein and in Section 4 above. In certain embodiments where two mass transfer limiting membranes exist, e.g., a first mass transfer limiting membrane and a second mass transfer limiting membrane, each membrane may be crosslinked with a different crosslinking agent. For example, but not limited to, a crosslinking agent may result in a membrane that either more restricts the diffusion of a particular compound, e.g., a test substance, through the membrane, or does not restrict the diffusion of a particular compound, e.g., by affecting the size of the pores in the membrane. For example, but not limited to, in a sensor configured to detect ketones and glucose, a mass transfer limiting membrane covering a ketone-responsive region may have a pore size that restricts the diffusion of larger compounds, e.g., glucose, through the membrane.

[0255] In certain embodiments, the crosslinking agents for use in this disclosure may include polyepoxides, carbodiimides, cyanuryl chlorides, triglycidylglycerols, N-hydroxysuccinimides, imide esters, epichlorohydrins, or derivative variants thereof. In certain embodiments, a membrane polymer covering one or more active regions may be crosslinked to a branched crosslinking agent, for example, which can reduce the amount of extract obtained from the mass transfer limiting membrane. Non-limiting examples of branched crosslinking agents include branched glycidyl ether crosslinking agents, for example, branched glycidyl ether crosslinking agents containing two, three, or more crosslinkable groups. In certain embodiments, the branched crosslinking agent may contain two or more crosslinkable groups, such as polyethylene glycol diglycidyl ether. In certain embodiments, the branched crosslinking agent may contain three or more crosslinkable groups, such as polyethylene glycol tetraglycidyl ether. In certain embodiments, the mass transfer limiting membrane may include polyvinylpyridine or a copolymer of vinylpyridine and styrene crosslinked to a branched glycidyl ether crosslinking agent containing two or three crosslinkable groups, such as polyethylene glycol tetraglycidyl ether or polyethylene glycol diglycidyl ether. In certain embodiments, epoxide groups such as polyepoxides, for example polyethylene glycol tetraglycidyl ether or polyethylene glycol diglycidyl ether, can form covalent bonds with pyridine or imidazole by ring-opening of the epoxide ring, resulting in a hydroxyalkyl group that crosslinks the crosslinking agent body with the heterocycle of the film polymer.

[0256] In certain embodiments, the crosslinking agent is polyethylene glycol diglycidyl ether (PEGDGE). In certain embodiments, PEGDGE used to facilitate crosslinking (e.g., intermolecular crosslinking) between two or more membrane polymer backbones may exhibit a wide range of suitable molecular weights. In certain embodiments, the molecular weight of PEGDGE may 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 can generally vary over a range within a given sample from which the average molecular weight is obtained. In certain embodiments, PEGDGE for use in this disclosure has an average molecular weight of about 200 to 1000, for example, about 400 (M n ) has. In a particular embodiment, the crosslinking agent is PEGDGE400.

[0257] In certain embodiments, 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 may 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 from which the average molecular weight is obtained.

[0258] In certain embodiments, polydimethylsiloxane (PDMS) may be incorporated into any of the mass transfer limiting membranes disclosed herein. In certain embodiments, the analyte sensor described herein may comprise a sensor tail portion 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 comprises at least one enzyme (optionally covalently bound to a first polymer and / or stabilizer) and an enzyme system that responds to the first analyte, e.g., an enzyme system that responds to ketones. For example, without limitation, the analyte sensor described herein may comprise a sensor tail portion having at least a first working platinum electrode, a ketone-responsive active region comprising an enzyme system having β-hydroxybutyrate dehydrogenase and NADH oxidase disposed on the surface of the first working electrode (where one or both 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 comprises 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 substance sensor of the present disclosure includes a sensor tail comprising 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, the first active region is located on the surface of the first working electrode and the second active region is located on the surface of the second working electrode, and the first and second active regions respond to different substances. For example, but not limited to, 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 membrane covers the first and second active regions, and the mass transfer limiting membrane comprises a bilayer portion covering the first active region and a uniform portion covering the second active region. In certain embodiments, each layer of the bilayer portion comprises a different membrane polymer. In certain embodiments, the bottom layer of the bilayer portion above the first active region comprises a different membrane polymer, such as a polyvinylpyridine polymer, than the uniform portion above the second active region.

[0260] In certain embodiments, when a first active region and a second active region configured to assay different test substances are placed on separate working electrodes, the mass transfer limiting membrane may have different permeability values ​​for the first and second test substances. For example, but not limited to, a mass transfer limiting membrane covering at least one of the active regions may include a mixture of the first and second membrane polymers, or two layers of the first and second membrane polymers. A uniform membrane may cover the active region not covered by the mixture or the two layers, and the uniform membrane may include only one of the first or second membrane polymers. Advantageously, the test substance sensor architecture disclosed herein allows a continuous membrane having a uniform membrane portion to be placed on the first active region of the test substance sensor, and a multi-component membrane portion to be placed on the second active region of the test substance sensor, thereby simultaneously leveling the permeability values ​​for each test substance and improving sensitivity and detection accuracy. In certain embodiments, continuous film deposition can be achieved by a continuous dip coating operation.

[0261] 6. Interference Domain In certain embodiments, the sensor of this disclosure, for example, the sensor tail, may further comprise interference domains. In certain embodiments, the interference domains may include polymer domains that restrict the flow of one or more interfering substances to the surface of the working electrode, for example. In certain embodiments, the interference domains may function as molecular sieves that allow the substance being measured by the working electrode and other substances to pass through, while preventing the passage of other substances, such as interfering substances. In certain embodiments, the interfering substances may affect the signal obtained at the working electrode. Non-limiting examples of interfering substances include acetaminophen, ascorbates, ascorbic acid, bilirubin, cholesterol, creatinine, dopamine, ephedrine, ibuprofen, L-dopa, methyldopa, salicylates, tetracycline, trazamide, tolbutamide, triglycerides, urea, and uric acid.

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

[0263] In certain embodiments, the interference domain includes a thin, non-swelling, hydrophobic membrane that restricts the diffusion of high molecular weight species. For example, but not limited to, the interference domain may be 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 domains may be deposited directly onto the working electrode, for example, on the platinum surface of the working electrode. In certain embodiments, the interference domains may have a thickness in the range of about 0.1 μm to about 1000 μm, for example, 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 domains may have a thickness of about 0.1 μm to about 10 μm, for example, about 0.5 μm to about 10 μm, about 1 μm to about 10 μm, about 1 μm to about 5 μm, or about 0.1 μm to about 5 μm. In certain embodiments, the sensor may be immersed in the interference domain solution two or more times. For example, but not limited to, the sensor (or working electrode) of the Disclosure may be immersed in the interference domain solution at least two, at least three, at least four, or at least five times to obtain a desired interference domain thickness.

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

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

[0267] In certain embodiments, the method may further include adding a membrane composition on top of an enzyme composition, for example, a cured enzyme composition. In certain embodiments, the membrane composition may include a polymer, for example, polyvinylpyridine and / or a crosslinking agent, for example, polyethylene glycol diglycidyl ether. In certain embodiments, the membrane is applied to 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 substance sensor may further include a second working electrode, and a method for manufacturing such a sensor includes, for example, depositing a second enzyme composition for detecting a second substance onto 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 film composition on top of the second enzyme composition.

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

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

[0271] Generally, the film thickness 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 sprayed onto the sensor, the volume of film solution sprayed onto the sensor, and any combination of these factors. In certain embodiments, the films described herein may have thicknesses ranging from about 0.1 micrometers (μm) to about 1000 μm, for example, 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 may be immersed in the film solution two or more times. For example, but not limited to, the sensor (or working electrode) of this disclosure may be immersed in the film solution at least two, at least three, at least four, 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 may have a thickness of about 0.1 μm to about 10 μm, for example, 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, in order to achieve the desired thickness of the active region and / or film, a series of droplets may be applied on top of each other without substantially increasing the diameter of the droplets to be applied (i.e., while maintaining the desired diameter or range). In certain embodiments, each single droplet may be applied, then allowed to cool or dry, and then one or more further droplets may be applied. For example, but not limited to, in order to achieve the desired thickness of the active region, at least one droplet, at least two droplets, at least three droplets, at least four droplets, or at least five droplets may be added on top of each other.

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

[0274] In certain embodiments, the Disclosure provides a method for detecting ketone levels in a subject that requires it. In certain embodiments, the Disclosure provides a method for detecting in vivo ketone levels in a subject. In certain embodiments, the Disclosure provides a method for detecting ketone levels in interstitial fluid in a subject. In certain embodiments, the Disclosure provides a method for detecting ketone levels in a subject with diabetes. In certain embodiments, the Disclosure provides a method for detecting ketone levels in a subject receiving a ketogenic diet. In certain embodiments, the Disclosure provides a method for detecting ketone levels in a subject in a state of ketosis or in a subject maintaining a state of ketosis. In certain embodiments, the test substance sensor of the Disclosure may be used to ensure that a subject adheres to a ketogenic diet. For example, but not limited to, the test substance sensor of the Disclosure may be used to measure the level of ketones in a sample to tell a subject to adjust or modify their diet to maintain ketosis. In certain embodiments, the Disclosure provides a method for detecting ketone levels in a subject at risk of developing ketoacidosis. In certain embodiments, the Disclosure provides a method for detecting ketone levels in subjects at risk of developing diabetic ketoacidosis. In certain embodiments, the sensors of the Disclosure may be used to monitor and / or prevent diabetic ketoacidosis. For example, but not limited to, the sensors of the Disclosure include a detection chemical for detecting ketones and glucose to monitor and / or prevent diabetic ketoacidosis in a subject, e.g., a subject with diabetes. Alternatively or further, the sensors of the Disclosure may be used in combination with a glucose sensor to monitor and / or prevent diabetic ketoacidosis. In certain embodiments, the sensors of the Disclosure may be used for applications to monitor ketone levels in a subject, e.g., to monitor adherence to a ketogenic diet, to maintain a ketotic state, and / or to monitor and / or prevent diabetic ketoacidosis.

[0275] In a particular embodiment, a method for detecting ketones includes (i) a sensor comprising (a) a sensor tail including at least a first working electrode, (b) a ketone-responsive active region disposed on the surface of the first working electrode and responding to ketones, for example at a low potential, the ketone-responsive active region comprising an enzyme system comprising ketone-responsive β-hydroxybutyrate dehydrogenase and NADH oxidase 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, 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 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 a particular embodiment, 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 approximately +0.2V to approximately +0.5V relative to an Ag / AgCl reference. In certain embodiments, the potential applied to the first working electrode is approximately +0.3V to approximately +0.4V relative to an Ag / AgCl reference, for example, approximately +0.35V relative to an Ag / AgCl reference.

[0276] In certain embodiments, the method of the present disclosure may include (i) exposing a test substance sensor comprising (a) a sensor tail including at least a first working electrode, (b) a ketone-responsive active region disposed on the surface of the first working electrode and responding to ketones, for example at a low potential, the ketone-responsive active region comprising an enzyme system comprising ketone-responsive β-hydroxybutyrate dehydrogenase and NADH oxidase and optionally a first polymer, and (c) a mass transfer limiting membrane covering the ketone-responsive active region and permeable to ketones, to a fluid containing 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 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 approximately +0.2V to approximately +0.5V relative to an Ag / AgCl reference. In certain embodiments, the potential applied to the first working electrode is approximately +0.3V to approximately +0.4V relative to an Ag / AgCl reference, for example, approximately +0.35V relative to an Ag / AgCl reference.

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

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

[0281] A3. A sensor for testing a substance, as described in any one of A to A2, wherein the working electrode is made of platinum. A4. A sensor for a test substance as described in any one of A to A2, wherein the ketone-responsive active region is further equipped with a stabilizer.

[0282] A5. A test substance sensor as described in A4, in which serum albumin is used as the stabilizer. A6. A sensor for a test substance according to any one of A to A5, wherein the ketone-responsive active region further comprises a crosslinking agent.

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

[0284] A9. A sensor for a substance to be tested 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] A10. A sensor for a substance to be tested according to A8 or A9, wherein the substance transfer limiting membrane comprises polyvinylpyridine. A11. A test substance sensor according to any one of A to A10, wherein the sensor tail is configured for insertion into tissue.

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

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

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

[0289] A15. A test substance sensor as described in any one of A to A14, wherein the ketone-responsive active region responds to ketones at a potential of approximately +0.2V to approximately +0.5V relative to an Ag / AgCl reference.

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

[0291] A17.(iv) A second working electrode, and (v) a second active region disposed on the surface of the second working electrode and responding to a second test substance different from ketones, further comprising a second active region comprising at least one enzyme that responds to the second test substance, according to any one of A to A16.

[0292] A18. The substance sensor according to A17, wherein a second portion of the mass transfer limiting membrane covers a second active region. A19. The sensor for a substance to be tested according to A17, wherein a second mass transfer limiting membrane covers a second active region.

[0293] A20. The sensor for a substance to be tested according to A17, wherein a second mass transfer limiting membrane covers a second active region and a first active region. A21. A test substance sensor according to any one of A17 to A20, wherein the second test substance comprises glucose, lactate, creatinine, or alcohol.

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

[0295] A24. A test substance sensor described in any one of A to A23, to be implanted in a person with diabetes. A25. A test substance sensor described in any one of A-A24, to be implanted in subjects who are experiencing or at risk of developing ketoacidosis.

[0296] A26. A test substance sensor described in one of A-A24, to be implanted in subjects consuming a ketogenic diet. A27. A sensor for the substance to be tested, as described in any one of A to A24, which is implanted in a subject that is in a state of ketosis or needs to maintain a state of ketosis.

[0297] A28. A sensor for a test substance as described in any one of A to A27, wherein hydrogen peroxide produced by the reaction of an enzyme system with a ketone in a ketone-responsive active region is detected at the working electrode.

[0298] A29. A sensor for a test substance according to any one of A to A28, wherein the ketone-responsive active region further comprises a polymer. A30. A sensor for testing a substance 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 a sensor for a test substance 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 which is above the redox potential of the ketone-responsive active region and is 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.

[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 is made of 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 described in 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 polyvinylpyridine, polyvinylimidazole, a copolymer of vinylpyridine and styrene, or a combination thereof.

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

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

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

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

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

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

[0310] B19. The method according to any one of B to B18, wherein the test substance sensor further comprises (d) a second working electrode and (e) a second active region disposed on the surface of the second working electrode that responds to a second test substance different from ketones.

[0311] B20. The method according to B19, wherein a second portion of the mass transfer limiting membrane covers a second active region. B21. The method according to B19, wherein a second mass transfer limiting membrane covers a second active region.

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

[0313] B24. The method according to any one of B to B23, wherein the fluid is interstitial fluid from the subject. B25. The method according to any one of B to B24, wherein a test substance sensor is implanted in a subject with diabetes.

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

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

[0316] C. A test substance sensor according to any one of A to A30 for use in the detection of ketones in subjects requiring a test substance sensor. C1. A test substance sensor for use in individuals with diabetes.

[0317] C2. A test substance sensor for use in subjects who are experiencing or at risk of developing ketoacidosis. C3. A test substance sensor for use in subjects consuming a ketogenic diet.

[0318] C4. A test substance sensor for use in cases where the subject is in a state of ketosis or needs to maintain a state of ketosis. Examples The subject matter of this disclosure is provided as an example of the subject matter disclosed herein and is not limited thereto, but will be better understood by referring to the following examples.

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

[0320] The oxidation characteristics of hydrogen peroxide and NADH in phosphate-buffered saline (PBS) were determined using a platinum (Pt) electrode. The solution was maintained at a temperature of 33°C. Linear scan voltammetry was performed using a CH Instruments CHI1030B potentiostat, and the results relative to an Ag / AgCl reference electrode were recorded. The results are shown in Figure 23. As shown in Figure 23, the oxidation potential of NADH is approximately +0.6V relative to the Ag / AgCl reference. Furthermore, Figure 23 shows that the oxidation of hydrogen peroxide becomes flat at a potential of +0.35V relative to the 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 embodiment provides a sensor for detecting β-hydroxybutyrate, which is used as a substitute for ketones in vivo. In this embodiment, the enzyme system shown in Figure 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 formulations shown in Table 1 were deposited onto the Pt electrode. A control sensor was also prepared using the same detection chemical formulation as in Table 1, except that NADHOx was not included in the formulation. The sensor was cured overnight. Following the curing process, the sensor was immersed in a mixture of polyvinylpyridine (PVP) and polyethylene glycol diglycidyl ether 400 (PEGDGE400). Next, the sensor was cured again overnight. Subsequently, a beaker test was performed in 100 mM PBS buffer at 33°C. The sensor current was recorded at +0.35V relative to an Ag / AgCl reference electrode using a CH Instruments CHI1030B potentiostat.

[0323] Figure 24 shows the current response for four NADHOx sensors and a control. As shown, the current increased over several minutes after exposure to a new concentration of β-hydroxybutyrate and then stabilized. It is also shown that this effect was not observed in the control sensor, indicating that hydrogen peroxide generation proceeds via the proposed detection mechanism. Figure 25 provides exemplary plots of the current response and β-hydroxybutyrate concentration for each of the NADHOx sensors and the control.

[0324] While the subject matter and its advantages disclosed herein have been described in detail, it should be understood that various modifications, substitutions, and variations can be implemented herein without departing from the technical idea and scope of the disclosed subject matter. Furthermore, the scope of this application is not intended to be limited to specific embodiments of the processes, machines, manufactures, and compositions of materials, methods, and processes described herein. As will be readily apparent to those skilled in the art from the disclosed subject matter, existing or future-developed processes, machines, manufactures, compositions of materials, methods, or processes that perform substantially the same function or achieve substantially the same results as the corresponding embodiments described herein can be utilized in accordance with the disclosed subject matter. Accordingly, the attached claims are intended to include within their scope such processes, machines, manufactures, compositions of materials, methods, or processes.

[0325] Various patents, patent applications, publications, product descriptions, protocols, and sequence accession numbers are cited in this application, and those inventions are incorporated herein by reference in their entirety for all purposes. The technical concepts included in this disclosure are described below. [Note 1] A test substance sensor for detecting ketones in vivo, (i) At least a first working electrode comprising a conductive material that oxidizes hydrogen peroxide, (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 generating hydrogen peroxide in the presence of a ketone, the enzyme system comprising β-hydroxybutyrate dehydrogenase and NADH oxidase, and the ketone-responsive active region not comprising superoxide dismutase, (iii) comprising a mass transfer limiting membrane that covers at least a portion of the ketone-responsive active region and is permeable to ketones, The ketone-responsive active region responds to ketones at a potential of +0.2V to +0.5V relative to an Ag / AgCl reference. The distal portion of the aforementioned test substance sensor is configured to be inserted into the target skin to detect ketones in vivo. [Note 2] The sensor for the substance to be tested, as described in Appendix 1, wherein the ketone-responsive active region does not contain an electron transfer agent. [Note 3] The sensor for a test substance according to Appendix 1 or 2, wherein the ketone-responsive active region further comprises a stabilizer for stabilizing the enzyme system. [Note 4] The substance sensor according to any one of the appendices 1 to 3, wherein the substance transfer limiting membrane comprises polyvinylpyridine, polyvinylimidazole, polyvinylpyridine copolymer, polyacrylate, polyurethane, polyetherurethane, or a combination thereof. [Note 5] The substance sensor for testing according to Appendix 4, wherein the substance transfer limiting membrane comprises polyvinylpyridine. [Note 6] (iv) The second working electrode and (v) A test substance sensor according to any one of appendices 1 to 5, further comprising a second active region disposed on the surface of the second working electrode and responding to a second test substance different from ketones, the second active region comprising at least one enzyme that responds to the second test substance. [Note 7] The sensor for testing a substance according to Appendix 6, further comprising a second mass transfer limiting membrane covering the second active region. [Note 8] The test substance sensor according to appendix 6 or 7, wherein the second test substance comprises glucose. [Note 9] A method for controlling the operation of a test substance sensor as described in Appendix 1, wherein the test substance sensor is introduced into tissue, and the control method is (i) Applying a potential to the first working electrode, (ii) Obtain a first signal that is above the redox potential of the ketone-responsive active region and is proportional to the concentration of ketones in the fluid in contact with the ketone-responsive active region, (iii) A control method comprising correlating the first signal with the concentration of ketones in the fluid. [Note 10] The control method according to Appendix 9, wherein the ketone-responsive active region does not contain an electron transfer agent. [Note 11] The control method according to appendix 9 or 10, wherein the conductive material comprises platinum. [Note 12] The control method according to any one of appendices 9 to 11, wherein the ketone-responsive active region further comprises a stabilizer for stabilizing the enzyme system. [Note 13] The control method according to any one of appendices 9 to 12, wherein the mass transfer limiting membrane comprises polyvinylpyridine, polyvinylimidazole, polyvinylpyridine copolymer, polyacrylate, polyurethane, polyetherurethane, or a combination thereof. [Note 14] The control method according to Appendix 13, wherein the mass transfer limiting membrane comprises polyvinylpyridine. [Note 15] The aforementioned sensor for the substance to be tested, The second working electrode, A second active region is located on the surface of the second working electrode and responds to a second test substance different from ketones, The control method according to any one of appendices 9 to 14, further comprising a second mass transfer limiting film covering the second active region. [Note 16] The test substance sensor as described in Appendix 7, wherein the second mass transfer limiting membrane also covers the ketone-responsive active region. [Note 17] The conductive material comprises platinum, as described in any one of appendices 1-8 and 16, for the substance sensor to be tested.

Claims

1. A test substance sensor for detecting ketones in vivo, (i) A first working electrode comprising a conductive material that oxidizes hydrogen peroxide to generate a signal correlated with 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 generating hydrogen peroxide in the presence of a ketone, the enzyme system comprising β-hydroxybutyrate dehydrogenase, oxidized nicotinamide adenine dinucleotide (NAD+), reduced nicotinamide adenine dinucleotide (NADH), and NADH oxidase, (iii) A mass transfer limiting membrane suitable for enabling inward diffusion of ketones and retaining NAD+ and NADH, comprising the mass transfer limiting membrane disposed on the ketone-responsive active region, The ketone-responsive active region responds to ketones at a potential of +0.2V to +0.5V relative to an Ag / AgCl reference. The distal portion of the aforementioned test substance sensor is configured to be inserted into the target skin to detect ketones in vivo.

2. The sensor for testing a substance according to claim 1, wherein the ketone-responsive active region does not contain an electron transfer agent.

3. The ketone-responsive active region does not contain superoxide dismutase, as described in claim 1 or 2.

4. The sensor for testing a substance according to any one of claims 1 to 3, wherein the ketone-responsive active region further comprises a stabilizer for stabilizing the enzyme system.

5. The substance sensor according to any one of claims 1 to 4, wherein the substance transfer limiting membrane comprises polyvinylpyridine, polyvinylimidazole, polyvinylpyridine copolymer, polyacrylate, polyurethane, polyetherurethane, or a combination thereof.

6. The substance sensor according to claim 5, wherein the substance transfer limiting membrane comprises polyvinylpyridine.

7. The sensor for testing a substance according to 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. The sensor for testing a substance according to any one of claims 1 to 7, wherein the conductive material comprises platinum.

9. The sensor for testing a substance according to any one of claims 1 to 8, wherein the ketone-responsive active region further comprises a first polymer, the first polymer comprising polyvinylpyridine, polyvinylimidazole, or a copolymer thereof.

10. The sensor for testing a substance according to claim 9, wherein one or both of the β-hydroxybutyrate dehydrogenase and the NADH oxidase are covalently bonded to the first polymer.

11. (iv) The second working electrode and (v) The test substance sensor according to any one of claims 1 to 10, further comprising: a second active region disposed on the surface of the second working electrode and responding to a second test substance different from ketones, the second active region comprising at least one enzyme that responds to the second test substance.

12. The sensor for a substance to be tested according to claim 11, further comprising a second mass transfer limiting film covering the second active region.

13. The test substance sensor according to claim 11 or 12, wherein the second test substance is glucose.

14. The sensor for testing a substance according to claim 13, wherein the at least one enzyme in the second active region is glucose oxidase or glucose dehydrogenase.

15. The sensor for a substance to be tested according to any one of claims 11 to 14, wherein the second active region comprises an electron transfer agent.

16. (i) Sensor electronic equipment and (ii) A sensor control device comprising: a test substance sensor according to any one of claims 1 to 15, the test substance sensor which obtains a signal correlated with the concentration of ketones and transmits the signal to the sensor electronic device.

17. The sensor control device according to claim 16, wherein the sensor electronic device is configured to correlate the signal with the concentration of ketones.

18. (i) A sensor for the substance to be tested according to any one of claims 1 to 15, (ii) A ketone detection system comprising (a) a sensor electronic device configured to correlate a signal obtained by the test substance sensor with the concentration of ketones, and (b) a reader device to transmit and display the concentration of ketones.

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 is (i) Applying a potential to the first working electrode, (ii) Obtain a signal proportional to the concentration of ketones in the fluid at an oxidation-reduction potential above the ketone-responsive activity region, (iii) A control method comprising correlating the signal with the concentration of ketones in the fluid.

20. The control method according to 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 is (i) Applying a potential to the first working electrode, (ii) Applying a potential to the second working electrode, (iii) Obtain a signal proportional to the concentration of ketones in the fluid at an oxidation-reduction potential above the ketone-responsive activity region, (iv) Obtain a second signal that is proportional to the concentration of the second test substance in the fluid, at an oxidation-reduction potential above the second active region. (v) Correlating the signal with the concentration of ketones in the fluid, (vi) A control method comprising correlating the second signal with the concentration of a second test substance in the fluid.

22. The control method according to claim 21, wherein the fluid is interstitial fluid.

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