Analyte sensors for detecting asparagine and aspartate and methods of use thereof

Abstract

To provide an analyte sensor for use in detecting aspartate and / or asparagine.SOLUTION: In certain embodiments, an aspartate-responsive active site of a presently disclosed analyte sensor includes an aspartate oxidase disposed upon a surface of a working electrode. In certain embodiments, an asparagine-responsive active site of a presently disclosed analyte sensor includes an enzyme system comprising an aspartate oxidase and an asparaginase disposed upon a surface of a working electrode. The present disclosure further provides methods for detecting aspartate and / or asparagine using the disclosed analyte sensors.SELECTED DRAWING: Figure 1A

Description

Technical Field

[0001] The subject matter described herein relates to a test substance sensor for detecting asparagine and / or aspartate, and methods of using the same.

Background Art

[0002] The detection of various test substances in an individual can be extremely important for monitoring the health of the individual, since a deviation from normal test substance levels can indicate a physiological condition. For example, monitoring glucose levels can enable people with diabetes to take appropriate corrective measures, including administration of medications or ingestion of specific foods or beverage products, to avoid serious physiological discomfort. Other test substances, such as aspartate and asparagine, may also desirably be monitored. In certain instances, it may be desirable to monitor two or more test substances to monitor one or more physiological conditions, particularly when an individual is suffering from a concurrent condition in which two or more test substances combine with each other to result in a simultaneous dysregulation.

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

[0004] Many analytes can be interesting targets for physiological analysis if an appropriate detection chemical can be identified. For this purpose, enzyme-based amperometric sensors configured to continuously assay glucose in vivo have been developed and improved in recent years to assist in monitoring the health of diabetic individuals. Other analytes that commonly undergo dysregulation in concert with glucose in diabetic individuals include, for example, glutamate and asparagine. Similarly, it may be desirable to monitor glutamate and asparagine independently of glucose dysregulation. However, implanted analyte sensors configured to detect glutamate or asparagine in vivo are not currently available. Accordingly, there is a need in the art for sensors for detecting asparagine or glutamate in vivo. SUMMARY OF THE INVENTION

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

[0006] To achieve these and other advantages, in accordance with the purpose of the disclosed subject matter, as embodied and broadly described, the disclosed subject matter includes an analyte sensor for detecting glutamate 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 disposed on the surface of the first working electrode for detecting an analyte and comprising aspartate oxidase, and a mass transfer 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 region comprises a layer of asparaginase disposed on a layer comprising aspartate oxidase. In certain embodiments, the analyte-responsive active region comprises a single layer comprising aspartate oxidase and asparaginase. In certain embodiments, one or more of aspartate oxidase and asparaginase are covalently bound to a polymer within the analyte-responsive active region.

[0008] In certain embodiments, the analyte-responsive activity further comprises an electron transfer agent. In certain embodiments, the mass transfer limiting membrane comprises a polyvinylpyridine-based polymer, polyvinylimidazole, polyacrylate, polyurethane, polyetherurethane, silicone, or a combination thereof. In certain embodiments, the mass transfer limiting membrane comprises polyvinylpyridine or polyvinylimidazole. In certain embodiments, the mass transfer limiting membrane comprises a polyvinylpyridine-based polymer. In certain embodiments, the mass transfer 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 comprising a sensor tail comprising at least a first working electrode, at least one aspartate-responsive active region disposed on the surface of the first working electrode and comprising aspartate oxidase, and a mass transfer 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 that is proportional to the concentration of aspartate in a fluid in contact with the aspartate-responsive active region at an oxidation-reduction potential of the aspartate-responsive active region or higher, 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 comprises a sensor tail comprising at least a first working electrode, an asparagine-responsive active region disposed on the surface of the first working electrode and comprising an enzyme system comprising asparaginic acid oxidase and asparaginase, and a test substance sensor comprising a mass transfer limiting membrane that covers at least the aspartate-responsive active region and is permeable to aspartate. In certain embodiments, the method further comprises applying a potential to the first working electrode, obtaining a first signal that is proportional to the concentration of asparagine in the fluid contacting the asparagine-responsive active region at a redox potential above that of 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 comprise an electron transfer agent. In certain embodiments, asparaginic acid oxidase is covalently bound to a polymer within the asparagine-responsive active region. In certain embodiments, asparaginase is covalently bound to a polymer within the asparagine-responsive active region. In certain embodiments, the asparagine-responsive active region comprises a layer of asparaginase disposed on a layer comprising asparaginic acid oxidase. In certain embodiments, the asparagine-responsive active region comprises a single layer comprising asparaginic acid 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 regarded 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.

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Mode for Carrying Out the Invention

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

[0014] The present disclosure provides a sensor chemistry suitable for detecting aspartic acid and / or asparagine over a range of physiologically relevant aspartate and / or asparagine concentrations. In certain embodiments, the present disclosure provides an analyte sensor that utilizes an enzyme system comprising at least two enzymes that can operate cooperatively to facilitate the detection of an analyte, such as asparagine. As used herein, the term "cooperatively" refers to a coupled enzyme reaction in which the product of a first enzyme reaction becomes the substrate of a second enzyme reaction, and the second enzyme reaction serves as a basis for measuring the concentration of the substrate (e.g., analyte) that reacted during the first enzyme reaction. In certain embodiments, the product and / or substrate of the reaction can be the reduced and / or oxidized form of a cofactor or coenzyme (e.g., FAD or NAD) of the enzymes of the enzyme system. Although defined with respect to two coupled enzyme reactions, it should be understood that three or more enzyme reactions can be similarly coupled in some instances. For example, the product of a first enzyme reaction can become the substrate of a second enzyme reaction, the product of the second enzyme reaction can become the substrate of a third enzyme reaction, and the third enzyme reaction serves as a basis for measuring the concentration of the substrate (e.g., analyte) that reacted during the first enzyme 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 sensor of the present disclosure has a sensitivity of from about 0.1 to about 10 nA / mM, such as from about 0.1 to about 10 nA / mM, from about 0.1 to about 9 nA / mM, from about 0.1 to about 8 nA / mM, from about 0.1 to about 7 nA / mM, from about 0.1 to about 6 nA / mM, from about 0.1 to about 5 nA / mM, from about 0.1 to about 4 nA / mM, from about 0.1 to about 3 nA / mM, from about 0.1 to about 2 nA / mM, or from about 0.1 to about 1 nA / mM.

[0016] For 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. Definitions II. Analyte Sensor 1. General Structure of the Analyte Sensor System 2. Enzyme 3. Redox mediator 4. Polymer backbone 5. Mass transfer limiting membrane 6. Interference domain 7. Manufacture III. Method of use IV. Exemplary embodiments I. Definitions As used herein, terms generally have their ordinary meanings within the context of the present disclosure and in the particular context in which each term is used. Particular 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 their methods of making and using.

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

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

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

[0020] As used herein, "analyte sensor" or "sensor" can refer to any device capable of receiving sensor information from a user, and includes, by way of example and not limitation, a body temperature sensor, a blood pressure sensor, a pulse or heart rate sensor, a glucose level sensor, an analyte sensor, a physical activity sensor, a body movement sensor, or any other sensor for collecting physical or biological information. Analytes measured by an analyte sensor can 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 bodily fluid or fluid derivative in which an analyte can be measured. Non-limiting examples of biological fluids include skin fluid, interstitial fluid, plasma, blood, lymph fluid, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage, amniotic fluid, sweat, and tears. In certain embodiments, the biological fluid is skin fluid or interstitial fluid.

[0022] As used herein, the term "electrolysis" refers to the direct electrolysis at an electrode or the electrooxidation or electroreduction of a compound through 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 "multicomponent membrane" refers to a membrane comprising two or more types of membrane polymers.

[0024] As used herein, the term "potassium-independent asparaginase" refers to an asparaginase that shows no change in catalytic activity in the presence of potassium, such as potassium ions (K+).

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

[0026] As used herein, the term "polyvinylpyridine-based polymer" refers to a polymer or copolymer comprising polyvinylpyridine (e.g., 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 either directly or through one or more additional electron transfer agents between the analyte or analyte-reducing enzyme or analyte-oxidizing enzyme and the electrode. In certain embodiments, a redox mediator comprising a polymer backbone may be referred to as a "redox polymer".

[0028] As used herein, the term "reference electrode" can refer to either a reference electrode or an electrode that functions as both a reference electrode and a counter electrode. Similarly, the term "counter electrode" as used herein can 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 film" refers to a film that contains one type of film polymer. II. Test Substance Sensor 1. General Structure of Test Substance Sensor System Before describing the subject matter in detail, it should be understood that the present disclosure is not limited to the specific embodiments described and can, of course, vary. Since the scope of the present disclosure is limited only by the appended claims, it should also be understood that the terms used herein are for the purpose of describing only specific embodiments and are not intended to be limiting.

[0030] The publications discussed herein are provided only for their disclosure prior to the filing date of the present application. Nothing in this specification should be construed as an admission that the present disclosure has the right to antedate such publications by virtue of prior disclosure. Further, the publication dates shown may be different from the actual publication dates, which may need to be independently confirmed.

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

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

[0033] Furthermore, for every embodiment 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, embodiments of sensor control devices are disclosed, and these devices can have one or more sensors, an analyte monitoring circuit (e.g., an analog circuit), a memory (e.g., for storing instructions), a power source, a communication circuit, a transmitter, a receiver, a processor, and / or a controller (e.g., for executing instructions) that can perform or facilitate the execution of any method step. These embodiments of sensor control devices can be used to perform the steps executed by the sensor control device from any and all of the methods described herein and can be capable of being used.

[0034] Furthermore, the systems and methods shown herein may be used for the operation of sensors used in an analyte monitoring system, for example, but not limited to, any purpose involving wellness, fitness, diet, research, information, or analyte detection over time. As used herein, an "analyte sensor" or "sensor" can refer to any device capable of receiving sensor information from a user, including, by way of example and not limitation, a body temperature sensor, a blood pressure sensor, a pulse or heart rate sensor, a glucose level sensor, an analyte sensor, a body activity sensor, a body movement sensor, or any other sensor for collecting physical or biological information. In certain embodiments, the analyte sensors of the present disclosure can measure aspartate and / or asparagine. In certain embodiments, the analyte sensors of the present disclosure can 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, etc.

[0035] As described above, some embodiments of systems, devices, and methods are provided herein that provide improved assembly and use of a skin sensor insertion device for use with an in vivo analyte monitoring system. In particular, some embodiments of the present disclosure are designed to improve 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, some embodiments include a skin sensor insertion mechanism with an increased firing speed and a slower tip retraction. In other embodiments, the tip retraction mechanism can be actuated by operation such that the tip is not retracted until the user pulls the applicator away from the skin. As a result, these embodiments can, among other advantages, reduce the likelihood of prematurely withdrawing 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. Some embodiments of the present disclosure also provide an improved insertion tip module that takes into account small-scale skin sensors and relatively shallow insertion paths present in the dermal layer of the subject. Additionally, some embodiments of the present disclosure are designed to prevent unwanted axial and / or rotational movement of the applicator component during sensor insertion. Thus, these embodiments can, among other advantages, reduce the likelihood of instability of the positioned skin sensor, irritation at the insertion site, damage to surrounding tissue, and rupture of capillaries resulting in contamination of the skin fluid by blood. Additionally, to reduce inaccurate sensor readings that may be caused by trauma at the insertion site, some embodiments of the present disclosure can reduce the end depth penetration of the needle relative to the sensor tip during insertion.

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

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

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

[0039] An in vivo monitoring system can include a sensor that contacts a user's body fluid and detects the level of an analyte contained therein while being disposed in vivo. The sensor can be part of a sensor control device that is present on the user's body and includes electronic devices and a power source that enable and control the detection of the analyte. The sensor control device and its variations can also be referred to as, to name a few, a "sensor control unit", an "on-body electronics" device or unit, an "on-body" device or unit, or a "sensor data communication" device or unit.

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

[0041] Sensor 104 is adapted to be at least partially inserted into a target tissue such as within the dermal or subcutaneous layer of the skin. Sensor 104 may include a sensor tail that is long enough to be inserted 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 region for detecting an analyte, such as glutamate, disposed on the working electrode. A counter electrode may be present in combination with at least one working electrode. Specific electrode configurations on the sensor tail are described in more detail below.

[0042] The active region can be configured to detect a specific analyte. In certain embodiments, the active region can be configured to detect aspartate and / or asparagine. In certain embodiments, the active region can be configured to detect aspartate. In certain embodiments, the active region can be configured to detect asparagine. In certain embodiments, the active region can be configured to detect two or more analytes. In certain embodiments, the active region can be configured to detect asparagine and / or aspartate, and / or an analyte different from asparagine and aspartate. In certain embodiments, analytes different from asparagine and aspartate can 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, etc.

[0043] In certain embodiments of the present disclosure, one or more analytes can be monitored in any body fluid of a subject, such as skin fluid, interstitial fluid, plasma, blood, lymph fluid, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage, or amniotic fluid. In certain embodiments, the analyte sensor of the present disclosure can 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] To facilitate introduction of the sensor 104 into tissue, an introducer may be present temporarily. In certain exemplary embodiments, the introducer may include a needle or similar tip. As will be readily recognized by those skilled in the art, other types of introducers, such as sheaths or blades, may be present in alternative embodiments. More specifically, the needle or other introducer may be present temporarily near the sensor 104 prior to tissue insertion and may then be withdrawn. 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 enable implantation of the sensor 104. After opening the access path, the needle or other introducer may be withdrawn so that it does not present a sharp hazard. In an exemplary embodiment, a suitable needle may be solid or hollow, angled or non-angled, and / or may have a circular or non-circular cross-section. In more specific embodiments, suitable needles may be similar to acupuncture needles in cross-sectional diameter and / or tip design, and these may have a cross-sectional diameter of about 250 microns (250 μm). However, a suitable needle may have a larger or smaller cross-sectional diameter if required 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 such that the needle first penetrates the tissue and opens an access path for the sensor 104. In certain embodiments, the sensor 104 may be present within the lumen or groove of the needle, and the needle similarly opens an access path for the sensor 104. In either case, the needle may be withdrawn following facilitation of sensor insertion.

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

[0047] C. Exemplary Data Receiver Device Architecture For purposes of illustration and not limitation, reference is made to an exemplary embodiment of a data receiver device 120 for use with the disclosed subject matter shown in FIG. 2B. The data receiver device 120 and related multipurpose data receiver device 130 include a test substance sensor 110 and components closely related to the discussion of its operation, and additional components may be included. In certain embodiments, the data receiver device 120 and multipurpose data receiver device 130 may be, or may include, components provided by a third party 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 a microcontroller 4010, a memory 4020, and a storage 4030, and includes an ASIC 4000 communicably connected to a communication module 4040. Power for the components of the data receiving device 120 can be delivered by a power module 4050, which may include a rechargeable battery as embodied herein. The data receiving device 120 may further include a display unit 4070 to facilitate verification of the test substance data received from the test substance sensor 110 or other devices (e.g., the user device 140 or the remote application server 150). The data receiving device 120 may include separate user interface components (e.g., physical keys, optical sensors, microphones, etc.).

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

[0051] As embodied herein, the on-board storage 4030 of the data receiving device 120 may store the analyte data received from the analyte sensor 110. Further, the data receiving device 120, the multi-purpose data receiving device 130, or the user device 140 may be configured to communicate with the remote application server 150 via 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. Next, the user computing device 140 (or the multi-purpose data receiving device 130) may 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 that is 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 to operate in conjunction with the analyte sensor 110 based on analyte data received from the analyte sensor 110. As an example, when the analyte sensor 110 is a glucose sensor, the data receiving device 120 may be or include an insulin pump or insulin injection pen. In conjunction, a compatible device 130 may adjust the insulin dosage for the user based on the glucose value received from the analyte sensor.

[0053] D. Exemplary Sensor Control Device FIGS. 2C and 2D are block schematic diagrams showing examples of embodiments of a sensor control device 102 having an analyte sensor 104 and sensor electronics 160 (including an analyte monitoring circuit) that may have most of the processing capabilities for rendering final result data suitable for display to a user. In FIG. 2C, a single semiconductor chip 161, which may be a custom application specific integrated circuit (ASIC), is shown. Inside 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 can be implemented as a transmitter, receiver, transceiver, passive circuit, or in other ways according to a communication protocol). In this embodiment, both the AFE 162 and the processor 166 are used as the analyte monitoring circuit, but in other embodiments, either circuit may perform the analyte monitoring function. The processor 166 can include one or more processors, microprocessors, controllers, and / or microcontrollers, each of which can be a separate chip or distributed among a number of different chips (and portions thereof).

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

[0055] FIG. 2D is similar to FIG. 2C, but instead includes two separate semiconductor chips 162 and 174 that can be packaged together or separately. Here, AFE 162 is resident on ASIC 161. Processor 166 is integrated with power management circuit 164 and communication circuit 168 on chip 174. AFE 162 includes memory 163 and chip 174 includes memory 165, which can be separated or distributed internally. In one exemplary embodiment, AFE 162 is combined with power management circuit 164 and processor 166 on one chip, and communication circuit 168 is on a separate chip. In another exemplary embodiment, both AFE 162 and communication circuit 168 are on one chip, and processor 166 and power management circuit 164 are on another chip. Note that other combinations of chips including three or more chips are possible, with each chip performing the described separate functions 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 the analyte sensor 110 for use with the disclosed subject matter shown in FIG. 2E. FIG. 2E shows a block schematic of an example of an analyte sensor 110 according to an exemplary embodiment that is 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, on-board memory 5020, and 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 particular 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 rewritable or one-time programmable (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 can be or include one or more modules for supporting the analyte sensor 110 that communicates with other devices of the analyte monitoring system 100. By way of non-limiting example only, an exemplary communication module 5040 can include a Bluetooth® Low Energy (“BLE”) module 5041. As used throughout this disclosure, Bluetooth® Low Energy (“BLE”) refers to a short-range communication protocol optimized to enable easy pairing of Bluetooth® devices by an end user. The communication module 5040 can transmit and receive data and commands via interaction with a communication module having a similar function of the data receiving device 120 or the user device 140. The communication module 5040 can include additional or alternative chip sets for use with similar short-range communication schemes, such as a personal area network according to the IEEE802.15 protocol, the IEEE802.11 protocol, and infrared communication according to the Infrared Data Association standard (IrDA).

[0059] To perform its function, the sensor 100 can further include appropriate sensing hardware 5060 suitable for its function. As embodied herein, the sensing hardware 5060 can include an analyte sensor disposed transcutaneously or subcutaneously in contact with a target body fluid. The analyte sensor can generate sensor data including values corresponding to the levels of one or more analytes in the body fluid.

[0060] E. Exemplary Assembly Process for the Sensor Control Device The components of the sensor control device 102 can be obtained by the user in a plurality of packages that require final assembly by the user before delivery to the appropriate user's location. FIGS. 3A through 3D show examples of embodiments of the assembly process for the sensor control device 102 by the user, including the preparation of the individual components before connecting the components to prepare the sensor for delivery. FIGS. 3E through 3F show examples of embodiments of the delivery of the sensor control device 102 to the appropriate user's location by selecting the appropriate delivery location and applying the device 102 to that location.

[0061] FIG. 3A is a proximal perspective view showing an example of an embodiment of a user preparing a container 810, here configured as a tray (although other packages may be used), for the assembly process. The user can achieve this preparation by removing the lid 812 from the tray 810 to expose the platform 808, for example, by peeling the non-adhesive portion of the lid 812 from the tray 810 such that the adhesive portion of the lid 812 is removed. The removal of the lid 812 can be appropriate in various embodiments while the platform 808 is sufficiently exposed within the tray 810. Next, the lid 812 can be placed sideways.

[0062] FIG. 3B is a side view showing an example of an embodiment of a user preparing the applicator device 150 for assembly. The applicator device 150 can be provided within a sterile package sealed by a cap 708. The preparation of the applicator device 150 can include separating the housing 702 from the cap 708 to expose the sheath 704 (FIG. 3C). This can be achieved by turning the cap 708 off (or separating it in another way) from the housing 702. Next, the cap 708 can be placed sideways.

[0063] Figure 3C is a proximal perspective view showing an example of an embodiment of a user inserting the applicator device 150 into the tray 810 during assembly. First, the user may align the housing orientation feature 1302 (or slot or recess) and the tray orientation feature 924 (contact or detent), and then insert the sheath 704 into the platform 808 inside the tray 810. When the sheath 704 is inserted into the platform 808, the sheath 704 is temporarily unlocked relative to the housing 702, and the platform 808 is temporarily unlocked relative to the tray 810. At this stage, removal of the applicator device 150 from the tray 810 will result in the same state as before the first insertion of the applicator device 150 into the tray 810 (i.e., this process is reversed or interrupted at this point and can then be repeated without consequence).

[0064] While the housing 702 is advancing distally, the sheath 704 is maintained in position within the platform 808 relative to the housing 702 and is coupled to the platform 808 to advance the platform 808 distally relative to the tray 810. This step unlocks the platform 808 and folds it into the tray 810. A fixed feature (not shown) within the tray 810 contacts and disengages the sheath 704 to prevent the sheath 704 from moving (relatively) while the sheath 704 is unlocked relative to the housing 702 and the housing 702 continues to advance the platform 808 distally. When the advancement of the housing 702 and the platform 808 is complete, the sheath 704 is permanently unlocked relative to the housing 702. A tip and sensor (not shown) within the tray 810 may be coupled to an electronics housing (not shown) within the housing 702 at the end of the distal advancement of the housing 702. The operation and interaction of the applicator device 150 and the tray 810 are further described below.

[0065] Figure 3D is a proximal perspective view showing an example of an embodiment in which the user removes the applicator device 150 from the tray 810 during assembly. The user can remove the applicator 150 from the tray 810 by advancing the housing 702 in the proximal direction relative to the tray 810 or by other operations having the same final effect as the disconnection between the applicator 150 and the tray 810. The applicator device 150 is removed with the sensor control device 102 (not shown) fully assembled therein (tip, sensor, electronics) and placed for delivery.

[0066] Figure 3E is a proximal perspective view showing an example of an embodiment in which the patient uses the applicator device 150 to apply the sensor control device 102 to a target area of the skin, such as the abdomen or other suitable location. By advancing the housing 702 in the distal direction, the sheath 704 is folded into the housing 702 and the sensor is applied to the target position such that the adhesive layer on the bottom side of the sensor control device 102 adheres to the skin. While the tip is automatically retracted when the housing 702 is fully advanced, a sensor (not shown) remains in place to measure the analyte level.

[0067] Figure 3F is a proximal perspective view showing an example of an embodiment of the patient with the sensor control device 102 in the applied position. Next, the user may remove the applicator 150 from the application site. The system 100 described with respect to FIGS. 3A - 3F and other parts of this specification can reduce or eliminate the possibility of accidental breakage, permanent deformation, or inaccurate assembly of the applicator components as compared to prior art systems. Instead of an indirect engagement via the sheath 704, the applicator housing 702 engages directly with the platform 808 while the sheath 704 unlocks, so the relative angle between the sheath 704 and the housing 702 does not result in breakage or permanent deformation of the arm or other components. The possibility of relatively large forces being applied during assembly (as in conventional devices) is reduced, and in turn, the possibility of assembly failure by the user is reduced.

[0068] F. Exemplary Sensor Applicator Device Figure 4A is a side view showing an example of an embodiment of an applicator device 150 coupled to a screw cap 708. This is an example of how the applicator 150 is shipped to and received by a user prior to assembly of the sensor by the user. Figure 4B is a side perspective view showing the applicator 150 and cap 708 after separation. Figure 4C is a perspective view showing an example of an embodiment of the distal end of the applicator device 150 with the electronic device housing 706 and adhesive patch 105 removed from the position where they are held on the sensor carrier 710 of the sheath 704 when the cap 708 is in a fixed position.

[0069] For purposes of illustration and not limitation, referring to FIGS. 4D through 4G, applicator device 20150 can be provided to the user as a single integrated assembly. FIGS. 4D and 4E respectively provide a top perspective view and a bottom perspective view of applicator device 20150, FIG. 4F provides an exploded view of applicator device 20150, and FIG. 4G provides a side cross-sectional view. The perspective views show how applicator 20150 is shipped to and received by the user. The exploded view and cross-sectional view show the components of applicator device 20150. Applicator device 20150 can include housing 20702, gasket 20701, sheath 20704, tip carrier 201102, spring 205612, sensor carrier 20710 (also referred to as a “pack carrier”), tip hub 205014, sensor control device (also referred to as a “pack”) 20102, adhesive patch 20105, desiccant 20502, cap 20708, serial label 20709, and tamper evidence form 20712. When received by the user, only housing 20702, cap 20708, tamper evidence form 20712, and label 20709 are visible. Tamper evidence form 20712 can be, for example, a sticker connected to each of housing 20702 and cap 20708, and tamper evidence form 20712 is irreparably damaged, for example, by removing the connection between housing 20702 and cap 20708, thereby indicating to the user that the connection between housing 20702 and cap 20708 has been previously removed. 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 of an embodiment of a tray 810 to which a sterilization lid 812 is removably connected, which can show how the package is shipped to and received by the user prior to assembly.

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

[0072] FIG. 6B is a proximal perspective view showing sensor module 504 in more detail. Here, the retaining arm extension 1834 of platform 808 releasably fixes sensor module 504 in place. Module 2200 is coupled to connector 2300, tip module 2500, and a sensor (not shown), such that, during assembly, these can be removed together as sensor module 504.

[0073] H. Exemplary Applicator and Sensor Control Device for One-Piece Architecture Referring again briefly to FIGS. 1A and 3A through 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, and thus the user must open each package and ultimately assemble the system. In some applications, 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 not compatible with other contents. More specifically, the sensor tray 202 including the plug assembly 207 including the sensor 110 and the tip 220 can be sterilized using radiation sterilization such as electron beam (i.e., “e-beam”) irradiation. Suitable radiation sterilization processes include, but are not limited to, electron beam (e-beam) irradiation, gamma ray irradiation, X-ray irradiation, or any combination thereof. However, radiation sterilization can damage the electrical components disposed within the electronics housing of the sensor control device 102. As a result, when it is necessary to sterilize the sensor applicator 102 including the electronics housing of the sensor control device 102, it can be sterilized by another method such as gas chemical sterilization using ethylene oxide, for example. However, gas chemical sterilization can damage the enzymes or other chemical substances and biological agents included on the sensor 110. Due to this sterilization incompatibility, the sensor tray 202 and the sensor applicator 102 are generally sterilized in separate sterilization processes and then packaged separately, which requires the user to ultimately assemble the components for use.

[0074] Figs. 7A and 7B are, respectively, an exploded top view and an exploded bottom view of a sensor control device 3702 according to one or more embodiments. A shell 3706 and a mount 3708 operate as opposing clam shell halves that contain or otherwise substantially encapsulate various electronic components of the sensor control device 3702. As shown, the sensor control device 3702 may include a printed circuit board assembly (PCBA) 3802 that includes a printed circuit board (PCB) 3804 to which a plurality of electronic modules 3806 are connected. Exemplary electronic modules 3806 include, but are not limited to, resistors, transistors, capacitors, inductors, diodes, and switches. Conventional sensor control devices generally stack PCB components on only one side of the PCB. In contrast, the PCB components 3806 in the sensor control device 3702 may be distributed across the surface areas (i.e., the top and bottom surfaces) of both sides of the PCB 3804.

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

[0076] The battery opening 3810 may be sized to receive and secure a battery 3812 defined within the PCB 3804 and configured to supply power to the sensor control device 3702. The axial battery contact 3814a and the radial battery contact 3814b may be coupled to the PCB 3804, extend into the battery opening 3810, and facilitate the transmission 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. Disposing the battery 3812 within the battery opening 3810 having the battery contacts 3814a, b serves to reduce the height H of the sensor control device 3702, which allows the PCB 3804 to be centrally located and its components to be dispersed on both sides (i.e., the top and bottom surfaces). This also helps to facilitate mounting the chamfer 3718 on top of the electronic device housing 3704.

[0077] The sensor 3716 may be centrally located relative to the PCB 3804 and may include a tail 3816, a flag 3818, and a neck 3820 interconnecting the tail 3816 and the flag 3818. The tail 3816 may extend through the central opening 3720 of the mount 3708 and may be configured to be received transcutaneously under the user's skin. Additionally, the tail 3816 may have an enzyme or other chemical substance included thereon to help facilitate analyte monitoring.

[0078] Flag 3818 may include a generally flat surface having one or more sensor contacts 3822 (three are shown in FIG. 7B) disposed thereon. The sensor contact(s) 3822 may be configured to align and engage with corresponding one or more circuit contacts 3824 (three are shown in FIG. 7A) provided on the PCB 3804. In some embodiments, the sensor contact(s) 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 contact(s) 3822 of the present disclosure provide a direct connection between the sensor 3716 and the PCB 3804 connection, which eliminates the need for prior art connectors and advantageously reduces the height H. Further, by eliminating the compliant carbon-impregnated polymer module, significant circuit resistance is eliminated, and thus circuit conductivity is improved.

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

[0080] The sensor control device 3702 may further include one or more electromagnetic shields shown as the first shield 3828a and the second shield. The shell 3706 may include 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). By pairing the first and second clocking receptacles 3830a, b with the first and second clocking posts 3832a, b respectively, the shell 3706 is properly aligned with the mount 3708.

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

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

[0083] Referring further to FIG. 7A, the mount 3708 may further include a plurality of carrier grip formations 3842 (two shown) defined about the outer periphery of the mount 3708. The carrier grip formations 3842 are axially offset from the bottom 3844 of the mount 3708, where a transfer adhesive (not shown) may be applied during assembly. In contrast to conventional sensor control devices generally including conical carrier grip formations that intersect the bottom of the mount, the carrier grip formations 3842 of the present disclosure are offset from the plane (i.e., the bottom 3844) to which the transfer adhesive is applied. This may be advantageous in helping to ensure that the delivery system does not inadvertently adhere to the transfer adhesive during assembly. Further, the carrier grip formations 3842 of the present disclosure eliminate the need for a wavy transfer adhesive, which simplifies the manufacture of the transfer adhesive and eliminates the need to accurately register the transfer adhesive to the mount 3708. This also increases the adhesive area and thus the adhesive strength.

[0084] Referring to FIG. 7B, the bottom 3844 of the mount 3708 may comprise a plurality of grooves 3846 or be otherwise defined, which are defined on or near the outer periphery of the mount 3708 and may be spaced equidistant from each other. A transfer adhesive (not shown) may be coupled to the bottom 3844, and the grooves 3846 may be configured to help carry (move) moisture away from the sensor control device 3702 and around the mount 3708 during use. In some embodiments, the spacing of the grooves 3846 may sandwich the module pocket 3838 (FIG. 7A) defined on the opposite (inner) side of the mount 3708. As will be appreciated, by alternating the positions of the grooves 3846 and the module pocket 3838, it is ensured that opposing features on either side of the mount 3708 do not extend into each other. This helps to maximize the use of material for the mount 3708, which in turn may help to maintain the minimum height H of the sensor control device 3702. The module pocket 3838 may also significantly reduce the mold sink and improve the flatness of the bottom 3844 to which the transfer adhesive adheres.

[0085] Still referring to FIG. 7B, the inner surface of the shell 3706 may also comprise or be otherwise defined with a plurality of pockets or recesses configured to accommodate various components of the sensor control device 3702 when the shell 3706 is paired with the mount 3708. For example, the inner surface of the shell 3706 may define an opposing battery locator 3848 that is disposed 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 device 3702 is assembled. The opposing battery locator 3848 extends a short distance within the inner surface of the shell 3706 and helps to reduce the overall height H of the sensor control device 3702.

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

[0087] Alternative sensor assembly / electronic device assembly connection approaches are shown in FIGS. 8A - 8C according to embodiments of the present disclosure. As shown, the sensor assembly 14702 includes a sensor 14704, a connector support 14706, and a tip 14708. In particular, a recess or receptacle 14710 may be defined at the bottom of the mount of the electronic device assembly 14712, providing a position where the sensor assembly 14702 can be received and coupled to the electronic device assembly 14712, thereby allowing the sensor control device to be fully assembled. The profile of the sensor assembly 14702 may be shaped to match or be complementary to the receptacle 14710, which includes an elastomeric sealing member 14714 (including a conductive material coupled to the circuit board and aligned with the electrical contacts of the sensor 14704). Thus, by driving the sensor assembly 14702 into the integrally formed recess 14710 of the electronic device assembly 14712, the on - body device 14714 shown in FIG. 8C is formed when the sensor assembly 14702 snap - fits or is otherwise adhered to the electronic device assembly 14712. This embodiment provides an integrated connector for the sensor assembly 14702 within the electronic device assembly 14712.

[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 hereby incorporated by reference in its entirety.

[0089] According to an embodiment of the present disclosure, the sensor control device 102 can be modified to provide a one-piece architecture that can receive a sterilization technique specially designed for a one-piece architecture sensor control device. The one-piece architecture enables the sensor applicator 150 and the sensor control device 102 to be shipped to the user in a single sealed package that does not require any final user assembly steps. Rather, the user 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 be shown to be advantageous in eliminating component parts, various manufacturing process steps, and user assembly steps. As a result, packaging and waste are reduced, and the potential for user error or system contamination is reduced.

[0090] Figures 9A and 9B are, respectively, a side view and a side cross-sectional view of an example embodiment of a sensor applicator 102 with an applicator cap 210 attached. More specifically, Figure 9A shows how the sensor applicator 102 is shipped to and received by the user, and Figure 9B shows the sensor control device 4402 disposed within the sensor applicator 102. Thus, the fully assembled sensor control device 4402 may already be assembled and installed within the sensor applicator 102 before being 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 attached within the sensor applicator 102, and the applicator cap 210 may then be coupled to the sensor applicator 102. In some embodiments, the applicator cap 210 may be threaded to the housing 208 and may include a tampering 4702. Rotating the applicator cap 210 relative to the housing 208 (e.g., loosening the screw) may cause the tampering 4702 to 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 can receive gaseous chemical sterilization 4704 configured to sterilize the electronic device housing 4404 and any other exposed portions of the sensor control device 4402. To achieve this, a chemical substance may be injected into a sterilization chamber 4706 defined cooperatively by the sensor applicator 102 and the interconnected cap 210. In some applications, the chemical substance can be injected into the sterilization chamber 4706 through one or more vents 4708 defined at the proximal end 610 of the applicator cap 210. Exemplary chemical substances that can be used for gaseous chemical sterilization 4704 include, but are not limited to, ethylene oxide, vaporized hydrogen peroxide, nitrogen oxides (e.g., nitrous oxide, nitrogen dioxide, etc.), and steam.

[0093] Since the distal portions of the sensor 4410 and the tip 4412 are sealed within the sensor cap 4416, the chemical substances used during the gaseous chemical sterilization process do not interact with the enzymes, chemical substances, and biological agents provided on other sensor components such as the tail 4524 and the membrane coating that regulates the flow of the analyte.

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

[0095] In some embodiments, the seal 4712 may comprise two or more layers of different materials. The first layer may be made from a synthetic material such as Tyvek® available from DuPont® (e.g., flash-spun high density polyethylene fibers). Tyvek® is highly durable and puncture resistant and allows the penetration of vapor. The Tyvek® layer can be applied prior to the gaseous chemical sterilization process and, following the gaseous chemical sterilization process, a foil or other vapor and moisture barrier layer can be sealed (e.g., heat-sealed) over the Tyvek® layer to prevent the entry of contaminants and moisture into the sterilization chamber 4706. In other embodiments, 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 capable of protecting 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 against external 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 can also create a dust-free environment during transportation and storage that prevents the adhesive patch 4714 from becoming soiled.

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

[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 the user to open multiple packages and ultimately assemble the sensor control device 5002 prior to application. Rather, upon receipt by the user, the sensor control device 5002 is already fully assembled and may be properly positioned within the sensor applicator 150 (FIG. 1A). To use the sensor control device 5002, the user need only open one barrier (e.g., applicator cap 708 of FIG. 3B) prior to promptly delivering the sensor control device 5002 to the target monitoring location for use.

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

[0100] As shown, the sensor control device 5002 includes an electronic device housing 5004 that includes a shell 5006 and a mount 5008 that can mate with the shell 5006. The shell 5006 may be secured to the mount 5008 via various methods such as snap-fit engagement, press-fit, sonic welding, one or more mechanical fasteners (e.g., screws), gaskets, adhesives, or any combination thereof. In some cases, the shell 5006 may 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 assist in the transdermal delivery of the sensor 5010 under the user's skin while the sensor control device 5002 is being applied. As shown, the corresponding portions of the sensor 5010 and the tip 5012 extend distally from the bottom of the electronic device housing 5004 (e.g., the 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 a mating member 5016 or may be defined in some other way. To connect the tip 5012 to the sensor control device 5002, the tip 5012 may be axially advanced through the electronic device housing 5004 until the tip hub 5014 engages the upper surface of the shell 5006 and the mating member 5016 extends distally from the bottom of the mount 5008. When the tip 5012 penetrates the electronic device housing 5004, the exposed portion of the sensor 5010 may be received within the hollow or concave (arcuate) portion of the tip 5012. The remaining portion of the sensor 5010 is disposed within the electronic device housing 5004.

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

[0103] The sensor cap 5018 can be removably coupled to the electronic device housing 5004 at or near the bottom of the mount 5008. More specifically, the sensor cap 5018 can 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 can define a set of male threads 5026a (FIG. 10B) that are engageable with a set of female threads 5026b (FIG. 10A) defined by the sensor cap 5018. In some embodiments, the male and female threads 5026a, b may comprise a flat thread profile (e.g., without helical curvature), which can prove advantageous when molding the components. Alternatively, the male and female threads 5026a, b can comprise a helical thread engagement. Thus, the sensor cap 5018 can be screwed onto the sensor control device 5002 at the mating member 5016 of the tip hub 5014. In other embodiments, the sensor cap 5018 can 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 a frangible member or substance that can be broken with minimal separating force (e.g., an axial or rotational force).

[0104] In some embodiments, the sensor cap 5018 can comprise a monolithic (single) structure that extends between first and second ends 5020a, b. However, in other embodiments, the sensor cap 5018 can comprise two or more components. In the illustrated embodiment, for example, the sensor cap 5018 can 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 can be configured to help seal the internal chamber 5022, as described in more detail below. In at least one embodiment, the seal ring 5028 can comprise an elastomeric O-ring. The desiccant cap 5030 can contain or comprise a desiccant that helps maintain a preferred humidity level within the internal chamber 5022. The desiccant cap 5030 can also define or otherwise comprise an engagement feature 5024 of the sensor cap 5018.

[0105] Figures 11A through 11C are sequential side cross-sectional views showing the assembly of sensor applicator 102 with sensor control device 5002 according to one or more embodiments. Once sensor control device 5002 is fully assembled, the sensor control device can be installed within sensor applicator 102. Referring to FIG. 11A, tip hub 5014 includes hub snap pole 5302 configured to assist in coupling sensor control device 5002 to sensor applicator 102, or may be otherwise defined. More specifically, sensor control device 5002 may be advanced into the interior of sensor applicator 102, and hub snap pole 5302 may be received by corresponding arm 5304 of tip carrier 5306 disposed within sensor applicator 102.

[0106] In FIG. 11B, sensor control device 5002 is received by tip carrier 5306 and is thus shown secured within sensor applicator 102. Once sensor control device 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 a plurality of opposing mating threads 5308 such that applicator cap 210 is screwed onto housing 208 in a clockwise (or counterclockwise) direction, thereby enabling applicator cap 210 to be secured to sensor applicator 102.

[0107] As shown, the sheath 212 is also positioned within the sensor applicator 102, and the sensor applicator 102 may include a sheath fixing mechanism 5310 configured to ensure that the sheath 212 is not folded prematurely during impact occurrence. In the illustrated embodiment, the sheath fixing mechanism 5310 may have 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 inside of the applicator cap 210, and one or more male threads 5312b may be defined or otherwise provided on the sheath 212. When the applicator cap 210 is screwed into the sensor applicator 102 at the thread 5308, the female and male threads 5312a, b may be configured to engage in a screwing manner. The female and male threads 5312a, b may have the same thread pitch as the thread 5308 that allows the applicator cap 210 to be screwed into the housing 208.

[0108] In FIG. 11C, it is shown where the applicator cap 210 is fully screwed (connected) into the housing 208. As shown, the applicator cap 210 further includes a cap post 5314 located at the center inside the applicator cap 210 and extending proximally from its bottom, which may be defined in other ways. The cap post 5314 may be configured to receive at least a portion of the sensor cap 5018 when the applicator cap 210 is screwed into the housing 208.

[0109] The sensor control device 5002 is mounted within the sensor applicator 102, and when the applicator cap 210 is properly secured, the sensor control device 5002 can receive gaseous chemical sterilization configured to sterilize the electronic device housing 5004 and any other exposed portions of the sensor control device 5002. Since the distal portions of the sensor 5010 and tip 5012 are sealed within the sensor cap 5018, the chemicals used during the gaseous chemical sterilization process cannot interact with the enzymes, chemicals, and biologic agents provided at the tail 5104 and other sensor components such as the membrane coating that regulates the influx of the analyte.

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

[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 positioning the sensor applicator 102 in the firing position.

[0112] In FIG. 12B, the applicator cap 210 is aligned with the housing 208 and advances toward the housing 208, whereby the sheath 212 is received within the applicator cap 210. Instead of rotating the applicator cap 210 relative to the housing 208, the threads of the applicator cap 210 may be snap - fitted to the corresponding threads of the housing 208 to couple the applicator cap 210 to the housing 208. An axial cut or slot 5703 (one shown) defined within the applicator cap 210 may allow a portion near the threads of the applicator cap 210 to flex outwardly to be snap - fitted in engagement with the threads of the housing 208. When the applicator cap 210 is snap - fitted to the housing 208, the sensor cap 5018 may, in response, be snap - fitted within the cap post 5314.

[0113] Similar to the embodiments of FIGS. 11A through 11C, the sensor applicator 102 can include a sheath fixation mechanism configured to ensure that the sheath 212 is not folded prematurely during impact. In the illustrated embodiment, the sheath fixation mechanism is defined near the base of the sheath 212 and includes one or more ribs 5704 (one shown) configured to interact with a shoulder 5708 defined near the base of the applicator cap 210 and one or more ribs 5706 (two shown). The rib 5704 can be configured to interlock between the rib 5706 and the shoulder 5708 while the applicator cap 210 is being attached to the housing 208. More specifically, when the applicator cap 210 is snap - fitted to the housing 208, the applicator cap 210 may be rotated (e.g., clockwise), which positions the rib 5704 of the sheath 212 between the rib 5706 and the shoulder 5708 of the applicator cap 210, thereby "locking" the applicator cap 210 in place until the user rotates the applicator cap 210 counter - clockwise and removes the applicator cap 210 for use. The engagement of the rib 5704 with the rib 5706 and the shoulder 5708 of the applicator cap 210 can also prevent the sheath 212 from being folded prematurely.

[0114] In FIG. 12C, the applicator cap 210 has been removed from the housing 208. Similar to the embodiments of FIGS. 12A through 12C, the applicator cap 210 may be removed by rotating the applicator cap 210 counter - clockwise, in response to which the cap post 5314 is rotated in the same direction, and, as generally described above, the sensor cap 5018 is unscrewed from the mating member 5016. Further, by removing the sensor cap 5018 from the sensor control device 5002, the distal portions of the sensor 5010 and the tip 5012 are exposed.

[0115] When the applicator cap 210 is rotated and removed from the housing 208, the rib 5704 defined on the sheath 212 can slidably engage with the upper portion of the rib 5706 defined on the applicator cap 210. The upper portion of the rib 5706 may provide a corresponding inclined surface that causes the sheath 212 to move upward when the applicator cap 210 is rotated. Moving the sheath 212 upward disengages it from engagement with the first detent 5702a and deflects it so that the sheath arm 5604 is received within the second detent 5702b. When the sheath 212 moves to the second detent 5702b, the radial shoulder 5614 moves out of radial engagement with the carrier arm(s) 5608, allowing the passive spring force of the spring 5612 to push the tip carrier 5306 upward and disengage the carrier arm(s) 5608 from engagement with the groove(s) 5610. As the tip carrier 5306 moves upward within the housing 208, the mating member 5016 can correspondingly retract until it is in the same plane, substantially the same plane, or a quasi-same plane as the bottom of the sensor control device 5002. At this point, the sensor applicator 102 is in the firing position. Thus, in this embodiment, removing the applicator cap 210 correspondingly retracts the mating member 5016.

[0116] I. Exemplary Firing Mechanisms for One-Piece and Two-Piece Applicators Figures 13A-13F show detailed examples of an internal device mechanism that "fires" applicator 216 to apply sensor control device 222 to a user and safely retracts tip 1030 into the used applicator 216. Collectively, these drawings represent an example of a sequence of driving tip 1030 (which supports a sensor coupled to sensor control device 222) into the user's skin, leaving the sensor in operative contact with the user's interstitial fluid and retracting the tip, and adhering the sensor control device to the user's skin with an adhesive. Modifications of such operations for use with alternative applicator assembly embodiments and components can be understood by those skilled in the art with reference to the same. Further, applicator 216 can be a sensor applicator having a one-piece architecture or a two-piece architecture as disclosed herein.

[0117] Referring now to FIG. 13A, sensor 1102 is supported within tip 1030, just above user's skin 1104. Rails 1106 (optionally, three of them) of upper guide section 1108 can be provided to control the movement of applicator 216 relative to sheath 318. Sheath 318 is held by a detent formation 1110 within applicator 216, and an appropriate downward force along the longitudinal axis of applicator 216 overcomes the resistance force provided by detent formation 1110 such that tip 1030 and sensor control device 222 can translate into (and up) user's skin 1104 along the longitudinal axis. Further, catch arm 1112 of sensor carrier 1022 engages tip retraction assembly 1024 to maintain tip 1030 in position relative to sensor control device 222.

[0118] In Figure 13B, when the user's force overcomes or exceeds the return stop form 1110, the sheath 318 is folded into the housing 314, driving the sensor control device 222 (along with related components) to translate downward 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 throughout the full stroke of the sensor / tip insertion process. The retention of the stop surface 1114 of the carrier arm 1112 against the complementary surface 1116 of the tip retraction assembly 1024 maintains the position of the member together with the fully biased telescopic spring 1118. According to an embodiment, rather than employing the user's force to translate the sensor control device 222 downward along the longitudinal axis as indicated by arrow L, a drive spring (e.g., but not limited to, a coil spring) may be actuated to drive a button (e.g., but not limited to, a push button) that drives the sensor control device 222 and is included in the housing 314.

[0119] In Figure 13C, the sensor 1102 and the tip 1030 have reached the depth of full insertion. Thereby, the carrier arm 1112 moves away from the inner diameter of the upper guide section 1108. Next, the compressive force of the coil telescopic spring 1118 drives the angled stop surface 1114 radially outward, releasing the force to drive the tip carrier 1102 of the tip retraction assembly 1024 and pull the tip 1030 (configured with slots or otherwise) away from the user and away from the sensor 1102 as indicated by arrow R in Figure 13D.

[0120] With the tip 1030 fully retracted as shown in Figure 13E, the upper guide section 1108 of the sheath 318 is fixed by the final fixing form 1120. As shown in Figure 13F, the used applicator assembly 216 is removed from the insertion site leaving the sensor control device 222 with the tip 1030 safely secured inside the 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 sense that both the insertion and retraction of the tip 1030 are automatically performed by the internal mechanism of the applicator 216. In other words, according to the present invention, the user is avoided from experiencing the sense of manually driving the tip 1030 against the user's skin. Therefore, when the user applies sufficient force to overcome the resistance from the detent form of the applicator 216, the resulting movement of the applicator 216 is recognized as an automatic response to the "induction" of the applicator. The user does not recognize that the user is supplying additional force to drive the tip 1030 to pierce the skin, even though all the driving force is provided by the user and no additional biasing / driving means are used to insert the tip 1030. As described above with reference to FIG. 13C, the retraction of the tip 1030 is automated by the coil expansion and contraction spring 1118 of the applicator 216.

[0122] Regarding any of the embodiments of the applicator described herein, and including, without limitation, any of its components including embodiments of the tip, tip module, and sensor module, one of ordinary skill in the art will understand that the embodiment can be sized and configured for use with a sensor configured to detect analyte levels in a body fluid within the epidermis, dermis, or subcutaneous tissue of a subject. In some embodiments, for example, both the tip and the distal portion of the analyte sensor disclosed herein can be sized and configured to be positioned at a specific end depth (i.e., the furthest point of penetration within a tissue or layer of the subject's body, e.g., within the epidermis, dermis, or subcutaneous tissue). Regarding some embodiments of the applicator, one of ordinary skill in the art will recognize that a particular embodiment of the tip can be sized and configured to be positioned at different end depths in the subject's body relative to the final end depth of the analyte sensor. In some embodiments, for example, the tip can be positioned at a first end depth within the epidermis of the subject prior to retraction, while the distal portion of the analyte sensor can be positioned at a second end depth within the dermis of the subject. In other embodiments, the tip can be positioned at a first end depth within the dermis of the subject prior to retraction, while the distal portion of the analyte sensor can be positioned at a second end depth within the subcutaneous tissue of the subject. In still other embodiments, the tip can be located at a first end depth prior to retraction and the analyte sensor can be located at a second end depth, where both the first end depth and the second end depth are within the same layer or tissue of the subject's body.

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

[0124] Further details of suitable devices, systems, methods, components, and their operations, along with related features, are described in International Publication No. WO2018 / 136898 by Rao et al., International Publication No. WO2019 / 236850 by Thomas et al., International Publication No. WO2019 / 236859 by Thomas et al., International Publication No. WO2019 / 236876 by Thomas et al., and U.S. Patent Publication No. 2020 / 0196919 filed on June 6, 2019, each of which is hereby incorporated by reference in its entirety. Further details regarding applicators, their components, and embodiments of their variations are described in U.S. Patent Publication Nos. 2013 / 0150691, 2016 / 0331283, and 2018 / 0235520, all of which are hereby incorporated by reference in their entirety for all purposes. Further details regarding tip modules, tips, their components, and embodiments of their variations are described in U.S. Patent Publication No. 2014 / 0171771, which is hereby incorporated by reference in its entirety for all purposes.

[0125] J. Exemplary method for calibrating a test substance sensor Biochemical sensors can be described by one or more sensing characteristics. A common sensing characteristic is shown as the sensitivity of the biochemical sensor, which is a measure of the responsiveness of the sensor to the concentration of a chemical substance or composition that the sensor is designed to detect. In an electrochemical sensor, this response can be in the form of current (current measurement) or charge (charge measurement). In other types of sensors, the response can be in different forms such as photon intensity (e.g., optical light). The sensitivity of a biochemical test substance sensor can 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 a current measurement analyte sensor. The in vitro sensitivity can be obtained by performing in vitro tests on the sensor at various analyte concentrations and then performing regression (e.g., linear or non-linear) or other curve fitting on the resulting data. In this example, the sensitivity of the analyte sensor is linear or substantially linear and 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, and the intercept generally corresponds to the background signal (e.g., noise). For a sensor having a linear or substantially linear response, the analyte level corresponding to a given current can be determined from the slope and intercept of the sensitivity. A sensor having non-linear sensitivity requires additional information to determine the analyte level resulting from the output current of the sensor, and those skilled in the art are familiar with methods for modeling non-linear sensitivity. In certain embodiments of the in vivo sensor, the in vitro sensitivity may be the same as the in vivo sensitivity, but in other embodiments, a transfer (or conversion) function is used to 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 that describe 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 by user interaction or by the system itself in an automated method after the sensor has been implanted in the user or patient. For example, if user intervention is required, the user performs an in vitro measurement (e.g., a blood glucose (BG) measurement using a finger stick 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 value to the in vivo signal and uses the difference to determine an estimate of the in vivo sensitivity of the sensor. The in vivo sensitivity can then be used in an algorithmic process to convert the data collected by the sensor into a value indicative of the user's analyte level. This process and other processes that require user action to perform calibration are referred to as "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 regular (e.g., daily) schedule, a variable schedule, or as needed) may be required to maintain accuracy. Embodiments described herein may incorporate some degree of user calibration for certain implementations, but generally this is undesirable as it requires the user to perform painful or otherwise burdensome BG measurements and may introduce user error.

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

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

[0131] The sensor can include calibration codes or parameters, which are derived or determined during one or more sensor manufacturing processes and, as part of the manufacturing process, are encoded or programmed in the data processing device of the analyte monitoring system, or can be provided to the sensor itself, for example, as a barcode, laser tag, RFID tag, or other machine-readable information provided by the sensor. When the code is provided to a receiver (or other data processing device), user calibration during in vivo use of the sensor can be eliminated, or the frequency of in vivo calibration during sensor wear can be reduced. In embodiments where the calibration code or parameters are provided to the sensor itself, the calibration code or parameters can be automatically transmitted or provided to the data processing device within the analyte monitoring system before or at the start of sensor use.

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

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

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

[0135] According to the disclosed subject matter, each sensor within a sensor lot (in some examples, excluding sample sensors used in in vitro testing) can be non-destructively inspected to determine or measure its properties such as the thickness of the membrane at one or more points of the sensor, and other properties including physical properties such as the surface area / volume of the active region 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 measuring device or system, and the determined sensor properties of each sensor within the sensor lot are compared to corresponding average values based on a sample sensor for possible correction of calibration parameters or codes assigned to each sensor. For example, in the case of a calibration parameter defined as sensor sensitivity, the sensitivity is approximately inversely proportional to the thickness of the membrane, such that, for example, in the case of a sensor having a measured membrane thickness that is approximately 4% greater than the average membrane thickness of sensors sampled from the same sensor lot as the sensor, in one embodiment the sensitivity assigned to that sensor is the average sensitivity determined from the sampled sensors divided by 1.04. Similarly, since the sensitivity is approximately proportional to the active region of the sensor, for a sensor having a measured active region that is approximately 3% smaller than the average active region of sensors sampled from the same sensor lot, the sensitivity assigned to that sensor is the average sensitivity multiplied by 0.97. The assigned sensitivity can be determined from the average sensitivity from the sampled sensors by a plurality of successive adjustments for each inspection or measurement of the sensor. In certain embodiments, the inspection or measurement of each sensor may further include a measurement of the uniformity or structure of the membrane in addition to the thickness of the membrane and / or the surface area or volume of the active sensing region.

[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 hereby 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 having a function of providing an interface for making 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 service provided by the communication module 5040 may include a generic access profile service, a generic attribute service, a generic access service, a device information service, a data transmission service, and a security service. The data transmission service may be a primary service used to transmit data such as sensor control data, sensor status data, analyte measurement data (past and present), and event log data. The sensor status data may include error data, current active time, and software status. The analyte measurement data may include information such as current and past raw measurement values, current and past values after being processed using appropriate algorithms or models, prediction and trend of measurement levels, comparison of other values with patient-specific average values, action requests determined by algorithms or models, and other similar types of data.

[0138] According to an aspect of the disclosed subject matter, as embodied herein, the sensor 110 may be configured to communicate with multiple devices simultaneously by adapting to the 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 be provided with software or firmware to enable multiple simultaneous connections between the sensor 110 as a central device and other devices as peripheral devices, or between the sensor 110 as a peripheral device when another device is the central device.

[0139] The connection between two devices using a communication protocol such as BLE and the subsequent communication session can be characterized by a similar physical channel operating between the two devices (e.g., sensor 110 and data receiving device 120). The physical channel can include a single channel or a series of channels, and can include, for example, but not limited to, using a common clock and a series of agreed channels determined by a channel or frequency hopping sequence. The communication session can use a similar amount of available communication spectrum, and multiple such communication sessions can exist in proximity. In certain embodiments, each set of devices in a communication session uses a different physical channel or series of channels to manage interference of similarly proximate devices.

[0140] By way of illustration and not limitation, exemplary embodiments of the processing for a sensor-receiver connection for use with the disclosed subject matter are referenced. First, sensor 110 repeatedly informs its surroundings of its connection information in the discovery of data receiving device 120. Sensor 110 can repeatedly inform periodically until a connection is established. Data receiving device 120 detects the advertising packet and performs scanning and filtering to connect via the data provided by sensor 120 in the advertising packet. Next, data receiving device 120 transmits a scan request command, and sensor 110 responds with a scan response packet providing additional details. Next, data receiving device 120 transmits a connection request using the Bluetooth® device address associated with data receiving device 120. Data receiving device 120 can also continuously request to establish a connection to sensor 110 having a specific Bluetooth® device address. Next, the devices establish an initial connection to enable data exchange. The devices start a process of initializing the data exchange service and perform a mutual authentication process.

[0141] During a first connection between the sensor 110 and the data receiving device 120, the data receiving device 120 may initialize service, characteristic, and attribute discovery processes. The data receiving device 120 may evaluate these features of the sensor 110 and save them for use during the next connection. Next, the device enables a notification about a customized security service used for mutual authentication of the sensor 110 and the data receiving device 120. The mutual authentication process can be automated and does not require user intervention. After the mutual authentication process is successfully completed, the sensor 110 sends a connection parameter update to request the data receiving device 120 to use connection parameter settings that are suitable for the sensor 110 and configured to maximize lifespan.

[0142] Next, the data receiving device 120 executes sensor control processing to backfill historical data, current data, event logs, and factory data. As an example, for each type of data, the data receiving device 120 sends a request to start a backfill process. The request may specify a range of records defined based on, for example, measurement values, timestamps, etc., as 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 receiving device 120. The sensor 110 may respond to the backfill request from the data receiving device 120 that all data has already been transmitted. When the backfill is complete, the data receiving device 120 may notify the sensor 110 that it is ready to receive normal measurement values. The sensor 110 may send measurement values over multiple notification results on a repeating basis. As embodied herein, the multiple notifications may be redundant notifications to ensure that the data is 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 sensor 110. The shutdown operation is performed, for example, when sensor 110 is in an error state, an insertion failure state, or a sensor expiration state. If sensor 110 is not in those states, sensor 110 may record the command and perform a shutdown when sensor 110 transitions to an error state or a sensor expiration state. Data receiving device 120 sends a properly formatted shutdown command to sensor 110. If sensor 110 is actively processing another command, sensor 110 responds with a standard error response indicating that sensor 110 is busy. Otherwise, sensor 110 sends a response upon receiving the command. Further, in order to notify that sensor 110 has received the command, sensor 110 sends a success notification via the sensor control characteristics. Sensor 110 registers the shutdown command. At the next appropriate opportunity (e.g., depending on the current sensor state as described herein), sensor 110 shuts down.

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

[0145] When entering state 6025, sensor 110 can save information about the authenticated device for communicating with the sensors set during operation, or can initialize the algorithm for performing and interpreting measurements from the detection hardware 5060. Sensor 110 can also initialize the life cycle timer involved in maintaining the active count of the operating time of sensor 110 and start communication with the authenticated device for transmitting the recorded data. While in the insertion detection state 6025, the sensor can enter state 6030, where sensor 110 checks whether the operating time is equal to a predetermined threshold. This operating time threshold can correspond to a timeout function for determining whether the insertion was successful. If the operating time reaches the threshold, sensor 110 proceeds to state 6035, where sensor 110 checks whether the average data read amount is greater than the threshold corresponding to the expected data read amount for inducing the detection of successful insertion. If the data read amount is lower than the threshold while in state 6035, the sensor proceeds to state 6040 corresponding to failed insertion. If the data read amount meets the threshold, the sensor proceeds to the active pairing state 6055.

[0146] The active pairing state 6055 of sensor 110 indicates the state while sensor 110 is operating normally by recording measurement values, processing the measurement values, and reporting them as necessary. While in the active pairing state 6055, sensor 110 attempts to transmit the measurement results or establish a connection with the receiving device 120. Sensor 110 also increases the operating time. When sensor 110 reaches a predetermined operating time threshold (for example, when the operating time reaches the predetermined threshold), sensor 110 transitions to the active expiration state 6065. The active expiration state 6065 of sensor 110 indicates the state while sensor 110 is operating over its maximum predetermined time.

[0147] While in the expired active state 6065, sensor 110 may generally perform operations related to the end of operation and ensure that the collected measurements are safely transmitted to the receiving device as needed. For example, while in the expired active state 6065, sensor 110 can transmit the collected data and, if the connection is not executable, increase attempts to find a nearby authenticated device and establish a connection with it. While in the expired active state 6065, sensor 110 may receive a shutdown command in state 6070. If the shutdown command is not received, sensor 110 may also check in state 6075 whether the operating time has exceeded a final operating threshold. The final operating threshold may be based on the battery life of sensor 110. The normal end state 6080 corresponds to the final operation of sensor 110 and ultimately shuts down sensor 110.

[0148] Before the sensor is activated, ASIC 5000 is in a low-power save mode state. For example, when an incident RF field (e.g., an NFC field) drives the voltage of the power supply to ASIC 5000 above a reset threshold, thereby causing sensor 110 to enter the wake-up state, the activation process may start. While in the wake-up state, ASIC 5000 enters the activation sequence state. Next, ASIC 5000 activates communication module 5040. Communication module 5040 is initialized and induces a power-on self-test. The power-on self-test may include ASIC 5000 communicating with communication module 5040 using a predetermined sequence of reading and writing data to verify that the memory and one-time programmable memory are not damaged.

[0149] When the ASIC5000 first enters the measurement mode, an insertion detection sequence is executed to verify that the sensor 110 is properly placed on the patient's body before appropriate measurements can be made. First, the sensor 110 interprets the command to activate the measurement setting process and causes the ASIC5000 to enter the measurement command mode. Next, the sensor 110 temporarily enters the measurement life cycle state and performs several consecutive measurements to check whether the insertion was successful. The communication module 5040 or the ASIC5000 evaluates the measurement results to determine the success of the insertion. If the insertion is considered successful, the sensor 110 enters the measurement state and the sensor 110 begins to perform normal measurements using the detection hardware 5060. If the sensor 110 determines that the insertion was not successful, the sensor 110 is induced into the insertion failure mode, the ASIC5000 is commanded to return to the save mode, while the communication module 5040 disables itself.

[0150] M. Exemplary Over-the-Air Update FIG. 1B further shows an example of an operating environment for providing an over-the-air (「OTA」) update for use with the techniques described herein. An operator of the analyte monitoring system 100 may bundle updates for the data receiving device 120 or the sensor 110 with updates for an application running on the multipurpose data receiving device 130. Using the available communication channels between the data receiving device 120, the multipurpose data receiving device 130, and the sensor 110, the multipurpose data receiving device 130 may receive normal updates for the data receiving device 120 or the sensor 110 and initiate the installation of the updates to the data receiving device 120 or the sensor 110. The application that enables the multipurpose data receiving device 130 to communicate with the analyte sensor 110, the data receiving device 120, and / or the remote application server 150 can update the software or firmware on the data receiving device 120 or the sensor 110 without wide area network capabilities, so the multipurpose data receiving device 130 functions as an installation or update platform for the data receiving device 120 or the sensor 110.

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

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

[0153] The data receiving device 120 or the sensor 110 receives data for updating and a command to start the update from the multi-purpose data receiving device 130. Next, the data receiving device 120 may install a firmware or software update. To install the update, the data receiving device 120 or the sensor 110 can put itself into a so-called "safe" mode with restricted operating functions or restart. When the update is complete, the data receiving device 120 or the sensor 110 re-enters the standard operating mode or is reset. The data receiving device 120 or the sensor 110 may perform one or more self-tests to determine that the firmware or software update has been successfully installed. The multi-purpose data receiving device 130 may receive a notification of update success. Next, the multi-purpose data receiving device 130 may report the confirmation of update success to the remote application server 150.

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

[0155] FIG. 16 is a schematic diagram showing an example of the operation and data flow for over-the-air (OTA) programming of the storage memory 5030 in the sensor device 100 and for the use of the memory after OTA programming in the execution of a process by the sensor device 110 according to the disclosed subject matter. In the example of OTA programming 500 shown in FIG. 5, a request is transmitted from an external device (e.g., data receiving device 130) to initiate OTA programming (or reprogramming). At 511, the communication module 5040 of the sensor device 110 receives an 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 an OTA programming command, the microcontroller 5010 verifies the OTA programming command. The microcontroller 5010 may determine, for example, whether the OTA programming command is signed with a proper digital signature token. If it is determined that the OTA programming command is valid, the microcontroller 5010 may set the sensor device to the OTA programming mode. At 532, the microcontroller 5010 may verify the OTA programming data. At 533, the microcontroller 5010 may reset the sensor device 110 to re-initialize the sensor device 110 to a programming state. When the sensor device 110 transitions to the OTA programming state, the microcontroller 5010 may start writing data to the rewritable memory 540 (e.g., memory 5020) of the sensor device at 534 and may start writing data to the OTP memory 550 (e.g., storage memory 5030) of the sensor device at 535. The data written by the microcontroller 5010 may be based on the verified OTA programming data. The microcontroller 5010 may write data and mark one or more programming blocks or regions of the OTP memory 550 as invalid or inaccessible. Data written to the free or unused portion of the OTP memory may be used to replace the invalidated or inaccessible programming blocks of the OTP memory 550. After the microcontroller 5010 writes data to each memory at 534 and 535, the microcontroller 5010 may perform one or more software integrity checks to ensure that no error was introduced into the programming blocks during the write process. If the microcontroller 5010 can determine that the data was written without error, the microcontroller 5010 may resume the normal operation of the sensor device.

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

[0158] N. Exemplary Security and Other Architectural Features As embodied herein, a first layer of security for communication between the analyte sensor 110 and other devices can be specified by the communication protocol used for communication and established based on a security protocol integrated into the communication protocol. Another layer of security can be based on a communication protocol that requires proximity of the communication devices. Additionally, certain packets and / or certain data contained within the packets can be encrypted, while other packets and / or data within the packets are encrypted in a different manner or not encrypted. Additionally or alternatively, application layer encryption can be used with one or more block ciphers or stream ciphers to establish mutual authentication and communication encryption with other devices within the analyte monitoring system 100.

[0159] The ASIC 5000 of the analyte sensor 110 can be configured to dynamically generate authentication and encryption keys using the data stored in the storage memory 5030. The storage memory 5030 can also be pre-programmed with a set of valid authentication and encryption keys for use with a particular class of devices. The ASIC 5000 can be further configured to perform an authentication process with other devices using the received data and apply the generated keys to the confidential data before transmitting the confidential data. The generated keys can be unique to the analyte sensor 110, unique to a pair of devices, unique to a communication session between the analyte sensor 110 and other devices, unique to a message transmitted during the communication session, or unique to a block of data included in the message.

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

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

[0162] The test substance data associated with the user is at least partially confidential data, because this information can be used for various purposes including health monitoring and drug administration decisions. In addition to user data, the test substance monitoring system 100 can perform enhanced security against the implementation by external parties for reverse engineering. The communication connection can be encrypted using device-specific or session-specific encryption keys. Encrypted or unencrypted communication between any two devices can be verified using a transmission integrity check incorporated into the communication. The operation of the test substance sensor 110 can be protected from tampering by restricting access to the read and write functions to the memory 5020 via the communication interface. The sensor can be configured to allow access only to known or "trusted" devices provided within a "white list", or only to devices that can provide a predetermined code associated with a user authenticated by the manufacturer or other means. The white list can indicate an exclusive range meaning that connection identifiers other than those included in the white list are not used, or a preferred range where the white list is searched first but other devices can still be used. The sensor 110 can further reject connection requests and shut down if the requester cannot complete the login process via the communication interface within a predetermined time (e.g., within 4 seconds). These features protect against specific service disruption attacks, especially service disruption attacks against the BLE interface.

[0163] As embodied herein, the test substance monitoring system 100 can use periodic key rotation to further reduce the possibility of key leakage and abuse. The key rotation strategy employed by the test substance monitoring system 100 can be designed to support backward compatibility of in-field or distributed devices. As an example, the test substance monitoring system 100 can adopt keys for downstream devices (e.g., devices in the field or devices that cannot be provided with the ability to perform updates) 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 message sequence diagram 600 for use with the disclosed subject matter, shown in FIG. 17 and illustrating an example of data exchange between a pair of devices, particularly between sensor 110 and data receiving device 120. Data receiving device 120 can be data receiving device 120 or multipurpose data receiving device 130, as implemented herein. In step 605, data receiving device 120 can send sensor activation command 605 to sensor 110, e.g., via a short-range communication protocol. Sensor 110 may be primarily in a dormant state prior to step 605 and conserve its battery until full activation is required. After activation, during step 610, sensor 110 can collect data or perform other operations as appropriate for sensor 110's sensing hardware 5060. In step 615, data receiving device 120 can initiate authentication request command 615. In response to authentication request command 615, both sensor 110 and data receiving device 120 can participate in mutual authentication process 620. Mutual authentication process 620 can involve the transfer of data, including challenge parameters that enable sensor 110 and data receiving device 120 to ensure that the other device is sufficiently compliant with the agreed-upon security framework described herein. Mutual authentication can be based on a mechanism for authenticating two or more entities to each other, with or without an online trusted third party, to verify the establishment of a secret key via a challenge response. Mutual authentication can be performed using two-pass authentication, three-pass authentication, four-pass authentication, or five-pass authentication, or similar versions thereof.

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

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

[0167] O. Exemplary Payload / Communication Frequency The analyte sensor 110 may be configured to attempt to change its discoverability behavior to increase the probability that the receiving device receives an appropriate data packet and / or the likelihood of providing a response signal, or to otherwise reduce the limitation that the response signal may not be received. Changing the discoverability behavior of the analyte sensor 110 may include, for example, but not limited to, changing the frequency at which connection data is included in data packets, changing the frequency at which data packets are generally transmitted, extending or shortening the broadcast window of data packets, changing the time after broadcast at which the analyte sensor 110 accepts a response or scan signal, including direct transmissions to one or more devices previously communicated with by the analyte sensor 110 (e.g., via one or more attempted transmissions) and / or one or more devices on a whitelist, changing the transmit power associated with the communication module when broadcasting data packets (e.g., to increase the range of the broadcast, reduce the energy consumed, and extend the life of the analyte sensor's battery), changing the speed at which data packets are prepared and broadcast, or including a combination of one or more other changes. Additionally or alternatively, the receiving device may similarly adjust parameters regarding the listening behavior of the device to increase the likelihood of receiving data packets containing connection data.

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

[0169] In certain embodiments, the discoverability behavior of the analyte sensor can be saved in a discoverability profile and changed based on one or more factors such as the state of the analyte sensor 110 and / or by applying rules based on the state of the analyte sensor 110. For example, when the battery level of the analyte sensor 110 drops below a certain amount, the rule can cause the analyte sensor 110 to reduce the power consumed by the broadcast process. As another example, configuration settings associated with broadcasting or otherwise transmitting packets can be adjusted based on ambient temperature, the temperature of the analyte sensor 110, or the temperature of specific components of the communication hardware of the analyte sensor 110. In addition to changing the transmit power, other parameters related to the transmit capabilities or processes of the communication hardware of the analyte sensor 110 can be changed, including, but not limited to, transmit speed, frequency, and timing. As another example, when the analyte data indicates that the subject is experiencing or is in the process of experiencing a negative health event, the rule can cause the analyte sensor 110 to increase its discoverability to warn the receiving device of the negative health event.

[0170] P. Exemplary Sensor Sensitivity Initialization / Adjustment Mechanism As embodied herein, a calibration mechanism for the detection hardware 5060 of the analyte sensor 110 can be adjusted based on external or ambient environmental characteristics and to compensate for attenuation of the detection hardware 5060 during periods when it is not in use (e.g., “storage time” prior to use). The calibration mechanism of the detection hardware 5060 can be adjusted autonomously by the sensor 110 (e.g., by operation of the ASIC 5000 to change characteristics within the memory 5020 or storage 5030), or can be adjusted by other devices of the analyte monitoring system 100.

[0171] As an example, the sensor sensitivity of the detection hardware 5060 can be adjusted based on external temperature data or time since manufacture. When the external temperature is monitored during storage of the sensor, the disclosed subject matter can adaptively change the compensation for sensor sensitivity over time as the device experiences changes in storage conditions. For purposes of illustration and not limitation, in an “active” storage mode where the analyte sensor 110 is periodically powered on to measure temperature, an adaptive sensitivity adjustment can be performed. These mechanisms can conserve the battery of the analyte device and extend the life of the analyte sensor. At each temperature measurement, the analyte sensor 110 can calculate a sensitivity adjustment for that period based on the measured temperature. Next, the temperature-weighted adjustment values can be accumulated over the active storage mode period to calculate a total sensor sensitivity adjustment value at the end of the active storage mode (e.g., upon insertion). Similarly, upon insertion, the sensor 110 can determine the time difference between the manufacture of the sensor 110 (which can be written to the storage 5030 of the ASIC 5000) or the detection hardware 5060, and can change the sensor sensitivity or other calibration mechanism according to one or more known attenuation rates or equations.

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

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

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

[0175] R. Exemplary alarm function The data transmitted between the sensor 110 and the data receiving device 120 may include raw measurement values or processed measurement values. The data transmitted between the sensor 110 and the data receiving device 120 may further include an alarm or notification for display to the user. The data receiving device 120 can display a notification to the user based on the raw measurement value or the processed measurement value, or convey it in another way, or display an alarm when received from the sensor 110. The alarm that can be triggered for display to the user includes a direct analyte value (e.g., a single reading that exceeds or does not meet a threshold), an analyte value trend (e.g., an average reading, gradient over a set period that exceeds or does not meet a threshold), an analyte value prediction (e.g., an algorithm calculation based on an analyte value that exceeds or does not meet a threshold), a sensor alert (e.g., a detected suspected malfunction), a communication alert (e.g., no communication between the sensor 110 and the data receiving device 120 over a threshold period, an unknown device attempting to start or failing to start a communication session with the sensor 110), a reminder (e.g., a reminder to charge the data receiving device 120, take a medication, or perform other activities), and alarms based on other alerts of a similar nature. By way of example and not limitation, as embodied herein, the alarm parameters described herein may be configurable by the user, or may be fixed during manufacture, or may be a combination of user-configurable and non-user-configurable parameters.

[0176] S. Exemplary Electrode Configuration A sensor configuration featuring a single active region configured for the detection of a corresponding single analyte may use a two-electrode or three-electrode detection motif, as further described herein with reference to FIGS. 18A through 18C. A sensor configuration featuring two different active regions for the detection of the same or different analytes, either on separate working electrodes or on the same working electrode, will be described separately later with reference to FIGS. 19A through 21C. Since the signal contributions from each active region can be readily determined, a sensor configuration having multiple working electrodes can be particularly advantageous for incorporating two different active regions within the same sensor tail.

[0177] When a single working electrode is present within an analyte sensor, a three-electrode sensor configuration can include a working electrode, a counter electrode, and a reference electrode. A related two-electrode sensor configuration 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 on top of each other (layered) and / or laterally spaced apart from each other on the sensor tail. A suitable sensor configuration may be of a substantially flat shape, a substantially cylindrical shape, or any suitable shape. In any of the sensor configurations disclosed herein, the various electrodes may be electrically insulated from each other by a dielectric material or a 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] FIG. 18A shows a schematic diagram of an exemplary two - electrode analyte sensor configuration suitable for use in the disclosure herein. As shown, the analyte sensor 200 includes a substrate 30212 disposed between a working electrode 214 and a counter / reference electrode 30216. Alternatively, the working electrode 214 and the counter / reference electrode 30216 can be disposed on the same side of the substrate 30212 with a dielectric material therebetween (configuration not shown). The active region 218 is disposed as at least one layer over at least a portion of the working electrode 214. The active region 218 can include a plurality of spots or a single spot configured for the detection of an analyte, as further discussed herein. In certain embodiments, the active region 218 is configured to detect aspartate and / or asparagine as described herein.

[0180] Referring further to FIG. 18A, a membrane 220 covers at least the active region 218. In certain embodiments, the membrane 220 can cover a portion or all of the working electrode 214 and / or the counter / reference electrode 30216, or the entire analyte sensor 200. One or both sides of the analyte sensor 200 can be covered by the membrane 220. The membrane 220 can include one or more polymer membrane materials having the ability to restrict the flux of the analyte to the active region 218 (i.e., the membrane 220 is a mass transfer - limiting membrane having some permeability to the analyte of interest). According to the disclosure herein, as further described below, the membrane 220 can be cross - linked by a cross - linking agent in certain sensor configurations. For example, but not limited to, the membrane 220 is cross - linked with the cross - linking agents described herein. The composition and thickness of the membrane 220 can vary to facilitate the desired analyte flux to the active region 218, thereby providing the desired signal intensity and stability. The analyte sensor 200 can be operable to assay the analyte by any of the electrochemical detection techniques of coulometry, amperometry, voltammetry, or potentiometry.

[0181] Figures 18B and 18C show schematic diagrams of exemplary three - electrode analyte sensors, which are also compatible for use in the disclosure herein. The three - electrode analyte sensor configuration can be similar to that shown as analyte sensor 200 in Figure 18A, except that it includes an additional electrode 217 within analyte sensors 201 and 202 (Figures 18B and 18C). With the additional electrode 217, the counter / reference electrode 30216 can then function as either a counter electrode or a reference electrode, and the additional electrode 217 serves other electrode functions not otherwise described. The working electrode 214 continues to perform its original function. The additional electrode 217 can be disposed on either the working electrode 214 or the electrode 30216, sandwiching a separation layer of dielectric material therebetween. For example, but not limited to, as shown in Figure 18B, dielectric layers 219a, 219b, and 219c separate electrodes 214, 30216, and 217 from each other and provide electrical insulation. Alternatively, as shown in Figure 18C, at least one of electrodes 214, 30216, and 217 may be positioned on the opposite surface of the substrate 30212. Thus, in certain embodiments, the electrode 214 (working electrode) and the electrode 30216 (counter electrode) may be disposed on opposite surfaces of the substrate 30212, and the electrode 217 (reference electrode) may be disposed on one of the electrodes 214 or 30216 and spaced therefrom by a dielectric material. A reference material layer 230 (e.g., Ag / AgCl) may be present on the electrode 217, and the position of the reference material layer 230 is not limited to the positions shown in Figures 18B and 18C. Similar to the sensor 200 shown in Figure 18A, the active regions 218 within analyte sensors 201 and 202 can include multiple spots or a single spot. Additionally, analyte sensors 201 and 202 can be operable to assay an analyte by any of the electrochemical detection techniques of coulometry, amperometry, voltammetry, or potentiometry.

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

[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, namely a first active region 218a and a second active region 218b, on the working electrode 214, which respond to the same or different analytes and are laterally spaced from each other on the surface of the working electrode 214. The active regions 218a and 218b may include a plurality of spots or a single spot configured for the detection of each analyte. The composition of the membrane 220 may vary in the active regions 218a and 218b or may be compositionally the same. The first active region 218a and the second active region 218b may be configured to detect their corresponding analytes at different working electrode potentials, as further discussed below.

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

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

[0186] Exemplary sensor configurations having a plurality of working electrodes, specifically two working electrodes, are described in more detail with reference to FIGS. 20 through 21C. The following description is primarily directed to sensor configurations having two working electrodes, but it should be understood that, by the extension of the disclosure herein, more than two working electrodes may be incorporated. Additional working electrodes may be used to endow the analyte sensor with additional sensing capabilities for detecting, for example, third and / or fourth analytes in addition to the first and second analytes.

[0187] Figure 20 shows a cross-sectional view of an exemplary analyte sensor configuration having two working electrodes, a reference electrode, and a counter electrode that is compatible for use in the disclosure of this specification. As shown, the analyte sensor 300 includes working electrodes 304 and 306 disposed on opposite surfaces of a substrate 302. A first active region 310a is disposed on the surface of working electrode 304, and a second active region 310b is disposed on the surface of working electrode 306. The counter electrode 320 is electrically insulated from the working electrode 304 by a dielectric layer 322, and the reference electrode 321 is electrically insulated from the working electrode 306 by a dielectric layer 323. Outer dielectric layers 30230 and 332 are disposed on the reference electrode 321 and the counter electrode 320, respectively. The membrane 340 can, according to various embodiments, cover at least the active regions 310a and 310b, and optionally, other components of the analyte sensor 300 or the entire analyte sensor 300 can be covered by the membrane 340. Further, the membrane 340 may be continuous, but can vary compositionally between a first membrane portion 340a and a second membrane portion 340b (i.e., over the active regions 310a and 310b) to allow for different permeability values to separately regulate the analyte flux at each location. In certain embodiments, different membrane formulations can be sprayed and / or printed on opposite surfaces of the analyte sensor 300. For example, without limitation, according to various embodiments, the first membrane portion 340a can cover at least the active region 310a, and the second membrane portion 340b can cover at least the active region 310b, other components of the analyte sensor 300, or the entire analyte sensor 300. Dip coating techniques can also be suitable, particularly for depositing at least a portion of a bilayer membrane on top of one of the active regions 310a and 310b. In certain embodiments, the membrane 340 may be the same or have a different composition in the active regions 310a and 310b. For example, without limitation, the membrane 340 may include a bilayer covering the active region 310a, a uniform membrane covering the active region 310b, or the membrane 340 may include a bilayer covering the active region 310b and a uniform membrane covering the active region 310a.In certain embodiments, according to certain embodiments of the present disclosure, one of the first membrane portion 340a and the second membrane portion 340b may comprise a bilayer membrane, and the other of the first membrane portion 340a and the second membrane portion 340b may comprise a single membrane polymer. In certain embodiments, the analyte sensor may include more than one membrane 340, for example, two or more membranes. For example, but not limited to, the analyte sensor may include membranes covering one or more active regions, such as 310a and 310b, and additional membranes covering the entire sensor as shown in FIG. 20. In such a configuration, the bilayer membrane may be formed over one or more active regions (e.g., 310a and 310b).

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

[0189] Similar to the analyte sensors 200, 201, and 202, the analyte sensor 300 may be operable to assay for aspartate and / or asparagine by any of the electrochemical detection techniques of coulometry, amperometry, voltammetry, or potentiometry.

[0190] An alternative sensor configuration having a plurality of working electrodes and different from the configuration shown in FIG. 20 may feature a counter / reference electrode instead of separate counter and reference electrodes 320, 321, and / or may feature a layer and / or film arrangement different from that explicitly shown. For example, but not limited to, the positions of counter electrode 320 and reference electrode 321 may be opposite to those shown in FIG. 20. Further, working electrodes 304 and 306 do not necessarily have to be on the opposite side of substrate 302 as shown in FIG. 20.

[0191] A suitable sensor configuration can feature electrodes that are substantially planar as a characteristic, but a sensor configuration featuring non-planar electrodes may also be advantageous and may be particularly suitable for use in the disclosure herein. In particular, substantially cylindrical electrodes arranged concentrically with each other can facilitate the deposition of a mass transfer limiting membrane, as described below. FIGS. 21A - 21C show perspective views of an analyte sensor featuring two working electrodes arranged concentrically with each other. It should be recognized that sensor configurations having a concentric electrode arrangement but no second working electrode are also possible in the present disclosure. In particular, concentric working electrodes spaced along the length of the sensor tail can facilitate film deposition by a continuous dip coating operation in a manner similar to that described above for a substantially planar sensor configuration.

[0192] FIG. 21A shows a perspective view of an exemplary sensor configuration in which a plurality of electrodes are substantially cylindrical and arranged concentrically 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 each other. In particular, the working electrode 410 is disposed on the surface of the central substrate 402, and the dielectric layer 412 is disposed on a portion of the working electrode 410 distal to the sensor tip 404. The working electrode 420 is disposed on the dielectric layer 412, and the dielectric layer 422 is disposed on a portion of the working electrode 420 distal to the sensor tip 404. The counter electrode 430 is disposed on the dielectric layer 422, and the dielectric layer 432 is disposed on a portion of the counter electrode 430 distal to the sensor tip 404. The reference electrode 440 is disposed on the dielectric layer 432, and the dielectric layer 442 is disposed on a portion of the reference electrode 440 distal to the sensor tip 404. Thus, the exposed surfaces of the working electrode 410, the working electrode 420, the counter electrode 430, and the reference electrode 440 are spaced apart from each other along the longitudinal axis B of the analyte sensor 400.

[0193] Referring further to FIG. 21A, a first active region 414a and a second active region 414b involved in different or the same analyte are disposed on the exposed surfaces of working electrodes 410 and 420, respectively, thereby enabling contact with a fluid for sensing to occur. In FIG. 21A, active regions 414a and 414b are shown as three separate spots, but it should be understood that in alternative sensor configurations, there may be fewer or more than three spots including a continuous layer of the active region. In certain embodiments, either or both of active regions 414a and 414b may be configured to detect aspartate. In certain embodiments, only one of the active regions of 414a and 414b is configured to detect aspartate. In certain embodiments, the other active region is configured to detect a second analyte different from aspartate. Non-limiting examples of the second analyte 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. In certain embodiments, only one of the active regions of 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 the second analyte 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, sensor 400 is partially covered by membrane 450 over working electrodes 410 and 420 and active regions 414a and 414b disposed thereon. FIG. 21B shows an alternative sensor configuration in which substantially all of sensor 401 is covered by membrane 450. Membrane 450 may be the same or compositionally different in active regions 414a and 414b. For example, membrane 450 may include a bilayer membrane covering active region 414a and a uniform membrane covering active region 414b.

[0195] It should be further understood that the positioning of the various electrodes in FIGS. 21A and 21B may be different from that explicitly shown. For example, the positions of the counter electrode 430 and the reference electrode 440 may be reversed from the configurations shown in FIGS. 21A and 21B. Similarly, the positions of the working electrodes 410 and 420 are not limited to the positions explicitly shown in FIGS. 21A and 21B. FIG. 21C shows an alternative to the sensor configuration shown in FIG. 21B, where the sensor 405 includes a counter electrode 430 and a reference electrode 440 that are positioned more proximally relative to the sensor tip 404, and working electrodes 410 and 420 that are positioned more distally relative to the sensor tip 404. A sensor configuration in which the working electrodes 410 and 420 are positioned more distally relative to the sensor tip 404 provides a larger surface area for the deposition of the active regions 414a and 414b (exemplarily showing five separate detection spots in FIG. 21C), thereby potentially being advantageous by facilitating an increase in signal strength in some cases. Similarly, the central substrate 402 can be omitted in any of the concentric sensor configurations disclosed herein, in which case the innermost electrode can instead support the subsequently deposited layers.

[0196] In certain embodiments, one or more electrodes of the analyte sensor described herein are wire electrodes, such as permeable wire electrodes. In certain embodiments, the sensor tail includes 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 enable the reaction of one or more enzymes and the analyte on the electrode. In certain embodiments, each electrode is formed from a thin wire having a diameter of from about 25.4 micrometers (0.001 inches) or less to about 254 micrometers (0.010 inches) or more. In certain embodiments, the working electrode has a diameter of from about 25.4 micrometers (0.001 inches) or less to about 254 micrometers (0.010 inches) or more, such as from about 50.8 micrometers (0.002 inches) to about 203 micrometers (0.008 inches), more preferably from 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, a conductive polymer, an alloy, or other conductive materials. In certain embodiments, the conductive material is a permeable conductive material. In certain embodiments, the electrodes can be formed by various manufacturing techniques (e.g., bulk metal processing, deposition of metal on a substrate, etc.), and the electrodes can be formed from plated wires (e.g., platinum on a steel wire) or bulk metals (e.g., platinum wire). In certain embodiments, the electrodes are formed from, for example, tantalum wire coated with a conductive material.

[0197] In certain embodiments, a reference electrode that can function as a reference electrode alone or as a dual electrode of a reference electrode and a counter electrode is formed from silver, silver / silver chloride, or the like. In certain embodiments, the reference electrode is juxtaposed and / or coiled with and / or around the working electrode. In certain embodiments, the reference electrode is helically wound around the working electrode. In certain embodiments, the wire assembly may be coated or adhered with an insulating material to provide an insulating attachment.

[0198] In certain embodiments, additional electrodes may be included at the sensor tail. For example, without limitation, a three-electrode system (working electrode, reference electrode, and counter electrode) and / or additional working electrodes (e.g., an electrode for detecting a second analyte). In certain embodiments where the sensor includes two working electrodes, the two working electrodes may be juxtaposed and a reference electrode may be disposed therearound (e.g., helically wound around the two or more working electrodes). In certain embodiments, the two or more working electrodes may extend parallel to each other. In certain embodiments, the reference electrode is wound around the working electrode and extends towards the distal end (i.e., the in vivo end) of the sensor tail. In certain embodiments, the reference electrode extends (e.g., helically) into the exposed region of the working electrode.

[0199] In certain embodiments, one or more working electrodes are helically wound around the reference electrode. In certain embodiments where two or more working electrodes are provided, the working electrodes may be formed in a double, triple, quadruple, etc. helical configuration (e.g., surrounding a reference electrode, an insulated rod, or other support structure) along the length of the sensor tail. In certain embodiments, the electrodes, e.g., two or more working electrodes, are formed coaxially. For example, without limitation, all the electrodes share the same central axis.

[0200] In certain embodiments, the working electrode comprises a tube with an insulator therebetween and a reference electrode disposed or wound therein. Alternatively, the reference electrode comprises a tube with an insulator therebetween and a working electrode disposed or wound therein. In certain embodiments, a polymer (e.g., insulating) rod is provided and one or more electrodes (e.g., one or more electrode layers) are disposed thereon (e.g., by electroplating). In certain embodiments, a metal (e.g., steel or tantalum) rod or wire coated with an insulating material (described herein) and having one or more working and reference electrodes disposed thereon is provided. For example, without limitation, the present disclosure provides a sensor comprising one or more tantalum wires, e.g., a sensor tail, and a conductive material is disposed on a portion of one or more of the tantalum wires to function as a working electrode. In certain embodiments, a platinum-coated tantalum wire is covered with an insulating material, and the insulating material is partially covered with a silver / silver chloride composition that functions as a reference and / or counter electrode.

[0201] In certain embodiments where the insulator is disposed on top of the working electrode (e.g., on the platinum surface of the electrode), a portion of the insulator can be peeled off or otherwise removed to expose the electroactive surface of the working electrode. For example, but not limited to, a portion of the insulator can be removed by hand, an excimer laser, chemical etching, laser ablation, grit blasting, or others. Alternatively, a portion of the electrode can be masked prior to deposition of the insulator to maintain an exposed electroactive surface area. In certain embodiments, the portion of the insulator that is peeled off and / or removed can be from about 0.1 mm (about 0.004 inches) or less in length to about 2 mm (about 0.078 inches) or more in length, for example from about 0.5 mm (about 0.02 inches) to about 0.75 mm (0.03 inches) in length. In certain embodiments, the insulator is a non-conductive polymer. In certain embodiments, the insulator comprises parylene, a fluorinated polymer, polyethylene terephthalate, polyvinylpyrrolidone, polyurethane, polyimide, and other non-conductive polymers. In certain embodiments, a glass or ceramic material can also be used 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] Some portions of the sensor including the active region are further described below. 2. Enzyme The active region of the analyte sensor of the present disclosure can be configured to detect an analyte. Non-limiting examples of analytes that can be detected using the disclosed analyte sensor include asparagine and aspartate. In certain embodiments, the active region of the analyte sensor of the present disclosure is configured to detect asparagine. In certain embodiments, the active region of the analyte sensor of the present disclosure is configured to detect aspartate.

[0203] In certain embodiments, the active region of the analyte sensor of the present disclosure can be configured to detect two or more analytes. For example, but not limited to, the active region can be configured to detect asparagine and aspartate. Alternatively, the active region 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, and each active region is configured to detect a different analyte. In certain embodiments, the first active region can be configured to detect asparagine, and the second active region can be configured to detect aspartate. In certain embodiments, the first active region can be configured to detect asparagine and / or aspartate, and the 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 can include an asparagine-responsive active region or an aspartate-responsive active region. In certain embodiments, the asparagine-responsive active region can include one or more enzymes for detecting asparagine. In certain embodiments, the aspartate-responsive active region can 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 shown enzyme reaction, aspartate oxidase (shown as AspOx in FIG. 22) can be used to detect aspartate in accordance with the disclosure herein. When the aspartate-responsive active region contains this enzyme system, aspartate oxidase can catalyze the oxidation of aspartate to oxaloacetate and the reduction of its coenzyme flavin adenine dinucleotide (FAD) to FADH2. Next, an electron transfer agent can mediate the electron transfer from FADH2 to the working electrode. The electrons transferred during this reaction provide the basis for aspartate detection at the working electrode. Next, the electrochemical signal obtained at the working electrode can be correlated with the amount of aspartate initially 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 specific 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. Next, the aspartate produced can be oxidized by aspartate oxidase as shown in FIGS. 22 and 23 and as 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 the electron transfer agent mediates the electron transfer from FADH2 to the working electrode. Next, the electrochemical signal obtained at the working electrode can be correlated with the amount of asparagine initially present in the sample.

[0208] Any suitable aspartate oxidase can be used in the analyte sensor of the present disclosure. Specific examples of aspartate oxidases suitable for use in the analyte sensors disclosed herein include, but are not limited to, potassium-dependent and potassium-independent aspartate oxidases. 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 oxidases include L-aspartate oxidases from Sulfolobus tokodaii, Escherichia coli, Arabidopsis thaliana, or Thermococcus litoralis. For example, but not limited to, the L-aspartate oxidase for use in the aspartate and asparagine sensors of the present disclosure is 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 entirety.

[0209] Any suitable asparaginase can be used in the analyte sensor of the present disclosure. Specific examples of asparaginases suitable for use in the analyte sensors 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 mung bean.

[0210] In certain embodiments, the present disclosure provides an analyte sensor for detecting aspartate. In certain embodiments, the analyte sensor for detecting aspartate can include a sensor tail portion comprising at least one working electrode and at least one aspartate-responsive active region disposed on the surface of the working electrode and containing 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 portion having at least one working electrode and at least one asparagine-responsive active region disposed on the surface of the working electrode and including an enzyme system comprising oxidase and hydrolase. In certain embodiments, the oxidase is aspartic acid oxidase. In certain embodiments, the hydrolase is asparaginase. In certain embodiments, the asparagine-responsive active region includes a first enzyme layer comprising oxidase, such as aspartic acid oxidase, and a second layer disposed on the first enzyme layer and comprising hydrolase, such as asparaginase. Alternatively or in addition, the oxidase, such as aspartic acid oxidase, and the hydrolase, such as asparaginase, are retained within the same enzyme layer. In certain embodiments, both aspartic acid oxidase and asparaginase present in the asparagine-responsive active region are potassium-independent.

[0212] In certain embodiments, the asparagine-responsive active region of the present disclosure can include an asparaginase to asparaginase oxidase ratio of from about 100:1 to about 1:100, such as from about 95:1 to about 1:95, from about 90:1 to about 1:90, from about 85:1 to about 1:85, from about 80:1 to about 1:80, from about 75:1 to about 1:75, from about 60:1 to about 1:60, from about 55:1 to about 1:55, from about 50:1 to about 1:50, from about 45:1 to about 1:45, from about 40:1 to about 1:40, from about 35:1 to about 1:35, from about 30:1 to about 1:30, from about 25:1 to about 1:25, from about 20:1 to about 1:20, from about 15:1 to about 1:15, from about 10:1 to about 1:10, from about 9:1 to about 1:9, from about 8:1 to about 1:8, from about 7:1 to about 1:7, from about 6:1 to about 1:6, from about 5:1 to about 1:5, from about 4:1 to about 1:4, from about 3:1 to about 1:3, or from about 2:1 to about 1:2. In certain embodiments, the asparagine-responsive active region can include an asparaginase to asparaginase oxidase ratio of from about 5:1 to about 1:5. In certain embodiments, the asparagine-responsive active region can include an asparaginase to asparaginase oxidase ratio of from about 2:1 to about 1:2. In certain embodiments, the asparagine-responsive active region can include an asparaginase to asparaginase oxidase ratio of about 1:1.

[0213] 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 wt% to about 80 wt% of aspartate oxidase, such as from about 10 wt% to about 75 wt%, from about 10 wt% to about 70 wt%, from about 10 wt% to about 65 wt%, from about 10 wt% to about 60 wt%, from about 10 wt% to about 55 wt%, from about 10 wt% to about 50 wt%, from about 10 wt% to about 45 wt%, from about 15 wt% to about 50 wt%, from about 15 wt% to about 45 wt%, from about 20 wt% to about 40 wt%, from about 20 wt% to about 35 wt%, from about 20 wt% to about 30 wt%. 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 wt% to about 50 wt% 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 wt% to about 45 wt% 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 wt% to about 35 wt% 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 wt% to about 35 wt% of aspartate oxidase.

[0214] In certain embodiments, the analyte-responsive active region, such as an asparagine-responsive active region, can comprise from about 0.1 wt% to about 50 wt% asparaginase, such as from about 0.1 wt% to about 45 wt%, from about 0.1 wt% to about 40 wt%, from about 0.1 wt% to about 35 wt%, from about 0.1 wt% to about 30 wt%, from about 0.1 wt% to about 25 wt%, from about 0.1 wt% to about 20 wt%, from about 0.1 wt% to about 15 wt%, from about 0.1 wt% to about 10 wt%, from about 0.5 wt% to about 45 wt%, from about 0.5 wt% to about 40 wt%, from about 0.5 wt% to about 30 wt%, from about 0.5 wt% to about 25 wt%, from about 0.5 wt% to about 20 wt%, from about 0.5 wt% to about 15 wt%, from about 0.5 wt% to about 10 wt%, from about 0.5 wt% to about 5 wt%, from about 20 wt% to about 40 wt%, from about 20 wt% to about 35 wt%, from about 20 wt% to about 30 wt%. In certain embodiments, the analyte-responsive active region, such as an asparagine-responsive active region, can comprise from about 0.1 wt% to about 20 wt% asparaginase. In certain embodiments, the analyte-responsive active region, such as an asparagine-responsive active region, can comprise from about 5 wt% to about 20 wt% asparaginase. In certain embodiments, the analyte-responsive active region, such as an asparagine-responsive active region, can comprise from about 0.1 wt% to about 5 wt% asparaginase. In certain embodiments, the analyte-responsive active region, such as an asparagine-responsive active region, can comprise from about 0.1 wt% to about 2 wt% asparaginase.

[0215] In certain embodiments, the aspartate-responsive active region or asparagine-responsive active region can further comprise, for example, a stabilizer for stabilizing 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 can comprise a stabilizer-to-asparaginase and / or aspartate oxidase ratio of from about 100:1 to about 1:100, such as from about 95:1 to about 1:95, from about 90:1 to about 1:90, from about 85:1 to about 1:85, from about 80:1 to about 1:80, from about 75:1 to about 1:75, from about 60:1 to about 1:60, from about 55:1 to about 1:55, from about 50:1 to about 1:50, from about 45:1 to about 1:45, from about 40:1 to about 1:40, from about 35:1 to about 1:35, from about 30:1 to about 1:30, from about 25:1 to about 1:25, from about 20:1 to about 1:20, from about 15:1 to about 1:15, from about 10:1 to about 1:10, from about 9:1 to about 1:9, from about 8:1 to about 1:8, from about 7:1 to about 1:7, from about 6:1 to about 1:6, from about 5:1 to about 1:5, from about 4:1 to about 1:4, from about 3:1 to about 1:3, or from about 2:1 to about 1:2, for example, a serum albumin to asparaginase and / or aspartate oxidase ratio. In certain embodiments, the aspartate-responsive active region or asparagine-responsive active region can comprise a stabilizer-to-asparaginase and / or aspartate oxidase ratio of from about 80:1 to about 1:80. In certain embodiments, the aspartate-responsive active region or asparagine-responsive active region can comprise a stabilizer-to-asparaginase and / or aspartate oxidase ratio of from about 50:1 to about 1:50. In certain embodiments, the aspartate-responsive active region or asparagine-responsive active region can comprise a stabilizer-to-asparaginase and / or aspartate oxidase ratio of from about 10:1 to about 1:10.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 from 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 stabilizer to asparaginase and / or aspartate oxidase from 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 stabilizer to asparaginase and / or aspartate oxidase from 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 stabilizer to asparaginase and / or aspartate oxidase from 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 from 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 from about 5 wt% to about 50 wt% stabilizer, such as from about 10 wt% to about 50 wt%, from about 15 wt% to about 45 wt%, from about 20 wt% to about 40 wt%, from about 20 wt% to about 35 wt%, from about 20 wt% to about 30 wt%. In certain embodiments, the aspartate-responsive active region or asparagine-responsive active region can comprise from about 5 wt% to about 40 wt% stabilizer. In certain embodiments, the aspartate-responsive active region or asparagine-responsive active region can comprise from about 5 wt% to about 35 wt% stabilizer. In certain embodiments, the aspartate-responsive active region or asparagine-responsive active region can comprise from about 5 wt% to about 30 wt% stabilizer. In certain embodiments, the aspartate-responsive active region or asparagine-responsive active region can comprise from about 10 wt% to about 30 wt% stabilizer.In certain embodiments, the aspartate-responsive active region or the asparagine-responsive active region can comprise from about 15 wt% to about 35 wt% stabilizer.

[0216] In certain embodiments, the analyte-responsive active region, e.g., the aspartate-responsive active region or the asparagine-responsive active region, can further comprise cofactors 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 limited to, a membrane coating the analyte-responsive active region can allow sufficient inward diffusion of the analyte to enable detection of the analyte while serving to retain the cofactor within the analyte-responsive active region.

[0217] In certain embodiments, the analyte sensor can include two working electrodes, e.g., a first active region disposed on a first working electrode and a second active region disposed on a second working electrode. In certain embodiments, the analyte sensors disclosed herein can be characterized by an aspartate-responsive active region and a second active region for detecting an analyte different from aspartate. In certain embodiments, the analyte sensors disclosed herein can be characterized by an asparagine-responsive active region and a second active region 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 region or an asparagine-responsive active region disposed on the surface of the first working electrode, and a second active region disposed on the surface of the second working electrode and configured to detect a different analyte, e.g., a second enzyme-responsive active region. Alternatively, in certain embodiments, the analyte sensor of the present disclosure can include a sensor tail having at least a first working electrode and a second working electrode, an aspartate-responsive active region disposed on the surface of the first working electrode, and an asparagine-responsive active region disposed on the surface of the second working electrode. In certain embodiments, when the sensor is configured to detect two or more analytes, detection of each analyte can include applying a potential to each working electrode such that distinct signals are obtained from each analyte. Next, the signals obtained from each analyte can be correlated to analyte concentration by use of a calibration curve or function, or by employing a look-up table. In certain embodiments, the correlation between the analyte signal and the analyte concentration can be performed by use of a processor.

[0218] In the configuration of certain other analyte sensors, the first active region and the second active region can be disposed on a single working electrode. The first signal can be obtained, for example, at a low potential from the first active region, and the second signal, which includes the contributions of the signals from both active regions, can be obtained at a high potential. Next, by subtracting the first signal from the second signal, it becomes possible to determine the signal contribution resulting from the second analyte. Next, the signal contribution from each analyte can be correlated with the analyte concentration in a manner similar to that described for the sensor configuration having a plurality of working electrodes. In a particular embodiment, an aspartate-responsive active region or an asparagine-responsive active region and a second active region (e.g., a second enzyme-responsive active region) configured to detect a different analyte are thus disposed on a single working electrode, one of the active regions can be configured to respond separately to facilitate the detection of each analyte. For example, either the aspartate / asparagine-responsive active region or the second active region that responds to the 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 can vary by changing the coverage (area or size) of the active region, the area ratio of the active regions relative to each other, the identity, the thickness and / or composition of the mass transfer limiting membrane covering the active region. Given the benefit of the disclosure herein, changes in these parameters can be readily implemented by those skilled in the art.

[0220] 3. Redox mediator In certain embodiments, the analyte sensors disclosed herein can include an electron transfer agent. For example, without limitation, at least one active region of the analyte sensors of the present disclosure can include an electron transfer agent. In certain embodiments, the analyte sensors of the present disclosure can include at least one aspartate-responsive active region that includes an electron transfer agent. In certain embodiments, the analyte sensors of the present disclosure can 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 can include two or more active regions, and only one active region (e.g., an aspartate-responsive active region or an asparagine-responsive active region) includes an electron transfer agent. In certain embodiments, the analyte sensor of the present disclosure can include two or more active regions, and each active region includes an electron transfer agent. For example, but not limited to, the analyte sensor of the present disclosure has 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 comprising a first electron transfer agent, and a second analyte-responsive active region disposed on the surface of the second working electrode and comprising 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 region, thereby generating a current indicative of the presence of a particular analyte. The amount of current generated is proportional to the amount of analyte present.

[0223] In certain embodiments, suitable electron transfer agents can include electroreducible and electrooxidizable ions, complexes, or molecules (such as quinones) having a redox potential that is several hundred millivolts higher or lower than the redox potential of the standard calomel electrode (SCE). In certain embodiments, the redox mediators can include osmium complexes and other transition metal complexes (e.g., as described in U.S. Pat. Nos. 6,134,461 and 6,605,200, which are hereby incorporated by reference in their entirety). Further examples of suitable redox mediators include those described in U.S. Pat. Nos. 6,736,957, 7,501,053, and 7,754,093, the disclosures of each of which are also hereby incorporated by reference in their entirety. Examples of other suitable redox mediators include metal compounds or complexes of ruthenium, osmium, iron (e.g., polyvinylferrocene or hexacyanoferrate), or cobalt (including their metallocene compounds). Suitable ligands for the metal complexes can also include multidentate ligands such as, for example, bipyridine, biimidazole, phenanthroline, or pyridyl(imidazole). Other suitable bidentate ligands can include, for example, amino acids, oxalic acid, acetylacetone, diaminoalkanes, or o-diaminoarenes. Any combination of monodentate, bidentate, tridentate, tetradentate, or higher dentate ligands can be present in the metal complex (such as 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 functional groups suitable for promoting covalent attachment to a polymer (referred to herein as the polymer backbone) within the active region, as further discussed below. For example, without limitation, an electron transfer agent for use in the present disclosure may comprise an electron transfer agent attached to a polymer. Non-limiting examples of suitable polymer-bound electron transfer agents include those described in U.S. Patent Nos. 8,444,834, 8,268,143, and 6,605,201, the disclosures of which are hereby incorporated by reference in their entireties. In certain embodiments, the electron transfer agent is a bidentate osmium complex attached to a polymer described herein. In certain embodiments, the electron transfer agent is a bidentate osmium complex attached to a polymer described herein, such as the polymer backbone described in Section 4 below. In certain embodiments, the polymer-bound electron transfer agent shown in FIG. 3 of U.S. Patent No. 8,444,834 may be used in the sensors of the present disclosure.

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

[0226] In certain embodiments, the analyte sensor of the present disclosure can include a sensor tail comprising a working electrode and an asparagine-responsive active region disposed on the surface of the working electrode and comprising asparaginase, aspartate oxidase, and an electron transfer agent. In certain embodiments, the asparagine-responsive active region includes 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 in addition, aspartate oxidase, asparaginase, and the electron transfer agent are retained within the same enzyme layer.

[0227] In certain embodiments, the aspartate-responsive active region or asparagine-responsive active region of the present disclosure can include a ratio of aspartate oxidase and / or asparaginase to redox mediator from about 100:1 to about 1:100, such as from about 95:1 to about 1:95, from about 90:1 to about 1:90, from about 85:1 to about 1:85, from about 80:1 to about 1:80, from about 75:1 to about 1:75, from about 60:1 to about 1:60, from about 55:1 to about 1:55, from about 50:1 to about 1:50, from about 45:1 to about 1:45, from about 40:1 to about 1:40, from about 35:1 to about 1:35, from about 30:1 to about 1:30, from about 25:1 to about 1:25, from about 20:1 to about 1:20, from about 15:1 to about 1:15, from about 10:1 to about 1:10, from about 9:1 to about 1:9, from about 8:1 to about 1:8, from about 7:1 to about 1:7, from about 6:1 to about 1:6, from about 5:1 to about 1:5, from about 4:1 to about 1:4, from about 3:1 to about 1:3, or from about 2:1 to about 1:2. In certain embodiments, the aspartate-responsive active region or asparagine-responsive active region can include a ratio of aspartate oxidase and / or asparaginase to redox mediator from about 7:1 to about 1:7. In certain embodiments, the aspartate-responsive active region or asparagine-responsive active region can include a ratio of aspartate oxidase and / or asparaginase to redox mediator from about 6:1 to about 1:6. In certain embodiments, the aspartate-responsive active region or asparagine-responsive active region can include a ratio of aspartate oxidase and / or asparaginase to redox mediator from about 5:1 to about 1:5. In certain embodiments, the aspartate-responsive active region or asparagine-responsive active region can include a ratio of aspartate oxidase and / or asparaginase to redox mediator from about 4:1 to about 1:4. In certain embodiments, the aspartate-responsive active region or asparagine-responsive active region can include a ratio of aspartate oxidase and / or asparaginase to redox mediator from about 3:1 to about 1:3.In certain embodiments, the aspartate-responsive active region or the asparagine-responsive active region can comprise a ratio of aspartate oxidase and / or asparaginase to redox mediator of from about 2:1 to about 1:2. In certain embodiments, the aspartate-responsive active region or the 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 the asparagine-responsive active region, can comprise from about 10 wt% to about 50 wt% redox mediator, e.g., from about 15 wt% to about 45 wt%, from about 20 wt% to about 40 wt%, from about 20 wt% to about 35 wt% or from about 20 wt% to about 30 wt% 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 from about 5 wt% to about 35 wt% 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 from about 10 wt% to about 35 wt% 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 from about 10 wt% to about 30 wt% 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 from about 15 wt% to about 35 wt% 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 bound. Any suitable polymer backbone may be present in the active region to facilitate detection of the analyte via covalent attachment of the enzyme and / or redox mediator thereto. Non-limiting examples of suitable polymers in the active region include, for example, polyvinylpyridines, such as poly(4-vinylpyridine) and / or poly(2-vinylpyridine), and polyvinylimidazoles, such as poly(N-vinylimidazole) and poly(1-vinylimidazole), or copolymers thereof, where a quaternized pyridine group serves as a binding site for the redox mediator or enzyme thereto. Exemplary copolymers that may be suitable for inclusion in the active region include those containing monomer units such as styrene, acrylamide, methacrylamide, or acrylonitrile. In certain embodiments, the polymer is a copolymer of vinylpyridine and styrene. In certain embodiments, the polymer that may be present in the active region includes polyurethane or a copolymer 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 hereby incorporated by reference in its entirety, such as poly(acrylic acid), styrene / maleic anhydride copolymer, methyl vinyl ether / maleic anhydride copolymer (GANTREZ polymer), poly(vinylbenzyl chloride), poly(allylamine), polylysine, poly(4-vinylpyridine) quaternized with a carboxypentyl group, and poly(sodium 4-styrenesulfonate). In certain embodiments where 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 of the analyte-responsive active region can be covalently bound to a polymer. In certain embodiments, one or more enzymes of 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 within the aspartate-responsive active region.

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

[0232] In certain embodiments, the membrane that coats the 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 enable detection of aspartate or asparagine. Suitable membrane polymers for coating the analyte-responsive active region are discussed further herein.

[0233] In certain embodiments, when a stabilizer is present in the active region, one or more enzymes within the region can be covalently bound to the stabilizer. For example, without limitation, one or more enzymes may be covalently bound to a stabilizer present in the active region, such as albumin. In certain embodiments, the aspartate oxidase present in the active region of the present disclosure may be covalently bound to a stabilizer. In certain embodiments, the asparaginase present in the active region of the present disclosure may be covalently bound to a stabilizer.

[0234] In certain embodiments, the covalent attachment of one or more enzymes and / or redox mediators to a polymer and / or stabilizer in a given active region can occur via cross-links introduced by a suitable cross-linking agent. In certain embodiments, cross-linking of a polymer and / or stabilizer to one or more enzymes and / or redox mediators can reduce the occurrence of delamination of the enzyme composition from the electrode. Suitable cross-linking agents can include one or more cross-linkable 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 cross-linking agent comprises one or more, two or more, three or more, or four or more epoxide groups. For example, but not by way of limitation, cross-linking agents for use in the present disclosure can include mono-, di-, tri-, and tetra-ethylene oxide. In certain embodiments, cross-linking agents for reaction with free amino groups in the enzyme (e.g., with free side-chain amines in lysine) can include cross-linking agents such as polyethylene glycol dibutyl ethers, polypropylene glycol dimethyl ethers, polyalkylene glycol allyl methyl ethers, polyethylene glycol diglycidyl ether (PEGDGE), or other polyepoxides, cyanuric chloride, N-hydroxysuccinimide, imido esters, epichlorohydrin, or derivatized variants thereof. In certain embodiments, the cross-linking agent is PEGDGE having an average molecular weight (M n ) of, for example, about 200 to 1000, for example about 400. In certain embodiments, the cross-linking agent is PEGDGE400. In certain embodiments, the cross-linking agent can be glutaraldehyde. In certain embodiments, cross-linking of the enzyme to the polymer is generally intermolecular. In certain embodiments, cross-linking 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, such as asparagine and / or asparaginase, of from about 100:1 to about 1:100. In certain embodiments, the aspartate and / or asparagine-responsive active region can comprise a ratio of crosslinker to one or more enzymes, such as asparagine and / or asparaginase, of from about 40:1 to about 1:40, such as from about 35:1 to about 1:35, from about 30:1 to about 1:30, from about 25:1 to about 1:25, from about 20:1 to about 1:20, from about 15:1 to about 1:15, from about 10:1 to about 1:10, from about 9:1 to about 1:9, from about 8:1 to about 1:8, from about 7:1 to about 1:7, from about 6:1 to about 1:6, from about 5:1 to about 1:5, from about 4:1 to about 1:4, from about 3:1 to about 1:3, from about 2:1 to about 1:2, or about 1:1. In certain embodiments, the aspartate and / or asparagine-responsive active region can comprise from about 5 wt% to about 50 wt% crosslinker, such as from about 5 wt% to about 45 wt%, from about 5 wt% to about 40 wt%, from about 5 wt% to about 35 wt%, from about 10 wt% to about 30 wt%, or from about 10 wt% to about 25 wt%.

[0236] 5. Mass transfer limiting membrane In certain embodiments, the analyte sensor disclosed herein further comprises a membrane that covers at least one active region, such as a first active region and / or a second active region and is permeable to one or more analytes. For example, but not limited to, the membrane is permeable to aspartate and / or asparagine. In certain embodiments, the membrane covers each of the active regions of the analyte sensor. Alternatively, a first membrane covers one of the active regions and a second membrane covers the second active region. Alternatively, a first membrane covers one of the active regions and a second membrane covers both the first and second active regions.

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

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

[0239] In certain embodiments, the mass transfer limiting film can include two or more layers, such as a bilayer or trilayer film. In certain embodiments, each layer can have a different concentration or thickness and can comprise different polymers or the same polymer. In certain embodiments, the first analyte-responsive active region may be covered by a multi-layer film, such as a bilayer film, and the second analyte-responsive active region may be covered by a single film. In certain embodiments, the first analyte-responsive active region may be covered by a multi-layer film, such as a bilayer film, and the second analyte-responsive active region may be covered by a multi-layer film, such as a bilayer film. In certain embodiments, the first analyte-responsive active region may be covered by a single film, and the second analyte-responsive active region may be covered by a multi-layer film, such as a bilayer film may be covered by a single film. In certain embodiments, the first analyte-responsive active region may be covered by a single film, and the second analyte-responsive active region may be covered by a single film.

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

[0241] In certain embodiments, the mass transfer limiting membrane can include polyvinylpyridine (e.g., poly(4-vinylpyridine) or poly(4-vinylpyridine)), polyvinylimidazole, a polyvinylpyridine copolymer (e.g., a copolymer of vinylpyridine and styrene), polyacrylate, polyurethane, polyetherurethane, silicone, polytetrafluoroethylene, polyethylene-co-tetrafluoroethylene, polyolefin, polyester, polycarbonate, biostable polytetrafluoroethylene, a homopolymer, copolymer, or terpolymer of polyurethane, polypropylene, polyvinyl chloride, polyvinylidene difluoride, polybutylene terephthalate, polymethyl methacrylate, polyetheretherketone, a cellulose polymer, polysulfone, and their block copolymers including, for example, diblock, triblock, alternating, random, and graft copolymers, or chemically related materials and others.

[0242] In certain embodiments, the membranes for use in the present disclosure, such as single-component membranes, may include polyvinylpyridine (e.g., poly(4-vinylpyridine) and / or poly(2-vinylpyridine)). In certain embodiments, the membranes for use in the present disclosure, such as single-component membranes, may include poly(4-vinylpyridine). In certain embodiments, the membranes for use in the present disclosure, such as single-component membranes, may include a copolymer of vinylpyridine and styrene. In certain embodiments, the membrane may comprise a polyvinylpyridine-co-styrene copolymer. For example, without limitation, the polyvinylpyridine-co-styrene copolymer for use in the present disclosure may include a polyvinylpyridine-co-styrene copolymer in which some of the pyridine nitrogen atoms are functionalized by uncrosslinked polyethylene glycol tails and some of the pyridine nitrogen atoms are functionalized by an alkylsulfonic acid, such as a 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 entire content of which is incorporated herein by reference. In certain embodiments, the polyvinylpyridine-based polymer has a molecular weight of from about 50 Da to about 500 kDa.

[0243] Suitable copolymers of vinyl pyridine and styrene can have a styrene content in the range of about 0.01% to about 50% mole percent, or about 0.05% to about 45% mole percent, or about 0.1% to about 40% mole percent, or about 0.5% to about 35% mole percent, or about 1% to about 30% mole percent, or about 2% to about 25% mole percent, or about 5% to about 20% mole percent. Substituted styrenes can likewise be used in similar amounts. Suitable copolymers of vinyl pyridine and styrene can 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 vinyl pyridine and styrene can have a molecular weight in the range of about 5 kDa to about 150 kDa, or about 10 kDa to about 125 kDa, or about 15 kDa to about 100 kDa, or about 20 kDa to about 80 kDa, or about 25 kDa to about 75 kDa, or about 30 kDa to about 60 kDa.

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

[0245] In certain embodiments, the membrane comprises a polyurethane membrane that includes regions that are both hydrophilic and hydrophobic. In certain embodiments, the hydrophobic polymer component is polyurethane, polyurethane urea, or poly(ether-urethane-urea). In certain embodiments, the polyurethane is a polymer produced by a condensation reaction of a diisocyanate and a difunctional hydroxyl-containing material. In certain embodiments, the polyurethane urea is a polymer produced by a condensation reaction of a diisocyanate and a difunctional amine-containing material. In certain embodiments, the diisocyanate for use herein includes, for example, an aliphatic diisocyanate containing from about 4 to about 8 methylene units, or a diisocyanate containing an alicyclic moiety. Further non-limiting examples of polymers that can be used for the production of the membranes of the sensors of the present disclosure include vinyl polymers, polyethers, polyesters, polyamides, inorganic polymers (such as polysiloxanes and polycarbosiloxanes), natural polymers (such as cellulose-based and protein-based materials), and mixtures (such as mixed or laminated structures), or combinations thereof. In certain embodiments, the hydrophilic polymer component is polyethylene oxide and / or polyethylene glycol. In certain embodiments, the hydrophilic polymer component is a polyurethane copolymer. For example, without limitation, the hydrophobic-hydrophilic copolymer component for use in the present disclosure is a polyurethane polymer comprising from about 10% to about 50%, such as about 20%, hydrophilic polyethylene oxide.

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

[0247] In certain embodiments, a hydrophilic or hydrophobic modifier can be used to “fine-tune” the permeability of the resulting membrane to the 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 there are multiple active regions, the mass transport limiting membrane can cover each active region including the choice of compositional changes in different active regions, which can be achieved via a continuous dip coating operation that produces bilayer membrane portions at the working electrodes placed in closer proximity to the sensor tip.

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

[0250] In certain embodiments, the composition of the mass transfer limiting membrane disposed on an analyte sensor having two active regions may be the same or different when the mass transfer limiting membrane covers each active region. For example, without limitation, the portion of the mass transfer limiting membrane covering the aspartate or asparagine-responsive active region may be multi-component, and / or the portion of the mass transfer limiting membrane covering the second analyte-responsive active region may be single-component. Instead, the portion of the mass transfer limiting membrane covering the aspartate or asparagine-responsive active region may be single-component, and / or the portion of the mass transfer limiting membrane covering the second analyte-responsive active region may be multi-component.

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

[0252] In certain embodiments, the mass transfer limiting membrane can comprise a membrane polymer crosslinked with a crosslinking agent disclosed herein and in Section 4 above. In certain embodiments where two mass transfer limiting membranes are present, e.g., a first mass transfer limiting membrane and a second mass transfer limiting membrane, each membrane can be crosslinked with a different crosslinking agent. For example, without limitation, the crosslinking agent can result in a membrane that more restricts the diffusion of a particular compound, e.g., an analyte in the membrane, or less restricts the diffusion of a particular compound, e.g., by affecting the size of the pores in the membrane. For example, without limitation, in a sensor configured to detect aspartate and / or asparagine, the mass transfer limiting membrane coating 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, the crosslinking agent for use in the present disclosure can include polyepoxides, carbodiimides, cyanuric chloride, triglycidyl glycerol (Gly3), N-hydroxysuccinimide, imido esters, epichlorohydrin, or derivatized variants thereof. In certain embodiments, the membrane polymer that coats one or more active regions can be crosslinked to a branched crosslinking agent that can, for example, reduce the amount of extract obtained from a mass transfer limiting membrane. Non-limiting examples of branched crosslinking agents include, for example, branched glycidyl ether crosslinking agents that include two, three, or more crosslinkable groups. In certain embodiments, the branched crosslinking agent can include two or more crosslinkable groups such as polyethylene glycol diglycidyl ether. In certain embodiments, the branched crosslinking agent can include three or more crosslinkable groups such as polyethylene glycol tetraglycidyl ether. In certain embodiments, the mass transfer limiting membrane can include polyvinyl pyridine or a copolymer of vinyl pyridine and styrene crosslinked to a branched glycidyl ether crosslinking agent that includes two or three crosslinkable groups such as polyethylene glycol tetraglycidyl ether or polyethylene glycol diglycidyl ether. In certain embodiments, epoxy groups such as polyepoxides, for example polyethylene glycol tetraglycidyl ether or polyethylene glycol diglycidyl ether, can form covalent bonds with pyridine or imidazole by ring opening of the epoxy ring, resulting in hydroxyalkyl groups that crosslink the body of the crosslinking agent and the heterocycle of the membrane polymer.

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

[0255] In certain embodiments, the crosslinking agent is polyethylene glycol diglycidyl ether (PEGDGE). In certain embodiments, the PEGDGE used to facilitate crosslinking (e.g., intermolecular crosslinking) between two or more membrane polymer backbones may exhibit a wide range of suitable molecular weights. In certain embodiments, the molecular weight of PEGDGE can range from about 100 g / mol to about 5000 g / mol. The number of ethylene glycol repeating units in each arm of PEGDGE may be the same or different and generally can vary over a range within a given sample for which an average molecular weight is obtained. In certain embodiments, the PEGDGE for use in the present disclosure has an average molecular weight (M n ) of about 200 - 1000, such as about 400. In certain embodiments, the crosslinking agent is PEGDGE400.

[0256] In certain embodiments, the polyethylene glycol tetra glycidyl ether used to facilitate crosslinking (e.g., intermolecular crosslinking) between two or more membrane polymer backbones can exhibit a wide range of suitable molecular weights. Up to four polymer backbones can be crosslinked by a single molecule of the polyethylene glycol tetra glycidyl ether crosslinking agent. In certain embodiments, the molecular weight of the polyethylene glycol tetra glycidyl ether can range from about 1000 g / mol to about 5000 g / mol. The number of ethylene glycol repeating units in each arm of the polyethylene glycol tetra glycidyl ether may be the same or different and generally can vary over a range within a given sample to obtain an average molecular weight. In certain embodiments, the mass transfer limiting membrane can be deposited directly on the active region.

[0257] In certain embodiments, polydimethylsiloxane (PDMS) can be incorporated into any of the mass transfer limiting membranes disclosed herein. In certain embodiments, the analyte sensor described herein can comprise a sensor tail comprising at least a first working electrode, a first analyte-responsive active region disposed on the surface of the first working electrode for detecting a first analyte, and a mass transfer limiting membrane that covers at least the first analyte-responsive active region and is permeable to the first analyte. In certain embodiments, the first analyte-responsive active region comprises an enzyme system that responds to a first analyte, for example, aspartate and / or asparagine, comprising at least one enzyme that responds to the first analyte. In certain embodiments, the first analyte-responsive active region comprises an electron transfer agent and / or a first polymer (optionally, an enzyme present within the first analyte-responsive active site is covalently bound to the first polymer).

[0258] In certain embodiments, the analyte sensor described herein can comprise a sensor tail having at least a first working electrode, at least one aspartate-responsive active region disposed on the surface of the first working electrode and comprising aspartate oxidase (the enzyme can optionally be covalently bound to a polymer), and a mass transfer limiting membrane that covers the aspartate-responsive active region and is permeable to aspartate. In certain embodiments, the mass transfer limiting membrane comprises a membrane polymer crosslinked with a branched glycidyl ether crosslinking agent having 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 can comprise a sensor tail having 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 including aspartate oxidase and asparaginase (one or both of the enzymes can optionally be covalently bound to a polymer), and a mass transfer limiting membrane that covers the asparagine-responsive active region and is permeable to asparagine. In certain embodiments, aspartate oxidase is present in a first layer and asparaginase is present in a second layer. In certain embodiments, aspartate oxidase and asparaginase are present in a single layer. In certain embodiments, the mass transfer limiting membrane comprises a membrane polymer crosslinked with a branched glycidyl ether crosslinking agent having 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 or a different analyte as the first active region. In certain embodiments, at least a portion of the mass transfer limiting membrane covering the first active region can cover the second active region. Alternatively or in addition, a second mass transfer limiting membrane can be used to cover the second active region. In certain embodiments, at least a portion of the second mass transfer limiting membrane covering the second active region can cover the first active region. In certain embodiments, the mass transfer limiting membrane covering the first active region has a composition different from that of the second mass transfer limiting membrane.

[0261] In certain embodiments, when a first active region and a second active region configured to assay different analytes are disposed on separate working electrodes, the mass transfer limiting membrane can have different permeability values for the first analyte and the second analyte. For example, without limitation, the mass transfer limiting membrane covering at least one of the active regions can include a mixture of a first membrane polymer and a second membrane polymer, or a bilayer of the first membrane polymer and the second membrane polymer. The active region not covered by the uniform membrane with the mixture or bilayer can be covered, and the uniform membrane can include 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 uniform membrane portion to be disposed over the first active region of the analyte sensor and a multi-component membrane portion to be disposed over the second active region of the analyte sensor, thereby simultaneously equalizing the permeability values for each analyte and improving sensitivity and detection accuracy. In certain embodiments, continuous film deposition can be performed by a continuous dip coating operation.

[0262] In certain embodiments, the mass transfer limiting membrane has a thickness in the range of from about 0.1 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, for example, the dry thickness. In certain embodiments, the mass transfer limiting membrane can have a thickness from about 0.1 μm to about 100 μm, such as from about 1 μm to about 90 μm, from about 1 μm to about 80 μm, from about 1 μm to about 70 μm, from about 1 μm to about 60 μm, from about 1 μm to about 50 μm, from about 1 μm to about 40 μm, from about 1 μm to about 30 μm, from about 1 μm to about 20 μm, from about 0.5 μm to about 10 μm, from about 1 μm to about 10 μm, from about 1 μm to about 5 μm or from about 0.1 μm to about 5 μm. In certain embodiments, the mass transfer limiting membrane can have a thickness from about 1 μm to about 100 μm.

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

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

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

[0266] In certain embodiments, the interference domain comprises a thin hydrophobic membrane that is non-swellable and restricts the diffusion of high molecular weight species. For example, without 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 can be deposited directly on the working electrode, for example, on the surface of a permeable working electrode. In certain embodiments, the interference domain has a thickness in the range of from about 0.1 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, for example, a dry thickness. In certain embodiments, the interference domain can have a thickness of from about 0.1 μm to about 100 μm, such as from about 1 μm to about 90 μm, from about 1 μm to about 80 μm, from about 1 μm to about 70 μm, from about 1 μm to about 60 μm, from about 1 μm to about 50 μm, from about 1 μm to about 40 μm, from about 1 μm to about 30 μm, from about 1 μm to about 20 μm, from about 0.5 μm to about 10 μm, from about 1 μm to about 10 μm, from about 1 μm to about 5 μm, or from about 0.1 μm to about 5 μm. In certain embodiments, the sensor can be immersed in the solution of the interference domain two or more times. For example, but not limited to, the sensor (or working electrode) of the present disclosure can be immersed in the solution of the interference domain at least 2 times, at least 3 times, at least 4 times, or at least 5 times to obtain the desired thickness of the interference domain.

[0268] 7. Manufacturing The present disclosure further provides a method for manufacturing the disclosed analyte sensor comprising 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 can further include adding a composition comprising an enzyme onto the surface of the working electrode to generate active sites on the working electrode. For example, but not limited to, the composition can include aspartate oxidase. In certain embodiments, the composition can further include asparaginase. In certain embodiments, the composition can further include an electron transfer agent. In certain embodiments, the composition can further include a crosslinking agent, such as polyethylene glycol diglycidyl ether, and a stabilizer, such as albumin, such as BSA. In certain embodiments, the method can further include curing the enzyme composition.

[0270] Instead, a first enzyme composition containing aspartic acid oxidase can be first deposited on the surface of the working electrode to generate a test substance-responsive active region on the working electrode. In certain embodiments, the first enzyme composition can further contain a redox mediator, a crosslinking agent such as polyethylene glycol diglycidyl ether, and / or a stabilizer such as albumin like BSA. In certain embodiments, the method can include curing the first enzyme composition to produce a first enzyme layer. In certain embodiments, the method can include depositing a second enzyme composition containing asparaginase on the surface of the first enzyme layer and curing the second enzyme composition to produce a second enzyme layer. In certain embodiments, the second enzyme composition can contain a crosslinking agent such as polyethylene glycol diglycidyl ether and / or a stabilizer such as albumin like BSA.

[0271] In certain embodiments, the method can further include adding a membrane composition on the cured enzyme composition(s). In certain embodiments, the membrane composition can include a polymer such as polyvinyl pyridine and / or a crosslinking agent such as polyethylene glycol diglycidyl ether. In certain embodiments, the method can include curing the polymer composition.

[0272] Generally, 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 immersed in the membrane solution or the membrane solution is sprayed onto the sensor, the volume of the membrane solution sprayed onto the sensor, etc., and any combination of these factors. In certain embodiments, the membranes described herein can have a thickness in the range of about 0.1 micrometers (μm) to about 1000 μm, such as about 1 μm to about 500 μm, about 10 μm to about 100 μm, or about 10 μm to about 100 μm. In certain embodiments, the sensor can be immersed in the membrane solution more than once. For example, but not limited to, the sensor (or working electrode) of the present disclosure can be immersed in the membrane solution at least 2 times, at least 3 times, at least 4 times, at least 5 times, or at least 6 times to obtain the desired membrane thickness.

[0273] In certain embodiments, the membrane may cover one or more active regions, and in certain embodiments, the active region may have a thickness of from about 0.1 μm to about 100 μm, such as from about 1 μm to about 90 μm, from about 1 μm to about 80 μm, from about 1 μm to about 70 μm, from about 1 μm to about 60 μm, from about 1 μm to about 50 μm, from about 1 μm to about 40 μm, from about 1 μm to about 30 μm, from about 1 μm to about 20 μm, from about 0.5 μm to about 10 μm, from about 1 μm to about 10 μm, from about 1 μm to about 5 μm, or from about 0.1 μm to about 5 μm. In certain embodiments, to achieve the desired thickness of the active region and / or the membrane, a series of droplets may be applied on top of each other without substantially increasing the diameter of the droplets being applied (i.e., while maintaining the desired diameter or range thereof). In certain embodiments, each single droplet may be applied, then allowed to cool or dry, and subsequently one or more additional droplets may be applied. For example, without limitation, to achieve the desired thickness of the active region, at least 1 droplet, at least 2 droplets, at least 3 droplets, at least 4 droplets, or at least 5 droplets may be added on top of each other.

[0274] III. Method of Use The present disclosure further provides a method of using the analyte sensor disclosed herein. In certain embodiments, the present disclosure provides a method for detecting an analyte. For example, without limitation, 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 comprises: (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 the surface of the working electrode and comprising aspartate oxidase and optionally a first polymer and / or a redox mediator, and (c) a mass transfer limiting membrane that covers the aspartate-responsive active region and is permeable to aspartate; (ii) applying a potential to the first working electrode; (iii) obtaining a first signal that is proportional to the concentration of aspartate in a fluid in contact with the aspartate-responsive active region at a potential above the redox potential of the aspartate-responsive active region; and (iv) correlating the first signal to the concentration of aspartate in a fluid, such as a body fluid.

[0276] In certain embodiments, a method for detecting aspartate can comprise: (i) exposing 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 the surface of the first working electrode and responsive to aspartate and comprising aspartate oxidase and optionally a first polymer and / or a redox mediator, and (c) a mass transfer limiting membrane that covers the aspartate-responsive active region and is permeable to aspartate to a fluid comprising aspartate; (ii) applying a potential to the first working electrode; (iii) obtaining a first signal that is proportional to the concentration of aspartate in the fluid at a potential above the redox potential of the aspartate-responsive active region; and (iv) correlating the first signal to the concentration of aspartate in the fluid, such as a body fluid.

[0277] In certain embodiments, a method for detecting asparagine comprises: (i) providing an analyte sensor comprising (a) a sensor tail comprising at least a first working electrode, (b) at least one asparagine-responsive active region disposed on the surface of the working electrode and comprising an enzyme system comprising aspartic acid oxidase, asparaginase, and optionally a first polymer and / or a redox mediator, and (c) a mass transfer limiting membrane that covers the asparagine-responsive active region and is permeable to asparagine. In certain embodiments, the method further comprises: (ii) applying a potential to the first working electrode; (iii) obtaining a first signal that is proportional to the concentration of asparagine in a fluid in contact with the asparagine-responsive active region, at a potential above the redox potential of the asparagine-responsive active region; and (iv) correlating the first signal to the concentration of asparagine in a fluid, such as a body fluid. In certain embodiments, the asparagine-responsive active region comprises an aspartic acid oxidase layer disposed on the first working electrode and an asparaginase layer disposed on the aspartic acid oxidase layer. In certain embodiments, the aspartic acid oxidase layer comprises aspartic acid oxidase and a redox mediator, and the asparaginase layer comprises asparaginase. Alternatively, the asparagine-responsive active region comprises aspartic acid oxidase and asparaginase in a single enzyme layer disposed on the first working electrode.

[0278] In certain embodiments, a method for detecting asparagine comprises: (i) exposing a test substance sensor to a fluid comprising asparagine, the test substance sensor comprising: (a) a sensor tail portion comprising at least a first working electrode; (b) an asparagine-responsive active region disposed on the surface of the first working electrode and comprising an enzyme system that responds to asparagine and includes asparagine oxidase, asparaginase, and optionally a first polymer and / or a redox mediator; and (c) a mass transfer limiting membrane that covers the asparagine-responsive active region and is permeable to asparagine, to the fluid comprising asparagine; (ii) applying a potential, such as a low potential, to the first working electrode; (iii) obtaining a first signal that is proportional to the concentration of asparagine in the fluid at an oxidation-reduction potential of the asparagine-responsive active region or higher; and (iv) correlating the first signal to the concentration of asparagine in a fluid, such as a body fluid. In certain embodiments, the asparagine-responsive active region comprises an asparagine oxidase layer disposed on the first working electrode and an asparaginase layer disposed on the asparagine oxidase layer. In certain embodiments, the asparagine oxidase layer comprises asparagine oxidase and a redox mediator, and the asparaginase layer comprises asparaginase. Alternatively, the asparagine-responsive active region comprises asparagine oxidase and asparaginase in a single enzyme layer disposed on the first working electrode.

[0279] In certain embodiments, the membrane polymer comprises polyvinyl pyridine or polyvinyl imidazole. In certain embodiments, the membrane polymer comprises a copolymer of vinyl pyridine and styrene. In certain embodiments, the mass transfer limiting membrane of the test substance sensor comprises a membrane polymer crosslinked with a branched crosslinking agent having three or more crosslinkable groups. In certain embodiments, the branched crosslinking agent comprises polyethylene glycol tetra glycidyl ether.

[0280] IV. Exemplary Embodiments A. In certain non-limiting embodiments, the subject matter of the present disclosure provides a substance sensor comprising: (i) a first working electrode; (ii) a substance-responsive active region disposed on the surface of the first working electrode and comprising aspartate oxidase for detecting a substance to be detected; and (iii) a mass transfer limiting membrane that at least covers the substance-responsive active region and is permeable to the substance to be detected.

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

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

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

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

[0285] A10. The substance sensor according to A9, wherein the substance to be detected is asparagine. A11. The substance sensor according to A9 or A10, wherein the asparaginase is covalently bonded to a polymer within the substance-responsive active region.

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

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

[0288] A15. The analyte 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 according to any one of A to A16, further comprising: (iv) a second working electrode; and (v) a second active region disposed on the surface of the second working electrode and comprising at least one enzyme that responds to a second analyte different from the first analyte and responds to the second analyte.

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

[0291] A20. The analyte sensor according to A17, further comprising a second mass transfer limiting membrane that covers the second active region and the first active region. A21. The second analyte 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, and is the analyte sensor according to any one of A17 to A20.

[0292] A22. The sensor tail is configured to be inserted into tissue, and is the analyte sensor according to any one of A to A21. A23. The sensor tail is configured to be inserted into tissue for detecting the level of an analyte in vivo, and is the analyte sensor according to any one of A to A22.

[0293] A24. 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, and is the analyte sensor according to any one of A9 to A23.

[0294] B. In certain non-limiting embodiments, the subject matter of the present disclosure is: (i) providing a test substance sensor, comprising: (a) a sensor tail portion comprising at least a first working electrode; (b) at least one aspartate-responsive active region disposed on the surface of the first working electrode and comprising aspartate oxidase; and (c) a mass transfer limiting membrane that covers at least the aspartate-responsive active region and is permeable to aspartate; (ii) applying a potential to the first working electrode; (iii) obtaining a first signal that is proportional to the concentration of aspartate in a fluid in contact with the aspartate-responsive active region at a potential above the redox potential of the aspartate-responsive active region; and (iv) correlating the first signal with the concentration of aspartate in the fluid, thereby providing a method for detecting aspartate.

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

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

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

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

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

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

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

[0302] B14. The method according to B13, wherein the second portion of the mass transfer limiting membrane covers the second active region. B15. The method according to B13, further comprising a second mass transfer limiting membrane that covers the second active region.

[0303] B16. The method according to B13, further comprising a second mass transfer limiting membrane that covers the second active region and the first active region. B17. The method according to any one of B13 to B16, wherein the second analyte 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] The sensor tail is configured for insertion into tissue by the method described in any one of B to B17. The sensor tail is configured for insertion into tissue to detect the level of aspartate in vivo by the method described in any one of B to B18.

[0305] C. In certain non-limiting embodiments, the subject matter of the present disclosure is (i) providing an analyte sensor comprising: (a) a sensor tail comprising at least a first working electrode; (b) an aspartate-responsive active region disposed on the surface of the first working electrode and comprising an enzyme system comprising aspartate oxidase and asparaginase; and (c) a mass transfer 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 that is proportional to the concentration of aspartate in a fluid in contact with the aspartate-responsive active region at a potential above the redox potential of the aspartate-responsive active region; and (iv) correlating the first signal to the concentration of aspartate in the fluid, thereby providing a method for detecting aspartate.

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

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

[0308] The method according to any one of C to C4, wherein the aspartate-responsive active region further comprises a cross-linking agent. The method according to C6, wherein the crosslinking agent is polyethylene glycol diglycidyl ether (PEGDGE).

[0309] The method according to any one of C1 to C7, wherein the mass transfer limiting membrane comprises a polyvinylpyridine-based polymer, polyvinylimidazole, polyacrylate, polyurethane, polyetherurethane, silicone, or a combination thereof.

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

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

[0312] The method according to any one of C1 to C12, further comprising: (d) a second working electrode; and (e) a second active region disposed on the surface of the second working electrode and comprising at least one enzyme that responds to a second analyte different from the first analyte and responds to the second analyte.

[0313] The method according to C13, wherein the second portion of the mass transfer limiting membrane covers the second active region. The method according to C13, further comprising a second mass transfer limiting membrane that covers the second active region.

[0314] The method according to C13, further comprising a second mass transfer limiting membrane that covers the second active region and the first active region. The method according to any one of C13 to C16, wherein the second analyte 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] The method according to any one of C to C17, wherein the sensor tail is configured for insertion into tissue. The method according to any one of C to C18, wherein the sensor tail is configured to be inserted into tissue to detect the level of asparagine in vivo.

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

[0317] The method according to any one of C to C19, wherein the asparagine-responsive active region comprises a single layer comprising aspartate oxidase and asparaginase. The method according to any one of C to C21, wherein aspartate oxidase is covalently bound to a polymer within the asparagine-responsive active region.

[0318] The method according to any one of C to C22, wherein asparaginase is covalently bound to a polymer within the asparagine-responsive active region. Examples The subject matter of the present disclosure is provided as an illustration of the subject matter disclosed herein and is not limiting, but will be better understood by reference to the following examples.

[0319] Example 1: Aspartate Sensor This example provides a sensor for detecting aspartate, which includes an enzyme system comprising aspartate oxidase and a redox mediator disclosed in FIG. 22. In this analyte sensor, the redox mediator is chemically cross-linked to the aspartate oxidase enzyme. The components in Table 1 were mixed and then deposited twice onto the working electrode. After curing, some sensors were immersed in a self-made polyvinylpyridine polymer film composition (denoted as 10Q5-Gly3) to form sensor membranes of different thicknesses (denoted as 1×6, 2×6, and 3×6, where the first number is the number of immersions in the dipping solution and the second number is the rate (mm / s) at which the sensor is pulled out of the dipping solution).

[0320]

Table 1

[0321] The sensors were tested at 33 °C under air and argon 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, and this reaction of oxygen competes with the oxidation reaction by the redox mediator, so it is an undesirable side reaction. As shown in FIG. 24, the sensors first showed a good response under air. The sensor membrane decreased the current, but not significantly. The subsequent argon purge did not change the current response of the sensors, indicating that the enzyme system is very efficient in 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 a few minutes and then stabilized after exposure to a new concentration of aspartate. FIG. 25B shows the concentration-versus-current curve, and FIG. 25C shows the calibration curve for each of the sensors, indicating that the response is nearly linear when the aspartate level is less than 1000 μM. In particular, R 2The value ranges from 0.9896 to 0.9939. Table 2 provides the sensitivity of the sensor tested with aspartate from 0 to 1000 μM.

[0323]

Table 2

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

[0325]

Table 3

[0326]

Table 4

[0327]

Table 5

[0328]

Table 6

[0329] In the case of enzyme loading with 3X and 7X, precipitation was found to occur in the formulation mixture, and thus such sensors were not prepared. To evaluate the signal response, 1X and 2X sensors were tested with various concentrations of aspartate (49.8 μM, 99.5 μM, 198 μM, 392 μM, 676 μM, and 952 μM). As shown in Figure 26, the current increased over a few minutes after exposure to the new concentration of aspartate and then stabilized. Furthermore, the sensor designated as 2X showed a strong response in the presence of the new aspartate concentration compared to the 1X sensor. More deposition paths also increased the sensor current for both 1X and 2X enzyme concentrations. Table 7 summarizes the sensitivities 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 sensors of Example 2 were tested with various amounts of redox mediator. In particular, as shown in Table 8, the sensors were modified by doubling the amount of redox mediator and enzyme compared to Table 4. In addition, the amount of redox mediator was decreased by 3X as shown in Table 9. Table 10 provides the composition of the aspartate sensing layer including a decrease in the amount of redox mediator and BSA.

[0332]

Table 8

[0333]

Table 9

[0334]

Table 10

[0335] Sensors with 2X amounts of enzyme and 2X amounts of redox mediator were tested with various concentrations of aspartate to evaluate the signal response. As shown in Figure 27, the current increased over a few minutes after exposure to the new concentration of aspartate and then stabilized. Table 11 provides the sensitivity (nA / mM) of the sensors tested with or without the 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 sensors without a membrane (PVP + P400).

[0336]

Table 11

[0337] Example 4 - Asparagine Sensor: Asparaginase Layer on Aspartate Detection Layer This example provides an example of an asparagine sensor that uses an enzyme system comprising asparaginase, aspartate oxidase, and a redox mediator, as shown in Figure 23. In this example, the asparagine sensor comprises an asparaginase layer on an aspartate detection layer. The sensor was prepared by depositing an asparaginase layer containing the components of Table 12 on top of the aspartate detection 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). Next, a subset of the sensors was immersed in the polyvinylpyridine polymer membrane solution as described in the previous examples.

[0338]

Table 12

[0339] Sensors with and without membranes 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 FIGS. 28A, 28B, and 28C, the current increased over several minutes after exposure to the new asparagine concentration and then stabilized, indicating that the sensors respond well to asparagine. FIG. 28B shows the average of each type of sensor shown in FIG. 28A. FIG. 28C compares the responses to aspartate and asparagine with the asparagine sensor fabricated in this example. The enzyme system of the asparagine sensor is expected to respond to aspartate as well, which is shown in FIG. 28C.

[0340] Example 5 - Asparagine Sensor: Asparaginase in the Aspartate Detection 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 the asparaginase is included in the aspartate detection layer. The asparaginase used was purchased from ProSpec (catalog number ENZ - 287). The sensor was prepared by depositing a mixture containing the components of Table 13 on the working electrode. Next, a subset of the sensors was immersed in the membrane solution.

[0341] [Table 13]

[0342] Sensors with and without membranes were tested in the presence of various asparagine concentrations. As shown in FIG. 29, the current increased over several minutes after exposure to the new aspartate concentration and then stabilized. These data indicate that asparagine sensors containing asparaginase in the aspartate detection layer also respond well to asparagine.

[0343] Although the disclosed subject matter and its advantages have been described in detail, it is to be understood that various changes, substitutions, and modifications can be made herein without departing from the spirit and scope of the disclosed subject matter. Further, the scope of this application is not intended to be limited to the particular embodiments of the processes, machines, manufactures, and compositions of matter, methods, and processes described herein. Those skilled in the art will readily appreciate that presently existing or later developed processes, machines, manufactures, compositions of matter, methods, or steps that perform substantially the same function or achieve substantially the same result as the corresponding embodiments described herein can be utilized in accordance with the disclosed subject matter. Accordingly, the appended claims are intended to include within their scope such processes, machines, manufactures, compositions of matter, methods, or steps.

[0344] Various patents, patent applications, publications, product descriptions, protocols, and sequence accession numbers are cited through this application, and the inventions thereof are hereby incorporated by reference in their entirety for all purposes.

Claims

1. A sensor for a substance to be tested, (i) at least one working electrode, (ii) At least one test substance-responsive active region for detecting the test substance, the test substance-responsive active region comprising aspartate oxidase, an electron transfer agent, a polymer, and a stabilizer, wherein the aspartate oxidase and the electron transfer agent are present in a weight ratio of 3:1 to 1:3, (iii) Interference domain and (iv) A test substance sensor comprising a mass transfer limiting membrane that covers at least the test substance-responsive active region and is permeable to the test substance, wherein the test substance sensor is configured to be inserted into tissue.

2. The test substance sensor according to claim 1, wherein the test substance is aspartate.

3. The test substance sensor according to claim 1, wherein the test substance-responsive active region further comprises asparaginase.

4. The test substance sensor according to claim 3, wherein the test substance is asparagine.

5. The test substance sensor according to claim 3, wherein one or more of the aspartate oxidase and the asparaginase are covalently bonded to the polymer.

6. The test substance sensor according to claim 3, wherein the test substance-responsive active region includes a layer containing asparaginase disposed on top of the layer containing aspartate oxidase.

7. The test substance sensor according to claim 3, wherein the test substance-responsive active region comprises a single layer containing the aspartate oxidase and the asparaginase.

8. The test substance sensor according to any one of claims 1 to 7, wherein the mass transfer limiting membrane comprises a polyvinylpyridine polymer, polyvinylimidazole, polyacrylate, polyurethane, polyetherurethane, silicone, or a combination thereof.

9. The test substance sensor according to claim 8, wherein the mass transfer limiting membrane comprises a polyvinylpyridine polymer or copolymer.

10. The test substance sensor according to any one of claims 1 to 7, wherein the interference domain is located between the working electrode and the test substance-responsive active region.

11. The test substance sensor according to claim 10, wherein the interference domain includes a polymer domain.

12. The test substance sensor according to claim 11, wherein the polymer domain comprises cellulose acetate, cellulose acetate butyrate, hydroxyethylcellulose, cellulose acetate phthalate, cellulose acetate propionate, or cellulose acetate trimellitate.

13. The test substance sensor according to any one of claims 1 to 7, wherein the interference domain is located between the test substance-responsive active region and the mass transfer limiting membrane.

14. The test substance sensor according to claim 12, wherein the interference domain comprises a hydrophobic and non-swelling membrane.

15. The test substance sensor according to any one of claims 1 to 7, wherein the stabilizer is albumin.

16. A method for detecting aspartate, (i) A step of exposing the test substance sensor according to any one of claims 1 to 2 to a fluid, (ii) A step of applying a potential to the working electrode, (iii) A step of obtaining a signal that is above the redox potential of the aspartate-responsive active region as the test substance-responsive active region and is proportional to the aspartate concentration in the fluid in contact with the aspartate-responsive active region, (iv) A step of correlating the signal with the concentration of aspartate in the fluid. A method that includes this.

17. A method for detecting asparagine, (i) A step of exposing the test substance sensor according to any one of claims 3 to 7 to a fluid, (ii) A step of applying a potential to the working electrode, (iii) A step of obtaining a signal that is above the redox potential of the asparagine-responsive active region, which is the test substance-responsive active region, and is proportional to the asparagine concentration in the fluid in contact with the active region, (iv) A step of correlating the signal with the asparagine concentration in the fluid. A method that includes this.

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