Continuous potassium sensor and method of use thereof

Abstract

To provide an analyte sensor for use in detecting potassium.SOLUTION: In certain embodiments, an analyte sensor of the present disclosure includes at least two asparagine-responsive active areas, where each asparagine-responsive active area includes an asparaginase that exhibits a particular potassium dependency. In certain embodiments, the analyte sensor of the present disclosure includes at least two aspartate-responsive active areas, where each aspartate-responsive active area includes an aspartate oxidase that exhibits a particular potassium dependency. The present disclosure further provides a method for monitoring potassium levels, e.g., in vivo potassium levels, using the disclosed analyte sensors.SELECTED DRAWING: Figure 1A

Description

[Technical field]

[0001] The subject matter described herein relates to analyte sensors for sensing potassium ions and methods of use thereof. [Background technology]

[0002] Detection of various analytes in an individual may be crucial for monitoring the health of an individual, since deviations from normal analyte levels may indicate a physiological condition. For example, monitoring glucose levels may allow people suffering from diabetes to take appropriate corrective measures, including administering medication or consuming certain food or beverage products, to avoid serious physiological harm. Other analytes, such as potassium, may be desirable for monitoring certain physiological conditions. In certain instances, it may be desirable to monitor more than one analyte to monitor a single or multiple physiological conditions, especially when a person suffers from a co-occurring condition in which two or more analytes are combined with each other and simultaneously dysregulated.

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

[0004] Many analytes represent interesting targets for physiological analysis, provided that suitable detection chemistries can be identified. To this end, enzyme-based amperometric sensors configured to continuously assay glucose in vivo have been developed and refined in recent years to aid in monitoring the health of diabetic individuals. Other analytes that commonly undergo dysregulation that coincides with glucose in diabetic individuals include, for example, potassium. Moreover, it may also be desirable to monitor potassium independent of glucose dysregulation. For example, potassium levels may be important to monitor in people suffering from various kidney or heart diseases. Implantable analyte sensors configured to detect potassium in vivo are not currently available. Thus, there is a need in the art for sensors to detect potassium in vivo. Summary of the Invention

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

[0006] To achieve these and other advantages, and in accordance with the objectives of the disclosed subject matter, as embodied and broadly described, the disclosed subject matter is directed to an analyte sensor for monitoring potassium levels in vivo.

[0007] In certain embodiments, the analyte sensor includes a sensor tail comprising at least a first working electrode and a second working electrode. In certain embodiments, the analyte sensor further includes a first analyte responsive active area disposed on a surface of the first working electrode and including a first aspartate oxidase, and a second analyte responsive active area disposed on a surface of the second working electrode and including a second aspartate oxidase. In certain embodiments, the analyte sensor further includes a first mass transport limiting membrane permeable to potassium overcoating the first analyte responsive active area and / or the second analyte responsive area. In certain embodiments, the first aspartate oxidase and the second aspartate oxidase have different potassium dependencies. For example, but not limited to, the first aspartate oxidase is potassium independent and the second aspartate oxidase is potassium dependent.

[0008] In certain embodiments, the first analyte-responsive active area further comprises a first asparaginase and the second analyte-responsive active area further comprises a second asparaginase. In certain embodiments, the first asparaginase and the second asparaginase have different potassium dependencies. For example, but not limited to, the first asparaginase is potassium-independent and the second asparaginase is potassium-dependent. In certain embodiments, the first aspartate oxidase and the second aspartate oxidase are both potassium-independent. In certain embodiments, the first analyte-responsive active area comprises a first enzyme layer comprising a first aspartate oxidase and a second enzyme layer comprising a first asparaginase disposed on the first enzyme layer. In certain embodiments, the second analyte responsive active area comprises a first enzyme layer comprising a second aspartate oxidase and a second enzyme layer comprising a second asparaginase disposed on the first enzyme layer. In certain embodiments, the first analyte responsive active area comprises a first enzyme layer comprising a first aspartate oxidase and a first asparaginase. In certain embodiments, the second analyte responsive active area comprises a first enzyme layer comprising an aspartate oxidase and a second asparaginase.

[0009] In certain embodiments, the first analyte responsive active area and / or the second analyte responsive active area further comprise an electron transfer agent and / or a stabilizing agent. In certain embodiments, the first mass transport limiting membrane comprises a polyvinylpyridine based polymer, polyvinylimidazole, polyacrylate, polyurethane, polyether urethane, silicone, or a combination thereof.

[0010] In certain embodiments, the analyte sensor includes a sensor tail comprising at least a first working electrode and a second working electrode. In certain embodiments, a first aspartate-responsive active area is disposed on a surface of the first working electrode and includes a potassium-dependent aspartate oxidase. In certain embodiments, a second aspartate-responsive active area is disposed on a surface of the second working electrode and includes a potassium-dependent aspartate oxidase. In certain embodiments, a first mass-transfer limiting membrane that is permeable to aspartate and potassium overcoats the first aspartate-responsive active area and / or the second aspartate-responsive active area. In certain embodiments, the first and / or second aspartate-responsive active area further include an electron transfer agent. For example, but not limited to, the first aspartate-responsive active area includes a first electron transfer agent and the second aspartate-responsive active area includes a second electron transfer agent. In certain embodiments, the potassium-dependent aspartate oxidase and / or the first electron transfer agent are covalently bound to a polymer in the first aspartate-responsive active area. In certain embodiments, the potassium-independent aspartate oxidase and / or the second electron transfer agent are covalently bound to a polymer in the second aspartate-responsive active area. In certain embodiments, the first and / or second aspartate-responsive active area further comprises a stabilizing agent, such as albumin. In certain embodiments, the first mass transport limiting membrane comprises a polyvinylpyridine-based polymer, polyvinylimidazole, polyacrylate, polyurethane, polyether urethane, silicone, or a combination thereof.

[0011] In certain embodiments, the analyte sensor includes a sensor tail with at least a first working electrode and a second working electrode. In certain embodiments, the analyte sensor further includes a first asparagine-responsive active area disposed on a surface of the first working electrode and including a first aspartate oxidase and a first asparaginase, and a second asparagine-responsive active area disposed on a surface of the second working electrode and including a second aspartate oxidase and a second asparaginase. In certain embodiments, the analyte sensor further includes a first mass transport limiting membrane permeable to asparagine overcoating the first asparagine-responsive active area and / or the second asparagine-responsive area. In certain embodiments, the first aspartate oxidase and the second aspartate oxidase are potassium independent. In certain embodiments, the first asparaginase and the second asparaginase have different potassium dependencies. For example, and without limitation, a first asparaginase is potassium independent and a second asparaginase is potassium dependent.

[0012] In certain embodiments, the first asparagine-responsive active area and / or the second asparagine-responsive active area further comprise an electron transfer agent. In certain embodiments, one or more of the enzymes in the first enzyme system are covalently bound to a polymer in the first asparagine-responsive active area, and / or one or more of the enzymes in the second enzyme system are covalently bound to a polymer in the second asparagine-responsive area. In certain embodiments, the first mass transport limiting membrane comprises a polyvinylpyridine-based polymer, polyvinylimidazole, polyacrylate, polyurethane, polyether urethane, silicone, or a combination thereof.

[0013] In certain embodiments, the first asparagine-responsive active area comprises a first enzyme layer comprising aspartate oxidase and a second layer comprising a first asparaginase disposed on the first enzyme layer. In certain embodiments, the first asparagine-responsive active area comprises an enzyme layer comprising aspartate oxidase and a first asparaginase. Alternatively or additionally, the second asparagine-responsive active area comprises a first enzyme layer comprising aspartate oxidase and a second layer comprising a second asparaginase disposed on the first enzyme layer. In certain embodiments, the second asparagine-responsive active area comprises an enzyme layer comprising aspartate oxidase and a second asparaginase.

[0014] In certain embodiments, the analyte sensor of the present disclosure includes a third working electrode and an active area disposed on a surface of the third working electrode and responsive to a second analyte different from potassium, the active area including at least one enzyme responsive to the second analyte. In certain embodiments, the second analyte is glutamate, glucose, ketone, 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. In certain embodiments, the second portion of the mass transport limiting membrane or the second mass transport limiting membrane overcoats the active area on the third working electrode. Alternatively, the second mass transport limiting membrane overcoats the active area on the third working electrode and / or overcoats the active area on the third working electrode and one or more of the asparagine-responsive active areas.

[0015] The present disclosure further provides a method for detecting potassium, e.g., potassium ions, in a fluid, e.g., a biological fluid. In certain embodiments, any analyte sensor of the present disclosure may be used to detect potassium, e.g., potassium ions, in a fluid, e.g., a biological fluid, of a subject. In certain embodiments, the analyte sensor is implanted in a subject at risk for or having a neurological condition or diabetes. In certain embodiments, the analyte sensor is implanted in the subject for about 15 days.

[0016] In certain embodiments, the method includes providing an analyte sensor, the analyte sensor including a sensor tail with at least a first working electrode and a second working electrode. In certain embodiments, the analyte sensor further includes a first analyte responsive active area disposed on a surface of the first working electrode and including a first aspartate oxidase, and a second analyte responsive active area disposed on a surface of the second working electrode and including a second aspartate oxidase. In certain embodiments, the analyte sensor further includes a first mass transport limiting membrane permeable to potassium overcoating the first analyte responsive active area and / or the second analyte responsive area. In certain embodiments, the first aspartate oxidase and the second aspartate oxidase have different potassium dependencies. For example, but not limited to, the first aspartate oxidase is potassium independent and the second aspartate oxidase is potassium dependent. In certain embodiments, the first analyte responsive active area further comprises a first asparaginase and the second analyte responsive active area further comprises a second asparaginase. In certain embodiments, the first asparaginase and the second asparaginase have different potassium dependencies. For example, but not limited to, the first asparaginase is potassium independent and the second asparaginase is potassium dependent. In certain embodiments, the method further comprises applying a potential to the first and second working electrodes, acquiring a first signal at or above a redox potential of the first analyte responsive active area, acquiring a second signal at or above a redox potential of the second analyte responsive activity, and correlating the first and second signals to a concentration of potassium in the fluid.

[0017] In certain embodiments, the method may include providing an analyte sensor including a sensor tail including at least a first working electrode and a second working electrode, a first aspartate-responsive active area including a potassium-dependent aspartate oxidase disposed on a surface of the first working electrode, a second aspartate-responsive active area including a potassium-independent aspartate oxidase disposed on a surface of the second working electrode, and a first mass transport limiting membrane permeable to aspartate and potassium overcoating the first aspartate-responsive active area and / or the second aspartate-responsive active area. In certain embodiments, the method further includes applying a potential to the first and second working electrodes, acquiring a first signal at or above a redox potential of the first aspartate-responsive active area, acquiring a second signal at or above a redox potential of the second aspartate-responsive activity, and correlating the first and second signals to a concentration of potassium in the fluid.

[0018] In certain embodiments, the method includes providing an analyte sensor, the analyte sensor including a sensor tail with at least a first working electrode and a second working electrode. In certain embodiments, the analyte sensor further includes a first asparagine-responsive active area disposed on a surface of the first working electrode and including an aspartate oxidase and a first asparaginase, and a second asparagine-responsive active area disposed on a surface of the second working electrode and including an aspartate oxidase and a second asparaginase. In certain embodiments, the first aspartate oxidase and the second aspartate oxidase are potassium-independent. In certain embodiments, the first asparaginase and the second asparaginase have different potassium dependencies. For example, but not limited to, the first asparaginase is potassium-independent and the second asparaginase is potassium-dependent. In certain embodiments, the analyte sensor further comprises a first mass transport limiting membrane permeable to asparagine overcoating the first asparagine-responsive active area and / or the second asparagine-responsive area. In certain embodiments, the method further comprises applying a potential to the first working electrode and the second working electrode. In certain embodiments, the method further comprises obtaining a first signal at or above the redox potential of the first asparagine-responsive active area. In certain embodiments, the method further comprises obtaining a second signal at or above the redox potential of the second asparagine-responsive active area. In embodiments, the method further comprises correlating the first signal and the second signal to a concentration of potassium ions in the fluid.

[0019] In certain embodiments, the first asparagine-responsive active area and the second asparagine-responsive active area of ​​the analyte sensor for use in the disclosed method further comprise an electron transfer agent.In certain embodiments, one or more of the enzymes in the first enzyme system are covalently bound to the polymer in the first asparagine-responsive active area, and / or one or more of the enzymes in the second enzyme system are covalently bound to the polymer in the second asparagine-responsive area.In certain embodiments, the first mass transport limiting membrane comprises a polyvinylpyridine-based polymer, polyvinylimidazole, polyacrylate, polyurethane, polyether urethane, silicone, or a combination thereof.

[0020] In certain embodiments, a first asparagine-responsive active area of ​​an analyte sensor for use in a method of the present disclosure includes a first enzyme layer including aspartate oxidase and a second layer including a first asparaginase disposed on the first enzyme layer. In certain embodiments, a first asparagine-responsive active area includes an enzyme layer including aspartate oxidase and a first asparaginase. In certain embodiments, a second asparagine-responsive active area of ​​an analyte sensor for use in a method of the present disclosure includes a first enzyme layer including aspartate oxidase and a second layer including a second asparaginase disposed on the first enzyme layer. In certain embodiments, a second asparagine-responsive active area includes an enzyme layer including aspartate oxidase and a second asparaginase. [Brief description of the drawings]

[0021] The following figures are included to illustrate certain aspects of the present disclosure and should not be considered as exclusive embodiments. The disclosed subject matter is capable of considerable modification, alteration, combinations, and equivalents in form and function without departing from the scope of the present disclosure. [Figure 1A] 1 is a system overview of a sensor applicator, a reader device, a monitoring system, a network, and a remote system. [Figure 1B]FIG. 1 illustrates an operating environment for an exemplary analyte monitoring system for use with the technology described herein. [Figure 2A] FIG. 2 is a block diagram illustrating an example embodiment of a reader device. [Figure 2B] 1 is a block diagram illustrating an example data receiving device for communicating with a sensor, in accordance with an example embodiment of the disclosed subject matter. [Figure 2C] FIG. 2 is a block diagram illustrating an exemplary embodiment of a sensor control device. [Figure 2D] Same as above. [Figure 2E] 1 is a block diagram illustrating an exemplary analyte sensor in accordance with an exemplary embodiment of the disclosed subject matter. [Figure 3A] FIG. 13 is a proximal perspective view showing an exemplary embodiment in which a user prepares a tray for assembly. [Figure 3B] 1A-1C are side views illustrating an exemplary embodiment in which a user prepares the applicator device for assembly. [Figure 3C] 13A-13C are proximal perspective views illustrating an exemplary embodiment in which a user inserts an applicator device into a tray during assembly. [Figure 3D] 13A-13C are proximal perspective views illustrating an exemplary embodiment in which a user removes the applicator device from a tray during assembly. [Figure 3E] 1 is a proximal perspective view illustrating an exemplary embodiment of a patient applying a sensor using an applicator device. [Figure 3F] 1A-1C are proximal perspective views illustrating an exemplary embodiment of a patient with a sensor applied and a used applicator device. [Figure 4A] 1A and 1B are side views illustrating an exemplary embodiment of an applicator device coupled with a cap. [Figure 4B] 1A-1C are side perspective views illustrating an exemplary embodiment of an applicator device and cap separated. [Figure 4C] 1 is a perspective view illustrating an exemplary embodiment of a distal end of an applicator device and an electronics housing. [Figure 4D] 1 is a top perspective view of an exemplary applicator device in accordance with the disclosed subject matter. [Figure 4E] FIG. 4E is a bottom perspective view of the applicator device of FIG. 4D. [Figure 4F] FIG. 4E is an exploded view of the applicator device of FIG. 4D. [Figure 4G] FIG. 4E is a side cutaway view of the applicator device of FIG. 4D. [Diagram 5] FIG. 1 is a proximal perspective view of an exemplary embodiment of a tray with a sterilization lid attached thereto. [Figure 6A] FIG. 13 is a proximal perspective cutaway view showing an exemplary embodiment of a tray with sensor delivery components. [Figure 6B] FIG. 13 is a proximal perspective view showing the sensor delivery components. [Figure 7A] FIG. 2 is an isometric exploded top view of an exemplary sensor control device. [Figure 7B] FIG. 2 is an isometric exploded bottom view of an exemplary sensor control device. [Figure 8A] 8A-8C are assembly and cross-sectional views of an on-body device including an integrated connector for a sensor assembly. [Figure 8B] Same as above. [Figure 8C] Same as above. [Figure 9A] 2D is a side view of an exemplary embodiment of the sensor applicator of FIG. 1A coupled with the cap of FIG. 2C. [Figure 9B] 2D is a cross-sectional side view of an exemplary embodiment of the sensor applicator of FIG. 1A coupled with the cap of FIG. 2C. [Figure 10A] FIG. 2 is an isometric view of another exemplary sensor control device. [Figure 10B] FIG. 2 is a side view of another exemplary sensor control device. [Figure 11A] 11A-11C are progressive cross-sectional side views illustrating the assembly of a sensor applicator with the sensor control device of FIGS. 10A-10B. [Figure 11B] Same as above. [Figure 11C] Same as above. [Figure 12A] 12A-12C are progressive cross-sectional side views illustrating assembly and disassembly of an exemplary embodiment of a sensor applicator with the sensor control device of FIGS. 10A-10B. [Figure 12B] Same as above. [Figure 12C] Same as above. [Figure 13A] 1A-1C show cross-sectional views illustrating an exemplary embodiment of an applicator during a deployment stage. [Figure 13B] Same as above. [Figure 13C] Same as above. [Figure 13D] Same as above. [Figure 13E] Same as above. [Figure 13F] Same as above. [Figure 14] 1 is a graph showing an example of the in vitro sensitivity of an analyte sensor. [Figure 15] 1A-1D illustrate example operational states of a sensor in accordance with an example embodiment of the disclosed subject matter. [Figure 16] FIG. 1 illustrates an example operation and data flow for wireless programming of a sensor in accordance with the disclosed subject matter. [Figure 17] 1 illustrates an example data flow for reliable exchange of data between two devices in accordance with the disclosed subject matter. [Figure 18A] 1 shows a cross-sectional view of an analyte sensor including a single active area. [Figure 18B] Same as above. [Figure 18C] Same as above. [Figure 19A] 1 shows a cross-sectional view of an analyte sensor including two active areas. [Figure 19B] Same as above. [Figure 19C] Same as above. [Figure 20] 1 shows a cross-sectional view of an analyte sensor including two active areas. [Figure 21A] FIG. 1 shows a perspective view of an analyte sensor including two active areas on separate working electrodes. [Figure 21B] Same as above. [Figure 21C]Same as above. [Figure 22] FIG. 1 shows a diagram of a particular enzyme system that can be used to monitor potassium in accordance with the present disclosure. [Figure 23] 7 shows a diagram of two particular enzyme systems that can be used in combination to monitor potassium according to the present disclosure: potassium ion-gated channel 701 includes an enzyme system that includes a potassium-dependent asparaginase, and potassium ion-independent channel 702 includes an enzyme system that includes a potassium-independent asparaginase. [Figure 24] 8 shows a diagram of two particular enzyme systems that may be used in combination to monitor potassium according to the present disclosure: A first potassium-dependent channel 801 includes an enzyme system that includes a first potassium-dependent asparaginase, and a second potassium-dependent channel 802 includes an enzyme system that includes a second potassium-dependent asparaginase, where the first and second potassium-dependent asparaginases exhibit different potassium dependencies from each other. [Figure 25A] 25 provides the current responses to asparagine at different concentrations for the two channels of FIG. 24 in the absence of potassium ions. [Figure 25B] 25 provides the current response to asparagine at a fixed concentration for the two channels of FIG. 24 in the presence of potassium ions. [Figure 26] 1 shows the current response of an analyte sensor of the present disclosure in the presence of various concentrations of asparagine and potassium nitrate. [Figure 27] FIG. 1 shows a diagram of a particular enzyme system that can be used to monitor potassium in accordance with the present disclosure. [Figure 28] 28 provides the current response of a sensor containing the enzyme system of FIG. 27 to 100 μM aspartate at different potassium concentrations. [Figure 29] 28 shows the current response of a sensor containing the enzyme system of FIG. 27 in the presence of various concentrations of aspartate and potassium. [Diagram 30] 28 shows the percentage increase in current response of a sensor containing the enzyme system of FIG. 27 in the presence of various concentrations of aspartate and potassium. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] The present disclosure generally describes analyte sensors that use enzymes, e.g., one or more enzymes for the detection of an analyte. For example, without limitation, the present disclosure provides analyte sensors that use one or more enzymes for the detection of potassium. In certain embodiments, the present disclosure provides multiple enzymes for the detection of an analyte, e.g., potassium. The present disclosure further provides methods of detecting one or more analytes, e.g., potassium, using the disclosed analyte sensors to monitor physiological conditions.

[0023] The present disclosure provides sensor chemistries suitable for monitoring potassium levels over a range of physiologically relevant potassium concentrations. In certain embodiments, the present disclosure provides sensor chemistries that utilize enzymes to monitor potassium concentrations, e.g., potassium ion concentrations, in a sample. In certain embodiments, the present disclosure provides sensor chemistries that utilize an enzyme system that includes at least two enzymes that can act in concert to monitor potassium concentrations, e.g., potassium ion concentrations, in a sample. In certain embodiments, two enzyme systems can be used to facilitate indirect measurement of a single analyte, such as potassium. As used herein, the term "in concert" refers to a coupled enzyme reaction in which the product of a first enzyme reaction becomes a substrate for a second enzyme reaction, which serves as a basis for measuring the concentration of a substrate (e.g., an analyte) reacted during the first enzyme reaction. In certain embodiments, the product and / or substrate of the reaction can be a cofactor or coenzyme of an enzyme of the enzyme system, e.g., reduced and / or oxidized forms of FAD or NAD. Although defined in terms of two coupled enzyme reactions, it should be understood that more than two enzyme reactions can be coupled in some instances as well. For example, the product of a first enzymatic reaction can be a substrate for a second enzymatic reaction, and the product of the second enzymatic reaction can be a substrate for a third enzymatic reaction, which serves as the basis for measuring the concentration of a substrate (e.g., an analyte) reacted during the first enzymatic reaction. A discussion of suitable enzyme systems for detecting, e.g., indirectly detecting, potassium in accordance with the disclosure herein follows.

[0024] For purposes of clarity, and not by way of limitation, the detailed description of the subject matter of the present disclosure is divided into the subsections that follow. I. Definitions; and II. Analyte sensors; 1. General structure of the analyte sensor system; 2. Enzymes; 3. Redox mediators; 4. Polymer backbone; 5. Mass-transfer limiting membrane; 6. Interference domain; 7. Manufacturing; III. Method of Use; and IV. Exemplary Embodiments.

[0025] I. Definition The terms used herein generally have their ordinary meanings in the art, within the context of this disclosure, and in the specific context in which each term is used. Certain terms are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner in describing the compositions and methods of the present disclosure and how to make and use them.

[0026] As used herein, the use of the words "a" or "an" when used in conjunction with the term "comprising" in the claims and / or specification may mean "one," but is also consistent with the meaning of "one or more," "at least one," and "one or more than one."

[0027] As used herein, the terms "comprise(s)," "include(s)," "having," "has," "can," "contain(s)," and variations thereof are intended to be open-ended transitional phrases, terms, or words that do not exclude additional acts or structures. The present disclosure also contemplates other embodiments that "comprising," "consisting of," and "consisting essentially of" the embodiments or elements presented herein, whether or not explicitly stated.

[0028] The term "about" or "approximately" means within an acceptable error range for a particular value as determined by a person skilled in the art, which depends in part on how the value is measured or determined, i.e., the limits of the measurement system. For example, "about" can mean within 3 or more than 3 standard deviations, according to the practice in the art. Alternatively, "about" can mean within 20%, preferably within 10%, more preferably within 5%, and even more preferably within 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, more preferably within 2-fold of a value.

[0029] As used herein, an "analyte sensor" or "sensor" may refer to any device capable of receiving sensor information from a user, including, by way of example and not limitation, a temperature sensor, a blood pressure sensor, a pulse or heart rate sensor, a glucose level sensor, an analyte sensor, a physical activity sensor, a physical movement sensor, or any other sensor for collecting physical or biological information. Analytes measured by an analyte sensor may include, by way of example and not limitation, glutamate, glucose, ketones, lactate, oxygen, hemoglobin A1C, albumin, alcohol, alkaline phosphatase, alanine transaminase, aspartate aminotransferase, bilirubin, blood urea nitrogen, calcium, carbon dioxide, chloride, creatinine, hematocrit, lactate, magnesium, oxygen, pH, phosphorus, potassium, asparagine, aspartate, sodium, total protein, uric acid, and the like.

[0030] As used herein, the term "biological fluid" refers to any bodily fluid or bodily fluid derivative in which an analyte can be measured. Non-limiting examples of biological fluids include dermal fluid, interstitial fluid, plasma, blood, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage, amniotic fluid, sweat, tears, etc. In certain embodiments, the biological fluid is dermal fluid or interstitial fluid.

[0031] The term "electrolysis," as used herein, refers to the electro-oxidation or electro-reduction of a compound either directly at an electrode or via one or more electron transfer agents (e.g., redox mediators or enzymes).

[0032] As used herein, the term "homogeneous membrane" refers to a membrane that includes a single type of membrane polymer. As used herein, the term "multi-component membrane" refers to a membrane that includes two or more membrane polymers.

[0033] As used herein, the term "potassium-independent aspartate oxidase" refers to an aspartate oxidase that does not exhibit any change in catalytic activity in the presence of potassium, e.g., potassium ions (K+).

[0034] As used herein, the term "potassium-dependent aspartate oxidase" refers to an aspartate oxidase that exhibits increased or decreased catalytic activity in the presence of potassium, e.g., potassium ions (K+). In certain embodiments, potassium-dependent aspartate oxidases include aspartate oxidases that require various concentrations of potassium, e.g., potassium ions (K+), for maximum catalytic activity.

[0035] As used herein, the term "potassium-independent asparaginase" refers to an asparaginase that does not exhibit any change in catalytic activity in the presence of potassium, e.g., potassium ions (K+).

[0036] As used herein, the term "potassium-dependent asparaginase" refers to an asparaginase that exhibits increased or decreased catalytic activity in the presence of potassium, e.g., potassium ions (K+). In certain embodiments, potassium-dependent asparaginase includes asparaginases that require various concentrations of potassium, e.g., potassium ions (K+), for maximum catalytic activity.

[0037] As used herein, the term "polyvinylpyridine-based polymer" refers to a polymer or copolymer that includes polyvinylpyridine (eg, poly(2-vinylpyridine) or poly(4-vinylpyridine)) or a derivative thereof.

[0038] As used herein, the term "redox mediator" refers to an electron transfer agent for carrying electrons between an analyte or an analyte reductase or analyte oxidase and an electrode, either directly or via one or more additional electron transfer agents. In certain embodiments, redox mediators that include a polymer backbone may also be referred to as "redox polymers."

[0039] As used herein, the term "reference electrode" may refer to either a reference electrode or an electrode that functions as both a reference electrode and a counter electrode. Similarly, as used herein, the term "counter electrode" may refer to both a counter electrode and a counter electrode that also functions as a reference electrode.

[0040] As used herein, the term "single-component membrane" refers to a membrane that includes one type of membrane polymer. II. Analyte Sensors 1. General structure of the analyte sensor system; Before describing the present subject matter in detail, it is to be understood that this disclosure is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

[0041] Publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[0042] In general, embodiments of the present disclosure include systems, devices, and methods for the use of an analyte sensor insertion applicator for use with an in vivo analyte monitoring system. The applicator can be provided to a user in a sterile package containing the electronics housing of the sensor control device. According to some embodiments, a structure separate from the applicator, such as a container, can also be provided to a user as a sterile package containing a sensor module and a sharps module. A user can couple the sensor module to the electronics housing and can couple the sharps to the applicator by an assembly process that includes inserting the applicator into the container in a specified manner. In other embodiments, the applicator, sensor control device, sensor module, and sharps module may be provided in a single package. The applicator may be used to place the sensor control device on the human body with the sensor in contact with the wearer's bodily fluids. The embodiments provided herein are improvements to reduce the likelihood that the sensor will be improperly inserted or damaged, or induce an adverse physiological response. Other improvements and advantages are provided as well. Various configurations of these devices are described in detail by way of embodiments that are merely examples.

[0043] Additionally, many embodiments include an in vivo analyte sensor that is structurally configured such that at least a portion of the sensor can be placed or positioned within the body of a user to obtain information regarding at least one analyte of the body. It should be noted, however, that the embodiments disclosed herein may be used with in vivo analyte monitoring systems that incorporate in vitro functionality, as well as simply in vitro or ex vivo analyte monitoring systems, including systems that are completely non-invasive.

[0044] Furthermore, for any and all embodiments of the methods disclosed herein, systems and devices capable of performing each of those embodiments are encompassed within the scope of the present disclosure. For example, sensor control device embodiments are disclosed, but these devices may have one or more sensors, analyte monitoring circuitry (e.g., analog circuitry), memory (e.g., for storing instructions), power sources, communication circuitry, transmitters, receivers, processors, and / or controllers (e.g., for executing instructions), which may perform or facilitate the performance of any and all method steps. These sensor control device embodiments may be used or may be capable of being used to implement steps performed by the sensor control device from any and all of the methods described herein.

[0045] Additionally, the systems and methods presented herein may be used for the operation of sensors used in analyte monitoring systems, such as, but not limited to, wellness, fitness, diet, research, information, or any purpose related to analyte sensing over time. As used herein, an "analyte sensor" or "sensor" may refer to any device capable of receiving sensor information from a user, including, by way of example only, but not limited to, a temperature sensor, a blood pressure sensor, a pulse or heart rate sensor, a glucose level sensor, an analyte sensor, a physical activity sensor, a physical movement sensor, or any other sensor for collecting physical or biological information. In certain embodiments, the analyte sensors of the present disclosure can further measure analytes including, but not limited to, glucose, ketones, lactate, oxygen, hemoglobin A1C, albumin, alcohol, alkaline phosphatase, alanine transaminase, aspartate aminotransferase, bilirubin, blood urea nitrogen, calcium, carbon dioxide, chloride, creatinine, hematocrit, lactate, magnesium, oxygen, pH, phosphorus, potassium, sodium, aspartate, asparagine, total protein, uric acid, and the like.

[0046] As mentioned above, several embodiments of systems, devices, and methods are described herein that provide improved assembly and use of skin sensor insertion devices for use with in vivo analyte monitoring systems. In particular, several embodiments of the present disclosure are designed to improve the method of sensor insertion with respect to in vivo analyte monitoring systems, and in particular to prevent premature retraction of the insertion sharp during the sensor insertion process. Some embodiments include a skin sensor insertion mechanism that, for example, increases the firing rate and slows the retraction of the sharp. In other embodiments, the sharp retraction mechanism may be motion actuated such that the sharp is not retracted until the user pulls the applicator away from the skin. As a result, these embodiments can reduce the possibility of premature withdrawal of the insertion sharp during the sensor insertion process, reduce the possibility of improper sensor insertion, and reduce the possibility of damaging the sensor during the sensor insertion process, to name a few advantages. Some embodiments of the present disclosure also provide an improved insertion sharps module that takes into account the small-scale skin sensors and the relatively shallow insertion path present in the dermis layer of a subject. In addition, some embodiments of the present disclosure are designed to prevent undesired axial and / or rotational movement of applicator components during sensor insertion. These embodiments can therefore reduce the likelihood of capillary breakage resulting in instability of the placed skin sensor, irritation at the insertion site, damage to surrounding tissue, and contamination of skin fluids with blood, to name a few advantages. Additionally, to mitigate inaccurate sensor readings that may be caused by trauma at the insertion site, some embodiments of the present disclosure can reduce the needle end penetration depth relative to the sensor tip during insertion.

[0047] However, before describing these aspects of the embodiments in detail, it is desirable to first describe examples of devices that may be present, for example, in an in vivo analyte monitoring system, as well as examples of their operation, all of which may be used in conjunction with the embodiments described herein.

[0048] There are various types of in vivo analyte monitoring systems. A "continuous analyte monitoring" system (or a "continuous glucose monitoring" system) may, for example, transmit data from a sensor control device to a reader device continuously and without prompting, e.g., automatically according to a schedule. As another example, a "flash analyte monitoring" system (or a "flash glucose monitoring" system or simply a "flash" system) may transfer data from a sensor control device in response to a scan or request for data by a reader device, such as using near field communication (NFC) or radio frequency identification (RFID) protocols. An in vivo analyte monitoring system may also operate without the need for finger-prick calibration.

[0049] In vivo analyte monitoring systems can be distinguished from "in vitro" systems, which contact a biological sample outside the body (or "ex vivo") and typically include a measurement device having a port for accepting an analyte test strip that carries a user's bodily fluid that can be analyzed to determine analyte levels in the user's blood.

[0050] An in-vivo monitoring system may include a sensor that contacts a user's bodily fluid while disposed in vivo and senses analyte levels contained therein. The sensor may be part of a sensor control device that resides on the user's body and contains electronics and a power source that enable and control analyte sensing. Sensor control devices and variations thereof may also be referred to as "sensor control units," "on-body electronics" devices or units, "on-body" devices or units, or "sensor data communication" devices or units, to name a few.

[0051] The in-vivo monitoring system may also include a device that receives the sensed analyte data from the sensor control device and processes and / or displays the sensed analyte data to a user in any number of forms. This device and variations thereof may be referred to as a "handheld reader device," "reader device" (or simply "reader"), "handheld electronic device" (or simply "handheld"), "portable data processing" device or unit, "data receiver," "receiver" device or unit (or simply "receiver"), or "remote" device or unit, to name a few. Other devices, such as personal computers, have also been utilized with or incorporated into in-vivo and in-vitro monitoring systems.

[0052] The sensor 104 is adapted to be at least partially inserted into a target tissue, such as into the dermis or subcutaneous layer of the skin. The sensor 104 may include a sensor tail of sufficient length to insert to a desired depth of a given tissue. The sensor tail may include at least one working electrode. In certain configurations, the sensor tail may include at least one active area for detecting an analyte disposed on the working electrode. A counter electrode may be present in combination with the at least one working electrode. Particular electrode configurations on the sensor tail are described in more detail below.

[0053] The active area may be configured to monitor a particular analyte, such as potassium, e.g., potassium ions. In certain embodiments, the active area may be configured to indirectly monitor potassium levels in a sample by detecting asparagine. For example, without limitation, the active area may be configured to indirectly measure potassium, e.g., potassium ions, by detecting asparagine using an enzyme system including potassium-independent asparaginase and / or potassium-dependent asparaginase. In certain embodiments, the active area may be configured to indirectly monitor potassium levels in a sample by detecting aspartate. For example, without limitation, the active area may be configured to indirectly measure potassium, e.g., potassium ions, by detecting aspartate using an enzyme system including potassium-independent aspartate oxidase and / or potassium-dependent aspartate oxidase.

[0054] In certain embodiments, the active areas may be configured to detect two or more analytes. In certain embodiments, the active areas may be configured to detect a second analyte. In certain embodiments, the sensor tail may include at least two active areas, one active area configured to detect potassium and a second active area configured to detect a second analyte. Non-limiting examples of the second analyte may be glucose, ketones, lactate, oxygen, hemoglobin A1C, albumin, alcohol, alkaline phosphatase, alanine transaminase, aspartate aminotransferase, bilirubin, blood urea nitrogen, calcium, carbon dioxide, chloride, creatinine, hematocrit, lactate, magnesium, oxygen, pH, phosphorus, potassium, sodium, aspartate, asparagine, total protein, uric acid, and the like.

[0055] In certain embodiments of the present disclosure, one or more analytes may be monitored in any biological fluid of interest (e.g., skin fluid, interstitial fluid, plasma, blood, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage, amniotic fluid, etc.). In certain embodiments, the analyte sensor of the present disclosure may be adapted to assay skin fluid or interstitial fluid to determine the concentration of one or more analytes in vivo. In certain embodiments, the biological fluid is interstitial fluid.

[0056] The introducer may be present temporarily to facilitate the introduction of the sensor 104 into the tissue. In certain exemplary embodiments, the introducer may include a needle or similar sharp. As will be readily recognized by one of ordinary skill in the art, other types of introducers, such as a sheath or blade, may be present in alternative embodiments. More specifically, the needle or other introducer may be present temporarily in proximity to the sensor 104 prior to tissue insertion and then withdrawn. While present, the needle or other introducer may facilitate the 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 the penetration of the epidermis as an access path to the dermis to allow the implantation of the sensor 104 to occur. After opening the access path, the needle or other introducer may be withdrawn such that it does not present a sharps hazard. Certain embodiments indicate that suitable needles may be solid or hollow, beveled or non-beveled, and / or circular or non-circular in cross section. In more particular embodiments, suitable needles may be comparable in cross-sectional diameter and / or tip design to acupuncture needles, which may have a cross-sectional diameter of about 250 microns. However, suitable needles may have larger or smaller cross-sectional diameters as required for a particular application.

[0057] In certain embodiments, the tip of the needle (while present) may be angled over the end of the sensor 104 so that the needle penetrates the tissue first, opening an access path for the sensor 104. In certain embodiments, the sensor 104 may reside within a lumen or channel of the needle, which similarly opens an access path for the sensor 104. In either case, the needle is withdrawn after facilitating insertion of the sensor. B. Exemplary Reader Devices 2A is a block diagram illustrating an example embodiment of a reader device configured as a smartphone, where the reader device 120 may include a display 122, an input component 121, and a processing core 206 including a communication processor 222 coupled to a memory 223 and an application processor 224 coupled to a memory 225. A separate memory 230, an RF transceiver 228 having an antenna 229, and a power source 226 having a power management module 238 may also be included. It may further include a multi-function transceiver 232 capable of communicating via Wi-Fi, NFC, Bluetooth, BTLE, and GPS via an antenna 234. As will be appreciated by those skilled in the art, these components are electrically and communicatively coupled in a manner to create a functional device.

[0058] C. Exemplary Data Receiving Device Architecture For purposes of illustration and not limitation, reference is made to an exemplary embodiment of a data receiving device 120 for use with the disclosed subject matter as shown in FIG. 2B. The data receiving device 120 and associated general-purpose data receiving device 130 include components germane to the description of the analyte sensor 110 and its operation, and may include additional components. In certain embodiments, the data receiving device 120 and general-purpose data receiving device 130 may be or include components provided by third parties, and are not necessarily limited to including devices made by the same manufacturer as the sensor 110.

[0059] 2B, the data receiving device 120 includes an ASIC 4000 that includes a microcontroller 4010, a memory 4020, and a storage 4030, and is communicatively coupled to a communication module 4040. Power for the components of the data receiving device 120 may be delivered by a power module 4050, which may include a rechargeable battery, as embodied herein. The data receiving device 120 may further include a display 4070 to facilitate review of analyte data received from the analyte sensor 110 or other devices (e.g., the user device 140 or the remote application server 150). The data receiving device 120 may include separate user interface components (e.g., physical keys, a light sensor, a microphone, etc.).

[0060] 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 interface with the analyte sensor 110, send commands to the analyte sensor 110, and receive data from the analyte sensor 110. As embodied herein, the data receiving device 120 may be configured to operate as an NFC scanner and a BLE endpoint via a particular module (e.g., the BLE module 4042 or the NFC module 4043) of the communication module 4040 with respect to the analyte sensor 110 as described herein. For example, the data receiving device 120 may use a first module of the communication module 4040 to issue commands (e.g., an activation command for a data broadcast mode of the sensor, a pairing command to identify the data receiving device 120) to the analyte sensor 110, and may use a second module of the communication module 4040 to receive data from and send data to the analyte sensor 110. The data receiving device 120 may be configured to communicate with the user device 140 via a universal serial bus (USB) module 4045 of the communication module 4040.

[0061] As another example, the communication module 4040 may include, for example, a cellular radio module 4044. The cellular radio module 4044 may include one or more radio transceivers for communicating using broadband cellular networks, including but not limited to third generation (3G), fourth generation (4G), and fifth generation (5G) networks. Additionally, the communication module 4040 of the data receiving device 120 may include a Wi-Fi radio module 4043 for communicating using wireless local area networks according to one or more of the IEEE 802.11 standards (e.g., 802.11a, 802.11b, 802.11g, 802.11n (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 a remote application server 150 to receive analyte data or provide updates or input received from a user (e.g., through 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.

[0062] 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. Additionally, the data receiving device 120, the general-purpose data receiving device 130, or the user device 140 may be configured to communicate with a remote application server 150 over a wide area network. As embodied herein, the analyte sensor 110 may provide data to the data receiving device 120 or the general-purpose data receiving device 130. The data receiving device 120 may transmit the data to the user computing device 140. The user computing device 140 (or the general-purpose data receiving device 130) may then transmit the data to the remote application server 150 for processing and analysis.

[0063] As embodied herein, the data receiving device 120 may further include sensing hardware 4060 similar to or extended from the sensing hardware 5060 of the analyte sensor 110. In certain embodiments, the data receiving device 120 may be configured in conjunction with the analyte sensor 110 to operate based on the analyte data received from the analyte sensor 110. As an example, if the analyte sensor 110 is a glucose sensor, the data receiving device 120 may be or include an insulin pump or insulin injection pen. In conjunction, the compatible device 130 may adjust an insulin dose for the user based on the glucose value received from the analyte sensor.

[0064] D. Exemplary Sensor Control Device 2C and 2D are block diagrams illustrating an exemplary embodiment of a sensor control device 102 having an analyte sensor 104 and sensor electronics 160 (including analyte monitoring circuitry) that may have most of the processing power for rendering final result data suitable for display to a user. In FIG. 2C, a single semiconductor chip 161 is shown, which may be a custom application specific integrated circuit (ASIC). Shown within the ASIC 161 are certain high level functional units including an analog front end (AFE) 162, a power management (or control) circuit 164, a processor 166, and a communication circuit 168 (which may be implemented as a transmitter, receiver, transceiver, passive circuitry, or otherwise following a communication protocol). In this embodiment, both the AFE 162 and the processor 166 are used as analyte monitoring circuitry, although in other embodiments, either circuitry may perform the analyte monitoring function. The processor 166 may include one or more processors, microprocessors, controllers, and / or microcontrollers, each of which may be a separate chip or may be distributed among (and portions of) several different chips.

[0065] The memory 163 is also included in the ASIC 161 and may be shared by the various functional units present in the ASIC 161 or distributed among two or more of them. The memory 163 may be a separate chip. The memory 163 may be a volatile and / or non-volatile memory. In this embodiment, the ASIC 161 is coupled to a power source 170, which may be a coin cell battery or the like. The AFE 162 interacts with the in-vivo analyte sensor 104, receives measurement data therefrom, and outputs the data in digital form to the processor 166, which then processes the data to arrive at final result glucose discrete values ​​and trend values, etc. This data may then be provided to the communication circuitry 168 for transmission via the antenna 171 to the reader device 120 (not shown), where, for example, minimal further processing is required by a resident software application to display the data.

[0066] FIG. 2D is similar to FIG. 2C, but instead includes two separate semiconductor chips 162 and 174, which may be packaged together or separately. Here, AFE 162 resides on ASIC 161. Processor 166 is integrated with power management circuitry 164 and communication circuitry 168 on chip 174. AFE 162 includes memory 163, and chip 174 includes memory 165, which may be separated or distributed within. In one exemplary embodiment, AFE 162 is combined with power management circuitry 164 and processor 166 on one chip, and communication circuitry 168 is on a separate chip. In another exemplary embodiment, both AFE 162 and communication circuitry 168 are on one chip, and processor 166 and power management circuitry 164 are on another chip. It should be noted that other chip combinations are possible, including three or more chips, with each chip performing separate functions as described, or sharing one or more functions for fail-safe redundancy.

[0067] For purposes of illustration and not limitation, reference is made to an exemplary embodiment of an analyte sensor 110 for use with the disclosed subject matter as shown in Figure 2E, which illustrates a block diagram of an exemplary analyte sensor 110 according to an exemplary embodiment that is compatible with the security architecture and communication schemes described herein.

[0068] As embodied herein, the analyte sensor 110 may include an application specific integrated circuit ("ASIC") 5000 communicatively coupled to a communication module 5040. The ASIC 5000 may include a microcontroller core 5010, an on-board memory 5020, and a storage memory 5030. The storage memory 5030 may store data used in the authentication and encryption security architecture. The storage memory 5030 may store programming instructions for the sensor 110. As embodied herein, a specific communication chipset may be incorporated into the ASIC 5000 (e.g., NFC transceiver 5025). The ASIC 5000 may receive power from a power module 5050, such as an on-board battery, or from an NFC pulse. The storage memory 5030 of the ASIC 5000 may be programmed to include information such as an identifier of the sensor 110 for identification and tracking purposes. The storage memory 5030 may also be programmed with configuration or calibration parameters for use by the sensor 110 and its various components. The storage memory 5030 may include rewriteable or one-time programming (OTP) memory. The storage memory 5030 may be updated using techniques described herein to extend the usefulness of the sensor 110.

[0069] As embodied herein, the communication module 5040 of the sensor 100 may be or include one or more modules for supporting the analyte sensor 110 to communicate with other devices of the analyte monitoring system 100. By way of example only and not limitation, the exemplary communication module 5040 may include a Bluetooth Low Energy ("BLE") module 5041. As used throughout this disclosure, Bluetooth Low Energy ("BLE") refers to a short-range communication protocol optimized to make Bluetooth device pairing simple for end users. The communication module 5040 may transmit and receive data and commands via interaction with a similarly capable communication module of the data receiving device 120 or user device 140. The communication module 5040 may include additional or alternative chipsets for use with similar short-range communication schemes, such as personal area networks according to the IEEE 802.15 protocol, IEEE 802.11 protocol, infrared communication according to the Infrared Data Association standard (IrDA), and the like.

[0070] To perform its functions, the sensor 100 may further include appropriate sensing hardware 5060 appropriate for that function. As embodied herein, the sensing hardware 5060 may include an analyte sensor disposed transcutaneously or subcutaneously in contact with the subject's bodily fluid. The analyte sensor may generate sensor data including a value corresponding to a level of one or more analytes in the bodily fluid.

[0071] E. Exemplary Assembly Process for a Sensor Control Device The components of the sensor control device 102 may be obtained by the user in multiple packages that require final assembly by the user prior to delivery to the appropriate user location. Figures 3A-3D show an exemplary embodiment of an assembly process for the sensor control device 102 by the user, including preparation of the separate components prior to combining the components to prepare the sensor for delivery. Figures 3E-3F show an exemplary embodiment of delivery of the sensor control device 102 to an appropriate user location by selecting an appropriate delivery location and applying the device 102 to that location.

[0072] 3A is a proximal perspective view showing an exemplary embodiment in which a user prepares a container 810, here configured as a tray (although other packaging may be used), for an assembly process. The user may accomplish this preparation by removing the lid 812 from the tray 810 to expose the platform 808, for example, by peeling a non-adhered portion of the lid 812 from the tray 810 such that the adhered portion of the lid 812 is removed. Removal of the lid 812 may be appropriate in various embodiments so long as the platform 808 is properly exposed within the tray 810. The lid 812 may then be set aside.

[0073] 3B is a side view illustrating an exemplary embodiment in which a user prepares the applicator device for assembly. Applicator device 150 may be provided in a sterile package sealed by cap 708. Preparing applicator device 150 may include separating housing 702 from cap 708 to expose sheath 704 (FIG. 3C). This may be accomplished by unscrewing (or otherwise separating) cap 708 from housing 702. Cap 708 may then be set aside.

[0074] 3C is a proximal perspective view showing an exemplary embodiment in which a user inserts applicator device 150 into tray 810 during assembly. First, a user may align housing orientation portion 1302 (or slot or recess) and tray orientation portion 924 (abutment or detent) and then insert sheath 704 into inner platform 808 of tray 810. Inserting sheath 704 into platform 808 temporarily unlocks sheath 704 relative to housing 702 and platform 808 relative to tray 810. Removal of applicator device 150 from tray 810 at this stage will result in the same condition as before initial insertion of applicator device 150 into tray 810 (i.e., the process can be reversed or interrupted at this point and then repeated without incident).

[0075] The sheath 704 may maintain its position within the platform 808 relative to the housing 702 while the housing 702 is advanced distally, thereby mating with the platform 808 and advancing the platform 808 distally relative to the tray 810. This step unlocks the platform 808 and folds it into the tray 810. The sheath 704 may contact and disengage a locking portion (not shown) within the tray 810 that unlocks the sheath 704 relative to the housing 702 and prevents the sheath 704 from moving (relatively) while the housing 702 continues to advance the platform 808 distally. At the end of the advancement of the housing 702 and platform 808, the sheath 704 is permanently unlocked relative to the housing 702. The sharps and sensors (not shown) within the tray 810 may be mated with the electronics housing (not shown) within the housing 702 at the end of the distal advancement of the housing 702. The operation and interaction of applicator device 150 and tray 810 are further described below.

[0076] 3D is a proximal perspective view showing an exemplary embodiment in which a user removes applicator device 150 from tray 810 during assembly. A user can remove applicator 150 from tray 810 by advancing housing 702 proximally relative to tray 810, or by other motion that has the same end effect of separating applicator 150 and tray 810. Applicator device 150 is removed with sensor control device 102 (not shown) fully assembled therein (sharps, sensor, electronics) and positioned for delivery.

[0077] 3E is a proximal perspective view showing an exemplary embodiment of a patient using applicator device 150 to apply sensor control device 102 to a target area of ​​skin, e.g., the abdomen or other suitable location. Distal advancement of housing 702 causes sheath 704 to collapse within housing 702, and the sensor is applied to the target location such that the adhesive layer on the bottom side of sensor control device 102 adheres to the skin. The sharp is automatically retracted when housing 702 is fully advanced, but the sensor (not shown) is left in place to measure the analyte level.

[0078] 3F is a proximal perspective view showing an exemplary embodiment of a patient with the sensor control device in place. The user can then remove the applicator 150 from the application site.

[0079] The system 100 described with respect to Figures 3A-3F and elsewhere herein can reduce or eliminate the possibility of accidental breakage, permanent deformation, or incorrect assembly of applicator components compared to prior art systems. Because the applicator housing 702 directly engages the platform 808 while the sheath 704 is unlocked, rather than indirect engagement via the sheath 704, the relative angle between the sheath 704 and the housing 702 does not result in breakage or permanent deformation of the arms or other components. The possibility of relatively large forces (as in conventional devices) during assembly is reduced, which in turn reduces the possibility of failed assembly by the user.

[0080] F. Exemplary Sensor Applicator Device FIGURE 4A is a side view showing an exemplary embodiment of applicator device 150 coupled with screw cap 708. This is an example of how applicator 150 is shipped to and received by a user prior to being assembled with a sensor by the user. FIGURE 4B is a side perspective view showing applicator device 150 and cap 708 after they have been separated. FIGURE 4C is a perspective view showing an exemplary embodiment of the distal end of applicator device 150 with electronics housing 706 and adhesive patch 105 removed from their positions within sensor carrier 710 of sheath 704 where they are held when cap 708 is in place.

[0081] 4D-G, for purposes of illustration and not limitation, the applicator device 20150 may be provided to a user as a single integrated assembly. Figures 4D and 4E provide perspective top and bottom views, respectively, of the applicator device 20150, Figure 4F provides an exploded view of the applicator device 20150, and Figure 4G provides a side cutaway view. The perspective views show how the applicator 20150 is shipped to and received by a user. The exploded and cutaway views show the components of the applicator device 20150. The applicator device 20150 may include a housing 20702, a gasket 20701, a sheath 20704, a sharps carrier 201102, a spring 205612, a sensor carrier 20710 (also referred to as a "puck carrier"), a sharps hub 205014, a sensor control device (also referred to as a "puck") 20102, an adhesive patch 20105, a desiccant 20502, a cap 20708, a serial label 20709, and a tamper evidence portion 20712. When received by a user, only the housing 20702, the cap 20708, the tamper evidence portion 20712, and the label 20709 are visible. The tamper evidence portion 20712 may be, for example, a sticker coupled to each of the housing 20702 and the cap 20708, and the tamper evidence portion 20712 may be, for example, irreparably damaged by separating the housing 20702 and the cap 20708, thereby indicating to a user that the housing 20702 and the cap 20708 have previously been separated. These features are described in more detail below.

[0082] G. Exemplary Tray and Sensor Module Assembly FIG. 5 is a proximal perspective view showing an exemplary embodiment of a tray 810 with a sterilization lid 812 removably coupled thereto, which may represent how the package is shipped to and received by a user prior to assembly.

[0083] 6A is a proximal perspective cutaway view showing the sensor delivery components within a tray 810. A platform 808 is slidably coupled within the tray 810. A desiccant 502 is stationary relative to the tray 810. A sensor module 504 is mounted within the tray 810.

[0084] 6B is a proximal perspective view showing the sensor module 504 in greater detail, where the retention arm extension 1834 of the platform 808 releasably secures the sensor module 504 in place. The module 2200 is coupled with the connector 2300, the sharps module 2500, and the sensor (not shown) so that they can be removed together as the sensor module 504 during assembly.

[0085] H. Exemplary Applicator and Sensor Control Device for One-Piece Architecture 1A and 3A-3G, in the case of a two-piece architecture system, the sensor tray 202 and the sensor applicator 102 are provided to the user as separate packages, thus requiring the user to open each package and ultimately assemble the system. In some applications, the separate sealed packages allow the sensor tray 202 and the sensor applicator 102 to be sterilized in separate sterilization processes that are specific to the contents of each package and incompatible with the contents of the others. More specifically, the sensor tray 202 including the plug assembly 207 including the sensor 110 and the sharps 220 may be sterilized using radiation sterilization, such as electron beam (or "e-beam") irradiation. Suitable radiation sterilization processes include, but are not limited to, electron beam (e-beam) irradiation, gamma irradiation, x-ray irradiation, or any combination thereof. However, radiation sterilization may damage electrical components disposed within the electronics housing of the sensor control device 102. As a result, if the sensor applicator 102, including the electronics housing of the sensor control device 102, needs to be sterilized, it may be sterilized by another method, such as gas chemical sterilization using, for example, ethylene oxide. However, gas chemical sterilization may damage enzymes or other chemicals and biologics contained on the sensor 110. Due to this sterilization incompatibility, the sensor tray 202 and the sensor applicator 102 are typically sterilized in separate sterilization processes and then packaged separately, which requires final assembly of the components for use by the user.

[0086] 7A and 7B are isometric exploded top and bottom views, respectively, of a sensor control device 3702 according to one or more embodiments. The shell 3706 and the mount 3708 operate as opposing clamshell halves that enclose 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 including a printed circuit board (PCB) 3804 to which a number of electronic modules 3806 are coupled. Exemplary electronic modules 3806 include, but are not limited to, resistors, transistors, capacitors, inductors, diodes, and switches. Conventional sensor control devices typically stack PCB components on only one side of the PCB. In contrast, the PCB components 3806 in the sensor control device 3702 may be distributed around the surface area of ​​both sides (i.e., top and bottom) of the PCB 3804.

[0087] In addition to the electronic module 3806, the PCBA 3802 may also include a data processing unit 3808 mounted on the PCB 3804. The data processing unit 3808 may comprise, for example, an application specific integrated circuit (ASIC) configured to implement 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 functions may include, but are not limited to, filtering and encoding data signals each corresponding to a sampled user's analyte level. The data processing unit 3808 may also include an antenna for communicating with or otherwise communicating with the reader device 106 (FIG. 1A).

[0088] The battery opening 3810 may be defined in the PCB 3804 and sized to receive and mount a battery 3812 configured to power the sensor control device 3702. The axial battery contact 3814a and the radial battery contact 3814b may be coupled to the PCB 3804 and extend into the battery opening 3810 to facilitate the transfer of power from the battery 3812 to the PCB 3804. As their names suggest, the axial battery contact 3814a may be configured to provide an axial contact for the battery 3812, while the radial battery contact 3814b may provide a radial contact for the battery 3812. Locating the battery 3812 in the battery opening 3810 with the battery contacts 3814a,b helps to reduce the height H of the sensor control device 3702, which allows the PCB 3804 to be centrally located and its components distributed on both sides (i.e., top and bottom). This also helps facilitate the chamfer 3718 provided on the electronics housing 3704 .

[0089] The sensor 3716 may be centrally disposed with respect to the PCB 3804 and may include a tail 3816, a flag 3818, and a neck 3820 interconnecting the tail 3816 and the flag 3818. The tail 3816 may extend through a central opening 3720 in the mount 3708 and be configured to be transdermally received beneath the skin of a user. Additionally, the tail 3816 may have an enzyme or other chemical included thereon to help facilitate analyte monitoring.

[0090] The flag 3818 may include a generally flat surface having one or more sensor contacts 3822 (three shown in FIG. 7B) disposed thereon. The sensor contacts 3822 may be configured to align and engage with corresponding one or more circuit contacts 3824 (three shown in FIG. 7A) provided on the PCB 3804. In some embodiments, the sensor contacts 3822 may include a carbon-impregnated polymer printed or otherwise digitally applied to the flag 3818. Conventional sensor control devices typically include a silicone rubber connector that encapsulates one or more compliant carbon-impregnated polymer modules that function as conductive contacts between the sensor and the PCB. In contrast, the sensor contacts 3822 of the present disclosure provide a direct connection between the sensor 3716 and the PCB 3804 connection, which eliminates the need for a prior art connector and advantageously reduces the height H. Additionally, by eliminating the compliant carbon-impregnated polymer modules, significant circuit resistance is eliminated, thus improving circuit conductivity.

[0091] The sensor control device 3702 may further include a compliant member 3826 that may be disposed between the flag 3818 and an inner surface of the shell 3706. More specifically, when the shell 3706 and the mount 3708 are assembled together, the compliant member 3826 may be configured to provide a passive biasing load against the flag 3818 that continuously engages the sensor contacts 3822 with corresponding circuit contacts 3824. In the illustrated embodiment, the compliant member 3826 is an elastomeric O-ring, but may alternatively comprise any other type of biasing device or mechanism, such as a compression spring, without departing from the scope of the present disclosure.

[0092] The sensor control device 3702 may further include one or more electromagnetic shields, shown as a first shield 3828a and a second shield. The shell 3706 may provide or define a first clocking receptacle 3830a (FIG. 7B) and a second clocking receptacle 3830b (FIG. 7B), and the mount 3708 may provide or define a first clocking post 3832a (FIG. 7A) and a second clocking post 3832b (FIG. 7A). The shell 3706 is properly aligned with the mount 3708 by mating the first and second clocking receptacles 3830a,b with the first and second clocking posts 3832a,b, respectively.

[0093] 7A , the inner surface of the mount 3708 may provide or define a number of pockets or recesses configured to accommodate various components of the sensor control device 3702 when the shell 3706 is mated to the mount 3708. For example, the inner surface of the mount 3708 may define a battery locator 3834 configured to accommodate a portion of the battery 3812 when the sensor control device 3702 is assembled. An adjacent contact pocket 3836 may be configured to accommodate a portion of the axial contact 3814a.

[0094] Additionally, a plurality of module pockets 3838 may be defined on the inner surface of the mount 3708 to accommodate various electronic modules 3806 disposed on the bottom of the PCB 3804. Additionally, a shield locator 3840 may be defined on the inner surface of the mount 3708 to accommodate at least a portion of the second shield 3828b when the sensor control device 3702 is assembled. The battery locator 3834, the contact pocket 3836, the module pocket 3838, and the shield locator 3840 all extend a short distance into 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).

[0095] 7A, the mount 3708 may further include a plurality of carrier grip portions 3842 (two are shown) defined around the circumference of the mount 3708. The carrier grip portions 3842 are axially offset from a bottom 3844 of the mount 3708 to allow a transfer adhesive (not shown) to be applied to the bottom of the mount during assembly. In contrast to conventional sensor control devices that typically include a conical carrier grip portion that intersects with the bottom of the mount, the carrier grip portions 3842 of the present disclosure are offset from the plane where the transfer adhesive is applied (i.e., the bottom 3844). This proves to be advantageous in helping to ensure that the delivery system does not inadvertently stick to the transfer adhesive during assembly. Additionally, the carrier grip portions 3842 of the present disclosure eliminate the need for a corrugated transfer adhesive, which simplifies the manufacture of the transfer adhesive as well as eliminates the need to precisely clock the transfer adhesive against the mount 3708. This also increases the bond area and therefore the bond strength.

[0096] With reference to FIG. 7B, the bottom 3844 of the mount 3708 may provide or otherwise define a number of grooves 3846, which may be defined at or near the periphery of the mount 3708 and spaced equidistant from one another. A transfer adhesive (not shown) may be coupled to the bottom 3844, and the grooves 3846 may be configured to help wick (transfer) moisture away from the sensor control device 3702 toward the periphery of the mount 3708 during use. In some embodiments, the spacing of the grooves 3846 may sandwich a module pocket 3838 (FIG. 7A) defined on the opposite (inner) side of the mount 3708. As will be appreciated, alternating the location of the grooves 3846 and the module pocket 3838 ensures that opposing features on either side of the mount 3708 do not extend into one another. This helps to maximize the use of material for the mount 3708, which may help to maintain a minimum height H of the sensor control device 3702. The module pocket 3838 can also significantly reduce mold sink and improve the flatness of the bottom 3844 where the transfer adhesive bonds.

[0097] 7B, the inner surface of the shell 3706 may also provide or define a number of pockets or recesses configured to accommodate various components of the sensor control device 3702 when the shell 3706 is mated to the mount 3708. For example, the inner surface of the shell 3706 may define an opposing battery locator 3848 positionable opposite the battery locator 3834 (FIG. 7A) of the mount 3708 and configured to accommodate a portion of the battery 3812 when the sensor control apparatus 3702 is assembled. The opposing battery locator 3848 extends a short distance into the inner surface of the shell 3706 and helps to reduce the overall height H of the sensor control device 3702.

[0098] A sharps and sensor locator 3852 may also be provided by or otherwise defined on an inner surface of the shell 3706. The sharps and sensor locator 3852 may be configured to receive both a sharp (not shown) and a portion of the sensor 3716. Additionally, the sharps and sensor locator 3852 may be configured to align and / or mate with a corresponding sharps and sensor locator 2054 ( FIG. 7A ) provided on an inner surface of the mount 3708.

[0099] According to an embodiment of the present disclosure, an alternative sensor assembly / electronics assembly connection approach is shown in Figures 8A-8C. As shown, the sensor assembly 14702 includes a sensor 14704, a connector support 14706, and a sharp 14708. In particular, a recess or receptacle 14710 may be defined in the bottom of the mount of the electronics assembly 14712, providing a location where the sensor assembly 14702 may be received and coupled to the electronics assembly 14712, thereby allowing the sensor control device to be fully assembled. The contours of the sensor assembly 14702 may be complementarily fitted or molded to the receptacle 14710, which includes an elastomeric sealing member 14714 (which includes 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 a recess 14710 integrally formed in the electronics assembly 14712, when the sensor assembly 14702 is snap-fit ​​or otherwise adhered to the electronics assembly 14712, the on-body device 14714 shown in FIG. 8C is formed. This embodiment provides an integrated connector for the sensor assembly 14702 in the electronics assembly 14712.

[0100] Further information regarding sensor assemblies is provided in U.S. Patent Application Publication No. 2013 / 0150691 and U.S. Patent Application Publication No. 2021 / 0204841, each of which is incorporated by reference in its entirety into this specification.

[0101] According to an embodiment of the present disclosure, the sensor control device 102 may be modified to provide a one-piece architecture that can undergo sterilization techniques specifically designed for the one-piece architecture sensor control device. The one-piece architecture allows the sensor applicator 150 and the sensor control device 102 to be shipped to the user in a single sealed package that does not require any final user assembly steps. Rather, the user only needs to open one package and then deliver the sensor control device 102 to the target monitoring location. The one-piece system architecture described herein may prove advantageous in eliminating components, various fabrication process steps, and user assembly steps. As a result, packaging and waste are reduced, as well as the possibility of user error or contamination of the system is mitigated.

[0102] 9A and 9B are a side view and a cross-sectional side view, respectively, of an exemplary embodiment of the sensor applicator 102 having an applicator cap 210 coupled thereto. More specifically, FIG. 9A illustrates how the sensor applicator 102 is shipped to and received by a user, and FIG. 9B illustrates the sensor control device 4402 disposed within the sensor applicator 102. Thus, a fully assembled sensor control device 4402 may already be assembled and installed within the sensor applicator 102 before being delivered to the user, thus eliminating any additional assembly steps that the user would otherwise have to perform.

[0103] The fully assembled sensor control device 4402 may be loaded into the sensor applicator 102, and the applicator cap 210 may then be coupled to the sensor applicator 102. In some embodiments, the applicator cap 210 may be threaded onto the housing 208 and may include a tamper ring 4702. When the applicator cap 210 is rotated (e.g., unscrewed) relative to the housing 208, the tamper ring 4702 may shear, thereby releasing the applicator cap 210 from the sensor applicator 102.

[0104] According to the present disclosure, while loaded into the sensor applicator 102, the sensor control device 4402 may be subjected to a gaseous chemical sterilization 4704 configured to sterilize the electronics housing 4404 and any other exposed portions of the sensor control device 4402. To accomplish this, a chemical may be injected into a sterilization chamber 4706 cooperatively defined by the sensor applicator 102 and the interconnected cap 210. In some applications, the chemical may be injected into the sterilization chamber 4706 via one or more vents 4708 defined in the proximal end 610 of the applicator cap 210. Exemplary chemicals that may be used for gaseous chemical sterilization 4704 include, but are not limited to, ethylene oxide, vaporized hydrogen peroxide, nitrogen oxides (e.g., nitrous oxide, nitrogen dioxide, etc.), and steam.

[0105] Because the sensor 4410 and the distal portion of the sharps 4412 are sealed within the sensor cap 4416, the chemicals used during the gas chemical sterilization process do not interact with the enzymes, chemicals, and biologics provided on the tail 4524 and other sensor components such as the membrane coating that regulates analyte influx.

[0106] Once the desired sterility assurance level has been achieved within the sterilization chamber 4706, the gaseous solution may be removed and the sterilization chamber 4706 may be vented. Venting may be accomplished by a series of vacuum pulls followed by circulating a gas (e.g., nitrogen) or filtered air through the sterilization chamber 4706. Once the sterilization chamber 4706 has been properly vented, the vent port 4708 may be blocked with a seal 4712 (shown in dashed lines).

[0107] In some embodiments, the seal 4712 may comprise two or more layers of different materials. The first layer may be made of a synthetic material (e.g., flash-spun high density polyethylene fibers) such as Tyvek® available from DuPont®. Tyvek® is highly durable and puncture resistant and allows vapor transmission. The Tyvek® layer may be applied prior to the gas-chemical sterilization process, and following the gas-chemical sterilization process, a foil or other vapor and moisture resistant material layer may be sealed (e.g., heat sealed) over the Tyvek® layer to prevent ingress of contaminants and moisture into the sterilization chamber 4706. In other embodiments, the seal 4712 may comprise only a single protective layer applied to the applicator cap 210. In such embodiments, the single layer may be gas permeable for the sterilization process, but may also be capable of protecting against moisture and other harmful elements once the sterilization process is complete.

[0108] With the seal 4712 in place, the applicator cap 210 provides a barrier to outside contamination, thereby maintaining a sterile environment for the assembled sensor control device 4402 until the user removes (unscrews) the applicator cap 210. The applicator cap 210 may also create a dust-free environment during shipping and storage that prevents the adhesive patch 4714 from becoming soiled.

[0109] 10A and 10B are isometric and side views, respectively, of another exemplary sensor control device 5002 in accordance with one or more embodiments of the present disclosure. The sensor control device 5002 may be similar in some respects to the sensor control device 102 of FIG. 1A and therefore may be best understood by referring to FIG. 1A. Additionally, the sensor control device 5002 may replace the sensor control device 102 of FIG. 1A and thus may be used with the sensor applicator 102 of FIG. 1A that can deliver the sensor control device 5002 to a target monitoring location on the skin of a user.

[0110] However, unlike the sensor control device 102 of FIG. 1A, the sensor control device 5002 may comprise a one-piece system architecture that does not require a user to open multiple packages and ultimately assemble the sensor control device 5002 prior to application. Rather, upon receipt by a user, the sensor control device 5002 may already be fully assembled and properly positioned within the sensor applicator 150 (FIG. 1A). To use the sensor control device 5002, the user only needs to open one barrier (e.g., applicator cap 708 of FIG. 3B) before quickly delivering the sensor control device 5002 to a target monitoring location for use.

[0111] As shown, the sensor control device 5002 includes an electronics housing 5004 that is generally disk-shaped and may have a circular cross-section. However, in other embodiments, the electronics housing 5004 may assume other cross-sectional shapes, such as oval or polygonal, without departing from the scope of the present disclosure. The electronics housing 5004 may be configured to house or otherwise contain various electrical components used to operate the sensor control device 5002. In at least one embodiment, an adhesive patch (not shown) may be disposed on the bottom of the electronics housing 5004. The adhesive patch may be similar to the adhesive patch 105 of FIG. 1A and may therefore aid in adhering the sensor control device 5002 to a user's skin for use.

[0112] As shown, the sensor control device 5002 includes an electronics housing 5004 including a shell 5006 and a mount 5008 matable with the shell 5006. The shell 5006 may be secured to the mount 5008 via a variety of methods, such as a snap-fit ​​engagement, an interference fit, sonic welding, one or more mechanical fasteners (e.g., screws), a gasket, an adhesive, or any combination thereof. In some cases, the shell 5006 may be secured to the mount 5008 to create a sealed interface therebetween.

[0113] The sensor control device 5002 may further include a sensor 5010 (partially visible) and a sharp 5012 (partially visible) used to aid in transdermal delivery of the sensor 5010 beneath the skin of a user during application of the sensor control device 5002. As shown, the sensor 5010 and corresponding portions of the sharp 5012 extend distally from a bottom (e.g., mount 5008) of the electronics housing 5004. The sharp 5012 may include a sharp hub 5014 configured to secure and carry the sharp 5012. As best seen in FIG. 10B, the sharp hub 5014 may include or otherwise define an engagement member 5016. To couple the sharp 5012 to the sensor control device 5002, the sharp 5012 may be advanced axially through the electronics housing 5004 until the sharp hub 5014 engages the top surface of the shell 5006 and the mating member 5016 extends distally from the bottom of the mount 5008. Once the sharp 5012 penetrates the electronics housing 5004, an exposed portion of the sensor 5010 may be received within a hollow or recessed (arcuate) portion of the sharp 5012. The remaining portion of the sensor 5010 is disposed within the interior of the electronics housing 5004.

[0114] The sensor control device 5002 may further include a sensor cap 5018, which is shown exploded or removed from the electronics housing 5004 in FIGS. 10A-10B. The sensor cap 5016 may be removably coupled to the sensor control device 5002 (e.g., electronics housing 5004) at or near the bottom of the mount 5008. The sensor cap 5018 may serve to provide a hermetic barrier that surrounds and protects the exposed portions of the sensor 5010 and sharps 5012 from gas 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 provide or otherwise define an engagement portion 5024. As described herein, the engagement portion 5024 may help to mate the sensor cap 5018 with a cap (e.g., applicator cap 708 of FIG. 3B) of a sensor applicator (e.g., sensor applicator 150 of FIGS. 1 and 3A-3G) and may help to remove the sensor cap 5018 from the sensor control device 5002 when the cap is removed from the sensor applicator.

[0115] The sensor cap 5018 may be removably coupled to the electronics housing 5004 at or near the bottom of the mount 5008. More specifically, the sensor cap 5018 may be removably coupled to a mating member 5016 extending distally from the bottom of the mount 5008. In at least one embodiment, for example, the mating member 5016 may define a set of male threads 5026a (FIG. 10B) that are matable with a set of female threads 5026b (FIG. 10A) defined by the sensor cap 5018. In some embodiments, the male and female threads 5026a,b may comprise a flat thread design (e.g., lack of helical curvature), which may prove advantageous in molding the part. Alternatively, the male and female threads 5026a,b may include a helical thread engagement. Thus, the sensor cap 5018 may be threadedly coupled to the sensor control device 5002 at the mating member 5016 of the sharps hub 5014. In other embodiments, the sensor cap 5018 may be removably coupled to the mating member 5016 via other types of engagement, including but not limited to an interference or friction fit, or a frangible member or material that can be broken with minimal separation force (e.g., axial or rotational force).

[0116] In some embodiments, the sensor cap 5018 may comprise a monolithic (single) structure extending between the first and second ends 5020a,b. However, in other embodiments, the sensor cap 5018 may comprise two or more components. In the illustrated embodiment, for example, the sensor cap 5018 may include a sealing ring 5028 positioned at the first end 5020a and a desiccant cap 5030 disposed at the second end 5020b. The sealing ring 5028 may be configured to help seal the internal chamber 5022, as described in more detail below. In at least one embodiment, the sealing ring 5028 may include an elastomeric O-ring. The desiccant cap 5030 may house or include a desiccant to help maintain a preferred humidity level within the internal chamber 5022. The desiccant cap 5030 may also define or otherwise provide an engagement portion 5024 for the sensor cap 5018.

[0117] 11A-11C are progressive cross-sectional side views illustrating the assembly of a sensor applicator 102 and a sensor control device 5002, according to one or more embodiments. Once the sensor control device 5002 is fully assembled, the sensor control device may be loaded into the sensor applicator 102. With reference to FIG. 11A, the sharps hub 5014 may include or otherwise define a hub snap tab 5302 configured to aid in coupling the sensor control device 5002 to the sensor applicator 102. More specifically, the sensor control device 5002 may be advanced into the sensor applicator 102, and the hub snap tab 5302 may be received by a corresponding arm 5304 of a sharps carrier 5306 positioned within the sensor applicator 102.

[0118] 11B, the sensor control device 5002 is shown received by a sharps carrier 5306 and thus secured within the sensor applicator 102. Once the sensor control device 5002 is loaded into the sensor applicator 102, the applicator cap 210 may be coupled to the sensor applicator 102. In some embodiments, the applicator cap 210 and the housing 208 may have an opposing, matable set of threads 5308 that allow the applicator cap 210 to be threaded onto the housing 208 in a clockwise (or counterclockwise) direction, thereby securing the applicator cap 210 to the sensor applicator 102.

[0119] As shown, the sheath 212 is also positioned within the sensor applicator 102, which may include a sheath locking mechanism 5310 configured to ensure that the sheath 212 does not prematurely collapse during an impact event. In the illustrated embodiment, the sheath locking mechanism 5310 may include a threaded engagement between the applicator cap 210 and the sheath 212. More specifically, one or more internal threads 5312a may be defined or otherwise provided on an inner surface of the applicator cap 210, and one or more external threads 5312b may be defined or otherwise provided on the sheath 212. The internal threads 5312a and external threads 5312b may be configured to thread together when the applicator cap 210 is threaded onto the sensor applicator 102 at the threads 5308. The female threads 5312a and male threads 5312b may have the same thread pitch as the threads 5308 that allow the applicator cap 210 to be screwed onto the housing 208.

[0120] 11C , applicator cap 210 is shown fully threaded (coupled) to housing 208. As shown, applicator cap 210 may further provide, or otherwise define, a cap post 5314 centrally located within and extending proximally from a bottom of applicator cap 210. Cap post 5314 may be configured to receive at least a portion of sensor cap 5018 when applicator cap 210 is threaded onto housing 208.

[0121] With the sensor control device 5002 loaded into the sensor applicator 102 and the applicator cap 210 properly secured, the sensor control device 5002 may then be subjected to a gas chemical sterilization configured to sterilize the electronics housing 5004 and any other exposed portions of the sensor control device 5002. Because the sensor 5010 and the distal portion of the sharps 5012 are sealed within the sensor cap 5018, the chemicals used during the gas chemical sterilization process cannot interact with the enzymes, chemicals, and biologics provided on the tail 5104 and other sensor components such as the membrane coating that regulates analyte influx.

[0122] 12A-12C are progressive cross-sectional side views illustrating assembly and disassembly of an alternative embodiment of a sensor applicator 102 having a sensor control device 5002, in accordance with one or more additional embodiments. The fully assembled sensor control device 5002 may be loaded into the sensor applicator 102 by coupling the hub snap tabs 5302 to the arms 5304 of a sharps carrier 5306 positioned within the sensor applicator 102, generally as described above.

[0123] 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 "lock" detent and the second detent 5702b may alternatively be referred to as a "fire" 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 described below, the sheath 212 may be actuated to move the sheath arm 5604 to the second detent 5702b, thereby placing the sensor applicator 102 in the fired position.

[0124] In FIG. 12B , applicator cap 210 is aligned with and advanced toward housing 208 such that sheath 212 is received within applicator cap 210. Instead of rotating applicator cap 210 relative to housing 208, threads of applicator cap 210 may be snapped into corresponding threads of housing 208 to couple applicator cap 210 to housing 208. Axial cuts or slots 5703 (one shown) defined in applicator cap 210 may allow a portion of applicator cap 210 near the threads to flex outwardly to snap into engagement with threads of housing 208. Once applicator cap 210 is snapped onto housing 208, sensor cap 5018 may correspondingly snap into cap post 5314.

[0125] 11A-11C , the sensor applicator 102 may include a sheath locking mechanism configured to ensure that the sheath 212 does not prematurely collapse during an impact event. In the illustrated embodiment, the sheath locking mechanism includes one or more ribs 5704 (one shown) defined near the base of the sheath 212 configured to interact with one or more ribs 5706 (two shown) and a shoulder 5708 defined near the base of the applicator cap 210. The rib 5704 may be configured to interlock between the rib 5706 and the shoulder 5708 during attachment of the applicator cap 210 to the housing 208. More specifically, once applicator cap 210 is snapped onto housing 208, applicator cap 210 may be rotated (e.g., clockwise), which positions rib 5704 of sheath 212 between rib 5706 and shoulder 5708 of applicator cap 210, thereby "locking" applicator cap 210 in place until a user removes applicator cap 210 for use by counter-rotating applicator cap 210. The engagement of rib 5704 between rib 5706 and shoulder 5708 of applicator cap 210 may also prevent sheath 212 from prematurely folding.

[0126] In Figure 12C, the applicator cap 210 has been removed from the housing 208. As with the embodiment of Figures 12A-12C, the applicator cap 210 can be removed by counter-rotating the applicator cap 210, which correspondingly rotates the cap post 5314 in the same direction to unscrew the sensor cap 5018 from the mating member 5016, generally as described above. Additionally, when the sensor cap 5018 is removed from the sensor control device 5002, the sensor 5010 and a distal portion of the sharp 5012 are exposed.

[0127] When the applicator cap 210 is unscrewed from the housing 208, the ribs 5704 defined on the sheath 212 may slidingly engage the tops of the ribs 5706 defined on the applicator cap 210. The tops of the ribs 5706 may provide corresponding sloped surfaces that result in upward movement of the sheath 212 as the applicator cap 210 is rotated, causing the sheath arms 5604 to flex out of engagement with the first detent 5702a and be received into the second detent 5702b. As the sheath 212 moves to the second detent 5702b, the radial shoulder 5614 moves out of radial engagement with the carrier arms 5608, thereby allowing the passive spring force of the springs 5612 to urge the sharps carrier 5306 upwardly, disengaging the carrier arms 5608 from engagement with the grooves 5610. As the sharps carrier 5306 moves upward within the housing 208, the engaging member 5016 may correspondingly retract until it is flush, substantially flush, or near-flush with the bottom of the sensor control device 5002. At this point, the sensor applicator 102 is in the fired position. Thus, in this embodiment, removal of the applicator cap 210 correspondingly retracts the engaging member 5016.

[0128] I. Exemplary Firing Mechanisms for One-Piece and Two-Piece Applicators 13A-13F show exemplary details of an embodiment of the internal device mechanism, including "firing" the applicator 216 to apply the sensor control device 222 to the user and safely retracting the sharp 1030 into the used applicator 216. Taken together, these figures depict an exemplary sequence of driving the sharp 1030 (carrying a sensor coupled to the sensor control device 222) into the user's skin, withdrawing the sharp while leaving the sensor in operative contact with the user's interstitial fluid, and adhering the sensor control device to the user's skin with an adhesive. Modifications of such activities for use with alternative applicator assembly embodiments and components can be understood by those skilled in the art with reference thereto. Additionally, the applicator 216 may be a sensor applicator having a one-piece or two-piece architecture as disclosed herein.

[0129] 13A, the sensor 1102 is supported within the sharp 1030, immediately above the user's skin 1104. Rails 1106 (optionally three of them) of the upper guide portion 1108 may be provided to control movement of the applicator 216 relative to the sheath 318. The sheath 318 is held by a detent portion 1110 within the applicator 216 such that an appropriate downward force along the longitudinal axis of the applicator 216 overcomes the resistance provided by the detent portion 1110, allowing the sharp 1030 and sensor control device 222 to translate along the longitudinal axis into (and onto) the user's skin 1104. Additionally, the catch arm 1112 of the sensor carrier 1022 engages the sharp retraction assembly 1024 to maintain the sharp 1030 in position relative to the sensor control device 222.

[0130] 13B, a user force is applied to overcome or override the detent portion 1110 and the sheath 318 collapses into the housing 314, driving the sensor control device 222 (along with associated components) to translate downward along the longitudinal axis as shown by arrow L. The inner diameter of the upper guide section 1108 of the sheath 318 constrains the position of the carrier arm 1112 throughout the entire stroke of the sensor / sharps insertion process. The retention of the stop surface 1114 of the carrier arm 1112 against the complementary surface 1116 of the sharps retraction assembly 1024 maintains the position of the member with the return spring 1118 fully biased. According to an embodiment, rather than using a user force to drive the sensor control device 222 to translate downward along the longitudinal axis as indicated by arrow L, the housing 314 may include a button (e.g., but not limited to, a push button) that activates a drive spring (e.g., but not limited to, a coil spring) to drive the sensor control device 222.

[0131] In Figure 13C, the sensor 1102 and sharp 1030 have reached full insertion depth, where the carrier arm 1112 clears the inner diameter of the upper guide portion 1108. The compressed force of the coil return spring 1118 then drives the angled stop surface 1114 radially outward, releasing the force to drive the sharps carrier 1102 of the sharps retraction assembly 1024, pulling the sharps 1030 (slotted or otherwise configured) away from the user and away from the sensor 1102, as shown by arrow R in Figure 13D.

[0132] As shown in Figure 13E, once the sharp 1030 is fully retracted, the upper guide portion 1108 of the sheath 318 is set with the final locking portion 1120. As shown in Figure 13F, the used applicator assembly 216 is removed from the insertion site leaving the sensor control device 222 behind and the sharp 1030 safely secured inside the applicator assembly 216. The used applicator assembly 216 is now ready for disposal.

[0133] The movement of the applicator 216 when applying the sensor control device 222 is designed to provide the user with the sensation that both the insertion and retraction of the sharp 1030 are performed automatically by the internal mechanisms of the applicator 216. In other words, the present invention avoids the user experiencing the sensation of manually driving the sharp 1030 into the skin. Thus, when the user applies sufficient force to overcome the resistance from the detent portion of the applicator 216, the resulting movement of the applicator 216 is perceived as an automatic response to the applicator being "triggered." The user does not perceive that the user is supplying additional force to drive the sharp 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 sharp 1030. As detailed above in FIG. 13C, retraction of the sharp 1030 is automated by the coil return spring 1118 of the applicator 216.

[0134] With respect to any of the applicator embodiments described herein and any of its components, including but not limited to sharps, sharps modules, and sensor modules embodiments, one skilled in the art will understand that said embodiments may be sized and configured for use with a sensor configured to sense an analyte level in a bodily fluid within the epidermis, dermis, or subcutaneous tissue of a subject. In some embodiments, for example, the sharps and distal portion of the analyte sensors disclosed herein may both be sized and configured to be positioned at a particular 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). With respect to some applicator embodiments, one skilled in the art will understand that certain embodiments of the sharps may be sized and configured to be positioned at different end depths within the subject's body relative to a final end depth of the analyte sensor. In some embodiments, for example, the sharps may be positioned at a first end depth within the subject's epidermis prior to retraction, and the distal portion of the analyte sensor may be positioned at a second end depth within the subject's dermis. In other embodiments, the sharp may be positioned at a first end depth within the dermis of the subject and the distal portion of the analyte sensor may be positioned at a second end depth within the subcutaneous tissue of the subject prior to retraction. In yet other embodiments, the sharp may be positioned at a first end depth and the analyte sensor may be positioned at a second end depth prior to retraction, both of which are in the same layer or tissue of the subject's body.

[0135] Additionally, with respect to any of the applicator embodiments described herein, one of skill in the art will understand that the analyte sensor, as well as one or more structural components coupled to the analyte sensor, including but not limited to one or more spring mechanisms, may be positioned within the applicator in an off-center position relative to one or more axes of the applicator. In some applicator embodiments, for example, the analyte sensor and spring mechanism may be positioned in a first off-center position relative to the applicator axis on a first side of the applicator, and the sensor electronics may be positioned in a second off-center position relative to the applicator axis on a second side of the applicator. In other applicator embodiments, the analyte sensor, spring mechanism, and sensor electronics may be positioned in an off-center position relative to the applicator axis on the same side. One of skill in the art will understand that other permutations and configurations in which any or all of the analyte sensor, spring mechanism, sensor electronics, and other components of the applicator are positioned in a central or off-center position relative to one or more axes of the applicator are possible and fully within the scope of the present disclosure.

[0136] Further details of suitable devices, systems, methods, components, and their operation with associated parts are described in International Publication No. WO 2018 / 136898 to Rao et al., International Publication No. WO 2019 / 236850 to Thomas et al., International Publication No. WO 2019 / 236859 to Thomas et al., International Publication No. WO 2019 / 236876 to Thomas et al., and U.S. Patent Application Publication No. 2020 / 0196919, filed June 6, 2019, each of which is incorporated by reference herein in its entirety. Further details regarding the applicator, its components, and variant embodiments thereof are described in U.S. Patent Application Publication Nos. 2013 / 0150691, 2016 / 0331283, and 2018 / 0235520, each of which is incorporated by reference in its entirety for all purposes. Further details regarding the sharps module, sharps, its components, and variant embodiments thereof are described in U.S. Patent Application Publication No. 2014 / 0171771, each of which is incorporated by reference in its entirety for all purposes.

[0137] J. Exemplary Methods for Calibrating Analyte Sensors A biochemical sensor can be described by one or more sensing properties. A common sensing property is called the sensitivity of a biochemical sensor, which is a measure of the sensor's responsiveness to the concentration of a chemical or composition that the sensor is designed to detect. For electrochemical sensors, this response can be in the form of a current (amperometric) or charge (coulometric). In other types of sensors, the response may be in a different form, such as photon intensity (e.g., optical light). The sensitivity of a biochemical analyte sensor can vary depending on several factors, including whether the sensor is in an in vitro or in vivo condition.

[0138] FIG. 14 is a graph showing the in vitro sensitivity of an amperometric analyte sensor. In vitro sensitivity can be obtained by in vitro testing a sensor at various analyte concentrations and then performing regression (e.g., linear or nonlinear) or other curve fitting on the resulting data. In this example, the sensitivity of the analyte sensor is linear or substantially linear and can be modeled according to the equation y=mx+b, where y is the electrical output current of the sensor, x is the analyte level (or concentration), m is the slope of the sensitivity, and b is the intercept of the sensitivity, where the intercept generally corresponds to the background signal (e.g., noise). For sensors with linear or substantially linear responses, the analyte level corresponding to a given current can be determined from the slope and intercept of the sensitivity. Sensors with nonlinear sensitivity require additional information to determine the analyte level resulting from the sensor's output current, and those skilled in the art are familiar with how to model nonlinear sensitivity. In certain embodiments of an in vivo sensor, the in vitro sensitivity may be the same as the in vivo sensitivity, while in other embodiments, a transfer (or transformation) function is used to convert the in vitro sensitivity to an in vivo sensitivity applicable to the intended in vivo use of the sensor.

[0139] Calibration is a technique for improving or maintaining accuracy by adjusting the measured output of a sensor to reduce the difference from the expected output of the sensor. One or more parameters describing the sensing characteristics of the sensor, such as the sensitivity of the sensor, are established for use in the calibration adjustment.

[0140] Certain in vivo analyte monitoring systems require calibration to be performed after the sensor is implanted in a user or patient, either by user intervention or by the system itself in an automated manner. For example, when user intervention is required, the user performs an in vitro measurement (e.g., a blood glucose (BG) measurement using a finger prick and an in vitro test strip) while the analyte sensor is implanted and inputs it into the system. The system then compares the in vitro measurement to the in vivo signal and uses the difference to determine an estimate of the in vivo sensitivity of the sensor. The in vivo sensitivity can then be used in an algorithmic process to convert the data collected by the sensor into a value indicative of the user's analyte level. This and other processes that require user action to perform a calibration are referred to as "user calibration." Systems may require user calibration due to instability in the sensitivity of the sensor, such that the sensitivity drifts or changes over time. Thus, multiple user calibrations (e.g., on a periodic (e.g., daily) schedule, a variable schedule, or as needed) may be required to maintain accuracy. While the embodiments described herein may incorporate some degree of user calibration for certain implementations, generally this is not preferred as it requires the user to perform painful or otherwise cumbersome BG measurements and can introduce user error.

[0141] Some in vivo analyte monitoring systems can periodically adjust calibration parameters through the use of automated measurements of sensor characteristics made by the system itself (e.g., processing circuitry running software). Repeated adjustment of the sensor's sensitivity based on variables measured by the system (not the user) is generally referred to as "system" (or automatic) calibration, and can be performed with or without user calibration, such as an early BG measurement. As with repeated user calibration, repeated system calibration is typically necessitated by drift in the sensor's sensitivity over time. Thus, although the embodiments described herein may be used with some degree of automatic system calibration, preferably the sensor's sensitivity is relatively stable over time such that post-implant calibration is not required.

[0142] Some in vivo analyte monitoring systems operate with sensors that are factory calibrated. Factory calibration refers to the determination or estimation of one or more calibration parameters prior to distribution to a user or healthcare professional (HCP). The calibration parameters may be determined by the sensor manufacturer (or the manufacturer of other components of the sensor control device, if the two entities are different). Many in vivo sensor manufacturing processes manufacture sensors in groups or batches called production lots, production stage lots, or simply lots. A single lot may contain thousands of sensors.

[0143] The sensor may include a calibration code or parameter that is derived or determined during one or more sensor manufacturing processes and may be coded or programmed into a data processing device of the analyte monitoring system as part of the manufacturing process, or may be provided on the sensor itself, for example, as a bar code, laser tag, RFID tag, or other machine-readable information provided to the sensor. If the code is provided to the receiver (or other data processing device), user calibration during in vivo use of the sensor may be unnecessary or the frequency of in vivo calibration during sensor wear may be reduced. In embodiments in which the calibration code or parameter is provided on the sensor itself, the calibration code or parameter may be automatically transmitted or provided to a data processing device in the analyte monitoring system prior to or at the start of sensor use.

[0144] Some in vivo analyte monitoring systems operate with sensors that can be one or more of factory calibrated, system calibrated, and / or user calibrated. For example, the sensor may be equipped with a calibration code or parameters that can enable factory calibration. If the information is provided to the receiver (e.g., entered by a user), the sensor may operate as a factory calibrated sensor. If the information is not provided to the receiver, the sensor may operate as a user calibrated sensor and / or a system calibrated sensor.

[0145] In further embodiments, programming or executable instructions may be provided or stored in a data processing device and / or receiver / controller unit of an analyte monitoring system to provide a time-varying adjustment algorithm for an in-vivo sensor during use. For example, a time-based predetermined or analytical curve or database may be generated based on retrospective statistical analysis of analyte sensors used in vivo and corresponding glucose level feedback, and may be configured to provide additional adjustments to one or more in-vivo sensor parameters to compensate for potential sensor drift in stability profiles or other factors.

[0146] In accordance with the disclosed subject matter, the analyte monitoring system may be configured to compensate or adjust the sensor sensitivity based on the sensor drift profile. A time-varying parameter β(t) may be defined or determined based on an analysis of the sensor behavior during in vivo use to determine the time-varying drift profile. In certain aspects, the compensation or adjustment to the sensor sensitivity may be programmed in a receiver unit, controller, or data processor of the analyte monitoring system such that the compensation or adjustment, or both, may be performed automatically and / or iteratively as sensor data is received from the analyte sensor. In accordance with the disclosed subject matter, the adjustment or compensation algorithm may be user-initiated or executed (rather than self-initiated or executed) such that the adjustment or compensation to the analyte sensor sensitivity profile is performed or executed upon user initiation or activation of a corresponding function or routine, or when the user enters a sensor calibration code.

[0147] According to the disclosed subject matter, each sensor in a sensor lot (in some cases not including the sample sensor used for in vitro testing) may be non-destructively inspected to determine or measure its characteristics, such as film thickness at one or more points on the sensor, and other characteristics, including physical characteristics such as surface area / volume of the active area, may be measured or determined. Such measurements or determinations may be performed in an automated manner, for example using an optical scanner or other suitable measurement device or system, and the determined sensor characteristics for each sensor in the sensor lot are compared to corresponding average values ​​based on the sample sensors for possible correction of the calibration parameters or codes assigned to each sensor. For example, for a calibration parameter defined as sensor sensitivity, the sensitivity is approximately inversely proportional to the film thickness, e.g., for a sensor having a measured film thickness that is about 4% greater than the average film thickness of sensors sampled from the same sensor lot as that sensor, the sensitivity assigned to that sensor in one embodiment is the average sensitivity determined from the sampled sensors divided by 1.04. Similarly, sensitivity is approximately proportional to the active area of ​​a sensor, so that for a sensor with a measured active area that is about 3% lower than the average active area of ​​sensors sampled from the same sensor lot, the assigned sensitivity for that sensor is the average sensitivity multiplied by 0.97. The assigned sensitivity may be determined from the average sensitivity from the sampled sensors by multiple successive adjustments for each inspection or measurement of the sensor. In certain embodiments, the inspection or measurement of each sensor may further include a measurement of the consistency or texture of the film in addition to the film thickness and / or surface area or volume of the active sensing area.

[0148] Further information regarding sensor calibration is provided in U.S. Patent Application Publication No. 2010 / 002302851 and U.S. Patent Application Publication No. 2019 / 0274598, each of which is incorporated by reference in its entirety.

[0149] 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 functions to provide an interface to make the BLE module 5041 available to the computing hardware of the sensor 110. These software functions may include a BLE logic interface and an interface parser. The BLE services provided by the communication module 5040 may include a generic access profile service, a generic attribute service, a generic access service, a device information service, a data transmission service, and a security service. The data transmission service may be a primary service used to transmit data such as sensor control data, sensor status data, analyte measurement data (past and present), and event log data. The sensor status data may include error data, current active time, and software status. The analyte measurement data may include information such as current and past raw measurements, current and past values ​​after processing using appropriate algorithms or models, predictions and trends of measurement levels, comparisons of other values ​​to patient-specific averages, calls to action determined by algorithms or models, and other similar types of data.

[0150] According to aspects of the disclosed subject matter, as embodied herein, the sensor 110 may be configured to communicate with multiple devices simultaneously by adapting the characteristics of the communication protocol or medium supported by the hardware and radio of the sensor 110. As an example, the BLE module 5041 of the communication module 5040 may comprise software or firmware to enable multiple simultaneous connections between the sensor 110 as a central device and other devices as peripheral devices, or as a peripheral device where another device is the central device.

[0151] A connection and subsequent communication session between two devices using a communication protocol such as BLE can be characterized by a similar physical channel operating between the two devices (e.g., the sensor 110 and the data receiving device 120). The physical channel may include a single channel or a set of channels, including, for example and without limitation, using a common clock and an agreed-upon set of channels determined by a channel or frequency hopping sequence. The communication sessions may use a similar amount of the available communication spectrum, and multiple such communication sessions may exist in close proximity. In certain embodiments, each set of devices in a communication session uses a different physical channel or set of channels to manage interference of devices in the same proximity.

[0152] For purposes of illustration and not limitation, reference is made to an exemplary embodiment of a procedure for sensor-receiver connection for use with the disclosed subject matter. First, the sensor 110 repeatedly advertises its connection information to its environment in search of the data receiving device 120. The sensor 110 may repeat advertising periodically until a connection is established. The data receiving device 120 detects the advertising packet and scans and filters for the sensor 120 to connect through the data provided in the advertising packet. Next, the data receiving device 120 sends a scan request command, and the sensor 110 responds with a scan response packet providing additional details. Next, the data receiving device 120 sends a connection request using a Bluetooth device address associated with the data receiving device 120. The data receiving device 120 may also continuously request to establish a connection to the sensor 110 with a particular Bluetooth device address. The devices can then establish an initial connection and begin data exchange. The devices begin the process of initializing the data exchange service and perform a mutual authentication procedure.

[0153] During the first connection between the sensor 110 and the data receiving device 120, the data receiving device 120 may initialize a service, characteristics, and attribute discovery procedure. The data receiving device 120 may evaluate these characteristics of the sensor 110 and store them for use during subsequent connections. The devices then enable notification of customized security services to be used for mutual authentication of the sensor 110 and the data receiving device 120. The mutual authentication procedure can be automated and does not require user intervention. Following successful completion of the mutual authentication procedure, the sensor 110 sends a connection parameter update requesting the data receiving device 120 to use the connection parameter settings preferred by the sensor 110 and configured to maximize longevity.

[0154] The data receiving device 120 then executes a sensor control procedure 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 initiate the backfill process. The request may specify a range of records defined, for example, based on measurements, timestamps, etc., as needed. The sensor 110 responds with the requested data until all previously untransmitted data in the memory of the sensor 110 is delivered to the data receiving device 120. The sensor 110 may respond to a backfill request from the data receiving device 120 that all data has already been transmitted. Once the backfill is complete, the data receiving device 120 may notify the sensor 110 that it is ready to receive periodic measurements. The sensor 110 may transmit measurements over multiple notification results on a recurring 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.

[0155] For purposes of illustration and not limitation, reference is made to an exemplary embodiment of a procedure for sending a shutdown command to the sensor 110. The shutdown operation is performed, for example, when the sensor 110 is in an error state, an insertion failure state, or a sensor expired state. If the sensor 110 is not in those states, the sensor 110 can log the command and perform the shutdown when the sensor 110 transitions to the error state or the sensor expired state. The data receiving device 120 sends a properly formatted shutdown command to the sensor 110. If the sensor 110 is actively processing another command, the sensor 110 responds with a standard error response indicating that the sensor 110 is busy. Otherwise, the sensor 110 sends a response once the command is received. Additionally, the sensor 110 sends a success notification via the sensor control property to confirm that the sensor 110 received the command. The sensor 110 registers the shutdown command. At the next appropriate opportunity (e.g., depending on the current sensor state, as described herein), the sensor 110 shuts down.

[0156] L. Exemplary Sensor States and Activations For purposes of illustration and not limitation, reference is made to an exemplary embodiment of a high-level depiction of a state machine representation 6000 of actions that may be taken by the sensor 110 as shown in FIG. 15. After initialization, the sensor enters a state 6005 associated with manufacturing the sensor 110. In the manufacturing state 6005, the sensor 110 may be configured for operation, e.g., the storage memory 5030 may be written to. At various times while in state 6005, the sensor 110 checks for a received command to proceed to the storage state 6015. Upon entering the storage state 6015, the sensor performs a software integrity check. While in the storage state 6015, the sensor may also receive a wake-up request command before proceeding to the insertion detection state 6025.

[0157] Upon entering state 6025, the sensor 110 may store information regarding devices authorized to communicate with the sensor set during startup or may initialize algorithms related to deriving and interpreting measurements from the sensing hardware 5060. The sensor 110 may also initialize a lifecycle timer responsible for maintaining an active count of the sensor's 110 operation time and may begin communicating with authorized devices to transmit recorded data. While in the insertion detection state 6025, the sensor may enter state 6030, in which the sensor 110 checks whether the operation time is equal to a predefined threshold. This operation time threshold may correspond to a timeout function for determining whether the insertion is successful. If the operation time reaches the threshold, the sensor 110 proceeds to state 6035, in which the sensor 110 checks whether the average data read is greater than a threshold amount corresponding to the expected amount of data read to trigger the detection of a successful insertion. If the amount of data read is lower than the threshold while in state 6035, the sensor proceeds to state 6040, corresponding to a failed insertion. If the amount of data read meets the threshold, the sensor proceeds to the Active Pair state 6055 .

[0158] The active pair state 6055 of the sensor 110 reflects the state while the sensor 110 is operating normally by recording measurements, processing the measurements, and reporting them accordingly. While in the active pair state 6055, the sensor 110 transmits measurements or attempts to establish a connection with the receiving device 120. The sensor 110 also increments the operating time. When the sensor 110 reaches a predetermined threshold operating time (e.g., the operating time reaches a predetermined threshold), the sensor 110 transitions to the active expired state 6065. The active expired state 6065 of the sensor 110 reflects the state while the sensor 110 has been operating for its maximum predetermined amount of time.

[0159] While in the active expired state 6065, the sensor 110 may generally perform actions related to finalization actions to ensure that collected measurements are securely transmitted to a receiving device as necessary. For example, while in the active expired state 6065, the sensor 110 may transmit collected data and, if a connection is not available, may increase efforts to discover and establish a connection with a nearby authenticated device. While in the active expired state 6065, the sensor 110 may receive a shutdown command in state 6070. If a shutdown command is not received, the sensor 110 may also check in state 6075 whether the operation time has exceeded a final operation threshold. The final operation threshold may be based on the battery life of the sensor 110. The normal termination state 6080 corresponds to a final operation of the sensor 110, ultimately shutting down the sensor 110.

[0160] Before the sensor is powered up, the ASIC 5000 is in a low power storage mode state. The power-up process can begin, for example, when a received RF field (e.g., an NFC field) drives the voltage of the power supply to the ASIC 5000 above a reset threshold, causing the sensor 110 to enter a wake-up state. While in the wake-up state, the ASIC 5000 enters a power-up sequence state. The ASIC 5000 then powers up the communication module 5040. The communication module 5040 is initialized and triggers a power-on self-test. The power-on self-test may include the ASIC 5000 communicating with the communication module 5040 using a prescribed sequence of reading and writing data to verify that the memory and one-time programmable memory are not corrupted.

[0161] When the ASIC 5000 first enters the measurement mode, an insertion detection sequence is performed to verify that the sensor 110 is properly placed on the patient's body before a proper measurement can be made. First, the sensor 110 interprets a command to initiate a measurement configuration process, causing the ASIC 5000 to enter a measurement command mode. The sensor 110 then enters a measurement lifecycle state temporarily, performing several consecutive measurements to test whether the insertion was successful. The communication module 5040 or the ASIC 5000 evaluates the measurement results to determine successful insertion. Once the insertion is deemed successful, the sensor 110 enters a measurement state, where the sensor 110 begins to take periodic measurements using the sensing hardware 5060. If the sensor 110 determines that the insertion was not successful, the sensor 110 is triggered into an insertion failure mode, and the ASIC 5000 is commanded to return to a storage mode, while the communication module 5040 disables itself.

[0162] M. Exemplary Over-the-Air Updates FIG. 1B further illustrates an exemplary operating environment for providing over-the-air ("OTA") updates for use with the techniques described herein. An operator of the analyte monitoring system 100 may bundle updates for the data receiving device 120 or the sensor 110 with updates for applications running on the general-purpose data receiving device 130. Using available communication channels between the data receiving device 120, the general-purpose data receiving device 130, and the sensor 110, the general-purpose data receiving device 130 can receive periodic updates for the data receiving device 120 or the sensor 110 and initiate installation of updates to the data receiving device 120 or the sensor 110. The general-purpose data receiving device 130 serves as an installation or update platform for the data receiving device 120 or the sensor 110, since applications that enable the general-purpose 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 software or firmware on the data receiving device 120 or the sensor 110 without wide area networking capabilities.

[0163] As embodied herein, a remote application server 150 operated by the manufacturer of the analyte sensor 110 and / or the operator of the analyte monitoring system 100 can provide software and firmware updates to the devices of the analyte monitoring system 100. In certain embodiments, the remote application server 150 can provide updated software and firmware to the user device 140 or directly to the general-purpose data receiving device. As embodied herein, the remote application server 150 can also provide application software updates to the application storefront server 160 using an interface provided by the application storefront. The general-purpose data receiving device 130 may periodically contact the application storefront server 160 to download and install updates.

[0164] After the multipurpose data receiving device 130 downloads the application update including the firmware or software update for the data receiving device 120 or the sensor 110, the data receiving device 120 or the sensor 110 and the multipurpose data receiving device 130 establish a connection. The multipurpose data receiving device 130 determines that a firmware or software update is available for the data receiving device 120 or the sensor 110. The multipurpose data receiving device 130 can prepare the software or firmware update for delivery to the data receiving device 120 or the sensor 110. As an example, the multipurpose data receiving device 130 may compress or segment data associated with the software or firmware update, or may encrypt or decrypt the firmware or software update, or perform an integrity check on the firmware or software update. The multipurpose data receiving device 130 transmits data for the firmware or software update to the data receiving device 120 or the sensor 110. The multipurpose data receiving device 130 may transmit a command to the data receiving device 120 or the sensor 110 to initiate the update. Additionally or alternatively, the multipurpose data receiving device 130 may provide notifications to a user of the multipurpose data receiving device 130 and may include instructions to facilitate the update, such as instructions to keep the data receiving device 120 and the multipurpose data receiving device 130 connected to and in close proximity to a power source until the update is complete.

[0165] The data receiving device 120 or the sensor 110 receives data for the update and a command to initiate the update from the multipurpose data receiving device 130. The data receiving device 120 can then install the firmware or software update. To install the update, the data receiving device 120 or the sensor 110 may place itself in a so-called "safe" mode with limited operational capabilities or reboot. Once the update is complete, the data receiving device 120 or the sensor 110 re-enters a standard operational 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 was successfully installed. The multipurpose data receiving device 130 can receive a notification of the successful update. The multipurpose data receiving device 130 can then report confirmation of the successful update to the remote application server 150.

[0166] In a particular embodiment, the storage memory 5030 of the sensor 110 includes a one-time programmable (OTP) memory. The term OTP memory may refer to a memory that includes access restrictions and security to facilitate a predetermined number of writes to a particular address or segment in the memory. The memory 5030 may be pre-arranged into a number of pre-allocated memory blocks or containers. The containers are pre-allocated to a fixed size. If the storage memory 5030 is a one-time programming memory, the containers may be considered to be in a non-programmable state. Additional containers that have not yet been written to may be placed in a programmable or writable state. Containerizing the storage memory 5030 in this manner may improve the transportability of code and data to be written to the storage memory 5030. Updating the software of a device (e.g., a sensor device described herein) stored in an OTP memory may be performed by replacing only the code in a particular previously written container or containers with updated code written to a new container or containers, rather than replacing the entire code in the memory. In a second embodiment, the memory is not pre-arranged. Instead, the space allocated for data is dynamically allocated or determined as needed. Containers of various sizes where updates are expected can be defined so that incremental updates can be issued.

[0167] 16 illustrates an example operation and data flow diagram for over-the-air (OTA) programming of a storage memory 5030 in a sensor device 100 and use of the memory after OTA programming in execution of a process by the sensor device 110 in accordance with the disclosed subject matter. In the example OTA programming 500 illustrated in FIG. 5, a request to initiate OTA programming (or reprogramming) is sent from an external device (e.g., data receiving device 130). At 511, a communication module 5040 of the sensor device 110 receives an OTA programming command. The communication module 5040 sends the OTA programming command to the microcontroller 5010 of the sensor device 110.

[0168] At 531, after receiving the OTA programming command, the microcontroller 5010 verifies the OTA programming command. The microcontroller 5010 may, for example, determine whether the OTA programming command is signed with a proper digital signature token. Upon determining that the OTA programming command is valid, the microcontroller 5010 may set the sensor device to an OTA programming mode. At 532, the microcontroller 5010 may verify the OTA programming data. At 533, the microcontroller 5010 may reset the sensor device 110 to reinitialize the sensor device 110 to a programming state. Once the sensor device 110 transitions to the OTA programming state, the microcontroller 5010 may begin writing data to the rewritable memory 540 (e.g., memory 5020) of the sensor device at 534 and write 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 to mark one or more programming blocks or regions of the OTP memory 550 as invalid or inaccessible. The data written to the free or unused portions of the OTP memory may be used to replace the invalidated or inaccessible programming blocks of the OTP memory 550. After the microcontroller 5010 has written the data to the respective memories at 534 and 535, the microcontroller 5010 may perform one or more software integrity checks to ensure that no errors were introduced into the programming blocks during the writing process. Once the microcontroller 5010 can determine that the data was written without error, the microcontroller 5010 can resume normal operation of the sensor device.

[0169] In the execution mode, the microcontroller 5010 can retrieve a programming manifest or profile from the rewritable memory 540 at 536. The programming manifest or profile may include a list of valid software programming blocks and may include a guide to program execution for the sensor 110. By following the programming manifest or profile, the microcontroller 5010 can determine which memory blocks of the OTP memory 550 are appropriate to execute and can avoid executing out-of-date or invalidated programming blocks or referencing out-of-date data. At 537, the microcontroller 5010 can selectively retrieve memory blocks from the OTP memory 550. At 538, the microcontroller 5010 can use the retrieved memory blocks by executing programming code stored in the memory or by using variables stored in the memory.

[0170] N. Exemplary Security and Other Architectural Features As embodied herein, a first layer of security for communications between the analyte sensor 110 and other devices may be established based on security protocols dictated by and built into the communications protocol used for communication. Another layer of security may be based on communications protocols that require proximity of the communicating devices. Additionally, certain packets and / or certain data contained within a packet may be encrypted while other packets and / or data within the packet are otherwise encrypted or remain unencrypted. Additionally or alternatively, application layer encryption may be used in conjunction with one or more block or stream ciphers to establish mutual authentication and communications encryption with other devices within the analyte monitoring system 100.

[0171] The ASIC 5000 of the analyte sensor 110 may be configured to dynamically generate authentication and encryption keys using data held in the storage memory 5030. The storage memory 5030 may also be pre-programmed with a set of valid authentication and encryption keys for use with a particular class of device. The ASIC 5000 may be further configured to perform an authentication procedure with the other device using the received data and apply a generated key to the sensitive data before transmitting the sensitive data. The generated key may be specific to the analyte sensor 110, specific to a pair of devices, specific to a communication session between the analyte sensor 110 and the other device, specific to a message transmitted during the communication session, or specific to a block of data contained within a message.

[0172] Both the sensor 110 and the data receiving device 120 can ensure the authorization of the other party in a communication session, for example, to issue commands or receive data. In certain embodiments, identity authentication may be performed through two features. First, the party asserting its identity provides a valid certificate signed by the device manufacturer or the operator of the analyte monitoring system 100. Second, authentication may be performed through the use of public and private keys and a shared secret derived therefrom or established by the device of the analyte monitoring system 100 or established by the operator of the analyte monitoring system 100. To verify the identity of the other party, a party can provide proof that the party has control of the private key.

[0173] The manufacturer of the analyte sensor 110, the data receiving device 120, or the provider of the application for the general-purpose data receiving device 130 can provide the information and programming necessary for the devices to communicate securely through secure programming and updates. For example, the manufacturer may provide information that can be used to generate cryptographic keys for each device, including a secure root key for the analyte sensor 110 and optionally the data receiving device 120, which can be used in combination with device-specific information and operational data (e.g., entropy-based random values) to generate cryptographic values ​​unique to the device, session, or data transmission, as needed.

[0174] Analyte data associated with a user is at least partially sensitive data, since this information may be used for a variety of purposes, including health monitoring and medication dosing decisions. In addition to user data, the analyte monitoring system 100 may implement security enhancements against efforts by external parties to reverse engineering. The communication connection may be encrypted using a device-specific or session-specific encryption key. Encrypted or unencrypted communications between any two devices may be verified with a transmission integrity check built into the communication. The operation of the analyte sensor 110 may be protected from tampering by restricting access to read and write functions to the memory 5020 via the communication interface. The sensor may be configured to allow access only to known or "trusted" devices provided in a "white list," or only to devices that can provide a predefined code associated with the manufacturer or otherwise authorized user. The white list may represent an exclusive scope, meaning that no connection identifiers other than those included in the white list are used, or a preferred scope, where the white list is searched first, but other devices may still be used. The sensor 110 may further reject a connection request and shut down if the requester fails to complete a login procedure over the communication interface within a predefined time (e.g., within 4 seconds). These properties protect against certain denial of service attacks, particularly those against BLE interfaces.

[0175] As embodied herein, the analyte monitoring system 100 may employ periodic key rotation to further reduce the likelihood of key leakage and exploitation. The key rotation strategy employed by the analyte monitoring system 100 may be designed to support backward compatibility of field-deployed or distributed devices. As an example, the analyte monitoring system 100 may use keys for downstream devices (e.g., devices that are in the field or cannot feasibly provide updates) that are designed to be compatible with multiple generations of keys used by upstream devices.

[0176] For purposes of illustration and not limitation, reference is made to an exemplary embodiment of a message sequence diagram 600 for use with the disclosed subject matter shown in FIG. 17, illustrating an exemplary data exchange between a pair of devices, specifically a sensor 110 and a data receiving device 120. The data receiving device 120 may be a data receiving device 120 or a general-purpose data receiving device 130, as embodied herein. In step 605, the data receiving device 120 may send a sensor wake-up command 605 to the sensor 110, for example via a short-range communication protocol. The sensor 110 may be primarily dormant prior to step 605 to conserve its battery until a full wake-up is required. After waking up during step 610, the sensor 110 may collect data or perform other operations as appropriate for the sensing hardware 5060 of the sensor 110. In step 615, the data receiving device 120 may initiate an authentication request command 615. In response to the authentication request command 615, both the sensor 110 and the data receiving device 120 may engage in a mutual authentication process 620. The mutual authentication process 620 may involve the transfer of data, including challenge parameters that enable the sensor 110 and the data receiving device 120 to ensure that the other device is capable of sufficient compliance with the agreed upon security framework described herein. Mutual authentication may be based on a mechanism for authenticating two or more entities to each other, with or without an online trusted third party, to verify the establishment of a secret key via challenge-response. Mutual authentication may be performed using two, three, four, or five pass authentication, or similar versions thereof.

[0177] Following a successful mutual authentication process 620, in step 625, the sensor 110 can provide the data receiving device 120 with a sensor secret 625. The sensor secret may include a sensor-specific value and can be derived from a random value generated during manufacture. The sensor secret can be encrypted before or during transmission to prevent third parties from accessing the secret. The sensor secret 625 may be encrypted via one or more of the keys generated by or in response to the mutual authentication process 620. In step 630, the data receiving device 120 can derive a sensor-specific encryption key from the sensor secret. The sensor-specific encryption key can further be session-specific. Thus, the sensor-specific encryption key can be determined by each device without being transmitted between the sensor 110 or the data receiving device 120. In step 635, the sensor 110 can encrypt the data contained in the payload. In step 640, the sensor 110 can transmit the encrypted payload 640 to the data receiving device 120 using the communication link established between the sensor 110 and the data receiving device 120 in an appropriate communication model. In step 645, the data receiving device 120 can decrypt the payload using the sensor-specific encryption key derived during step 630. Following step 645, the sensor 110 can deliver additional (including newly collected) data, and the data receiving device 120 can process the received data appropriately.

[0178] As described herein, the sensor 110 may be a device with limited processing power, battery supply, and storage. The encryption techniques (e.g., selection of cryptographic algorithms or implementations of algorithms) used by the sensor 110 may be selected based at least in part on these limitations. The data receiving device 120 may be a more powerful device with fewer limitations of this nature. Thus, the data receiving device 120 may employ more advanced and computationally intensive encryption techniques, such as cryptographic algorithms and implementations.

[0179] O. Exemplary Payloads / Communication Frequencies The analyte sensor 110 may be configured to modify its discoverability behavior to attempt to increase the probability that a receiving device receives a proper data packet and / or provides an acknowledgment signal or reduce limitations that may otherwise prevent the acknowledgment signal from being received. Modifying the discoverability behavior of the analyte sensor 110 may include, for example, but not limited to, modifying how often connection data is included in a data packet, modifying how often data packets are transmitted in general, lengthening or shortening the broadcast window of a data packet, modifying the amount of time the analyte sensor 110 listens for an acknowledgment signal or a scan signal after broadcasting, including direct transmissions (e.g., through one or more attempted transmissions) to one or more devices that previously communicated with the analyte sensor 110 and / or one or more devices on a whitelist, modifying the transmit power associated with the communication module when broadcasting a data packet (e.g., to increase the range of the broadcast or reduce energy consumed and extend the battery life of the analyte sensor), modifying the rate at which data packets are prepared and broadcast, or a combination of one or more other modifications. Additionally or alternatively, the receiving device may similarly adjust parameters related to the device's listening behavior to increase the likelihood of receiving data packets containing the connection data.

[0180] As embodied herein, the analyte sensor 110 may be configured to broadcast data packets using two types of windows. The first window refers to the rate at which the analyte sensor 110 is configured to operate its communications hardware. The second window refers to the rate at which the analyte sensor 110 is configured to actively transmit (e.g., broadcast) data packets. As an example, the first window may indicate that the analyte sensor 110 operates its communications hardware to transmit and / or receive data packets (including connection data) during the first 2 seconds of each 60 second period. The second window may indicate that during each 2 second window, the analyte sensor 110 transmits a data packet every 60 milliseconds. The remaining time during the 2 second window, the analyte sensor 110 is scanning. The analyte sensor 110 may lengthen or shorten either window to modify the discoverability behavior of the analyte sensor 110.

[0181] In certain embodiments, the discoverability behavior of the analyte sensor may be stored in a discoverability profile, and modifications may be made based on one or more factors, such as the state of the analyte sensor 110, and / or by applying rules based on the state of the analyte sensor 110. For example, when the battery level of the analyte sensor 110 falls below a certain amount, the rule may cause the analyte sensor 110 to reduce the power consumed by the broadcast process. As another example, configuration settings associated with broadcasting or otherwise transmitting packets may be adjusted based on the ambient temperature, the temperature of the analyte sensor 110, or the temperature of certain components of the communication hardware of the analyte sensor 110. In addition to modifying the transmission power, other parameters associated with the transmission capabilities or processes of the communication hardware of the analyte sensor 110 may be modified, including, but not limited to, the transmission rate, frequency, and timing. As another example, when the analyte data indicates that the subject is experiencing or about to experience a negative health event, the rule may cause the analyte sensor 110 to increase its discoverability and alert receiving devices of the negative health event.

[0182] P. Exemplary Sensor Sensitivity Initialization / Adjustment Features As embodied herein, certain calibration characteristics for the sensing hardware 5060 of the analyte sensor 110 may be adjusted based on external or interval environmental characteristics, as well as to compensate for decay of the sensing hardware 5060 during periods of not being used (e.g., "storage time" prior to use). The calibration characteristics of the sensing hardware 5060 may be adjusted autonomously by the sensor 110 (e.g., by operation of the ASIC 5000 to modify characteristics in the memory 5020 or storage 5030) or may be adjusted by other devices of the analyte monitoring system 100.

[0183] As an example, the sensor sensitivity of the sensing hardware 5060 may be adjusted based on external temperature data or time since manufacture. When external temperature is monitored during storage of the sensor, the disclosed subject matter may adaptively change compensation to the sensor sensitivity over time as the device experiences changing storage conditions. By way of example and not limitation, adaptive sensitivity adjustments may be performed in an "active" storage mode in which the analyte sensor 110 periodically wakes up to measure temperature. These features may conserve the battery of the analyte device and extend the life of the analyte sensor. At each temperature measurement, the analyte sensor 110 may calculate a sensitivity adjustment for that period based on the measured temperature. The temperature weighted adjustment values ​​may then be accumulated over the active storage mode period to calculate a total sensor sensitivity adjustment value at the end of the active storage mode (e.g., upon insertion). Similarly, upon insertion, the sensor 110 can determine the time difference between manufacture of the sensor 110 (which can be written to the storage 5030 of the ASIC 5000) or the sensing hardware 5060 and modify the sensor sensitivity or other calibration characteristics according to one or more known decay rates or formulas.

[0184] Additionally, for purposes of illustration and not limitation, as embodied herein, the sensor sensitivity adjustment may account for other sensor conditions, such as sensor drift. The sensor sensitivity adjustment may be hard-coded into the sensor 110 during manufacturing, for example, based on an estimate of how much the average sensor drifts in the case of sensor drift. The sensor 110 may use a calibration function having time-varying functions for sensor offset and gain, which may account for drift over the period of wear of the sensor. Thus, the sensor 110 may utilize a device-dependent function that describes the drift of the sensor 110 over time, and a function used to convert interstitial current to interstitial glucose, which may represent sensor sensitivity and may be device-specific combined with a baseline glucose profile. Such functions to account for sensor sensitivity and drift may improve the accuracy of the sensor 110 over the period of wear without user calibration.

[0185] Q. Exemplary Model-Based Analyte Measurements The sensor 110 detects raw measurements from the sensing hardware 5060. On-sensor processing can be performed, such as by one or more models trained to interpret the raw measurements. The models may be machine learning models trained off-device to detect, predict, or interpret the raw measurements to detect, predict, or interpret the levels of one or more analytes. Additional trained models may act on the output of the machine learning models trained to interact with the raw measurements. As an example, the models may be used to detect, predict, or recommend events based on the raw measurements and the type of analyte detected by the sensing hardware 5060. The events may include the initiation or completion of a physical activity, a meal, the application of a medical procedure or medication, an emergency health event, and other events of a similar nature.

[0186] The model may be provided to the sensor 110, data receiving device 120, or general-purpose data receiving device 130 during manufacturing or during a firmware or software update. The model may be periodically refined, such as by the manufacturer of the sensor 110 or the operator of the analyte monitoring system 100, based on data received from the sensor 110 and the data receiving device of an individual user or a population of users. In certain embodiments, the sensor 110 includes sufficient computer components to support further training or refinement of the machine learning model, such as based on unique characteristics of the user to which the sensor 110 is attached. The machine learning model may include models trained using or incorporating, by way of example and not limitation, decision tree analysis, gradient boosting, ada boosting, artificial neural networks or variations thereof, linear discriminant analysis, nearest neighbor analysis, support vector machines, supervised or unsupervised classification, and the like. The model may also include algorithmic or rule-based models in addition to machine learning models. Model-based processing may be performed by the data receiving device 120 or other devices, including the general-purpose data receiving device 130, upon receiving data from the sensor 110 (or other downstream devices).

[0187] R. Exemplary Alarm Functions The data transmitted between the sensor 110 and the data receiving device 120 may include raw measurements or processed measurements. The data transmitted between the sensor 110 and the data receiving device 120 may further include alarms or notifications for display to a user. The data receiving device 120 may display or otherwise convey notifications to a user based on the raw measurements or processed measurements, or may display alarms as they are received from the sensor 110. Alarms that may be triggered for display to a user include alarms based on direct analyte values ​​(e.g., a single reading that exceeds or does not meet a threshold), analyte value trends (e.g., average readings over a set period of time that exceed or do not meet a threshold, slope), analyte value predictions (e.g., an algorithmic calculation based on an analyte value exceeds or does not meet a threshold), sensor warnings (e.g., a suspected malfunction is detected), communication warnings (e.g., no communication between the sensor 110 and the data receiving device 120 for a threshold period of time, an unknown device attempting to initiate or failing to initiate a communication session with the sensor 110), reminders (e.g., a reminder to charge the data receiving device 120, take medication, or perform other activities), and other warnings of a similar nature. For purposes of illustration 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-settable and non-user-settable parameters.

[0188] S. Exemplary Electrode Configurations Sensor configurations featuring a single active area configured for detection of a corresponding single analyte may use a two-electrode or three-electrode detection motif, as further described herein with reference to Figures 18A-18C. Sensor configurations featuring two different active areas for detection of the same or different analytes, either on separate working electrodes or on the same working electrode, are described separately below with reference to Figures 19A-21C. Sensor configurations having multiple working electrodes may be particularly advantageous for incorporating two different active areas within the same sensor tail, since the signal contribution from each active area can be more easily determined.

[0189] When a single working electrode is present in the analyte sensor, a three-electrode sensor configuration can include a working electrode, a counter electrode, and a reference electrode. A related two-electrode sensor configuration can include a working electrode and a second electrode, which can function as both a counter electrode and a reference electrode (i.e., a counter / reference electrode). The various electrodes can be at least partially stacked (layered) on one another and / or laterally spaced apart from one another on the sensor tail. A suitable sensor configuration can be substantially planar, or substantially cylindrical, or any other suitable shape. In any sensor configuration disclosed herein, the various electrodes may be electrically insulated from one another by a dielectric material or similar insulator.

[0190] Analyte sensors featuring multiple working electrodes may also include at least one additional electrode. If one additional electrode is present, the one additional electrode may function as a counter / reference electrode for each of the multiple working electrodes. If 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.

[0191] 18A shows a 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 area 218 is disposed as at least one layer over at least a portion of the working electrode 214. The active area 218 may include multiple spots or a single spot configured for detection of an analyte, as discussed further herein. In certain embodiments, the active area 218 is configured to detect potassium, as described herein.

[0192] 18A, the membrane 220 overcoats at least the active area 218. In certain embodiments, the membrane 220 may also overcoat some 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 may be overcoated with the membrane 220. The membrane 220 may include one or more polymeric membrane materials capable of limiting the flow of analytes to the active area 218 (i.e., the membrane 220 is a mass transport limiting membrane having some permeability to the analyte of interest). In accordance with the disclosure herein, as further described below, the membrane 220 may be crosslinked with a branched crosslinker in certain sensor configurations. For example, but not limited to, the membrane 220 is crosslinked with a crosslinker described herein, such as a branched glycidyl ether. The composition and thickness of the membrane 220 may be varied to facilitate the flow of desired analytes to the active area 218, thereby providing desired signal strength and stability. The analyte sensor 200 may be operable to assay an analyte, for example potassium, by either coulometric, amperometric, voltammetric, or potentiometric electrochemical detection.

[0193] 18B and 18C show diagrams of an exemplary three-electrode analyte sensor configuration, which are also suitable for use in the disclosure herein. The three-electrode analyte sensor configuration may be similar to that shown for the analyte sensor 200 of FIG. 18A, except that the analyte sensors 201 and 202 (FIGS. 18B and 18C) include an additional electrode 217. With the additional electrode 217, the counter / reference electrode 30216 may then function as either a counter electrode or a reference electrode, and the additional electrode 217 performs other electrode functions not otherwise considered. The working electrode 214 continues to perform its original function. The additional electrode 217 may be disposed on either the working electrode 214 or the electrode 30216, with a separating layer of dielectric material therebetween. For example, but not by way of limitation, as shown in FIG. 18B, the dielectric layers 219a, 219b, and 219c separate the electrodes 214, 30216, and 217 from one another and provide electrical insulation. Alternatively, at least one of the electrodes 214, 30216, and 217 may be located on the opposite side of the substrate 30212, as shown in FIG. 18C. Thus, in certain embodiments, the electrode 214 (working electrode) and the electrode 30216 (counter electrode) may be located on the opposite side of the substrate 30212, and the electrode 217 (reference electrode) is located 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 location of the reference material layer 230 is not limited to the locations depicted in FIGS. 18B and 18C. Similar to the sensor 200 shown in FIG. 18A, the active area 218 in the analyte sensors 201 and 202 may include multiple spots or a single spot. Additionally, analyte sensors 201 and 202 may be operable to assay an analyte, for example potassium, by either coulometric, amperometric, voltammetric, or potentiometric electrochemical detection methods.

[0194] Similar to analyte sensor 200, membrane 220 may also overcoat active area 218 and other sensor components in analyte sensors 201 and 202, thereby functioning as a mass transport limiting membrane. In certain embodiments, additional electrode 217 may be overcoated with membrane 220. Although FIGS. 18B and 18C show electrodes 214, 30216, and 217 as being overcoated with membrane 220, it should be appreciated that in certain embodiments, only working electrode 214 is overcoated. Furthermore, the thickness of membrane 220 on each of electrodes 214, 30216, and 217 may be the same or different. As in the two-electrode analyte sensor configuration (FIG. 18A), one or both sides of analyte sensors 201 and 202 may be overcoated with membrane 220 in the sensor configuration of FIGS. 18B and 18C, or the entire analyte sensors 201 and 202 may be overcoated. Thus, the three-electrode sensor configuration shown in Figures 18B and 18C should be understood as not limiting the embodiments disclosed herein, and alternative electrode and / or layer configurations remain within the scope of the present disclosure.

[0195] FIG. 19A illustrates an exemplary configuration of a sensor 203 having a single working electrode with two different active areas disposed thereon. FIG. 19A is similar to FIG. 18A, except that there are two active areas on the working electrode 214, a first active area 218a and a second active area 218b, which respond to the same or different analytes and are laterally spaced apart from each other on the surface of the working electrode 214. The active areas 218a and 218b may include multiple spots or a single spot configured for detection of each analyte. The composition of the membrane 220 may vary in the active areas 218a and 218b or may be compositionally the same. The first active area 218a and the second active area 218b may be configured to detect their corresponding analytes at different working electrode potentials, as further described below. In certain embodiments, either or both of the active areas 218a and 218b may be configured to detect potassium. In certain embodiments, either or both of active areas 218a and 218b may be configured to detect potassium by using aspartate oxidase. In certain embodiments, either or both of active areas 218a and 218b may be configured to detect potassium by using aspartate oxidase and asparaginase. In certain embodiments, only one active area of ​​218a and 218b is configured to detect potassium. In certain embodiments, the other active area is configured to detect a second analyte different from potassium. Non-limiting examples of second analytes are described herein.

[0196] Figures 19B and 19C show cross-sectional views of exemplary three-electrode sensor configurations for sensors 204 and 205, respectively, each featuring a single working electrode having a first active area 218a and a second active area 218b disposed thereon. Figures 19B and 19C are otherwise similar to Figures 18B and 18C and may be better understood by reference thereto. As with Figure 19A, the composition of membrane 220 may vary in active areas 218a and 218b or may be compositionally the same.

[0197] Exemplary sensor configurations having multiple working electrodes, specifically two working electrodes, are described in further detail with reference to Figures 20-21C. The following description is primarily directed to sensor configurations having two working electrodes, but it should be understood that more than two working electrodes may be incorporated through extension of the disclosure herein. Additional working electrodes may be used to impart additional sensing capabilities to the analyte sensor, for detection of not only the first and second analytes, but also, for example, a third and / or fourth analyte.

[0198] 20 illustrates a cross-sectional view of an exemplary analyte sensor configuration having two working electrodes, a reference electrode, and a counter electrode suitable for use in the present disclosure. As shown, the analyte sensor 300 includes working electrodes 304 and 306 disposed on opposite sides of a substrate 302. A first active area 310a is disposed on the surface of the working electrode 304, and a second active area 310b is disposed on the surface of the working electrode 306. The counter electrode 320 is electrically insulated from the working electrode 304 by a dielectric layer 322, and the reference electrode 321 is electrically insulated from the working electrode 306 by a dielectric layer 323. Outer dielectric layers 302, 30 and 332 are disposed on the reference electrode 321 and the counter electrode 320, respectively. A membrane 340 can overcoat at least the active areas 310a and 310b according to various embodiments, and other components of the analyte sensor 300 or the entire analyte sensor 300 are optionally overcoated with the membrane 340. In certain embodiments, the membrane 340 may be continuous but compositionally varied within the first membrane portion 340a and the second membrane portion 340b (i.e., over the active areas 310a and 310b) to provide different permeability values ​​for differentially regulating analyte flow at each location. For example, and without limitation, according to various embodiments, the first membrane portion 340a can overcoat at least the active area 310a and the second membrane portion 340b can overcoat at least the active area 310b, along with other components of the analyte sensor 300 or the entirety of the analyte sensor 300.

[0199] In certain embodiments, different membrane formulations may be sprayed and / or printed on the opposite sides of the analyte sensor 300. Dip-coating techniques may also be suitable, particularly for depositing at least a portion of the bilayer membrane on one of the active areas 310a and 310b. In certain embodiments, the membrane 340 may be the same or different in composition in the active areas 310a and 310b. For example, but not limited to, the membrane 340 may include a bilayer overcoating active area 310a and be a homogeneous membrane overcoating active area 310b, or the membrane 340 may include a bilayer overcoating active area 310b and be a homogeneous membrane overcoating active area 310a. In certain embodiments, one of the first membrane portion 340a and the second membrane portion 340b may include a bilayer membrane and the other of the first membrane portion 340a and the second membrane portion 340b may include a single membrane polymer, according to certain embodiments of the present disclosure. In certain embodiments, the analyte sensor can include more than one membrane 340, such as two or more membranes. For example, without limitation, the analyte sensor may include a membrane overcoating one or more active areas, such as 310a and 310b, and an additional membrane overcoating the entire sensor, as shown in Figure 20. In such a configuration, a bilayer membrane may be formed over one or more active areas (e.g., 310a and 310b).

[0200] In certain embodiments, either or both of the active areas 310a and 310b may be configured to detect potassium, for example, by using aspartate oxidase. In certain embodiments, either or both of the active areas 310a and 310b may further include asparaginase. In certain embodiments, only one of the active areas 310a and 310b is configured to detect potassium, for example, by using aspartate oxidase. In certain embodiments, only one of the active areas 310a and 310b may further include asparaginase. In certain embodiments, the other active area is configured to detect a second analyte.

[0201] Alternative sensor configurations having multiple working electrodes and that differ from the configuration shown in Figure 20 may feature counter / reference electrodes instead of separate counter and reference electrodes 320, 321 and / or may feature layer and / or membrane arrangements that differ from those explicitly depicted. For example, and not by way of limitation, the positioning of the counter electrode 320 and reference electrode 321 may be reversed from that shown in Figure 20. Additionally, the working electrodes 304 and 306 do not necessarily need to be on opposite sides of the substrate 302 in the manner shown in Figure 20.

[0202] Although preferred sensor configurations may feature electrodes that are substantially planar in character, it is understood that sensor configurations featuring non-planar electrodes may be advantageous and particularly suitable for use in the present disclosure. In particular, substantially cylindrical electrodes arranged concentrically with respect to one another may facilitate deposition of a mass transport limiting membrane, as described below. In particular, concentric working electrodes spaced apart along the length of the sensor tail may facilitate membrane deposition by successive dip coating operations in a manner similar to that described above for substantially planar sensor configurations. Figures 21A-21C show perspective views of an analyte sensor featuring two working electrodes arranged concentrically with respect to one another. It is understood that sensor configurations having a concentric electrode arrangement, but lacking a second working electrode, are also possible in the present disclosure.

[0203] 21A shows a perspective view of an exemplary sensor configuration in which multiple electrodes are substantially cylindrical and concentrically arranged with respect to one another around a central substrate. As shown, the analyte sensor 400 includes a central substrate 402 around which all of the electrodes and dielectric layers are concentrically arranged with respect to one another. In particular, a working electrode 410 is disposed on a surface of the central substrate 402, and a dielectric layer 412 is disposed on a portion of the working electrode 410 distal from the sensor tip 404. A working electrode 420 is disposed on the dielectric layer 412, and a dielectric layer 422 is disposed on a portion of the working electrode 420 distal from the sensor tip 404. A counter electrode 430 is disposed on the dielectric layer 422, and a dielectric layer 432 is disposed on a portion of the counter electrode 430 distal from the sensor tip 404. A reference electrode 440 is disposed on the dielectric layer 432, and a dielectric layer 442 is disposed on a portion of the reference electrode 440 distal from the sensor tip 404. Thus, the exposed surfaces of working electrode 410 , working electrode 420 , counter electrode 430 , and reference electrode 440 are spaced apart from one another along longitudinal axis B of analyte sensor 400 .

[0204] 21A, a first active area 414a and a second active area 414b responsive to different analytes or the same analyte are disposed on the exposed surfaces of the working electrodes 410 and 420, respectively, thereby allowing contact with the fluid to occur for sensing. Although the active areas 414a and 414b are shown as three separate spots in FIG. 21A, it should be understood that fewer or more than three spots including a continuous layer of active areas may be present in alternative sensor configurations described herein. In certain embodiments, either or both of the active areas 414a and 414b may be configured to detect potassium. In certain embodiments, either or both of the active areas 414a and 414b may be configured to detect potassium by using an enzyme system including aspartate oxidase and, optionally, asparaginase. In certain embodiments, only one active area of ​​414a and 414b is configured to detect potassium. In certain embodiments, only one active area, 414a and 414b, is configured to detect potassium by using aspartate oxidase and, optionally, asparaginase, while the other active area is configured to detect a second analyte.

[0205] In Fig. 21A, the sensor 400 is partially coated with a membrane 450 on the working electrodes 410 and 420 and the active areas 414a and 414b disposed thereon. Fig. 21B shows an alternative sensor configuration in which substantially the entire sensor 401 is overcoated with a membrane 450. The membrane 450 may be compositionally the same or different in the active areas 414a and 414b. For example, the membrane 450 may include a bilayer overcoating active area 414a and a homogenous membrane overcoating active area 414b.

[0206] It should be further understood that the positioning of the various electrodes in FIG. 21A and FIG. 21B may differ from that explicitly shown. For example, the positions of the counter electrode 430 and the reference electrode 440 may be reversed from the configuration shown in FIG. 21A and FIG. 21B. Similarly, the positions of the working electrodes 410 and 420 are not limited to those explicitly depicted in FIG. 21A and FIG. 21B. FIG. 21C shows an alternative sensor configuration to that shown in FIG. 21B, where the sensor 405 contains the counter electrode 430 and the reference electrode 440 located more proximally to the sensor tip 404, and the working electrodes 410 and 420 located more distally to the sensor tip 404. A sensor configuration in which the working electrodes 410 and 420 are located more distally to the sensor tip 404 may be advantageous by providing a larger surface area for deposition of the active areas 414a and 414b (five separate sensing spots exemplarily shown in FIG. 21C), thereby facilitating increased signal strength in some cases. Similarly, the central substrate 402 may be omitted in any of the concentric sensor configurations disclosed herein, and the innermost electrode may instead support subsequently deposited layers.

[0207] In certain embodiments, one or more electrodes of the analyte sensors described herein are wire electrodes, e.g., permeable wire electrodes. In certain embodiments, the sensor tail comprises a working electrode and a reference electrode spirally wound around the working electrode. In certain embodiments, an insulator is disposed between the working electrode and the reference electrode. In certain embodiments, a portion of the electrode is exposed to allow reaction of one or more enzymes with the analyte on the electrode. In certain embodiments, each electrode is formed from a thin wire having a diameter of about 0.001 inches or less to about 0.010 inches or more. In certain embodiments, the working electrode has a diameter of about 0.001 inch or less to about 0.010 inch or more, for example, about 0.002 inch to about 0.008 inch, more preferably about 0.004 inch to about 0.005 inch. In certain embodiments, the electrode is formed from a plated insulator, a plated wire, or a bulk conductive material. In certain embodiments, the working electrode comprises a wire formed from a conductive material, such as platinum, platinum-iridium, palladium, graphite, gold, carbon, a conductive polymer, an alloy, or the like. In certain embodiments, the conductive material is a transparent conductive material. In certain embodiments, the electrodes may be formed by various manufacturing techniques (e.g., bulk metal processing, deposition of metal on a substrate, etc.), and the electrodes may be formed from plated wire (e.g., platinum on steel wire) or bulk metal (e.g., platinum wire). In certain embodiments, the electrodes are formed, for example, from tantalum wire coated with a conductive material.

[0208] In certain embodiments, the reference electrode can function as the reference electrode alone or as a dual reference and counter electrode and is formed from silver, silver / silver chloride, etc. In certain embodiments, the reference electrode is juxtaposed with the working electrode and / or is aligned with or around the working electrode. In certain embodiments, the reference electrode is spirally wound around the working electrode. In certain embodiments, the assembly of wires may be coated or glued together with an insulating material to provide an insulating attachment.

[0209] In certain embodiments, additional electrodes, such as wire electrodes, may be included in the sensor tail. For example, but not limited to, a three-electrode system (working, reference, and counter electrodes) and / or additional working electrodes (e.g., an electrode for detecting a second analyte) may be included in the sensor tail. In certain embodiments where the sensor comprises two working electrodes, the two working electrodes may be juxtaposed around which the reference electrode is disposed (e.g., spirally wound around the two or more working electrodes). In certain embodiments, the two or more working electrodes may extend parallel to one another. In certain embodiments, the reference electrode is coiled around one or more working electrodes and extends toward the distal end (i.e., the in vivo end) of the sensor tail. In certain embodiments, the reference electrode extends (e.g., spirally) to the exposed area of ​​one or more working electrodes.

[0210] In certain embodiments, one or more working electrodes are spirally wound around the reference electrode. In certain embodiments where more than one working electrode is provided, the working electrodes may be formed in a double, triple, quadruple, etc. spiral configuration along the length of the sensor tail (e.g., surrounding the reference electrode, an insulating rod, or other support structure). In certain embodiments, the electrodes, e.g., two or more working electrodes, are formed coaxially. For example, but not by way of limitation, the electrodes all share the same central axis.

[0211] In certain embodiments, the working electrode comprises a tube having a reference electrode disposed or coiled therein with an insulator therebetween. Alternatively, the reference electrode comprises a tube having a working electrode disposed or coiled therein with an insulator therebetween. 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 is provided and coated with an insulating material (described herein) and one or more working electrodes and a reference electrode are disposed thereon. For example, without limitation, the present disclosure provides a sensor, e.g., a sensor tail, comprising one or more tantalum wires, and a conductive material is disposed on a portion of the one or more tantalum wires to function as a working electrode. In certain embodiments, a platinum-coated tantalum wire is covered with an insulating material, and the insulating material is partially covered with a silver / silver chloride composition to function as a reference electrode and / or a counter electrode.

[0212] In certain embodiments where an insulator is disposed on the working electrode (e.g., on the platinum surface of the electrode), a portion of the insulator may be stripped or otherwise removed to expose the electroactive surface of the working electrode. For example, but not limited to, a portion of the insulator may be removed by hand, excimer laser, chemical etching, laser ablation, grit blasting, and the like. Alternatively, a portion of the electrode may be masked prior to depositing the insulator to maintain exposed electroactive surface area. In certain embodiments, the stripped and / or removed portion of the insulator may be about 0.1 mm (about 0.004 inches) or less to about 2 mm (about 0.078 inches) or more in length, for example, about 0.5 mm (about 0.02 inches) to about 0.75 mm (0.03 inches) in length. In certain embodiments, the insulator is a non-conductive polymer. In certain embodiments, the insulator includes parylene, fluorinated polymers, polyethylene terephthalate, polyvinylpyrrolidone, polyurethane, polyimide, and other non-conductive polymers. In certain embodiments, glass or ceramic materials may 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.

[0213] Several portions of the sensor, including the active area, are described further below. 2. Enzymes The active area of ​​the analyte sensor of the present disclosure can be configured to detect an analyte. In certain embodiments, the analyte sensor of the present disclosure is configured to measure potassium, such as potassium ions, in a sample. For example, but not by way of limitation, the active area of ​​the analyte sensor of the present disclosure is configured to detect potassium ions.

[0214] In certain embodiments, the analyte sensor of the present disclosure is configured to indirectly detect potassium ions by comparing signals obtained from at least one, e.g., at least two, aspartate-responsive active areas, in certain embodiments, the current signal from two aspartate-responsive active areas can be correlated to the concentration of potassium ions, as discussed below.

[0215] In certain embodiments, the analyte sensor of the present disclosure is configured to indirectly detect potassium ions by comparing signals obtained from at least one, e.g., at least two, asparagine-responsive active areas. In certain embodiments, the current signal from two asparagine-responsive active areas can be correlated to the concentration of potassium ions, as discussed below.

[0216] In certain embodiments, the active area of ​​the present disclosure is disposed on a portion of the working electrode. For example, but not limited to, the active area is disposed on the portion of the working electrode in a spot pattern, e.g., two or more spots on the working electrode. In certain embodiments, the active area is disposed on the portion of the working electrode in a slot pattern. In certain embodiments, the active area is disposed across the entire length of the working electrode or in a continuous pattern on the working electrode.

[0217] In certain embodiments, the active area of ​​the present disclosure may have a thickness of about 0.1 μm to about 100 μm, e.g., about 1 μm to about 90 μm, about 1 μm to about 80 μm, about 1 μm to about 70 μm, about 1 μm to about 60 μm, about 1 μm to about 50 μm, about 1 μm to about 40 μm, about 1 μm to about 30 μm, about 1 μm to about 20 μm, about 0.5 μm to about 10 μm, about 1 μm to about 10 μm, about 1 μm to about 5 μm, or about 0.1 μm to about 5 μm. In certain embodiments, the active area of ​​the present disclosure may have a thickness of about 0.01 mm. 2 ~ approx. 2.0 mm 2 , for example, about 0.1 mm 2 ~ approx. 1.0 mm 2 or about 0.2 mm 2 ~about 0.5mm 2 It has an area of.

[0218] It should be understood that the sensitivity (output current) of the analyte sensor to each analyte can be varied by varying the coverage (area or size) of the active areas, the area ratio of the active areas to each other, the identity, thickness, and / or composition of the mass transport limiting membrane overcoating the active areas. Modification of these parameters can be readily made by one of ordinary skill in the art given the benefit of the disclosure herein.

[0219] A. Aspartate-responsive active areas In certain embodiments, the analyte sensor of the present disclosure includes at least one active area, e.g., two active areas, configured to detect aspartate to facilitate indirect measurement of potassium ion concentration in a sample. Particular enzymes that can be used to detect aspartate are shown in FIG. 27. In certain embodiments, the enzyme present in the aspartate-responsive active area is aspartate oxidase. As shown in FIG. 27, aspartate oxidase can catalyze the oxidation of aspartate to oxaloacetate and the reduction of its coenzyme flavin adenine dinucleotide (FAD) to FADH2. An electron transfer agent can then mediate the electron transfer from FADH2 to the working electrode. The electrochemical signal obtained at the working electrode can then be correlated with the amount of potassium ions initially present in the sample.

[0220] Specific examples of aspartate oxidase suitable for use in the analyte sensor disclosed herein include, but are not limited to, potassium-dependent aspartate oxidase and potassium-independent aspartate oxidase. In certain embodiments, the aspartate oxidase used in the enzyme system is potassium-dependent. For example, but not limited to, the activity of aspartate oxidase depends on the potassium ion concentration, thereby allowing the indirect determination of the potassium level in the sample. In certain embodiments, the aspartate oxidase used in the enzyme system is potassium-independent. Non-limiting examples of aspartate oxidase for use in the present disclosure include L-aspartate oxidase from Thermococcus, Pyrococcus, Sulfolobus, and extremely halophilic species. In certain embodiments, the L-aspartate oxidase is from Sulfolobus tokodaii or Thermococcus litoralis. For example, but not by way of limitation, L-aspartate oxidase for use in the potassium sensor of the present disclosure may be from any of the methods described 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., J. Biol. Chem. 2010, 144:111-112 (2013), and the like. al., Extremophiles 22(1):59-71 (2018), and Hao et al., Plant Science 271:133-142 (2018), the contents of each of which are incorporated herein by reference in their entireties.

[0221] In certain embodiments, the aspartate-responsive active area may comprise an aspartate oxidase, e.g., a potassium-independent aspartate oxidase or a potassium-independent aspartate oxidase. In certain embodiments, the aspartate-responsive active area may comprise an enzyme system consisting essentially of aspartate oxidase. In certain embodiments, the aspartate-responsive active area may comprise an enzyme system consisting of aspartate oxidase. In certain embodiments, the aspartate-responsive active area may comprise from about 10% to about 80% by weight of aspartate oxidase, e.g., from about 15% to about 75% by weight, from about 20% to about 70% by weight, from about 25% to about 65% by weight, from about 20% to about 60% by weight, from about 20% to about 55% by weight, from about 20% to about 50% by weight, from about 20% to about 45% by weight, from about 20% to about 40% by weight, from about 20% to about 35% by weight, or from about 20% to about 30% by weight. In certain embodiments, the active area may comprise from about 10% to about 40% by weight of aspartate oxidase. In certain embodiments, the aspartate-responsive active area may comprise from about 15% to about 35% by weight of aspartate oxidase. In certain embodiments, the asparagine-responsive active area may comprise about 20% to about 30% by weight of aspartate oxidase.

[0222] In certain embodiments, the aspartate-responsive active area may further comprise a stabilizing agent. In certain embodiments, the stabilizing agent is used for enzyme stabilization. For example, but not limited to, the stabilizing agent may be albumin, such as serum albumin. Non-limiting examples of serum albumin include bovine serum albumin and human serum albumin. In certain embodiments, the stabilizing agent is human serum albumin. In certain embodiments, the stabilizing agent is bovine serum albumin. In certain embodiments, the aspartate-responsive active area may comprise a ratio of stabilizer to aspartate oxidase of about 40:1 to about 1:40, e.g., about 35:1 to about 1:35, about 30:1 to about 1:30, about 25:1 to about 1:25, about 20:1 to about 1:20, about 15:1 to about 1:15, about 10:1 to about 1:10, about 9:1 to about 1:9, about 8:1 to about 1:8, about 7:1 to about 1:7, about 6:1 to about 1:6, about 5:1 to about 1:5, about 4:1 to about 1:4, about 3:1 to about 1:3, about 2:1 to about 1:2, or about 1:1. In certain embodiments, the aspartate-responsive active area may comprise a ratio of stabilizer to aspartate oxidase of about 5:1 to about 1:5. In certain embodiments, the aspartate-responsive active area may comprise a ratio of stabilizer to aspartate oxidase of about 4:1 to about 1:4. In certain embodiments, the aspartate-responsive active area may comprise a ratio of stabilizer to aspartate oxidase of about 3:1 to about 1:3. In certain embodiments, the aspartate-responsive active area may comprise a ratio of stabilizer to aspartate oxidase of about 2:1 to about 1:2.

[0223] In certain embodiments, the disclosed analyte sensor for indirect measurement of potassium may include a first potassium-dependent channel including a first aspartate-responsive active area and a second potassium-dependent channel including a second aspartate-responsive active area. In certain embodiments, each channel is a working electrode. In certain embodiments, the first potassium-dependent channel includes a first potassium-dependent aspartate oxidase. In certain embodiments, the second potassium-dependent channel includes a second potassium-dependent aspartate oxidase. In such embodiments, the first potassium-dependent aspartate oxidase and the second potassium-dependent aspartate oxidase exhibit different dependencies on potassium and thus produce different signals from each other in the presence of the same potassium concentration. In certain embodiments, the difference in signals obtained from the first potassium-dependent channel and the second potassium-dependent channel can be correlated to the concentration of potassium ions in the sample.

[0224] In certain embodiments, the analyte sensor of the present disclosure for indirect measurement of potassium may include a first potassium-dependent channel and a second potassium-dependent channel. In certain embodiments, each channel is a working electrode. In certain embodiments, the first potassium-dependent channel 801 includes a first potassium-dependent aspartate oxidase. In certain embodiments, the second potassium-dependent channel includes a second potassium-dependent aspartate oxidase. In such embodiments, the first potassium-dependent aspartate oxidase and the second potassium-dependent aspartate oxidase exhibit different dependencies on potassium and thus produce different signals from each other in the presence of the same potassium concentration. In certain embodiments, the difference in signals obtained from the two channels can be correlated to the concentration of potassium ions in the sample.

[0225] In certain embodiments, the analyte sensor of the present disclosure may include a sensor tail including at least two working electrodes. In certain embodiments, an aspartate-responsive active area (e.g., a first aspartate-responsive active area) is disposed on a surface of a first working electrode, and an aspartate-responsive active area (e.g., a second aspartate-responsive active area) is disposed on a surface of a second working electrode, and each aspartate-responsive active area includes an aspartate oxidase. In certain embodiments, the first aspartate-responsive active area includes a potassium-independent aspartate oxidase, and the second aspartate-responsive active area includes a potassium-dependent aspartate oxidase. Alternatively, the first aspartate-responsive active area includes a first potassium-dependent aspartate oxidase, and the second aspartate-responsive active area includes a second potassium-dependent aspartate oxidase, and the aspartate oxidases have different dependencies on potassium ion concentration.

[0226] A. Asparagine-responsive active area In certain embodiments, the analyte sensor of the present disclosure includes at least one active area, e.g., two active areas, configured to detect asparagine to facilitate indirect measurement of potassium ion concentration in a sample. A particular enzyme system that can be used to detect asparagine is shown in FIG. 22. As shown in FIG. 22, asparaginase catalyzes the hydrolysis of asparagine to produce aspartate. Aspartate oxidase can then catalyze the oxidation of aspartate to oxaloacetate and the reduction of its coenzyme flavin adenine dinucleotide (FAD) to FADH2. An electron transfer reagent can then mediate the transfer of electrons from FADH2 to the working electrode.

[0227] Specific examples of asparaginases suitable for use in the analyte sensors disclosed herein include, but are not limited to, potassium-dependent asparaginases and potassium-independent asparaginases. In certain embodiments, the asparaginase used in the enzyme system is potassium-dependent. In certain embodiments, the asparaginase used in the enzyme system is potassium-independent. Non-limiting examples of potassium-independent and potassium-dependent asparaginases are described 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 selected from the group consisting of Staphylococcus vulgaris ( The asparaginase is a potassium-dependent (PvAspG1) and / or potassium-independent (PvAspG-T2) asparaginase from P. aureus. In certain embodiments, the asparaginase comprises an amino acid substitution at position 118 or 117 of PvAspG1 and PvAspG-T2, respectively.

[0228] In certain embodiments, the asparagine-responsive active area may comprise an enzyme system including aspartate oxidase and asparaginase. In certain embodiments, the asparagine-responsive active area may comprise an enzyme system consisting essentially of aspartate oxidase and asparaginase. In certain embodiments, the asparagine-responsive active area may comprise an enzyme system consisting of aspartate oxidase and asparaginase. In certain embodiments, the asparagine-responsive active area is from about 100:1 to about 1:100, e.g., 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 , about 30:1 to about 1:30, about 25:1 to about 1:25, about 20:1 to about 1:20, about 15:1 to about 1:15, about 10:1 to about 1:10, about 9:1 to about 1:9, about 8:1 to about 1:8, about 7:1 to about 1:7, about 6:1 to about 1:6, about 5:1 to about 1:5, about 4:1 to about 1:4, about 3:1 to about 1:3, or about 2:1 to about 1:2. In certain embodiments, the asparagine-responsive active area may comprise a ratio of aspartate oxidase to asparaginase of about 90:1 to about 1:90. In certain embodiments, the asparagine-responsive active area may comprise a ratio of aspartate oxidase to asparaginase of about 100:1 to about 2:1. In certain embodiments, the asparagine-responsive active area may comprise a ratio of aspartate oxidase to asparaginase of about 100:1 to about 50:1.In certain embodiments, the asparagine-responsive active area may comprise a ratio of aspartate oxidase to asparaginase of about 90:1 to about 70:1.In certain embodiments, the asparagine-responsive active area is from about 0.1% to about 80% by weight, e.g., from about 0.1% to about 75% by weight, from about 0.1% to about 70% by weight, from about 0.1% to about 65% by weight, from about 0.1% to about 60% by weight, from about 0.1% to about 55% by weight, from about 0.1% to about 50% by weight, from about 0.1% to about 45% by weight, from about 0.1% to about 40% by weight, from about 0.1% to about 35% by weight, from about 0.1% to about 30% by weight, from about 0.1% to about 25% by weight, from about 0.1% to about 20% by weight, from about 0.1% to about 50% by weight, from about 0.1% to about 50% by weight, from about 0.1% to about 50% by weight, from about 0.1% to about 60% by weight, from about 0.1% to about 75% by weight, from about 0.1% to about 70% by weight, from about 0.1% to about 50% by weight, from about 0.1% to about 60% by weight, from about 0.1% to about 55% by weight, from about 0.1% to about 50% by weight, from about 0.1% to about 45% by weight, from about 0.1% to about 40% by weight, from about 0.1% to about 35% by weight, from about 0.1% to about 30% by weight, from about 0.1% to about 25% by weight, from about 0.1% to about 20% by weight, from about 0.1% to about 50% by weight, from about 0.1% to about 50 In certain embodiments, the active area may comprise from about 0.1% to about 15% by weight, from about 0.1% to about 10% by weight, from about 0.1% to about 5% by weight, from about 15% to about 75% by weight, from about 20% to about 70% by weight, from about 25% to about 65% by weight, from about 20% to about 60% by weight, from about 20% to about 55% by weight, from about 20% to about 50% by weight, from about 20% to about 45% by weight, from about 20% to about 40% by weight, from about 20% to about 35% by weight, or from about 20% to about 30% by weight of one or more enzymes, e.g., aspartate oxidase and / or asparaginase. In certain embodiments, the active area may comprise from about 0.1% to about 40% by weight of one or more enzymes, e.g., aspartate oxidase and / or asparaginase. In certain embodiments, the active area may comprise from about 1% to about 40% by weight of one or more enzymes, such as aspartate oxidase and / or asparaginase. In certain embodiments, the active area may comprise from about 1% to about 35% by weight of one or more enzymes, such as aspartate oxidase and / or asparaginase. In certain embodiments, the active area may comprise from about 1% to about 30% by weight of one or more enzymes, such as aspartate oxidase and / or asparaginase. In certain embodiments, the active area may comprise from about 1% to about 25% by weight of one or more enzymes, such as aspartate oxidase and / or asparaginase. In certain embodiments, the asparagine responsiveness may comprise from about 15% to about 35% by weight of one or more enzymes, such as aspartate oxidase and / or asparaginase.In certain embodiments, the asparagine-responsiveness comprises about 20% to about 30% by weight of one or more enzymes, such as aspartate oxidase and / or asparaginase.

[0229] In certain embodiments, the asparagine-responsive active area may further comprise a stabilizing agent, for example, to stabilize the enzyme. For example, but not limited to, the stabilizing agent may be albumin, such as serum albumin. Non-limiting examples of serum albumin include bovine serum albumin and human serum albumin. In certain embodiments, the stabilizing agent is human serum albumin. In certain embodiments, the stabilizing agent is bovine serum albumin. In certain embodiments, the asparagine-responsive active area may comprise a ratio of stabilizer to asparaginase and / or aspartate oxidase of about 40:1 to about 1:40, e.g., about 35:1 to about 1:35, about 30:1 to about 1:30, about 25:1 to about 1:25, about 20:1 to about 1:20, about 15:1 to about 1:15, about 10:1 to about 1:10, about 9:1 to about 1:9, about 8:1 to about 1:8, about 7:1 to about 1:7, about 6:1 to about 1:6, about 5:1 to about 1:5, about 4:1 to about 1:4, about 3:1 to about 1:3, about 2:1 to about 1:2, or about 1:1. In certain embodiments, the asparagine-responsive active area may comprise a ratio of stabilizer to asparaginase and / or aspartate oxidase of about 5:1 to about 1:5. In certain embodiments, the asparagine-responsive active area may comprise a ratio of stabilizer to asparaginase and / or aspartate oxidase of about 4:1 to about 1:4. In certain embodiments, the asparagine-responsive active area may comprise a ratio of stabilizer to asparaginase and / or aspartate oxidase of about 3:1 to about 1:3. In certain embodiments, the asparagine-responsive active area may comprise a ratio of stabilizer to asparaginase and / or aspartate oxidase of about 2:1 to about 1:2. In certain embodiments, the asparagine-responsive active area may comprise a ratio of stabilizer to asparaginase and / or aspartate oxidase of about 1:1. In certain embodiments, the active area, e.g., an aspartate-responsive active area and / or an asparagine-responsive active area, may comprise from about 10% to about 50% by weight of a stabilizer, e.g., from about 15% to about 45% by weight, from about 20% to about 40% by weight, from about 20% to about 35% by weight, or from about 20% to about 30% by weight.In certain embodiments, the asparagine-responsive active area may comprise from about 15% to about 35% by weight of the stabilizing agent.

[0230] In certain embodiments, an analyte sensor of the present disclosure for indirect measurement of potassium is shown in FIG. 23. As shown in FIG. 23, the analyte sensor may include a potassium-dependent channel 701 and a potassium-independent channel 702. In certain embodiments, each channel is a working electrode. In certain embodiments, the potassium-dependent channel 701 includes an enzyme system including a potassium-dependent asparaginase. In certain embodiments, the potassium-independent channel 702 includes an enzyme system including a potassium-independent asparaginase. In certain embodiments, the potassium-dependent asparaginase is a wild-type form of asparaginase, and the potassium-independent asparaginase is a mutant form of the same asparaginase. In certain embodiments, the current signals obtained from the channels 701 and 702 can be compared to obtain the concentration of potassium ions in the sample.

[0231] In certain embodiments, an analyte sensor of the present disclosure for indirect measurement of potassium is shown in FIG. 24. As shown in FIG. 24, the analyte sensor may include a first potassium-dependent channel 801 and a second potassium-dependent channel 802. In certain embodiments, each channel is a working electrode. In certain embodiments, the first potassium-dependent channel 801 includes an enzyme system including a first potassium-dependent asparaginase. In certain embodiments, the second potassium-dependent channel 802 includes an enzyme system including a second potassium-dependent asparaginase. In such embodiments, the first potassium-dependent asparaginase and the second potassium-dependent asparaginase exhibit different dependencies on potassium and therefore produce different signals from each other in the presence of the same potassium concentration. In certain embodiments, the difference in signals obtained from channels 801 and 802 can be correlated to the concentration of potassium ions in the sample.

[0232] In certain embodiments, potassium ion concentration can be determined using an analyte sensor including the enzyme system of Figure 23 or Figure 24, as shown in Figures 25A and 25B. For example, and without limitation, in the absence of potassium ions, each of the channels (e.g., each of channels 801 and 802) has a linear response to increasing asparagine concentration, as shown in 25A, which can be described by the following equation:

[0233]

number

[0234]

number

[0235] where I1 and I2 correspond to the current output, C is the asparagine concentration, and k1 and k2 correspond to the slopes of the lines in the two channels. The two constants k1 and k2 can be obtained from a standard dose-response calibration in beakers with different levels of asparagine.

[0236] Figure 25B shows the sensor response in the presence of increasing potassium ion concentrations. As shown in Figure 25B, the current response is nonlinear and can be described by the following equation:

[0237]

number

[0238]

number

[0239] where I'1 and I'2 correspond to the current outputs and I 1-0 and I 2-0 corresponds to the sensor response when the potassium ion concentration is 0. Also note that:

[0240]

number

[0241] To determine the concentration of potassium ions in a sample, the system of equations 3-5 must be solved. This system is determined by the potassium concentration, I 1-0 , and I 2-0 It consists of three equations with three unknowns, including

[0242] In certain embodiments, the asparagine-responsive active area may comprise an asparaginase, e.g., a potassium-independent asparaginase or a potassium-dependent asparaginase. In certain embodiments, the asparagine-responsive active area may further comprise an aspartate oxidase, e.g., a potassium-independent aspartate oxidase. Alternatively, an asparagine-responsive active area comprising an asparaginase, e.g., a potassium-independent asparaginase or a potassium-dependent asparaginase, may be disposed on an aspartate-responsive active area, as described above in Section 2A.

[0243] In certain embodiments, the analyte sensor of the present disclosure may include a sensor tail including at least two working electrodes. In certain embodiments, an asparagine-responsive active area (e.g., a first asparagine-responsive active area) is disposed on a surface of a first working electrode, and an asparagine-responsive active area (e.g., a second asparagine-responsive active area) is disposed on a surface of a second working electrode, each asparagine-responsive active area including an enzyme system including aspartate oxidase and asparaginase. In certain embodiments, the first asparagine-responsive active area includes a potassium-independent asparaginase, and the second asparagine-responsive active area includes a potassium-dependent asparaginase. Alternatively, the first asparagine-responsive active area includes a first potassium-dependent asparaginase, and the second asparagine-responsive active area includes a second potassium-dependent asparaginase, and the asparaginases have different dependencies on potassium ion concentration.

[0244] In certain embodiments, the asparagine-responsive active area comprises a first enzyme layer comprising aspartate oxidase and a second layer comprising asparaginase disposed on the first enzyme layer. Alternatively or additionally, the aspartate oxidase and asparaginase are held within the same enzyme layer.

[0245] C. Active areas responsive to additional analytes In certain embodiments, the analyte sensor of the present disclosure may include an active area for detecting a second analyte. For example, but not limited to, the analyte sensor may be configured to detect potassium 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. In certain embodiments, such an analyte sensor may include a third working electrode configured to detect a different analyte by including an analyte-responsive active area disposed on a surface of the third working electrode.

[0246] In certain embodiments, the active sites present on the third working electrode of the analyte sensor of the present disclosure may include one or more enzymes that can be used to detect glucose. For example, without limitation, the analyte sensor of the present disclosure may include an active area including one or more enzymes for detecting glucose, disposed on, for example, the third working electrode. In certain embodiments, the analyte sensor may include active sites for detecting glucose that include glucose oxidase and / or glucose dehydrogenase.

[0247] In certain embodiments, the active sites present on the third working electrode of the analyte sensor of the present disclosure may include one or more enzymes that can be used to detect ketones. For example, without limitation, the analyte sensor of the present disclosure may include an active area including one or more enzymes, e.g., an enzyme system, for detecting ketones, disposed on, e.g., the third working electrode. In certain embodiments, the analyte sensor may include an active site for detecting ketones that includes β-hydroxybutyrate dehydrogenase. In certain embodiments, the analyte sensor may include an active site for detecting ketones that includes β-hydroxybutyrate dehydrogenase and diaphorase.

[0248] In certain embodiments, the active sites present on the third working electrode of the analyte sensor of the present disclosure may include one or more enzymes that can be used to detect lactate. For example, without limitation, the analyte sensor of the present disclosure may include an active area that includes one or more enzymes, e.g., an enzyme system, for detecting lactate, disposed, e.g., on the third working electrode. In certain embodiments, the analyte sensor may include active sites that include lactate dehydrogenase and / or lactate oxidase.

[0249] In certain embodiments, the active sites present on the third working electrode of the analyte sensor of the present disclosure may include one or more enzymes that can be used to detect alcohol. For example, without limitation, the analyte sensor of the present disclosure may include an active area that includes one or more enzymes, e.g., an enzyme system, for detecting alcohol, disposed, e.g., on the third working electrode. In certain embodiments, the analyte sensor may include an active site that includes alcohol dehydrogenase.

[0250] In certain embodiments, when a sensor is configured to detect two or more analytes on two different working electrodes, detection of each analyte may include applying a potential to each working electrode separately such that a separate signal is obtained from each analyte. The signal obtained from each analyte may then be correlated to the analyte concentration through the use of a calibration curve or function or by using a look-up table. In certain embodiments, the correlation of the analyte signal to the analyte concentration may be performed through the use of a processor.

[0251] 3. Redox mediators In certain embodiments, the analyte sensors disclosed herein may include an electron transfer agent. In certain embodiments, one or more active areas of the analyte sensors of the present disclosure may include an electron transfer agent. For example, without limitation, the analyte sensors disclosed herein may include two active areas, both of which include an electron transfer agent. Alternatively, the analyte sensors of the present disclosure may include two or more active areas, with only one active area including an electron transfer agent.

[0252] In certain embodiments, the analyte sensor of the present disclosure may include at least one aspartate-responsive active area that includes an electron transfer agent. In certain embodiments, the analyte sensor of the present disclosure may include at least one asparagine-responsive active area that includes an electron transfer agent. In certain embodiments, the analyte sensor of the present disclosure may include two aspartate-responsive active areas, each aspartate-responsive active area including an electron transfer agent. In certain embodiments, the analyte sensor of the present disclosure may include two asparagine-responsive active areas, each asparagine-responsive active area including an electron transfer agent. In certain embodiments, the electron transfer agents in the two asparagine-responsive active areas or the two aspartate-responsive active areas may be the same or different. In certain embodiments, the analyte sensor of the present disclosure may include two or more active areas, and only one active area, for example, the asparagine-responsive active area or the aspartate-responsive active area, includes an electron transfer agent.

[0253] A suitable electron transfer agent can facilitate the transfer of electrons to the adjacent working electrode after the analyte undergoes an enzymatic redox reaction in the corresponding active area, thereby generating a current indicative of the presence of that particular analyte, the amount of current generated being proportional to the amount of analyte present.

[0254] In certain embodiments, suitable electron transfer agents may include electroreducible or electrooxidizable ions, complexes, or molecules (e.g., quinones) that have redox potentials several hundred millivolts higher or lower than the redox potential of a standard calomel electrode (SCE). In certain embodiments, redox mediators may include osmium complexes and other transition metal complexes, such as those described in U.S. Pat. Nos. 6,134,461 and 6,605,200, which are incorporated herein by reference in their entireties. Further examples of suitable redox mediators include those described in U.S. Pat. Nos. 6,736,957, 7,501,053, and 7,754,093, the disclosures of each of which are also incorporated herein by reference in their entireties. Other examples of suitable redox mediators include metal compounds or complexes (including, for example, metallocene compounds thereof) of ruthenium, osmium, iron (e.g., polyvinylferrocene or hexacyanoferrate), or cobalt. Suitable ligands for metal complexes can also include bidentate or higher dentate ligands, such as, for example, bipyridine, biimidazole, phenanthroline, or pyridyl (imidazole). Other suitable bidentate ligands can include, for example, amino acids, oxalic acid, acetylacetone, diaminoalkanes, or o-diaminoarenes. Any combination of monodentate, bidentate, tridentate, tetradentate or higher ligands can be present in a 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.

[0255] In certain embodiments, the electron transfer agent disclosed herein may include suitable functional groups to facilitate covalent attachment to the polymer (also referred to herein as the polymer backbone) in the active area, as discussed further below. For example, without limitation, the electron transfer agent for use in the present disclosure may include a polymer-bound electron transfer agent. Suitable non-limiting examples of polymer-bound electron transfer agents include those described in U.S. Pat. Nos. 8,444,834, 8,268,143, and 6,605,201, the disclosures of which are incorporated herein by reference in their entirety. In certain embodiments, the electron transfer agent is a bidentate osmium complex bound to a polymer described herein. In certain embodiments, the electron transfer agent is a bidentate osmium complex bound to a polymer described herein, such as a polymer backbone described in Section 4 below. In certain embodiments, the polymer-bound electron transfer agent shown in FIG. 3 of U.S. Pat. No. 8,444,834 may be used in the sensor of the present disclosure.

[0256] In certain embodiments, an analyte sensor of the present disclosure for monitoring the level of potassium, e.g., potassium ions, may include a sensor tail including at least two or more working electrodes, e.g., a first working electrode and a second working electrode. In certain embodiments, a first asparagine-responsive active area including an asparaginase (e.g., a first asparaginase), an aspartate oxidase, and an electron transfer agent is disposed on a surface of the first working electrode. In certain embodiments, a second asparagine-responsive active area including an asparaginase (e.g., a second asparaginase), an aspartate oxidase, and an electron transfer agent is disposed on a surface of the second working electrode. In certain embodiments, the two asparaginases differ in potassium dependency. For example, but not limited to, the first asparaginase may be potassium-dependent and the second asparaginase may be potassium-independent. In certain embodiments, the first asparaginase may be potassium-independent and the second asparaginase may be potassium-dependent. In certain embodiments, the first and second asparaginases are both potassium dependent, but have different potassium dependencies. In certain embodiments, the signal measured from the two asparagine-responsive active areas can be correlated to the concentration of potassium ions in the sample being analyzed.

[0257] In certain embodiments, the asparagine-responsive active area is from about 100:1 to about 1:100, e.g., 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 , about 25:1 to about 1:25, about 20:1 to about 1:20, about 15:1 to about 1:15, about 10:1 to about 1:10, about 9:1 to about 1:9, about 8:1 to about 1:8, about 7:1 to about 1:7, about 6:1 to about 1:6, about 5:1 to about 1:5, about 4:1 to about 1:4, about 3:1 to about 1:3, about 2:1 to about 1:2, or about 1:1. In certain embodiments, the asparagine-responsive active area may comprise a ratio of aspartate oxidase and / or asparaginase to redox mediator of about 5:1 to about 1:5. In certain embodiments, the asparagine-responsive active area may comprise a ratio of aspartate oxidase and / or asparaginase to redox mediator of about 4:1 to about 1:4. In certain embodiments, the asparagine-responsive active area may comprise a ratio of aspartate oxidase and / or asparaginase to redox mediator of about 3:1 to about 1:3. In certain embodiments, the asparagine-responsive active area may comprise a ratio of aspartate oxidase and / or asparaginase to redox mediator of about 2:1 to about 1:2. In certain embodiments, the asparagine-responsive active area may comprise a ratio of aspartate oxidase and / or asparaginase to redox mediator of about 1:1.

[0258] In certain embodiments, an analyte sensor of the present disclosure for monitoring potassium, e.g., potassium ion levels, may include a sensor tail including at least two or more working electrodes, e.g., a first working electrode and a second working electrode. In certain embodiments, a first aspartate-responsive active area including an aspartate oxidase (e.g., a first aspartate oxidase) and an electron transfer agent (e.g., a first electron transfer agent) is disposed on a surface of the first working electrode. In certain embodiments, a second aspartate-responsive active area including an aspartate oxidase (e.g., a second aspartate oxidase) and an electron transfer agent (e.g., a second electron transfer agent) is disposed on a surface of the second working electrode. In certain embodiments, the two aspartate oxidases differ in potassium dependency. For example, but not limited to, the first aspartate oxidase may be potassium-dependent and the second aspartate oxidase may be potassium-independent. In certain embodiments, the first aspartate oxidase may be potassium independent and the second aspartate oxidase may be potassium dependent. In certain embodiments, the first and second aspartate oxidases are both potassium dependent, but have different potassium dependencies. In certain embodiments, the signal measured from the two aspartate responsive active areas may be correlated to the concentration of potassium ions in the sample being analyzed.

[0259] In certain embodiments, the aspartate-responsive active area is from about 100:1 to about 1:100, e.g., 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 ... The ratio of aspartate oxidase to redox mediator may be from about 0:1 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-responsive active area may comprise a ratio of aspartate oxidase to redox mediator of from about 5:1 to about 1:5. In certain embodiments, the aspartate-responsive active area may comprise a ratio of aspartate oxidase to redox mediator of about 4:1 to about 1:4. In certain embodiments, the aspartate-responsive active area may comprise a ratio of aspartate oxidase to redox mediator of about 3:1 to about 1:3. In certain embodiments, the aspartate-responsive active area may comprise a ratio of aspartate oxidase to redox mediator of about 2:1 to about 1:2. In certain embodiments, the aspartate-responsive active area may comprise a ratio of aspartate oxidase to redox mediator of about 1:1.

[0260] 4. Polymer backbone; In certain embodiments, one or more active sites for facilitating analyte detection may include a polymer to which an enzyme and / or a redox mediator is covalently attached. Any suitable polymer backbone may be present in the active area to facilitate detection of an analyte via covalent attachment of an enzyme and / or a redox mediator. Non-limiting examples of suitable polymers in the active area include polyvinylpyridine, e.g., poly(4-vinylpyridine) or poly(2-vinylpyridine), and polyvinylimidazole, e.g., poly(N-vinylimidazole) and poly(1-vinylimidazole), or copolymers thereof, e.g., quaternized pyridine groups serve as attachment points for redox mediators or enzymes. Exemplary copolymers that may be suitable for inclusion in the active area include those that contain monomer units such as, for example, styrene, acrylamide, methacrylamide, or acrylonitrile. 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. In certain embodiments, the polymer that may be present in the active area includes polyurethane or its copolymer, and / or polyvinylpyrrolidone. Further non-limiting examples of polymers that may be present in the active area include, but are not limited to, those described in U.S. Pat. No. 6,605,200, the entirety of which is incorporated herein by reference, such as poly(acrylic acid), styrene / maleic anhydride copolymer, methyl vinyl ether / maleic anhydride copolymer (GANTREZ polymer), poly(vinylbenzyl chloride), poly(allylamine), polylysine, poly(4-vinylpyridine) quaternized with carboxypentyl groups, and poly(sodium 4-styrenesulfonate). In certain embodiments in which the analyte sensor includes two active sites, the polymer in each active area may be the same or different.

[0261] In certain embodiments, when an enzyme system having multiple enzymes is present in a given active area, all of the multiple enzymes may be covalently bound to the polymer. In certain other embodiments, only a portion of the multiple enzymes are covalently bound to the polymer. For example, without limitation, one or more enzymes in the enzyme system may be covalently bound to the polymer and at least one enzyme may be non-covalently bound to the polymer such that the non-covalently bound enzyme is physically retained within the polymer. In certain embodiments, aspartate oxidase and / or asparaginase may be covalently bound to a polymer in an analyte-responsive active area of ​​a disclosed analyte sensor. In certain embodiments, aspartate oxidase may be covalently bound to a polymer in an asparagine-responsive active area of ​​a disclosed analyte sensor. In certain embodiments, asparaginase may be covalently bound to a polymer in an asparagine-responsive active area of ​​a disclosed analyte sensor. In certain embodiments, aspartate oxidase may be covalently bound to a polymer and asparaginase may be non-covalently bound to a polymer. In certain embodiments, 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, they may be physically retained within the asparagine-responsive active area. In certain embodiments, a membrane overcoating the asparagine-responsive active area may help retain one or more enzymes within the asparagine-responsive active area while still allowing sufficient inward diffusion of asparagine to allow detection of asparagine. Suitable membrane polymers for overcoating the analyte-responsive active area are further described herein.

[0262] In certain embodiments, when a stabilizing agent is present in the active area, one or more enzymes in the area may be covalently bound to the stabilizing agent. For example, but not limited to, one or more enzymes, such as aspartate oxidase and / or asparaginase, may be covalently bound to a stabilizing agent, such as albumin, present in the active area. In certain embodiments, aspartate oxidase present in the active area of ​​the present disclosure may be covalently bound to a stabilizing agent. In certain embodiments, asparaginase present in the active area of ​​the present disclosure may be covalently bound to a stabilizing agent.

[0263] In certain embodiments, the covalent bonding of one or more enzymes and / or redox mediators to the polymer and / or stabilizing agent in a given active area may occur via crosslinking introduced by a suitable crosslinking agent. In certain embodiments, crosslinking of the polymer and / or stabilizing agent to the one or more enzymes and / or redox mediators may reduce the occurrence of delamination of the enzyme composition from the electrode. Suitable crosslinking agents may include one or more crosslinkable functional groups, such as, but not limited to, vinyl, alkoxy, acetoxy, enoxy, oxime, amino, hydroxyl, cyano, halo, acrylate, epoxide, and isocyanate groups. In certain embodiments, the crosslinking agent includes one or more, two or more, three or more, or four or more epoxide groups. For example, but not limited to, crosslinking agents for use in the present disclosure may include mono-, di-, tri-, and tetra-ethylene oxide. In certain embodiments, crosslinkers for reaction with free amino groups in an enzyme (e.g., free side chain amines in lysine) can include crosslinkers such as, for example, polyethylene glycol dibutyl ether, polypropylene glycol dimethyl ether, polyalkylene glycol allyl methyl ether, polyethylene glycol diglycidyl ether (PEGDGE) or other polyepoxides, cyanuric chloride, N-hydroxysuccinimide, imidoesters, epichlorohydrin, or derivatized variants thereof. In certain embodiments, the crosslinker has an average molecular weight (M) of, for example, about 200 to 1,000, e.g., about 400. n) is PEGDGE. In certain embodiments, the crosslinker is PEGDGE400. In certain embodiments, the crosslinker can be glutaraldehyde. In certain embodiments, the crosslinking of the enzyme to the polymer is generally intermolecular. In certain embodiments, the crosslinking of the enzyme to the polymer is generally intramolecular.

[0264] In certain embodiments, the active area may comprise a ratio of crosslinker to one or more enzymes, e.g., aspartate oxidase, asparaginase, or both, of about 50:1 to about 1:50, e.g., about 45:1 to about 1:45, about 40:1 to about 1:40, about 35:1 to about 1:35, about 30:1 to about 1:30, about 25:1 to about 1:25, about 20:1 to about 1:20, about 15:1 to about 1:15, about 10:1 to about 1:10, about 9:1 to about 1:9, about 8:1 to about 1:8, about 7:1 to about 1:7, about 6:1 to about 1:6, about 5:1 to about 1:5, about 4:1 to about 1:4, about 3:1 to about 1:3, about 2:1 to about 1:2, or about 1:1. In certain embodiments, the active area may comprise a ratio of about 50:1 to about 1:50 of crosslinker to one or more enzymes, e.g., aspartate oxidase, asparaginase, or both. In certain embodiments, the active area may comprise a ratio of about 50:1 to about 30:1 of crosslinker to one or more enzymes, e.g., aspartate oxidase, asparaginase, or both. In certain embodiments, the active area may comprise a ratio of about 50:1 to about 1:1 of crosslinker to one or more enzymes, e.g., aspartate oxidase, asparaginase, or both. In certain embodiments, the active area may comprise a ratio of about 2:1 to about 1:2 of crosslinker to one or more enzymes, e.g., aspartate oxidase, asparaginase, or both. In certain embodiments, the active area may comprise a ratio of about 1:1 of crosslinker to one or more enzymes, e.g., aspartate oxidase, asparaginase, or both. In certain embodiments, the active area may comprise a ratio of about 5:1 to about 1:5 crosslinker to one or more enzymes, e.g., aspartate oxidase, asparaginase, or both. In certain embodiments, the active area may comprise a ratio of about 2:1 to about 1:2 crosslinker to one or more enzymes, e.g., aspartate oxidase, asparaginase, or both. In certain embodiments, the active area may comprise a ratio of about 1:1 crosslinker to one or more enzymes, e.g., aspartate oxidase, asparaginase, or both.In certain embodiments, the active area, e.g., the aspartate-responsive active area and / or the asparagine-responsive active area, may comprise from about 5% to about 20% by weight of crosslinker, e.g., from about 10% to about 20% by weight or from about 10% to about 15% by weight. In certain embodiments, the glutamate-responsive active area may comprise from about 10% to about 20% by weight of crosslinker.

[0265] 5. Mass-transfer limiting membrane In certain embodiments, the analyte sensors disclosed herein further comprise a membrane permeable to the analyte overcoating at least one active area, e.g., the first active area and / or the second active area, present on the working electrode of the analyte sensor. In certain embodiments, the analyte sensors disclosed herein further comprise a membrane permeable to potassium overcoating at least one active area, e.g., the first active area and / or the second active area, present on the working electrode of the analyte sensor.

[0266] In certain embodiments, the membrane overcoating the analyte-responsive active area can function as a mass transport limiting membrane and / or to improve biocompatibility. The mass transport limiting membrane can act as a diffusion limiting barrier to reduce the mass transport rate of the analyte. For example, but not limited to, using a mass transport limiting membrane to limit the access of the analyte (e.g., aspartate, asparagine, and / or potassium) to the analyte-responsive active area can help avoid sensor overload (saturation), thereby improving detection performance and accuracy.

[0267] In certain embodiments, the mass transport limiting membrane may be homogeneous and single-component (containing a single membrane polymer). Alternatively, the mass transport limiting membrane may be multi-component (containing two or more different membrane polymers). In certain embodiments, the multi-component membrane may exist as a multi-layer membrane or as a homogeneous blend of two or more membrane polymers. A homogeneous blend may be produced by combining two or more membrane polymers in a solution and then depositing the solution onto the working electrode (e.g., dip coating). A multi-layer membrane may be produced by sequentially depositing the membrane polymers onto the working electrode, e.g., by dip coating.

[0268] In certain embodiments, the mass transport limiting membrane may include two or more layers, for example, a bilayer or trilayer membrane. In certain embodiments, each layer may include a different polymer or the same polymer in different concentrations or thicknesses. In certain embodiments, the first analyte responsive active area may be covered by a multilayer membrane, for example, a bilayer membrane, and the second analyte responsive active area may be covered by a single membrane. In certain embodiments, the first analyte responsive active area may be covered by a multilayer membrane, for example, a bilayer membrane, and the second analyte responsive active area may be covered by a multilayer membrane, for example, a bilayer membrane. In certain embodiments, the first analyte responsive active area may be covered by a single membrane and the second analyte responsive active area may be covered by a multilayer membrane, for example, a bilayer membrane, and covered by a single membrane. In certain embodiments, the first analyte responsive active area may be covered by a single membrane and the second analyte responsive active area may be covered by a single membrane.

[0269] In certain embodiments, the composition of the mass transport limiting membrane disposed on an analyte sensor having two active areas may be the same or different when the mass transport limiting membrane overcoats each active area. For example, but not by way of limitation, the portion of the mass transport limiting membrane overcoating the asparagine-responsive active area or the aspartate-responsive active area used for the indirect measurement of potassium may be multicomponent and / or the portion of the mass transport limiting membrane overcoating the second analyte-responsive active area may be single component. Alternatively, the portion of the mass transport limiting membrane overcoating the asparagine-responsive active area or the aspartate-responsive active area used for the indirect measurement of potassium ions may be single component and / or the portion of the mass transport limiting membrane overcoating the second analyte-responsive active area may be multicomponent.

[0270] In certain embodiments, the mass transport limiting membrane may comprise a polymer containing a heterocyclic nitrogen group. In certain embodiments, the mass transport limiting membrane may comprise a polyvinylpyridine-based polymer. Non-limiting examples of polyvinylpyridine-based polymers are disclosed in U.S. Patent Application Publication No. 2003 / 0042137 (e.g., Formula 2b), the contents of which are incorporated herein by reference in their entirety.

[0271] In certain embodiments, the mass transport limiting membrane may comprise polyvinylpyridine (e.g., poly(4-vinylpyridine) or poly(4-vinylpyridine)), polyvinylimidazole, polyvinylpyridine copolymers (e.g., copolymers of vinylpyridine and styrene), polyacrylates, polyurethanes, polyetherurethanes, homopolymers, copolymers or terpolymers of polyurethanes, silicones, polytetrafluoroethylene, polyethylene-co-tetrafluoroethylene, polyolefins, polyesters, polycarbonates, biostable polytetrafluoroethylene, polypropylene, polyvinyl chloride, polyvinylidene difluoride, polybutylene terephthalate, polymethyl methacrylate, polyether ether ketone, cellulosic polymers, polysulfones and block copolymers thereof (including, for example, diblock, triblock, alternating, random and graft copolymers or chemically related materials, etc.).

[0272] In certain embodiments, a membrane for use in the present disclosure, e.g., a single-component membrane, may comprise polyvinylpyridine (e.g., poly(4-vinylpyridine) and / or poly(2-vinylpyridine)). In certain embodiments, a membrane for use in the present disclosure, e.g., a single-component membrane, may comprise poly(4-vinylpyridine). In certain embodiments, a membrane for use in the present disclosure, e.g., a single-component membrane, may comprise a copolymer of vinylpyridine and styrene. In certain embodiments, a membrane may comprise a polyvinylpyridine-co-styrene copolymer. For example, but not limited to, a polyvinylpyridine-co-styrene copolymer for use in the present disclosure may comprise a polyvinylpyridine-co-styrene copolymer in which a portion of the pyridine nitrogen atoms are functionalized with non-crosslinked polyethylene glycol tails and a portion of the pyridine nitrogen atoms are functionalized with alkylsulfonic acid, e.g., propylsulfonic acid groups. In certain embodiments, a derivatized polyvinylpyridine-co-styrene copolymer for use as a membrane polymer may be a 10Q5 polymer as described in U.S. Patent No. 8,761,857, the contents of which are incorporated herein by reference in their entirety. In certain embodiments, the polyvinylpyridine-based polymer has a molecular weight of about 50 Da to about 500 kDa.

[0273] Suitable copolymers of vinylpyridine and styrene may have a styrene content ranging from about 0.01% to about 50% mole percent, or from about 0.05% to about 45% mole percent, or from about 0.1% to about 40% mole percent, or from about 0.5% to about 35% mole percent, or from about 1% to about 30% mole percent, or from about 2% to about 25% mole percent, or from about 5% to about 20% mole percent. Substituted styrenes can be used in similar amounts as well. Suitable copolymers of vinylpyridine and styrene may have a molecular weight of 5 kDa or more, or about 10 kDa or more, or about 15 kDa or more, or about 20 kDa or more, or about 25 kDa or more, or about 30 kDa or more, or about 40 kDa or more, or about 50 kDa or more, or about 75 kDa or more, or about 90 kDa or more, or about 100 kDa or more. In non-limiting examples, suitable copolymers of vinylpyridine and styrene may have a molecular weight ranging from about 5 kDa to about 150 kDa, or from about 10 kDa to about 125 kDa, or from about 15 kDa to about 100 kDa, or from about 20 kDa to about 80 kDa, or from about 25 kDa to about 75 kDa, or from about 30 kDa to about 60 kDa.

[0274] In certain embodiments, the membrane may comprise polymers such as, but not limited to, poly(styrene co-maleic anhydride) crosslinked with poly(propylene glycol)-block-poly(ethylene glycol)-block-poly(propylene glycol) bis(2-aminopropyl ether), dodecylamine, and poly(propylene glycol)-block-poly(ethylene glycol)-block-poly(propylene glycol)(2-aminopropyl ether); poly(N-isopropylacrylamide); copolymers of poly(ethylene oxide) and poly(propylene oxide); or combinations thereof.

[0275] In certain embodiments, the membrane comprises a polyurethane membrane that includes both hydrophilic and hydrophobic regions. In certain embodiments, the hydrophobic polymer component is a polyurethane, polyurethane-urea, or poly(ether-urethane-urea). In certain embodiments, the polyurethane is a polymer produced by the condensation reaction of a diisocyanate with a difunctional hydroxyl-containing material. In certain embodiments, the polyurethane-urea is a polymer produced by the condensation reaction of a diisocyanate with a difunctional amine-containing material. In certain embodiments, diisocyanates for use herein include, for example, aliphatic diisocyanates containing about 4 to about 8 methylene units, or diisocyanates containing alicyclic moieties. Further non-limiting examples of polymers that may be used for the production of the membrane of the sensor of the present disclosure include vinyl polymers, polyethers, polyesters, polyamides, inorganic polymers (e.g., polysiloxanes and polycarbosiloxanes), natural polymers (e.g., cellulose and protein-based materials), and mixtures (e.g., blends or layered structures), or combinations thereof. In certain embodiments, the hydrophilic polymer component is polyethylene oxide and / or polyethylene glycol. In certain embodiments, the hydrophilic polymer component is a polyurethane copolymer. For example, and without limitation, a hydrophobic-hydrophilic copolymer component for use in the present disclosure is a polyurethane polymer containing about 10% to about 50%, for example 20%, of hydrophilic polyethylene oxide.

[0276] In certain embodiments, the membrane comprises a silicone polymer / hydrophobic-hydrophilic polymer blend.In certain embodiments, the hydrophobic-hydrophilic polymer for use in the blend can be any suitable hydrophobic-hydrophilic polymer, such as, but not limited to, polyvinylpyrrolidone, polyhydroxyethyl methacrylate, polyvinyl alcohol, polyacrylate, such as, for example, polyethers such as polyethylene glycol or polypropylene oxide, and copolymers thereof (including, for example, diblock copolymers, triblock copolymers, alternating copolymers, random copolymers, comb copolymers, star copolymers, dendritic copolymers, and graft copolymers).In certain embodiments, the hydrophobic-hydrophilic polymer is a copolymer of poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO). Non-limiting examples of copolymers of PEO and PPO include PEO-PPO diblock copolymers, PPO-PEO-PPO triblock copolymers, PEO-PPO-PEO triblock copolymers, alternating block copolymers of PEO-PPO, random copolymers of ethylene oxide and propylene oxide, and blends thereof. In certain embodiments, the copolymers may be substituted with hydroxy substituents.

[0277] In certain embodiments, hydrophilic or hydrophobic modifiers may be used to "fine tune" the permeability of the resulting membrane to the analyte of interest. In certain embodiments, hydrophilic modifiers (e.g., poly(ethylene) glycol, hydroxyl or polyhydroxyl modifiers, and the like, and any combination thereof) may be used to enhance the biocompatibility of the polymer or the resulting membrane.

[0278] In certain embodiments where multiple active areas are present, the mass transport limiting membrane may overcoat each active area, including the option of compositional variations on the different active areas, which can be accomplished by successive dip coating operations to produce portions of the bilayer membrane on the working electrode located closer to the sensor tip.

[0279] In certain embodiments where there are multiple active areas, a separate mass transport limiting membrane may overcoat each active area. For example, but not by way of limitation, a mass transport limiting membrane may be disposed on a first active area, e.g., an aspartate-responsive active area or an asparagine-responsive active area, and a separate second mass transport limiting membrane may overcoat the second active area. In certain embodiments, the two mass transport limiting membranes are spatially separated and do not overlap each other. In certain embodiments, the first mass transport limiting membrane does not overlap the second mass transport limiting membrane, and the second mass transport limiting membrane does not overlap the first mass transport limiting membrane. Alternatively, the second mass transport limiting membrane overlaps the first mass transport limiting membrane. In certain embodiments, the first mass transport limiting membrane comprises a different polymer than the second mass transport limiting membrane. Alternatively, the first mass transport limiting membrane comprises the same polymer as the second mass transport limiting membrane. In certain embodiments, the first mass transport limiting membrane comprises the same polymer as the second mass transport limiting membrane, but a different crosslinker.

[0280] In certain embodiments of the present disclosure, the asparagine-responsive active area used for the indirect measurement of potassium ions may be overcoated with a multi-component membrane comprising polyvinylpyridine and polyvinylpyridine-co-styrene copolymer, either as a bilayer membrane or a homogeneous blend, and the second analyte-responsive active area may be overcoated with a single-component membrane comprising polyvinylpyridine or polyvinylpyridine-co-styrene copolymer. Alternatively, the asparagine-responsive active area used for the indirect measurement of potassium ions may be overcoated with a single-component membrane comprising polyvinylpyridine or polyvinylpyridine-co-styrene copolymer, and the second analyte-responsive active area may be overcoated with a multi-component membrane comprising polyvinylpyridine and polyvinylpyridine-co-styrene copolymer, either as a bilayer membrane or a homogeneous blend.

[0281] In certain embodiments of the present disclosure, the aspartate-responsive active area used for the indirect measurement of potassium ions may be overcoated with a multi-component membrane comprising polyvinylpyridine and polyvinylpyridine-co-styrene copolymer, either as a bilayer membrane or a homogeneous blend, and the second analyte-responsive active area may be overcoated with a single-component membrane comprising polyvinylpyridine or polyvinylpyridine-co-styrene copolymer. Alternatively, the aspartate-responsive active area used for the indirect measurement of potassium ions may be overcoated with a single-component membrane comprising polyvinylpyridine or polyvinylpyridine-co-styrene copolymer, and the second analyte-responsive active area may be overcoated with a multi-component membrane comprising polyvinylpyridine and polyvinylpyridine-co-styrene copolymer, either as a bilayer membrane or a homogeneous blend.

[0282] Polydimethylsiloxane (PDMS) can be incorporated into any of the mass transport limiting membranes disclosed herein. In certain embodiments, the mass transport limiting membrane may comprise a membrane polymer crosslinked with a crosslinking agent as disclosed herein and in section 4 above. In certain embodiments where there are two mass transport limiting membranes (e.g., a first mass transport limiting membrane and a second mass transport limiting membrane), each membrane may be crosslinked with a different crosslinking agent. For example, but not by way of limitation, the crosslinking agent may result in a membrane that is more restrictive to the diffusion of a particular compound, e.g., an analyte within the membrane, or less restrictive to the diffusion of a particular compound, e.g., by affecting the size of the pores within the membrane. For example, but not by way of limitation, in a sensor configured to detect potassium, the mass transport limiting membrane overcoating the analyte responsive area may have a pore size that limits the diffusion of compounds larger than potassium through the membrane.

[0283] In certain embodiments, crosslinkers for use in the present disclosure may include polyepoxides, carbodiimides, cyanuric chloride, triglycidyl glycerol, N-hydroxysuccinimide, imidoesters, epichlorohydrin, or derivatized variants thereof. In certain embodiments, the membrane polymer overcoating one or more active areas may be crosslinked with a branched crosslinker, which may, for example, reduce the amount of extractables obtained from the mass transport limiting membrane. Non-limiting examples of branched crosslinkers include branched glycidyl ether crosslinkers, for example, branched glycidyl ether crosslinkers that contain two or more crosslinkable groups. In certain embodiments, the branched crosslinker may contain two or more crosslinkable groups, such as polyethylene glycol diglycidyl ether. In certain embodiments, the branched crosslinker may contain three or more crosslinkable groups, such as polyethylene glycol tetraglycidyl ether. In certain embodiments, the mass transport limiting membrane may comprise polyvinylpyridine or a copolymer of vinylpyridine and styrene crosslinked with a branched glycidyl ether crosslinker containing two or three crosslinkable groups, such as polyethylene glycol tetraglycidyl ether or polyethylene glycol diglycidyl ether. In certain embodiments, the epoxide groups of the polyepoxide, e.g., polyethylene glycol tetraglycidyl ether or polyethylene glycol diglycidyl ether, may form covalent bonds with pyridine or imidazole via epoxide ring opening, resulting in hydroxyalkyl groups bridging the body of the crosslinker to the heterocycle of the membrane polymer.

[0284] In certain embodiments, the crosslinker is polyethylene glycol diglycidyl ether (PEGDGE). In certain embodiments, the PEGDGE used to facilitate crosslinking (e.g., intermolecular crosslinking) between two or more membrane polymer backbones may exhibit a wide range of suitable molecular weights. In certain embodiments, the molecular weight of the PEGDGE may range from about 100 g / mol to about 5,000 g / mol. The number of ethylene glycol repeat units in each arm of the PEGDGE may be the same or different, and typically may vary over a range within a given sample to give an average molecular weight. In certain embodiments, the PEGDGE for use in the present disclosure has an average molecular weight (M) of about 200 to 1,000, e.g., about 400. n In certain embodiments, the crosslinker is PEGDGE400.

[0285] In certain embodiments, the polyethylene glycol tetraglycidyl ether used to facilitate crosslinking (e.g., intermolecular crosslinking) between two or more membrane polymer backbones may exhibit a wide range of suitable molecular weights. Up to four polymer backbones may be crosslinked with a single molecule of polyethylene glycol tetraglycidyl ether crosslinker. In certain embodiments, the molecular weight of the polyethylene glycol tetraglycidyl ether may range from about 1,000 g / mol to about 5,000 g / mol. The number of ethylene glycol repeat units in each arm of the polyethylene tetraglycidyl ether may be the same or different, and typically may vary over a range within a given sample to give an average molecular weight. In certain embodiments, the mass transport limiting membrane may be deposited directly on the active area.

[0286] In certain other embodiments, the membrane polymer overcoating one or more active areas may be crosslinked with a branched crosslinker containing three or more crosslinkable groups (e.g., polyethylene glycol tetraglycidyl ether), which, as described above, may reduce the amount of extractables that may be obtained from the mass transport limiting membrane. In certain embodiments, the mass transport limiting membrane may comprise a copolymer of vinylpyridine and styrene crosslinked with a branched glycidyl ether crosslinker containing three crosslinkable groups, such as polyvinylpyridine or polyethylene glycol tetraglycidyl ether. In certain embodiments, the epoxide group of the polyethylene glycol tetraglycidyl ether may form a covalent bond with pyridine or imidazole via epoxide ring opening, resulting in a hydroxyalkyl group that bridges the body of the crosslinker to the heterocycle of the membrane polymer.

[0287] In certain embodiments, an analyte sensor described herein for monitoring levels of potassium, e.g., potassium ions, may include a sensor tail including at least one working electrode, an asparagine-responsive active area disposed on a surface of the working electrode, and a mass transport limiting membrane permeable to asparagine and potassium overcoating at least a first active area. In certain embodiments, the asparagine-responsive active area includes asparaginase, aspartate oxidase, an electron transfer agent, and optionally a polymer. In certain embodiments, one or both enzymes are covalently attached to the polymer.

[0288] In certain embodiments, an analyte sensor of the present disclosure for monitoring potassium, e.g., potassium ion levels, may include a sensor tail including at least two or more working electrodes, e.g., a first working electrode and a second working electrode. In certain embodiments, a first asparagine-responsive active area including asparaginase, aspartate oxidase, and an electron transfer agent is disposed on a surface of the first working electrode. In certain embodiments, a second asparagine-responsive active area including asparaginase, aspartate oxidase, and an electron transfer agent is disposed on a surface of the second working electrode. In certain embodiments, the analyte sensor further includes a mass transport limiting membrane permeable to asparagine and / or potassium overcoating at least one of the asparagine-responsive active areas. In certain embodiments, a mass transport limiting membrane permeable to asparagine and / or potassium overcoats both asparagine-responsive active areas. Alternatively, a first mass transport limiting membrane permeable to asparaginase overcoats the first asparagine-responsive active area, and a second mass transport limiting membrane permeable to asparaginase overcoats the second asparagine-responsive active area. In certain embodiments, the first mass transport limiting membrane and the second mass transport limiting membrane comprise the same polymer. Alternatively, the first mass transport limiting membrane and the second mass transport limiting membrane comprise different polymers.

[0289] In certain embodiments, an analyte sensor described herein for monitoring levels of potassium, e.g., potassium ions, may include a sensor tail including at least one working electrode, an aspartate-responsive active area disposed on a surface of the working electrode, and a mass transport limiting membrane permeable to aspartate and potassium overcoating at least a first active area. In certain embodiments, the aspartate-responsive active area includes aspartate oxidase, an electron transfer agent, and optionally a polymer. In certain embodiments, the enzyme is covalently bound to the polymer.

[0290] In certain embodiments, an analyte sensor of the present disclosure for monitoring the level of potassium, e.g., potassium ions, may include a sensor tail including at least two or more working electrodes, e.g., a first working electrode and a second working electrode, each working electrode having an aspartate-responsive active area. For example, but not limited to, a first aspartate-responsive active area including aspartate oxidase and an electron transfer agent is disposed on a surface of the first working electrode. In certain embodiments, a second aspartate-responsive active area including aspartate oxidase and an electron transfer agent is disposed on a surface of the second working electrode. In certain embodiments, the analyte sensor further includes a mass transport limiting membrane permeable to aspartate and / or potassium overcoating at least one of the aspartate-responsive active areas. In certain embodiments, a mass transport limiting membrane permeable to aspartate and / or potassium overcoats both aspartate-responsive active areas. Alternatively, a first mass transport limiting membrane permeable to aspartate and / or potassium overcoats the first aspartate-responsive active area, and a second mass transport limiting membrane permeable to aspartate and / or potassium overcoats the second aspartate-responsive active area. In certain embodiments, the first mass transport limiting membrane and the second mass transport limiting membrane comprise the same polymer. Alternatively, the first mass transport limiting membrane and the second mass transport limiting membrane comprise different polymers.

[0291] In certain embodiments, an analyte sensor of the present disclosure for monitoring potassium, e.g., potassium ion levels, may include a sensor tail including at least three or more working electrodes, e.g., a first working electrode, a second working electrode, and a third working electrode, where two of the three working electrodes are configured to detect different analytes. In certain embodiments, a first aspartate-responsive active area including a potassium-dependent aspartate oxidase and an electron transfer agent is disposed on a surface of the first working electrode. In certain embodiments, a second aspartate-responsive active area including a potassium-independent aspartate oxidase and an electron transfer agent is disposed on a surface of the second working electrode. In certain embodiments, an additional analyte-responsive active area including an enzyme for detecting a second analyte is disposed on a surface of the third working electrode. In certain embodiments, the analyte sensor further includes a mass transport limiting membrane that is permeable to aspartate and / or potassium overcoating at least one, e.g., both, of the aspartate-responsive active areas. In certain embodiments, a mass transport limiting membrane that is permeable to aspartate and / or potassium overcoats one or more aspartate-responsive active areas and a second analyte-responsive active area. Alternatively, a first mass transport limiting membrane that is permeable to aspartate and / or potassium overcoats at least one, e.g., both, of the aspartate-responsive active areas, and a second mass transport limiting membrane that is permeable to the second analyte overcoats the second analyte-responsive active area. In certain embodiments, a second mass transport limiting membrane that is permeable to the second analyte overcoats at least a portion of at least one or both of the aspartate-responsive active areas.

[0292] In certain embodiments, an analyte sensor of the present disclosure for monitoring potassium, e.g., potassium ion levels, may include a sensor tail including at least three or more working electrodes, e.g., a first working electrode, a second working electrode, and a third working electrode, where two of the three working electrodes are configured to detect different analytes. In certain embodiments, a first asparagine-responsive active area including aspartate oxidase, potassium-dependent asparaginase, and an electron transfer agent is disposed on a surface of the first working electrode. In certain embodiments, a second asparagine-responsive active area including aspartate oxidase, potassium-independent asparaginase, and an electron transfer agent is disposed on a surface of the second working electrode. In certain embodiments, an additional analyte-responsive active area including an enzyme for detecting a second analyte is disposed on a surface of the third working electrode. In certain embodiments, the analyte sensor further includes a mass transport limiting membrane that is permeable to asparagine and / or potassium overcoating at least one, e.g., both, of the asparagine-responsive active areas. In certain embodiments, a mass transport limiting membrane that is permeable to asparagine and / or potassium overcoats one or more asparagine-responsive active areas and a second analyte-responsive active area. Alternatively, a first mass transport limiting membrane that is permeable to asparagine and / or potassium overcoats at least one, e.g., both, of the asparagine-responsive active areas, and a second mass transport limiting membrane that is permeable to the second analyte overcoats the second analyte-responsive active area. In certain embodiments, a second mass transport limiting membrane that is permeable to the second analyte overcoats at least a portion of at least one or both of the asparagine-responsive active areas.

[0293] In certain embodiments, the mass transport limiting membrane has a thickness, e.g., a dry thickness, in the range of about 0.1 μm to about 1,000 μm, e.g., about 1 μm to about 500 μm, about 10 μm to about 100 μm, or about 10 μm to about 100 μm. In certain embodiments, the mass transport limiting membrane may have a thickness of about 0.1 μm to about 100 μm, e.g., about 1 μm to about 90 μm, about 1 μm to about 80 μm, about 1 μm to about 70 μm, about 1 μm to about 60 μm, about 1 μm to about 50 μm, about 1 μm to about 40 μm, about 1 μm to about 30 μm, about 1 μm to about 20 μm, about 0.5 μm to about 10 μm, about 1 μm to about 10 μm, about 1 μm to about 5 μm, or about 0.1 μm to about 5 μm. In certain embodiments, the mass transport limiting membrane may have a thickness of about 1 μm to about 100 μm. In certain embodiments, the sensor may be immersed in the mass transport limiting membrane solution more than once. For example, but not by way of limitation, the sensor (or working electrode) of the present disclosure may be immersed in the mass transport limiting membrane solution at least two times, at least three times, at least four times, or at least five times to obtain the desired mass transport limiting membrane thickness.

[0294] 6. Interference Domains In certain embodiments, the sensor of the present disclosure, e.g., the sensor tail, may further include an interference domain. In certain embodiments, the interference domain may include, for example, a polymer domain that restricts the flow of one or more interferents to the surface of the working electrode. In certain embodiments, the interference domain may function as a molecular sieve that allows the passage of the analyte and other substances measured by the working electrode while preventing the passage of other substances, such as interferents. In certain embodiments, the interferents may affect the signal obtained at the working electrode. Non-limiting examples of interferents include acetaminophen, ascorbate, ascorbic acid, bilirubin, cholesterol, creatinine, dopamine, ephedrine, ibuprofen, L-dopa, methyldopa, salicylate, tetracycline, tolazamide, tolbutamide, triglycerides, urea, and uric acid.

[0295] In certain embodiments, the interference domain is located between the working electrode and one or more active areas, such as the asparagine-responsive active area or the aspartate-responsive active area. In certain embodiments, non-limiting examples of polymers that can be used in the interference domain include polyurethanes, polymers with pendant ionic groups, and polymers with controlled pore size. In certain embodiments, the interference domain is formed from one or more cellulose derivatives. Non-limiting examples of cellulose derivatives include polymers such as cellulose acetate, cellulose acetate butyrate, 2-hydroxyethyl cellulose, cellulose acetate phthalate, cellulose acetate propionate, and cellulose acetate trimellitate.

[0296] In certain embodiments, the interference domain is part of the mass transport limiting membrane and is not a separate membrane. In certain embodiments, the interference domain is disposed between the mass limiting membrane and one or more analyte responsive active areas.

[0297] In certain embodiments, the interference domain comprises a thin, hydrophobic membrane that is non-swelling and limits the diffusion of high molecular weight species, for example, and without limitation, the interference domain can be permeable to relatively low molecular weight substances, but restrict the passage of higher molecular weight substances.

[0298] In certain embodiments, the interference domain may 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, e.g., a dry thickness, in the range of about 0.1 μm to about 1,000 μm, for example, about 1 μm to about 500 μm, about 10 μm to about 100 μm, or about 10 μm to about 100 μm. In certain embodiments, the interference domain may have a thickness of about 0.1 μm to about 100 μm, e.g., about 1 μm to about 90 μm, about 1 μm to about 80 μm, about 1 μm to about 70 μm, about 1 μm to about 60 μm, about 1 μm to about 50 μm, about 1 μm to about 40 μm, about 1 μm to about 30 μm, about 1 μm to about 20 μm, about 0.5 μm to about 10 μm, about 1 μm to about 10 μm, about 1 μm to about 5 μm, or about 0.1 μm to about 5 μm. In certain embodiments, the sensor may be immersed in the interference domain solution more than once. For example, but not limited to, a sensor (or working electrode) of the present disclosure may be immersed in the interference domain solution at least two times, at least three times, at least four times, or at least five times to obtain the desired interference domain thickness.

[0299] 7.Manufacturing The present disclosure further provides a method of manufacturing an analyte sensor of the present disclosure comprising one or more active sites. In certain embodiments, the method includes screen printing a working electrode, e.g., a carbon working electrode, for example, by using a carbon ink.

[0300] In certain embodiments, the method may further include adding a composition comprising an enzyme onto the surface of the working electrode to generate an analyte-responsive active area on the working electrode. For example, without limitation, the composition may include an aspartate oxidase, e.g., potassium-dependent aspartate oxidase or potassium-independent aspartate oxidase. In certain embodiments, the composition may further include an asparaginase, e.g., potassium-dependent asparaginase or potassium-independent asparaginase. In certain embodiments, the composition may further include a redox mediator. In certain embodiments, the composition may further include a crosslinker, e.g., polyethylene glycol diglycidyl ether, and a stabilizer, e.g., albumin, such as BSA. In certain embodiments, the method may further include curing the enzyme composition.

[0301] Alternatively, a first enzyme composition comprising aspartate oxidase may be first deposited on the surface of the working electrode to generate an analyte-responsive active area on the working electrode. In certain embodiments, the first enzyme composition may further comprise a redox mediator, a cross-linking agent, e.g., polyethylene glycol diglycidyl ether, and / or a stabilizing agent, e.g., albumin, such as BSA. In certain embodiments, the method may include curing the first enzyme composition to generate the first enzyme layer. In certain embodiments, the method may include depositing a second enzyme composition comprising asparaginase on the surface of the first enzyme layer and curing the second enzyme composition to generate the first second layer. In certain embodiments, the second enzyme composition may comprise a cross-linking agent, e.g., polyethylene glycol diglycidyl ether, and / or a stabilizing agent, e.g., albumin, such as BSA.

[0302] In an analyte sensor having two working electrodes, a second analyte-responsive active area may be generated on the second working electrode using the methods described herein. In certain embodiments, in an analyte sensor having two working electrodes, a second aspartate-responsive active area or a second asparagine-responsive active area may be generated on the second working electrode using the methods described herein. In certain embodiments, the first aspartate-responsive active area comprises potassium-independent aspartate oxidase and the second aspartate-responsive active area comprises potassium-dependent aspartate oxidase. In certain embodiments, the first asparagine-responsive active area comprises potassium-independent asparaginase and the second asparagine-responsive active area comprises potassium-dependent asparaginase.

[0303] In an analyte sensor having three working electrodes, an active area responsive to a second analyte may be generated on the third working electrode using the methods described herein. Non-limiting examples of the second analyte may be glucose, ketones, lactate, oxygen, hemoglobin A1C, albumin, alcohol, alkaline phosphatase, alanine transaminase, aspartate aminotransferase, bilirubin, blood urea nitrogen, calcium, carbon dioxide, chloride, creatinine, hematocrit, lactate, magnesium, oxygen, pH, phosphorus, potassium, sodium, aspartate, asparagine, total protein, uric acid, hormones such as cortisol, and the like.

[0304] In certain embodiments, the method may further include adding a film composition onto the hardened enzyme composition. In certain embodiments, the film composition may include a polymer, such as a polyvinylpyridine-based polymer, such as polyvinylpyridine, and / or a cross-linking agent, such as polyethylene glycol diglycidyl ether. In certain embodiments, the method may include hardening the polymer composition.

[0305] In general, the thickness of the membrane is controlled by the concentration of the membrane solution, the number of droplets of membrane solution applied, the number of times the sensor is dipped into the membrane solution or sprayed with the membrane solution, the amount of membrane solution sprayed onto the sensor, and the like, and any combination of these factors. In certain embodiments, the membranes described herein may have a thickness ranging from about 0.1 micrometers (μm) to about 1,000 μm, e.g., from about 1 μm to about 500 μm, from about 10 μm to about 100 μm, or from about 10 μm to about 100 μm. In certain embodiments, the sensor may be dipped into the membrane solution more than once. For example, but not limited to, the sensor (or working electrode) of the present disclosure may be dipped into the membrane solution at least two times, at least three times, at least four times, or at least five times to obtain the desired membrane thickness.

[0306] In certain embodiments, the membrane may cover one or more active areas, and in certain embodiments, the active areas may have a thickness of about 0.1 μm to about 100 μm, e.g., about 1 μm to about 90 μm, about 1 μm to about 80 μm, about 1 μm to about 70 μm, about 1 μm to about 60 μm, about 1 μm to about 50 μm, about 1 μm to about 40 μm, about 1 μm to about 30 μm, about 1 μm to about 20 μm, about 0.5 μm to about 10 μm, about 1 μm to about 10 μm, about 1 μm to about 5 μm, or about 0.1 μm to about 5 μm. In certain embodiments, a series of droplets may be applied on top of each other to achieve a desired thickness of the active areas and / or membrane without substantially increasing the diameter of the droplets applied (i.e., while maintaining the desired diameter or range). In certain embodiments, each single drop may be applied and then cooled or dried, followed by application of one or more additional drops, for example, but not limited to, at least one drop, at least two drops, at least three drops, at least four drops, or at least five drops added on top of each other to achieve a desired thickness of the active area.

[0307] III.How to use The present disclosure further provides a method of using the analyte sensor disclosed herein. In certain embodiments, the present disclosure provides a method for monitoring the level of potassium, e.g., potassium ions, in a sample. For example, but not limited to, a subject in need of potassium monitoring may be a subject at risk of developing or has developed one or more disorders and / or conditions associated with dysregulated levels of potassium as described herein. In certain embodiments, a subject in need of potassium monitoring may be a subject at risk of developing or has developed one or more disorders and / or conditions associated with potassium deficiency as described herein. In certain embodiments, a subject in need of potassium monitoring may be a subject at risk of developing or has developed one or more disorders and / or conditions associated with elevated levels of potassium as described herein.

[0308] In certain embodiments, the potassium sensor of the present disclosure can be used to continuously monitor potassium levels in subjects at risk for or having a neurological disorder, such as Parkinson's disease, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis or Lou Gehrig's disease (ALS), and multiple sclerosis (MS). Further examples of diseases and disorders associated with potassium dysregulation include autism, cancer, heart failure, myocardial infarction, and atherosclerosis as disclosed in Udensi and Tchounwou, Int. JClin. xp. Physiol. 4(3):111-122 (2017), the contents of which are incorporated herein by reference in their entirety.

[0309] In certain embodiments, the potassium sensor of the present disclosure may be implanted in a subject at risk of developing or having one or more disorders and / or conditions associated with potassium deficiency. In certain embodiments, the potassium sensor of the present disclosure may be implanted in a subject for up to about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, or about 20 days. In certain embodiments, the potassium sensor of the present disclosure may be implanted in a subject for up to about 15 days.

[0310] In certain embodiments, a method for monitoring potassium includes the steps of: (i) providing an analyte sensor comprising: (a) a sensor tail including at least a first working electrode and a second electrode; (b) a first asparagine-responsive active area disposed on a surface of the first working electrode and including an enzyme system including aspartate oxidase and a first asparaginase and optionally a first polymer; (c) a second asparagine-responsive active area disposed on a surface of the second working electrode and including an enzyme system including aspartate oxidase and a second asparaginase and optionally a second polymer; and (d) an asparagine-permeable mass transport limiting membrane overcoating the first and / or second asparagine-responsive active areas. In certain embodiments, the method further comprises (ii) applying a potential to the first working electrode, (iii) acquiring a first signal at or above the redox potential of the first asparagine-responsive active area, (iv) applying a potential to the second working electrode, (v) acquiring a second signal at or above the redox potential of the second asparagine-responsive active area, and (vi) correlating the first and second signals to the concentration of potassium, e.g., potassium ions, in the fluid. In certain embodiments, the first and / or second asparagine-responsive active area further comprises an electron transfer agent. In certain embodiments, the asparaginase of the first asparagine-responsive active area is potassium-dependent and the asparaginase of the second asparagine-responsive active area is potassium-independent. Alternatively, both asparaginases are potassium-dependent but exhibit different potassium dependencies.

[0311] In certain embodiments, a method of the present disclosure for monitoring potassium may include (i) exposing an analyte sensor comprising: (a) a sensor tail comprising at least a first working electrode and a second working electrode; (b) a first asparagine-responsive active area disposed on a surface of the first working electrode and comprising an enzyme system comprising aspartate oxidase and asparaginase and optionally a first polymer; (c) a second aspartate-responsive active area disposed on a surface of the second working electrode and comprising an enzyme system comprising aspartate oxidase and asparaginase and optionally a second polymer; and (d) an asparagine-permeable mass transport limiting membrane overcoating the first and / or second asparagine-responsive active areas to a fluid comprising potassium ions and asparagine. In certain embodiments, the method further comprises (ii) applying a potential to the first and second working electrodes, (iii) acquiring a first signal at or above the redox potential of the first asparagine-responsive active area, (iv) acquiring a second signal at or above the redox potential of the second asparagine-responsive active area, and (v) correlating the first and second signals to the concentration of potassium ions in the fluid. In certain embodiments, the first and / or second asparagine-responsive active area further comprises an electron transfer agent. In certain embodiments, the asparaginase of the first asparagine-responsive active area is potassium-dependent and the asparaginase of the second asparagine-responsive active area is potassium-independent. Alternatively, both asparaginases are potassium-dependent but exhibit different potassium dependencies.

[0312] In certain embodiments, a method for monitoring potassium includes providing an analyte sensor including: (i) (a) a sensor tail including at least a first working electrode and a second electrode; (b) a first aspartate-responsive active area disposed on a surface of the first working electrode and including a first aspartate oxidase (e.g., a potassium-dependent aspartate oxidase) and optionally a first polymer; (c) a second aspartate-responsive active area disposed on a surface of the second working electrode and including a second aspartate oxidase (e.g., a potassium-independent aspartate oxidase) and optionally a second polymer; and (d) an aspartate-permeable mass transport limiting membrane overcoating the first and / or second aspartate-responsive active areas. In certain embodiments, the method further comprises (ii) applying a potential to the first working electrode, (iii) acquiring a first signal at or above the redox potential of the first aspartate-responsive active area, (iv) applying a potential to the second working electrode, (v) acquiring a second signal at or above the redox potential of the second aspartate-responsive active area, and (vi) correlating the first and second signals to the concentration of potassium, e.g., potassium ions, in the fluid. In certain embodiments, the first and / or second aspartate-responsive active area further comprises an electron transfer agent. In certain embodiments, the aspartate oxidase of the first aspartate-responsive active area is potassium-dependent and the aspartate oxidase of the second aspartate-responsive active area is potassium-independent. Alternatively, both aspartate oxidases are potassium-dependent but exhibit different potassium dependencies. In certain embodiments, the membrane polymer comprises polyvinylpyridine or polyvinylimidazole. In certain embodiments, the membrane polymer comprises a copolymer of vinylpyridine and styrene. In certain embodiments, the mass transport limiting membrane of the analyte sensor comprises a membrane polymer crosslinked with a branched crosslinker comprising three or more crosslinkable groups. In certain embodiments, the branched crosslinker comprises polyethylene glycol tetraglycidyl ether.

[0313] In certain embodiments, a method of the present disclosure for monitoring potassium may include (i) exposing an analyte sensor comprising: (a) a sensor tail comprising at least a first working electrode and a second working electrode; (b) a first aspartate-responsive active area disposed on a surface of the first working electrode and comprising a first aspartate oxidase and optionally a first polymer; (c) a second aspartate-responsive active area disposed on a surface of the second working electrode and comprising a second aspartate oxidase and optionally a second polymer; and (d) a mass transport limiting membrane permeable to aspartate and potassium ions overcoating the first and / or second aspartate-responsive active areas to a fluid comprising potassium ions and aspartate. In certain embodiments, the method further comprises (ii) applying a potential to the first and second working electrodes; (iii) acquiring a first signal at or above the redox potential of the first aspartate-responsive active area, e.g., proportional to the concentration of aspartate in the fluid; (iv) acquiring a second signal at or above the redox potential of the second aspartate-responsive active area, e.g., proportional to the concentration of aspartate in the fluid; and (v) correlating the first and second signals to the concentration of potassium ions in the fluid. In certain embodiments, the first and / or second aspartate-responsive active area further comprises an electron transfer agent. In certain embodiments, the aspartate oxidase of the first aspartate-responsive active area is potassium-dependent and the aspartate oxidase of the second aspartate-responsive active area is potassium-independent. Alternatively, both aspartate oxidases are potassium-dependent but exhibit different potassium dependencies.

[0314] In certain embodiments, the present disclosure further provides a method for detecting potassium and a second analyte. For example, but not limited to, the method of the present disclosure may further include providing an analyte sensor including an active area and / or detecting a second analyte by exposing an analyte sensor including an active area to a fluid, e.g., a body fluid, including aspartate, potassium, and a second analyte. In certain embodiments, an analyte sensor for use in a method for detecting potassium and a second analyte may further include a third working electrode and an active area disposed on a surface of the third working electrode and responsive to a second analyte different from the first analyte, the third active area including at least one enzyme responsive to the second analyte and optionally a second polymer and / or an electron transfer agent, and a portion, e.g., a second portion, of the mass transport limiting membrane overcoats the active area. Alternatively, the active areas may be overcoated with a second mass transport limiting membrane that is separate and / or different from the mass transport limiting membrane that overcoats at least one of the aspartate-responsive active areas. In certain embodiments, the second mass transport limiting membrane may overcoat a portion of at least one of the aspartate-responsive active areas.

[0315] IV. Illustrative Embodiments A. In certain non-limiting embodiments, the subject matter of the present disclosure comprises: (i) a sensor tail including at least a first working electrode; (ii) a first analyte responsive active area comprising a first aspartate oxidase disposed on a surface of the working electrode; and The present invention provides an analyte sensor for detecting potassium levels, comprising:

[0316] A1. The analyte sensor of A, further comprising a first mass transport limiting membrane, permeable to potassium, overcoating the first analyte responsive active area. A2. The analyte sensor of A or A1, wherein the first aspartate oxidase is a potassium-dependent aspartate oxidase.

[0317] A3. The analyte sensor of A or A1, wherein the first aspartate oxidase is a potassium-independent aspartate oxidase. A4. The analyte sensor of any one of A-A3, wherein the first analyte responsive active area further comprises a first asparaginase.

[0318] A5. The analyte sensor of A4, wherein the first asparaginase is a potassium-dependent aspartate oxidase. A6. The analyte sensor of A4, wherein the first asparaginase is a potassium-independent aspartate oxidase.

[0319] A7. The analyte sensor of any one of A-A6, further comprising a second working electrode and a second analyte responsive active area disposed on a surface of the second working electrode, the second analyte responsive active area comprising a second aspartate oxidase.

[0320] A8. The analyte sensor of A7, wherein the second aspartate oxidase is a potassium-dependent aspartate oxidase. A9. The analyte sensor of A7, wherein the second aspartate oxidase is a potassium-independent aspartate oxidase.

[0321] A10. The analyte sensor of any one of A7 to A9, wherein the first aspartate oxidase and the second aspartate oxidase exhibit different potassium dependencies. A11. The analyte sensor of any one of A7-A10, wherein the second analyte responsive active area further comprises a second asparaginase.

[0322] A12. The analyte sensor of A11, wherein the first asparaginase is a potassium-dependent aspartate oxidase. A13. The analyte sensor of A11, wherein the second asparaginase is a potassium-independent aspartate oxidase.

[0323] A14. The analyte sensor according to any one of A11 to A13, wherein the first asparaginase and the second asparaginase exhibit different potassium dependencies. A15. The analyte sensor of any one of A1-A14, wherein the first analyte responsive active area and / or the second analyte responsive active area further comprises an electron transfer agent.

[0324] A16. The analyte sensor of any one of A-A15, wherein the first analyte responsive active area and / or the second analyte responsive active area further comprises a stabilizing agent. A17. The analyte sensor according to A16, wherein the stabilizing agent is albumin.

[0325] A18. The analyte sensor according to A17, wherein the albumin is bovine serum albumin. A19. The analyte sensor of any one of A-A17, wherein the first analyte responsive active area and / or the second analyte responsive active area further comprises a polymer.

[0326] A20. The analyte sensor of A19, wherein (i) a first aspartate oxidase and / or a first asparaginase is covalently bound to the polymer, and / or (ii) a second aspartate oxidase and / or a second asparaginase is covalently bound to the polymer.

[0327] A21. The analyte sensor of any one of A19-A20, wherein the first and / or second electron transfer agent is covalently attached to the polymer. A22. The analyte sensor according to A15-A21, wherein the first and second electron transfer agents are the same.

[0328] A23. The analyte sensor of any one of A-A22, wherein the first mass transport limiting membrane overcoats the second analyte responsive active area. A24. The analyte sensor of any one of A-A23, wherein the first mass transport limiting membrane comprises a polyvinylpyridine-based polymer, polyvinylimidazole, polyacrylate, polyurethane, polyether urethane, silicone, or a combination thereof.

[0329] A25. The analyte sensor of A24, wherein the first mass transport limiting membrane comprises a polyvinylpyridine-based polymer. A26. The analyte sensor of A24, wherein the first mass transport limiting membrane comprises polyurethane.

[0330] A27. The analyte sensor of A24, wherein the first mass transport limiting membrane comprises silicone. A28. The analyte sensor of A24, wherein the first mass transport limiting membrane comprises polyvinylpyridine.

[0331] A29. The analyte sensor of A24, wherein the first mass transport limiting membrane comprises a copolymer of vinylpyridine and styrene. A30. The analyte sensor of A-A29, further comprising a third working electrode and a third analyte responsive active area responsive to a second analyte other than potassium disposed on a surface of the third working electrode, the third analyte responsive active area c...

Claims

1. (i) a sensor tail including at least a first working electrode and a second working electrode; (ii) a first analyte-responsive active area disposed on a surface of the first working electrode, the first analyte-responsive active area including a first enzyme layer comprising a first aspartate oxidase and a second enzyme layer comprising a first potassium-dependent asparaginase; (iii) a second analyte-responsive active area disposed on a surface of the second working electrode, the second analyte-responsive active area comprising a first enzyme layer comprising a second aspartate oxidase and a second enzyme layer comprising a second potassium-dependent asparaginase; Including, 1. An analyte sensor for measuring potassium levels, wherein the second enzyme layer of the first analyte-responsive active area is disposed on the first enzyme layer of the first analyte-responsive active area and / or the second enzyme layer of the second analyte-responsive active area is disposed on the first enzyme layer of the second analyte-responsive active area.

2. 10. The analyte sensor of claim 1, wherein the first analyte-responsive active area and / or the second analyte-responsive active area further comprises an electron transfer agent.

3. The analyte sensor of claim 1 or 2, wherein the first analyte-responsive active area and / or the second analyte-responsive active area further comprises a stabilizing agent.

4. The analyte sensor of claim 3 , wherein the stabilizing agent is albumin.

5. The analyte sensor of claim 4 , wherein the albumin is bovine serum albumin.

6. The analyte sensor of any one of claims 1 to 5, wherein the first analyte-responsive active area and / or the second analyte-responsive active area further comprises a polymer.

7. 7. The analyte sensor of claim 6, wherein the first potassium-dependent asparaginase is covalently bound to the polymer and / or the second potassium-dependent asparaginase is covalently bound to the polymer.

8. 8. The analyte sensor of claim 6 or 7, wherein the first and / or second electron transfer agents are covalently attached to the polymer.

9. The analyte sensor of any one of claims 2 to 8, wherein the first and second electron transfer agents are the same.

10. 10. The analyte sensor of claim 1, further comprising a first mass transport limiting membrane permeable to potassium overcoating the first analyte-responsive active area and / or the second analyte-responsive active area.

11. 11. The analyte sensor of claim 10, wherein the first mass transport limiting membrane comprises a polyvinylpyridine-based polymer, polyvinylimidazole, polyacrylate, polyurethane, polyetherurethane, silicone, or a combination thereof.

12. 11. The analyte sensor of claim 10, wherein the first mass transport limiting membrane comprises a polyvinylpyridine-based polymer.

13. 11. The analyte sensor of claim 10, wherein the first mass transport limiting membrane comprises a copolymer of vinylpyridine and styrene.

14. (iv) a third working electrode; and (v) a third active area responsive to a second analyte different from potassium, disposed on a surface of the third working electrode, the third active area including at least one enzyme responsive to the second analyte; The analyte sensor of any one of claims 1 to 13, comprising:

15. 15. The analyte sensor of claim 14, further comprising a second portion of the mass transport limiting membrane overcoating the third active area.

16. 15. The analyte sensor of claim 14, further comprising a second mass transport limiting membrane overcoating the third active area.

17. 17. The analyte sensor of claim 16, wherein the second mass transport limiting membrane further overcoats the first analyte-responsive active area and / or the second analyte-responsive active area.

18. 18. The analyte sensor of claim 16 or 17, wherein the second mass transport limiting membrane comprises a polymer that is not in the first mass transport limiting membrane.

19. A method for measuring potassium levels in a fluid using an analyte sensor according to any one of claims 1 to 18 implanted in a subject.

20. (i) applying a potential to the first working electrode and the second working electrode; (ii) obtaining a first signal at or above the redox potential of the first analyte-responsive active area; (iii) obtaining a second signal at or above the redox potential of the second analyte-responsive active area; (iv) correlating the first signal and the second signal to a concentration of potassium ions in the fluid; 20. The method of claim 19, comprising:

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