Analyte sensor for detecting asparagine and aspartate and method of use thereof

Abstract

The present disclosure provides an analyte sensor for use in detecting aspartate and / or asparagine. In certain embodiments, the aspartate-responsive active site of the disclosed analyte sensor comprises aspartate oxidase disposed on the surface of the working electrode. In certain embodiments, the asparagine-responsive active site of the disclosed analyte sensor comprises an enzyme system comprising aspartate oxidase and asparaginase disposed on the surface of the working electrode. The present disclosure further provides methods for detecting aspartate and / or asparagine using the disclosed analyte sensors.

Description

[Technical field]

[0001] The subject matter described herein relates to analyte sensors for detecting asparagine and / or aspartate, and methods of use thereof. [Background technology]

[0002] The detection of various analytes in an individual may be crucial for monitoring the health of an individual, since deviations from normal analyte levels may indicate physiological conditions. For example, monitoring glucose levels may allow people suffering from diabetes to take appropriate corrective measures, including administering medicines or taking certain food or beverage products, to avoid serious physiological problems. It may also be desirable to monitor other analytes, such as aspartate and asparagine. In certain instances, it may be desirable to monitor two or more analytes to monitor one or more physiological conditions, especially when a person suffers from a co-morbid condition in which two or more analytes are combined with each other to cause dysregulation at the same time.

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

[0004] Many analytes are interesting targets for physiological analysis if suitable detection chemistries can be identified. To this end, enzyme-based amperometric sensors configured to continuously assay glucose in vivo have been developed and improved in recent years to aid in monitoring the health of diabetic individuals. Other analytes that commonly undergo dysregulation that coincides with glucose in diabetic individuals include, for example, aspartate and asparagine. Similarly, it may be desirable to monitor aspartate and asparagine independently of glucose dysregulation. However, implanted analyte sensors configured to detect aspartate or asparagine in vivo are not currently available. Thus, there is a need in the art for sensors to detect asparagine or aspartate in vivo. Summary of the Invention

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

[0006] To achieve these and other advantages, and in accordance with the objects of the disclosed subject matter, as embodied and broadly described, the disclosed subject matter includes an analyte sensor for detecting aspartate and / or asparagine. In certain embodiments, the analyte sensor includes a sensor tail having at least a first working electrode, an analyte-responsive active region comprising aspartate oxidase disposed on a surface of the first working electrode for detecting the analyte, and a mass transport limiting membrane covering at least the analyte-responsive active region and permeable to the analyte.

[0007] In certain embodiments, the analyte is aspartate. In certain embodiments, the analyte responsive activity further comprises asparaginase, and the analyte is asparagine. In certain embodiments, the analyte responsive active area comprises a layer comprising asparaginase disposed on a layer comprising aspartate oxidase. In certain embodiments, the analyte responsive active area comprises a single layer comprising aspartate oxidase and asparaginase. In certain embodiments, one or more of the aspartate oxidase and asparaginase are covalently attached to a polymer within the analyte responsive active area.

[0008] In certain embodiments, the analyte responsive activity further comprises an electron transfer agent. In certain embodiments, the mass transport limiting membrane comprises a polyvinylpyridine-based polymer, polyvinylimidazole, polyacrylate, polyurethane, polyetherurethane, silicone, or a combination thereof. In certain embodiments, the mass transport limiting membrane comprises polyvinylpyridine or polyvinylimidazole. In certain embodiments, the mass transport limiting membrane comprises a polyvinylpyridine-based polymer. In certain embodiments, the mass transport limiting membrane comprises a copolymer of vinylpyridine and styrene.

[0009] The present disclosure further provides a method for detecting aspartate. In certain embodiments, the method may include providing an analyte sensor including a sensor tail comprising at least a first working electrode, at least one aspartate-responsive active region comprising aspartate oxidase disposed on the surface of the first working electrode, and a mass transport limiting membrane covering at least the aspartate-responsive active region and permeable to aspartate. In certain embodiments, the method further includes applying a potential to the first working electrode, obtaining a first signal at or above the redox potential of the aspartate-responsive active region and proportional to the concentration of aspartate in a fluid contacting the aspartate-responsive active region, and correlating the first signal to the concentration of aspartate in the fluid.

[0010] The present disclosure further provides a method for detecting asparagine. In certain embodiments, the method can include providing an analyte sensor including a sensor tail comprising at least a first working electrode, an asparagine-responsive active region comprising an enzyme system comprising aspartate oxidase and asparaginase disposed on a surface of the first working electrode, and a mass transport limiting membrane covering at least the aspartate-responsive active region and permeable to aspartate. In certain embodiments, the method further includes applying a potential to the first working electrode, obtaining a first signal at or above the redox potential of the asparagine-responsive active region and proportional to the concentration of asparagine in a fluid contacting the asparagine-responsive active region, and correlating the first signal to the concentration of asparagine in the fluid.

[0011] In certain embodiments, the aspartate-responsive active region and / or the asparagine-responsive active region further comprises an electron transfer agent. In certain embodiments, the aspartate oxidase is covalently bound to a polymer in the asparagine-responsive active region. In certain embodiments, the asparaginase is covalently bound to a polymer in the asparagine-responsive active region. In certain embodiments, the asparagine-responsive active region comprises a layer comprising asparaginase disposed on a layer comprising aspartate oxidase. In certain embodiments, the asparagine-responsive active region comprises a single layer comprising aspartate oxidase and asparaginase. [Brief description of the drawings]

[0012] The following figures are included to illustrate certain aspects of the present disclosure and should not be considered as exclusive embodiments. The disclosed subject matter is capable of numerous modifications, variations, combinations, and equivalents in form and function without departing from the scope of the present disclosure. [Figure 1A] 1 is a system overview of a sensor applicator, a reader device, a monitoring system, a network, and a remote system. [Figure 1B]1 is a schematic diagram illustrating the operating environment of an example analyte monitoring system for use with the technology described herein. [Figure 2A] 1 is a schematic block diagram illustrating an example embodiment of a reader device. [Figure 2B] 1 is a block diagram illustrating an example of a data receiving device for communicating with a sensor according to an exemplary embodiment of the disclosed subject matter. [Figure 2C] 1 is a schematic block diagram illustrating an example embodiment of a sensor control device. [Figure 2D] 1 is a schematic block diagram illustrating an example embodiment of a sensor control device. [Figure 2E] 1 is a block diagram illustrating an example of an analyte sensor according to an exemplary embodiment of the disclosed subject matter. [Figure 3A] 13A-13C are proximal perspective views illustrating an example embodiment of a user preparing a tray for assembly. [Figure 3B] 11A-11C are side views illustrating an example embodiment of a user preparing an applicator device for assembly. [Figure 3C] 13A-13C are proximal perspective views illustrating an example embodiment of a user inserting an applicator device into a tray during assembly. [Figure 3D] 11A-11C are proximal perspective views illustrating an example embodiment of a user removing the applicator device from the tray during assembly. [Figure 3E] 1A-1C are proximal perspective views illustrating an example embodiment of a patient applying a sensor with an applicator device. [Figure 3F] 1A and 1B are proximal perspective views showing an example embodiment of a patient with an applied sensor and a used applicator device. [Figure 4A] 1A-1C are side views of an example embodiment of an applicator device coupled with a cap. [Figure 4B] 1 is a side perspective view of an example embodiment of an applicator device and cap separated. [Figure 4C] 1 is a perspective view illustrating an example embodiment of an applicator device and a distal end of an electronics housing. [Figure 4D]FIG. 1 is a top perspective view of an exemplary applicator device in accordance with the disclosed subject matter. [Figure 4E] FIG. 4E is a bottom perspective view of the applicator device of FIG. 4D. [Figure 4F] FIG. 4E is an exploded view of the applicator device of FIG. 4D. [Figure 4G] 4E is a side cross-sectional view of the applicator device of FIG. 4D. [Diagram 5] FIG. 13 is a proximal perspective view showing an example embodiment of a tray with an associated sterilization lid. [Figure 6A] 1A-1C are proximal perspective cross-sectional views illustrating an example embodiment of a tray having a sensor delivery component. [Figure 6B] FIG. 13 is a proximal perspective view showing the sensor delivery component. [Figure 7A] FIG. 2 illustrates an isometric exploded top view of an exemplary sensor control device. [Figure 7B] FIG. 2 illustrates an isometric exploded bottom view of an exemplary sensor control device. [Figure 8A] 1A-1C are assembly and cross-sectional views of an on-body device including an integrated connector for sensor assembly. [Figure 8B] 1A-1C are assembly and cross-sectional views of an on-body device including an integrated connector for sensor assembly. [Figure 8C] 1A-1C are assembly and cross-sectional views of an on-body device including an integrated connector for sensor assembly. [Figure 9A] 2D is a side view of an example embodiment of the sensor applicator of FIG. 1A coupled with the cap of FIG. 2C. [Figure 9B] 2D is a side cross-sectional view of an example embodiment of the sensor applicator of FIG. 1A coupled with the cap of FIG. 2C. [Figure 10A] FIG. 2 is an isometric view of another example sensor control device. [Figure 10B] FIG. 13 is a side view of another example sensor control device. [Figure 11A] 10A-10B. FIG. 11 is a side cross-sectional view showing a staged assembly of a sensor applicator having the sensor control device of FIGS. [Figure 11B]10A-10B. FIG. 11 is a side cross-sectional view showing a staged assembly of a sensor applicator having the sensor control device of FIGS. [Figure 11C] 10A-10B. FIG. 11 is a side cross-sectional view showing a staged assembly of a sensor applicator having the sensor control device of FIGS. [Figure 12A] 10A-10B , depicting step-by-step cross-sectional side views illustrating assembly and disassembly of an example embodiment of a sensor applicator having the sensor control device of FIGS. [Figure 12B] 10A-10B , depicting step-by-step cross-sectional side views illustrating assembly and disassembly of an example embodiment of a sensor applicator having the sensor control device of FIGS. [Figure 12C] 10A-10B , depicting step-by-step cross-sectional side views illustrating assembly and disassembly of an example embodiment of a sensor applicator having the sensor control device of FIGS. [Figure 13A] 1A-1D show cross-sectional views illustrating an example embodiment of an applicator during a placement stage. [Figure 13B] 1A-1D show cross-sectional views illustrating an example embodiment of an applicator during a placement stage. [Figure 13C] 1A-1D show cross-sectional views illustrating an example embodiment of an applicator during a placement stage. [Figure 13D] 1A-1D show cross-sectional views illustrating an example embodiment of an applicator during a placement stage. [Figure 13E] 1A-1D show cross-sectional views illustrating an example embodiment of an applicator during a placement stage. [Figure 13F] 1A-1D show cross-sectional views illustrating an example embodiment of an applicator during a placement stage. [Figure 14] 1 is a graph showing an example of the in vitro sensitivity of an analyte sensor. [Figure 15] 1 is a diagram illustrating example operational states of a sensor according to an exemplary embodiment of the disclosed subject matter. [Figure 16] 1 is a diagram illustrating an example of operations and data flow for over-the-air programming of a sensor in accordance with the disclosed subject matter. [Figure 17] 1 is a diagram illustrating an example of data flow for reliable exchange of data between two devices in accordance with the disclosed subject matter. [Figure 18A] 1 shows a schematic cross-sectional view of an analyte sensor including a single active area. [Figure 18B] 1 shows a schematic cross-sectional view of an analyte sensor including a single active area. [Figure 18C] 1 shows a schematic cross-sectional view of an analyte sensor including a single active area. [Figure 19A] 1 shows a schematic cross-sectional view of an analyte sensor that includes two active areas. [Figure 19B] 1 shows a schematic cross-sectional view of an analyte sensor that includes two active areas. [Figure 19C] 1 shows a schematic cross-sectional view of an analyte sensor that includes two active areas. [Figure 20] 1 shows a schematic cross-sectional view of an analyte sensor that includes two active areas. [Figure 21A] FIG. 1 shows a perspective view of an analyte sensor including two active areas on separate working electrodes. [Figure 21B] FIG. 1 shows a perspective view of an analyte sensor including two active areas on separate working electrodes. [Figure 21C] FIG. 1 shows a perspective view of an analyte sensor including two active areas on separate working electrodes. [Figure 22] 1 shows a schematic diagram of a particular enzyme system that can be used to detect aspartate according to the present disclosure. [Figure 23] 1 shows a schematic diagram of a particular enzyme system that can be used to detect asparagine according to the present disclosure. [Figure 24] 1 shows the current of an aspartate sensor of the present disclosure under air and argon. [Figure 25A] 1 shows the current response of aspartate analyte sensors of the present disclosure including various sensor membranes for aspartate detection. [Figure 25B] 1 shows the current response of aspartate analyte sensors of the present disclosure including various sensor membranes for aspartate detection. [Figure 25C] 1 shows the current response of aspartate analyte sensors of the present disclosure including various sensor membranes for aspartate detection. [Figure 26]1 shows the current response of aspartate analyte sensors of the present disclosure containing various amounts of sensing chemical for the detection of aspartate. [Figure 27] 1 shows the current response of an electrode containing an enzyme system for aspartate detection with higher loading of enzyme and redox mediator and with and without a membrane. [Figure 28A] 1 shows the current response of an electrode containing an enzyme system for the detection of asparagine. [Figure 28B] 1 shows the current response of an electrode containing an enzyme system for the detection of asparagine. [Figure 28C] 1 shows the current response of an electrode containing an enzyme system for the detection of asparagine. [Figure 29] 1 shows the current response of an electrode containing an enzyme system for asparagine detection with and without a membrane. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] The present disclosure generally describes analyte sensors that use one or more enzymes for the detection of an analyte. For example, but not limited to, the present disclosure provides analyte sensors for the detection of an analyte, such as aspartate and / or asparagine. The present disclosure further provides methods of detecting one or more analytes using the disclosed analyte sensors.

[0014] The present disclosure provides sensor chemistries suitable for detecting aspartate and / or asparagine over a range of physiologically relevant aspartate and / or asparagine concentrations. In certain embodiments, the present disclosure provides analyte sensors that utilize an enzyme system comprising at least two enzymes that can operate in concert to facilitate detection of an analyte, such as asparagine. As used herein, the term "in concert" refers to a coupled enzymatic reaction in which the product of a first enzymatic reaction becomes a substrate for a second enzymatic reaction, which serves as a basis for measuring the concentration of the substrate (e.g., analyte) reacted during the first enzymatic reaction. In certain embodiments, the product and / or substrate of the reaction can be a reduced and / or oxidized form of a cofactor or coenzyme (e.g., FAD or NAD) of an enzyme of the enzyme system. Although defined in terms of two coupled enzymatic reactions, it should be understood that three or more enzymatic reactions can be coupled in some instances as well. For example, the product of a first enzymatic reaction can be a substrate for a second enzymatic reaction, and the product of the second enzymatic reaction can be a substrate for a third enzymatic reaction, which serves as the basis for measuring the concentration of a substrate (e.g., an analyte) reacted during the first enzymatic reaction. A discussion of suitable enzymes and enzyme systems for detecting aspartate and / or asparagine in accordance with the disclosure herein follows.

[0015] In certain embodiments, the analyte sensors of the present disclosure have a sensitivity of about 0.1 to about 10 nA / mM, e.g., about 0.1 to about 10 nA / mM, about 0.1 to about 9 nA / mM, about 0.1 to about 8 nA / mM, about 0.1 to about 7 nA / mM, about 0.1 to about 6 nA / mM, about 0.1 to about 5 nA / mM, about 0.1 to about 4 nA / mM, about 0.1 to about 3 nA / mM, about 0.1 to about 2 nA / mM, or about 0.1 to about 1 nA / mM.

[0016] For purposes of clarity, and not by way of limitation, the detailed description of the subject matter of the present disclosure is divided into the following subsections. I. Definition II. Test substance sensor 1. General structure of the test substance sensor system 2. Enzymes 3. Redox mediators 4. Polymer backbone 5. Mass-transfer limiting membrane 6. Interference Domains 7.Manufacturing III.How to use IV. Illustrative Embodiments I. Definition The terms used herein generally have their ordinary meanings in the art, within the context of this disclosure and in the specific context in which each term is used. Certain terms are discussed below or elsewhere in the specification to provide additional guidance to the practitioner in describing the compositions and methods of the present disclosure and how to make and use them.

[0017] As used herein, the use of the words "a" or "an" when used in conjunction with the term "comprising" in the claims and / or specification may mean "one," but is also consistent with the meanings of "one or more," "at least one," and "one or more than one."

[0018] As used herein, the terms "comprise(s)," "include(s)," "having," "has," "can," "contain(s)," and variations thereof are intended to be open-ended transitional phrases, terms, or words that do not exclude additional acts or structures. The present disclosure also contemplates other embodiments that "comprise," "consist," and "consist essentially of" the embodiments or elements set forth herein, whether or not expressly stated.

[0019] The term "about" or "approximately" means within an acceptable error range for a particular value as determined by a person skilled in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 3 or more than 3 standard deviations, according to the practice in the art. Alternatively, "about" can mean within 20%, preferably within 10%, more preferably within 5%, and even more preferably within 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold of a value.

[0020] As used herein, an "analyte sensor" or "sensor" may refer to any device capable of receiving sensor information from a user, including, by way of example and not limitation, a temperature sensor, a blood pressure sensor, a pulse or heart rate sensor, a glucose level sensor, an analyte sensor, a physical activity sensor, a physical movement sensor, or any other sensor for collecting physical or biological information. Analytes measured by an analyte sensor may include, by way of example and not limitation, glutamate, glucose, ketones, lactate, oxygen, hemoglobin A1C, albumin, alcohol, alkaline phosphatase, alanine transaminase, aspartate aminotransferase, bilirubin, blood urea nitrogen, calcium, carbon dioxide, chloride, creatinine, hematocrit, lactate, magnesium, oxygen, pH, phosphorus, potassium, asparagine, aspartate, sodium, total protein, uric acid, and the like.

[0021] As used herein, the term "biological fluid" refers to any body fluid or body fluid derivative in which an analyte can be measured. Non-limiting examples of biological fluids include dermal fluid, interstitial fluid, plasma, blood, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage, amniotic fluid, sweat, and tears, etc. In certain embodiments, the biological fluid is dermal fluid or interstitial fluid.

[0022] The term "electrolysis," as used herein, refers to the electrooxidation or electroreduction of a compound either directly at an electrode or via one or more electron transfer agents (e.g., redox mediators or enzymes).

[0023] As used herein, the term "homogeneous membrane" refers to a membrane comprising one type of membrane polymer. As used herein, the term "multi-component membrane" refers to a membrane comprising two or more membrane polymers.

[0024] As used herein, the term "potassium-independent asparaginase" refers to an asparaginase that does not exhibit any change in catalytic activity in the presence of potassium, e.g., potassium ions (K+).

[0025] As used herein, the term "potassium-dependent asparaginase" refers to an asparaginase that exhibits increased or decreased catalytic activity in the presence of potassium, e.g., potassium ions (K+). In certain embodiments, potassium-dependent asparaginase includes asparaginases that require different concentrations of potassium, e.g., potassium ions (K+), for maximum catalytic activity.

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

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

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

[0029] As used herein, the term "single-component membrane" refers to a membrane that includes one type of membrane polymer. II. Test substance sensor 1. General structure of the test substance sensor system Before describing the present subject matter in detail, it is to be understood that this disclosure is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

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

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

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

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

[0034] Additionally, the systems and methods provided herein may be used for the operation of sensors used in analyte monitoring systems, such as, but not limited to, wellness, fitness, diet, research, information, or any purpose involving analyte detection over time. As used herein, an "analyte sensor" or "sensor" may refer to any device capable of receiving sensor information from a user, including, by way of example and not limitation, a temperature sensor, a blood pressure sensor, a pulse or heart rate sensor, a glucose level sensor, an analyte sensor, a physical activity sensor, a physical movement sensor, or any other sensor for collecting physical or biological information. In certain embodiments, the analyte sensors of the present disclosure may measure aspartate and / or asparagine. In certain embodiments, the analyte sensors of the present disclosure may further measure analytes including, but not limited to, glutamate, glucose, ketones, lactate, oxygen, hemoglobin A1C, albumin, alcohol, alkaline phosphatase, alanine transaminase, aspartate aminotransferase, bilirubin, blood urea nitrogen, calcium, carbon dioxide, chloride, creatinine, hematocrit, lactate, magnesium, oxygen, pH, phosphorus, potassium, sodium, aspartate, asparagine, total protein, uric acid, and the like.

[0035] As mentioned above, several embodiments of systems, devices, and methods are described herein that provide improved assembly and use of a skin sensor insertion device for use with an in vivo analyte monitoring system. In particular, several embodiments of the present disclosure are designed to improve the method of sensor insertion with respect to an in vivo analyte monitoring system, and in particular to prevent premature retraction of the insertion tip during the sensor insertion process. For example, several embodiments include a skin sensor insertion mechanism with an increased firing rate and slower tip retraction. In other embodiments, the tip retraction mechanism may be actuated by motion such that the tip is not retracted until the user pulls the applicator away from the skin. As a result, these embodiments may reduce the likelihood of premature withdrawal of the insertion tip during the sensor insertion process, reduce the likelihood of improper sensor insertion, and reduce the likelihood of damaging the sensor during the sensor insertion process, to name a few advantages. Several embodiments of the present disclosure also provide an improved insertion tip module that takes into account the small-scale skin sensors and the relatively shallow insertion path present in the dermis layer of a subject. Additionally, several embodiments of the present disclosure are designed to prevent undesired axial and / or rotational movement of applicator parts during sensor insertion. These embodiments may thus reduce the likelihood of capillary breakage resulting in instability of the positioned skin sensor, irritation at the insertion site, damage to surrounding tissue, and contamination of skin fluid with blood, to name a few advantages. Additionally, to mitigate inaccurate sensor readings that may be caused by trauma at the insertion site, some embodiments of the present disclosure may reduce needle end depth penetration relative to the sensor tip during insertion.

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

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

[0038] In vivo analyte monitoring systems may be distinguished from "in vitro" systems, which contact a biological sample outside the body (or "ex vivo") and generally include a meter having a port for accepting an analyte test strip carrying a user's bodily fluid that can be analyzed to determine the user's blood analyte level.

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

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

[0041] The sensor 104 is adapted to be at least partially inserted into a target tissue, such as into the dermis or subcutaneous layer of the skin. The sensor 104 may include a sensor tail of sufficient length to insert to a desired depth within a given tissue. The sensor tail may include at least one working electrode. In certain configurations, the sensor tail may include at least one active area for detecting an analyte, such as glutamate, disposed on the working electrode. A counter electrode may be present in combination with the at least one working electrode. Particular electrode configurations on the sensor tail are described in more detail below.

[0042] The active area may be configured to detect a particular analyte. In certain embodiments, the active area may be configured to detect aspartate and / or asparagine. In certain embodiments, the active area may be configured to detect aspartate. In certain embodiments, the active area may be configured to detect asparagine. In certain embodiments, the active area may be configured to detect more than one analyte. In certain embodiments, the active area may be configured to detect asparagine and / or aspartate, and / or an analyte different from asparagine and aspartate. In certain embodiments, the analyte different from asparagine and aspartate may be glucose, ketones, lactate, oxygen, hemoglobin A1C, albumin, alcohol, alkaline phosphatase, alanine transaminase, aspartate aminotransferase, bilirubin, blood urea nitrogen, calcium, carbon dioxide, chloride, creatinine, hematocrit, lactate, magnesium, oxygen, pH, phosphorus, potassium, sodium, aspartate, asparagine, total protein, uric acid, and the like.

[0043] In certain embodiments of the present disclosure, one or more analytes may be monitored in any bodily fluid of a subject, such as skin fluid, interstitial fluid, plasma, blood, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage, or amniotic fluid, etc. In certain embodiments, the analyte sensor of the present disclosure may be adapted to assay skin fluid or interstitial fluid to determine the concentration of one or more analytes in vivo. In certain embodiments, the biological fluid is interstitial fluid.

[0044] An introducer may be temporarily present to facilitate introduction of the sensor 104 into the tissue. In certain exemplary embodiments, the introducer may include a needle or similar pointed tip. As will be readily recognized by one of ordinary skill in the art, other types of introducers, such as a sheath or blade, may be present in alternative embodiments. More specifically, the needle or other introducer may be temporarily present near the sensor 104 prior to tissue insertion and then withdrawn. While present, the needle or other introducer may facilitate insertion of the sensor 104 into the tissue by opening an access path for the sensor 104 to follow. For example, but not by way of limitation, according to one or more embodiments, the needle may facilitate penetration of the epidermis as an access path to the dermis to allow implantation of the sensor 104 to occur. After opening the access path, the needle or other introducer may be withdrawn such that it does not present a sharps hazard. In exemplary embodiments, a suitable needle may be solid or hollow, beveled or non-beveled, and / or circular or non-circular in cross section. In more specific embodiments, suitable needles may be similar in cross-sectional diameter and / or tip design to acupuncture needles, which may have a cross-sectional diameter of about two hundred fifty microns (250 μm). However, suitable needles may have larger or smaller cross-sectional diameters as needed for a particular application.

[0045] In certain embodiments, the tip of the needle (while present) may be angled beyond the end of the sensor 104, so that the needle penetrates the tissue first, opening an access path for the sensor 104. In certain embodiments, the sensor 104 may reside within a lumen or groove of the needle, which likewise opens an access path for the sensor 104. In either case, the needle may be subsequently withdrawn after facilitating sensor insertion.

[0046] B. Exemplary Reader Device 2A is a block schematic diagram illustrating an example of an embodiment of a reader device configured as a smartphone. Here, the reader device 120 may include a display 122, an input component 121, and a processing core 206 including a communication processor 222 connected to a memory 223 and an application processor 224 connected to a memory 225. Also included may be a separate memory 230, an RF transceiver 228 with an antenna 229, and a power supply 226 with a power management module 238. Further included may be a multi-function transceiver 232 that may communicate with an antenna 234 via Wi-Fi, NFC, Bluetooth, BTLE, and GPS. As will be appreciated by those skilled in the art, these components are electrically and communicatively connected in a manner to create a functional device.

[0047] C. Exemplary Data Receiving Device Architecture For purposes of illustration and not limitation, reference is made to an exemplary embodiment of a data receiving device 120 for use with the disclosed subject matter shown in FIG. 2B. The data receiving device 120 and associated general-purpose data receiving device 130 include components germane to the discussion of the analyte sensor 110 and its operation, and may include additional components. In certain embodiments, the data receiving device 120 and general-purpose data receiving device 130 may be or include components provided by third parties, and are not necessarily limited to including devices made by the same manufacturer as the sensor 110.

[0048] As shown in FIG. 2B, the data receiving device 120 includes an ASIC 4000 that includes a microcontroller 4010, a memory 4020, and a storage 4030, and is communicatively coupled to a communication module 4040. Power for the components of the data receiving device 120 can be delivered by a power module 4050, which may include a rechargeable battery as embodied herein. The data receiving device 120 may further include a display 4070 to facilitate review of analyte data received from the analyte sensor 110 or other devices (e.g., the user device 140 or the remote application server 150). The data receiving device 120 may include separate user interface components (e.g., a physical key, a light sensor, a microphone, etc.).

[0049] The communication module 4040 may include a BLE module 4041 and an NFC module 4042. The data receiving device 120 may be configured to wirelessly connect to the analyte sensor 110, send commands to the analyte sensor 110, and receive data from the analyte sensor 110. As embodied herein, the data receiving device 120 may be configured to operate as an NFC scanner and a BLE endpoint via a specific module (e.g., the BLE module 4042 or the NFC module 4043) of the communication module 4040 with respect to the analyte sensor 110 described herein. For example, the data receiving device 120 may use a first module of the communication module 4040 to issue commands (e.g., an activation command for a data broadcast mode of the sensor, a pairing command for identifying the data receiving device 120) to the analyte sensor 110, and use a second module of the communication module 4040 to receive data from and send data to the analyte sensor 110. The data receiving device 120 may be configured to communicate with the user device 140 via a universal serial bus (USB) module 4045 of the communication module 4040.

[0050] As another example, the communication module 4040 may include, for example, a cellular radio module 4044. The cellular radio module 4044 may include one or more radio transceivers for communicating using broadband cellular networks, including but not limited to third generation (3G), fourth generation (4G), and fifth generation (5G) networks. Furthermore, the communication module 4040 of the data receiving device 120 may include a Wi-Fi radio module 4043 for communicating using wireless local area networks according to one or more of the IEEE 802.11 standards (e.g., 802.11a, 802.11b, 802.11g, 802.11n (aka Wi-Fi4), 802.11ac (aka Wi-Fi5), 802.11ax (aka Wi-Fi6)). Using the cellular radio module 4044 or the Wi-Fi radio module 4043, the data receiving device 120 may communicate with the remote application server 150 to receive analyte data or provide updates or input received from a user (e.g., via one or more user interfaces). Although not shown, the communications module 5040 of the analyte sensor 120 may similarly include a cellular radio module or a Wi-Fi radio module.

[0051] As embodied herein, the on-board storage 4030 of the data receiving device 120 may store analyte data received from the analyte sensor 110. Additionally, the data receiving device 120, the multi-purpose data receiving device 130, or the user device 140 may be configured to communicate with a remote application server 150 over a wide area network. As embodied herein, the analyte sensor 110 may provide data to the data receiving device 120 or the multi-purpose data receiving device 130. The data receiving device 120 may transmit the data to the user computing device 140. The user computing device 140 (or the multi-purpose data receiving device 130) may then transmit the data to the remote application server 150 for processing and analysis.

[0052] As embodied herein, the data receiving device 120 may further include sensing hardware 4060 similar to or extended from the sensing hardware 5060 of the analyte sensor 110. In certain embodiments, the data receiving device 120 may be configured in conjunction with the analyte sensor 110 to operate based on the analyte data received from the analyte sensor 110. As an example, if the analyte sensor 110 is a glucose sensor, the data receiving device 120 may be or include an insulin pump or an insulin injection pen. In conjunction, the compatible device 130 may adjust insulin dosage for the user based on the glucose value received from the analyte sensor.

[0053] D. Exemplary Sensor Control Device 2C and 2D are block schematic diagrams illustrating an example embodiment of a sensor controller 102 having an analyte sensor 104 and sensor electronics 160 (including analyte monitoring circuitry), which may have most of the processing power for rendering final result data suitable for display to a user. In FIG. 2C, a single semiconductor chip 161 is shown, which may be a custom application specific integrated circuit (ASIC). Within the ASIC 161, certain high level functional units are shown, including an analog front end (AFE) 162, a power management (or control) circuit 164, a processor 166, and a communication circuit 168 (which may be implemented as a transmitter, receiver, transceiver, passive circuitry, or in other ways according to a communication protocol). In this embodiment, both the AFE 162 and the processor 166 are used as analyte monitoring circuitry, although in other embodiments, either circuitry may perform the analyte monitoring function. The processor 166 may include one or more processors, microprocessors, controllers, and / or microcontrollers, each of which may be a separate chip or distributed among many different chips (and portions thereof).

[0054] The memory 163 is also included in the ASIC 161 and may be shared by the various functional units present in the ASIC 161 or distributed between two or more of them. The memory 163 may also be a separate chip. The memory 163 may be a volatile and / or non-volatile memory. In this embodiment, the ASIC 161 is connected to a power source 170, which may be a button cell battery or the like. The AFE 162 interfaces with the in-vivo analyte sensor 104 to receive measurement data therefrom and outputs the data in digital form to the processor 166, which then processes the data to arrive at final result glucose discrete values ​​and trend values, etc. This data may then be provided to the communication circuitry 168 for transmission via the antenna 171 to the reader device 120 (not shown), where minimal further processing is required, for example, by a resident software application to display the data.

[0055] FIG. 2D is similar to FIG. 2C, but instead includes two separate semiconductor chips 162 and 174, which may be packaged together or separately. Here, AFE 162 resides on ASIC 161. Processor 166 is integrated with power management circuitry 164 and communication circuitry 168 on chip 174. AFE 162 includes memory 163, and chip 174 includes memory 165, which may be separated or distributed within. In one exemplary embodiment, AFE 162 is combined with power management circuitry 164 and processor 166 on one chip, and communication circuitry 168 is on a separate chip. In another exemplary embodiment, both AFE 162 and communication circuitry 168 are on one chip, and processor 166 and power management circuitry 164 are on another chip. It should be noted that other chip combinations are possible, including more than two chips, with each chip performing separate functions as described, or sharing one or more functions for fail-safe redundancy.

[0056] For purposes of illustration and not limitation, reference is made to an exemplary embodiment of an analyte sensor 110 for use with the disclosed subject matter shown in Figure 2E, which illustrates a block diagram of an example analyte sensor 110 according to an exemplary embodiment compatible with the security architecture and communication schemes described herein.

[0057] As embodied herein, the analyte sensor 110 may include an application specific integrated circuit ("ASIC") 5000 communicatively coupled to a communication module 5040. The ASIC 5000 may include a microcontroller core 5010, an on-board memory 5020, and a storage memory 5030. The storage memory 5030 may store data used in the authentication and encryption security architecture. The storage memory 5030 may store programming instructions for the sensor 110. As embodied herein, a specific communication chipset may be incorporated into the ASIC 5000 (e.g., NFC transceiver 5025). The ASIC 5000 may receive power from a power module 5050, such as an on-board battery, or from an NFC pulse. The storage memory 5030 of the ASIC 5000 may be programmed to include information such as an identifier of the sensor 110 for identification and tracking purposes. The storage memory 5030 may also be programmed with configuration or calibration parameters for use by the sensor 110 and its various components. The storage memory 5030 may include rewriteable or one-time programming (OTP) memory. The storage memory 5030 may be updated using the techniques described herein to extend the usefulness of the sensor 110.

[0058] As embodied herein, the communication module 5040 of the sensor 100 may be or include one or more modules for supporting the analyte sensor 110 in communication with other devices of the analyte monitoring system 100. By way of example only and not limitation, an exemplary communication module 5040 may include a Bluetooth® Low Energy ("BLE") module 5041. As used throughout this disclosure, Bluetooth® Low Energy ("BLE") refers to a short-range communication protocol optimized to simplify pairing of Bluetooth® devices for end users. The communication module 5040 may transmit and receive data and commands via interaction with a similarly capable communication module of the data receiving device 120 or user device 140. The communication module 5040 may include additional or alternative chipsets for use with similar short-range communication schemes, such as personal area networks according to the IEEE 802.15 protocol, IEEE 802.11 protocol, infrared communication according to the Infrared Data Association standard (IrDA), and the like.

[0059] To perform its functions, the sensor 100 may further include appropriate sensing hardware 5060 appropriate for that function. As embodied herein, the sensing hardware 5060 may include an analyte sensor disposed transcutaneously or subcutaneously in contact with the subject's bodily fluid. The analyte sensor may generate sensor data including a value corresponding to a level of one or more analytes in the bodily fluid.

[0060] E. Exemplary Assembly Process for a Sensor Control Unit The parts of the sensor control device 102 may be acquired by the user in multiple packages that require final assembly by the user prior to delivery to the appropriate user location. Figures 3A-3D show an example embodiment of an assembly process for the sensor control device 102 by the user, including preparation of separate parts before joining the parts to prepare the sensor for delivery. Figures 3E-3F show an example embodiment of delivery of the sensor control device 102 to the appropriate user location by selecting the appropriate delivery location and applying the device 102 to that location.

[0061] 3A is a proximal perspective view showing an example of an embodiment of a user preparing a container 810, here configured as a tray (but other packages may be used), for an assembly process. The user may accomplish this preparation by removing the lid 812 from the tray 810 to expose the platform 808, for example, by peeling a non-adhered portion of the lid 812 from the tray 810 such that an adhered portion of the lid 812 is removed. Removal of the lid 812 may be appropriate in various embodiments while the platform 808 is fully exposed within the tray 810. The lid 812 may then be set aside.

[0062] 3B is a side view illustrating an example embodiment of a user preparing applicator device 150 for assembly. Applicator device 150 may be provided in a sterile package sealed by cap 708. Preparing applicator device 150 may include separating housing 702 from cap 708 to expose sheath 704 (FIG. 3C). This may be accomplished by unscrewing (or otherwise separating) cap 708 from housing 702. Cap 708 may then be set aside.

[0063] 3C is a proximal perspective view showing an example embodiment of a user inserting applicator device 150 into tray 810 during assembly. First, a user may insert sheath 704 into inner platform 808 of tray 810 after aligning housing orientation feature 1302 (or slot or recess) and tray orientation feature 924 (abutment or detent). Inserting sheath 704 into platform 808 temporarily unlocks sheath 704 relative to housing 702 and also temporarily unlocks platform 808 relative to tray 810. At this stage, removal of applicator device 150 from tray 810 will result in the same condition as before the initial insertion of applicator device 150 into tray 810 (i.e., the process may be reversed or interrupted at this point and then repeated without consequence).

[0064] While the housing 702 is advanced distally, the sheath 704 is maintained in position relative to the housing 702 within the platform 808 and couples with the platform 808 to advance the platform 808 distally relative to the tray 810. This process unlocks the platform 808 and folds it into the tray 810. The sheath 704 contacts and disengages a locking feature (not shown) in the tray 810 that prevents the sheath 704 from moving (relatively) while the housing 702 continues to advance the platform 808 distally. When the advancement of the housing 702 and platform 808 is completed, the sheath 704 is permanently unlocked relative to the housing 702. A tip and sensor (not shown) in the tray 810 may couple with an electronics housing (not shown) in the housing 702 at the end of the distal advancement of the housing 702. The operation and interaction of applicator device 150 and tray 810 are described further below.

[0065] 3D is a proximal perspective view illustrating an example embodiment in which the user removes the applicator device 150 from the tray 810 during assembly. The user can remove the applicator 150 from the tray 810 by advancing the housing 702 proximally relative to the tray 810, or by other action that has the same end effect as decoupling 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.

[0066] 3E is a proximal perspective view of an example embodiment in which a patient uses applicator device 150 to apply sensor control unit 102 to a target area of ​​skin, such as the abdomen or other suitable location. Housing 702 is advanced distally, collapsing sheath 704 into housing 702 and applying the sensor to the target location such that the adhesive layer on the bottom side of sensor control unit 102 adheres to the skin. When housing 702 is fully advanced, the tip is automatically retracted while the sensor (not shown) is left in place to measure the analyte level.

[0067] 3F is a proximal perspective view showing an example of a patient embodiment with the sensor control unit 102 in application position. The user may then remove the applicator 150 from the application site. 3A-3F and elsewhere herein, the system 100 may reduce or eliminate the possibility of accidental breakage, permanent deformation, or incorrect assembly of applicator parts compared to prior art systems. Because the applicator housing 702 directly engages the platform 808 while the sheath 704 is unlocked, rather than indirect engagement 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 arms or other parts. The possibility of relatively large forces (as in conventional devices) during assembly is reduced, which in turn reduces the possibility of failed assembly by the user.

[0068] F. Exemplary Sensor Applicator Device FIGURE 4A is a side view of an example embodiment of applicator device 150 coupled with a screw cap 708. This is an example of how applicator 150 may be shipped to and received by a user prior to assembly of the sensor by the user. FIGURE 4B is a side perspective view of applicator 150 and cap 708 after they have been separated. FIGURE 4C is a perspective view of an example embodiment of the distal end of applicator device 150 with electronics housing 706 and adhesive patch 105 removed from their positions in sensor carrier 710 of sheath 704 while cap 708 is in place.

[0069] 4D-4G, for purposes of illustration and not limitation, the applicator device 20150 may be provided to a user as a single integrated assembly. Figures 4D and 4E provide top and bottom perspective views, respectively, of the applicator device 20150, Figure 4F provides an exploded view of the applicator device 20150, and Figure 4G provides a side cross-sectional view. The perspective views show how the applicator 20150 is shipped to and received by a user. The exploded and cross-sectional views show the components of the applicator device 20150. The applicator device 20150 may include a housing 20702, a gasket 20701, a sheath 20704, a tip carrier 201102, a spring 205612, a sensor carrier 20710 (also referred to as a "puck carrier"), a tip hub 205014, a sensor control device (also referred to as a "puck") 20102, an adhesive patch 20105, a desiccant 20502, a cap 20708, a serial label 20709, and a tamper evidence feature 20712. When received by a user, only the housing 20702, the cap 20708, the tamper evidence feature 20712, and the label 20709 are visible. The tamper evidence feature 20712 may be, for example, a sticker coupled to each of the housing 20702 and the cap 20708, such that the tamper evidence feature 20712 may be irreparably damaged, for example, by disconnecting the housing 20702 from the cap 20708, thereby indicating to a user that the housing 20702 and the cap 20708 have previously been disconnected. These features are described in more detail below.

[0070] G. Exemplary Tray and Sensor Module Assembly FIG. 5 is a proximal perspective view showing an example embodiment of a tray 810 with a sterilization lid 812 removably coupled thereto, which may illustrate how the package may be shipped to and received by a user prior to assembly.

[0071] 6A is a proximal perspective cross-sectional view showing the sensor delivery components within a tray 810. A platform 808 is slidably coupled within the tray 810. A desiccant 502 is fixed relative to the tray 810. A sensor module 504 is mounted within the tray 810.

[0072] 6B is a proximal perspective view showing the sensor module 504 in greater detail, where the retention arm extensions 1834 of the platform 808 releasably secure the sensor module 504 in place. The module 2200 is coupled to the connector 2300, tip module 2500, and sensor (not shown) so that they can be removed together as the sensor module 504 during assembly.

[0073] H. Exemplary Applicator and Sensor Control Unit for a One-Piece Architecture Briefly referring back to FIGS. 1A and 3A-3G, in the case of a two-piece architecture system, the sensor tray 202 and the sensor applicator 102 are provided to the user as separate packages, thus requiring the user to open each package and ultimately assemble the system. In some applications, the separate sealed packages allow the sensor tray 202 and the sensor applicator 102 to be sterilized in separate sterilization processes that are specific to the contents of each package and incompatible with the contents of the other. More specifically, the sensor tray 202, including the plug assembly 207, including the sensor 110 and the tip 220, may be sterilized using radiation sterilization, such as electron beam (i.e., "e-beam") irradiation. Suitable radiation sterilization processes include, but are not limited to, electron beam (e-beam) irradiation, gamma irradiation, x-ray irradiation, or any combination thereof. However, radiation sterilization may damage electrical components located within the electronics housing of the sensor control device 102. As a result, if the sensor applicator 102, including the electronics housing of the sensor control device 102, needs to be sterilized, it may be sterilized by another method, such as gas chemical sterilization using, for example, ethylene oxide. However, gas chemical sterilization may damage the enzymes or other chemicals and biologics contained on the sensor 110. Because of this sterilization incompatibility, the sensor tray 202 and the sensor applicator 102 are typically sterilized in separate sterilization processes and then packaged separately, which requires final assembly of the parts for use by the user.

[0074] 7A and 7B are top and bottom exploded views, respectively, of a sensor control device 3702 according to one or more embodiments. The shell 3706 and the 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 that includes a printed circuit board (PCB) 3804 to which a number of electronic modules 3806 are connected. Exemplary electronic modules 3806 include, but are not limited to, resistors, transistors, capacitors, inductors, diodes, and switches. Conventional sensor control devices typically stack PCB components on only one side of the PCB. In contrast, the PCB components 3806 in the sensor control device 3702 may be distributed over both surface areas (i.e., top and bottom surfaces) of the PCB 3804.

[0075] In addition to the electronic module 3806, the PCBA 3802 may also include a data processing unit 3808 mounted on the PCB 3804. The data processing unit 3808 may comprise, for example, an application specific integrated circuit (ASIC) configured to perform one or more functions or routines associated with the operation of the sensor controller 3702. More specifically, the data processing unit 3808 may be configured to perform data processing functions, which may include, but are not limited to, filtering and encoding data signals, each of which corresponds to a sampled analyte level of a user. The data processing unit 3808 may also include an antenna for communicating with or otherwise communicating with the reader device 106 (FIG. 1A).

[0076] The battery opening 3810 may be defined within the PCB 3804 and sized to receive and secure a battery 3812 configured to power the sensor controller 3702. The axial battery contact 3814a and the radial battery contact 3814b may be coupled to the PCB 3804 and extend within the battery opening 3810 to facilitate the transfer of power from the battery 3812 to the PCB 3804. As their names suggest, the axial battery contact 3814a may be configured to provide an axial contact to the battery 3812, and the radial battery contact 3814b may provide a radial contact to the battery 3812. Locating the battery 3812 within the battery opening 3810 with the battery contacts 3814a,b helps reduce the height H of the sensor controller 3702, which allows the PCB 3804 to be centrally located and its components to be distributed on both sides (i.e., top and bottom). This also helps make it easier to mount the chamfer 3718 onto the electronics housing 3704 .

[0077] The sensor 3716 may be centrally disposed with respect to the PCB 3804 and may include a tail 3816, a flag 3818, and a neck 3820 interconnecting the tail 3816 and the flag 3818. The tail 3816 may extend through a central opening 3720 in the mount 3708 and be configured to be transdermally received beneath the skin of a user. Additionally, the tail 3816 may have an enzyme or other chemical contained thereon to help facilitate analyte monitoring.

[0078] The flag 3818 may include a generally flat surface having one or more sensor contacts 3822 (three shown in FIG. 7B ) disposed thereon. The sensor contacts 3822 may be configured to align and engage with one or more corresponding circuit contacts 3824 (three shown in FIG. 7A ) provided on the PCB 3804. In some embodiments, the sensor contacts 3822 may include a carbon-impregnated polymer printed or otherwise digitally applied to the flag 3818. Conventional sensor control devices generally include a silicone rubber connector that encapsulates one or more compliant carbon-impregnated polymer modules that function as conductive contacts between the sensor and the PCB. In contrast, the sensor contacts 3822 of the present disclosure provide a direct connection between the sensor 3716 and the PCB 3804 connection, which eliminates the need for a prior art connector and advantageously reduces the height H. Additionally, by eliminating the compliant carbon-impregnated polymer modules, significant circuit resistance is eliminated, thus improving circuit conductivity.

[0079] The sensor control device 3702 may further include a compliant member 3826 that may be disposed between the flag 3818 and an inner surface of the shell 3706. More specifically, when the shell 3706 and the mount 3708 are assembled together, the compliant member 3826 may be configured to provide a passive biasing load against the flag 3818 that continuously engages the sensor contacts 3822 with corresponding circuit contacts 3824. In the illustrated embodiment, the compliant member 3826 is an elastomeric O-ring, but may alternatively comprise any other type of biasing device or mechanism, such as a compression spring, without departing from the scope of the present disclosure.

[0080] The sensor control device 3702 may further include one or more electromagnetic shields, shown as a first shield 3828a and a second shield. The shell 3706 may include or otherwise define a first clocking receptacle 3830a (FIG. 7B) and a second clocking receptacle 3830b (FIG. 7B), and the mount 3708 may include or otherwise define a first clocking post 3832a (FIG. 7A) and a second clocking post 3832b (FIG. 7A). The shell 3706 is properly aligned with the mount 3708 by mating the first and second clocking receptacles 3830a,b with the first and second clocking posts 3832a,b, respectively.

[0081] 7A , the inner surface of the mount 3708 may include or otherwise define a number of pockets or recesses configured to accommodate various components of the sensor control unit 3702 when the shell 3706 is mated to the mount 3708. For example, the inner surface of the mount 3708 may define a battery locator 3834 configured to accommodate a portion of the battery 3812 when the sensor control unit 3702 is assembled. An adjacent contact pocket 3836 may be configured to accommodate a portion of the axial contact 3814a.

[0082] Additionally, a plurality of module pockets 3838 may be defined on an inner surface of the mount 3708 to accommodate various electronic modules 3806 disposed on the bottom of the PCB 3804. Additionally, a shield locator 3840 may be defined on an inner surface of the mount 3708 to accommodate at least a portion of the second shield 3828b when the sensor control device 3702 is assembled. The battery locator 3834, contact pocket 3836, module pocket 3838, and shield locator 3840 all extend a short distance into the inner surface of the mount 3708, such that an overall height H of the sensor control device 3702 may be reduced as compared to conventional sensor control devices. The module pockets 3838 may also help minimize the diameter of the PCB 3804 by allowing PCB components to be disposed on both sides (i.e., top and bottom).

[0083] 7A, the mount 3708 may further include a plurality of carrier grip features 3842 (two shown) defined around the circumference of the mount 3708. The carrier grip features 3842 are axially offset from a bottom 3844 of the mount 3708 where a transfer adhesive (not shown) may be applied during assembly. In contrast to conventional sensor control devices that typically include a conical carrier grip feature that intersects with the bottom of the mount, the carrier grip features 3842 of the present disclosure are offset from the plane where the transfer adhesive is applied (i.e., the bottom 3844). This may prove advantageous to help ensure that the delivery system does not inadvertently adhere to the transfer adhesive during assembly. Additionally, the carrier grip features 3842 of the present disclosure eliminate the need for a corrugated transfer adhesive, which simplifies the manufacture of the transfer adhesive and eliminates the need to precisely register the transfer adhesive to the mount 3708. This also increases the bond area and therefore the bond strength.

[0084] 7B, the bottom 3844 of the mount 3708 may include or otherwise define a number of grooves 3846, which may be defined at or near the perimeter of the mount 3708 and spaced equidistant from one another. A transfer adhesive (not shown) may be coupled to the bottom 3844, and the grooves 3846 may be configured to help wick (migrate) moisture away from the sensor control unit 3702 toward the perimeter of the mount 3708 during use. In some embodiments, the spacing of the grooves 3846 may sandwich a module pocket 3838 (FIG. 7A) defined on the opposite (inner) side of the mount 3708. As will be appreciated, alternating the location of the grooves 3846 and the module pockets 3838 ensures that opposing features on either side of the mount 3708 do not extend into one another. This helps to maximize the use of material for the mount 3708, which may help to maintain a minimum height H of the sensor control unit 3702. The module pocket 3838 may also significantly reduce mold sink and improve the flatness of the bottom 3844 where the transfer adhesive adheres.

[0085] 7B, the inner surface of the shell 3706 may also include or otherwise define a number of pockets or recesses configured to accommodate various components of the sensor control unit 3702 when the shell 3706 is mated to the mount 3708. For example, the inner surface of the shell 3706 may define an opposing battery locator 3848 positionable opposite the battery locator 3834 (FIG. 7A) of the mount 3708 and configured to accommodate a portion of the battery 3812 when the sensor control unit 3702 is assembled. The opposing battery locator 3848 extends a short distance into the inner surface of the shell 3706 and helps to reduce the overall height H of the sensor control unit 3702.

[0086] A tip and sensor locator 3852 may also be provided or otherwise defined on an inner surface of the shell 3706. The tip and sensor locator 3852 may be configured to receive both a tip (not shown) and a portion of the sensor 3716. Additionally, the tip and sensor locator 3852 may be configured to align and / or mate with a corresponding tip and sensor locator 2054 ( FIG. 7A ) provided on an inner surface of the mount 3708.

[0087] An alternative sensor assembly / electronics assembly connection approach is shown in Figures 8A-8C according to an embodiment of the present disclosure. As shown, the sensor assembly 14702 includes a sensor 14704, a connector support 14706, and a tip 14708. In particular, a recess or receptacle 14710 may be defined in the bottom of the mount of the electronics assembly 14712 to provide a location in which the sensor assembly 14702 may be received and coupled to the electronics assembly 14712 to fully assemble the sensor control device. The profile of the sensor assembly 14702 may be shaped in a manner that matches or is complementary to the receptacle 14710, which includes an elastomeric sealing member 14714 (coupled to a circuit board and including a conductive material aligned with the electrical contacts of the sensor 14704). 8C is formed when the sensor assembly 14702 is snap-fit ​​or otherwise attached to the electronics assembly 14712. This embodiment provides an integrated connector for the sensor assembly 14702 within the electronics assembly 14712.

[0088] Further information regarding sensor assemblies is provided in U.S. Publication No. 2013 / 0150691 and U.S. Publication No. 2021 / 0204841, each of which is incorporated by reference in its entirety into this specification.

[0089] According to an embodiment of the present disclosure, the sensor control device 102 may be modified to provide a one-piece architecture that can undergo sterilization techniques specifically designed for the one-piece architecture sensor control device. The one-piece architecture allows the sensor applicator 150 and the sensor control device 102 to be shipped to the user in a single sealed package that does not require any final user assembly steps. Rather, the user only needs to open one package and then deliver the sensor control device 102 to the target monitoring location. The one-piece system architecture described herein may prove advantageous in eliminating components, various manufacturing process steps, and user assembly steps. As a result, packaging and waste are reduced, and the possibility of user error or contamination of the system is mitigated.

[0090] Figures 9A and 9B are side and cross-sectional views, respectively, of an example embodiment of a sensor applicator 102 coupled with an applicator cap 210. More specifically, Figure 9A illustrates how the sensor applicator 102 is shipped to and received by a user, and Figure 9B illustrates the sensor control unit 4402 disposed within the sensor applicator 102. Thus, a fully assembled sensor control unit 4402 may already be assembled and installed within the sensor applicator 102 before it is delivered to the user, thereby eliminating any additional assembly steps that the user would otherwise have to perform.

[0091] The fully assembled sensor control device 4402 may be installed into the sensor applicator 102, and the applicator cap 210 may then be coupled to the sensor applicator 102. In some embodiments, the applicator cap 210 may be threaded onto the housing 208 and may include a tamper ring 4702. When the applicator cap 210 is rotated (e.g., unscrewed) relative to the housing 208, the tamper ring 4702 may shear, thereby releasing the applicator cap 210 from the sensor applicator 102.

[0092] According to the present disclosure, while attached to the sensor applicator 102, the sensor control device 4402 may be subjected to gaseous chemical sterilization 4704 configured to sterilize the electronics housing 4404 and any other exposed portions of the sensor control device 4402. To accomplish this, a chemical may be injected into a sterilization chamber 4706 cooperatively defined by the sensor applicator 102 and the interconnected cap 210. In some applications, the chemical may be injected into the sterilization chamber 4706 via one or more vents 4708 defined in the proximal end 610 of the applicator cap 210. Exemplary chemicals that may 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 steam.

[0093] The sensor 4410 and the distal portion of the tip 4412 are sealed within the sensor cap 4416 so that the chemicals used during the gas chemical sterilization process do not interact with the enzymes, chemicals, and biological agents provided on the other sensor components, such as the tail 4524 and the membrane coating that regulates the influx of the analyte.

[0094] Once the desired level of sterility assurance has been achieved within the sterilization chamber 4706, the gas solution may be removed and the sterilization chamber 4706 may be vented. Venting may be accomplished by a series of vacuums followed by circulation of a gas (e.g., nitrogen) or filtered air through the sterilization chamber 4706. Once the sterilization chamber 4706 is properly vented, the vent 4708 may be closed with a seal 4712 (shown in dotted lines).

[0095] In some embodiments, the seal 4712 may comprise two or more layers of different materials. A first layer may be made of a synthetic material (e.g., flash-spun high density polyethylene fibers) such as Tyvek® available from DuPont®. Tyvek® is highly durable and puncture resistant while allowing vapor penetration. The Tyvek® layer may be applied prior to the gas-chemical sterilization process, and following the gas-chemical sterilization process, a foil or other vapor and moisture resistant material layer may be sealed (e.g., heat sealed) over the Tyvek® layer to prevent ingress of contaminants and moisture into the sterilization chamber 4706. In other embodiments, the seal 4712 may comprise only a single protective layer applied to the applicator cap 210. In such embodiments, the single layer may be gas permeable for the sterilization process, but may also be able to protect against moisture and other harmful elements once the sterilization process is complete.

[0096] With the seal 4712 in place, the applicator cap 210 provides a barrier to outside contamination, thereby maintaining a sterile environment for the assembled sensor control device 4402 until the user removes (unscrews) the applicator cap 210. The applicator cap 210 may also create a dust-free environment during shipping and storage that prevents the adhesive patch 4714 from becoming soiled.

[0097] 10A and 10B are isometric and side views, respectively, of another example of a sensor control device 5002 in accordance with one or more embodiments of the present disclosure. The sensor control device 5002 may be similar in some respects to the sensor control device 102 of FIG. 1A and, therefore, may be best understood by referring to FIG. 1A. Additionally, the sensor control device 5002 may replace the sensor control device 102 of FIG. 1A and, as a result, may be used in conjunction with the sensor applicator 102 of FIG. 1A, which may deliver the sensor control device 5002 to a target monitoring location on the skin of a user.

[0098] However, unlike the sensor control device 102 of FIG. 1A, the sensor control device 5002 may comprise a one-piece system architecture that does not require a user to open multiple packages and ultimately assemble the sensor control device 5002 prior to application. Rather, upon receipt by a user, the sensor control device 5002 may already be fully assembled and properly positioned within the sensor applicator 150 (FIG. 1A). To use the sensor control device 5002, the user only needs to open one barrier (e.g., applicator cap 708 of FIG. 3B) before quickly delivering the sensor control device 5002 to a target monitoring location for use.

[0099] As shown, the sensor control unit 5002 includes an electronics housing 5004 that is generally disk-shaped and may have a circular cross-section. However, in other embodiments, the electronics housing 5004 may exhibit other cross-sectional shapes, such as oval or polygonal, without departing from the scope of the present disclosure. The electronics housing 5004 may be configured to house or otherwise contain various electrical components used to operate the sensor control unit 5002. In at least one embodiment, an adhesive patch (not shown) may be disposed on the bottom of the electronics housing 5004. The adhesive patch may be similar to the adhesive patch 105 of FIG. 1A and may therefore aid in adhering the sensor control unit 5002 to a user's skin for use.

[0100] As shown, the sensor control device 5002 includes an electronics housing 5004 that includes a shell 5006 and a mount 5008 that can mate with the shell 5006. The shell 5006 can be secured to the mount 5008 via a variety of methods, such as a snap-fit ​​engagement, an interference fit, sonic welding, one or more mechanical fasteners (e.g., screws), a gasket, an adhesive, or any combination thereof. In some cases, the shell 5006 can be secured to the mount 5008 such that a sealed interface is created therebetween.

[0101] The sensor control device 5002 may further include a sensor 5010 (partially visible) and a tip 5012 (partially visible) that are used to aid in transdermal delivery of the sensor 5010 beneath the skin of a user during application of the sensor control device 5002. As shown, corresponding portions of the sensor 5010 and tip 5012 extend distally from a bottom of the electronics housing 5004 (e.g., mount 5008). The tip 5012 may include a tip hub 5014 configured to secure and deliver the tip 5012. As best seen in FIG. 10B, the tip hub 5014 may include or otherwise define a mating member 5016. To couple the tip 5012 to the sensor control device 5002, the tip 5012 may be advanced axially through the electronics housing 5004 until the tip hub 5014 engages the top surface of the shell 5006 and the mating member 5016 extends distally from the bottom of the mount 5008. Once the tip 5012 penetrates the electronics housing 5004, an exposed portion of the sensor 5010 may be received within a hollow or concave (arcuate) portion of the tip 5012. The remainder of the sensor 5010 is disposed within the interior of the electronics housing 5004.

[0102] The sensor control unit 5002 may further include a sensor cap 5018, which is shown exploded or separated from the electronics housing 5004 in FIGS. 10A-10B. The sensor cap 5016 may be removably coupled to the sensor control unit 5002 (e.g., electronics housing 5004) at or near the bottom of the mount 5008. The sensor cap 5018 may serve to provide a sealed barrier surrounding and protecting the exposed portions of the sensor 5010 and tip 5012 from gaseous chemical sterilization. As shown, the sensor cap 5018 may comprise a generally cylindrical body having a first end 5020a and a second end 5020b opposite the first end 5020a. The first end 5020a may be open to provide access into an inner chamber 5022 defined within the body. In contrast, the second end 5020b may be closed and may comprise or otherwise define an engagement feature 5024. As described herein, the engagement feature 5024 may help to fit the sensor cap 5018 to a cap (e.g., applicator cap 708 of FIG. 3B) of a sensor applicator (e.g., sensor applicator 150 of FIGS. 1A and 3A-3G) and may help to remove the sensor cap 5018 from the sensor control device 5002 when the cap is removed from the sensor applicator.

[0103] The sensor cap 5018 may be removably coupled to the electronics housing 5004 at or near the bottom of the mount 5008. More specifically, the sensor cap 5018 may be removably coupled to a mating member 5016 that extends distally from the bottom of the mount 5008. In at least one embodiment, for example, the mating member 5016 may define a set of male threads 5026a (FIG. 10B) that are matable with a set of female threads 5026b (FIG. 10A) defined by the sensor cap 5018. In some embodiments, the male and female threads 5026a,b may comprise a flat thread design (e.g., no helical curvature), which may prove advantageous in molding the part. Alternatively, the male and female threads 5026a,b may comprise a helical thread engagement. Thus, the sensor cap 5018 may be threadably coupled to the sensor control device 5002 at the mating member 5016 of the tip hub 5014. In other embodiments, the sensor cap 5018 may be removably coupled to the mating member 5016 via other types of engagement including, but not limited to, an interference fit or friction fit, or frangible members or materials that can be broken with minimal separation force (e.g., axial or rotational force).

[0104] In some embodiments, the sensor cap 5018 may comprise 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 illustrated embodiment, for example, the sensor cap 5018 may include a seal ring 5028 disposed at the first end 5020a and a desiccant cap 5030 disposed at the second end 5020b. The seal ring 5028 may be configured to help seal the internal chamber 5022, as described in more detail below. In at least one embodiment, the seal ring 5028 may comprise an elastomeric O-ring. The desiccant cap 5030 may contain or comprise a desiccant that helps maintain a preferred humidity level within the internal chamber 5022. The desiccant cap 5030 may also define or otherwise comprise an engagement feature 5024 of the sensor cap 5018.

[0105] 11A-11C are step-by-step cross-sectional side views illustrating the assembly of the sensor applicator 102 with a sensor control device 5002, according to one or more embodiments. Once the sensor control device 5002 is fully assembled, it may be installed within the sensor applicator 102. With reference to FIG. 11A, the tip hub 5014 may include or otherwise define a hub snap pole 5302 configured to aid in coupling the sensor control device 5002 to the sensor applicator 102. More specifically, the sensor control device 5002 may be advanced into the sensor applicator 102, and the hub snap pole 5302 may be received by a corresponding arm 5304 of a tip carrier 5306 disposed within the sensor applicator 102.

[0106] 11B, the sensor control unit 5002 is shown received by tip carrier 5306 and thus secured within sensor applicator 102. Once sensor control unit 5002 is installed within sensor applicator 102, applicator cap 210 may be coupled to sensor applicator 102. In some embodiments, applicator cap 210 and housing 208 may have opposing matable threads 5308 that allow applicator cap 210 to be threaded into housing 208 in a clockwise (or counterclockwise) direction, thereby securing applicator cap 210 to sensor applicator 102.

[0107] As shown, the sheath 212 is also positioned within the sensor applicator 102, which may include a sheath locking mechanism 5310 configured to ensure that the sheath 212 does not collapse prematurely during an impact event. In the embodiment shown, the sheath locking mechanism 5310 may comprise a threaded engagement between the applicator cap 210 and the sheath 212. More specifically, one or more female threads 5312a may be defined or otherwise provided on the interior of the applicator cap 210 and one or more male threads 5312b may be defined or otherwise provided on the sheath 212. The female and male threads 5312a,b may be configured to threadably engage when the applicator cap 210 is threaded onto the sensor applicator 102 at the threads 5308. The female and male threads 5312 a,b may have the same thread pitch as the threads 5308 that allow the applicator cap 210 to be screwed onto the housing 208 .

[0108] 11C , applicator cap 210 is shown fully threaded (coupled) to housing 208. As shown, applicator cap 210 further includes, or may be otherwise defined as, a cap post 5314 centrally located within and extending proximally from a bottom of applicator cap 210. Cap post 5314 may be configured to receive at least a portion of sensor cap 5018 when applicator cap 210 is threaded onto housing 208.

[0109] Once the sensor control unit 5002 is installed in the sensor applicator 102 and the applicator cap 210 is properly secured, the sensor control unit 5002 may undergo a gas chemical sterilization configured to sterilize the electronics housing 5004 and any other exposed portions of the sensor control unit 5002. Because the sensor 5010 and the distal portion of the tip 5012 are sealed within the sensor cap 5018, the chemicals used during the gas chemical sterilization process cannot interact with other sensor components, such as the enzymes, chemicals, and biologics provided in the tail 5104, and the membrane coating that regulates the influx of the analyte.

[0110] 12A through 12C are step-by-step cross-sectional side views illustrating the assembly and disassembly of an alternative embodiment of a sensor applicator 102 with a sensor control unit 5002, according to one or more further embodiments. As generally described above, the fully assembled sensor control unit 5002 may be attached to the sensor applicator 102 by coupling the hub snap pole 5302 into the arm 5304 of a tip carrier 5306 disposed within the sensor applicator 102.

[0111] In the illustrated embodiment, the sheath arm 5604 of the sheath 212 may be configured to interact with a first detent 5702a and a second detent 5702b defined within the housing 208. The first detent 5702a may alternatively be referred to as a "locking" detent and the second detent 5702b may alternatively be referred to as a "firing" detent. When the sensor control device 5002 is initially installed within the sensor applicator 102, the sheath arm 5604 may be received within the first detent 5702a. As discussed below, the sheath 212 may be actuated to move the sheath arm 5604 to the second detent 5702b, thereby placing the sensor applicator 102 in a fired position.

[0112] 12B, applicator cap 210 is aligned with and advanced toward housing 208 such that sheath 212 is received within applicator cap 210. Instead of rotating applicator cap 210 relative to housing 208, threads of applicator cap 210 may snap into corresponding threads of housing 208 to couple applicator cap 210 to housing 208. Axial cuts or slots 5703 (one shown) defined in applicator cap 210 may allow a portion of applicator cap 210 near the threads to flex outwardly and snap into engagement with threads of housing 208. Once applicator cap 210 is snapped onto housing 208, sensor cap 5018 may snap into cap post 5314 accordingly.

[0113] 11A-11C , the sensor applicator 102 may include a sheath fixation mechanism configured to ensure that the sheath 212 does not prematurely collapse during an impact event. In the embodiment shown, the sheath fixation mechanism includes one or more ribs 5704 (one shown) defined near a base of the sheath 212 and configured to interact with one or more ribs 5706 (two shown) and a shoulder 5708 defined near a base of the applicator cap 210. The rib 5704 may be configured to interlock between the rib 5706 and the shoulder 5708 during attachment of the applicator cap 210 to the housing 208. More specifically, once applicator cap 210 is snapped onto housing 208, applicator cap 210 may be rotated (e.g., clockwise) which positions rib 5704 of sheath 212 between rib 5706 and shoulder 5708 of applicator cap 210, thereby "locking" applicator cap 210 in place until a user counter-rotates applicator cap 210 and removes applicator cap 210 for use. Engagement of rib 5704 with rib 5706 and shoulder 5708 of applicator cap 210 may also prevent sheath 212 from prematurely collapsing.

[0114] 12C, the applicator cap 210 has been removed from the housing 208. Similar to the embodiment of FIGS. 12A-12C, the applicator cap 210 may be removed by counter-rotating the applicator cap 210, which in turn rotates the cap post 5314 in the same direction to unscrew the sensor cap 5018 from the fitting 5016, generally as described above. Additionally, removing the sensor cap 5018 from the sensor control device 5002 exposes the sensor 5010 and a distal portion of the tip 5012.

[0115] When applicator cap 210 is unscrewed from housing 208, rib 5704 defined on sheath 212 may slidingly engage an upper portion of rib 5706 defined on applicator cap 210. The upper portion of rib 5706 may provide a corresponding ramp surface that results in upward movement of sheath 212 as applicator cap 210 is rotated, which causes sheath arm 5604 to flex out of engagement with first detent 5702a to be received in second detent 5702b. As the sheath 212 moves to the second detent 5702b, the radial shoulder 5614 moves out of radial engagement with the carrier arms 5608, which allows the passive spring force of the spring 5612 to urge the tip carrier 5306 upwardly, disengaging the carrier arms 5608 from engagement with the grooves 5610. As the tip carrier 5306 moves upwardly within the housing 208, the mating member 5016 may correspondingly retract until it is flush, substantially flush, or near-flush with the bottom of the sensor control device 5002. At this point, the sensor applicator 102 is in the fired position. Thus, in this embodiment, removing the applicator cap 210 correspondingly retracts the mating member 5016.

[0116] I. Exemplary Firing Mechanisms for One-Piece and Two-Piece Applicators 13A-13F show detailed example embodiments of internal device mechanisms that "fire" the applicator 216 to apply the sensor control unit 222 to a user, and safely retract the tip 1030 into the used applicator 216. Together, these figures represent an example sequence for driving the tip 1030 (carrying a sensor coupled to the sensor control unit 222) into the user's skin, retracting the tip leaving the sensor in operative contact with the user's interstitial fluid, and adhesively adhering the sensor control unit to the user's skin. Modifications of such operations for use with alternative applicator assembly embodiments and components may be understood by those skilled in the art with reference to the same. Additionally, the applicator 216 may be a sensor applicator having a one-piece or two-piece architecture as disclosed herein.

[0117] 13A, the sensor 1102 is supported in the tip 1030, immediately above the user's skin 1104. Rails 1106 (optionally three of them) of the upper guide section 1108 may be provided to control movement of the applicator 216 relative to the sheath 318. The sheath 318 is held by detent features 1110 in the applicator 216, and an appropriate downward force along the longitudinal axis of the applicator 216 may overcome the resistance provided by the detent features 1110 to translate the tip 1030 and sensor control unit 222 along the longitudinal axis into (and onto) the user's skin 1104. Additionally, a catch arm 1112 of the sensor carrier 1022 engages the tip retraction assembly 1024 to maintain the tip 1030 in position relative to the sensor control unit 222.

[0118] In FIG. 13B, a user force is applied to overcome or overcome the detent feature 1110, causing the sheath 318 to collapse into the housing 314, driving the sensor control device 222 (along with associated components) to translate downwardly along the longitudinal axis as indicated by arrow L. The inner diameter of the upper guide section 1108 of the sheath 318 limits the position of the carrier arm 1112 through the full stroke of the sensor / tip insertion process. The retention of the stop surface 1114 of the carrier arm 1112 against the complementary surface 1116 of the tip retraction assembly 1024 together with a fully biased return spring 1118 maintains the position of the member. According to an embodiment, rather than employing a user force to translate the sensor control device 222 downwardly along the longitudinal axis as indicated by arrow L, the housing 314 may include a button (e.g., but not limited to, a push button) that activates a drive spring (e.g., but not limited to, a coil spring) to drive the sensor control device 222.

[0119] In Figure 13C, the sensor 1102 and tip 1030 have reached their fully inserted depth, causing the carrier arm 1112 to move off the inner diameter of the upper guide section 1108. The compression force of the coil return spring 1118 then drives the angled stop surface 1114 radially outward, releasing the force and driving the tip carrier 1102 of the tip retraction assembly 1024 to pull the (slotted or otherwise configured) tip 1030 outward from the user and away from the sensor 1102, as shown by arrow R in Figure 13D.

[0120] With tip 1030 fully retracted, as shown in Figure 13E, upper guide section 1108 of sheath 318 is secured by final locking feature 1120. As shown in Figure 13F, the used applicator assembly 216 is removed from the insertion site leaving behind sensor control device 222 with tip 1030 safely secured inside applicator assembly 216. The used applicator assembly 216 is now ready for disposal.

[0121] The operation of the applicator 216 when applying the sensor control device 222 is designed to provide the user with the sensation that both insertion and retraction of the tip 1030 are performed automatically by the internal mechanisms of the applicator 216. In other words, the present invention avoids the user experiencing the sensation of manually driving the tip 1030 into the user's skin. Thus, when the user applies sufficient force to overcome the resistance from the detent features of the applicator 216, the resulting movement of the applicator 216 is perceived as an automatic response to the applicator being "triggered". The user is not aware that the user is supplying additional force to drive the tip 1030 to pierce the skin, even though all of the driving force is provided by the user and no additional biasing / driving means are used to insert the tip 1030. Retraction of the tip 1030 is automated by the coil return spring 1118 of the applicator 216, as described above in FIG. 13C.

[0122] With respect to any of the applicator embodiments described herein and any of its components, including but not limited to the tip, tip module, and sensor module embodiments, one of ordinary skill in the art will understand that the embodiments may be sized and configured for use with a sensor configured to sense an analyte level in a bodily fluid within the epidermis, dermis, or subcutaneous tissue of a subject. In some embodiments, for example, the tip and distal portion of the analyte sensor disclosed herein may both be sized and configured to be positioned at a particular distal depth (i.e., the furthest point of penetration within a tissue or layer of a subject's body, e.g., within the epidermis, dermis, or subcutaneous tissue). With respect to some applicator embodiments, one of ordinary skill in the art will recognize that certain embodiments of the tip may be sized and configured to be positioned at a different distal depth in the subject's body relative to the final distal depth of the analyte sensor. In some embodiments, for example, the tip may be positioned at a first distal depth within the subject's epidermis, while the distal portion of the analyte sensor may be positioned at a second distal depth within the subject's dermis prior to retraction. In other embodiments, the tip may be positioned at a first distal depth within the dermis of the subject prior to retraction, while a distal portion of the analyte sensor may be positioned at a second distal depth within the subcutaneous tissue of the subject. In yet other embodiments, the tip may be positioned at a first distal depth and the analyte sensor at a second distal depth prior to retraction, where both the first distal depth and the second distal depth are within the same layer or tissue of the subject's body.

[0123] Additionally, with respect to any of the applicator embodiments described herein, one of ordinary skill in the art will appreciate that the analyte sensor and one or more structural components coupled to the analyte sensor, including but not limited to one or more spring mechanisms, may be disposed within the applicator in an eccentric position relative to one or more axes of the applicator. In some applicator embodiments, for example, the analyte sensor and spring mechanism may be disposed in a first eccentric position relative to the applicator axis on a first side of the applicator, and the sensor electronics may be disposed in a second eccentric position relative to the applicator axis on a second side of the applicator. In other applicator embodiments, the analyte sensor, spring mechanism, and sensor electronics may be disposed in an eccentric position relative to the applicator axis on the same side. One of ordinary skill in the art will appreciate that other permutations and configurations in which any or all of the analyte sensor, spring mechanism, sensor electronics, and other parts of the applicator are disposed in central or eccentric positions relative to one or more axes of the applicator are possible and fully within the scope of the present disclosure.

[0124] Further details of suitable devices, systems, methods, components, and their operation, along with associated features, are described in Rao et al., International Publication No. WO 2018 / 136898, Thomas et al., International Publication No. WO 2019 / 236850, Thomas et al., International Publication No. WO 2019 / 236859, Thomas et al., International Publication No. WO 2019 / 236876, and U.S. Patent Publication No. 2020 / 0196919, filed June 6, 2019, each of which is incorporated herein by reference in its entirety. Further details regarding embodiments of the applicators, their components, and variations thereof are described in U.S. Patent Publication Nos. 2013 / 0150691, 2016 / 0331283, and 2018 / 0235520, all of which are incorporated herein by reference in their entirety for all purposes. Further details regarding embodiments of the tip module, tips, their components, and variations thereof are described in U.S. Patent Publication No. 2014 / 0171771, which is incorporated by reference in its entirety and for all purposes.

[0125] J. Exemplary Methods for Calibrating Analyte Sensors A biochemical sensor may be described by one or more sensing characteristics. A common sensing characteristic is indicated as the sensitivity of a biochemical sensor, which is a measure of the sensor's responsiveness to the concentration of a chemical or composition that the sensor is designed to detect. In electrochemical sensors, this response may be in the form of a current (amperometric) or charge (coulometric). In other types of sensors, the response may be in a different form, such as photon intensity (e.g., optical light). The sensitivity of a biochemical analyte sensor may vary depending on several factors, including whether the sensor is in an in vitro or in vivo state.

[0126] FIG. 14 is a graph showing the in vitro sensitivity of an amperometric analyte sensor. In vitro sensitivity may be obtained by in vitro testing a sensor at various analyte concentrations and then performing regression (e.g., linear or nonlinear) or other curve fitting on the resulting data. In this example, the sensitivity of the analyte sensor is linear or substantially linear and may be modeled according to the equation y=mx+b, where y is the electrical output current of the sensor, x is the analyte level (or concentration), m is the slope of the sensitivity, and b is the intercept of the sensitivity, where the intercept generally corresponds to the background signal (e.g., noise). For sensors with linear or substantially linear responses, the analyte level corresponding to a given current can be determined from the slope and intercept of the sensitivity. Sensors with nonlinear sensitivity require additional information to determine the analyte level resulting from the sensor's output current, and those skilled in the art are familiar with how to model nonlinear sensitivity. In certain embodiments of an in vivo sensor, the in vitro sensitivity may be the same as the in vivo sensitivity, while in other embodiments, a transfer (or transformation) function is used to convert the in vitro sensitivity to an in vivo sensitivity applicable to the intended in vivo use of the sensor.

[0127] Calibration is a technique for improving or maintaining accuracy by adjusting the measured output of a sensor to reduce the difference from the expected output of the sensor. One or more parameters describing the sensing characteristics of the sensor, such as its sensitivity, are established for use in the calibration adjustment.

[0128] Certain in vivo analyte monitoring systems require calibration to be performed after the sensor is implanted in a user or patient, either by user interaction or by the system itself in an automated manner. For example, if user intervention is required, the user performs an in vitro measurement (e.g., a blood glucose (BG) measurement using a fingerstick and an in vitro test strip) and inputs this into the system while the analyte sensor is implanted. The system then compares the in vitro measurement to the in vivo signal and uses the difference to determine an estimate of the in vivo sensitivity of the sensor. The in vivo sensitivity may then be used in an algorithmic process to convert the data collected by the sensor into a value indicative of the user's analyte level. This and other processes requiring user action to perform a calibration are denoted "user calibration." The system may require user calibration due to instability in the sensitivity of the sensor, such that the sensitivity drifts or changes over time. Thus, multiple user calibrations (e.g., on a periodic (e.g., daily) schedule, a variable schedule, or as needed) may be required to maintain accuracy. Although the embodiments described herein may incorporate some degree of user calibration for particular implementations, in general this is not preferred as it requires the user to perform painful or otherwise cumbersome BG measurements and can introduce user error.

[0129] Some in vivo analyte monitoring systems can periodically adjust calibration parameters by using automated measurements of sensor characteristics made by the system itself (e.g., processing circuitry running software). Repeated adjustment of the sensor's sensitivity based on variables measured by the system (not the user) is generally denoted "system" (or automatic) calibration, and can be performed with or without user calibration, such as an early BG measurement. As with repeated user calibration, repeated system calibration is generally necessitated by drift in the sensor's sensitivity over time. Thus, although the embodiments described herein can be used with some degree of automatic system calibration, preferably the sensor's sensitivity is relatively stable over time such that post-implant calibration is not required.

[0130] Some in vivo analyte monitoring systems operate with sensors that are factory calibrated. Factory calibration refers to the determination or estimation of one or more calibration parameters prior to distribution to a user or healthcare practitioner (HCP). The calibration parameters may be determined by the sensor manufacturer (or the manufacturer of other parts of the sensor control device, if the two entities are different). Many in vivo sensor manufacturing processes manufacture sensors in groups or batches called production lots, production stage lots, or simply lots. A single lot may contain thousands of sensors.

[0131] The sensor may include a calibration code or parameter that is derived or determined during one or more sensor manufacturing processes and may be coded or programmed into a data processing device of the analyte monitoring system as part of the manufacturing process, or may be provided on the sensor itself, for example as a bar code, laser tag, RFID tag, or other machine-readable information carried by the sensor. If the code is provided to a receiver (or other data processing device), user calibration during in vivo use of the sensor may be unnecessary or the frequency of in vivo calibration during sensor wear may be reduced. In embodiments in which the calibration code or parameter is provided on the sensor itself, the calibration code or parameter may be automatically transmitted or provided to a data processing device in the analyte monitoring system prior to or upon initiation of sensor use.

[0132] Some in vivo analyte monitoring systems operate with sensors that may be one or more of factory calibrated, system calibrated, and / or user calibrated. For example, a sensor may include a calibration code or parameters that may enable factory calibration. If the information is provided to the receiver (e.g., entered by a user), the sensor may operate as a factory calibrated sensor. If the information is not provided to the receiver, the sensor may operate as a user calibrated sensor and / or a system calibrated sensor.

[0133] In further aspects, programming or executable instructions may be provided or stored in the data processor and / or receiver / controller unit of the analyte monitoring system to provide time-varying adjustment algorithms for the in-vivo sensor during use. For example, based on retrospective statistical analysis of analyte sensors used in vivo and corresponding glucose level feedback, a time-based predetermined or analytical curve or database may be generated and configured to provide further adjustments to one or more in-vivo sensor parameters or other factors that compensate for potential sensor drift in the stability profile.

[0134] In accordance with the disclosed subject matter, an analyte monitoring system may be configured to compensate or adjust sensor sensitivity based on a sensor drift profile. A time-varying parameter β(t) may be defined or determined based on an analysis of sensor behavior during in vivo use, and a time-varying drift profile may be determined. In certain aspects, the compensation or adjustment to the sensor sensitivity may be programmed in a receiver unit, controller, or data processor of the analyte monitoring system, such that the compensation or adjustment, or both, may be performed automatically and / or iteratively as sensor data is received from the analyte sensor. In accordance with the disclosed subject matter, the adjustment or compensation algorithm may be user-initiated or executed (rather than self-initiated or executed), such that the adjustment or compensation to the analyte sensor sensitivity profile is implemented or executed upon user initiation or activation of a corresponding function or routine, or when the user enters a sensor calibration code.

[0135] In accordance with the disclosed subject matter, each sensor in a sensor lot (in some examples not including the sample sensor used for in vitro testing) can be non-destructively inspected to determine or measure its characteristics, such as film thickness at one or more points on the sensor, and other characteristics, including physical characteristics such as surface area / volume of the active area, can be measured or determined. Such measurements or determinations may be performed in an automated manner, for example using an optical scanner or other suitable measurement device or system, and the determined sensor characteristics of each sensor in the sensor lot are compared to corresponding average values ​​based on the sample sensors for possible correction of the calibration parameters or codes assigned to each sensor. For example, for a calibration parameter defined as sensor sensitivity, the sensitivity is approximately inversely proportional to the film thickness, such that, for example, for a sensor having a measured film thickness that is about 4% greater than the average film thickness of sensors sampled from the same sensor lot as the sensor, in one embodiment the sensitivity assigned to the sensor is the average sensitivity determined from the sampled sensors divided by 1.04. Similarly, sensitivity is approximately proportional to the active area of ​​a sensor, so that for a sensor with a measured active area that is about 3% smaller than the average active area of ​​sensors sampled from the same sensor lot, the assigned sensitivity for that sensor is the average sensitivity multiplied by 0.97. The assigned sensitivity may be determined from the average sensitivity from the sampled sensors by multiple successive adjustments for each inspection or measurement of the sensor. In certain embodiments, the inspection or measurement of each sensor may further include a measurement of the uniformity or structure of the film in addition to the thickness of the film and / or the surface area or volume of the active sensing area.

[0136] Further information regarding sensor calibration is provided in U.S. Publication No. 2010 / 00230285 and U.S. Publication No. 2019 / 0274598, each of which is incorporated by reference in its entirety.

[0137] K. Exemplary Bluetooth Communication Protocol The storage memory 5030 of the sensor 110 may include software blocks related to the communication protocol of the communication module. For example, the storage memory 5030 may include a BLE service software block whose functions provide an interface 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 generic access profile service, a generic attribute service, a generic access service, a device information service, a data transmission service, and a security service. The data transmission service may be a primary service used to transmit data such as sensor control data, sensor status data, analyte measurement data (past and present), and event log data. The sensor status data may include error data, current active time, and software state. The analyte measurement data may include information such as current and past raw measurements, current and past values ​​after processing using appropriate algorithms or models, predictions and trends of measurement levels, comparison of other values ​​to patient-specific averages, calls to action determined by algorithms or models, and other similar types of data.

[0138] In accordance with aspects of the disclosed subject matter, as embodied herein, the sensor 110 may be configured to communicate with multiple devices simultaneously by adapting the characteristics of the communication protocol or medium supported by the hardware and radio of the sensor 110. As an example, the BLE module 5041 of the communication module 5040 may comprise software or firmware to enable multiple simultaneous connections between the sensor 110 as a central device and other devices as peripheral devices, or the sensor 110 as a peripheral device when another device is the central device.

[0139] A connection and subsequent communication session between two devices using a communication protocol such as BLE may be characterized by a similar physical channel operating between the two devices (e.g., the sensor 110 and the data sink 120). The physical channel may include a single channel or a set of channels, including, for example and without limitation, using a common clock and channel or an agreed-upon set of channels determined by a frequency hopping sequence. The communication sessions may use a similar amount of the available communication spectrum, and multiple such communication sessions may exist in close proximity. In certain embodiments, each set of devices in a communication session uses a different physical channel or set of channels to manage interference with devices that are also in close proximity.

[0140] For purposes of illustration and not limitation, reference is made to an exemplary embodiment of a process for sensor-receiver connection for use with the disclosed subject matter. First, the sensor 110 repeatedly advertises its connection information to its surroundings in search of the data receiving device 120. The sensor 110 can periodically repeat the advertisement until a connection is established. The data receiving device 120 detects the advertising packet and scans and filters for the sensor 120 to connect via the data provided in the advertising packet. Next, the data receiving device 120 sends a scan request command, and the sensor 110 responds with a scan response packet providing additional details. Next, the data receiving device 120 sends a connection request using the Bluetooth device address associated with the data receiving device 120. The data receiving device 120 may also continuously request to establish a connection to the sensor 110 with a particular Bluetooth device address. The device then establishes the initial connection to allow data exchange to begin. The device starts the process of initializing the data exchange service and performs a mutual authentication process.

[0141] During the first connection between the sensor 110 and the data sink 120, the data sink 120 may initialize a service, characteristic, and attribute discovery process. The data sink 120 may evaluate these characteristics of the sensor 110 and store them for use during the next connection. The device then enables notification of customized security services to be used for mutual authentication of the sensor 110 and the data sink 120. The mutual authentication process may be automated and does not require user intervention. After the mutual authentication process is successfully completed, the sensor 110 sends a connection parameter update requesting the data sink 120 to use connection parameter settings that are suitable for the sensor 110 and configured to maximize longevity.

[0142] The data sink 120 then executes a sensor control process to backfill the historical data, current data, event log, and factory data. As an example, for each type of data, the data sink 120 sends a request to initiate the backfill process. The request may specify a range of records defined, for example, based on measurements, timestamps, etc., as needed. The sensor 110 responds with the requested data until all previously untransmitted data in the memory of the sensor 110 is provided to the data sink 120. The sensor 110 may respond to a backfill request from the data sink 120 that all data has already been transmitted. Once the backfill is complete, the data sink 120 may notify the sensor 110 that it is ready to receive normal measurements. The sensor 110 may transmit measurements over multiple notifications results on a repeating basis. As embodied herein, the multiple notifications may be redundant notifications to ensure that the data is transmitted correctly. Alternatively, the multiple notifications may constitute a single payload.

[0143] For purposes of illustration and not limitation, reference is made to an exemplary embodiment of a process for sending a shutdown command to the sensor 110. The shutdown operation is performed, for example, when the sensor 110 is in an error state, an insertion failure state, or a sensor expired state. If the sensor 110 is not in those states, the sensor 110 may record the command and perform the shutdown when the sensor 110 transitions to the error state or the sensor expired state. The data sink 120 sends a properly formatted shutdown command to the sensor 110. If the sensor 110 is actively processing another command, the sensor 110 responds with a standard error response indicating that the sensor 110 is busy. Otherwise, the sensor 110 sends a response upon receiving the command. Additionally, the sensor 110 sends a success notification via the sensor control property to inform the sensor 110 that it has received the command. The sensor 110 registers the shutdown command. At the next appropriate opportunity (e.g., depending on the current sensor state, as described herein), the sensor 110 shuts down.

[0144] L. Exemplary Sensor States and Actuation For purposes of illustration and not limitation, reference is made to an exemplary embodiment of a high level depiction of a state machine representation 6000 of actions that may be taken by the sensor 110 shown in FIG. 15. After initialization, the sensor enters a state 6005 associated with manufacturing the sensor 110. In the manufacturing state 6005, the sensor 110 may be configured for operation, e.g., the storage memory 5030 may be written to. At various times while in state 6005, the sensor 110 checks for a received command to proceed to a save state 6015. Upon entering the save state 6015, the sensor performs a software integrity check. While in the save state 6015, the sensor may also receive an actuation request command before proceeding to the insertion detection state 6025.

[0145] Upon entering state 6025, the sensor 110 may store information about devices authorized to communicate with the sensor set during operation or may initialize algorithms related to performing and interpreting measurements from the sensing hardware 5060. The sensor 110 may also initialize a life cycle timer responsible for maintaining an active count of the sensor's 110 operation time and may begin communication with authorized devices to transmit recorded data. While in the insertion detection state 6025, the sensor may enter state 6030, where the sensor 110 checks whether the operation time is equal to a predefined threshold. This operation time threshold may correspond to a timeout function to determine whether the insertion is successful. If the operation time reaches the threshold, the sensor 110 proceeds to state 6035, where the sensor 110 checks whether the average data read volume is greater than a threshold corresponding to the expected data read volume to trigger the detection of a successful insertion. If the data read volume is lower than the threshold while in state 6035, the sensor proceeds to state 6040, corresponding to an insertion failure. If the data read volume meets the threshold, the sensor proceeds to active pair state 6055.

[0146] The active pair state 6055 of the sensor 110 indicates a state during which the sensor 110 is operating normally by recording measurements, processing the measurements, and reporting them as necessary. While in the active pair state 6055, the sensor 110 attempts to transmit measurements or establish a connection with the receiving device 120. The sensor 110 also increases the operating time. When the sensor 110 reaches a predefined operating time threshold (e.g., the operating time reaches a predefined threshold), the sensor 110 transitions to the active expired state 6065. The active expired state 6065 of the sensor 110 indicates a state during which the sensor 110 is operating for its maximum predefined time.

[0147] While in the active expired state 6065, the sensor 110 may perform operations generally related to the termination of operations and ensuring that collected measurements have been securely transmitted to a receiving device as necessary. For example, while in the active expired state 6065, the sensor 110 may transmit collected data and, if a connection is not feasible, may increase attempts to discover and establish a connection with a nearby authenticated device. While in the active expired state 6065, the sensor 110 may receive a shutdown command in state 6070. If a shutdown command is not received, the sensor 110 may also check in state 6075 whether the operation time has exceeded a final operation threshold. The final operation threshold may be based on the battery life of the sensor 110. The normal termination state 6080 corresponds to a final operation of the sensor 110, ultimately shutting down the sensor 110.

[0148] Before the sensor is activated, the ASIC 5000 is in a low power storage mode state. For example, the activation process may begin when an incident RF field (e.g., an NFC field) drives the voltage of the power supply to the ASIC 5000 above a reset threshold, which causes the sensor 110 to proceed to a wake-up state. While in the wake-up state, the ASIC 5000 enters an activation sequence state. The ASIC 5000 then starts the communication module 5040. The communication module 5040 is initialized and triggers a power-on self-test. The power-on self-test may include the ASIC 5000 communicating with the communication module 5040 using a predetermined sequence of reading and writing data to verify that the memory and one-time programmable memory are not corrupted.

[0149] When the ASIC 5000 enters the measurement mode for the first time, an insertion detection sequence is performed to verify that the sensor 110 is properly placed on the patient's body before a proper measurement can be made. First, the sensor 110 interprets a command to activate a measurement setup process, causing the ASIC 5000 to enter a measurement command mode. The sensor 110 then enters a measurement lifecycle state temporarily, performing several consecutive measurements to check if the insertion was successful. The communication module 5040 or the ASIC 5000 evaluates the measurement results to determine the successful insertion. If the insertion is deemed successful, the sensor 110 enters a measurement state, where the sensor 110 begins to perform normal measurements using the sensing hardware 5060. If the sensor 110 determines that the insertion was not successful, the sensor 110 is triggered into an insertion failure mode, and the ASIC 5000 is commanded to return to a storage mode, while the communication module 5040 disables itself.

[0150] M. Exemplary Over-the-Air Updates 1B further illustrates an example of an operating environment for providing over-the-air ("OTA") updates for use with the techniques described herein. An operator of the analyte monitoring system 100 may bundle updates for the data sink 120 or sensor 110 into updates for applications running on the multipurpose data sink 130. Using available communication channels between the data sink 120, the multipurpose data sink 130, and the sensor 110, the multipurpose data sink 130 may receive regular updates for the data sink 120 or sensor 110 and initiate installation of updates to the data sink 120 or sensor 110. The multipurpose data sink 130 serves as an installation or update platform for the data sink 120 or sensor 110, since applications that enable the multipurpose data sink 130 to communicate with the analyte sensor 110, the data sink 120, and / or the remote application server 150 can update software or firmware on the data sink 120 or sensor 110 without wide area network capabilities.

[0151] As embodied herein, a remote application server 150 operated by the manufacturer of the analyte sensor 110 and / or the operator of the analyte monitoring system 100 may provide software and firmware updates to the devices of the analyte monitoring system 100. In certain embodiments, the remote application server 150 may provide updated software and firmware to the user device 140 or directly to the general-purpose data receiving device. As embodied herein, the remote application server 150 may also provide application software updates to the application storefront server 160 using an interface provided by the application storefront. The general-purpose data receiving device 130 may periodically contact the application storefront server 160 to download and install updates.

[0152] After the multipurpose data receiving device 130 downloads the application update including the firmware or software update for the data receiving device 120 or the sensor 110, the data receiving device 120 or the sensor 110 and the multipurpose data receiving device 130 establish a connection. The multipurpose data receiving device 130 determines that a firmware or software update is available for the data receiving device 120 or the sensor 110. The multipurpose data receiving device 130 may prepare the software or firmware update for delivery to the data receiving device 120 or the sensor 110. As an example, the multipurpose data receiving device 130 may compress or split data associated with the software or firmware update, may encrypt or decrypt the firmware or software update, or may perform an integrity check of the firmware or software update. The multipurpose data receiving device 130 transmits data for the firmware or software update to the data receiving device 120 or the sensor 110. The multipurpose data receiving device 130 may also transmit a command to the data receiving device 120 or the sensor 110 to initiate the update. Additionally or alternatively, the multipurpose data receiving device 130 may provide a notification to a user of the multipurpose data receiving device 130 and may include instructions to facilitate the update, such as instructions to keep the data receiving device 120 and the multipurpose data receiving device 130 connected to a power source and in close proximity until the update is complete.

[0153] The data receiving device 120 or the sensor 110 receives data for the update and a command to start the update from the multipurpose data receiving device 130. The data receiving device 120 may then install the firmware or software update. To install the update, the data receiving device 120 or the sensor 110 may put itself into a so-called "safe" mode with limited operating capabilities, or may reboot. Once the update is complete, the data receiving device 120 or the sensor 110 may re-enter a standard operating mode or be reset. The data receiving device 120 or the sensor 110 may perform one or more self-tests to determine that the firmware or software update was successfully installed. The multipurpose data receiving device 130 may receive a notification of the successful update. The multipurpose data receiving device 130 may then report confirmation of the successful update to the remote application server 150.

[0154] In a particular embodiment, the storage memory 5030 of the sensor 110 includes a one-time programmable (OTP) memory. The term OTP memory may refer to a memory that includes access restrictions and security to facilitate a predetermined number of writes to a particular address or segment in the memory. The memory 5030 may be pre-populated into a number of pre-allocated memory blocks or containers. The containers are pre-allocated to a fixed size. If the storage memory 5030 is a one-time programmable memory, the containers may be considered to be in a non-programmable state. Additional containers that have not yet been written to may be in a programmable or writable state. Containerizing the storage memory 5030 in this manner may improve the transportability of code and data to be written to the storage memory 5030. Updating the software of a device (e.g., a sensor device described herein) stored in an OTP memory may be performed by replacing only the code in one or more specific previously written containers with updated code written to a new container or containers, rather than replacing the entire code in the memory. In a second embodiment, the memory is not pre-populated. Instead, the space allocated for data is dynamically allocated or determined as needed. Containers of various sizes can be defined in which updates are expected, allowing incremental updates to be issued.

[0155] 16 is a schematic diagram illustrating an example of operations and data flows for over-the-air (OTA) programming of a storage memory 5030 in a sensor device 100 and use of the memory after OTA programming in execution of a process by the sensor device 110 in accordance with the disclosed subject matter. In the example of OTA programming 500 shown in FIG. 5, a request is sent from an external device (e.g., data receiving device 130) to initiate OTA programming (or reprogramming). At 511, a communication module 5040 of the sensor device 110 receives the OTA programming command. The communication module 5040 transmits the OTA programming command to the microcontroller 5010 of the sensor device 110.

[0156] At 531, after receiving the OTA programming command, the microcontroller 5010 verifies the OTA programming command. The microcontroller 5010 may, for example, determine whether the OTA programming command is signed with a proper digital signature token. Upon determining that the OTA programming command is valid, the microcontroller 5010 may set the sensor device to an OTA programming mode. At 532, the microcontroller 5010 may verify the OTA programming data. At 533, the microcontroller 5010 may reset the sensor device 110 to reinitialize the sensor device 110 to a programming state. Once the sensor device 110 transitions to the OTA programming state, the microcontroller 5010 may begin writing data to the rewritable memory 540 (e.g., memory 5020) of the sensor device at 534 and to the OTP memory 550 (e.g., storage memory 5030) of the sensor device at 535. The data written by the microcontroller 5010 may be based on the verified OTA programming data. The microcontroller 5010 may write data to mark one or more programming blocks or regions of the OTP memory 550 as invalid or inaccessible. The data written to the free or unused portions of the OTP memory may be used to replace the invalidated or inaccessible programming blocks of the OTP memory 550. After the microcontroller 5010 has written the data to the respective memories at 534 and 535, the microcontroller 5010 may perform one or more software integrity checks to ensure that no errors were introduced into the programming blocks during the writing process. Once the microcontroller 5010 is able to determine that the data was written without error, the microcontroller 5010 may resume normal operation of the sensor device.

[0157] At 536, in the execution mode, the microcontroller 5010 can retrieve a programming manifest or profile from the rewritable memory 540. The programming manifest or profile can include a list of valid software programming blocks and can include a guide to program execution for the sensor 110. By following the programming manifest or profile, the microcontroller 5010 can determine which memory blocks of the OTP memory 550 are appropriate to execute and can avoid executing out-of-date or invalidated programming blocks or referencing out-of-date data. At 537, the microcontroller 5010 can selectively retrieve memory blocks from the OTP memory 550. At 538, the microcontroller 5010 can use the retrieved memory blocks by executing programming code stored in the memory or by using variables stored in the memory.

[0158] N. Exemplary Security and Other Architectural Features As embodied herein, a first layer of security for communications between analyte sensor 110 and other devices may be established based on a security protocol dictated by and integrated into the communications protocol used for communication. Another layer of security may be based on communications protocols requiring proximity of the communicating devices. Additionally, certain packets and / or certain data contained within packets may be encrypted, while other packets and / or data within packets are otherwise encrypted or unencrypted. Additionally or alternatively, application layer encryption may be used in conjunction with one or more block or stream ciphers to establish mutual authentication and communications encryption with other devices in analyte monitoring system 100.

[0159] The ASIC 5000 of the analyte sensor 110 may be configured to dynamically generate authentication and encryption keys using data held in the storage memory 5030. The storage memory 5030 may also be pre-programmed with a set of valid authentication and encryption keys for use with a particular class of device. The ASIC 5000 may be further configured to perform an authentication process with the other device using the received data and apply a generated key to the sensitive data before transmitting the sensitive data. The generated key may be specific to the analyte sensor 110, specific to a pair of devices, specific to a communication session between the analyte sensor 110 and the other device, specific to a message sent during the communication session, or specific to a block of data contained within a message.

[0160] Both the sensor 110 and the data receiving device 120 may ensure the authentication of the other party in a communication session, for example to issue commands or receive data. In certain embodiments, identity authentication may be performed through two mechanisms. First, the party asserting its identity provides a valid certificate signed by the device manufacturer or the operator of the analyte monitoring system 100. Second, authentication may be performed through the use of public and private keys established by the device of the analyte monitoring system 100 or by the operator of the analyte monitoring system 100, and a shared secret key derived therefrom. To verify the identity of the other party, a party may provide proof that the party has control of the private key.

[0161] The manufacturer of the analyte sensor 110, data receiving device 120, or a provider of an application for the general-purpose data receiving device 130 may provide the information and programming necessary for the devices to communicate securely through secure programming and updates. For example, the manufacturer may provide information that can be used to generate encryption keys for each device, including a secure root key for the analyte sensor 110 and optionally the data receiving device 120, which may be used in combination with device specific information and operational data (e.g., entropy-based random values) to generate encryption values ​​specific to the device, session, or data transmission, as needed.

[0162] The analyte data associated with the user is at least partially sensitive data, since this information may be used for a variety of purposes, including health monitoring and medication administration decisions. In addition to the user data, the analyte monitoring system 100 may implement security enhancements against reverse engineering by external parties. The communication connection may be encrypted using a device-specific or session-specific encryption key. Encrypted or unencrypted communication between any two devices may be verified using a transmission integrity check built into the communication. The operation of the analyte sensor 110 may be protected from tampering by restricting access to read and write functions to the memory 5020 via the communication interface. The sensor may be configured to allow access only to known or "trusted" devices provided in a "white list" or to devices that can provide a predefined code associated with the manufacturer or an otherwise authenticated user. The white list may indicate an exclusive scope, meaning that no connection identifiers other than those included in the white list may be used, or a preferred scope, where the white list is searched first but other devices may still be used. The sensor 110 may further reject and shut down a connection request if the requester fails to complete the login process over the communication interface within a predetermined time (e.g., within 4 seconds). These properties protect against certain denial of service attacks, particularly those against BLE interfaces.

[0163] As embodied herein, analyte monitoring system 100 may use periodic key rotation to further reduce the likelihood of key leakage and misuse. The key rotation strategy employed by analyte monitoring system 100 may be designed to support backward compatibility of field-deployed or distributed devices. As an example, analyte monitoring system 100 may employ keys for downstream devices (e.g., devices that are in the field or cannot feasibly provide updates) that are designed to be compatible with multiple generations of keys used by upstream devices.

[0164] For purposes of illustration and not limitation, reference is made to an exemplary embodiment of a message sequence diagram 600 for use with the disclosed subject matter shown in FIG. 17, illustrating an example of data exchange between a pair of devices, specifically a sensor 110 and a data receiving device 120. The data receiving device 120 may be a data receiving device 120 or a general-purpose data receiving device 130 as embodied herein. In step 605, the data receiving device 120 may transmit a sensor activation command 605 to the sensor 110, for example via a short-range communication protocol. The sensor 110 may be primarily dormant prior to step 605, conserving its battery until full activation is required. After activation, during step 610, the sensor 110 may collect data or perform other operations as appropriate for the sensing hardware 5060 of the sensor 110. In step 615, the data receiving device 120 may initiate an authentication request command 615. In response to the authentication request command 615, both the sensor 110 and the data sink 120 may engage in a mutual authentication process 620. The mutual authentication process 620 may involve the transfer of data, including challenge parameters that allow the sensor 110 and the data sink 120 to ensure that the other device is capable of fully complying with the agreed upon security framework described herein. Mutual authentication may be based on mechanisms for authenticating two or more entities to each other, with or without an online trusted third party, to verify the establishment of a secret key via a challenge response. Mutual authentication may be performed using two-pass, three-pass, four-pass or five-pass authentication, or similar versions thereof.

[0165] Following a successful mutual authentication process 620, in step 625, the sensor 110 may provide the data receiving device 120 with a sensor secret 625. The sensor secret may include a sensor-specific value and may be derived from a random value generated during manufacturing. The sensor secret may be encrypted before or during transmission to prevent a third party from accessing the secret. The sensor secret 625 may be encrypted via one or more of the keys generated by or in response to the mutual authentication process 620. In step 630, the data receiving device 120 may derive a sensor-specific encryption key from the sensor secret. The sensor-specific encryption key may further be session-specific. Thus, the sensor-specific encryption key may be determined by each device without being transmitted between the sensor 110 or the data receiving device 120. In step 635, the sensor 110 may encrypt the data contained in the payload. In step 640, using the communication link established between the appropriate communication model of the sensor 110 and the data receiving device 120, the sensor 110 may transmit the encrypted payload 640 to the data receiving device 120. In step 645, the data sink 120 may decrypt the payload using the sensor-specific encryption key derived during step 630. Following step 645, the sensor 110 may deliver additional (including newly collected) data, and the data sink 120 may process the received data appropriately.

[0166] As described herein, the sensor 110 may be a device with limited processing power, battery supply, and storage. The encryption techniques (e.g., selection of cryptographic algorithms or implementations of algorithms) used by the sensor 110 may be selected based at least in part on these limitations. The data receiving device 120 may be a more powerful device with fewer limitations of this nature. Thus, the data receiving device 120 may employ more sophisticated and computationally intensive encryption techniques, such as cryptographic algorithms and implementations.

[0167] O. Exemplary Payloads / Communication Frequencies The analyte sensor 110 may be configured to modify its discoverability behavior to attempt to increase the probability that a receiving device will receive a proper data packet and / or provide a response signal, or to otherwise reduce limitations that may prevent a response signal from being received. Modifying the discoverability behavior of the analyte sensor 110 may include, for example, without limitation, modifying the frequency at which connection data is included in a data packet, modifying the frequency at which data packets are generally transmitted, lengthening or shortening the broadcast window of a data packet, modifying the time at which the analyte sensor 110 will accept a response or scan signal after broadcasting, including direct transmissions to one or more devices that previously communicated with the analyte sensor 110 (e.g., via one or more attempted transmissions) and / or one or more devices on a whitelist, modifying the transmit power associated with the communications module when broadcasting a data packet (e.g., to increase the range of a broadcast or reduce energy consumed and extend the battery life of the analyte sensor), modifying the rate at which data packets are prepared and broadcast, or a combination of one or more other modifications. Additionally or alternatively, the receiving device may similarly adjust parameters related to the device's listening behavior in order to increase the likelihood of receiving data packets containing the connection data.

[0168] As embodied herein, the analyte sensor 110 may be configured to broadcast data packets using two types of windows. The first window indicates the rate at which the analyte sensor 110 is configured to operate its communications hardware. The second window indicates the rate at which the analyte sensor 110 is configured to actively transmit (e.g., broadcast) data packets. As an example, the first window may indicate that the analyte sensor 110 operates its communications hardware to transmit and / or receive data packets (including connection data) during the first 2 seconds of each 60 second period. The second window may indicate that during each 2 second window, the analyte sensor 110 transmits a data packet every 60 milliseconds. The remaining time during the 2 second window, the analyte sensor 110 is scanning. The analyte sensor 110 may lengthen or shorten either window to change the discoverability behavior of the analyte sensor 110.

[0169] In certain embodiments, the discoverability behavior of the analyte sensor may be stored in a discoverability profile and may be altered based on one or more factors, such as the state of the analyte sensor 110, and / or by applying rules based on the state of the analyte sensor 110. For example, when the battery level of the analyte sensor 110 falls below a certain amount, a rule may cause the analyte sensor 110 to reduce the power consumed by the broadcast process. As another example, configuration settings associated with broadcasting or otherwise transmitting packets may be adjusted based on the ambient temperature, the temperature of the analyte sensor 110, or the temperature of a particular part of the communication hardware of the analyte sensor 110. In addition to altering the transmission power, other parameters associated with the transmission capabilities or processes of the communication hardware of the analyte sensor 110 may be altered, including, but not limited to, transmission rate, frequency, and timing. As another example, when the analyte data indicates that a subject is experiencing or is about to experience a negative health event, a rule may cause the analyte sensor 110 to increase its discoverability to alert a receiving device of the negative health event.

[0170] P. Exemplary Sensor Sensitivity Initialization / Adjustment Mechanism As embodied herein, certain calibration mechanisms for the sensing hardware 5060 of the analyte sensor 110 may be adjusted based on external or interval environmental characteristics and to compensate for decay of the sensing hardware 5060 during periods of non-use (e.g., "storage time" before use). The calibration mechanisms of the sensing hardware 5060 may be adjusted autonomously by the sensor 110 (e.g., by operation of the ASIC 5000 to modify characteristics in the memory 5020 or storage 5030) or may be adjusted by other devices in the analyte monitoring system 100.

[0171] As an example, the sensor sensitivity of the sensing hardware 5060 may be adjusted based on external temperature data or time since manufacture. When external temperature is monitored during storage of the sensor, the disclosed subject matter may adaptively change compensation to the sensor sensitivity over time as the device experiences changing storage conditions. By way of example and not limitation, adaptive sensitivity adjustments may be performed in an "active" storage mode in which the analyte sensor 110 periodically wakes up to measure temperature. These mechanisms may conserve the battery of the analyte device and extend the life of the analyte sensor. At each temperature measurement, the analyte sensor 110 may calculate a sensitivity adjustment for that period based on the measured temperature. The temperature weighted adjustments may then be accumulated over the active storage mode period to calculate a total sensor sensitivity adjustment at the end of the active storage mode (e.g., upon insertion). Similarly, upon insertion, the sensor 110 may determine the time difference between manufacture of the sensor 110 (which may be written to the storage 5030 of the ASIC 5000) or the sensing hardware 5060 and alter the sensor sensitivity or other calibration mechanisms according to one or more known attenuation rates or formulas.

[0172] Additionally, for purposes of illustration and not limitation, as embodied herein, the sensor sensitivity adjustment may account for other sensor conditions, such as sensor drift. The sensor sensitivity adjustment may be hard-coded into the sensor 110 during manufacturing, for example, based on an estimate of how much the average sensor drifts in the case of sensor drift. The sensor 110 may use a calibration function having time-varying functions for sensor offset and gain, which may account for drift over the period of sensor wear. Thus, the sensor 110 may utilize a device-dependent function that describes the drift of the sensor 110 over time, and a function used to convert interstitial current to interstitial glucose, which may indicate sensor sensitivity and may be device-specific combined with a baseline glucose profile. Such a function to account for sensor sensitivity and drift may improve the accuracy of the sensor 110 over the period of wear without user calibration.

[0173] Q. Exemplary Model-Based Analyte Measurements The sensor 110 detects raw measurements from the sensing hardware 5060. On-sensor processing may be performed, such as by one or more models trained to interpret the raw measurements. The models may be machine learning models trained off-device to detect, predict, or interpret the raw measurements to detect, predict, or interpret the levels of one or more analytes. Additional trained models may operate on the output of the machine learning models trained to interact with the raw measurements. As an example, the models may be used to detect, predict, or recommend an event based on the raw measurements and the type of analyte(s) detected by the sensing hardware 5060. The events may include the initiation or completion of a physical activity, a meal, the application of a medical procedure or medication, an emergency health event, and other events of a similar nature.

[0174] The model may be provided to the sensor 110, data receiving device 120, or general-purpose data receiving device 130 during manufacturing or during a firmware or software update. The model may be periodically refined, such as by the manufacturer of the sensor 110 or an operator of the analyte monitoring system 100, based on data received from the sensor 110 and the data receiving device of an individual user or multiple users collectively. In certain embodiments, the sensor 110 includes sufficient computational components to support further training or refinement of the machine learning model, such as based on unique characteristics of the user to which the sensor 110 is attached. The machine learning model may include models trained using or incorporating, by way of example and not limitation, decision tree analysis, gradient boosting, ada boosting, artificial neural networks or variations thereof, linear discriminant analysis, nearest neighbor analysis, support vector machines, supervised or unsupervised classification, and the like. The model may also include algorithmic or rule-based models in addition to machine learning models. Model-based processing may be performed by other devices, including the data receiving device 120 or general-purpose data receiving device 130, upon receiving data from the sensor 110 (or other downstream devices).

[0175] R. Exemplary Alarm Functions The data transmitted between the sensor 110 and the data sink 120 may include raw measurements or processed measurements. The data transmitted between the sensor 110 and the data sink 120 may further include alarms or notifications for display to a user. The data sink 120 may display or otherwise convey notifications to a user based on the raw measurements or processed measurements, or may display alarms as received from the sensor 110. Alarms that may be triggered for display to a user include alarms based on direct analyte values ​​(e.g., a single reading that exceeds or does not meet a threshold), analyte value trends (e.g., average readings over a set period of time that exceed or do not meet a threshold, slope), analyte value predictions (e.g., algorithmic calculations based on analyte values ​​that exceed or do not meet a threshold), sensor alerts (e.g., a suspected malfunction detected), communication alerts (e.g., no communication between the sensor 110 and the data receiving device 120 for a threshold period of time, an unknown device attempting to initiate or failing to initiate a communication session with the sensor 110), reminders (e.g., a reminder to charge the data receiving device 120, take medication, or perform other activities), and other alerts of a similar nature. For purposes of example and not limitation, as embodied herein, the alarm parameters described herein may be configurable by the user, or may be fixed during manufacture, or may be a combination of user-configurable and non-user-configurable parameters.

[0176] S. Exemplary Electrode Configurations Sensor configurations featuring a single active area configured for detection of a corresponding single analyte may use a two-electrode or three-electrode detection motif, as described further herein with reference to Figures 18A-18C. Sensor configurations featuring two different active areas for detection of the same or different analytes, either on separate working electrodes or on the same working electrode, are described separately below with reference to Figures 19A-21C. Sensor configurations having multiple working electrodes may be particularly advantageous for incorporating two different active areas within the same sensor tail, since the signal contribution from each active area can be readily determined.

[0177] When a single working electrode is present in the analyte sensor, a three-electrode sensor configuration may include a working electrode, a counter electrode, and a reference electrode. A related two-electrode sensor configuration may include a working electrode and a second electrode, which may function as both a counter electrode and a reference electrode (i.e., a counter / reference electrode). The various electrodes may be at least partially stacked (layered) on one another and / or spaced laterally from one another on the sensor tail. A suitable sensor configuration may be substantially planar, substantially cylindrical, or any suitable shape. In any of the sensor configurations disclosed herein, the various electrodes may be electrically insulated from one another by a dielectric material or similar insulator.

[0178] An analyte sensor featuring multiple working electrodes may similarly include at least one additional electrode. When one additional electrode is present, the one additional electrode may function as a counter / reference electrode for each of the multiple working electrodes. When two additional electrodes are present, one of the additional electrodes may function as a counter electrode for each of the multiple working electrodes, and the other of the additional electrodes may function as a reference electrode for each of the multiple working electrodes.

[0179] 18A shows a schematic diagram of an exemplary two-electrode analyte sensor configuration suitable for use in the present disclosure. As shown, the analyte sensor 200 includes a substrate 30212 disposed between a working electrode 214 and a counter / reference electrode 30216. Alternatively, the working electrode 214 and the counter / reference electrode 30216 may be disposed on the same side of the substrate 30212 with a dielectric material therebetween (configuration not shown). The active area 218 is disposed as at least one layer over at least a portion of the working electrode 214. The active area 218 may include multiple spots or a single spot configured for detection of an analyte, as discussed further herein. In certain embodiments, the active area 218 is configured to detect aspartate and / or asparagine, as described herein.

[0180] 18A, membrane 220 covers at least active area 218. In certain embodiments, membrane 220 may cover some or all of working electrode 214 and / or counter / reference electrode 30216, or the entire analyte sensor 200. One or both sides of analyte sensor 200 may be covered with membrane 220. Membrane 220 may include one or more polymeric membrane materials capable of limiting analyte flux to active area 218 (i.e., membrane 220 is a mass transport limiting membrane having some permeability to the analyte of interest). In accordance with the disclosure herein, membrane 220 may be crosslinked with branched crosslinkers in certain sensor configurations, as described further below. For example, but not limited to, membrane 220 is crosslinked with crosslinkers described herein. The composition and thickness of membrane 220 may be varied to facilitate a desired analyte flux to active area 218, thereby providing a desired signal strength and stability. Analyte sensor 200 may be operable to assay the analyte by any of the following electrochemical detection techniques: coulometry, amperometry, voltammetry, or potentiometry.

[0181] 18B and 18C show schematic diagrams of exemplary three-electrode analyte sensor configurations, which are also suitable for use in the present disclosure. The three-electrode analyte sensor configuration may be similar to that shown as analyte sensor 200 in FIG. 18A, except for the inclusion of an additional electrode 217 in analyte sensors 201 and 202 (FIGS. 18B and 18C). With the additional electrode 217, counter / reference electrode 30216 may then function as either a counter electrode or a reference electrode, and additional electrode 217 performs other electrode functions not otherwise described. Working electrode 214 continues to perform its original function. Additional electrode 217 may be disposed on either working electrode 214 or electrode 30216, with a separating layer of dielectric material therebetween. For example, but not by way of limitation, as shown in FIG. 18B, dielectric layers 219a, 219b, and 219c separate electrodes 214, 30216, and 217 from one another and provide electrical insulation. Alternatively, at least one of the electrodes 214, 30216, and 217 may be located on the opposite side of the substrate 30212, as shown in FIG. 18C. Thus, in certain embodiments, the electrode 214 (working electrode) and the electrode 30216 (counter electrode) may be disposed on the opposite side of the substrate 30212, and the electrode 217 (reference electrode) is disposed on one of the electrodes 214 or 30216 and separated therefrom by a dielectric material. A reference material layer 230 (e.g., Ag / AgCl) may be present on the electrode 217, and the location of the reference material layer 230 is not limited to the location shown in FIG. 18B and FIG. 18C. Similar to the sensor 200 shown in FIG. 18A, the active area 218 in the analyte sensors 201 and 202 may include multiple spots or a single spot. Additionally, analyte sensors 201 and 202 may be operable to assay the analyte by any of the following electrochemical detection techniques: coulometry, amperometry, voltammetry, or potentiometry.

[0182] Similar to analyte sensor 200, membrane 220 may also cover active area 218 and other sensor components in analyte sensors 201 and 202, thereby functioning as a mass transport limiting membrane. In certain embodiments, additional electrode 217 may be covered by membrane 220. Although FIGS. 18B and 18C show electrodes 214, 30216, and 217 as being covered by membrane 220, it should be appreciated that in certain embodiments, only working electrode 214 is covered. Furthermore, the thickness of membrane 220 on each of electrodes 214, 30216, and 217 may be the same or different. As in the two-electrode analyte sensor configuration (FIG. 18A), one or both faces of analyte sensors 201 and 202 may be covered by membrane 220 in the sensor configuration of FIGS. 18B and 18C, or the entire analyte sensors 201 and 202 may be covered. Thus, the three-electrode sensor configuration shown in Figures 18B and 18C should be understood as not limiting the embodiments disclosed herein, and alternative electrode and / or layer configurations remain within the scope of the present disclosure.

[0183] FIG. 19A shows an exemplary configuration of a sensor 203 having a single working electrode with two different active regions disposed thereon. FIG. 19A is similar to FIG. 18A, except that there are two active regions on the working electrode 214, a first active region 218a and a second active region 218b, which are responsive to the same or different analytes and are laterally spaced apart from each other on the surface of the working electrode 214. The active regions 218a and 218b may include multiple spots or a single spot configured for detection of each analyte. The composition of the membrane 220 may vary in the active regions 218a and 218b or may be compositionally the same. The first active region 218a and the second active region 218b may be configured to detect their corresponding analytes at different working electrode potentials from each other, as discussed further below.

[0184] In certain embodiments, either one or both of active regions 218a and 218b may be configured to detect aspartate. In certain embodiments, only one of active regions 218a and 218b is configured to detect aspartate. In certain embodiments, the other active region is configured to detect a second analyte different from aspartate. In certain embodiments, either one or both of active regions 218a and 218b may be configured to detect asparagine. In certain embodiments, only one of active regions 218a and 218b is configured to detect asparagine. In certain embodiments, the other active region is configured to detect a second analyte different from asparagine. Non-limiting examples of second analytes are described herein. In certain embodiments, one of active regions 218a and 218b is configured to detect aspartate and the other active region is configured to detect asparagine.

[0185] Figures 19B and 19C show cross-sectional views of exemplary three-electrode sensor configurations for sensors 204 and 205, respectively, each featuring a single working electrode having a first active area 218a and a second active area 218b disposed thereon. Figures 19B and 19C are otherwise similar to Figures 18B and 18C and may be better understood by reference thereto. As with Figure 19A, the composition of membrane 220 may vary in active areas 218a and 218b or may be compositionally the same.

[0186] Exemplary sensor configurations having multiple working electrodes, specifically two working electrodes, are described in further detail with reference to Figures 20 through 21C. The following description is primarily directed to sensor configurations having two working electrodes, but it should be understood that extensions of the disclosure herein may incorporate more than two working electrodes. Additional working electrodes may be used to provide the analyte sensor with further sensing capabilities for detection of not only the first and second analytes, but also, for example, a third and / or fourth analyte.

[0187] 20 illustrates a cross-sectional view of an exemplary analyte sensor configuration having two working electrodes, a reference electrode, and a counter electrode suitable for use in the present disclosure. As shown, the analyte sensor 300 includes working electrodes 304 and 306 disposed on opposite sides of a substrate 302. A first active area 310a is disposed on the surface of the working electrode 304, and a second active area 310b is disposed 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 302, 303 and 332 are disposed on the reference electrode 321 and the counter electrode 320, respectively. A membrane 340 can cover at least the active areas 310a and 310b according to various embodiments, with other components of the analyte sensor 300 or the entire analyte sensor 300 optionally being covered by the membrane 340. Additionally, the membrane 340 may be continuous but compositionally varied in the first membrane portion 340a and the second membrane portion 340b (i.e., over the active areas 310a and 310b) to allow different permeability values ​​for separately adjusting the analyte flux at each location. In certain embodiments, different membrane formulations may be sprayed and / or printed on opposite sides of the analyte sensor 300. For example, and without limitation, according to various embodiments, the first membrane portion 340a may cover at least the active area 310a and the second membrane portion 340b may cover at least the active area 310b, other components of the analyte sensor 300 or the entire analyte sensor 300. Dip coating techniques may also be suitable, particularly to deposit at least a portion of the bilayer membrane on one of the active areas 310a and 310b. In certain embodiments, the membrane 340 may be the same or different in composition in the active areas 310a and 310b. For example, and without limitation, film 340 may include a bilayer covering active area 310a and may be a uniform film covering active area 310b, or film 340 may include a bilayer covering active area 310b and may be a uniform film covering active area 310a.In certain embodiments, 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, according to certain embodiments of the present disclosure. In certain embodiments, the analyte sensor may include more than one membrane 340, such as two or more membranes. For example, without limitation, the analyte sensor may include a membrane covering one or more active areas, such as 310a and 310b, and an additional membrane covering the entire sensor as shown in FIG. 20. In such a configuration, a bilayer membrane may be formed over one or more active areas (e.g., 310a and 310b).

[0188] In certain embodiments, either one or both of active regions 310a and 310b may be configured to detect aspartate. In certain embodiments, only one of active regions 310a and 310b is configured to detect aspartate. In certain embodiments, the other active region is configured to detect a second analyte different from aspartate. In certain embodiments, either one or both of active regions 310a and 310b may be configured to detect asparagine. In certain embodiments, only one of active regions 310a and 310b is configured to detect asparagine. In certain embodiments, the other active region is configured to detect a second analyte different from asparagine. Non-limiting examples of second analytes are described herein. In certain embodiments, one of active regions 310a and 310b is configured to detect aspartate and the other active region is configured to detect asparagine.

[0189] Similar to analyte sensors 200, 201, and 202, analyte sensor 300 may be operable to assay aspartate and / or asparagine by any of the following electrochemical detection techniques: coulometric, amperometric, voltammetric, or potentiometric.

[0190] Alternative sensor configurations having multiple working electrodes and that differ from the configuration shown in Figure 20 may feature counter / reference electrodes in place of separate counter and reference electrodes 320, 321, and / or different layer and / or film arrangements than those explicitly shown. For example, but not by way of limitation, the locations of counter electrode 320 and reference electrode 321 may be opposite those shown in Figure 20. Additionally, working electrodes 304 and 306 need not necessarily be on opposite sides of substrate 302 as in the location shown in Figure 20.

[0191] Although preferred sensor configurations may feature electrodes that are substantially planar in nature, it should be appreciated that sensor configurations featuring non-planar electrodes may be advantageous and may be particularly suitable for use in the present disclosure. In particular, substantially cylindrical electrodes arranged concentrically with respect to one another may facilitate deposition of a mass transport limiting film, as described below. Figures 21A-21C show perspective views of an analyte sensor featuring two working electrodes arranged concentrically with respect to one another. It should be appreciated that sensor configurations having a concentric electrode arrangement but no second working electrode are also possible in the present disclosure. In particular, concentric working electrodes spaced apart along the length of the sensor tail may facilitate film deposition by successive dip coating operations in a manner similar to that described above for the substantially planar sensor configuration.

[0192] 21A shows a perspective view of an exemplary sensor configuration in which multiple electrodes are substantially cylindrical and arranged concentrically with respect to one another with respect to a central substrate. As shown, the analyte sensor 400 includes a central substrate 402 around which all of the electrodes and dielectric layers are arranged concentrically with respect to one another. In particular, a working electrode 410 is disposed on a surface of the central substrate 402, with a dielectric layer 412 disposed over a portion of the working electrode 410 distal to the sensor tip 404. A working electrode 420 is disposed over the dielectric layer 412, with a dielectric layer 422 disposed over a portion of the working electrode 420 distal to the sensor tip 404. A counter electrode 430 is disposed over the dielectric layer 422, with a dielectric layer 432 disposed over a portion of the counter electrode 430 distal to the sensor tip 404. A reference electrode 440 is disposed over the dielectric layer 432, with a dielectric layer 442 disposed over a portion of the reference electrode 440 distal to the sensor tip 404. Thus, the exposed surfaces of working electrode 410 , working electrode 420 , counter electrode 430 , and reference electrode 440 are spaced apart from one another along the longitudinal axis B of analyte sensor 400 .

[0193] 21A, a first active area 414a and a second active area 414b associated with different analytes or the same analyte are disposed on the exposed surfaces of the working electrodes 410 and 420, respectively, thereby allowing contact with the fluid for sensing to occur. Although the active areas 414a and 414b are shown as three separate spots in FIG. 21A, it should be understood that there may be fewer or more than three spots comprising a continuous layer of active areas in alternative sensor configurations. In certain embodiments, either one or both of the active areas 414a and 414b may be configured to detect aspartate. In certain embodiments, only one of the active areas 414a and 414b is configured to detect aspartate. In certain embodiments, the other active area is configured to detect a second analyte different from aspartate. In certain embodiments, either one or both of the active areas 414a and 414b may be configured to detect asparagine. In certain embodiments, only one active region, 414a and 414b, is configured to detect asparagine. In certain embodiments, the other active region is configured to detect a second analyte different from asparagine. Non-limiting examples of second analytes are described herein. In certain embodiments, one of active regions 414a and 414b is configured to detect aspartate and the other active region is configured to detect asparagine.

[0194] In Fig. 21A, the sensor 400 is partially covered by a membrane 450 over the working electrodes 410 and 420 and the active areas 414a and 414b disposed thereon. Fig. 21B illustrates an alternative sensor configuration in which substantially all of the sensor 401 is covered by a membrane 450. The membrane 450 may be the same or compositionally different in the active areas 414a and 414b. For example, the membrane 450 may include a bilayer membrane covering the active area 414a or may be a uniform membrane covering the active area 414b.

[0195] It should be further understood that the positioning of the various electrodes in Figures 21A and 21B may differ from that explicitly shown. For example, the positions of the counter electrode 430 and the reference electrode 440 may be reversed from the configuration shown in Figures 21A and 21B. Similarly, the positions of the working electrodes 410 and 420 are not limited to those explicitly shown in Figures 21A and 21B. Figure 21C illustrates an alternative sensor configuration to that shown in Figure 21B, where the sensor 405 includes the counter electrode 430 and the reference electrode 440 located more proximally relative to the sensor tip 404, and the working electrodes 410 and 420 located more distally relative to the sensor tip 404. A sensor configuration in which the working electrodes 410 and 420 are located more distally relative to the sensor tip 404 may be advantageous by providing a larger surface area for deposition of the active regions 414a and 414b (five separate sensing spots are illustratively shown in Figure 21C), thereby facilitating increased signal strength in some cases. Similarly, the center substrate 402 may be omitted in any of the concentric sensor configurations disclosed herein, in which case the innermost electrode may instead support subsequently deposited layers.

[0196] In certain embodiments, one or more electrodes of the analyte sensors described herein are wire electrodes, e.g., permeable wire electrodes. In certain embodiments, the sensor tail comprises a working electrode and a reference electrode spirally wound around the working electrode. In certain embodiments, an insulator is disposed between the working electrode and the reference electrode. In certain embodiments, a portion of the electrode is exposed to allow reaction of one or more enzymes with the analyte on the electrode. In certain embodiments, each electrode is formed from a thin wire having a diameter of about 25.4 micrometers (0.001 inches) or less to about 254 micrometers (0.010 inches) or more. In certain embodiments, the working electrode has a diameter of about 25.4 micrometers (0.001 inches) or less to about 254 micrometers (0.010 inches) or more, e.g., about 50.8 micrometers (0.002 inches) to about 203 micrometers (0.008 inches), more preferably 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 conductive material is a transparent conductive material. In certain embodiments, the electrodes can be formed by a variety of manufacturing techniques (e.g., bulk metal processing, deposition of metal on a substrate, etc.), and the electrodes can be formed from plated wires (e.g., platinum on a steel wire) or bulk metals (e.g., platinum wire). In certain embodiments, the electrodes are formed from, for example, tantalum wire coated with a conductive material.

[0197] In certain embodiments, the reference electrode, which may function as a reference electrode alone or as a dual reference and counter electrode, is formed from silver, silver / silver chloride, etc. In certain embodiments, the reference electrode is juxtaposed and / or twisted with or around the working electrode. In certain embodiments, the reference electrode is spirally wrapped around the working electrode. In certain embodiments, the assembly of wires may be coated or glued with an insulating material to provide an insulating attachment.

[0198] In certain embodiments, additional electrodes may be included in the sensor tail. For example, but not limited to, a three-electrode system (working, reference, and counter electrodes) and / or additional working electrodes (e.g., an electrode for detecting a second analyte). In certain embodiments where the sensor comprises two working electrodes, the two working electrodes may be juxtaposed and the reference electrode may be disposed therearound (e.g., spirally wrapped around the two or more working electrodes). In certain embodiments, the two or more working electrodes may extend parallel to one another. In certain embodiments, the reference electrode is wrapped around the working electrodes and extends toward the distal end (i.e., the in vivo end) of the sensor tail. In certain embodiments, the reference electrode extends (e.g., spirally) into the exposed area of ​​the working electrodes.

[0199] In certain embodiments, one or more working electrodes are spirally wound around the reference electrode. In certain embodiments where more than one working electrode is provided, the working electrodes may be formed in a double, triple, quadruple, etc. spiral configuration (e.g., surrounding a reference electrode, an insulated rod, or other supporting structure) along the length of the sensor tail. In certain embodiments, the electrodes, e.g., two or more working electrodes, are formed coaxially. For example, but not by way of limitation, the electrodes all share the same central axis.

[0200] In certain embodiments, the working electrode comprises a tube with an insulator therebetween and within which the reference electrode is disposed or wound. Alternatively, the reference electrode comprises a tube with an insulator therebetween and within which the working electrode is disposed or wound. In certain embodiments, a polymeric (e.g., insulating) rod is provided and one or more electrodes (e.g., one or more electrode layers) are disposed thereon (e.g., by electroplating). In certain embodiments, a metal (e.g., steel or tantalum) rod or wire is provided that is coated with an insulating material (as described herein) and has one or more working and reference electrodes disposed thereon. For example, but not by way of limitation, the present disclosure provides a sensor, e.g., a sensor tail, that comprises one or more tantalum wires, with a conductive material disposed over a portion of the one or more tantalum wires to function as the working electrode. In certain embodiments, a platinum-coated tantalum wire is coated with an insulating material that is partially coated with a silver / silver chloride composition that functions as the reference and / or counter electrode.

[0201] In certain embodiments where an insulator is disposed on the working electrode (e.g., on the platinum surface of the electrode), a portion of the insulator can be stripped or otherwise removed to expose the electroactive surface of the working electrode. For example, but not limited to, a portion of the insulator can be removed by hand, excimer laser, chemical etching, laser ablation, grit blasting, and the like. Alternatively, a portion of the electrode can be masked prior to deposition of the insulator to maintain an exposed electroactive surface area. In certain embodiments, the stripped and / or removed portion of the insulator can be about 0.1 mm (about 0.004 inches) or less to about 2 mm (about 0.078 inches) or more in length, such as about 0.5 mm (about 0.02 inches) to about 0.75 mm (about 0.03 inches) in length. In certain embodiments, the insulator is a non-conductive polymer. In certain embodiments, the insulator comprises parylene, fluorinated polymers, polyethylene terephthalate, polyvinylpyrrolidone, polyurethane, polyimide, and other non-conductive polymers. In certain embodiments, glass or ceramic materials can also be used for the insulator layer. In certain embodiments, the insulator comprises parylene. In certain embodiments, the insulator comprises polyurethane. In certain embodiments, the insulator comprises polyurethane and polyvinylpyrrolidone.

[0202] Several portions of the sensor, including the active area, are further described below. 2. Enzymes The active area of ​​the disclosed analyte sensor may be configured to detect an analyte. Non-limiting examples of analytes that may be detected using the disclosed analyte sensors include asparagine and aspartate. In certain embodiments, the active area of ​​the disclosed analyte sensor is configured to detect asparagine. In certain embodiments, the active area of ​​the disclosed analyte sensor is configured to detect aspartate.

[0203] In certain embodiments, the active area of ​​the analyte sensor of the present disclosure can be configured to detect two or more analytes. For example, and without limitation, the active area can be configured to detect asparagine and aspartate. Alternatively, the active area can be configured to detect asparagine and / or aspartate and a second analyte. Non-limiting examples of the second analyte include glutamate, glucose, ketones, lactate, oxygen, hemoglobin A1C, albumin, alcohol, alkaline phosphatase, alanine transaminase, aspartate aminotransferase, bilirubin, blood urea nitrogen, calcium, carbon dioxide, chloride, creatinine, hematocrit, lactate, magnesium, oxygen, pH, phosphorus, potassium, sodium, aspartate, asparagine, total protein, uric acid, and the like.

[0204] In certain embodiments, the analyte sensor of the present disclosure can include two or more active regions, each active region configured to detect a different analyte. In certain embodiments, a first active region can be configured to detect asparagine and a second active region can be configured to detect aspartate. In certain embodiments, a first active region can be configured to detect asparagine and / or aspartate, and a second active region can be configured to detect a second analyte, such as glutamate, glucose, ketones, lactate, oxygen, hemoglobin A1C, albumin, alcohol, alkaline phosphatase, alanine transaminase, aspartate aminotransferase, bilirubin, blood urea nitrogen, calcium, carbon dioxide, chloride, creatinine, hematocrit, lactate, magnesium, oxygen, pH, phosphorus, potassium, sodium, aspartate, asparagine, total protein, and / or uric acid.

[0205] In certain embodiments, the analyte sensor of the present disclosure may include an asparagine-responsive active region or an aspartate-responsive active region. In certain embodiments, the asparagine-responsive active region may include one or more enzymes for detecting asparagine. In certain embodiments, the aspartate-responsive active region may include one or more enzymes for detecting aspartate.

[0206] In certain embodiments, the analyte sensor of the present disclosure includes at least one active region configured to detect aspartate. A particular enzyme system that can be used to detect aspartate is shown in FIG. 22. In the enzymatic reaction shown, aspartate oxidase (shown as AspOx in FIG. 22) can be used to detect aspartate according to the disclosure herein. When the aspartate-responsive active region contains this enzyme system, the aspartate oxidase can catalyze the oxidation of aspartate to oxaloacetate and the reduction of its coenzyme flavin adenine dinucleotide (FAD) to FADH2. An electron transfer agent can then mediate the transfer of electrons from FADH2 to the working electrode. The electrons transferred during this reaction provide the basis for aspartate detection at the working electrode. The electrochemical signal obtained at the working electrode can then be correlated with the amount of aspartate originally present in the sample.

[0207] In certain embodiments, the analyte sensor of the present disclosure includes at least one active region configured to detect asparagine. A particular enzyme system that can be used to detect asparagine is shown in FIG. 23. As shown in FIG. 23, the enzyme system for detecting aspartate shown in FIG. 22 can be modified to detect asparagine by the addition of asparaginase. In certain embodiments, the enzyme system can include aspartate oxidase and asparaginase. As shown in FIG. 23, asparaginase catalyzes the hydrolysis of asparagine to produce aspartate. The produced aspartate can then be oxidized by aspartate oxidase as shown in FIGS. 22 and 23 and described above. For example, but not limited to, aspartate oxidase catalyzes the oxidation of aspartate to oxaloacetate and the reduction of its coenzyme FAD to FADH2, and then an electron transfer agent mediates the transfer of electrons from FADH2 to the working electrode. The electrochemical signal obtained at the working electrode can then be correlated with the amount of asparagine originally present in the sample.

[0208] Any suitable aspartate oxidase can be used in the analyte sensor of the present disclosure. Particular examples of aspartate oxidase suitable for use in the analyte sensor disclosed herein include, but are not limited to, potassium-dependent and potassium-independent aspartate oxidase. In certain embodiments, the aspartate oxidase used in the enzyme system is potassium-dependent. In certain other embodiments, the aspartate oxidase used in the enzyme system is potassium-independent. Non-limiting examples of aspartate oxidase include L-aspartate oxidase from Sulfolobus tokodaii, Escherichia coli, Arabidopis thaliana, or Thermococcus litoralis. For example, but not by way of limitation, L-aspartate oxidases for use in the aspartate and asparagine sensors of the present disclosure are disclosed in Nasu et al., J. of Biological Chemistry 257(2):626-32 (1982); Bifulco et al., Appl. Microbiol. Biotechnol. 97(16):7285-95 (2013); Washio et al., Extremophiles 22(1):59-71 (2018); and Hao et al., Plant Science 271:133-142 (2018), the contents of which are incorporated herein by reference in their entireties.

[0209] Any suitable asparaginase can be used in the analyte sensor of the present disclosure. Particular examples of asparaginases suitable for use in the analyte sensor disclosed herein include, but are not limited to, potassium-dependent and potassium-independent asparaginases. In certain embodiments, the asparaginase used in the enzyme system is potassium-dependent. In certain other embodiments, the asparaginase used in the enzyme system is potassium-independent. Non-limiting examples of potassium-independent and dependent asparaginases are disclosed in Ajewole et al., FEBS Journal 285(8):1528-1539 (2018) and Bejger et al., Acta Crystallogr. D. Biol. Crystallogr. 70(Pt 7):1854-72 (2014), the contents of which are incorporated herein by reference in their entirety. In certain embodiments, the asparaginase for use in the present disclosure is potassium-dependent (PvAspG1) and / or potassium-independent (PvAspG-T2) asparaginase from Phaseolus vulgaris.

[0210] In certain embodiments, the present disclosure provides an analyte sensor for detecting aspartate. In certain embodiments, the analyte sensor for detecting aspartate may include a sensor tail with at least one working electrode and at least one aspartate-responsive active region disposed on a surface of the working electrode, the active region including aspartate oxidase.

[0211] The present disclosure further provides an analyte sensor for detecting asparagine. In certain embodiments, the analyte sensor for detecting asparagine may include a sensor tail comprising at least one working electrode and at least one asparagine-responsive active region disposed on the surface of the working electrode and comprising an enzyme system comprising an oxidase and a hydrolase. In certain embodiments, the oxidase is aspartate oxidase. In certain embodiments, the hydrolase is asparaginase. In certain embodiments, the asparagine-responsive active region comprises a first enzyme layer comprising an oxidase, e.g., aspartate oxidase, and a second layer disposed on the first enzyme layer comprising a hydrolase, e.g., asparaginase. Alternatively or in addition, the oxidase, e.g., aspartate oxidase, and the hydrolase, e.g., asparaginase, are held within the same enzyme layer. In certain embodiments, both the aspartate oxidase and the asparaginase present in the asparagine-responsive active region are potassium-independent.

[0212] In certain embodiments, the asparagine-responsive active region of the disclosure has a molecular weight of about 100:1 to about 1:100, e.g., about 95:1 to about 1:95, about 90:1 to about 1:90, about 85:1 to about 1:85, about 80:1 to about 1:80, about 75:1 to about 1:75, about 60:1 to about 1:60, about 55:1 to about 1:55, about 50:1 to about 1:50, about 45:1 to about 1:45, about 40:1 to about 1:40, about 35:1 to about 1:3 1 to about 1:5, 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, or about 2:1 to about 1:2. In certain embodiments, the asparagine-responsive activity region can comprise a ratio of aspartate oxidase to asparaginase of about 5:1 to about 1:5. In certain embodiments, the asparagine-responsive activity region can comprise a ratio of aspartate oxidase to asparaginase of about 2:1 to about 1:2. In certain embodiments, an asparagine-responsive activity region can comprise about a 1:1 ratio of aspartate oxidase to asparaginase.

[0213] In certain embodiments, the analyte-responsive active region, e.g., an aspartate-responsive active region or an asparagine-responsive active region, can comprise from about 10% to about 80% by weight, e.g., from about 10% to about 75% by weight, from about 10% to about 70% by weight, from about 10% to about 65% by weight, from about 10% to about 60% by weight, from about 10% to about 55% by weight, from about 10% to about 50% by weight, from about 10% to about 45% by weight, from about 15% to about 50% by weight, from about 15% to about 45% by weight, from about 20% to about 40% by weight, from about 20% to about 35% by weight, or from about 20% to about 30% by weight of aspartate oxidase. In certain embodiments, the analyte responsive active region, e.g., the aspartate responsive active region or the asparagine responsive active region, can comprise from about 10% to about 50% by weight of aspartate oxidase. In certain embodiments, the analyte responsive active region, e.g., the aspartate responsive active region or the asparagine responsive active region, can comprise from about 15% to about 45% by weight of aspartate oxidase. In certain embodiments, the analyte responsive active region, e.g., the aspartate responsive active region or the asparagine responsive active region, can comprise from about 15% to about 35% by weight of aspartate oxidase. In certain embodiments, the analyte responsive active region, e.g., the aspartate responsive active region or the asparagine responsive active region, can comprise from about 10% to about 35% by weight of aspartate oxidase.

[0214] In certain embodiments, the analyte-responsive active region, e.g., the asparagine-responsive active region, is present in an amount of from about 0.1% to about 50% by weight, e.g., from about 0.1% to about 45% by weight, from about 0.1% to about 40% by weight, from about 0.1% to about 35% by weight, from about 0.1% to about 30% by weight, from about 0.1% to about 25% by weight, from about 0.1% to about 20% by weight, from about 0.1% to about 15% by weight, from about 0.1% to about 10% by weight, from about The asparaginase may comprise 0.5% to about 45% by weight, about 0.5% to about 40% by weight, about 0.5% to about 30% by weight, about 0.5% to about 25% by weight, about 0.5% to about 20% by weight, about 0.5% to about 15% by weight, about 0.5% to about 10% by weight, about 0.5% to about 5% by weight, about 20% to about 40% by weight, about 20% to about 35% by weight, or about 20% to about 30% by weight of asparaginase. In certain embodiments, the analyte-responsive active region, e.g., the asparagine-responsive active region, may comprise about 0.1% to about 20% by weight of asparaginase. In certain embodiments, the analyte-responsive active region, e.g., the asparagine-responsive active region, may comprise about 5% to about 20% by weight of asparaginase. In certain embodiments, the analyte-responsive active region, e.g., the asparagine-responsive active region, can comprise from about 0.1% to about 5% by weight of asparaginase. In certain embodiments, the analyte-responsive active region, e.g., the asparagine-responsive active region, can comprise from about 0.1% to about 2% by weight of asparaginase.

[0215] In certain embodiments, the aspartate-responsive active region or the asparagine-responsive active region can further comprise a stabilizer, for example, to stabilize one or more enzymes. For example, but not limited to, the stabilizer can be albumin, such as serum albumin. Non-limiting examples of serum albumin include bovine serum albumin and human serum albumin. In certain embodiments, the stabilizer is human serum albumin. In certain embodiments, the stabilizer is bovine serum albumin. In certain embodiments, the aspartate-responsive active region or asparagine-responsive active region of the present disclosure has a ribozyme activity of about 100:1 to about 1:100, e.g., about 95:1 to about 1:95, about 90:1 to about 1:90, about 85:1 to about 1:85, about 80:1 to about 1:80, about 75:1 to about 1:75, about 60:1 to about 1:60, about 55:1 to about 1:55, about 50:1 to about 1:50, about 45:1 to about 1:45, about 40:1 to about 1:40, about 35:1 to about 1:35, The ratio of stabilizer, e.g., serum albumin to asparaginase and / or aspartate oxidase, may be 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, or about 2:1 to about 1:2. In certain embodiments, the aspartate-responsive active region or asparagine-responsive active region may comprise a ratio of stabilizer to asparaginase and / or aspartate oxidase of about 80:1 to about 1:80. In certain embodiments, an aspartate-responsive active region or an asparagine-responsive active region can comprise a ratio of stabilizer to asparaginase and / or aspartate oxidase of about 50:1 to about 1:50.In certain embodiments, an aspartate-responsive active region or an asparagine-responsive active region can comprise a ratio of stabilizer to asparaginase and / or aspartate oxidase of about 10:1 to about 1:10.In certain embodiments, an aspartate-responsive active region or asparagine-responsive active region can comprise a ratio of stabilizer to asparaginase and / or aspartate oxidase of about 7:1 to about 1:7. In certain embodiments, an aspartate-responsive active region or asparagine-responsive active region can comprise a ratio of stabilizer to asparaginase and / or aspartate oxidase of about 6:1 to about 1:6. In certain embodiments, an aspartate-responsive active region or asparagine-responsive active region can comprise a ratio of stabilizer to asparaginase and / or aspartate oxidase of about 5:1 to about 1:5. In certain embodiments, an aspartate-responsive active region or asparagine-responsive active region can comprise a ratio of stabilizer to asparaginase and / or aspartate oxidase of about 4:1 to about 1:4. In certain embodiments, the aspartate-responsive active region or asparagine-responsive active region can comprise a ratio of stabilizer to asparaginase and / or aspartate oxidase of about 2:1 to about 1:2. In certain embodiments, the aspartate-responsive active region or asparagine-responsive active region can comprise a ratio of stabilizer to asparaginase and / or aspartate oxidase of about 1:1. In certain embodiments, the aspartate-responsive active region or asparagine-responsive active region can comprise about 5% to about 50% by weight of stabilizer, e.g., about 10% to about 50% by weight, about 15% to about 45% by weight, about 20% to about 40% by weight, about 20% to about 35% by weight, about 20% to about 30% by weight. In certain embodiments, the aspartate-responsive active region or asparagine-responsive active region can comprise about 5% to about 40% by weight of stabilizer. In certain embodiments, the aspartate-responsive active region or asparagine-responsive active region may comprise about 5% to about 35% by weight of a stabilizer. In certain embodiments, the aspartate-responsive active region or asparagine-responsive active region may comprise about 5% to about 30% by weight of a stabilizer. In certain embodiments, the aspartate-responsive active region or asparagine-responsive active region may comprise about 10% to about 30% by weight of a stabilizer.In certain embodiments, the aspartate-responsive active region or asparagine-responsive active region can comprise from about 15% to about 35% by weight of a stabilizer.

[0216] In certain embodiments, the analyte-responsive active region, e.g., an aspartate-responsive active region or an asparagine-responsive active region, can further comprise a cofactor for one or more enzymes present in the analyte-responsive active region. In certain embodiments, the cofactor is NAD(P) or FAD. In certain embodiments, the cofactor can be physically retained within the analyte-responsive active region. For example, but not by way of limitation, a membrane covering the analyte-responsive active region can help retain the cofactor within the analyte-responsive active region while allowing sufficient inward diffusion of the analyte to allow detection of the analyte.

[0217] In certain embodiments, the analyte sensor can include two working electrodes, e.g., a first active area disposed on the first working electrode and a second active area disposed on the second working electrode. In certain embodiments, the analyte sensor disclosed herein can feature an aspartate-responsive active area and a second active area for detecting an analyte different from aspartate. In certain embodiments, the analyte sensor disclosed herein can feature an asparagine-responsive active area and a second active area for detecting an analyte different from aspartate. For example, without limitation, such an analyte sensor can include a sensor tail having at least a first working electrode and a second working electrode, an aspartate-responsive active area or an asparagine-responsive active area disposed on the surface of the first working electrode, and a second active area, e.g., a second enzyme-responsive active area, disposed on the surface of the second working electrode and configured to detect a different analyte. Alternatively, in certain embodiments, the analyte sensor of the present disclosure may include a sensor tail having at least a first working electrode and a second working electrode, an aspartate-responsive active region disposed on a surface of the first working electrode, and an asparagine-responsive active region disposed on a surface of the second working electrode. In certain embodiments, when the sensor is configured to detect more than one analyte, detection of each analyte may include applying a potential to each working electrode, respectively, such that a separate signal is obtained from each analyte. The signal obtained from each analyte may then be correlated to an analyte concentration by use of a calibration curve or function, or by employing a look-up table. In certain embodiments, the correlation of the analyte signal to the analyte concentration may be performed by use of a processor.

[0218] In certain other analyte sensor configurations, the first and second active regions may be disposed on a single working electrode. A first signal may be obtained from the first active region, for example at a low potential, and a second signal, including signal contributions from both active regions, may be obtained at a high potential. Subtracting the first signal from the second signal then allows the signal contribution resulting from the second analyte to be determined. The signal contribution from each analyte may then be correlated with the analyte concentration in a manner similar to that described for the sensor configurations having multiple working electrodes. In certain embodiments, when an aspartate- or asparagine-responsive active region and a second active region configured to detect a different analyte (e.g., a second enzyme-responsive active region) are disposed on a single working electrode in this manner, one of the active regions may be configured to respond separately to facilitate detection of each analyte. For example, either the aspartate / asparagine-responsive active region or a second active region responsive to a second analyte can generate a signal independently of the other active region.

[0219] It should also be understood that the sensitivity (output current) of the analyte sensor directed to each analyte may be varied by varying the coverage (area or size) of the active regions, the area ratio of the active regions to one another, the identity, the thickness and / or composition of the mass transport limiting membrane covering the active regions. Variations in these parameters can be readily implemented by one of ordinary skill in the art given the benefit of the disclosure herein.

[0220] 3. Redox mediators In certain embodiments, the analyte sensors disclosed herein may include an electron transfer agent. For example, but not limited to, at least one active region of the analyte sensors of the present disclosure may include an electron transfer agent. In certain embodiments, the analyte sensors of the present disclosure may include at least one aspartate-responsive active region that includes an electron transfer agent. In certain embodiments, the analyte sensors of the present disclosure may include at least one asparagine-responsive active region that includes an electron transfer agent.

[0221] In certain embodiments, the analyte sensor of the present disclosure may include two or more active regions, with only one active region (e.g., an aspartate-responsive active region or an asparagine-responsive active region) including an electron transfer agent. In certain embodiments, the analyte sensor of the present disclosure may include two or more active regions, with each active region including an electron transfer agent. For example, without limitation, the analyte sensor of the present disclosure may include a sensor tail having at least a first working electrode and a second working electrode, an aspartate-responsive active region or an asparagine-responsive active region disposed on the surface of the first working electrode and including a first electron transfer agent, and a second analyte-responsive active region disposed on the surface of the second working electrode and including at least one enzyme responsive to a second analyte and a second electron transfer agent. In certain embodiments, the first electron transfer agent and the second electron transfer agent are the same. Alternatively, the first electron transfer agent and the second electron transfer agent are different.

[0222] A suitable electron transfer agent can facilitate the transfer of electrons to an adjacent working electrode after the analyte undergoes an enzymatic redox reaction within the corresponding active area, thereby generating a current indicative of the presence of a particular analyte, the amount of current generated being proportional to the amount of analyte present.

[0223] In certain embodiments, suitable electron transfer agents may include electroreducible and electrooxidizable ions, complexes, or molecules (e.g., quinones) that have redox potentials several hundred millivolts higher or lower than the redox potential of a standard calomel electrode (SCE). In certain embodiments, redox mediators may include osmium complexes and other transition metal complexes, such as those described in U.S. Pat. Nos. 6,134,461 and 6,605,200, which are incorporated herein by reference in their entireties. Further examples of suitable redox mediators include those described in U.S. Pat. Nos. 6,736,957, 7,501,053, and 7,754,093, the disclosures of each of which are also incorporated herein by reference in their entireties. Other examples of suitable redox mediators include metal compounds or complexes of ruthenium, osmium, iron (e.g., polyvinylferrocene or hexacyanoferrate), or cobalt, including, for example, metallocene compounds thereof. Suitable ligands for metal complexes may also include bidentate or higher ligands, such as, for example, bipyridine, biimidazole, phenanthroline, or pyridyl (imidazole). Other suitable bidentate ligands may include, for example, amino acids, oxalic acid, acetylacetone, diaminoalkanes, or o-diaminoarenes. Any combination of monodentate, bidentate, tridentate, tetradentate, or higher dentate ligands may be present in a metal complex (e.g., an osmium complex) to achieve a complete coordination sphere. In certain embodiments, the electron transfer agent is an osmium complex. In certain embodiments, the electron transfer agent is osmium complexed with a bidentate ligand.

[0224] In certain embodiments, the electron transfer agents disclosed herein may comprise suitable functional groups to facilitate covalent attachment to a polymer (referred to herein as a polymer backbone) in the active region, as discussed further below. For example, without limitation, the electron transfer agents for use in the present disclosure may include polymer-bound electron transfer agents. Non-limiting examples of suitable polymer-bound electron transfer agents include those described in U.S. Pat. Nos. 8,444,834, 8,268,143, and 6,605,201, the disclosures of which are incorporated herein by reference in their entireties. In certain embodiments, the electron transfer agent is a bidentate osmium complex bound to a polymer described herein. In certain embodiments, the electron transfer agent is a bidentate osmium complex bound to a polymer described herein, such as a polymer backbone described in Section 4 below. In certain embodiments, the polymer-bound electron transfer agents shown in FIG. 3 of U.S. Pat. No. 8,444,834 may be used in the sensors of the present disclosure.

[0225] In certain embodiments, an analyte sensor of the present disclosure can include a sensor tail comprising a working electrode and an aspartate-responsive active region disposed on a surface of the working electrode and comprising aspartate oxidase and an electron transfer agent.

[0226] In certain embodiments, an analyte sensor of the present disclosure can include a sensor tail comprising a working electrode and an asparagine-responsive active region disposed on a surface of the working electrode and comprising asparaginase, aspartate oxidase, and an electron transfer agent. In certain embodiments, the asparagine-responsive active region comprises a first enzyme layer comprising aspartate oxidase and an electron transfer agent, and a second layer comprising asparaginase and disposed on the first enzyme layer. Alternatively or additionally, the aspartate oxidase, asparaginase, and electron transfer agent are held within the same enzyme layer.

[0227] In certain embodiments, the aspartate-responsive active region or asparagine-responsive active region of the present disclosure has a ribozyme activity of about 100:1 to about 1:100, e.g., about 95:1 to about 1:95, about 90:1 to about 1:90, about 85:1 to about 1:85, about 80:1 to about 1:80, about 75:1 to about 1:75, about 60:1 to about 1:60, about 55:1 to about 1:55, about 50:1 to about 1:50, about 45:1 to about 1:45, about 40:1 to about 1:40, 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, or about 2:1 to about 1:2. In certain embodiments, the aspartate-responsive active region or asparagine-responsive active region may comprise a ratio of aspartate oxidase and / or asparaginase to redox mediator of about 7:1 to about 1:7. In certain embodiments, the aspartate-responsive active region or asparagine-responsive active region can comprise a ratio of aspartate oxidase and / or asparaginase to redox mediator of about 6:1 to about 1:6. In certain embodiments, the aspartate-responsive active region or asparagine-responsive active region can comprise a ratio of aspartate oxidase and / or asparaginase to redox mediator of about 5:1 to about 1:5. In certain embodiments, the aspartate-responsive active region or asparagine-responsive active region can comprise a ratio of aspartate oxidase and / or asparaginase to redox mediator of about 4:1 to about 1:4. In certain embodiments, the aspartate-responsive active region or asparagine-responsive active region can comprise a ratio of aspartate oxidase and / or asparaginase to redox mediator of about 3:1 to about 1:3.In certain embodiments, the aspartate-responsive active region or asparagine-responsive active region can comprise a ratio of aspartate oxidase and / or asparaginase to redox mediator of about 2:1 to about 1:2. In certain embodiments, the aspartate-responsive active region or asparagine-responsive active region can comprise a ratio of aspartate oxidase and / or asparaginase to redox mediator of about 1:1. In certain embodiments, the analyte-responsive active region, e.g., the aspartate-responsive active region or asparagine-responsive active region, can comprise about 10% to about 50% by weight of redox mediator, e.g., about 15% to about 45%, about 20% to about 40%, about 20% to about 35%, or about 20% to about 30% by weight of redox mediator. In certain embodiments, the analyte-responsive active region, e.g., the aspartate-responsive active region or the asparagine-responsive active region, can comprise about 5% to about 35% by weight of the redox mediator. In certain embodiments, the analyte-responsive active region, e.g., the aspartate-responsive active region or the asparagine-responsive active region, can comprise about 10% to about 35% by weight of the redox mediator. In certain embodiments, the analyte-responsive active region, e.g., the aspartate-responsive active region or the asparagine-responsive active region, can comprise about 10% to about 30% by weight of the redox mediator. In certain embodiments, the analyte-responsive active region, e.g., the aspartate-responsive active region or the asparagine-responsive active region, can comprise about 15% to about 35% by weight of the redox mediator.

[0228] 4. Polymer backbone In certain embodiments, one or more active sites for facilitating detection of an analyte may include a polymer to which an enzyme and / or a redox mediator is covalently attached. Any suitable polymer backbone may be present in the active region to facilitate detection of an analyte via covalent attachment of an enzyme and / or a redox mediator thereto. Non-limiting examples of suitable polymers in the active region include polyvinylpyridines, such as poly(4-vinylpyridine) and / or poly(2-vinylpyridine), and polyvinylimidazoles, such as poly(N-vinylimidazole) and poly(1-vinylimidazole), or copolymers thereof, for example, where quaternized pyridine groups serve as attachment points for a redox mediator or enzyme thereto. Exemplary copolymers that may be suitable for inclusion in the active region include those that include monomer units such as, for example, styrene, acrylamide, methacrylamide, or acrylonitrile. In certain embodiments, the polymer is a copolymer of vinylpyridine and styrene. In certain embodiments, polymers that may be present in the active region include polyurethane or copolymers thereof and / or polyvinylpyrrolidone. Further non-limiting examples of polymers that may be present in the active region include 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(vinylbenzyl chloride), poly(allylamine), polylysine, poly(4-vinylpyridine) quaternized with carboxypentyl groups, and poly(sodium 4-styrenesulfonate). In certain embodiments in which the analyte sensor includes two active sites, the polymers in each active region may be the same or different.

[0229] In certain embodiments, the polymer is a polyvinylpyridine-based polymer. In certain embodiments, the polymer is polyvinylpyridine or a copolymer thereof. In certain embodiments, the polymer is a copolymer of vinylpyridine and styrene.

[0230] In certain embodiments, one or more enzymes in the analyte-responsive active region can be covalently bound to a polymer. In certain embodiments, one or more enzymes in the aspartate-responsive active region can be covalently bound to a polymer. For example, but not limited to, aspartate oxidase can be covalently bound to a polymer in the aspartate-responsive active region.

[0231] In certain embodiments, when an enzyme system including multiple enzymes is present in a given active area, all of the multiple enzymes may be covalently bound to the polymer. In certain other embodiments, only a portion of the multiple enzymes are covalently bound to the polymer. For example, but not limited to, one or more enzymes in the enzyme system may be covalently bound to the polymer and at least one enzyme may be non-covalently bound to the polymer, thereby physically retaining the non-covalently bound enzyme within the polymer. For example, but not limited to, aspartate oxidase may be covalently bound to a polymer in the asparagine-responsive active area of ​​the disclosed analyte sensor. In certain embodiments, aspartate oxidase may be covalently bound to a polymer in the aspartate-responsive active area of ​​the disclosed analyte sensor. In certain embodiments, asparaginase may be covalently bound to a polymer in the asparagine-responsive active area. In certain embodiments, aspartate oxidase may be covalently bound to a polymer and asparaginase may be non-covalently bound to a polymer. Alternatively, the asparaginase may be covalently bound to the polymer and the aspartate oxidase may be non-covalently bound to the polymer. In certain embodiments where one or more enzymes are not covalently bound, the enzymes may be physically retained within the aspartate- or asparagine-responsive active region.

[0232] In certain embodiments, a membrane coating an aspartate- or asparagine-responsive active region can help retain one or more enzymes within the aspartate- or asparagine-responsive active region while still allowing sufficient inward diffusion of aspartate or asparagine to allow detection of aspartate or asparagine. Suitable membrane polymers for coating analyte-responsive active regions are discussed further herein.

[0233] In certain embodiments, when a stabilizer is present in the active region, one or more enzymes in the region may be covalently bound to the stabilizer. For example, but not limited to, one or more enzymes may be covalently bound to a stabilizer, such as albumin, present in the active region. In certain embodiments, aspartate oxidase present in the active region of the present disclosure may be covalently bound to a stabilizer. In certain embodiments, asparaginase present in the active region of the present disclosure may be covalently bound to a stabilizer.

[0234] In certain embodiments, the covalent bonding of one or more enzymes and / or redox mediators to the polymer and / or stabilizer in a given active area can occur via crosslinking introduced by a suitable crosslinking agent. In certain embodiments, crosslinking of the polymer and / or stabilizer to the one or more enzymes and / or redox mediators can reduce the occurrence of delamination of the enzyme composition from the electrode. Suitable crosslinking agents can include one or more crosslinkable functional groups, such as, but not limited to, vinyl, alkoxy, acetoxy, enoxy, oxime, amino, hydroxyl, cyano, halo, acrylate, epoxide, and isocyanate groups. In certain embodiments, the crosslinking agent comprises one or more, two or more, three or more, or four or more epoxide groups. For example, but not limited to, crosslinking agents for use in the present disclosure can include mono-, di-, tri-, and tetra-ethylene oxide. In certain embodiments, crosslinkers for reaction with free amino groups in an enzyme (e.g., with the free side chain amine in lysine) can include crosslinkers such as, for example, polyethylene glycol dibutyl ethers, polypropylene glycol dimethyl ethers, polyalkylene glycol allyl methyl ethers, polyethylene glycol diglycidyl ether (PEGDGE), or other polyepoxides, cyanuric chloride, N-hydroxysuccinimide, imidoesters, epichlorohydrin, or derivatized variants thereof. In certain embodiments, the crosslinker has an average molecular weight (M) of, for example, about 200 to 1000, e.g., about 400. n ) is PEGDGE. In certain embodiments, the crosslinker is PEGDGE400. In certain embodiments, the crosslinker can be glutaraldehyde. In certain embodiments, the crosslinking of the enzyme to the polymer is generally intermolecular. In certain embodiments, the crosslinking of the enzyme to the polymer is generally intramolecular.

[0235] In certain embodiments, the aspartate- and / or asparagine-responsive active region can comprise a ratio of crosslinker to one or more enzymes in the active region, e.g., asparagine and / or aspartate oxidase, of about 100:1 to about 1:100. In certain embodiments, the aspartate- and / or asparagine-responsive active region may comprise a ratio of crosslinker to one or more enzymes of the active region, e.g., asparagine and / or aspartate oxidase, of about 40:1 to about 1:40, e.g., about 35:1 to about 1:35, about 30:1 to about 1:30, about 25:1 to about 1:25, about 20:1 to about 1:20, about 15:1 to about 1:15, about 10:1 to about 1:10, about 9:1 to about 1:9, about 8:1 to about 1:8, about 7:1 to about 1:7, about 6:1 to about 1:6, about 5:1 to about 1:5, about 4:1 to about 1:4, about 3:1 to about 1:3, about 2:1 to about 1:2, or about 1:1. In certain embodiments, the aspartate- and / or asparagine-responsive active region can comprise from about 5% to about 50% by weight of crosslinker, e.g., from about 5% to about 45% by weight, from about 5% to about 40% by weight, from about 5% to about 35% by weight, from about 10% to about 30% by weight, or from about 10% to about 25% by weight.

[0236] 5. Mass-transfer limiting membrane In certain embodiments, the analyte sensor disclosed herein further comprises a membrane covering at least one active area, e.g., the first active area and / or the second active area, and permeable to one or more analytes. For example, but not by way of limitation, the membrane is permeable to aspartate and / or asparagine. In certain embodiments, the membrane covers each of the active areas of the analyte sensor. Alternatively, the first membrane covers one of the active areas and the second membrane covers the second active area. Alternatively, the first membrane covers one of the active areas and the second membrane covers both the first and second active areas.

[0237] In certain embodiments, the membrane covering the analyte-responsive active region may function as a mass transport limiting membrane and / or to improve biocompatibility. The mass transport limiting membrane may act as a diffusion limiting barrier to reduce the rate of mass transport of the analyte. For example, but not limited to, the mass transport limiting membrane may help to avoid sensor overloading (saturation) by restricting the access of the analyte, such as aspartate or asparagine, to the analyte-responsive active region, thereby improving detection performance and accuracy.

[0238] In certain embodiments, the mass transport limiting membrane may be homogeneous and may be single component (comprising a single membrane polymer). Alternatively, the mass transport limiting membrane may be multi-component (comprising two or more different membrane polymers). In certain embodiments, the multi-component membrane may exist as a bilayer membrane or as a homogeneous mixture of two or more membrane polymers. A homogeneous mixture may be deposited by mixing two or more membrane polymers in a solution and then depositing (e.g., dip coating) the solution onto the working electrode.

[0239] In certain embodiments, the mass transport limiting membrane may include two or more layers, for example, a bilayer or trilayer membrane. In certain embodiments, each layer may comprise different polymers or the same polymer, but at different concentrations or thicknesses. In certain embodiments, the first analyte responsive active area may be covered by a multilayer membrane, for example, a bilayer membrane, and the second analyte responsive active area may be covered by a single membrane. In certain embodiments, the first analyte responsive active area may be covered by a multilayer membrane, for example, a bilayer membrane, and the second analyte responsive active area may be covered by a multilayer membrane, for example, a bilayer membrane. In certain embodiments, the first analyte responsive active area may be covered by a single membrane and the second analyte responsive active area may be covered by a multilayer membrane, for example, a bilayer membrane may be covered by a single membrane. In certain embodiments, the first analyte responsive active area may be covered by a single membrane and the second analyte responsive active area may be covered by a single membrane.

[0240] In certain embodiments, the mass transport limiting membrane may include a polymer containing a heterocyclic nitrogen group. In certain embodiments, the mass transport limiting membrane may include polyvinylpyridine, polyvinylimidazole, copolymers of vinylpyridine and styrene, polyurethane or polyetherurethane, or chemically related materials, or membranes made of silicone, etc. In certain embodiments, the mass transport limiting membrane may include a polyvinylpyridine-based polymer. Non-limiting examples of polyvinylpyridine-based polymers are disclosed in U.S. Patent Publication No. 2003 / 0042137 (e.g., Formula 2b), the contents of which are incorporated herein by reference in their entirety.

[0241] In certain embodiments, the mass transport limiting membrane can comprise polyvinylpyridine (e.g., poly(4-vinylpyridine) or poly(4-vinylpyridine), polyvinylimidazole, polyvinylpyridine copolymers (e.g., copolymers of vinylpyridine and styrene), polyacrylates, polyurethanes, polyetherurethanes, silicones, polytetrafluoroethylene, polyethylene-co-tetrafluoroethylene, polyolefins, polyesters, polycarbonates, biostable polytetrafluoroethylene, homopolymers, copolymers, or terpolymers of polyurethanes, polypropylenes, polyvinyl chloride, polyvinylidene difluoride, polybutylene terephthalate, polymethyl methacrylate, polyether ether ketones, cellulose polymers, polysulfones, and block copolymers thereof, including, for example, diblock, triblock, alternating, random, and graft copolymers, or other chemically related materials.

[0242] In certain embodiments, membranes, e.g., single component membranes, for use in the present disclosure may include polyvinylpyridine (e.g., poly(4-vinylpyridine) and / or poly(2-vinylpyridine). In certain embodiments, membranes, e.g., single component membranes, for use in the present disclosure may include poly(4-vinylpyridine). In certain embodiments, membranes, e.g., single component membranes, for use in the present disclosure may include a copolymer of vinylpyridine and styrene. In certain embodiments, the membrane may comprise a polyvinylpyridine-co-styrene copolymer. For example, but not limited to, polyvinylpyridine-co-styrene copolymers for use in the present disclosure may include The polymer may include a polyvinylpyridine-co-styrene copolymer in which a portion of the pyridine nitrogen atoms are functionalized with a non-crosslinked polyethylene glycol tail and a portion of the pyridine nitrogen atoms are functionalized with an alkylsulfonic acid, e.g., propylsulfonic acid group. In certain embodiments, the derivatized polyvinylpyridine-co-styrene copolymer for use as a membrane polymer may be the 10Q5 polymer described in U.S. Patent No. 8,761,857, the contents of which are incorporated herein by reference in their entirety. In certain embodiments, the polyvinylpyridine-based polymer has a molecular weight of about 50 Da to about 500 kDa.

[0243] Suitable copolymers of vinylpyridine and styrene may have a styrene content ranging from about 0.01% to about 50% mole percent, or from about 0.05% to about 45% mole percent, or from about 0.1% to about 40% mole percent, or from about 0.5% to about 35% mole percent, or from about 1% to about 30% mole percent, or from about 2% to about 25% mole percent, or from about 5% to about 20% mole percent. Substituted styrenes may be used in similar amounts as well. Suitable copolymers of vinylpyridine and styrene may have a molecular weight of 5 kDa or more, or about 10 kDa or more, or about 15 kDa or more, or about 20 kDa or more, or about 25 kDa or more, or about 30 kDa or more, or about 40 kDa or more, or about 50 kDa or more, or about 75 kDa or more, or about 90 kDa or more, or about 100 kDa or more. In non-limiting examples, suitable copolymers of vinylpyridine and styrene can have a molecular weight ranging from about 5 kDa to about 150 kDa, or from about 10 kDa to about 125 kDa, or from about 15 kDa to about 100 kDa, or from about 20 kDa to about 80 kDa, or from about 25 kDa to about 75 kDa, or from about 30 kDa to about 60 kDa.

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

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

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

[0247] In certain embodiments, hydrophilic or hydrophobic modifiers can be used to "fine tune" the permeability of the resulting membrane to an analyte of interest. In certain embodiments, hydrophilic modifiers such as poly(ethylene) glycol, hydroxyl or polyhydroxyl modifiers, and any combination thereof, can be used to improve the biocompatibility of the polymer or the resulting membrane.

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

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

[0250] In certain embodiments, the composition of a mass transport limiting membrane disposed on an analyte sensor having two active regions may be the same or different when a mass transport limiting membrane covers each active region. For example, but not by way of limitation, the portion of the mass transport limiting membrane covering the aspartate- or asparagine-responsive active region may be multi-component and / or the portion of the mass transport limiting membrane covering the second analyte-responsive active region may be single-component. Alternatively, the portion of the mass transport limiting membrane covering the aspartate- or asparagine-responsive active region may be single-component and / or the portion of the mass transport limiting membrane covering the second analyte-responsive active region may be multi-component.

[0251] In certain embodiments, the membrane, e.g., the single-component membrane, can comprise polyvinylpyridine. In certain embodiments, the membrane, e.g., the single-component membrane, can comprise a copolymer of vinylpyridine and styrene. In certain embodiments of the present disclosure, the aspartate or asparagine-responsive active area can be coated with a multi-component membrane comprising at least two polymers, e.g., polyvinylpyridine and polyvinylpyridine-co-styrene copolymer, either as a bilayer membrane or a homogeneous mixture, and the second analyte-responsive active area can be coated with a membrane comprising a single polymer, e.g., polyvinylpyridine-co-styrene copolymer.

[0252] In certain embodiments, the mass transport limiting membrane can comprise a membrane polymer crosslinked with a crosslinking agent as disclosed herein and in Section 4 above. In certain embodiments where there are two mass transport limiting membranes, e.g., a first mass transport limiting membrane and a second mass transport limiting membrane, each membrane can be crosslinked to a different crosslinking agent. For example, but not by way of limitation, the crosslinking agent can result in a membrane that is more restrictive of the diffusion of a particular compound, e.g., an analyte, in the membrane, e.g., by affecting the size of the pores in the membrane, or that is less restrictive of the diffusion of a particular compound. For example, but not by way of limitation, in a sensor configured to detect aspartate and / or asparagine, the mass transport limiting membrane covering the aspartate and / or asparagine active region can have a pore size that restricts the diffusion of compounds larger than aspartate and / or asparagine through the membrane.

[0253] In certain embodiments, crosslinkers for use in the present disclosure may include polyepoxides, carbodiimides, cyanuric chloride, triglycidylglycerol (Gly3), N-hydroxysuccinimide, imide esters, epichlorohydrin, or derivatized variants thereof. In certain embodiments, the membrane polymers covering one or more active regions may be crosslinked to a branched crosslinker that may, for example, reduce the amount of extractables obtained from the mass transport limiting membrane. Non-limiting examples of branched crosslinkers include branched glycidyl ether crosslinkers, including, for example, branched glycidyl ether crosslinkers that include two, three, or more crosslinkable groups. In certain embodiments, the branched crosslinker may include two or more crosslinkable groups, such as polyethylene glycol diglycidyl ether. In certain embodiments, the branched crosslinker may include three or more crosslinkable groups, such as polyethylene glycol tetraglycidyl ether. In certain embodiments, the mass transport limiting membrane may comprise polyvinylpyridine or a copolymer of vinylpyridine and styrene crosslinked to a branched glycidyl ether crosslinker containing two or three crosslinkable groups, such as polyethylene glycol tetraglycidyl ether or polyethylene glycol diglycidyl ether. In certain embodiments, the epoxide group of a polyepoxide, such as polyethylene glycol tetraglycidyl ether or polyethylene glycol diglycidyl ether, can form a covalent bond with a pyridine or imidazole by opening the epoxide ring, resulting in a hydroxyalkyl group bridging the body of the crosslinker with the heterocycle of the membrane polymer.

[0254] In certain other embodiments, the membrane polymer covering one or more active regions may be crosslinked with a branched crosslinker containing three or more crosslinkable groups (e.g., polyethylene glycol tetraglycidyl ether), which, as described above, may reduce the amount of extractables that may be obtained from the mass transport limiting membrane. In certain embodiments, the mass transport limiting membrane may comprise a polyvinylpyridine or a copolymer of vinylpyridine and styrene crosslinked to a branched glycidyl ether crosslinker containing three crosslinkable groups, such as polyethylene glycol tetraglycidyl ether. In certain embodiments, the epoxide group of the polyethylene glycol tetraglycidyl ether may form a covalent bond with pyridine or imidazole by opening the epoxide ring, resulting in a hydroxyalkyl group bridging the body of the crosslinker with the heterocycle of the membrane polymer.

[0255] In certain embodiments, the crosslinker is polyethylene glycol diglycidyl ether (PEGDGE). In certain embodiments, the PEGDGE used to facilitate crosslinking (e.g., intermolecular crosslinking) between two or more membrane polymer backbones may exhibit a wide range of suitable molecular weights. In certain embodiments, the molecular weight of the PEGDGE may range from about 100 g / mol to about 5000 g / mol. The number of ethylene glycol repeat units in each arm of the PEGDGE may be the same or different, and generally may vary over a range within a given sample from which the average molecular weight is obtained. In certain embodiments, the PEGDGE for use in the present disclosure has an average molecular weight (M n In certain embodiments, the crosslinker is PEGDGE400.

[0256] In certain embodiments, the polyethylene glycol tetraglycidyl ether used to promote cross-linking (e.g., intermolecular cross-linking) between two or more membrane polymer backbones may exhibit a wide range of suitable molecular weights. Up to four polymer backbones may be cross-linked by a single molecule of polyethylene glycol tetraglycidyl ether cross-linker. In certain embodiments, the molecular weight of the polyethylene glycol tetraglycidyl ether may range from about 1000 g / mol to about 5000 g / mol. The number of ethylene glycol repeat units in each arm of the polyethylene glycol tetraglycidyl ether may be the same or different, and generally may vary over a range within a given sample for which the average molecular weight is obtained. In certain embodiments, the mass transport limiting membrane may be deposited directly on the active region.

[0257] In certain embodiments, polydimethylsiloxane (PDMS) may be incorporated into any of the mass transport limiting membranes disclosed herein. In certain embodiments, the analyte sensor described herein may comprise a sensor tail comprising at least a first working electrode, a first analyte-responsive active area disposed on a surface of the first working electrode for detecting the first analyte, and a mass transport limiting membrane covering at least the first analyte-responsive active area and permeable to the first analyte. In certain embodiments, the first analyte-responsive active area comprises an enzyme system responsive to a first analyte, e.g., aspartate and / or asparagine, including at least one enzyme responsive to the first analyte. In certain embodiments, the first analyte-responsive active area comprises an electron transfer agent and / or a first polymer (optionally, the enzyme present in the first analyte-responsive active site is covalently attached to the first polymer).

[0258] In certain embodiments, the analyte sensor described herein can comprise a sensor tail comprising at least a first working electrode, at least one aspartate-responsive active region disposed on a surface of the first working electrode and comprising aspartate oxidase (the enzyme can optionally be covalently bound to a polymer), and a mass transport limiting membrane covering the aspartate-responsive active region and permeable to aspartate. In certain embodiments, the mass transport limiting membrane comprises a membrane polymer crosslinked with a branched glycidyl ether crosslinker comprising three or more crosslinkable groups. In certain embodiments, the aspartate-responsive active region further comprises an electron transfer agent.

[0259] In certain embodiments, the analyte sensor described herein may comprise a sensor tail comprising at least a first working electrode, at least one asparagine-responsive active region disposed on the surface of the first working electrode and comprising an enzyme system comprising aspartate oxidase and asparaginase (one or both enzymes can be optionally covalently bound to the polymer), and a mass transport limiting membrane covering the asparagine-responsive active region and permeable to asparagine. In certain embodiments, the aspartate oxidase is present in a first layer and the asparaginase is present in a second layer. In certain embodiments, the aspartate oxidase and asparaginase are present in a single layer. In certain embodiments, the mass transport limiting membrane comprises a membrane polymer crosslinked with a branched glycidyl ether crosslinker comprising three or more crosslinkable groups. In certain embodiments, the asparagine-responsive active region further comprises an electron transfer agent.

[0260] In certain embodiments, the analyte sensor of the present disclosure can include a second active region configured to detect the same analyte as the first active region or a different analyte. In certain embodiments, at least a portion of the mass transport limiting membrane covering the first active region can cover the second active region. Alternatively or in addition, a second mass transport limiting membrane can be used to cover the second active region. In certain embodiments, at least a portion of the second mass transport limiting membrane covering the second active region can cover the first active region. In certain embodiments, the mass transport limiting membrane covering the first active region is of a different composition than the second mass transport limiting membrane.

[0261] In certain embodiments, when a first active area and a second active area configured to assay different analytes are placed on separate working electrodes, the mass transport limiting membrane may have different permeability values ​​for the first analyte and the second analyte. For example, but not by way of limitation, the mass transport limiting membrane covering at least one of the active areas may include a mixture of a first membrane polymer and a second membrane polymer, or a bilayer of a first membrane polymer and a second membrane polymer. A homogeneous membrane may cover the active areas not covered by the mixture or bilayer, the homogeneous membrane including only one of the first membrane polymer or the second membrane polymer. Advantageously, the architecture of the analyte sensor disclosed herein allows for a continuous membrane having a homogeneous membrane portion to be placed on the first active area of ​​the analyte sensor, and a multi-component membrane portion to be placed on the second active area of ​​the analyte sensor, whereby the permeability values ​​for each analyte are simultaneously equalized, improving sensitivity and detection accuracy. In certain embodiments, successive film depositions can be accomplished by successive dip coating operations.

[0262] In certain embodiments, the mass transport limiting membrane has a thickness, e.g., a dry thickness, in the range of about 0.1 μm to about 1000 μm, e.g., about 1 μm to about 500 μm, about 10 μm to about 100 μm, or about 10 μm to about 100 μm. In certain embodiments, the mass transport limiting membrane can have a thickness of about 0.1 μm to about 100 μm, e.g., about 1 μm to about 90 μm, about 1 μm to about 80 μm, about 1 μm to about 70 μm, about 1 μm to about 60 μm, about 1 μm to about 50 μm, about 1 μm to about 40 μm, about 1 μm to about 30 μm, about 1 μm to about 20 μm, 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 mass transport limiting membrane can have a thickness of about 1 μm to about 100 μm.

[0263] 6. Interference Domains In certain embodiments, the sensor of the present disclosure, e.g., the sensor tail, may further comprise an interference domain. In certain embodiments, the interference domain may include, for example, a polymer domain that restricts the flow of one or more interferents to the surface of the working electrode. In certain embodiments, the interference domain may function as a molecular sieve that allows the passage of the analyte and other substances measured by the working electrode while preventing the passage of other substances, such as interferents. In certain embodiments, the interferents may affect the signal obtained at the working electrode. Non-limiting examples of interferents include acetaminophen, ascorbate, ascorbic acid, bilirubin, cholesterol, creatinine, dopamine, ephedrine, ibuprofen, L-dopa, methyldopa, salicylate, tetracycline, tolazamide, tolbutamide, triglycerides, urea, and uric acid.

[0264] In certain embodiments, the interference domain is located between the working electrode and one or more active regions, such as the aspartate-responsive active region and / or the asparagine-responsive active region. In certain embodiments, non-limiting examples of polymers that can be used in the interference domain include polyurethanes, polymers with pendant ionic groups, and polymers with controlled pore size. In certain embodiments, the interference domain is formed from one or more cellulosic derivatives. Non-limiting examples of cellulosic derivatives include polymers such as cellulose acetate, cellulose acetate butyrate, 2-hydroxyethyl cellulose, cellulose acetate phthalate, cellulose acetate propionate, cellulose acetate trimellitate, and the like.

[0265] In certain embodiments, the interference domain is part of the mass transport limiting membrane and is not a separate membrane, hi certain embodiments, the interference domain is located between one or more active regions and the mass transport limiting membrane.

[0266] In certain embodiments, the interference domain comprises a thin, hydrophobic membrane that is non-swelling and limits the diffusion of high molecular weight species, for example, but not by way of limitation, the interference domain can be permeable to relatively low molecular weight substances while restricting the passage of higher molecular weight substances.

[0267] In certain embodiments, the interference domain may be deposited directly onto the working electrode, e.g., onto the surface of a permeable working electrode. In certain embodiments, the interference domain has a thickness, e.g., a dry thickness, in the range of about 0.1 μm to about 1000 μm, e.g., about 1 μm to about 500 μm, about 10 μm to about 100 μm, or about 10 μm to about 100 μm. In certain embodiments, the interference domain can have a thickness of about 0.1 μm to about 100 μm, e.g., about 1 μm to about 90 μm, about 1 μm to about 80 μm, about 1 μm to about 70 μm, about 1 μm to about 60 μm, about 1 μm to about 50 μm, about 1 μm to about 40 μm, about 1 μm to about 30 μm, about 1 μm to about 20 μm, about 0.5 μm to about 10 μm, about 1 μm to about 10 μm, about 1 μm to about 5 μm, or about 0.1 μm to about 5 μm. In certain embodiments, the sensor can be immersed in the solution of the interference domain more than once. For example, but not by way of limitation, the sensor (or working electrode) of the present disclosure can be immersed in the solution of the interference domain at least twice, at least three times, at least four times, or at least five times to obtain the desired thickness of the interference domain.

[0268] 7.Manufacturing The present disclosure further provides methods for manufacturing the disclosed analyte sensors that include one or more active sites for detecting aspartate or asparagine.

[0269] In certain embodiments, the method includes screen printing the working electrode. In certain embodiments, the method may further include adding a composition comprising an enzyme onto the surface of the working electrode to generate active sites on the working electrode. For example, but not limited to, the composition may include aspartate oxidase. In certain embodiments, the composition may further include asparaginase. In certain embodiments, the composition may further include an electron transfer agent. In certain embodiments, the composition may further include a crosslinker, e.g., polyethylene glycol diglycidyl ether, and a stabilizer, e.g., albumin, such as BSA. In certain embodiments, the method may further include curing the enzyme composition.

[0270] Alternatively, a first enzyme composition comprising aspartate oxidase can be first deposited on the surface of the working electrode to generate an analyte-responsive active area on the working electrode. In certain embodiments, the first enzyme composition can further comprise a redox mediator, a cross-linking agent, e.g., polyethylene glycol diglycidyl ether, and / or a stabilizer, e.g., albumin, such as BSA. In certain embodiments, the method can include curing the first enzyme composition to generate the first enzyme layer. In certain embodiments, the method can include depositing a second enzyme composition comprising asparaginase on the surface of the first enzyme layer and curing the second enzyme composition to generate the second enzyme layer. In certain embodiments, the second enzyme composition can include a cross-linking agent, e.g., polyethylene glycol diglycidyl ether, and / or a stabilizer, e.g., albumin, such as BSA.

[0271] In certain embodiments, the method may further include adding a film composition over the cured enzyme composition(s). In certain embodiments, the film composition may include a polymer, such as polyvinylpyridine, and / or a crosslinker, such as polyethylene glycol diglycidyl ether. In certain embodiments, the method may include curing the polymer composition.

[0272] In general, the thickness of the membrane is controlled by the concentration of the membrane solution, the number of droplets of the membrane solution applied, the number of times the sensor is dipped into the membrane solution or sprayed onto the sensor, the volume of the membrane solution sprayed onto the sensor, and the like, and any combination of these factors. In certain embodiments, the membranes described herein may have a thickness ranging from about 0.1 micrometers (μm) to about 1000 μm, such as from about 1 μm to about 500 μm, from about 10 μm to about 100 μm, or from about 10 μm to about 100 μm. In certain embodiments, the sensor may be dipped into the membrane solution more than once. For example, and without limitation, the sensor (or working electrode) of the present disclosure may be dipped into the membrane solution at least two times, at least three times, at least four times, at least five times, or at least six times to obtain a desired membrane thickness.

[0273] In certain embodiments, the film may cover one or more active regions, and in certain embodiments, the active regions may have a thickness of about 0.1 μm to about 100 μm, e.g., about 1 μm to about 90 μm, about 1 μm to about 80 μm, about 1 μm to about 70 μm, about 1 μm to about 60 μm, about 1 μm to about 50 μm, about 1 μm to about 40 μm, about 1 μm to about 30 μm, about 1 μm to about 20 μm, about 0.5 μm to about 10 μm, about 1 μm to about 10 μm, about 1 μm to about 5 μm, or about 0.1 μm to about 5 μm. In certain embodiments, a series of droplets may be applied on top of one another to achieve a desired thickness of the active regions and / or film without substantially increasing the diameter of the applied droplets (i.e., while maintaining the desired diameter or range). In certain embodiments, each single drop may be applied and then allowed to cool or dry, followed by application of one or more additional drops, for example, but not limited to, at least one drop, at least two drops, at least three drops, at least four drops, or at least five drops added on top of each other to achieve the desired thickness of the active region.

[0274] III.How to use The present disclosure further provides a method of using the analyte sensor disclosed herein. In certain embodiments, the present disclosure provides a method for detecting an analyte. For example, but not limited to, the present disclosure provides a method for detecting one or more analytes, including aspartate and / or asparagine, for example, in a subject in need thereof. In certain embodiments, the present disclosure provides a method for detecting aspartate. In certain embodiments, the present disclosure provides a method for detecting asparagine.

[0275] In certain embodiments, a method for detecting aspartate includes (i) providing an analyte sensor including (a) a sensor tail including at least a first working electrode, (b) at least one aspartate-responsive active region disposed on a surface of the working electrode and including aspartate oxidase and optionally a first polymer and / or a redox mediator, and (c) a mass transport limiting membrane covering the aspartate-responsive active region and permeable to aspartate. In certain embodiments, the method further includes (ii) applying a potential to the first working electrode, (iii) obtaining a first signal at or above the redox potential of the aspartate-responsive active region and proportional to the concentration of aspartate in a fluid contacting the aspartate-responsive active region, and (iv) correlating the first signal to the concentration of aspartate in the fluid, e.g., a bodily fluid.

[0276] In certain embodiments, a method for detecting aspartate may include (i) exposing an analyte sensor to a fluid comprising aspartate, the analyte sensor comprising: (a) a sensor tail comprising at least a first working electrode; (b) at least one aspartate-responsive active region disposed on a surface of the first working electrode and responsive to aspartate, the aspartate oxidase and optionally a first polymer and / or a redox mediator; and (c) a mass transport limiting membrane covering the aspartate-responsive active region and permeable to aspartate; (ii) applying a potential to the first working electrode; (iii) obtaining a first signal at or above the redox potential of the aspartate-responsive active region, the first signal being proportional to a concentration of aspartate in the fluid; and (iv) correlating the first signal to a concentration of aspartate in the fluid, e.g., a bodily fluid.

[0277] In certain embodiments, a method for detecting asparagine includes (i) providing an analyte sensor including (a) a sensor tail including at least a first working electrode, (b) at least one asparagine-responsive active region disposed on a surface of the working electrode and including an enzyme system including aspartate oxidase, asparaginase, and optionally a first polymer and / or a redox mediator, and (c) a mass transport limiting membrane covering the asparagine-responsive active region and permeable to asparagine. In certain embodiments, the method further includes (ii) applying a potential to the first working electrode, (iii) obtaining a first signal at or above the redox potential of the asparagine-responsive active region and proportional to the concentration of asparagine in a fluid contacting the asparagine-responsive active region, and (iv) correlating the first signal to the concentration of asparagine in the fluid, e.g., a bodily fluid. In certain embodiments, the asparagine-responsive active area comprises an aspartate oxidase layer disposed on the first working electrode and an asparaginase layer disposed on the aspartate oxidase layer. In certain embodiments, the aspartate oxidase layer comprises aspartate oxidase and a redox mediator, and the asparaginase layer comprises asparaginase. Alternatively, the asparagine-responsive active area comprises aspartate oxidase and asparaginase in a single enzyme layer disposed on the first working electrode.

[0278] In certain embodiments, a method for detecting asparagine includes (i) exposing an analyte sensor to a fluid comprising asparagine, the analyte sensor comprising: (a) a sensor tail comprising at least a first working electrode; (b) an asparagine-responsive active region disposed on a surface of the first working electrode and responsive to asparagine and comprising an enzyme system comprising aspartate oxidase, asparaginase, and optionally a first polymer and / or a redox mediator; and (c) a mass transport limiting membrane covering the asparagine-responsive active region and permeable to asparagine; (ii) applying a potential, e.g., a low potential, to the first working electrode; (iii) obtaining a first signal at or above the redox potential of the asparagine-responsive active region, the first signal being proportional to the concentration of asparagine in the fluid; and (iv) correlating the first signal to the concentration of asparagine in the fluid, e.g., a bodily fluid. In certain embodiments, the asparagine-responsive active area comprises an aspartate oxidase layer disposed on the first working electrode and an asparaginase layer disposed on the aspartate oxidase layer. In certain embodiments, the aspartate oxidase layer comprises aspartate oxidase and a redox mediator, and the asparaginase layer comprises asparaginase. Alternatively, the asparagine-responsive active area comprises aspartate oxidase and asparaginase in a single enzyme layer disposed on the first working electrode.

[0279] In certain embodiments, the membrane polymer comprises polyvinylpyridine or polyvinylimidazole. In certain embodiments, the membrane polymer comprises a copolymer of vinylpyridine and styrene. In certain embodiments, the mass transport limiting membrane of the analyte sensor comprises a membrane polymer crosslinked with a branched crosslinker comprising three or more crosslinkable groups. In certain embodiments, the branched crosslinker comprises polyethylene glycol tetraglycidyl ether.

[0280] IV. Illustrative Embodiments A. In certain non-limiting embodiments, the presently disclosed subject matter provides an analyte sensor comprising: (i) a first working electrode; (ii) an analyte-responsive active region disposed on a surface of the first working electrode, the analyte-responsive active region comprising aspartate oxidase for detecting the analyte; and (iii) a mass transport limiting membrane covering at least the analyte-responsive active region and permeable to the analyte.

[0281] A1. The analyte sensor of A, wherein the analyte responsive active region further comprises an electron transfer agent. A2. The analyte sensor of A or A1, wherein the analyte-responsive active region further comprises a stabilizer.

[0282] A3. The analyte sensor of any one of A2, wherein the stabilizer comprises albumin. A4. The test substance sensor according to A3, wherein the albumin is serum albumin. A5. The test substance sensor according to A4, wherein the serum albumin is human serum albumin.

[0283] A6. The analyte sensor of any one of A to A5, wherein the aspartate oxidase is covalently bound to a polymer within the analyte responsive active region. A7. The analyte sensor of any one of A to A6, wherein the analyte-responsive active region further comprises a cross-linking agent.

[0284] A8. The analyte sensor according to any one of A to A7, wherein the analyte is aspartate. A9. The analyte sensor of any one of A to A8, wherein the analyte responsive active region further comprises asparaginase.

[0285] A10. The test substance sensor according to A9, wherein the test substance is asparagine. A11. The analyte sensor of A9 or A10, wherein the asparaginase is covalently bound to a polymer within the analyte-responsive active region.

[0286] A12. The analyte sensor of any one of A to A11, wherein the mass transport limiting membrane comprises a polyvinylpyridine based polymer, polyvinylimidazole, polyacrylate, polyurethane, polyether urethane, silicone, or a combination thereof.

[0287] A13. The analyte sensor according to A12, wherein the mass transport limiting membrane comprises a polyvinylpyridine-based polymer. A14. The test substance sensor according to A13, wherein the polyvinylpyridine-based polymer is poly(2-vinylpyridine) or poly(4-vinylpyridine).

[0288] A15. The test substance sensor according to A13, wherein the polyvinylpyridine-based polymer is a polyvinylpyridine copolymer. A16. The analyte sensor according to A15, wherein the polyvinylpyridine copolymer is a copolymer of vinylpyridine and styrene.

[0289] A17. The analyte sensor of any one of A to A16, further comprising: (iv) a second working electrode; and (v) a second active area disposed on the surface of the second working electrode, responsive to a second analyte different from the first analyte, and comprising at least one enzyme responsive to the second analyte.

[0290] A18. The analyte sensor of A17, wherein a second portion of the mass transport limiting membrane covers the second active area. A19. The analyte sensor according to A17, further comprising a second mass transport limiting membrane covering the second active area.

[0291] A20. The analyte sensor according to A17, further comprising a second mass transport limiting membrane covering the second active area and the first active area. A21. A test substance sensor described in any one of A17 to A20, wherein the second test substance is selected from the group consisting of glucose, ketones, lactate, oxygen, hemoglobin A1C, albumin, alcohol, alkaline phosphatase, alanine transaminase, aspartate aminotransferase, bilirubin, blood urea nitrogen, calcium, carbon dioxide, chloride, creatinine, hematocrit, lactate, magnesium, oxygen, pH, phosphorus, potassium, sodium, aspartate, asparagine, total protein, uric acid, and combinations thereof.

[0292] A22. The analyte sensor of any one of A to A21, wherein the sensor tail is configured to be inserted into tissue. A23. The analyte sensor of any one of A to A22, wherein the sensor tail is configured to be inserted into tissue to detect analyte levels in vivo.

[0293] A24. An analyte sensor according to any one of A9 to A23, wherein the analyte responsive active region comprises a layer comprising asparaginase disposed on a layer comprising aspartate oxidase, or the analyte responsive active region comprises a single layer comprising aspartate oxidase and asparaginase.

[0294] B. In certain non-limiting embodiments, the presently disclosed subject matter provides methods for detecting aspartate comprising: (i) providing an analyte sensor comprising: (a) a sensor tail comprising at least a first working electrode; (b) at least one aspartate-responsive active region disposed on a surface of the first working electrode and comprising aspartate oxidase; and (c) a mass transport limiting membrane covering at least the aspartate-responsive active region and permeable to aspartate; (ii) applying a potential to the first working electrode; (iii) obtaining a first signal at or above the redox potential of the aspartate-responsive active region, the first signal being proportional to the concentration of aspartate in a fluid contacting the aspartate-responsive active region; and (iv) correlating the first signal to the concentration of aspartate in the fluid.

[0295] B1. The method of B, wherein the aspartate-responsive active region further comprises an electron transfer agent. B2. The method of B or B1, wherein the aspartate responsive active region further comprises a stabilizer.

[0296] B3. The method of B2, wherein the stabilizer comprises albumin. B4. The method of B3, wherein the albumin is serum albumin. B5. The method of B4, wherein the serum albumin is human serum albumin.

[0297] B6. The method of any one of B to B5, wherein the aspartate oxidase is covalently attached to a polymer within an analyte-responsive active region. B7. The method of any one of B to B6, wherein the aspartate-responsive activity region further comprises a cross-linking agent.

[0298] B8. The method of any one of B to B7, wherein the mass transport limiting membrane comprises a polyvinylpyridine based polymer, a polyvinylimidazole, a polyacrylate, a polyurethane, a polyether urethane, a silicone, or a combination thereof.

[0299] B9. The method of B8, wherein the mass transport limiting membrane comprises a polyvinylpyridine-based polymer. B10. The method of B9, wherein the polyvinylpyridine-based polymer is poly(2-vinylpyridine) or poly(4-vinylpyridine).

[0300] B11. The method of B9, wherein the polyvinylpyridine-based polymer is a polyvinylpyridine copolymer. B12. The method of B11, wherein the polyvinylpyridine copolymer is a copolymer of vinylpyridine and styrene.

[0301] B13. The method of any one of B to B12, further comprising: (d) a second working electrode; and (e) a second active area disposed on a surface of the second working electrode, responsive to a second analyte different from the first analyte, and comprising at least one enzyme responsive to the second analyte.

[0302] B14. The method of B13, wherein a second portion of the mass transport limiting membrane covers a second active area. B15. The method of B13, further comprising a second mass transport limiting membrane covering the second active area.

[0303] B16. The method of B13, further comprising a second mass transport limiting membrane covering the second active region and the first active region. B17. The method of any one of B13 to B16, wherein the second test substance is selected from the group consisting of glucose, ketones, lactate, oxygen, hemoglobin A1C, albumin, alcohol, alkaline phosphatase, alanine transaminase, aspartate aminotransferase, bilirubin, blood urea nitrogen, calcium, carbon dioxide, chloride, creatinine, hematocrit, lactate, magnesium, oxygen, pH, phosphorus, potassium, sodium, aspartate, asparagine, total protein, uric acid, and combinations thereof.

[0304] B18. The method of any one of B to B17, wherein the sensor tail is configured for insertion into tissue. B19. The method of any one of B to B18, wherein the sensor tail is configured to be inserted into tissue to detect aspartate levels in vivo.

[0305] C. In certain non-limiting embodiments, the presently disclosed subject matter provides methods for detecting asparagine comprising: (i) providing an analyte sensor comprising: (a) a sensor tail comprising at least a first working electrode; (b) an asparagine-responsive active region disposed on a surface of the first working electrode and comprising an enzyme system comprising aspartate oxidase and asparaginase; and (c) a mass transport limiting membrane covering at least the asparagine-responsive active region and permeable to asparagine; (ii) applying a potential to the first working electrode; (iii) obtaining a first signal at or above the redox potential of the asparagine-responsive active region, the first signal being proportional to an aspartate concentration in a fluid contacting the asparagine-responsive active region; and (iv) correlating the first signal to the asparagine concentration in the fluid.

[0306] C1. The method of C, wherein the asparagine-responsive active region further comprises an electron transfer agent. C2. The method of C or C1, wherein the asparagine-responsive active region further comprises a stabilizer.

[0307] C3. The method of any one of C2, wherein the stabilizer comprises albumin. C4. The method of C3, wherein the albumin is human serum albumin. C5. The method of any one of C to C4, wherein the asparaginase is covalently attached to a polymer within the analyte-responsive active region.

[0308] C6. The method of any one of C to C4, wherein the asparagine-responsive activity region further comprises a cross-linker. C7. The method of C6, wherein the crosslinker is polyethylene glycol diglycidyl ether (PEGDGE).

[0309] C8. The method of any one of C to C7, wherein the mass transport limiting membrane comprises a polyvinylpyridine based polymer, a polyvinylimidazole, a polyacrylate, a polyurethane, a polyether urethane, a silicone, or a combination thereof.

[0310] C9. The method of C8, wherein the mass transport limiting membrane comprises a polyvinylpyridine-based polymer. C10. The method of C9, wherein the polyvinylpyridine-based polymer is poly(2-vinylpyridine) or poly(4-vinylpyridine).

[0311] C11. The method of C9, wherein the polyvinylpyridine-based polymer is a polyvinylpyridine copolymer. C12. The method of C11, wherein the polyvinylpyridine copolymer is a copolymer of vinylpyridine and styrene.

[0312] C13. The method of any one of C to C12, further comprising: (d) a second working electrode; and (e) a second active area disposed on a surface of the second working electrode, responsive to a second analyte different from the first analyte, and comprising at least one enzyme responsive to the second analyte.

[0313] C14. The method of C13, wherein a second portion of the mass transport limiting membrane covers a second active area. C15. The method of C13, further comprising a second mass transport limiting membrane covering the second active area.

[0314] C16. The method of C13, further comprising a second mass transport limiting membrane covering the second active region and the first active region. C17. The method of any one of C13 to C16, wherein the second test substance is selected from the group consisting of glucose, ketones, lactate, oxygen, hemoglobin A1C, albumin, alcohol, alkaline phosphatase, alanine transaminase, aspartate aminotransferase, bilirubin, blood urea nitrogen, calcium, carbon dioxide, chloride, creatinine, hematocrit, lactate, magnesium, oxygen, pH, phosphorus, potassium, sodium, aspartate, asparagine, total protein, uric acid, and combinations thereof.

[0315] C18. The method of any one of C to C17, wherein the sensor tail is configured for insertion into tissue. C19. The method of any one of C to C18, wherein the sensor tail is configured to be inserted into tissue to detect levels of asparagine in vivo.

[0316] C20. The method of any one of C to C19, wherein the asparagine-responsive active region comprises a layer comprising asparaginase disposed above a layer comprising aspartate oxidase.

[0317] C21. The method of any one of C to C19, wherein the asparagine-responsive activity region comprises a monolayer comprising aspartate oxidase and asparaginase. C22. The method of any one of C to C21, wherein the aspartate oxidase is covalently attached to a polymer within an asparagine-responsive active region.

[0318] C23. The method of any one of C to C22, wherein the asparaginase is covalently attached to a polymer within an asparagine-responsive active region. Working Example The presently disclosed subject matter will be better understood by reference to the following examples, which are provided by way of illustration and not limitation of the presently disclosed subject matter.

[0319] Example 1: Aspartate Sensor This example provides a sensor for detecting aspartate, comprising an enzyme system comprising aspartate oxidase and a redox mediator as disclosed in FIG. 22. In this analyte sensor, the redox mediator is chemically crosslinked to the aspartate oxidase enzyme. The components in Table 1 were mixed and then deposited twice on the working electrode. After curing, some of the sensors were dipped into a homemade polyvinylpyridine polymer film composition (labeled 10Q5-Gly3) to form sensor films of different thicknesses (labeled 1×6, 2×6, and 3×6, where the first number is the number of dipping times into the dipping solution and the second number is the speed (mm / s) at which the sensor was withdrawn from the dipping solution).

[0320] [Table 1]

[0321] The sensor was tested under air and argon at 33° C. in the presence of 0.95 mM aspartate in 100 mM PBS solution to evaluate the effect of oxygen. Oxidase is an enzyme that catalyzes the oxidation of its substrate by oxygen, which is an undesirable side reaction because it competes with the oxidation reaction by the redox mediator. As shown in FIG. 24, the sensor initially showed a good response under air. The sensor membrane reduced the current, but not significantly. Subsequent argon purging did not change the current response of the sensor, indicating that the enzyme system is very efficient at transferring electrons from the oxidation of aspartate to the redox mediator rather than to oxygen.

[0322] The sensors were also tested with various concentrations of aspartate to evaluate the signal response. As shown in FIG. 25A, the current increased over the course of several minutes after exposure to a new concentration of aspartate and then stabilized. FIG. 25B shows the concentration vs. current curves, and FIG. 25C shows the calibration curves for each of the sensors, showing that the response is close to linear when the aspartate level is below 1000 μM. Notably, R 2The values ​​range from 0.9896 to 0.9939. Table 2 provides the sensitivity of the sensors tested with aspartate from 0 to 1000 μM.

[0323] [Table 2]

[0324] Example 2: Aspartate Sensor with Varying Amounts of Enzyme This example provides a sensor for detecting aspartate, comprising an enzyme system comprising aspartate oxidase and a redox mediator as shown in FIG. 22. In this analyte sensor, a redox mediator and an aspartate oxidase enzyme were loaded on the aspartate sensing layer, and the amount of enzyme was varied. The concentration of enzyme deposited on the sensor was varied 1X, 2X, 3X, and 7X. Table 3 provides the chemical composition of the sensor designated 1X in FIG. 26, Table 4 provides the chemical composition of the sensor designated 2X in FIG. 26, Table 5 provides the chemical composition of the sensor designated 3X, and Table 6 provides the chemical composition of the sensor designated 7X. The sensor designated 2X contains twice the amount of enzyme as the sensor designated 1X. For all formulations, the sensors were fabricated by depositing a mixture containing the components in Table 1 on the sensor working electrode two, four, or six times, which are labeled as 2 passes, 4 passes, and 6 passes, respectively, in FIG. 26 and Table 7. Other curing and membrane dipping procedures were the same as those described in Example 1. The membrane polymer used was polyvinylpyridine and the crosslinker was PEGDGE400 (denoted as PVP+P400).

[0325] [Table 3]

[0326] [Table 4]

[0327] [Table 5]

[0328] [Table 6]

[0329] It was found that 3X and 7X enzyme loading caused precipitation in the formulation mixture, and therefore such sensors were not prepared. To evaluate the signal response, 1X and 2X sensors were tested with various concentrations of aspartate (49.8μM, 99.5μM, 198μM, 392μM, 676μM and 952μM). As shown in FIG. 26, the current increased over the course of several minutes after exposure to the new concentration of aspartate and then stabilized. Furthermore, the sensor designated as 2X showed a stronger response in the presence of the new aspartate concentration compared to the 1X sensor. More deposition passes also increased the sensor current for both 1X and 2X enzyme concentrations. Table 7 summarizes the sensitivity of the sensors tested with and without the membrane (PVP+P400).

[0330] [Table 7]

[0331] Example 3 - Aspartate sensors with various amounts of redox mediator The aspartate sensor of Example 2 was tested with various amounts of redox mediator. In particular, the sensor was modified by doubling the amount of redox mediator and enzyme as shown in Table 8 compared to Table 4. In addition, the amount of redox mediator was reduced by 3X as shown in Table 9. Table 10 provides the composition of the aspartate sensing layer including reduced amounts of redox mediator and BSA.

[0332] [Table 8]

[0333] [Table 9]

[0334] [Table 10]

[0335] Sensors with 2X the amount of enzyme and 2X the amount of redox mediator were tested with various concentrations of aspartate to evaluate the signal response. As shown in FIG. 27, the current increased over the course of several minutes after exposure to the new concentration of aspartate and then stabilized. Table 11 provides the sensitivity (nA / mM) of the sensors tested with and without a membrane. As shown in Table 11, the amount of redox mediator has a higher impact on sensitivity than the amount of enzyme. This effect is more pronounced in the sensor without a membrane (PVP+P400).

[0336] [Table 11]

[0337] Example 4 - Asparagine sensor: Asparaginase layer on top of aspartate sensing layer This example provides an example of an asparagine sensor using an enzyme system comprising asparaginase, aspartate oxidase, and a redox mediator, as shown in FIG. 23. In this example, the asparagine sensor comprises an asparaginase layer on an aspartate sensing layer. The sensor was prepared by depositing an asparaginase layer comprising the components in Table 12 on the aspartate sensing layer composition described in Examples 1 and 2. The asparaginase used in this sensor was purchased from Sigma (catalog number A3809) and ProSpec (catalog number ENZ-287). A subset of the sensors was then immersed in a polyvinylpyridine polymer film solution as described in the previous examples.

[0338] [Table 12]

[0339] The membrane and non-membrane sensors were tested in the presence of various asparagine concentrations, including 50 μM, 100 μM, 200 μM, 400 μM, 700 μM, and 1000 μM. As shown in Figures 28A, 28B, and 28C, the current increases for several minutes after exposure to the new asparagine concentration before stabilizing, indicating that the sensor responds well to asparagine. Figure 28B shows the average of each type of sensor shown in Figure 28A. Figure 28C compares the response to aspartate and asparagine with the asparagine sensor made in this example. It is expected that the enzyme system of the asparagine sensor should also respond to aspartate, which is shown in Figure 28C.

[0340] Example 5 - Asparagine sensor: Asparaginase in aspartate sensing layer This example provides an example of an asparagine sensor using an enzyme system comprising asparaginase, aspartate oxidase, and a redox mediator (shown in FIG. 23), where asparaginase is included in the aspartate sensing layer. The asparaginase used was purchased from ProSpec (catalog number ENZ-287). The sensors were prepared by depositing a mixture containing the components in Table 13 onto the working electrode. A subset of the sensors was then immersed in the membrane solution.

[0341] [Table 13]

[0342] The sensors with and without the membrane were tested in the presence of various asparagine concentrations. As shown in Figure 29, the current increased over the course of several minutes after exposure to a new concentration of aspartate and then stabilized. These data indicate that the asparagine sensor containing asparaginase in the aspartate sensing layer also responds well to asparagine.

[0343] Although the disclosed subject matter and its advantages have been described in detail, it should be understood that various changes, substitutions, and alterations can be made herein without departing from the spirit and scope of the disclosed subject matter. Moreover, the scope of the present application is not intended to be limited to the particular embodiments of the processes, machines, manufacture, and compositions of matter, methods, and processes described herein. As one skilled in the art will readily appreciate from the disclosed subject matter of the presently disclosed subject matter, any now existing or later developed process, machine, manufacture, composition of matter, method, or step that performs substantially the same function or achieves substantially the same results as the corresponding embodiments described herein may be utilized in accordance with the presently disclosed subject matter. Accordingly, it is intended that the appended claims include within their scope such processes, machines, manufacture, compositions of matter, methods, or steps.

[0344] Various patents, patent applications, publications, product descriptions, protocols, and sequence accession numbers are cited throughout this application, the inventions of which are incorporated herein by reference in their entireties for all purposes.

Claims

1. 1. An analyte sensor configured for insertion into tissue, comprising: (i) at least a working electrode; (ii) at least one analyte responsive active area disposed on a surface of the working electrode for detecting an analyte, the analyte responsive active area comprising aspartate oxidase and an electron transfer agent, the weight ratio of the aspartate oxidase to the electron transfer agent being between 3:1 and 1:3; (iii) a mass transport limiting membrane permeable to the analyte and covering at least the analyte-responsive active area.

2. 10. The analyte sensor of claim 1, wherein the mass transport limiting membrane comprises a polyvinylpyridine based polymer, a polyvinylimidazole, a polyacrylate, a polyurethane, a polyether urethane, a silicone, or a combination thereof.

3. 3. The analyte sensor of claim 1, wherein the mass transport limiting membrane comprises a polyvinylpyridine-based polymer.

4. The analyte sensor according to claim 1 , wherein the analyte is an aspartate.

5. The analyte sensor of claim 1 , wherein the analyte responsive active region further comprises asparaginase.

6. The analyte sensor of claim 5 , wherein the analyte is asparagine.

7. The test substance sensor of claim 5 or 6, wherein the test substance responsive active region comprises a polymer, and one or more of the aspartate oxidase and the asparaginase are covalently bonded to the polymer.

8. 8. The analyte sensor of claim 5, wherein the analyte responsive active region comprises a layer comprising the asparaginase disposed on a layer comprising the aspartate oxidase, or comprises a single layer comprising the aspartate oxidase and the asparaginase.

9. A method for detecting aspartate, comprising: (i) exposing the analyte sensor of any one of claims 1 to 4 to a fluid; (ii) applying a potential to the working electrode; (iii) obtaining a signal at or above the redox potential of the analyte-responsive active region, the signal being proportional to the concentration of aspartate in the fluid in contact with the analyte-responsive active region; (iv) correlating the signal with a concentration of the aspartate in the fluid.

10. The method of claim 9, wherein the test substance responsive active region comprises a polymer, and the aspartate oxidase is covalently bonded to the polymer.

11. 11. The method of claim 9 or 10, wherein the mass transport limiting membrane comprises a polyvinylpyridine based polymer, a polyvinylimidazole, a polyacrylate, a polyurethane, a polyether urethane, a silicone, or a combination thereof.

12. A method for detecting asparagine, comprising: (i) exposing the analyte sensor according to any one of claims 5 to 8 to a fluid; (ii) applying a potential to the working electrode; (iii) obtaining a signal at or above the redox potential of the analyte-responsive active region, the signal being proportional to the concentration of asparagine in a fluid in contact with the analyte-responsive active region; (iv) correlating the signal with a concentration of the asparagine in the fluid.

13. The method of claim 12, wherein the test substance responsive active region comprises a polymer, and the aspartate oxidase and / or asparaginase are covalently bonded to the polymer.

14. 14. The method of claim 12 or 13, wherein the mass transport limiting membrane comprises a polyvinylpyridine based polymer, a polyvinylimidazole, a polyacrylate, a polyurethane, a polyether urethane, a silicone, or a combination thereof.

15. 15. The method of any one of claims 12 to 14, wherein the analyte responsive active area comprises a layer comprising the asparaginase disposed above a layer comprising the aspartate oxidase.

16. 15. The method of claim 12, wherein the analyte responsive active area comprises a monolayer comprising the aspartate oxidase and asparaginase.

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