Temperature-insensitive membranes for analyte sensors.

Abstract

The present disclosure provides membrane structures that exhibit reduced temperature sensitivity. Analyte sensors comprising the membranes are also provided herein. The present disclosure further provides methods for detecting analytes using the sensors.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 481,498, filed January 25, 2023, the contents of which are incorporated herein by reference in their entirety. Field The presently disclosed subject matter relates to membrane structures for analyte sensors and methods for preparing analyte sensors having such membrane structures. [Background technology]

[0002] Detection of various analytes in an individual can sometimes be important for monitoring the health status of an individual, as deviations from normal analyte levels can be indicative of a physiological condition. For example, monitoring glucose levels can enable people with diabetes to take appropriate corrective action, including administering medication or consuming certain food or beverage products to avoid significant physiological damage. Other analytes may be desirable for monitoring other physiological conditions. In certain cases, it may be desirable to monitor more than one analyte to monitor multiple physiological conditions, especially when a person suffers from coexisting conditions that result in the simultaneous dysregulation of two or more analytes that are interrelated. Continuous analyte monitoring can be performed using one or more sensors that remain at least partially implanted in an individual's tissue, for example, transdermally, subcutaneously, or intravenously, allowing analysis to be performed in vivo. The implanted sensors can collect analyte data as needed, on a set schedule, or continuously, depending on the individual's specific health needs and / or previously measured analyte levels. Analyte monitoring with in vivo implanted sensors may be a desirable approach for individuals with severe analyte dysregulation and / or rapidly fluctuating analyte levels, but it may also be beneficial for other individuals. However, in vivo analyte monitoring systems can be adversely affected by temperature. For example, many enzymes and / or other components, such as membranes in in vivo analyte systems, are sensitive to temperature changes. Such sensitivity can result in inaccurate analyte information at different temperatures. Thus, there is a need in the art to develop analyte sensors and methods of in vivo analyte monitoring that are insensitive to temperature changes, eg, in vivo temperature changes. Summary of the Invention

[0003] The objects and advantages of the disclosed subject matter will be set forth in and obvious from the description which follows, and will be learned by practicing 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 the appended drawings. To achieve these and other advantages, and in accordance with the purpose of the disclosed subject matter as embodied and broadly described, the disclosed subject matter includes a membrane structure including an enzyme layer and a membrane disposed adjacent to the enzyme layer (e.g., disposed above the enzyme layer), the membrane comprising a copolymer of at least a first monomer and a second monomer. In certain embodiments, the first monomer comprises acrylamide. In certain embodiments, the first monomer is acrylamide. For example, but not by way of limitation, the first monomer comprises an N-alkylacrylamide. In certain embodiments, the first monomer is an N-alkylacrylamide. In certain embodiments, the alkyl of the N-alkylacrylamide is a C1-C6 linear or branched alkyl group or a C3-C6 cycloalkyl group. In certain embodiments, the alkyl of the N-alkylacrylamide is a branched alkyl group. In certain embodiments, the first monomer is N-isopropylacrylamide. In certain embodiments, the second monomer comprises a heterocycle-containing moiety. In certain embodiments, the heterocycle of the heterocycle-containing component is selected from the group consisting of furan, thiophene, pyrrole, pyridine, pyrimidine, imidazole, oxadiazole, isoxazole, oxazole, pyrazole, isothiazole, thiazole, pyrazine, isoquinoline, quinoline, benzofuran, benzimidazole, or a derivative thereof. In certain embodiments, the heterocycle is pyridine or a derivative thereof. In certain embodiments, the second monomer is a vinylpyridine, such as 4-vinylpyridine or 2-vinylpyridine, or a derivative thereof. In certain embodiments, the second monomer is 4-vinylpyridine or a derivative thereof. In certain embodiments, the second monomer is vinylimidazole or a derivative thereof. In certain embodiments, the second monomer is vinylimidazole, such as 1-vinylimidazole, or a derivative thereof.

[0004] In certain embodiments, the copolymer of the membrane structure comprises about 20 mer% to about 70 mer% of the first monomer. In certain embodiments, the copolymer of the membrane structure comprises about 30 mer% to about 60 mer% of the first monomer. In certain embodiments, the copolymer of the membrane structure comprises about 40 mer% to about 60 mer% of the first monomer. In certain embodiments, the copolymer of the membrane structure comprises about 30 mer% to about 80 mer% of the second monomer. In certain embodiments, the copolymer of the membrane structure comprises about 40 mer% to about 70 mer% of the second monomer. In certain embodiments, the copolymer of the membrane structure comprises about 30 mer% to about 65 mer% of the second monomer. In certain embodiments, the copolymer of the membrane structure comprises about 30 mer% to about 50 mer% of the second monomer.

[0005] In certain embodiments, the copolymer has Formula I: [ka] wherein m and n are each a positive integer. In certain embodiments, the ratio of m to n, e.g., the molar ratio, is about 100:1 to about 1:100. In certain embodiments, the ratio of m to n, e.g., the molar ratio, is about 4:1 to about 4:1. In certain embodiments, the ratio of m to n, e.g., the molar ratio, is about 1:1 to about 1:100, e.g., about 1:1 to about 1:50, about 1:1 to about 1:40, about 1:1 to about 1:30, about 1:1 to about 1:20, about 1:1 to about 1:10, about 1:1 to about 1:5, about 1:1 to about 1:4, or about 1:1 to about 1:2. In certain embodiments, the ratio of m to n, e.g., the molar ratio, is about 1:1 to about 1:4. In certain embodiments, the ratio of m to n, e.g., the molar ratio, is about 1:1 to about 1:3. In certain embodiments, the ratio of m to n, e.g., the molar ratio, is about 1:1 to about 1:2. In certain embodiments, the ratio of m to n, e.g., the molar ratio, is about 1:1 to about 100:1, e.g., about 1:1 to about 50:1, about 1:1 to about 40:1, about 1:1 to about 30:1, about 1:1 to about 20:1, about 1:1 to about 10:1, about 1:1 to about 5:1, about 1:1 to about 4:1, or about 1:1 to about 2:1. In certain embodiments, the ratio of m to n, e.g., the molar ratio, is about 1:1 to about 4:1. In certain embodiments, m is in the range of about 1 to about 90, e.g., about 1 to about 80 or about 1 to 50. In certain embodiments, n is in the range of about 1 to about 90, e.g., about 1 to about 70, or about 1 to about 50. In certain embodiments, m is in the range of about 1 to 50 (e.g., about 20 to 50) and n is in the range of about 1 to 70 (e.g., about 40 to 70). In certain embodiments, m is about 80 and n is about 22, or m is about 65 and n is about 35, or m is about 50 and n is about 50, or m is about 40 and n is about 60. In certain embodiments, m is about 40 and n is about 60.

[0006] In certain embodiments, the membrane structure includes one or more cross-linking agents. In certain embodiments, the cross-linking agent is selected from the group consisting of polyethylene glycol diglycidyl ether, polyethylene glycol tetraglycidyl ether, and polyetheramine. In certain embodiments, the membrane structure includes, for example, about 10% to about 20% by mass of the cross-linking agent. In certain embodiments, the present disclosure provides an analyte sensor comprising: (i) a sensor tail including at least a first working electrode; (ii) a first active area disposed on the surface of the first working electrode; and (iii) a mass transport limiting membrane overcoating at least the first active area and permeable to a first analyte. In certain embodiments, the first active area comprises an electron transfer agent. In certain embodiments, the first analyte is selected from the group consisting of glucose, ketone, potassium, creatinine, glutamate, lactate, creatine, sarcosine, and ascorbate. In certain embodiments, the first analyte is glucose. In certain embodiments, the first analyte is lactate.

[0007] In certain embodiments, the mass transport limiting membrane comprises a copolymer of at least a first monomer, the copolymer comprising a first monomer and a second monomer. In certain embodiments, the second monomer comprises acrylamide. For example, but not by way of limitation, the mass transport limiting membrane comprises a copolymer of at least a first monomer, the copolymer comprising acrylamide and a second monomer. In certain embodiments, the second monomer comprises a heterocycle-containing moiety. In certain embodiments, the acrylamide is an N-alkylacrylamide or a derivative thereof. In certain embodiments, the alkyl of the N-alkylacrylamide is a C1-C6 linear or branched alkyl group or a C3-C6 cycloalkyl group. In certain embodiments, the alkyl of the N-alkylacrylamide is a branched alkyl group. In certain embodiments, the N-alkylacrylamide is N-isopropylacrylamide or a derivative thereof. In certain embodiments, the heterocycle of the heterocycle-containing component is selected from the group consisting of furan, thiophene, pyrrole, pyridine, pyrimidine, imidazole, oxadiazole, isoxazole, oxazole, pyrazole, isothiazole, thiazole, pyrazine, isoquinoline, quinoline, benzofuran, benzimidazole, or a derivative thereof. In certain embodiments, the second monomer is a vinylpyridine, such as 4-vinylpyridine or 2-vinylpyridine, or a derivative thereof. In certain embodiments, the second monomer is 4-vinylpyridine or a derivative thereof. In certain embodiments, the second monomer is vinylimidazole or a derivative thereof. In certain embodiments, the second monomer is vinylimidazole, such as 1-vinylimidazole, or a derivative thereof.

[0008] In certain embodiments, the copolymer of the mass transport limiting membrane comprises from about 20 mer% to about 70 mer% of the first monomer. In certain embodiments, the copolymer of the mass transport limiting membrane comprises from about 30 mer% to about 60 mer% of the first monomer. In certain embodiments, the copolymer of the membrane structure comprises from about 40 mer% to about 60 mer% of the first monomer. In certain embodiments, the copolymer of the mass transport limiting membrane comprises from about 30 mer% to about 80 mer% of the second monomer. In certain embodiments, the copolymer of the mass transport limiting membrane comprises from about 40 mer% to about 70 mer% of the second monomer. In certain embodiments, the copolymer of the membrane structure comprises from about 30 mer% to about 65 mer% of the second monomer. In certain embodiments, the copolymer of the membrane structure comprises from about 30 mer% to about 50 mer% of the second monomer. In certain embodiments, the copolymer of the mass transport limiting membrane has Formula I: [ka] wherein m and n are each a positive integer. In certain embodiments, the ratio of m to n is about 100:1 to about 1:100. In certain embodiments, the ratio of m to n, e.g., a molar ratio, is about 4:1 to about 1:4. In certain embodiments, the ratio of m to n is about 1:1 to about 1:100, e.g., about 1:1 to about 1:50, about 1:1 to about 1:40, about 1:1 to about 1:30, about 1:1 to about 1:20, about 1:1 to about 1:10, about 1:1 to about 1:5, or about 1:1 to about 1:2. In certain embodiments, the ratio of m to n, e.g., the molar ratio, is about 1:1 to about 100:1, e.g., about 1:1 to about 50:1, about 1:1 to about 40:1, about 1:1 to about 30:1, about 1:1 to about 20:1, about 1:1 to about 10:1, about 1:1 to about 5:1, about 1:1 to about 4:1, or about 1:1 to about 2:1. In certain embodiments, the ratio of m to n, e.g., the molar ratio, is about 1:1 to about 4:1. In certain embodiments, the ratio of m to n, e.g., the molar ratio, is about 1:1 to about 1:4. In certain embodiments, the ratio of m to n, e.g., the molar ratio, is about 1:1 to about 1:3. In certain embodiments, the ratio of m to n, e.g., the molar ratio, is about 1:1 to about 1:2. In certain embodiments, the ratio of m to n, e.g., the molar ratio, is about 4:1 to about 1:3. In certain embodiments, the ratio of m to n, e.g., the molar ratio, is about 4:1 to about 1:2. In certain embodiments, m is in the range of about 1 to about 90, e.g., about 1 to about 80, or about 1 to 50, and n is in the range of about 1 to 90, e.g., about 1 to about 70, or about 1 to 50. In certain embodiments, m is in the range of about 1 to 50 (e.g., about 20 to 50), and n is in the range of about 1 to 70 (e.g., about 40 to 70). In certain embodiments, m is about 80 and n is about 22, or m is about 65 and n is about 35, or m is about 50 and n is about 50, or m is about 40 and n is about 60. In certain embodiments, m is about 40 and n is about 60.

[0009] In certain embodiments, the analyte sensor further includes a second working electrode and a second active area disposed on the surface of the second working electrode and responsive to a second analyte different from the first analyte, the second active area including at least one enzyme responsive to the second analyte. In certain embodiments, the mass transport limiting membrane directly overcoats the second active area, a second portion of the mass transport limiting membrane overcoats the second active area, or a second, separate mass transport limiting membrane overcoats the second active area. In certain embodiments, the second, separate mass transport limiting membrane comprises a different polymer than the first, separate mass transport limiting membrane. In certain embodiments, the second, separate mass transport limiting membrane comprises the same polymer as the first, separate mass transport limiting membrane. In certain embodiments, the second active area comprises an electron transfer agent. In certain embodiments, the second analyte is different from the first analyte. In certain embodiments, the first analyte is glucose. In certain embodiments, the second analyte is selected from the group consisting of glucose, glutamate, lactate, creatine, sarcosine, and ascorbate.

[0010] In certain embodiments, the present disclosure further provides a method for detecting an analyte, the method comprising: providing an analyte sensor as disclosed herein; acquiring a first signal at or above the redox potential of the first active area, the first signal being proportional to the concentration of the first analyte in a fluid in contact with the first active area; and correlating the first signal with the concentration of the first analyte in the fluid. In certain embodiments, the first active area comprises one or more enzymes responsive to the first analyte. Alternatively, the first active area does not comprise an enzyme, and the analyte undergoes a redox reaction at a working electrode. In certain embodiments, the present disclosure provides a method for detecting at least two analytes, comprising the steps of acquiring a first signal proportional to the concentration of a first analyte in a fluid contacting the first active area at or above the redox potential of the first active area, acquiring a second signal proportional to the concentration of a second analyte in a fluid contacting the second active area at or above the redox potential of the second active area, correlating the first signal with the concentration of the first analyte in the fluid, and correlating the second signal with the concentration of the second analyte in the fluid. In certain embodiments, the first active area contains one or more enzymes responsive to the first analyte and / or the second active area contains one or more enzymes responsive to the second analyte.

[0011] The present disclosure further provides a copolymer of at least a first monomer comprising an acrylamide and a second monomer comprising a heterocycle-containing component. In certain embodiments, the acrylamide is an N-alkylacrylamide or a derivative thereof. In certain embodiments, the alkyl of the N-alkylacrylamide is a C1-C6 linear or branched alkyl group or a C3-C6 cycloalkyl group. In certain embodiments, the alkyl of the N-alkylacrylamide is a branched alkyl group. In certain embodiments, the N-alkylacrylamide is N-isopropylacrylamide or a derivative thereof. In certain embodiments, the heterocycle of the heterocycle-containing component is selected from the group consisting of furan, thiophene, pyrrole, pyridine, pyrimidine, imidazole, oxadiazole, isoxazole, oxazole, pyrazole, isothiazole, thiazole, pyrazine, isoquinoline, quinoline, benzofuran, benzimidazole, or a derivative thereof. In certain embodiments, the second monomer is a vinylpyridine, such as 4-vinylpyridine or 2-vinylpyridine, or a derivative thereof. In certain embodiments, the second monomer is 4-vinylpyridine or a derivative thereof. In certain embodiments, the second monomer is vinylimidazole or a derivative thereof. In certain embodiments, the second monomer is vinylimidazole, such as 1-vinylimidazole, or a derivative thereof.

[0012] In certain embodiments, the copolymers of the present disclosure comprise from about 20 mer% to about 70 mer% of the first monomer. In certain embodiments, the copolymers of the mass transport limiting membrane comprise from about 40 mer% to about 60 mer% of the first monomer. In certain embodiments, the copolymers of the mass transport limiting membrane comprise from about 30 mer% to about 60 mer% of the first monomer. In certain embodiments, the copolymers of the mass transport limiting membrane comprise from about 30 mer% to about 80 mer% of the second monomer. In certain embodiments, the copolymers of the mass transport limiting membrane comprise from about 40 mer% to about 80 mer% of the second monomer. In certain embodiments, the copolymers of the mass transport limiting membrane comprise from about 30 mer% to about 65 mer% of the second monomer. In certain embodiments, the copolymers of the mass transport limiting membrane comprise from about 30 mer% to about 50 mer% of the second monomer.

[0013] In certain embodiments, the copolymers of the present disclosure have Formula I: [ka] wherein m and n are each a positive integer. In certain embodiments, the ratio of m to n is about 100:1 to about 1:100. In certain embodiments, the ratio of m to n, e.g., the molar ratio, is about 4:1 to about 1:4. In certain embodiments, the ratio of m to n is about 1:1 to about 1:100, e.g., about 1:1 to about 1:50, about 1:1 to about 1:40, about 1:1 to about 1:30, about 1:1 to about 1:20, about 1:1 to about 1:10, about 1:1 to about 1:5, or about 1:1 to about 1:2. In certain embodiments, the ratio of m to n, e.g., the molar ratio, is about 1:1 to about 100:1, e.g., about 1:1 to about 50:1, about 1:1 to about 40:1, about 1:1 to about 30:1, about 1:1 to about 20:1, about 1:1 to about 10:1, about 1:1 to about 5:1, about 1:1 to about 4:1, or about 1:1 to about 2:1. In certain embodiments, the ratio of m to n, e.g., the molar ratio, is about 1:1 to about 4:1. In certain embodiments, the ratio of m to n, e.g., the molar ratio, is about 1:1 to about 1:4. In certain embodiments, the ratio of m to n, e.g., the molar ratio, is about 1:1 to about 1:3. In certain embodiments, the ratio of m to n, e.g., the molar ratio, is about 1:1 to about 1:2. In certain embodiments, the ratio of m to n, e.g., the molar ratio, is about 4:1 to about 1:3. In certain embodiments, the ratio of m to n, e.g., the molar ratio, is about 4:1 to about 1:2. In certain embodiments, m is in the range of about 1 to about 90, e.g., about 1 to about 80 or about 1 to 50, and n is in the range of about 1 to about 90, e.g., about 1 to about 70 or about 1 to 50. In certain embodiments, m is in the range of about 1 to 50 (e.g., about 20 to 50) and n is in the range of about 1 to 70 (e.g., about 40 to 70). In certain embodiments, m is about 80 and n is about 22, or m is about 65 and n is about 35, or m is about 50 and n is about 50, or m is about 40 and n is about 60. In certain embodiments, m is about 40 and n is about 60. The present disclosure further provides membranes comprising the copolymers of the present disclosure.

[0014] The present disclosure further provides an analyte sensor comprising the membrane structure described herein. The present disclosure provides for the use of the analyte sensors described herein to detect the concentration of an analyte, for example, in a fluid. The following figures are included to illustrate certain aspects of the present disclosure and should not be considered exclusive embodiments. The disclosed subject matter is capable of considerable modification, alteration, combination, and equivalents in form and function without departing from the scope of the present disclosure. [Brief explanation of the drawings]

[0015] [Figure 1] 1 provides a diagram of an illustrative sensing system that can incorporate the analyte sensors of the present disclosure. [Figures 2A-2C] 1 provides a cross-sectional view of an analyte sensor including a single active area. [Figure 3A-3C] 1 shows a cross-sectional view of an analyte sensor comprising two active areas above a working electrode. [Figure 4] 1 shows a cross-sectional view of an analyte sensor containing two active areas. [Figures 5A-5C] FIG. 1 shows a perspective view of an analyte sensor comprising two active areas on separate working electrodes. [Figure 6] 1 provides a graph of sensor signal (nA) over time in the temperature range of 22° C. to 42° C. for a control glucose sensor having a poly(4-vinylpyridine-co-styrene) polymer-containing membrane over a working electrode with a glucose oxidase (GOX)-containing enzyme layer. [Figure 7] 1 provides a graph of sensor signal (nA) over time over a temperature range of 22° C. to 42° C. for glucose sensors having membrane compositions including poly(4-vinylpyridine-co-N-isopropylacrylamide) polymers with varying amounts of N-isopropylacrylamide and a control glucose sensor having a poly(4-vinylpyridine-co-styrene) polymer-containing membrane. [Figure 8]1 provides a graph of sensor signal (nA) over time over a temperature range of 22°C to 42°C for glucose sensors having membrane compositions including poly(4-vinylpyridine-co-N-isopropylacrylamide) polymer with varying amounts of crosslinker and a control glucose sensor having a poly(4-vinylpyridine-co-styrene) polymer-containing membrane. [Figure 9] 1 provides a graph of sensor signal (nA) over time in the temperature range of 22°C to 42°C for glucose sensors having membrane compositions including poly(4-vinylpyridine-co-N-isopropylacrylamide) polymer with varying amounts of N-isopropylacrylamide on a working electrode with a GOX-containing enzyme layer and a control glucose sensor having a poly(4-vinylpyridine-co-styrene) polymer-containing membrane on a GOX enzyme layer. [Figure 10] 1 provides a graph of sensor signal (nA) over time over a temperature range of 22°C to 42°C for glucose sensors having membrane compositions including poly(4-vinylpyridine-co-N-isopropylacrylamide) polymer with varying amounts of N-isopropylacrylamide on a working electrode with a FADGDH-containing enzyme layer and a control glucose sensor having a poly(4-vinylpyridine-co-styrene) polymer-containing membrane on a GOX enzyme layer. [Figure 11] FIG. 1 provides a graph of sensor signal (nA) over time over a temperature range of 22° C. to 42° C. for lactate sensors having membrane compositions comprising poly(4-vinylpyridine-co-N-isopropylacrylamide) polymer with varying amounts of N-isopropylacrylamide on top of a working electrode with a lactate oxidase (LOX)-containing enzyme layer and a control lactate sensor having a control membrane on the LOX-containing enzyme layer. DETAILED DESCRIPTION OF THE INVENTION

[0016] The present disclosure provides membranes for analyte sensors that are temperature independent, such that analyte detection is not adversely affected by temperature changes. In certain embodiments, the sensitivity of analyte sensors comprising membranes of the present disclosure remains constant or the change in sensitivity is not clinically significant (little or no change, either increasing or decreasing) over a range of temperatures. In certain embodiments, the analyte permeability through the membranes of the present disclosure remains constant, or the change in analyte permeability is not clinically significant (little or no change, either increasing or decreasing) over a range of temperatures. In certain embodiments, the membranes of the present disclosure have an analyte diffusion rate (i.e., analyte flux) that shows little or no change at different temperatures and / or in response to changes in temperature. In certain embodiments, the change in analyte diffusion rate through the membranes of the present disclosure in response to a change in temperature is less than about 5%, e.g., less than about 1%. In certain embodiments, the analyte diffusion rate through the membranes of the present disclosure does not change at all in response to a change in temperature. In certain embodiments, the membranes of the present disclosure withstand changes in analyte permeability at different temperatures over an extended period of time. For example, but not by way of limitation, the membranes of the present disclosure withstand changes in analyte permeability at different temperatures over at least the in vivo lifetime (service life or use period) of the membrane, e.g., at least 1 week, at least 2 weeks, at least 3 weeks, or at least 4 weeks.

[0017] In certain embodiments, the sensitivity of an analyte sensor including a membrane of the present disclosure shows little or no change at different temperatures and / or in response to temperature changes. In certain embodiments, the change in sensitivity of an analyte sensor including a membrane of the present disclosure is less than about 5%, e.g., less than about 1%, in response to temperature changes. In certain embodiments, the sensitivity of an analyte sensor including a membrane of the present disclosure does not change at all in response to temperature changes. In certain embodiments, an analyte sensor including a membrane of the present disclosure tolerates changes in sensitivity at different temperatures over an extended period of time. For example, but not limited to, an analyte sensor including a membrane of the present disclosure tolerates changes in sensitivity at different temperatures over at least the in vivo lifetime (use or usage period) of the membrane, e.g., at least 1 week, at least 2 weeks, at least 3 weeks, or at least 4 weeks.

[0018] For clarity, but not by way of limitation, the detailed description of the presently disclosed subject matter is divided into the following subsections. I. Definition, II. Analyte sensors; 1. General structure of the analyte sensor system; 2. Enzymes, 3. Redox mediators, 4. Polymer backbone, 5. Mass transport limiting membranes, and 6. Interference domain, III. Method of Use, IV. METHODS OF MANUFACTURING; AND V. Illustrative Embodiments

[0019] I. Definition The terms used herein generally have their ordinary meaning in the art, within the context of this disclosure and within the specific context in which each term is used. Certain terms are discussed below or elsewhere in the specification to provide further guidance to the practitioner regarding the description of the compositions and methods of the present disclosure and how to make and use them. For purposes of reading this specification, the following definitions apply, and whenever appropriate, terms used in the singular include the plural and vice versa. In the event that any definition set forth below conflicts with any document incorporated herein by reference, the definition set forth below shall control. As used herein, the use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and / or specification can mean "one," but is also consistent with the meanings of "one or more," "at least one," and "one or more." For example, but not by way of limitation, reference to "an" or "the" "analyte" encompasses a single analyte as well as combinations and / or mixtures of two or more different analytes.

[0020] The terms "comprise(s), "include(s), "having," "has," "can," and "contain(s)," and variations thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not exclude additional actions or structures. The present disclosure also contemplates other embodiments that "comprise," "consist," and "consist essentially of" the embodiments or elements presented herein, whether or not explicitly described. As used herein, the term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within or more than three times the standard deviation per practice in the art. Alternatively, "about" can mean within 20%, preferably up to 10%, more preferably up to 5%, and even more preferably up to 1% of a given value.

[0021] The term "biological fluid," as used herein, 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, lymph, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, amniotic fluid, sweat, tears, and the like. In certain embodiments, the biological fluid is dermal fluid or interstitial fluid. The term "crosslinker," as used herein, refers to a molecule containing at least two reactive groups that can link two or more polymers together or link two or more portions of the same polymer together. As described herein, linking two or more different polymers together is an intermolecular crosslink, and linking two or more portions of the same polymer is an intramolecular crosslink. In certain embodiments of the present disclosure, the crosslinker of interest is capable of simultaneous intermolecular and intramolecular crosslinking. As used herein, the term "hydrophilic" means having an affinity for and being able to absorb water. As used herein, the terms "enzyme composition" and "sensing chemistry" are used interchangeably and refer to a composition used to detect and / or measure an analyte. In certain non-limiting embodiments, the enzyme composition can include one or more enzymes, polymers, redox mediators, crosslinkers, etc.

[0022] The term "lower critical solution temperature" is used herein in its conventional sense to mean the temperature below which the components of a mixture are miscible. In certain embodiments, the LCST may depend on pressure (e.g., increasing pressure can increase the LCST), degree of polymerization, polydispersity (e.g., distribution of molecular weights in the polymer), branching of the polymer, etc. The term "polymer," as used herein, refers to a molecular structure comprising repeating structural units called monomers. These subunits are typically connected by covalent chemical bonds. As will be readily recognized by one of ordinary skill in the art, a polymer can be branched or unbranched. In certain embodiments, a polymer is a homopolymer, which is a polymer formed by polymerization of a single type of monomer. In certain other embodiments, a polymer is a heteropolymer or copolymer, comprising two or more different types of monomers. As used herein, the term "redox mediator" refers to an electron transfer agent for transporting electrons between an analyte or analyte reductase or analyte oxidase and an electrode, either directly or via one or more additional electron transfer agents. In certain embodiments, a redox mediator comprising a polymer backbone may also be referred to as a "redox polymer."

[0023] The term "reference electrode," as used herein, can refer to a reference electrode or an electrode that functions as both a reference electrode and a counter electrode. Similarly, the term "counter electrode," as used herein, refers to both a counter electrode and a counter electrode that also functions as a reference electrode. As used herein, the terms "temperature insensitive," "constant or same analyte permeability," "low temperature sensitivity," "temperature independent," and similar terms are used interchangeably herein to refer to a membrane or analyte sensor in which the diffusion rate of an analyte through an analyte-permeable membrane does not change (increase or decrease) by more than 5% per °C, e.g., 4.5% per °C, 4.0% per °C, 3.5% per °C, 3.0% per °C, 2.5% per °C, 2.0% per °C, 1.5% per °C, 1.0% per °C, 0.5% per °C, 0.01% per °C, or less, with a standard deviation of about 1%, in response to a temperature change from 20°C to 50°C. In certain embodiments, the diffusion rate of an analyte, such as the diffusion of glucose, across a membrane of the present disclosure is constant (within the parameters described herein) over a temperature range of 20°C to 50°C or 20°C to 45°C, including temperatures of, for example, 22°C, 27°C, 32°C, 37°C, and 45°C.

[0024] As used herein, "analyte sensor" or "sensor" may refer to any device capable of receiving sensory information from a user, including, by way of illustration 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 body movement sensor, or any other sensor for gathering physical or biological information. Analytes measured by an analyte sensor may include, by way of example and not limitation, 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, aspartate, asparagine, magnesium, oxygen, pH, phosphorus, potassium, sodium, total protein, uric acid, and the like. The term "reactive group," as used herein, refers to a functional group of a molecule that can react with another compound, e.g., a polymer, to link at least a portion of the other compound, e.g., a polymer, to the molecule. Non-limiting examples of reactive groups include carboxy, activated ester, sulfonyl halide, sulfonate ester, isocyanate, isothiocyanate, epoxide, aziridine, halide, aldehyde, ketone, amine, acrylamide, thiol, acyl azide, acyl halide, hydrazine, hydroxylamine, alkyl halide, imidazole, pyridine, phenol, alkyl sulfonate, halotriazine, imido ester, maleimide, hydrazide, hydroxy, and photoreactive azidoaryl groups. Activated esters, as used herein and understood in the art, include, but are not limited to, succinimidyl, benzotriazolyl, or esters of aryls substituted with electron-withdrawing groups such as sulfo, nitro, cyano, or halo groups, or carboxylic acids activated by carbodiimides.

[0025] As used herein, the term "multi-component membrane" means a membrane that includes two or more types of membrane polymers. As used herein, the term "single-component membrane" means a membrane that includes one type of membrane polymer. As used herein, the term "polyvinylpyridine-based polymer" means a polymer or copolymer that includes polyvinylpyridine (eg, poly(2-vinylpyridine) or poly(4-vinylpyridine)) or a derivative thereof. The term "alkyl," as used herein, means a straight or branched saturated aliphatic hydrocarbon. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, and the like. Unless otherwise noted, the term "alkyl" includes both alkyl and cycloalkyl groups.

[0026] II. Analyte Sensors 1. General structure of an 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. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the present disclosure is not entitled to antedate such publication by 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.

[0027] In general, embodiments of the present disclosure include systems, devices, and methods for using 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 that includes the electronics housing of a sensor control device. According to certain embodiments, a structure separate from the applicator, such as a container, can also be provided to a user in a sterile package that includes a sensor module and a needle module. The user can connect the sensor module to the electronics housing and connect the needle to the applicator through an assembly process that includes inserting the applicator into the container in a specific manner. In certain embodiments, the applicator, sensor control device, sensor module, and needle module can be provided in a single package. The applicator can be used to position the sensor control device on the human body so that the sensor contacts the wearer's bodily fluids. The embodiments provided herein are improvements to reduce the likelihood of the sensor being improperly inserted or damaged, or of eliciting an adverse physiological response. Other improvements and advantages are also provided. Various configurations of these devices are described in detail by way of exemplary embodiments only. Additionally, many embodiments include in vivo analyte sensors, where at least a portion of the sensor is located or structurally configured to be capable of being located on the body of a user to obtain information about at least one analyte in the body. However, it should be noted that the embodiments disclosed herein can be used with in vivo analyte monitoring systems that incorporate in vitro capabilities, as well as purely in vitro or ex vivo analyte monitoring systems, including completely non-invasive systems.

[0028] Furthermore, for any and all embodiments of the methods disclosed herein, systems and devices capable of implementing each of these embodiments are included within the scope of the present disclosure. For example, sensor control device embodiments are disclosed, which may include one or more sensors, analyte monitoring circuitry (e.g., analog circuitry), memory (e.g., for storing instructions), a power source, communications circuitry, a transmitter, a receiver, a processor, and / or a controller (e.g., for executing instructions) capable of performing or facilitating the execution of any and all method steps. These sensor control device embodiments can be used to perform steps performed by the sensor control device from any and all methods described herein. Additionally, the systems and methods presented herein can be used for any purpose, including the operation of sensors used in analyte monitoring systems, such as, but not limited to, health, wellness, diet, research, information, or sensing of analytes over time. As used herein, "analyte sensor" or "sensor" can mean any device capable of receiving sensor information from a user, and includes, by way of illustration 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 body movement sensor, or any other sensor for collecting physical or biological information. In certain embodiments, the analyte sensors of the present disclosure can measure additional analytes including, but not limited to, glutamate, glucose, ketones, lactate, oxygen, hemoglobin A1C, albumin, alcohol, alkaline phosphatase, alanine transaminase, aspartate aminotransferase, bilirubin, blood urea nitrogen, calcium, carbon dioxide, chloride, creatinine, creatine, hematocrit, magnesium, oxygen, pH, phosphorus, potassium, sodium, total protein, uric acid, and others.

[0029] However, before describing aspects of these embodiments in detail, it is first desirable to describe examples of devices that may be present, for example, in an in vivo analyte monitoring system, as well as examples of their operation, all of which may be used with the embodiments described herein. There are various types of in vivo analyte monitoring systems. For example, a "continuous analyte monitoring" system (or "continuous glucose monitoring" system) can continuously transmit data from a sensor control device to a reader device without prompting, e.g., automatically according to a schedule. As another example, a "flash analyte monitoring" system (or "flash glucose monitoring" system or simply "flash" system) can transfer data from a sensor control device in response to a scan or data request by a reader device equipped with a protocol such as near-field communication (NFC) or radio frequency identification (RFID). In vivo analyte monitoring systems can also operate without the need for fingerstick calibration.

[0030] In vivo analyte monitoring systems can be distinguished from "in vitro" systems, which include a measurement device having a port for receiving an analyte test strip that contacts a biological sample outside the body (i.e., "ex vivo") and typically carries a user's bodily fluid that can be analyzed to determine the analyte level in the user's blood. An in-vivo monitoring system may include a sensor that contacts a user's bodily fluid while positioned in vivo and senses the level of an analyte contained therein. The sensor may be part of a sensor control device that resides on the user's body and includes electronic circuitry and a power source that enables and controls the sensing of the analyte. Sensor control devices and variations thereof may also be referred to as "sensor control units," "on-body electronics" devices or units, "on-body" devices or units, or "sensor data communication" devices or units, to name a few.

[0031] An in-vivo monitoring system may also include a device that accepts sensed analyte data from the sensor control device, processes the sensed analyte data, and / or presents it to a user in any number of forms. This device and variations thereof may be referred to as a "handheld reader device," a "reader device" (or simply "reader"), a "handheld electronic circuit" (or simply "handheld"), a "portable data processing" device or unit, a "data receiver," a "receiver" device or unit (or simply "receiver"), or a "remote" device or unit, to name a few. Other devices, such as personal computers, have also been utilized in conjunction with or incorporated into in-vivo and in-vitro monitoring systems.

[0032] FIG. 1 provides a schematic diagram of an illustrative sensing system that can incorporate the analyte sensors of the present disclosure. As shown, sensing system 100 includes a sensor control device 102 and a reader device 120 configured to communicate with each other via a local communication path or link 140, which may be wired or wireless, unidirectional or bidirectional, and encrypted or unencrypted. According to certain embodiments, reader device 120 may review analyte concentrations and alarms or notifications determined by sensor 104 or its associated processor, as well as provide an output medium that allows for one or more inputs from a user. Reader device 120 may be a general-purpose smartphone or a dedicated electronic reader device. While only one reader device 120 is shown, multiple reader devices 120 may be present in certain cases. Reader device 120 can communicate with remote terminal 170 and / or trusted computer system 180 via communication paths / links 141 and / or 142, each of which may also be wired or wireless, unidirectional or bidirectional, and encrypted or unencrypted. Reader device 120 may also or alternately communicate with network 150 (e.g., a mobile telephone network, the Internet, or a cloud server) via communication path / link 151. Network 150 may further be communicatively coupled to remote terminal 170 via communication path / link 152 and / or to trusted computer system 180 via communication path / link 153. Alternately, sensor 104 may communicate directly with remote terminal 170 and / or trusted computer system 180 without the presence of an intervening reader device 120. For example, but not by way of limitation, in certain embodiments, sensor 104 may communicate with remote terminal 170 and / or trusted computer system 180 via a direct communication link to network 150, as described in U.S. Patent Application Publication No. 2011 / 0213225, which is incorporated herein by reference in its entirety.Any suitable electronic communication protocol may be used for each communication path or link, such as near field communication (NFC), radio frequency identification (RFID), BLUETOOTH® or BLUETOOTH® low energy protocol, WiFi, or other. According to certain embodiments, the remote terminal 170 and / or trusted computer system may be accessible by individuals other than the primary user who are interested in the user's analyte levels. The reader device 120 may include a display 122 and optional input component 121. According to certain embodiments, the display 122 may include a touchscreen interface.

[0033] The sensor control device 102 includes a sensor housing 103 that can house circuitry and a power source for operating the sensor 104. The power source and / or active circuitry can optionally be omitted. A processor (not shown) can be communicatively coupled to the sensor 104, the processor being physically located within the sensor housing 103 or the reader device 120. According to certain embodiments, the sensor 104 protrudes from an underside of the sensor housing 103 and extends through an adhesive layer 105 adapted to adhere the sensor housing 103 to a tissue surface, such as skin. The sensor 104 is adapted to be at least partially inserted into a tissue of interest, such as the skin or the subcutaneous layer of the skin. The sensor 104 may include a sensor tail of sufficient length to insert to a desired depth into a given tissue. The sensor tail may include at least one working electrode. In certain embodiments, the sensor tail may include two working electrodes. In certain configurations, the sensor tail may include an active area for detecting an analyte, for example, on the working electrode. There may be a counter electrode in combination with the at least one working electrode. Specific electrode configurations on the sensor tail are described in more detail below. Active areas can be configured to detect specific analytes, such as glucose, glutamate, creatinine, creatine, sarcosine, ascorbate, and combinations thereof. For example, but not by way of limitation, a glucose-responsive active area can include a glucose-responsive enzyme, a glutamate-responsive active area can include a glutamate-responsive enzyme, a creatine-responsive active area can include a creatine-responsive enzyme system, a creatinine-responsive active area can include a creatinine-responsive enzyme system, a sarcosine-responsive active area can include a sarcosine-responsive enzyme system, and an ascorbate-responsive active area can include an ascorbate-responsive enzyme system.

[0034] As also described in more detail below, the active area can be overcoated with a membrane. 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 rate of mass transport of the analyte. For example, but not by way of limitation, limiting the access of the analyte to the analyte-responsive active area with a mass transport limiting membrane helps avoid sensor overload (saturation), thereby improving detection performance and accuracy. In certain embodiments, the membrane comprises a copolymer of the present disclosure, e.g., as described in Section II.5. For example, but not by way of limitation, the mass transport limiting membrane can comprise a copolymer comprising a first monomer, e.g., an acrylamide (e.g., an N-alkylacrylamide), and a second monomer comprising a heterocycle-containing moiety, e.g., a vinylpyridine, e.g., 4-vinylpyridine. In certain embodiments, the N-alkylacrylamide is N-isopropylacrylamide. In certain embodiments of the present disclosure, one or more analytes can be monitored in any biological fluid of interest, such as skin fluid, interstitial fluid, plasma, blood, lymph, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, amniotic fluid, etc. In certain embodiments, the analyte sensors of the present disclosure are adapted to assay skin fluid or interstitial fluid, and the concentration of one or more analytes can be determined in vivo. In certain embodiments, the biological fluid is interstitial fluid.

[0035] Still referring to FIG. 1 , the sensor 104 can automatically transmit data to the reader device 120. For example, but not by way of limitation, analyte concentration data can be automatically and periodically transmitted at a certain frequency, such as when the data is acquired or after a certain period of time has elapsed, with the data stored in memory until transmission (e.g., every minute, every five minutes, or some other predetermined time). In certain embodiments, the sensor 104 can communicate with the reader device 120 in a non-automated manner and not on a set schedule. For example, but not by way of limitation, data can be transmitted from the sensor 104 using RFID technology when the sensor's electronics come within range of the reader device 120. The data can remain stored in the sensor's 104 memory until transmitted to the reader device 120. That is, a user need not constantly maintain close proximity to the reader device 120, but can instead upload data at their convenience. In certain embodiments, a combination of automated and non-automated data transfer can be performed. For example, but not by way of limitation, data transfer may continue on an automated basis until the reader device 120 is no longer within range of the sensor 104 .

[0036] An introducer may be transiently present to facilitate the introduction of the sensor 104 into the tissue. In certain illustrative embodiments, the introducer may include a needle or similar needle-like object. As those skilled in the art will readily recognize, in alternative embodiments, other types of introducers may be present, such as a sheath or blade. More specifically, the needle or other introducer may be transiently present near the sensor 104 prior to insertion into the tissue and then removed. While present, the needle or other introducer may facilitate the insertion of the sensor 104 into the tissue by opening an access path through which the sensor 104 travels. For example, without limitation, according to one or more embodiments, the needle may facilitate penetration of the epidermis as an access path to the dermis, allowing implantation of the sensor 104 to occur. After opening the access path, the needle or other introducer may be removed so as not to present a needle-like hazard. In certain embodiments, suitable needles may be solid or hollow, beveled or unbeveled, and / or circular or non-circular in cross section. In more particular embodiments, suitable needles may have a cross-sectional diameter and / or tip shape similar to an acupuncture needle having a cross-sectional diameter of about 250 μm, although suitable needles may have a larger or smaller cross-sectional diameter if needed for a particular application.

[0037] In certain embodiments, the tip of the needle (while present) is angled beyond the end of the sensor 104, allowing the needle to penetrate the tissue first and open an access path for the sensor 104. In certain embodiments, the sensor 104 resides within a lumen or channel of the needle, which likewise allows the needle to open an access path for the sensor 104. In either case, the needle is removed after facilitating insertion of the sensor. Sensor configurations featuring a single active area configured to detect a single corresponding analyte can employ two-electrode or three-electrode detection motifs, as further described herein with reference to Figures 2A-2C. Sensor configurations featuring two different active areas for detecting the same or different analytes on separate working electrodes or on the same working electrode are described separately thereafter with reference to Figures 3A-5C. Sensor configurations with multiple working electrodes can be particularly advantageous for incorporating two different active areas within the same sensor tail, as the signal contribution from each active area can be more easily determined.

[0038] Where a single working electrode is present in an analyte sensor, a three-electrode sensor configuration may include a working electrode, a counter electrode, and a reference electrode. A related two-electrode sensor configuration includes a working electrode and a second electrode, in which the second electrode may function as both the counter electrode and the reference electrode (i.e., the counter / reference electrode). The various electrodes may be at least partially stacked (layered) on top of each other and / or spaced apart laterally on the sensor tail. Suitable sensor configurations may be substantially planar in shape, substantially cylindrical in shape, or any suitable shape. In any of the sensor configurations disclosed herein, the various electrodes may be electrically insulated from each other by a dielectric material or similar insulator. Analyte sensors featuring multiple working electrodes can also include at least one additional electrode. If one additional electrode is present, it can function as a counter / reference electrode for each of the multiple working electrodes. If two additional electrodes are present, one of the additional electrodes can function as a counter electrode for each of the multiple working electrodes, and the other of the additional electrodes can function as a reference electrode for each of the multiple working electrodes.

[0039] FIG. 2A shows a schematic diagram of an illustrative two-electrode analyte sensor configuration suitable for use in the present disclosure. As shown, the analyte sensor 200 includes a substrate 212 disposed between a working electrode 214 and a counter / reference electrode 216. Alternately, the working electrode 214 and the counter / reference electrode 216 can be located on the same side of the substrate 212 with a dielectric material interposed therebetween (configuration not shown). An active area 218 is disposed as at least one layer on at least a portion of the working electrode 214. The active area 218 can include multiple spots or a single spot configured to detect the analyte (e.g., at a low potential of the working electrode), as discussed further herein. In certain embodiments, the active area 218 can include an electron transfer agent as described herein. 2A , film 220 overcoats at least active area 218. In certain embodiments, film 220 comprises a copolymer of the present disclosure. In certain embodiments, film 220 comprises a copolymer including a first monomer, such as an acrylamide (e.g., an N-alkylacrylamide), and a second monomer. In certain embodiments, the second monomer can include a heterocycle-containing component (e.g., furan, thiophene, pyrrole, pyridine, pyrimidine, imidazole, oxadiazole, isoxazole, oxazole, pyrazole, isothiazole, thiazole, pyrazine, isoquinoline, quinoline, benzofuran, benzimidazole, or a derivative thereof). For example, but not by way of limitation, film 220 comprises a copolymer including a first monomer, such as an acrylamide (e.g., an N-alkylacrylamide), and a second monomer including a heterocycle-containing component, such as a vinylpyridine, e.g., 4-vinylpyridine. In certain embodiments, the N-alkylacrylamide is N-isopropylacrylamide.

[0040] In certain embodiments, the membrane 220 may overcoat part or all of the working electrode 214 and / or the counter / reference electrode 216, 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 comprise one or more polymeric membrane materials capable of limiting the flux of analyte to the active area 218 (i.e., the membrane 220 is a mass transport limiting membrane with some permeability to the analyte of interest). The composition and thickness of the membrane 220 may be varied to facilitate the flux of the desired analyte to the active area 218, thereby providing the desired signal strength and stability. The analyte sensor 200 may be operable to assay the analyte by any of the following electrochemical detection techniques: coulometric, amperometric, voltammetric, or potentiometric.

[0041] 2B and 2C show schematic diagrams of illustrative three-electrode analyte sensor configurations, also suitable for use in the present disclosure. The three-electrode analyte sensor configuration may be similar to that shown for analyte sensor 200 of FIG. 2A (FIGS. 2B and 2C), except that analyte sensors 201 and 202 include an additional electrode 217. With the additional electrode 217, counter / reference electrode 216 can function as a counter electrode or a reference electrode, and the additional electrode 217 fulfills the function of the other electrode not otherwise fulfilled. Working electrode 214 continues to fulfill its original function. The additional electrode 217 can be disposed on working electrode 214 or electrode 216 with a separating layer of dielectric material therebetween. For example, but not by way of limitation, as shown in FIG. 2B, dielectric layers 219a, 219b, and 219c separate electrodes 214, 216, and 217 from one another and provide electrical insulation. Alternatively, as shown in FIG. 2C , at least one of electrodes 214, 216, and 217 may be located on opposite sides of substrate 212. That is, in certain embodiments, electrode 214 (working electrode) and electrode 216 (counter electrode) are located on opposite sides of substrate 212, and electrode 217 (reference electrode) may be located on one of electrodes 214 or 216 and separated therefrom by a dielectric material. A reference material layer 230 (e.g., Ag / AgCl) may be present on electrode 217, and the location of reference material layer 230 is not limited to that shown in FIGS. 2B and 2C . Similar to sensor 200 shown in FIG. 2A , active area 218 in analyte sensors 201 and 202 may include multiple spots or a single spot. In certain embodiments, active area 218 may include a redox mediator disclosed herein. Additionally, analyte sensors 201 and 202 may be operable to assay analytes by any of the following electrochemical detection techniques: coulometric, amperometric, voltammetric, or potentiometric.

[0042] Similar to analyte sensor 200, membrane 220 may overcoat active area 218 and other sensor components in analyte sensors 201 and 202, thereby acting as a mass transport limiting membrane. In certain embodiments, additional electrode 217 may be overcoated with membrane 220. While FIGS. 2B and 2C depict electrodes 214, 216, 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 may be the same or different for each of electrodes 214, 216, and 217. Similar to the two-electrode analyte sensor configuration (FIG. 2A), in the sensor configuration of FIGS. 2B and 2C, one or both sides of analyte sensors 201 and 202 may be overcoated with membrane 220, or the entire analyte sensors 201 and 202 may be overcoated. Therefore, it should be understood that the three-electrode sensor configuration shown in Figures 2B and 2C is a non-limiting example of the embodiments disclosed herein, and that alternative electrode and / or layer configurations remain within the scope of the present disclosure.

[0043] FIG. 3A shows an illustrative configuration of a sensor 203 having a single working electrode with two distinct active areas disposed therein. FIG. 3A is similar to FIG. 2A except that there are two active areas on the working electrode 214: a first active area 218a and a second active area 218b. These active areas are associated with the same or different analytes and are laterally separated from one another on the surface of the working electrode 214. The active areas 218a and 218b may include multiple spots or a single spot configured to detect each analyte. The composition of the membrane 220 may vary or be compositionally identical in the active areas 218a and 218b. 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 discussed further below.

[0044] 3B and 3C show cross-sectional schematic views of illustrative three-electrode sensor configurations for sensors 204 and 205, respectively, each featuring a single working electrode with a first active area 218a and a second active area 218b disposed thereon. FIGS. 3B and 3C are otherwise similar to FIGS. 2B and 2C and can be better understood by reference thereto. As with FIG. 3A, the composition of membrane 220 may vary or be compositionally identical in active areas 218a and 218b. In certain embodiments, both active areas 218a and 218b may include a redox mediator described herein. In certain embodiments, only one of active areas 218a and 218b may include a redox mediator described herein. For example, but not by way of limitation, only active area 218a includes a redox mediator described herein. In certain embodiments, only active area 218b includes a redox mediator described herein. In certain embodiments, both active areas 218a and 218b include a redox mediator as described herein. In certain embodiments, the electron transfer agent present in active area 218a is different from the redox mediator present in 218b. Alternatively, the electron transfer agent present in active area 218a is the same redox mediator present in 218b.

[0045] Illustrative sensor configurations having multiple working electrodes, specifically two working electrodes, are described in more detail with reference to Figures 4-5C. While the following description is primarily directed to sensor configurations having two working electrodes, it should be recognized that extensions of the disclosure herein can incorporate three or more working electrodes. The use of working electrodes can provide additional sensing capabilities to the analyte sensor beyond just the first analyte and the second analyte.

[0046] 4 shows a cross-sectional schematic diagram of an illustrative analyte sensor configuration suitable for use in the present disclosure, having two working electrodes, a reference electrode, and a counter electrode. As shown, analyte sensor 300 includes working electrodes 304 and 306 disposed on opposite sides of substrate 302. A first active area 310a is disposed on the surface of working electrode 304, and a second active area 310b is disposed on the surface of working electrode 306. Counter electrode 320 is electrically insulated from working electrode 304 by dielectric layer 322, and reference electrode 321 is electrically insulated from working electrode 306 by dielectric layer 323. Outer dielectric layers 330 and 332 are positioned over reference electrode 321 and counter electrode 320, respectively. According to various embodiments, a membrane 340 may overcoat at least active areas 310a and 310b, along with other components of analyte sensor 300 or the entire analyte sensor 300. In certain embodiments, the membrane 340 comprises a copolymer of the present disclosure. For example, but not by way of limitation, the membrane 340 comprises a copolymer including a first monomer, such as an acrylamide (e.g., an N-alkylacrylamide), and a second monomer including a heterocycle-containing component, such as a vinylpyridine, e.g., 4-vinylpyridine. In certain embodiments, the N-alkylacrylamide is N-isopropylacrylamide.

[0047] In certain embodiments, the membrane 340 may be continuous but may vary in composition over the active area 310a and / or the active area 310b to provide different permeability values ​​at each location and differentially control analyte flux. For example, but not by way of limitation, one or more electrodes may be overcoated with a first membrane portion 340a and / or a second membrane portion 340b. In certain embodiments, different membrane formulations may be sprayed and / or printed on opposing sides of the analyte sensor 300. Dip-coating techniques may also be suitable, particularly for depositing at least a portion of a bilayer membrane over one of the active areas 310a and 310b. In certain embodiments, the membrane 340 may be identical or may vary in composition over the active areas 310a and 310b. For example, but not by way of limitation, the membrane 340 may include a bilayer overcoating the active area 310a and may be a homogeneous membrane overcoating the active area 310b, or the membrane 340 may include a bilayer overcoating the active area 310b and may be a homogeneous membrane overcoating the active area 310a. In certain embodiments, one of the first and second membrane portions may include a bilayer membrane, while the other of the first and second membrane portions may include a single membrane polymer, according to certain embodiments of the present disclosure. In certain embodiments, the analyte sensor may include more than one membrane 340, e.g., two or more membranes. For example, but not by way of limitation, the analyte sensor may include a membrane overcoating one or more active areas, e.g., 310a and 310b, and an additional membrane overcoating the entire sensor, as shown in FIG. 4 . In such a configuration, a bilayer membrane may be formed over one or more active areas, e.g., 310a and 310b. In certain embodiments, the two membranes may have different polymer compositions. For example, but not by way of limitation, a first film can include a copolymer of the present disclosure and a second film can include a different polymer. In certain embodiments, either active area 310a or 310b can include an electron transfer agent described herein. In certain embodiments, only one of active areas 310a or 310b can include a redox mediator described herein.For example, but not by way of limitation, only active area 310a includes a redox mediator described herein. In certain embodiments, only active area 310b includes a redox mediator described herein. In certain embodiments, both active areas 310a and 310b include a redox mediator described herein. In certain embodiments, the redox mediator present in active area 310a is different from the electron transfer agent present in 310b. Alternatively, the redox mediator present in active area 310a is the same electron transfer agent present in 310b.

[0048] Alternative sensor configurations having multiple working electrodes and differing from the configuration shown in Figure 4 may feature counter / reference electrodes instead of separate counter and reference electrodes 320 and 321, and / or may feature layer and / or film arrangements that differ from those explicitly illustrated. For example, without limitation, the positioning of counter electrode 320 and reference electrode 321 may be reversed from that shown in Figure 4. Additionally, working electrodes 304 and 306 need not necessarily be on opposite sides of substrate 302 in the manner shown in Figure 4. While preferred sensor configurations may feature electrodes of a substantially planar nature, it should be recognized 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 coaxially with one another may facilitate deposition of a mass transport limiting membrane, as described herein below. For example, but not by way of limitation, coaxial working electrodes spaced apart along the length of the sensor tail may facilitate deposition of the membrane by sequential dip-coating operations in the same manner as described above for substantially planar sensor configurations. Figures 5A-5C show perspective views of analyte sensors featuring two working electrodes arranged coaxially with one another. It should be recognized that sensor configurations having a coaxial electrode arrangement but lacking a second working electrode are also possible in the present disclosure.

[0049] 5A shows a perspective view of an illustrative sensor configuration in which multiple electrodes are substantially cylindrical and coaxially arranged relative to a central substrate. As shown, the analyte sensor 400 includes a central substrate 402 around which all of the electrodes and dielectric layers are coaxially arranged relative to one another. In particular, a working electrode 410 is disposed on the surface of the central substrate 402, and a dielectric layer 412 is disposed over a portion of the working electrode 410 distal to the sensor tip 404. A working electrode 420 is disposed over the dielectric layer 412, and a dielectric layer 422 is disposed over a portion of the working electrode 420 distal to the sensor tip 404. A counter electrode 430 is disposed over the dielectric layer 422, and a dielectric layer 432 is disposed over a portion of the counter electrode 430 distal to the sensor tip 404. A reference electrode 440 is disposed over the dielectric layer 432, and a dielectric layer 442 is disposed over a portion of the reference electrode 440 distal to the sensor tip 404. Thus, the exposed surfaces of working electrode 410 , working electrode 420 , counter electrode 430 , and reference electrode 440 are spaced apart from one another along the longitudinal axis B of analyte sensor 400 .

[0050] Still referring to FIG. 5A , first and second active areas 414a and 414b associated with different analytes are disposed on the exposed surfaces of working electrodes 410 and 420, respectively, allowing contact with the fluid for sensing. While active areas 414a and 414b are depicted as three discrete spots in FIG. 5A , it should be appreciated that fewer or more than three spots may be present in alternative sensor configurations, including a continuous layer of active areas. In certain embodiments, either one of active areas 414a and 414b may include an electron transfer agent described herein. In certain embodiments, only one of active areas 414a and 414b may include a redox mediator described herein. For example, but not by way of limitation, only active area 414a includes a redox mediator described herein. In certain embodiments, only active area 414b includes a redox mediator described herein. In certain embodiments, both active areas 414a and 414b include a redox mediator described herein. In certain embodiments, the redox mediator present in active area 414a is different from the electron transfer agent present in 414b, or alternatively, the redox mediator present in active area 414a is the same electron transfer agent present in 414b.

[0051] In FIG. 5A, the sensor 400 is partially coated with a membrane 450 over the working electrodes 410 and 420 and the active areas 414a and 414b disposed thereon. FIG. 5B shows an alternative sensor configuration in which substantially the entire sensor 401 is overcoated with the membrane 450. The membrane 450 may be the same as or compositionally different from the active areas 414a and 414b. For example, the membrane 450 may include a bilayer overcoating the active area 414a or may be a homogeneous membrane overcoating the active area 414b. In certain embodiments, the membrane 450 comprises a copolymer of the present disclosure. For example, but not by way of limitation, the membrane 450 may comprise a copolymer including a first monomer, such as an acrylamide (e.g., an N-alkylacrylamide), and a second monomer including a heterocycle-containing moiety, such as a vinylpyridine, e.g., 4-vinylpyridine.

[0052] It should be further recognized that the positioning of the various electrodes in Figures 5A and 5B may differ from that explicitly illustrated. For example, the locations of the counter electrode 430 and reference electrode 440 may be reversed from the configurations illustrated in Figures 5A and 5B. Similarly, the locations of the working electrodes 410 and 420 are not limited to those explicitly illustrated in Figures 5A and 5B. Figure 5C illustrates an alternative sensor configuration to that shown in Figure 5B, in which the sensor 405 includes the counter electrode 430 and reference electrode 440 located proximal to the sensor tip 404, and the working electrodes 410 and 420 located distal to the sensor tip 404. A sensor configuration in which the working electrodes 410 and 420 are located distal to the sensor tip 404 may be advantageous by providing a larger surface area for deposition of the active areas 414a and 414b (five discrete sensing spots illustratively shown in Figure 5C), thereby facilitating increased signal strength in some instances. Similarly, the central substrate 402 can be omitted in any of the coaxial sensor configurations disclosed herein and can instead support a layer onto which the innermost electrodes are subsequently deposited.

[0053] 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 includes a working electrode and a reference electrode spirally wound around the working electrode. In certain embodiments, an insulator is disposed between the working electrode and the reference electrode. In certain embodiments, a portion of the electrode is exposed to allow reaction of one or more enzymes with the analyte on the electrode. In certain embodiments, each electrode is formed from a fine wire having a diameter of about 0.001 inch or less to about 0.010 inch 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, e.g., about 0.002 inch to about 0.008 inch, or about 0.004 inch to about 0.005 inch. In certain embodiments, the electrodes are formed from plated insulators, plated wire, or bulk conductive material. In certain embodiments, the working electrode comprises a wire formed from platinum, platinum-iridium, palladium, graphite, gold, carbon, a conductive polymer, an alloy, or other conductive material. In certain embodiments, the conductive material is a transparent conductive material. In certain embodiments, the electrode can be formed by various manufacturing techniques (e.g., bulk metal processing, deposition of metal on a substrate, etc.). The electrode can be formed from a plated wire (e.g., platinum or steel wire) or a bulk metal (e.g., platinum wire). In certain embodiments, the electrode can be formed from, for example, a platinum-coated tantalum wire.

[0054] In certain embodiments, the reference electrode, which can function as a reference electrode alone or as a dual electrode countered by a reference, is formed from silver, silver / silver chloride, or the like. In certain embodiments, the reference electrode is juxtaposed to and / or intertwined with or surrounding the working electrode. In certain embodiments, the reference electrode is spirally wound around the working electrode. In certain embodiments, the wire assembly may be coated with or glued together with an insulating material to provide an insulating bond. In certain embodiments, additional electrodes can be included in the sensor tail. For example, but not limited to, a three-electrode system (working electrode, reference electrode, and counter electrode) and / or an additional working electrode (e.g., an electrode for detecting a second analyte). In certain embodiments where the sensor includes two working electrodes, the two working electrodes may be juxtaposed, with the reference electrode disposed therearound (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 wrapped around the working electrode and extends toward the distal end (i.e., the in vivo end) of the sensor tail. In certain embodiments, the reference electrode extends (e.g., spirally) into the exposed region of the working electrode.

[0055] In certain embodiments, one or more working electrodes are spirally wound around the reference electrode. In certain embodiments where two or more working electrodes are provided, the working electrodes can be formed in a double, triple, quadruple, or more spiral configuration along the length of the sensor tail (e.g., surrounding the reference electrode, 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. In certain embodiments, the working electrode comprises a tube within which a reference electrode, including an insulator, is disposed or wound. Alternatively, the reference electrode comprises a tube within which a working electrode, including an insulator, is disposed or wound. In certain embodiments, a polymeric (e.g., insulating) rod is provided, and one or more electrodes (e.g., one or more electrode layers) are disposed thereon (e.g., by electroplating). In certain embodiments, a metal (e.g., steel or tantalum) rod or wire is provided that is coated with an insulating material (as described herein) and has one or more working and reference electrodes disposed thereon. For example, but not by way of limitation, the present disclosure provides sensors, e.g., sensor tails, comprised of one or more tantalum wires, with a conductive material disposed over a portion of the one or more tantalum wires to function as the working electrode. In certain embodiments, a platinum-clad tantalum wire is coated with an insulating material, which is partially coated with a silver / silver chloride composition to function as the reference and / or counter electrode.

[0056] In certain embodiments where an insulator is disposed on a working electrode (e.g., on the platinum surface of the electrode), a portion of the insulator can be stripped or otherwise removed to expose the electrically active surface of the working electrode. For example, but not limited to, the portion of the insulator can be removed manually, by excimer laser, chemical etching, laser ablation, grit blasting, or other methods. Alternatively, to maintain an exposed electrically active surface area, a portion of the electrode can be masked before depositing the insulator. In certain embodiments, the portion of the insulator that is stripped and / or removed can be about 0.1 mm or less to about 2 mm or more in length, e.g., about 0.5 mm to about 0.75 mm 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 can also be used in the insulating layer. In certain embodiments, the insulator includes parylene. In certain embodiments, the insulator includes polyurethane. In one particular embodiment, the insulator comprises polyurethane and polyvinylpyrrolidone. The portions of the sensor that comprise the active area are further described below.

[0057] 2. Enzymes The active area of ​​the analyte sensors of the present disclosure can be configured to detect one or more analytes. In certain embodiments, the active area comprises one or more enzymes for detecting the analytes. In certain embodiments, the analyte sensors of the present disclosure can include two or more active areas, each configured to detect the same or different analytes.

[0058] In certain embodiments, the active area of ​​the sensors of the present disclosure may include one or more enzymes for detecting analytes including, but not limited to, 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, aspartate, asparagine, magnesium, oxygen, pH, phosphorus, potassium, sodium, total protein, uric acid, and the like. Additional analytes include acetoacetate, fructosamine, amylase, cholesterol, chorionic gonadotropin, creatine kinase (e.g., CK-MB), creatine, DNA, RNA, growth factors, growth hormone, hormones (e.g., thyroid-stimulating hormone), steroids, vitamins (e.g., ascorbic acid), neurochemicals (e.g., acetylcholine, norepinephrine, and dopamine), sarcosine, prostate-specific antigen, prothrombin, thrombin, troponin, pirbec, thrombin ... ester, acetaldehyde, ascorbate, galactose, L-xylono-1,4-lactone, glutathione disulfide, hydrogen peroxide, linoleate, 1,3-bisphosphoglycerate, 6-phospho-D-glucono-1,5-lactone, pharmaceuticals (e.g., antibiotics (e.g., gentamicin, vancomycin, etc.), digitoxin, digoxin, theophylline, insulin, and warfarin), drugs of abuse (e.g., analgesics, depressants, stimulants, and hallucinogens), and antibodies. In certain embodiments, the analyte is glucose, a ketone, glutamate, lactate, creatinine, sarcosine, and / or ascorbate. In certain embodiments, the analyte is glucose. In certain embodiments, the analyte is a ketone. In certain embodiments, the analyte is glutamate. In certain embodiments, the analyte is lactate. In certain embodiments, the analyte is creatinine. In certain embodiments, the analyte is sarcosine. In certain embodiments, the analyte is alcohol. In certain embodiments, the analyte is ascorbate. In certain embodiments, the analyte is potassium.

[0059] In certain embodiments, the enzyme may be an oxidoreductase. In certain embodiments, the oxidoreductase may be an enzyme belonging to enzyme class 1. For example, but not by way of limitation, the enzyme may belong to enzyme class 1.1, e.g., 1.1.1, 1.1.3, or 1.4, e.g., 1.4.3. In certain embodiments, the enzyme may be an NAD(P)+-dependent dehydrogenase. In certain embodiments, the enzyme may be an NAD(P)+-dependent dehydrogenase. In certain embodiments, the enzyme may be a flavin adenine dinucleotide (FAD)-dependent oxidoreductase. In certain embodiments, the enzyme may be a hydrolase. In certain embodiments, the hydrolase may be an enzyme belonging to enzyme class 3. For example, but not by way of limitation, the enzyme may belong to enzyme class 3.5, e.g., 3.5.2 or 3.5.3. In certain embodiments, the analyte-responsive active area of ​​an analyte sensor of the present disclosure, e.g., present on a working electrode, can include one or more enzymes that can be used to detect glucose. For example, but not by way of limitation, an analyte sensor of the present disclosure can include an active area containing one or more enzymes for detecting glucose, disposed, e.g., on a first working electrode. In certain embodiments, the analyte sensor can include an active site containing glucose oxidase and / or glucose dehydrogenase for detecting glucose. In certain embodiments, the glucose dehydrogenase can be pyrroloquinoline quinone (PQQ) or a cofactor-dependent glucose dehydrogenase, e.g., flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenase or nicotinamide adenine dinucleotide (NAD)-dependent glucose dehydrogenase. In certain embodiments, the active area can further include diaphorase. In certain embodiments, the enzyme for detecting glucose is FAD-dependent glucose oxidase.

[0060] In certain embodiments, the analyte-responsive active area of ​​an analyte sensor of the present disclosure, e.g., present on a working electrode, can include one or more enzymes that can be used to detect ketones. For example, but not by way of limitation, an analyte sensor of the present disclosure can include an active area including one or more enzymes, e.g., an enzyme system, for detecting ketones, disposed, e.g., on a first working electrode. In certain embodiments, the ketone-responsive active area can include an enzyme system including multiple enzymes that can act in concert to facilitate the detection of ketones, as described in U.S. Patent Publication No. 2020 / 0237275 (the contents of which are incorporated herein by reference in their entirety). In certain embodiments, the analyte sensor can include an active site including β-hydroxybutyrate dehydrogenase for detecting ketones. In certain embodiments, the active area can further include diaphorase. In certain embodiments, the analyte sensor can include β-hydroxybutyrate dehydrogenase and diaphorase for detecting ketones.

[0061] In certain embodiments, the analyte-responsive active area of ​​an analyte sensor of the present disclosure, e.g., present on a working electrode, can include one or more enzymes that can be used to detect lactate. For example, but not by way of limitation, an analyte sensor of the present disclosure can include an active area, e.g., an enzyme system, for detecting lactate, disposed on, e.g., a first working electrode. In certain embodiments, the lactate-responsive active area can include an enzyme system comprising multiple enzymes that can act in concert to facilitate the detection of lactate, as described in U.S. Publication No. 2019 / 0320947 (the contents of which are incorporated herein by reference in their entirety). In certain embodiments, the analyte sensor can include an active site comprising lactate dehydrogenase and / or lactate oxidase. In certain embodiments, the active area can further include diaphorase. In certain embodiments, the analyte sensor can include lactate oxidase and diaphorase.

[0062] In certain embodiments, the analyte-responsive active area of ​​the analyte sensor of the present disclosure, e.g., present on the working electrode, can include one or more enzymes that can be used to detect alcohol. For example, but not by way of limitation, the analyte sensor of the present disclosure can include an active area, e.g., an enzyme system, for detecting alcohol, disposed, e.g., on the first working electrode. In certain embodiments, the ethanol-responsive active area can include an enzyme system including multiple enzymes that can act in concert to facilitate the detection of ethanol, as in U.S. Patent Publication No. 2020 / 0237277 (the contents of which are incorporated herein by reference in their entirety). In certain embodiments, the analyte sensor can include an active site that includes an alcohol dehydrogenase or a ketoreductase. In certain embodiments, the analyte-responsive active area of ​​an analyte sensor of the present disclosure, e.g., present on a working electrode, can include one or more enzymes that can be used to detect creatinine. For example, but not by way of limitation, an analyte sensor of the present disclosure can include an active area, e.g., an enzyme system, for detecting creatinine, disposed, e.g., on a first working electrode. In certain embodiments, the creatinine-responsive active area can include an enzyme system including multiple enzymes that can act in concert to facilitate the detection of creatinine, as described in U.S. Patent Publication No. 2020 / 0241015 (the contents of which are incorporated herein by reference in their entirety). In certain embodiments, the analyte sensor can include an active site including amidohydrolase, creatine amidinohydrolase, and / or sarcosine oxidase.

[0063] In certain embodiments, the analyte-responsive active area of ​​an analyte sensor of the present disclosure, e.g., on a working electrode, can include one or more enzymes that can be used to detect glutamate. For example, but not by way of limitation, an analyte sensor of the present disclosure can include an active area, e.g., on a first working electrode, that includes one or more enzymes, e.g., an enzyme system, for detecting glutamate. In certain embodiments, the analyte sensor can include an active site that includes glutamate dehydrogenase or glutamate oxidase. In certain embodiments, the sensors of the present disclosure do not include an analyte-responsive active area that includes an enzyme. In certain embodiments, the sensors of the present disclosure include a working electrode that does not have an enzyme disposed thereon or that includes an inactive enzyme disposed thereon, e.g., an enzyme that lacks enzymatic activity (e.g., toward the analyte of interest). In certain embodiments, such sensors can be used to detect analytes that can be directly oxidized at the working electrode. For example, but not by way of limitation, a sensor of the present disclosure for detecting ascorbate does not include an enzyme on the working electrode. In certain embodiments, ascorbate is directly oxidized at the working electrode, resulting in a signal that correlates with the level of ascorbate in a biological fluid in contact with the sensor.

[0064] In certain embodiments, a working electrode containing no enzyme or an inactive enzyme can be used to detect background signals. In certain embodiments, the background signal includes signals caused by chemical species other than the analyte of interest present in the sample, such as signals caused by interferents. In certain embodiments, the background signal is a signal caused by one or more interferents. 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. In certain embodiments, the background signal can be used to calibrate, filter, and / or normalize signals obtained from a second working electrode (configured to detect the analyte) present on the same analyte sensor. In certain embodiments, the signal from a working electrode without an enzyme (or with an inactive enzyme) can be subtracted from the signal obtained from a working electrode configured to detect an analyte to determine the signal contribution from the analyte.

[0065] In certain embodiments, one or more enzymes may be present in the active area in various amounts. For example, but not by way of limitation, the enzyme may be present in the active area in an amount of from about 0.05 μg to about 20 μg, e.g., from about 0.1 μg to about 15 μg, from about 0.1 μg to about 10 μg, from about 0.1 μg to about 5 μg, from about 1 μg to about 20 μg, from about 1 μg to about 15 μg, from about 1 μg to about 10 μg, or from about 1 μg to about 5 μg. In certain embodiments, the enzyme is present in the active area in an amount of from about 0.01% to about 50% by weight of the active area composition. For example, but not by way of limitation, the enzyme may be present in the active area at about 0.1% to about 45% by weight, about 0.1% to about 40% by weight, about 0.1% to about 35% by weight, about 0.1% to about 30% by weight, about 0.1% to about 25% by weight, about 0.1% to about 20% by weight, about 0.1% to about 15% by weight, about 0.1% to about 10% by weight, or about 1% to about 10% by weight, or any value therebetween, based on the weight of the total active area composition.

[0066] In certain embodiments, the analyte-responsive active area may further comprise a stabilizer, e.g., to stabilize the enzyme. For example, but not by way of limitation, the stabilizer may be albumin, e.g., serum albumin. Non-limiting examples of serum albumin include bovine serum albumin and human serum albumin. In certain embodiments, the stabilizer is human serum albumin. In certain embodiments, the stabilizer is bovine serum albumin. In certain embodiments, the analyte-responsive active area may comprise about 5% to about 50% by weight of the stabilizer, e.g., about 10% to about 50% by weight, about 15% to about 45% by weight, about 20% to about 40% by weight, about 20% to about 35% by weight, or about 20% to about 30% by weight. In certain embodiments, the analyte-responsive active area may comprise about 5% to about 40% by weight of the stabilizer. In certain embodiments, the analyte-responsive active area may comprise about 5% to about 35% by weight of the stabilizer. In certain embodiments, the analyte-responsive active area can include about 5% to about 30% by weight of stabilizer. In certain embodiments, the analyte-responsive active area can include about 10% to about 30% by weight of stabilizer. In certain embodiments, the analyte-responsive active area can include about 15% to about 35% by weight of stabilizer.

[0067] In certain embodiments, the analyte-responsive active area, e.g., the analyte-responsive active area, may further comprise a cofactor or coenzyme for one or more enzymes present in the analyte-responsive active area. In certain embodiments, the cofactor is nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP) (collectively referred to herein as "NAD(P)"). In certain embodiments, the coenzyme is FAD. In certain embodiments, the analyte-responsive active area may comprise from about 1% to about 50% by weight of the cofactor, e.g., from about 10% to about 50% by weight, from about 15% to about 45% by weight, from about 20% to about 40% by weight, from about 20% to about 35% by weight, from about 20% to about 30% by weight, from about 1% to about 20% by weight, from about 1% to about 10% by weight, or from about 1% to about 5% by weight. In certain embodiments, the analyte-responsive active area can include about 1% to about 20% by weight of the cofactor. In certain embodiments, the analyte-responsive active area can include about 1% to about 10% by weight of the cofactor. In certain embodiments, the analyte-responsive active area can include about 15% to about 35% by weight of the cofactor. In certain embodiments, the cofactor, such as NAD(P) or FAD, can be physically retained within the analyte-responsive active area. For example, but not by way of limitation, a membrane overcoating the analyte-responsive active area can help retain the cofactor within the analyte-responsive active area while allowing sufficient diffusion of the analyte into the area to enable its detection.

[0068] In certain embodiments, the analyte-responsive active area is disposed on a portion of the working electrode. For example, but not by way of limitation, the analyte-responsive active area is disposed on a portion of the working electrode in a spotted pattern, such as two or more spots, three or more spots, four or more spots, five or more spots, or six or more spots on the working electrode. In certain embodiments, the analyte-responsive active area is disposed on a portion of the working electrode in a slotted pattern. In certain embodiments, the analyte-responsive active area is disposed over the entire length of the working electrode or in a continuous pattern on the working electrode. Non-limiting examples of depositing multiple reagent compositions on an electrode surface and forming a discontinuous or continuous perimeter around each reagent composition are described in U.S. Pat. No. 10,327,677, the disclosure of which is incorporated herein by reference. In certain embodiments, when two or more active areas are present in a sensor, the enzymes can be the same or different. For example, but not by way of limitation, when a sensor includes first and second active areas, the enzyme in the first active area and the enzyme in the second active area can be the same. In certain other embodiments, when a sensor includes a first active area and a second active area, the enzyme(s) in the first active area and the enzyme(s) in the second active area can be different, e.g., to detect different analytes or the same analyte.

[0069] In certain embodiments, an analyte sensor can include two working electrodes, e.g., a first active area disposed on a first working electrode and a second active area disposed on a second working electrode. In certain embodiments, the analyte sensors disclosed herein can feature a first analyte-responsive active area and a second active area for detecting an analyte different from the first analyte. For example, but not by way of limitation, such an analyte sensor can include a sensor tail having at least a first working electrode and a second working electrode, a first analyte-responsive active area disposed on the surface of the first working electrode, and a second active area, e.g., a second analyte-responsive active area, configured to detect a different analyte disposed on the surface of the second working electrode. In certain embodiments, when a sensor is configured to detect more than one analyte, detecting each analyte can include applying a potential to each working electrode separately, thereby obtaining a separate signal from each analyte. The signal obtained from each analyte can then be correlated to the analyte concentration using a calibration curve or function or by employing a lookup table. In certain embodiments, the correlation of the analyte signal with the analyte concentration can be performed using a processor.

[0070] In certain other analyte sensor configurations, a first active area and a second active area can be disposed on a single working electrode. A first signal can be obtained from the first active area, and a second signal can be obtained that includes signal contributions from both active areas. In certain embodiments, a first signal can be obtained from the first active area, e.g., at a low potential, and a second signal that includes signal contributions from both active areas can be obtained at a high potential. Subtracting the first signal from the second signal can then allow for determination of the signal contribution arising from the second analyte. The signal contribution from each analyte can then be correlated with the analyte concentration in a manner similar to that described for sensor configurations with multiple working electrodes. In certain embodiments, when a first active area and a second active area configured to detect different analytes, e.g., a second analyte-responsive active area, are disposed on a single working electrode in this manner, one of the active areas can be configured to be individually interrogated to facilitate detection of each analyte. For example, a first analyte-responsive active area or a second active area responsive to a second analyte can produce a signal independently of the other active area.

[0071] It should also be recognized 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. Variations of these parameters can be readily implemented by one of ordinary skill in the art given the benefit of the disclosure herein. In certain embodiments, the active area of ​​the present disclosure can have a thickness of about 0.1 μm to about 100 μm, e.g., about 1 μm to about 90 μm, about 1 μm to about 80 μm, about 1 μm to about 70 μm, about 1 μm to about 60 μm, about 1 μm to about 50 μm, about 1 μm to about 40 μm, about 1 μm to about 30 μm, about 1 μm to about 20 μm, about 0.5 μm to about 10 μm, about 1 μm to about 10 μm, about 1 μm to about 5 μm, or about 0.1 μm to about 5 μm. In certain embodiments, a series of droplets can be applied on top of each other to achieve a desired thickness of the active area without substantially increasing the diameter of the applied droplets (i.e., while maintaining the desired diameter or range).

[0072] 3. Redox mediators In certain embodiments, the analyte sensors disclosed herein may include an electron transfer agent, such as a redox mediator. In certain embodiments, one or more active areas of the analyte sensors disclosed herein may include an electron transfer agent, such as a redox mediator. In certain embodiments, the analyte-responsive active area can include one or more electron transfer agents. For example, but not by way of limitation, an analyte sensor of the present disclosure can include a sensor tail including at least a first working electrode and an analyte-responsive active area disposed on a surface of the first working electrode, the analyte-responsive active area including an electron transfer agent and one or more enzymes responsive to the analyte. In certain embodiments, the analyte sensors of the present disclosure may include two or more active areas, each of which includes an electron transfer agent, or 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. Suitable electron transfer agents for use in the analyte sensors of the present disclosure can facilitate the transfer of electrons to an 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 is proportional to the amount of analyte present. For example, but not by way of limitation, the electron transfer agent transfers electrons between the working electrodes via an oxidoreductase, such as an NAD(P)-dependent oxidoreductase.

[0073] In certain embodiments, suitable electron transfer agents can include electroreducible and electrooxidizable ions, complexes, or molecules (e.g., quinones) with redox potentials that are several hundred millivolts above or below the redox potential of a standard calomel electrode (SCE). In certain embodiments, electron transfer agents can include osmium complexes and other transition metal complexes, such as those described in U.S. Pat. Nos. 6,134,461 and 6,605,200, the disclosures of 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 electron transfer agents include metal compounds or complexes of ruthenium, osmium, iron (e.g., polyvinylferrocene or hexacyanoferrate), or cobalt, including, for example, metallocene compounds thereof. Suitable ligands for the metal complex may also include, for example, bidentate or higher dentate ligands, such as bipyridine, biimidazole, phenanthroline, or pyridyl (imidazole). Other suitable bidentate ligands may include, for example, amino acids, oxalic acid, acetylacetone, diaminoalkanes, or o-diaminoarenes. Any combination of monodentate, bidentate, tridentate, tetradentate, or higher dentate ligands may be present in the metal 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. In certain embodiments, the electron transfer agent is osmium complexed with a tridentate ligand.

[0074] In certain embodiments, the electron transfer agents disclosed herein can include suitable functionality to facilitate covalent attachment to a polymer (also referred to herein as the polymer backbone) in the active area, as discussed further below. For example, but not by way of limitation, electron transfer agents for use in the present disclosure can include polymer-bound electron transfer agents, such as redox polymers. 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, and WO 2022 / 147496, the disclosures of which are incorporated herein by reference in their entireties. In certain embodiments, the electron transfer agent is a bidentate osmium complex bound to a polymer described herein, such as the polymer backbone described in Section II.4 below. In certain embodiments, the polymer-bound electron transfer agent shown in Figure 3 of U.S. Pat. No. 8,444,834 (referred to as "X7") can be used in the sensors of the present disclosure. In certain embodiments, the electron transfer agent is a tridentate osmium complex attached to a polymer backbone as described herein, such as those described in Section II.4 below. In certain embodiments, the polymer-attached electron transfer agents described in WO 2022 / 147496 can be used in the sensors of the present disclosure.

[0075] 4. Polymer backbone In certain embodiments, one or more active sites for facilitating analyte detection may comprise 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 analyte detection by covalent attachment to an enzyme and / or a redox mediator. Non-limiting examples of suitable polymers within the active area include polyvinylpyridines, such as poly(4-vinylpyridine), and polyvinylimidazoles, such as poly(N-vinylimidazole) and poly(1-vinylimidazole), or copolymers thereof, e.g., in which quaternized pyridine groups serve as attachment points for redox mediators or enzymes. In certain embodiments, the polymer is a poly(4-vinylpyridine)-based polymer or a derivative thereof. Non-limiting polymers for use in the present disclosure are disclosed in U.S. Pat. No. 8,444,834.

[0076] Illustrative copolymers that may be suitable for inclusion in the active area include copolymers containing monomer units such as styrene, acrylamide, methacrylamide, or acrylonitrile. In certain embodiments, the polymer is a copolymer of vinylpyridine and styrene. Further non-limiting examples of polymers that may be present in the active area include those described in U.S. Pat. No. 6,605,200, incorporated herein by reference in its entirety, such as poly(acrylic acid), styrene / maleic anhydride copolymer, methyl vinyl ether / maleic anhydride copolymer (GANTREZ polymer), poly(vinylbenzyl chloride), poly(allylamine), polylysine, poly(4-vinylpyridine) quaternized with carboxypentyl groups, and poly(sodium 4-styrenesulfonate). In certain embodiments in which the analyte sensor includes two active sites, the polymers in each active area can be the same or different. In certain embodiments, the enzyme in a given active area can be immobilized. In certain embodiments, the enzyme in the active area is covalently bound to the polymer. Alternatively, or in addition, the enzyme in the active area can be non-covalently associated with the polymer, whereby the non-covalently bound enzyme is physically retained within the polymer.

[0077] In certain embodiments, covalent attachment of one or more enzymes and / or redox mediators to the polymer in a given active area can occur through crosslinks introduced by a suitable crosslinker. Suitable crosslinkers for reaction with free amino groups in the enzyme (e.g., with the free side chain amines on lysines) can include, for example, polyethylene glycol diglycidyl ether (PEGDGE) or other polyepoxides, cyanuric chloride, glutaraldehyde, N-hydroxysuccinimide, imidoesters, epichlorohydrin, or derivatized variants thereof. In certain embodiments, the crosslinker has an average molecular weight (M), for example, of about 200 to 1,000, e.g., about 400. n ) . In certain embodiments, the crosslinker is PEGDGE400. In certain embodiments, the crosslinker may be glutaraldehyde. Suitable crosslinkers for reaction with free carboxylic acid groups in the enzyme may include, for example, carbodiimides. In certain embodiments, crosslinking of the enzyme to the polymer is generally intermolecular. In certain embodiments, crosslinking of the enzyme to the polymer is generally intramolecular.

[0078] 5. Mass transport limiting membrane In certain embodiments, the analyte sensors disclosed herein include a membrane directly overcoating at least one active area, e.g., a first active area. In certain embodiments, the analyte sensors disclosed herein include a membrane directly overcoating at least two active areas of the analyte sensor, e.g., a first active area and / or a second active area. In certain embodiments, the active area is disposed on a working electrode. In certain embodiments, two active areas are disposed on one working electrode. Alternatively, two active areas are disposed on two separate working electrodes. In certain embodiments, the membrane is permeable to the analyte or analytes to be detected in one or more active areas. The membranes of the present disclosure include polymer membranes with diffusivities that are less sensitive to temperature. In certain embodiments, the membranes have the same diffusivity for a given analyte over a predetermined temperature range. For example, but not by way of limitation, the membranes have the same diffusivity for glucose over a predetermined temperature range. In certain embodiments, the membranes have the same diffusivity for lactose over a predetermined temperature range. In certain embodiments, the diffusion rate of an analyte through a membrane depends on the lower critical solution temperature (LCST) of the membrane. At temperatures above the LCST, one or more polymers of the membranes of the present disclosure may be immiscible (e.g., one or more polymers may solidify or crystallize), which may result in a decrease in analyte diffusion through the membrane. In certain embodiments, the decrease in diffusivity of the flux-limiting membrane can offset the increase in diffusivity with increasing temperature, thereby allowing the flux-limiting membrane to have the same diffusivity for an analyte over a temperature range of interest.

[0079] In certain embodiments, the diffusion rate of an analyte through a membrane of the present disclosure changes by less than about 10% in response to a temperature change. In certain embodiments, the diffusion rate of an analyte through a membrane of the present disclosure changes by less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% in response to a temperature change. In certain embodiments, the diffusion rate of an analyte through a membrane of the present disclosure changes by less than about 5% in response to a temperature change. In certain embodiments, the diffusion rate of an analyte through a membrane of the present disclosure changes by less than about 4% in response to a temperature change. In certain embodiments, the diffusion rate of an analyte through a membrane of the present disclosure changes by less than about 3% in response to a temperature change. In certain embodiments, the diffusion rate of an analyte through a membrane of the present disclosure changes by less than about 2% in response to a temperature change. In certain embodiments, the diffusion rate of an analyte through a membrane of the present disclosure changes by less than about 1% in response to a temperature change. In certain embodiments, the change in temperature is, for example, about 30°C, for example, about 25°C, or about 20°C (e.g., Celsius). In certain embodiments, the change in temperature is, for example, about 30°C, for example, about 25°C, or up to about 20°C (e.g., Celsius). In certain embodiments, the diffusion rate of an analyte through a membrane of the present disclosure changes by less than about 5% in response to a change in temperature, for example, a change of about 20°C (e.g., Celsius). In certain embodiments, the diffusion rate of an analyte through a membrane of the present disclosure changes by less than about 4% in response to a change in temperature, for example, a change of about 20°C (e.g., Celsius). In certain embodiments, the diffusion rate of an analyte through a membrane of the present disclosure changes by less than about 3% in response to a change in temperature, for example, a change of about 20°C (e.g., Celsius). In certain embodiments, the diffusion rate of an analyte through a membrane of the present disclosure changes by less than about 2% in response to a change in temperature, for example, a change of about 20°C (e.g., Celsius). In certain embodiments, the diffusion rate of an analyte through a membrane of the present disclosure changes by less than about 1% in response to a change in temperature, for example, a change of about 20° C. (eg, degrees Celsius).

[0080] In certain embodiments, the diffusion rate of an analyte through a membrane of the present disclosure changes by less than about 10% in response to a change in temperature from about 20° C. to about 45° C., e.g., from about 22° C. to about 42° C. In certain embodiments, the diffusion rate of an analyte through a membrane of the present disclosure changes by less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% in response to a change in temperature from about 20° C. to about 45° C., e.g., from about 22° C. to about 42° C. In certain embodiments, the diffusion rate of an analyte through a membrane of the present disclosure changes by less than about 5% in response to a change in temperature from about 20° C. to about 45° C., e.g., from about 22° C. to about 42° C. In certain embodiments, the diffusion rate of an analyte through a membrane of the present disclosure changes by less than about 4% in response to a change in temperature from about 20° C. to about 45° C., e.g., from about 22° C. to about 42° C. In certain embodiments, the diffusion rate of an analyte through a membrane of the present disclosure changes by less than about 3% in response to a change in temperature from about 20° C. to about 45° C., e.g., from about 22° C. to about 42° C. In certain embodiments, the diffusion rate of an analyte through a membrane of the present disclosure changes by less than about 2% in response to a change in temperature from about 20° C. to about 45° C., e.g., from about 22° C. to about 42° C. In certain embodiments, the diffusion rate of an analyte through a membrane of the present disclosure changes by less than about 1% in response to a change in temperature from about 20° C. to about 45° C., e.g., from about 22° C. to about 42° C.

[0081] In certain embodiments, the change in sensitivity of an analyte sensor comprising a membrane of the present disclosure in response to a change in temperature, for example, a temperature change of about 20° C. (e.g., degrees Celsius), is less than about 10%. In certain embodiments, the change in sensitivity of an analyte sensor comprising a membrane of the present disclosure is less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1%. In certain embodiments, the change in sensitivity of an analyte sensor comprising a membrane of the present disclosure in response to a change in temperature is less than about 5%. In certain embodiments, the change in sensitivity of an analyte sensor comprising a membrane of the present disclosure in response to a change in temperature is less than about 4%. In certain embodiments, the change in sensitivity of an analyte sensor comprising a membrane of the present disclosure in response to a change in temperature is less than about 3%. In certain embodiments, the change in sensitivity of an analyte sensor comprising a membrane of the present disclosure in response to a change in temperature is less than about 2%. In certain embodiments, the change in sensitivity of an analyte sensor comprising a membrane of the present disclosure in response to a change in temperature is less than about 1%.

[0082] In certain embodiments, an in vivo analyte sensor comprising one or more membranes of the present disclosure retains its initial sensitivity over time. In certain embodiments, the initial sensitivity of the analyte sensor is the sensitivity (e.g., average sensitivity) of the analyte sensor observed during the first 6 to 24 hours after insertion of the analyte sensor into a subject. In certain embodiments, the initial sensitivity of the analyte sensor is the sensitivity (e.g., average sensitivity) of the analyte sensor observed during manufacture (and / or in vitro testing) of the analyte sensor or a batch of analyte sensors. In certain embodiments, even when exposed to temperature changes, the sensor retains at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% of the initial sensitivity of the analyte sensor over time. In certain embodiments, even after exposure to temperature changes, the sensor retains about 85% to about 100%, e.g., about 90% to about 98%, of its initial sensitivity after 1 day or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, 7 days or more, 10 days or more, 14 days or more, 15 days or more, 1 month or more, 2 months or more, 4 months or more, 6 months or more, 9 months or more, or 1 year or more. In certain embodiments, even after exposure to temperature changes, the sensor retains about 85% to about 100%, e.g., about 90% to about 98%, or about 90% to about 100% of its initial sensitivity after about 5 days or more. In certain embodiments, even after exposure to temperature changes, the sensor retains about 85% to about 100%, e.g., about 90% to about 98%, or about 90% to about 100% of its initial sensitivity after about 10 days or more. In certain embodiments, even after exposure to temperature changes, the sensor retains about 85% to about 100%, e.g., about 90% to about 98%, or about 90% to about 100% of its initial sensitivity after about 14 days or more. In certain embodiments, even after exposure to temperature changes, the sensor retains about 85% to about 100%, e.g., about 90% to about 98%, or about 90% to about 100% of its initial sensitivity after about 15 days or more.In certain embodiments, an in vivo analyte sensor comprising one or more membranes of the present disclosure retains its initial sensitivity for 15 days or more when exposed to temperature changes.

[0083] In certain embodiments, an analyte sensor of the present disclosure has a sensitivity (e.g., initial sensitivity) of about 0.1 nA / mM or greater. In certain embodiments, an in vivo analyte sensor comprising one or more membranes of the present disclosure has a sensitivity of about 0.1 nA / mM or greater, about 0.5 nA / mM or greater, about 1 nA / mM or greater, about 1.5 nA / mM or greater, about 2 nA / mM or greater, about 2.5 nA / mM or greater, about 5 nA / mM or greater, about 7.5 nA / mM or greater, about 10 nA / mM or greater, about 12.5 nA / mM or greater, or about 15 nA / mM or greater. In certain embodiments, the membrane can absorb about 5% to about 95% of its mass in water, e.g., about 5% to about 95%, about 5% to about 90%, about 5% to about 85%, about 10% to about 95%, about 15% to about 95%, about 20% to about 95%, about 25% to about 95%, about 30% to about 95%, about 5% to about 30%, about 5% to about 35%, about 5% to about 25%, or about 5% to about 20%. In certain embodiments, the change in the amount of water absorbed by a membrane of the present disclosure in response to a change in temperature is less than about 5%, e.g., less than about 1%.

[0084] In certain embodiments, the membranes of the present disclosure that exhibit reduced temperature sensitivity comprise a copolymer of at least two different types of monomers. In certain embodiments, the copolymer has alternating monomer subunits. In certain other embodiments, the copolymer may be a block copolymer, which comprises two or more types of homopolymer subunits linked by covalent bonds. The copolymers of the present disclosure include block copolymers. In certain embodiments, the copolymer may be a graft polymer. In certain embodiments, the copolymer may be a random copolymer. In certain embodiments, the membranes of the present disclosure may include polymers containing heterocycle-containing components, e.g., monomers. In certain embodiments, the copolymers of the present disclosure include heterocycle-containing monomers. In certain embodiments, the copolymers of the present disclosure include heterocycles. In certain embodiments, heterocycles refer to cyclic moieties containing one or more heteroatoms, such as nitrogen (N), phosphate (P), oxygen (O), sulfur (S), and silicon (Si). In certain embodiments, the heterocycles include one or more nitrogens. In certain embodiments, the heterocycles include one nitrogen.

[0085] In certain embodiments, the heterocycle is selected from the group consisting of furan, thiophene, pyrrole, pyridine, pyrimidine, imidazole, oxadiazole, isoxazole, oxazole, pyrazole, isothiazole, thiazole, pyrazine, isoquinoline, quinoline, benzofuran, benzimidazole, or derivatives thereof. In certain embodiments, the heterocycle is furan or a derivative thereof. In certain embodiments, the heterocycle is thiophene or a derivative thereof. In certain embodiments, the heterocycle is pyrrole or a derivative thereof. In certain embodiments, the heterocycle is pyrimidine or a derivative thereof. In certain embodiments, the heterocycle is oxadiazole or a derivative thereof. In certain embodiments, the heterocycle is isoxazole or a derivative thereof. In certain embodiments, the heterocycle is oxazole or a derivative thereof. In certain embodiments, the heterocycle is pyrazole or a derivative thereof. In certain embodiments, the heterocycle is isothiazole or a derivative thereof. In certain embodiments, the heterocycle is thiazole or a derivative thereof. In certain embodiments, the heterocycle is pyrazine or a derivative thereof. In certain embodiments, the heterocycle is isoquinoline or a derivative thereof. In certain embodiments, the heterocycle is quinoline or a derivative thereof. In certain embodiments, the heterocycle is benzofuran or a derivative thereof. In certain embodiments, the heterocycle is benzimidazole or a derivative thereof. In certain embodiments, the heterocycle is imidazole or a derivative thereof. In certain embodiments, the heterocycle is pyridine or a derivative thereof. In certain embodiments, the monomers disclosed herein, e.g., derivatives of heterocycles, include forms of monomers that include one or more substituents and / or functional groups, e.g., alkene functional groups, e.g., vinyl functional groups.

[0086] In certain embodiments, the membrane of the present disclosure may include a polymer, such as a copolymer, containing a pyridine component or a derivative thereof. In certain embodiments, the heterocycle-containing monomer is vinylpyridine or a derivative thereof. Non-limiting examples of vinylpyridines include 2-vinylpyridine and 4-vinylpyridine. In certain embodiments, the heterocycle-containing monomer is 4-vinylpyridine. In certain embodiments, the heterocycle-containing monomer is 2-vinylpyridine. In certain embodiments, the membrane of the present disclosure may include a polymer, such as a copolymer, containing an imidazole component. In certain embodiments, the heterocycle-containing monomer is vinylimidazole. Non-limiting examples of vinylimidazole include 1-vinylimidazole (also referred to as N-vinylimidazole), 2-vinylimidazole, and 4-vinylimidazole. In certain embodiments, the heterocycle-containing monomer is 1-vinylimidazole. In certain embodiments, the heterocycle-containing monomer is 2-vinylimidazole. In certain embodiments, the heterocycle-containing monomer is 4-vinylimidazole.

[0087] In certain embodiments, the polymer, e.g., copolymer, may comprise a heterocycle-containing component, e.g., a monomer, in an amount of at least about 3%, at least about 5%, at least about 7%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% by weight of the total copolymer. In certain embodiments, the polymer, e.g., copolymer, may comprise a heterocycle-containing component, e.g., 4-vinylpyridine, in an amount of at least about 30% by weight of the total copolymer. In certain embodiments, a polymer, e.g., a copolymer, may contain a heterocycle-containing component, e.g., 4-vinylpyridine, in an amount of at least about 40% by weight of the total copolymer. In certain embodiments, a polymer, e.g., a copolymer, may contain a heterocycle-containing component, e.g., 4-vinylpyridine, in an amount of at least about 50% by weight of the total copolymer. In certain embodiments, a polymer, e.g., a copolymer, may contain a heterocycle-containing component, e.g., 4-vinylpyridine, in an amount of at least about 60% by weight of the total copolymer. In certain embodiments, a polymer, e.g., a copolymer, may contain a heterocycle-containing component, e.g., 4-vinylpyridine, in an amount of at least about 70% by weight of the total copolymer. In certain embodiments, a polymer, e.g., a copolymer, may contain a heterocycle-containing component, e.g., 4-vinylpyridine, in an amount of at least about 80% by weight of the total copolymer. In certain embodiments, a polymer, e.g., a copolymer, may contain a heterocycle-containing component, e.g., 4-vinylpyridine, in an amount of from about 30% to about 80% by weight of the total copolymer. In certain embodiments, the polymer, e.g., copolymer, can include a heterocycle-containing component, e.g., 4-vinylpyridine, in an amount of about 40% to about 80% by weight of the total copolymer. In certain embodiments, the polymer, e.g., copolymer, can include a heterocycle-containing component, e.g., 4-vinylpyridine, in an amount of about 40% to about 70% by weight of the total copolymer.In certain embodiments, a polymer, e.g., a copolymer, can contain a heterocycle-containing component, e.g., 4-vinylpyridine, in an amount of about 30% to about 65% by weight of the total copolymer. In certain embodiments, a polymer, e.g., a copolymer, can contain a heterocycle-containing component, e.g., 4-vinylpyridine, in an amount of about 10% to about 50% by weight of the total copolymer. In certain embodiments, a polymer, e.g., a copolymer, can contain a heterocycle-containing component, e.g., 4-vinylpyridine, in an amount of about 20% to about 50% by weight of the total copolymer. In certain embodiments, a polymer, e.g., a copolymer, can contain a heterocycle-containing component, e.g., 4-vinylpyridine, in an amount of about 30% to about 50% by weight of the total copolymer. In certain embodiments, a polymer, e.g., a copolymer, can contain a heterocycle-containing component, e.g., 4-vinylpyridine, in an amount of about 35% to about 45% by weight of the total copolymer. In certain embodiments, the polymer, eg, copolymer, can include a heterocycle-containing component, eg, 4-vinylpyridine, in an amount of about 40% by weight of the total copolymer.

[0088] In certain embodiments, a polymer, e.g., a copolymer, can contain at least about 3%, at least about 5%, at least about 7%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% mole percent (mer%) of a heterocycle-containing component, e.g., a monomer. In certain embodiments, a polymer, e.g., a copolymer, can contain at least about 80% mole percent of a heterocycle-containing component, e.g., a monomer. In certain embodiments, a polymer, e.g., a copolymer, can contain at least about 70% mole percent of a heterocycle-containing component, e.g., a monomer. In certain embodiments, a polymer, e.g., a copolymer, may contain at least about 65% mole percent of a heterocycle-containing component, e.g., a monomer. In certain embodiments, a polymer, e.g., a copolymer, may contain at least about 60% mole percent of a heterocycle-containing component, e.g., a monomer. In certain embodiments, a polymer, e.g., a copolymer, may contain at least about 55% mole percent of a heterocycle-containing component, e.g., a monomer, such as 4-vinylpyridine. In certain embodiments, a polymer, e.g., a copolymer, may contain at least about 50% mole percent of a heterocycle-containing component, e.g., a monomer, such as 4-vinylpyridine. In certain embodiments, a polymer, e.g., a copolymer, may contain at least about 45% mole percent of a heterocycle-containing component, e.g., a monomer. In certain embodiments, a polymer, e.g., a copolymer, may contain at least about 40% mole percent of a heterocycle-containing component, e.g., a monomer, such as 4-vinylpyridine. In certain embodiments, the polymer, eg, copolymer, can include at least about 35% mole percent of a heterocycle-containing component, eg, a monomer, such as 4-vinylpyridine.In certain embodiments, a polymer, e.g., a copolymer, can contain a heterocycle-containing component, e.g., a monomer, such as 4-vinylpyridine, at a mole percent of at least about 30%. In certain embodiments, a polymer, e.g., a copolymer, can contain a heterocycle-containing component, e.g., a monomer, such as 4-vinylpyridine, at a mole percent of at least about 25%. In certain embodiments, a polymer, e.g., a copolymer, can contain a heterocycle-containing component, e.g., 4-vinylpyridine, at a mole percent of about 30% to about 80% of the total copolymer. In certain embodiments, a polymer, e.g., a copolymer, can contain a heterocycle-containing component, e.g., 4-vinylpyridine, at a mole percent of about 40% to about 80% of the total copolymer. In certain embodiments, a polymer, e.g., a copolymer, can contain a heterocycle-containing component, e.g., 4-vinylpyridine, at a mole percent of about 40% to about 70% of the total copolymer. In certain embodiments, a polymer, e.g., a copolymer, can contain a heterocycle-containing component, e.g., 4-vinylpyridine, at a molar percentage of about 30% to about 65% of the total copolymer. In certain embodiments, a polymer, e.g., a copolymer, can contain a heterocycle-containing component, e.g., 4-vinylpyridine, at a molar percentage of about 10% to about 50% of the total copolymer. In certain embodiments, a polymer, e.g., a copolymer, can contain a heterocycle-containing component, e.g., 4-vinylpyridine, at a molar percentage of about 20% to about 50% of the total copolymer. In certain embodiments, a polymer, e.g., a copolymer, can contain a heterocycle-containing component, e.g., 4-vinylpyridine, at a molar percentage of about 30% to about 50% of the total copolymer. In certain embodiments, a polymer, e.g., a copolymer, can contain a heterocycle-containing component, e.g., 4-vinylpyridine, at a molar percentage of about 35% to about 45% of the total copolymer. In certain embodiments, the polymer, eg, copolymer, can include a heterocycle-containing component, eg, 4-vinylpyridine, at a mole percent of about 40% of the total copolymer.

[0089] In certain embodiments, the membranes of the present disclosure can include an acrylamide component, e.g., a monomer. For example, but not by way of limitation, polymers, e.g., copolymers, of the present disclosure can include an acrylamide component, e.g., a monomer. In certain embodiments, the acrylamide can be an N-alkyl acrylamide. In certain embodiments, the alkyl group is a C1-C6 linear or branched alkyl group or a C3-C6 cycloalkyl group. In certain embodiments, the alkyl group is a C1-C6 linear alkyl group (e.g., a C1-C6 linear alkyl group). Non-limiting examples of C1-C6 linear alkyl groups include methyl, ethyl, propyl, butyl, pentyl, and hexyl. In certain embodiments, the C1-C6 linear alkyl group includes a methyl group. In certain embodiments, the alkyl group is a C3-C6 cycloalkyl group. Non-limiting examples of C3-C6 cycloalkyl groups include cyclopropane, cyclobutene, cyclopentane, cyclohexane, and the like. In certain embodiments, the alkyl group is a branched alkyl group. Non-limiting examples of branched alkyl groups include isopropyl, isobutyl, sec-butyl, tert-butyl, tert-pentyl, neopentyl, isopentyl, sec-pentyl, 3-pentyl, sec-isopentyl, active pentyl, isohexyl, tert-hexyl, neohexyl, sec-hexyl, etc. In certain embodiments, the branched alkyl group is an isopentyl group. In certain embodiments, the branched alkyl group is a tert-butyl group.

[0090] In certain embodiments, the N-alkylacrylamide is methylacrylamide, N-ethylacrylamide, N-isopropylacrylamide, or N-tert-butylacrylamide. In certain embodiments, the copolymer included in the membrane of the present disclosure comprises methylacrylamide. In certain embodiments, the copolymer included in the membrane of the present disclosure comprises N-ethylacrylamide. In certain embodiments, the copolymer included in the membrane of the present disclosure comprises N-tert-butylacrylamide. In certain embodiments, the copolymer included in the membrane of the present disclosure comprises N-isopropylacrylamide.

[0091] In certain embodiments, the polymer, e.g., copolymer, may comprise an acrylamide component, e.g., a monomer, in an amount of at least about 3% by weight, at least about 5% by weight, at least about 7% by weight, at least about 10% by weight, at least about 15% by weight, at least about 20% by weight, at least about 25% by weight, at least about 30% by weight, at least about 35% by weight, at least about 40% by weight, at least about 45% by weight, at least about 50% by weight, at least about 55% by weight, at least about 60% by weight, at least about 65% by weight, at least about 70% by weight, at least about 75% by weight, at least about 80% by weight, at least about 85% by weight, at least about 90% by weight, or at least about 95% by weight of the total copolymer. In certain embodiments, the acrylamide component is an N-alkylacrylamide. In certain embodiments, the N-alkylacrylamide is N-isopropylacrylamide. In certain embodiments, the polymer, e.g., copolymer, may comprise an acrylamide component, e.g., an N-alkylacrylamide, in an amount of at least about 20% by weight of the total copolymer. In certain embodiments, the polymer, e.g., copolymer, may contain an acrylamide component, e.g., N-alkylacrylamide, in an amount of at least about 25% by weight of the total copolymer. In certain embodiments, the polymer, e.g., copolymer, may contain an acrylamide component, e.g., N-alkylacrylamide, in an amount of at least about 30% by weight of the total copolymer. In certain embodiments, the polymer, e.g., copolymer, may contain an acrylamide component, e.g., N-alkylacrylamide, in an amount of at least about 35% by weight of the total copolymer. In certain embodiments, the polymer, e.g., copolymer, may contain an acrylamide component, e.g., N-alkylacrylamide, in an amount of at least about 40% by weight of the total copolymer. In certain embodiments, the polymer, e.g., copolymer, may contain an acrylamide component, e.g., N-alkylacrylamide, in an amount of at least about 45% by weight of the total copolymer. In certain embodiments, the polymer, e.g., copolymer, may contain an acrylamide component, e.g., N-alkylacrylamide, in an amount of at least about 50% by weight of the total copolymer.In certain embodiments, the polymer, e.g., copolymer, may comprise an acrylamide component, e.g., N-alkylacrylamide, in an amount of at least about 55% by weight of the total copolymer. In certain embodiments, the polymer, e.g., copolymer, may comprise an acrylamide component, e.g., N-alkylacrylamide, in an amount of at least about 60% by weight of the total copolymer. In certain embodiments, the polymer, e.g., copolymer, may comprise an acrylamide component, e.g., N-alkylacrylamide, in an amount of at least about 65% by weight of the total copolymer. In certain embodiments, the polymer, e.g., copolymer, may comprise an acrylamide component, e.g., N-alkylacrylamide, in an amount of at least about 70% by weight of the total copolymer. In certain embodiments, the polymer, e.g., copolymer, may comprise an acrylamide component, e.g., N-alkylacrylamide, in an amount of at least about 75% by weight of the total copolymer. In certain embodiments, the polymer, e.g., copolymer, may comprise an acrylamide component, e.g., N-alkylacrylamide, in an amount of at least about 80% by weight of the total copolymer. In certain embodiments, the polymer, e.g., copolymer, comprises an acrylamide component, e.g., N-alkylacrylamide, in an amount of about 50% by weight of the total copolymer. In certain embodiments, the polymer, e.g., copolymer, comprises an acrylamide component, e.g., N-alkylacrylamide, in an amount of about 55% by weight of the total copolymer. In certain embodiments, the polymer, e.g., copolymer, comprises an acrylamide component, e.g., N-alkylacrylamide, in an amount of about 60% by weight of the total copolymer. In certain embodiments, the polymer, e.g., copolymer, may comprise an acrylamide component, e.g., N-alkylacrylamide, in an amount of about 10% to about 80% by weight of the total copolymer. In certain embodiments, the polymer, e.g., copolymer, may comprise an acrylamide component, e.g., N-alkylacrylamide, in an amount of about 20% to about 80% by weight of the total copolymer. In certain embodiments, the polymer, e.g., copolymer, may comprise an acrylamide component, e.g., N-alkylacrylamide, in an amount of about 20% to about 70% by weight of the total copolymer.In certain embodiments, the polymer, e.g., copolymer, may contain an acrylamide component, e.g., N-alkylacrylamide, in an amount of about 30% to about 80% by weight of the total copolymer. In certain embodiments, the polymer, e.g., copolymer, may contain an acrylamide component, e.g., N-alkylacrylamide, in an amount of about 40% to about 80% by weight of the total copolymer. In certain embodiments, the polymer, e.g., copolymer, may contain an acrylamide component, e.g., N-alkylacrylamide, in an amount of about 50% to about 80% by weight of the total copolymer. In certain embodiments, the polymer, e.g., copolymer, may contain an acrylamide component, e.g., N-alkylacrylamide, in an amount of about 50% to about 70% by weight of the total copolymer. In certain embodiments, the polymer, e.g., copolymer, may contain an acrylamide component, e.g., N-alkylacrylamide, in an amount of about 30% to about 60% by weight of the total copolymer. In certain embodiments, the polymer, e.g., copolymer, can include an acrylamide component, e.g., N-alkylacrylamide, in an amount of about 30% to about 50% by weight of the total copolymer. In certain embodiments, the polymer, e.g., copolymer, can include an acrylamide component, e.g., N-alkylacrylamide, in an amount of about 30% to about 65% by weight of the total copolymer. In certain embodiments, the polymer, e.g., copolymer, can include an acrylamide component, e.g., N-alkylacrylamide, in an amount of about 55% to about 65% by weight of the total copolymer.

[0092] In certain embodiments, the polymer, e.g., copolymer, can comprise an N-isopropylacrylamide component, e.g., monomer, in an amount of at least about 3%, at least about 5%, at least about 7%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% by weight of the total copolymer. In certain embodiments, the polymer, e.g., copolymer, can comprise N-isopropylacrylamide in an amount of at least about 20% by weight of the total copolymer. In certain embodiments, the polymer, e.g., copolymer, may comprise N-isopropylacrylamide in an amount of at least about 25% by weight of the total copolymer. In certain embodiments, the polymer, e.g., copolymer, may comprise N-isopropylacrylamide in an amount of at least about 30% by weight of the total copolymer. In certain embodiments, the polymer, e.g., copolymer, may comprise N-isopropylacrylamide in an amount of at least about 35% by weight of the total copolymer. In certain embodiments, the polymer, e.g., copolymer, may comprise N-isopropylacrylamide in an amount of at least about 40% by weight of the total copolymer. In certain embodiments, the polymer, e.g., copolymer, may comprise N-isopropylacrylamide in an amount of at least about 45% by weight of the total copolymer. In certain embodiments, the polymer, e.g., copolymer, may comprise N-isopropylacrylamide in an amount of at least about 50% by weight of the total copolymer. In certain embodiments, the polymer, e.g., copolymer, may comprise N-isopropylacrylamide in an amount of at least about 55% by weight of the total copolymer. In certain embodiments, the polymer, eg, copolymer, can include N-isopropylacrylamide in an amount of at least about 60% by weight of the total copolymer.In certain embodiments, the polymer, e.g., copolymer, may comprise N-isopropylacrylamide in an amount of at least about 65% by weight of the total copolymer. In certain embodiments, the polymer, e.g., copolymer, may comprise N-isopropylacrylamide in an amount of at least about 70% by weight of the total copolymer. In certain embodiments, the polymer, e.g., copolymer, may comprise N-isopropylacrylamide in an amount of at least about 75% by weight of the total copolymer. In certain embodiments, the polymer, e.g., copolymer, may comprise N-isopropylacrylamide in an amount of at least about 80% by weight of the total copolymer. In certain embodiments, the polymer, e.g., copolymer, may comprise N-isopropylacrylamide in an amount of about 50% by weight of the total copolymer. In certain embodiments, the polymer, e.g., copolymer, may comprise N-isopropylacrylamide in an amount of about 55% by weight of the total copolymer. In certain embodiments, the polymer, e.g., copolymer, may comprise N-isopropylacrylamide in an amount of about 60% by weight of the total copolymer. In certain embodiments, the polymer, e.g., copolymer, may comprise N-isopropylacrylamide in an amount of about 10% to about 80% by weight of the total copolymer. In certain embodiments, the polymer, e.g., copolymer, may comprise N-isopropylacrylamide in an amount of about 20% to about 80% by weight of the total copolymer. In certain embodiments, the polymer, e.g., copolymer, may comprise N-isopropylacrylamide in an amount of about 30% to about 80% by weight of the total copolymer. In certain embodiments, the polymer, e.g., copolymer, may comprise N-isopropylacrylamide in an amount of about 40% to about 80% by weight of the total copolymer. In certain embodiments, the polymer, e.g., copolymer, may comprise N-isopropylacrylamide in an amount of about 40% to about 70% by weight of the total copolymer. In certain embodiments, the polymer, e.g., copolymer, may comprise N-isopropylacrylamide in an amount of about 50% to about 80% by weight of the total copolymer. In certain embodiments, the polymer, eg, copolymer, can include N-isopropylacrylamide in an amount of about 50% to about 70% by weight of the total copolymer.In certain embodiments, the polymer, e.g., copolymer, can include N-isopropylacrylamide in an amount of about 30% to about 50% by weight of the total copolymer. In certain embodiments, the polymer, e.g., copolymer, can include N-isopropylacrylamide in an amount of about 30% to about 65% by weight of the total copolymer. In certain embodiments, the polymer, e.g., copolymer, can include N-isopropylacrylamide in an amount of about 55% to about 65% by weight of the total copolymer.

[0093] In certain embodiments, the polymer, e.g., copolymer, may contain an acrylamide component, e.g., a monomer, at a molar percentage of at least about 3%, at least about 5%, at least about 7%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%. In certain embodiments, the acrylamide component is an N-alkylacrylamide. In certain embodiments, the N-alkylacrylamide is N-isopropylacrylamide. In certain embodiments, the polymer, e.g., copolymer, may contain an acrylamide component, e.g., an N-alkylacrylamide, at a molar percentage of at least about 20%. As shown in Table 2, membranes containing copolymers containing at least 20% molar percent of acrylamide (e.g., N-alkylacrylamide) result in membranes with reduced temperature sensitivity (e.g., compared to control membranes). In certain embodiments, a polymer, e.g., a copolymer, can contain an acrylamide component, e.g., N-alkylacrylamide, at a molar percent of at least about 25%. In certain embodiments, a polymer, e.g., a copolymer, can contain an acrylamide component, e.g., N-alkylacrylamide, at a molar percent of at least about 30%. In certain embodiments, a polymer, e.g., a copolymer, can contain an acrylamide component, e.g., N-alkylacrylamide, at a molar percent of at least about 35%. In certain embodiments, a polymer, e.g., a copolymer, can contain an acrylamide component, e.g., N-alkylacrylamide, at a molar percent of at least about 40%. In certain embodiments, a polymer, e.g., a copolymer, can contain an acrylamide component, e.g., N-alkylacrylamide, at a molar percent of at least about 45%. In certain embodiments, the polymer, eg, copolymer, can include an acrylamide component, eg, an N-alkylacrylamide, in a mole percent of at least about 50%.In certain embodiments, a polymer, e.g., a copolymer, may contain an acrylamide component, e.g., an N-alkylacrylamide, at a molar percentage of at least about 55%. In certain embodiments, a polymer, e.g., a copolymer, may contain an acrylamide component, e.g., an N-alkylacrylamide, at a molar percentage of at least about 60%. In certain embodiments, a polymer, e.g., a copolymer, may contain an acrylamide component, e.g., an N-alkylacrylamide, at a molar percentage of at least about 65%. In certain embodiments, a polymer, e.g., a copolymer, may contain an acrylamide component, e.g., an N-alkylacrylamide, at a molar percentage of at least about 70%. In certain embodiments, a polymer, e.g., a copolymer, may contain an acrylamide component, e.g., an N-alkylacrylamide, at a molar percentage of at least about 75%. In certain embodiments, a polymer, e.g., a copolymer, may contain an acrylamide component, e.g., an N-alkylacrylamide, at a molar percentage of at least about 80%. In certain embodiments, the polymer, e.g., copolymer, comprises an acrylamide component, e.g., N-alkylacrylamide, at a molar percentage of about 50%. As shown in Table 2, membranes comprising copolymers comprising at least 50% molar percentage of acrylamide (e.g., N-alkylacrylamide) result in membranes with significantly reduced temperature sensitivity (e.g., compared to control membranes). In certain embodiments, the polymer, e.g., copolymer, may comprise at least about 55% molar percentage of the acrylamide component, e.g., N-alkylacrylamide. In certain embodiments, the polymer, e.g., copolymer, may comprise at least about 60% molar percentage of the acrylamide component, e.g., N-alkylacrylamide. In certain embodiments, the polymer, e.g., copolymer, may comprise at least about 65% molar percentage of the acrylamide component, e.g., N-alkylacrylamide. In certain embodiments, the polymer, e.g., copolymer, may comprise from about 10% to about 80% molar percentage of the total copolymer.In certain embodiments, the polymer, e.g., copolymer, may comprise an acrylamide component, e.g., N-alkylacrylamide, at a molar percentage of about 20% to about 80% of the total copolymer. In certain embodiments, the polymer, e.g., copolymer, may comprise an acrylamide component, e.g., N-alkylacrylamide, at a molar percentage of about 30% to about 80% by weight of the total copolymer. In certain embodiments, the polymer, e.g., copolymer, may comprise an acrylamide component, e.g., N-alkylacrylamide, at a molar percentage of about 40% to about 80% by weight of the total copolymer. In certain embodiments, the polymer, e.g., copolymer, may comprise an acrylamide component, e.g., N-alkylacrylamide, at a molar percentage of about 50% to about 80% of the total copolymer. In certain embodiments, the polymer, e.g., copolymer, may comprise an acrylamide component, e.g., N-alkylacrylamide, at a molar percentage of about 20% to about 70% of the total copolymer. In certain embodiments, a polymer, e.g., a copolymer, can contain an acrylamide component, e.g., an N-alkylacrylamide, at a molar percentage of about 30% to about 70% of the total copolymer. In certain embodiments, a polymer, e.g., a copolymer, can contain an acrylamide component, e.g., an N-alkylacrylamide, at a molar percentage of about 40% to about 70% of the total copolymer. In certain embodiments, a polymer, e.g., a copolymer, can contain an acrylamide component, e.g., an N-alkylacrylamide, at a molar percentage of about 50% to about 70% of the total copolymer. In certain embodiments, a polymer, e.g., a copolymer, can contain an acrylamide component, e.g., an N-alkylacrylamide, at a molar percentage of about 30% to about 50% of the total copolymer. In certain embodiments, a polymer, e.g., a copolymer, can contain an acrylamide component, e.g., an N-alkylacrylamide, at a molar percentage of about 30% to about 60% of the total copolymer. In certain embodiments, the polymer, eg, copolymer, can include an acrylamide component, eg, an N-alkylacrylamide, at a mole percent of about 30% to about 65% of the total copolymer.In certain embodiments, the polymer, e.g., copolymer, can include an acrylamide component, e.g., an N-alkylacrylamide, at a mole percent of about 55% to about 65% of the total copolymer. In certain embodiments, the polymer, e.g., copolymer, can include an acrylamide component, e.g., an N-alkylacrylamide, at a mole percent of about 60% of the total copolymer.

[0094] In certain embodiments, a polymer, e.g., a copolymer, may contain N-isopropylacrylamide at a molar percentage of at least about 3%, at least about 5%, at least about 7%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%. In certain embodiments, a polymer, e.g., a copolymer, may contain N-isopropylacrylamide at a molar percentage of at least about 20%. In certain embodiments, a polymer, e.g., a copolymer, may contain N-isopropylacrylamide at a molar percentage of at least about 25%. In certain embodiments, a polymer, e.g., a copolymer, may contain N-isopropylacrylamide at a molar percentage of at least about 30%. In certain embodiments, a polymer, e.g., a copolymer, may comprise N-isopropylacrylamide at a molar percentage of at least about 35%. In certain embodiments, a polymer, e.g., a copolymer, may comprise N-isopropylacrylamide at a molar percentage of at least about 40%. In certain embodiments, a polymer, e.g., a copolymer, may comprise N-isopropylacrylamide at a molar percentage of at least about 45%. In certain embodiments, a polymer, e.g., a copolymer, may comprise N-isopropylacrylamide at a molar percentage of at least about 50%. In certain embodiments, a polymer, e.g., a copolymer, may comprise N-isopropylacrylamide at a molar percentage of at least about 55%. In certain embodiments, a polymer, e.g., a copolymer, may comprise N-isopropylacrylamide at a molar percentage of at least about 60%. In certain embodiments, a polymer, e.g., a copolymer, may comprise N-isopropylacrylamide at a molar percentage of at least about 65%.In certain embodiments, a polymer, e.g., a copolymer, can comprise N-isopropylacrylamide at a molar percentage of at least about 70%. In certain embodiments, a polymer, e.g., a copolymer, can comprise N-isopropylacrylamide at a molar percentage of at least about 75%. In certain embodiments, a polymer, e.g., a copolymer, can comprise N-isopropylacrylamide at a molar percentage of at least about 80%. In certain embodiments, a polymer, e.g., a copolymer, can comprise N-isopropylacrylamide at a molar percentage of from about 10% to about 80% of the total copolymer. In certain embodiments, a polymer, e.g., a copolymer, can comprise N-isopropylacrylamide at a molar percentage of from about 20% to about 80% of the total copolymer. In certain embodiments, a polymer, e.g., a copolymer, can comprise N-isopropylacrylamide at a molar percentage of from about 30% to about 80% by weight of the total copolymer. In certain embodiments, the polymer, e.g., copolymer, may comprise N-isopropylacrylamide in an amount of about 40% to about 80% by weight of the total copolymer. In certain embodiments, the polymer, e.g., copolymer, may comprise N-isopropylacrylamide in an amount of about 50% to about 80% by mole percent of the total copolymer. In certain embodiments, the polymer, e.g., copolymer, may comprise N-isopropylacrylamide in an amount of about 30% to about 50% by weight of the total copolymer. In certain embodiments, the polymer, e.g., copolymer, may comprise N-isopropylacrylamide in an amount of about 30% to about 60% by weight of the total copolymer. In certain embodiments, the polymer, e.g., copolymer, may comprise N-isopropylacrylamide in an amount of about 20% to about 70% by weight of the total copolymer. In certain embodiments, the polymer, e.g., copolymer, may comprise N-isopropylacrylamide in an amount of about 30% to about 70% by weight of the total copolymer. In certain embodiments, the polymer, eg, copolymer, can include N-isopropylacrylamide in an amount of about 40% to about 70% by weight of the total copolymer.In certain embodiments, the polymer, e.g., copolymer, may comprise N-isopropylacrylamide at a molar percentage of about 50% to about 70% of the total copolymer. In certain embodiments, the polymer, e.g., copolymer, may comprise N-isopropylacrylamide in an amount of about 30% to about 65% by weight of the total copolymer. In certain embodiments, the polymer, e.g., copolymer, may comprise N-isopropylacrylamide at a molar percentage of about 55% to about 65% of the total copolymer. In certain embodiments, the polymer, e.g., copolymer, may comprise N-isopropylacrylamide at a molar percentage of about 60% of the total copolymer.

[0095] In certain embodiments, the copolymer of the presently disclosed membrane can include a heterocycle component, e.g., a heterocycle-containing monomer, and an acrylamide component, e.g., an acrylamide-containing monomer, in a ratio, e.g., a molar ratio, of about 10:1 to about 1:10. In certain embodiments, the heterocycle is pyridine (e.g., 4-vinylpyridine). In certain embodiments, the acrylamide component is an N-alkylacrylamide, e.g., isopropylacrylamide. In certain embodiments, the copolymers of the membranes of the present disclosure can include a ratio, e.g., a molar ratio, of a heterocycle component, e.g., a heterocycle-containing monomer, to an acrylamide component, e.g., an acrylamide-containing monomer, of about 9:1 to about 1:9, about 8:1 to about 1:8, about 7:1 to about 1:7, about 6:1 to about 1:6, about 5:1 to about 1:5, about 4:1 to about 1:4, about 3:1 to about 1:3, about 2:1 to about 1:2, about 1:1, about 5:1 to about 1:1, about 4:1 to about 1:1, about 2:1 to about 1:1, about 5:1 to about 1:2, about 4:1 to about 1:2, or about 3:1 to about 1:2. In certain embodiments, the copolymer of the presently disclosed membrane can include a heterocyclic component, such as a heterocyclic component-containing monomer (e.g., 4-vinylpyridine), and an acrylamide component, such as an acrylamide-containing monomer (N-alkylacrylamide), in a ratio, e.g., a molar ratio, of about 5:1 to about 1:3. In certain embodiments, the copolymer of the presently disclosed membrane can include a heterocyclic component, such as a heterocyclic component-containing monomer (e.g., 4-vinylpyridine), and an acrylamide component, such as an acrylamide-containing monomer (N-alkylacrylamide), in a ratio, e.g., a molar ratio, of about 4:1 to about 1:2. In certain embodiments, the copolymer of the presently disclosed membrane can include a heterocyclic component, such as a heterocyclic component-containing monomer (e.g., 4-vinylpyridine), and an acrylamide component, such as an acrylamide-containing monomer (N-alkylacrylamide), in a ratio, e.g., a molar ratio, of about 4:1 to about 1:1. In certain embodiments, the copolymers of the membranes of the present disclosure can include a heterocycle component, such as a heterocycle-containing monomer (e.g., 4-vinylpyridine), and an acrylamide component, such as an acrylamide-containing monomer (N-alkylacrylamide), in a ratio, e.g., molar ratio, of about 2:1 to about 1:1.In certain embodiments, the copolymer of the membrane of the present disclosure can include a heterocyclic component, e.g., a heterocyclic-containing monomer, and an acrylamide component, e.g., an acrylamide-containing monomer, in a ratio, e.g., molar ratio, of about 1:1. As shown in Tables 2, 6, 7, and 9, membranes including copolymers including a heterocyclic component, e.g., a heterocyclic-containing monomer (e.g., vinylpyridine), and an acrylamide component, e.g., an acrylamide-containing monomer (e.g., N-alkylacrylamide), in a ratio, e.g., molar ratio, of about 1:1 result in membranes that are significantly less temperature sensitive (e.g., compared to control membranes). In certain embodiments, the copolymer of the membrane of the present disclosure can include a heterocyclic component, e.g., a heterocyclic-containing monomer, and an acrylamide component, e.g., an acrylamide-containing monomer, in a ratio, e.g., molar ratio, of about 2:1, e.g., 1.8:1. As shown in Table 2, membranes including copolymers containing a heterocyclic component, e.g., a heterocyclic-containing monomer (e.g., vinylpyridine), and an acrylamide component, e.g., an acrylamide-containing monomer (e.g., N-alkylacrylamide), in a ratio, e.g., molar ratio, of about 2:1 result in membranes that are less temperature sensitive (e.g., compared to a control membrane). In certain embodiments, the copolymers of the disclosed membranes may contain a heterocyclic component, e.g., a heterocyclic-containing monomer, and an acrylamide component, e.g., an acrylamide-containing monomer, in a ratio, e.g., molar ratio, of about 3:1. In certain embodiments, the copolymers of the disclosed membranes may contain a heterocyclic component, e.g., a heterocyclic-containing monomer, and an acrylamide component, e.g., an acrylamide-containing monomer, in a ratio, e.g., molar ratio, of about 4:1. As shown in Table 2, membranes including copolymers containing a heterocyclic component, e.g., a heterocyclic-containing monomer (e.g., vinylpyridine), and an acrylamide component, e.g., an acrylamide-containing monomer (e.g., N-alkylacrylamide), in a ratio, e.g., molar ratio, of about 4:1 result in membranes that are less temperature sensitive (e.g., compared to a control membrane). In certain embodiments, the copolymers of the membranes of the present disclosure can include a heterocycle component, such as a heterocycle-containing monomer (e.g., 4-vinylpyridine), and an acrylamide component, such as an acrylamide-containing monomer (N-alkylacrylamide), in a ratio, e.g., molar ratio, of about 4:1 to about 1:4.In certain embodiments, the copolymer of the presently disclosed membrane can include a heterocyclic component, such as a heterocyclic component-containing monomer (e.g., 4-vinylpyridine), and an acrylamide component, such as an acrylamide-containing monomer (N-alkylacrylamide), in a ratio, e.g., a molar ratio, of about 3:1 to about 1:3. In certain embodiments, the copolymer of the presently disclosed membrane can include a heterocyclic component, such as a heterocyclic component-containing monomer (e.g., 4-vinylpyridine), and an acrylamide component, such as an acrylamide-containing monomer (N-alkylacrylamide), in a ratio, e.g., a molar ratio, of about 2:1 to about 1:2. In certain embodiments, the copolymer of the presently disclosed membrane can include a heterocyclic component, such as a heterocyclic component-containing monomer (e.g., 4-vinylpyridine), and an acrylamide component, such as an acrylamide-containing monomer (N-alkylacrylamide), in a ratio, e.g., a molar ratio, of about 1:1 to about 1:3. In certain embodiments, the copolymers of the membranes of the present disclosure can include a heterocycle component, e.g., a heterocycle-containing monomer (e.g., 4-vinylpyridine), and an acrylamide component, e.g., an acrylamide-containing monomer (e.g., N-alkylacrylamide), in a ratio, e.g., molar ratio, of about 1:1 to about 1:2. As shown in Tables 6, 7, and 9, membranes including copolymers including a heterocycle component, e.g., a heterocycle-containing monomer (e.g., vinylpyridine), and an acrylamide component, e.g., an acrylamide-containing monomer (e.g., N-alkylacrylamide), in a ratio, e.g., molar ratio, of about 1:1.5, exhibit reduced temperature sensitivity (e.g., compared to a control membrane).

[0096] In certain embodiments, the membranes of the present disclosure may include a copolymer of a heterocyclic component, such as a heterocyclic-containing monomer, and an acrylamide component, such as an acrylamide-containing monomer. In certain embodiments, the membranes of the present disclosure may include a copolymer of a heterocyclic component, such as a heterocyclic-containing monomer, and an N-alkylacrylamide. In certain embodiments, the membranes of the present disclosure may include a copolymer of a vinylpyridine (e.g., 4-vinylpyridine) and an acrylamide component. In certain embodiments, the membranes of the present disclosure may include a copolymer of a vinylpyridine (e.g., 4-vinylpyridine) and an N-alkylacrylamide, such as N-isopropylacrylamide. In certain embodiments, the membranes of the present disclosure may include a copolymer of 4-vinylpyridine and N-isopropylacrylamide. In certain embodiments, the membranes of the present disclosure can include a copolymer of vinylimidazole (e.g., 1-vinylimidazole) and an acrylamide component. In certain embodiments, the membranes of the present disclosure can include a copolymer of vinylimidazole (e.g., 1-vinylimidazole) and an N-alkylacrylamide, such as N-isopropylacrylamide. In certain embodiments, the membranes of the present disclosure can include a copolymer of 1-vinylimidazole and N-isopropylacrylamide. In certain embodiments, the membranes of the present disclosure may include poly(4-vinylpyridine-co-N-isopropylacrylamide).

[0097] In certain embodiments, poly(4-vinylpyridine-co-N-isopropylacrylamide) comprises 4-vinylpyridine in a percentage ranging from about 0.01% to about 80%, about 0.01% to about 75%, about 0.01% to about 70%, about 0.01% to about 65%, about 0.01% to about 60%, about 0.01% to about 55%, about 0.01% to about 50%, about 0.05% to about 45%, about 0.1% to about 40%, about 0.5% to about 35%, about 1% to about 30%, about 2% to about 25%, about 5% to about 20%, about 10% to about 60%, about 20% to about 60%, about 30% to about 60%, about 30% to about 50%, or about 35% to about 45% of the total weight of the copolymer. In certain embodiments, poly(4-vinylpyridine-co-N-isopropylacrylamide) comprises 4-vinylpyridine in a percentage ranging from about 30% to about 50% of the total weight of the copolymer. In certain embodiments, poly(4-vinylpyridine-co-N-isopropylacrylamide) comprises 4-vinylpyridine in a mole percent ranging from about 0.01% to about 60%, 0.01% to about 50%, about 0.05% to about 45%, about 0.1% to about 40%, about 0.5% to about 35%, about 1% to about 30%, about 2% to about 25%, about 5% to about 20%, about 10% to about 60%, about 20% to about 60%, about 30% to about 60%, about 30% to about 50%, or about 35% to about 45%. In certain embodiments, poly(4-vinylpyridine-co-N-isopropylacrylamide) comprises 4-vinylpyridine at a molar percentage ranging from about 1% to about 80%, about 1% to about 75%, about 1% to about 70%, about 1% to about 65%, or about 1% to about 60%, e.g., about 20% to about 50%, or about 30% to about 50%. In certain embodiments, poly(4-vinylpyridine-co-N-isopropylacrylamide) comprises 4-vinylpyridine at a molar percentage ranging from about 30% to about 50%.

[0098] In certain embodiments, poly(4-vinylpyridine-co-N-isopropylacrylamide) comprises N-isopropylacrylamide in a percentage ranging from about 0.01% to about 70%, about 0.01% to about 65%, about 0.01% to about 60%, about 0.01% to about 55%, about 0.01% to about 50%, about 0.05% to about 45%, about 0.1% to about 40%, about 0.5% to about 35%, about 1% to about 30%, about 2% to about 25%, about 5% to about 20%, about 10% to about 70%, about 20% to about 70%, about 30% to about 70%, about 40% to about 70%, about 50% to about 70%, or about 55% to about 65% of the total weight of the copolymer. In certain embodiments, poly(4-vinylpyridine-co-N-isopropylacrylamide) comprises N-isopropylacrylamide in a percentage ranging from about 50% to about 70% of the total weight of the copolymer. In certain embodiments, poly(4-vinylpyridine-co-N-isopropylacrylamide) copolymer comprises N-isopropylacrylamide in a percentage ranging from about 0.01% to about 50%, about 0.05% to about 45%, about 0.1% to about 40%, about 0.5% to about 35%, about 1% to about 30%, about 2% to about 25%, or about 5% to about 20% of the total weight of the copolymer. In certain embodiments, poly(4-vinylpyridine-co-N-isopropylacrylamide) comprises N-isopropylacrylamide in a mole percent ranging from about 0.01% to about 70%, from about 0.01% to about 65%, from about 0.01% to about 60%, from about 0.01% to about 55%, from about 0.01% to about 50%, from about 0.05% to about 45%, from about 0.1% to about 40%, from about 0.5% to about 35%, from about 1% to about 30%, from about 2% to about 25%, from about 5% to about 20%, from about 10% to about 70%, from about 20% to about 70%, from about 30% to about 70%, from about 40% to about 70%, from about 50% to about 70%, or from about 55% to about 65%. In certain embodiments, poly(4-vinylpyridine-co-N-isopropylacrylamide) comprises N-isopropylacrylamide at a molar percentage ranging from about 1% to about 70%, e.g., from about 1% to about 50%. In certain embodiments, poly(4-vinylpyridine-co-N-isopropylacrylamide) comprises N-isopropylacrylamide at a molar percentage ranging from about 50% to about 70%.

[0099] The molecular weight of copolymers containing heterocyclic and acrylamide components, such as poly(4-vinylpyridine-co-N-isopropylacrylamide) polymers, can vary. In certain embodiments, copolymers, such as poly(4-vinylpyridine-co-N-isopropylacrylamide), have a molecular weight of about 5 kDa to about 500 kDa. In certain embodiments, copolymers, such as poly(4-vinylpyridine-co-N-isopropylacrylamide), have a molecular weight of about 100 kDa to about 500 kDa, about 150 kDa to about 500 kDa, about 200 kDa to about 500 kDa, about 300 kDa to about 500 kDa, about 400 kDa to about 500 kDa, about 50 kDa to about 500 kDa, or about 50 kDa. to about 400 kDa, about 50 kDa to about 400 kDa, about 50 kDa to about 400 kDa, about 50 kDa to about 300 kDa, about 50 kDa to about 200 kDa, about 50 kDa to about 100 kDa, about 100 kDa to about 400 kDa, about 100 kDa to about 300 kDa, or about 200 kDa to about 400 kDa. For example, but not by way of limitation, the molecular weight of a copolymer, such as poly(4-vinylpyridine-co-N-isopropylacrylamide), can be 5 kDa or greater, or about 10 kDa or greater, or about 15 kDa or greater, or about 20 kDa or greater, or about 25 kDa or greater, or about 30 kDa or greater, or about 40 kDa or greater, or about 50 kDa or greater, or about 75 kDa or greater, or about 90 kDa or greater, or about 100 kDa or greater. In certain embodiments, the molecular weight of a copolymer comprising a heterocyclic component and an acrylamide component, such as poly(4-vinylpyridine-co-N-isopropylacrylamide) polymer, has a molecular weight of about 100 kDa to about 500 kDa.

[0100] In certain embodiments, the poly(4-vinylpyridine-co-N-isopropylacrylamide) copolymer has Formula I: [ka] Formula I In certain embodiments, m and n represent positive integers. Depending on the desired membrane properties, the ratio of m to n, e.g., the molar ratio, may be about 100:1 to about 1:100, e.g., about 100:1 to about 1:90, about 100:1 to about 1:80, about 100:1 to about 1:70, about 100:1 to about 1:60, about 100:1 to about 1:50, about 100:1 to about 1:40, about 100:1 to about 1:30, about 100:1 to about 1:20, about 100:1 to about 1:10, about 90:1 to about 1:100, about 80:1 to about 1:100, about 70:1 to about 1:100, about 60:1 to about 1: The ratio may vary within the range of about 1:100, about 50:1 to about 1:100, about 40:1 to about 1:100, about 30:1 to about 1:100, about 20:1 to about 1:100, about 10:1 to about 1:100, about 90:1 to about 1:90, about 80:1 to about 1:80, about 70:1 to about 1:70, about 60:1 to about 1:60, about 50:1 to about 1:50, about 40:1 to about 1:40, about 30:1 to about 1:30, about 20:1 to about 1:20, about 10:1 to about 1:10, about 5:1 to about 1:5, or about 5:1 to about 2:1. In certain embodiments, the ratio of m to n, e.g., the molar ratio, may be between about 1:1 and about 1:100, e.g., about 1:1 to about 1:95, about 1:1 to about 1:80, about 1:1 to about 1:75, about 1:1 to about 1:50, about 1:1 to about 1:25, about 1:1 to about 1:10, about 1:1 to about 1:5, about 1:1 to about 1:3, or about 1:1 to about 1:2. In certain embodiments, the ratio of m to n, e.g., the molar ratio, is about 1:1.5. In certain embodiments, the ratio of m to n, e.g., the molar ratio, is about 1:1.5. In certain embodiments, the ratio of m to n, e.g., the molar ratio, is in the range of about 1:1 to 100:1, e.g., about 1:1 to 95:1, about 1:1 to 80:1, about 1:1 to about 75:1, about 1:1 to about 50:1, about 1:1 to about 25:1, about 1:1 to about 10:1, about 1:1 to about 5:1, about 1:1 to about 4:1, about 1:1 to about 3:1, or about 1:1 to about 2:1. In certain embodiments, the ratio of m to n, e.g., the molar ratio, is in the range of about 4:1 to about 1:1. In certain embodiments, the ratio of m to n, e.g., the molar ratio, is in the range of about 4:1 to about 1:4.In certain embodiments, the ratio of m to n, e.g., the molar ratio, is in the range of about 3:1 to about 1:3. In certain embodiments, the ratio of m to n, e.g., the molar ratio, is in the range of about 2:1 to about 1:2. In certain embodiments, the ratio of m to n, e.g., the molar ratio, is in the range of about 1:1 to about 1:4. In certain embodiments, the ratio of m to n, e.g., the molar ratio, is in the range of about 1:1 to about 1:3. In certain embodiments, the ratio of m to n, e.g., the molar ratio, is in the range of about 1:1 to about 1:2. As shown in Table 2, membranes comprising copolymers including a heterocycle component, e.g., a heterocycle-containing monomer (e.g., vinylpyridine), and an acrylamide component, e.g., an acrylamide-containing monomer (e.g., N-alkylacrylamide), in a ratio, e.g., molar ratio, of about 4:1 to about 1:1 result in membranes that are significantly less temperature sensitive (e.g., compared to a control membrane). As shown in Tables 6, 7, and 9, membranes containing copolymers of a heterocycle component, e.g., a heterocycle-containing monomer (e.g., vinylpyridine) and an acrylamide component, e.g., an acrylamide-containing monomer (e.g., N-alkylacrylamide), in a ratio, e.g., molar ratio, of about 1:1 to about 1:2, have significantly less temperature sensitivity (e.g., compared to a control membrane). As shown in Tables 2, 6, 7, and 9, membranes containing copolymers of a heterocycle component, e.g., a heterocycle-containing monomer (e.g., vinylpyridine) and an acrylamide component, e.g., an acrylamide-containing monomer (e.g., N-alkylacrylamide), in a ratio, e.g., molar ratio, of about 4:1 to about 1:2, have significantly less temperature sensitivity (e.g., compared to a control membrane).

[0101] In certain embodiments, m can be in the range of about 1 to about 90. In certain embodiments, m can be in the range of about 1 to about 10. In certain embodiments, m can be in the range of about 10 to about 90. In certain embodiments, n can be in the range of about 1 to about 90. In certain embodiments, n can be in the range of about 1 to about 10. In certain embodiments, n can be in the range of about 10 to about 90. In certain embodiments, m can be in the range of about 1 to about 10, and n can be in the range of about 1 to about 10. In certain embodiments, m can be in the range of about 10 to about 90, and n can be in the range of about 10 to about 90. In certain embodiments, m can be in the range of about 40 to about 90, and n can be in the range of about 10 to about 60. In certain embodiments, m is about 80, and n is about 22. In certain embodiments, m is about 65, and n is about 35. In certain embodiments, m is about 50, and n is about 50. In certain embodiments, m is about 4, and n is about 6.

[0102] In certain embodiments, the membranes of the present disclosure include one or more crosslinkers (crosslinking agents) that crosslink the polymer backbones present in the membrane. In certain embodiments of the present disclosure, the crosslinking agents of interest can simultaneously provide intermolecular and intramolecular crosslinks. Non-limiting examples of crosslinkers for use herein are disclosed in Section II.4. In certain embodiments, the crosslinker can be a crosslinker having two, three, or four epoxide functional groups. In certain embodiments, the crosslinker can be a crosslinker having two epoxide functional groups. In certain embodiments, the crosslinker can be a crosslinker having three epoxide functional groups. In certain embodiments, the crosslinker can be a crosslinker having four epoxide functional groups. In certain embodiments, the crosslinker can be a branched glycidyl ether crosslinker. For example, but not by way of limitation, the crosslinker can be a branched glycidyl ether crosslinker containing two or more crosslinkable groups, such as, but not limited to, polyethylene glycol diglycidyl ether or polyethylene glycol tetraglycidyl ether. In certain embodiments, the crosslinker is polyethylene glycol diglycidyl ether, hi certain embodiments, the crosslinker is a polyetheramine crosslinker.

[0103] The ratio of copolymer, e.g., poly(4-vinylpyridine-co-N-isopropylacrylamide) polymer to crosslinker can vary depending on the desired diffusion characteristics of the membrane. In certain embodiments, the ratio, e.g., mass ratio, of copolymer, e.g., poly(4-vinylpyridine-co-N-isopropylacrylamide) polymer to crosslinker is about 1000:1 to about 5:1, e.g., about 900:1 to about 5:1, about 800:1 to about 5:1, about 700:1 to about 5:1, about 600:1 to about 5:1, about 500:1 to about 5:1, about 400:1 to about 5:1, about 300:1 to about 5:1, about 200:1 to about 5:1, about 100:1 to about 5:1, about 50:1 to about 5:1, about 10:1 to about 5:1, about 1:1 to about 5:1, or about 1000:1. to about 10:1, about 1000:1 to about 20:1, about 1000:1 to about 50:1, about 1000:1 to about 100:1, about 1000:1 to about 500:1, about 500:1 to about 20:1, about 500:1 to about 50:1, about 500:1 to about 100:1, about 500:1 to about 200:1, about 500:1 to about 400:1, about 200:1 to about 10:1, about 200:1 to about 20:1, about 200:1 to about 50:1, about 200:1 to about 100:1, about 100:1 to about 10:1, about 100:1 to about 20:1, or about 100:1 to about 50:1. In certain embodiments, the ratio, e.g., weight ratio, of copolymer, e.g., poly(4-vinylpyridine-co-N-isopropylacrylamide) polymer to crosslinker can range from about 10:1 to about 5:1. As shown in Table 3, membranes including ratios, e.g., weight ratios, of copolymer (e.g., poly(4-vinylpyridine-co-N-isopropylacrylamide)) and crosslinker resulted in membranes with significantly less temperature sensitivity (e.g., compared to control membranes). In certain embodiments, the ratio, e.g., weight ratio, of copolymer, e.g., poly(4-vinylpyridine-co-N-isopropylacrylamide) polymer to crosslinker can range from 1:1 to 1:100, 1:1 to 1:95, 1:1 to 1:80, 1:1 to 1:75, 1:1 to 1:50, 1:1 to 1:25, 1:1 to 1:10, 1:1 to 1:5, 1:1 to 1:3, and 1:1 to 1:2.In certain other embodiments, the ratio, e.g., weight ratio, of poly(4-vinylpyridine-co-N-isopropylacrylamide) polymer to crosslinker is in the range of 1:1 to 100:1, 1:1 to 95:1, 1:1 to 80:1, 1:1 to 75:1, 1:1 to 50:1, 1:1 to 25:1, 1:1 to 10:1, 1:1 to 5:1, 1:1 to 3:1, or 1:1 to 2:1. In certain embodiments, the ratio, e.g., weight ratio, of copolymer, e.g., poly(4-vinylpyridine-co-N-isopropylacrylamide) polymer to crosslinker is 4:1. In certain embodiments, the ratio, e.g., weight ratio, of copolymer, e.g., poly(4-vinylpyridine-co-N-isopropylacrylamide) polymer to crosslinker is 5:1. In certain embodiments, the ratio of copolymer, e.g., poly(4-vinylpyridine-co-N-isopropylacrylamide) polymer to crosslinker, e.g., a weight ratio of 10:1. In certain embodiments, the ratio of copolymer, e.g., poly(4-vinylpyridine-co-N-isopropylacrylamide) polymer to crosslinker, e.g., a weight ratio of 20:1. In certain embodiments, the ratio of copolymer, e.g., poly(4-vinylpyridine-co-N-isopropylacrylamide) polymer to crosslinker, e.g., a weight ratio of 50:1. In certain embodiments, the ratio of copolymer, e.g., poly(4-vinylpyridine-co-N-isopropylacrylamide) polymer to crosslinker, e.g., a weight ratio of 100:1. In certain embodiments, the ratio of copolymer, e.g., poly(4-vinylpyridine-co-N-isopropylacrylamide) polymer to crosslinker, e.g., a weight ratio of 200:1. In certain embodiments, the ratio of copolymer, e.g., poly(4-vinylpyridine-co-N-isopropylacrylamide) polymer to crosslinker, e.g., by weight, is 300:1. In certain embodiments, the ratio of copolymer, e.g., poly(4-vinylpyridine-co-N-isopropylacrylamide) polymer to crosslinker, e.g., by weight, is 400:1. In certain embodiments, the ratio of copolymer, e.g., poly(4-vinylpyridine-co-N-isopropylacrylamide) polymer to crosslinker, e.g., by weight, is 500:1.In certain embodiments, the ratio of copolymer, e.g., poly(4-vinylpyridine-co-N-isopropylacrylamide) polymer to crosslinker, e.g., a weight ratio, is 600:1. In certain embodiments, the ratio of copolymer, e.g., poly(4-vinylpyridine-co-N-isopropylacrylamide) polymer to crosslinker, e.g., a weight ratio, is 700:1. In certain embodiments, the ratio of copolymer, e.g., poly(4-vinylpyridine-co-N-isopropylacrylamide) polymer to crosslinker, e.g., a weight ratio, is 800:1. In certain embodiments, the ratio of copolymer, e.g., poly(4-vinylpyridine-co-N-isopropylacrylamide) polymer to crosslinker, e.g., a weight ratio, is 900:1. In certain embodiments, the ratio of copolymer, e.g., poly(4-vinylpyridine-co-N-isopropylacrylamide) polymer to crosslinker, e.g., a weight ratio, is 1000:1. In certain embodiments, the membranes of the present disclosure comprise a crosslinker at a weight percent of about 10% or more, e.g., about 15% or more, or about 18% or more. In certain embodiments, the membranes of the present disclosure comprise a crosslinker at a weight percent of about 10% to about 20%. In certain embodiments, the membranes of the present disclosure comprise a crosslinker at a weight percent of about 10% to about 30%.

[0104] Generally, the thickness of the film is controlled by the concentration of the film solution, the number of droplets of the film solution applied, the number of times the sensor is immersed in the film solution or sprayed onto the sensor, the volume of the film solution sprayed onto the sensor, and any combination of these factors. In certain embodiments, the films described herein can have a thickness, e.g., a dry thickness, in the range of about 0.1 μm to about 1000 μm, e.g., about 1 μm to about 500 μm, or about 10 μm to about 100 μm. In certain embodiments, the films described herein can have a thickness, e.g., a dry thickness, in the range of about 1 μm to about 50 μm. In certain embodiments, the films described herein can have a thickness, e.g., a dry thickness, in the range of about 5 μm to about 50 μm. In certain embodiments, the films described herein can have a thickness, e.g., a dry thickness, in the range of about 5 μm to about 40 μm. In certain embodiments, the films described herein can have a thickness, e.g., a dry thickness, in the range of about 10 μm to about 40 μm. In certain embodiments, the membranes described herein can have a thickness, e.g., a dry thickness, ranging from about 20 μm to about 30 μm. In certain embodiments, the membranes of the present disclosure have a thickness, e.g., a dry thickness, of about 20 μm. In certain embodiments, the membranes of the present disclosure have a thickness, e.g., a dry thickness, of about 30 μm.

[0105] In certain embodiments, the mass transport limiting membrane can be homogeneous and single-component (including single-membrane copolymers of two or more polymers or monomers as described herein). For example, but not by way of limitation, a membrane of the present disclosure can consist of, or consist essentially of, a copolymer of the present disclosure, such as poly(4-vinylpyridine-co-N-isopropylacrylamide) polymer. In certain embodiments, a membrane of the present disclosure can include a copolymer of the present disclosure, such as poly(4-vinylpyridine-co-N-isopropylacrylamide) polymer, and a crosslinker. In certain embodiments, a membrane of the present disclosure can consist of, or consist essentially of a copolymer of the present disclosure, such as poly(4-vinylpyridine-co-N-isopropylacrylamide) polymer, and a crosslinker.

[0106] Alternatively, the mass transport limiting membrane may be multicomponent (comprising two or more different membrane polymers, e.g., copolymers, e.g., as a composite). For example, but not by way of limitation, a membrane of the present disclosure may comprise a copolymer of the present disclosure, e.g., poly(4-vinylpyridine-co-N-isopropylacrylamide) polymer, and at least one additional polymer, e.g., a second polymer or copolymer. In certain embodiments, a membrane of the present disclosure may comprise a copolymer of the present disclosure, e.g., poly(4-vinylpyridine-co-N-isopropylacrylamide) polymer, a second polymer, and a crosslinker. Non-limiting examples of the second polymer include polyurethane, a silicone-based polymer, e.g., polydimethylsiloxane (PDMS), or a styrene-based polymer, e.g., poly(4-vinylpyridine-co-styrene) copolymer. In certain embodiments, the multi-component membrane can exist as a multilayer membrane, for example, a bilayer membrane or a trilayer membrane. In certain embodiments, the multi-component membrane can exist as a homogeneous mixture of two or more membrane polymers, for example, the copolymers of the present disclosure. In certain embodiments, the homogeneous mixture can be deposited by combining two or more membrane polymers in a solution and then depositing the solution on the working electrode, for example, by dip coating. In certain embodiments, a multilayer membrane can be deposited on the analyte-responsive active area by depositing a first layer, for example, by dip coating, and then depositing a second layer on the first layer, for example, by dip coating, to form a bilayer membrane. In certain embodiments, a third layer can be deposited on the second layer, for example, by dip coating, to form a trilayer membrane.

[0107] In certain embodiments, an analyte sensor of the present disclosure can include a sensor tail including at least a first working electrode, a first active area disposed on the surface of the first working electrode, and a mass transport limiting membrane directly overcoating the at least first active area and permeable to the first analyte. In certain embodiments, the first active area includes a first polymer, and at least one enzyme is covalently bound to the first polymer and responds to the first analyte. In certain embodiments, the mass transport limiting membrane is a membrane with low temperature dependency, such as a membrane disclosed herein, such that analyte detection is not adversely affected by temperature changes. In certain embodiments, the mass transport limiting membrane includes a polymer containing a heterocycle-containing moiety (e.g., pyridine) and an acrylamide moiety (e.g., N-isopropylacrylamide), such as poly(4-vinylpyridine-co-N-isopropylacrylamide) polymer. In certain embodiments, the mass transport limiting membrane further includes a crosslinker. In certain embodiments, the mass transport limiting membrane includes poly(4-vinylpyridine-co-N-isopropylacrylamide) polymer and a crosslinker.

[0108] In certain embodiments, the analyte sensor includes two active areas, and a membrane overcoats at least one of the active areas of the analyte sensor. In certain embodiments, a membrane overcoats each of the active areas of the analyte sensor. Alternatively, a first membrane overcoats one of the active areas, and a second membrane overcoats the second active area. In certain embodiments, at least one of the mass transport limiting membranes includes a polymer containing a heterocycle-containing moiety (e.g., pyridine) and an acrylamide moiety (e.g., N-isopropylacrylamide), such as poly(4-vinylpyridine-co-N-isopropylacrylamide) polymer. In certain embodiments, at least one of the mass transport limiting membranes includes poly(4-vinylpyridine-co-N-isopropylacrylamide) polymer and a crosslinker. In certain embodiments, the second mass transport limiting membrane includes a different polymer than the first mass transport limiting membrane.

[0109] 6. Interference Domains In certain embodiments, a sensor of the present disclosure, e.g., a 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, allowing the passage of the analyte and other substances to be measured by the working electrode while preventing the passage of other substances, such as interferents. In certain embodiments, 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. In certain embodiments, the interference domain is located between the working electrode and one or more active areas. In certain embodiments, non-limiting examples of polymers that can be used in the interference domain include polyurethane, polymers with pendant ionic groups, and polymers with controlled pore sizes. In certain embodiments, the interference domain is formed from one or more cellulose derivatives. Non-limiting examples of cellulose derivatives include cellulose acetate, cellulose acetate butyrate, 2-hydroxyethyl cellulose, cellulose acetate phthalate, cellulose acetate propionate, cellulose acetate, and trimellitate.

[0110] In certain embodiments, the interference domain is located between one or more active zones and the mass transport limiting membrane, hi 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 comprises a thin, hydrophobic membrane that is non-swelling and restricts the diffusion of high molecular weight species. For example, but not by way of limitation, the interference domain may be permeable to relatively low molecular weight substances such as hydrogen peroxide, while restricting the passage of high molecular weight substances such as ketones, glucose, acetaminophen, and / or ascorbic acid. In certain embodiments, the interference domain can be deposited directly onto the working electrode, for example, on the surface of a permeable working electrode. In certain embodiments, the interference domain can be deposited directly onto the active. In certain embodiments, the interference domain has a thickness, e.g., a dry thickness, ranging from about 0.1 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 interference domain can have a thickness of about 0.1 μm to about 10 μm, e.g., from about 0.5 μm to about 10 μm, from about 1 μm to about 10 μm, from about 1 μm to about 5 μm, or from about 0.1 μm to about 5 μm. In certain embodiments, the sensor can be dipped into a solution of the interference domain two or more times. For example, but not by way of limitation, a sensor (or working electrode) of the present disclosure may be dipped into the interference domain solution at least two times, at least three times, at least four times, or at least five times to achieve a desired interference domain thickness.

[0111] III. Method of Use The present disclosure further provides methods of use of the analyte sensors disclosed herein. In certain embodiments, the present disclosure provides methods for detecting an analyte. In certain embodiments, the present disclosure provides methods for detecting two or more analytes disclosed herein, e.g., a first analyte and a second analyte. For example, but not by way of limitation, the one or more analytes may be glucose, lactate, ketones (e.g., ketone bodies), glutamine, alcohol, aspartate, asparagine, potassium, glutamate, creatinine, acetoacetate, fructosamine, amylase, bilirubin, cholesterol, chorionic gonadotropin, creatine kinase (e.g., CK-MB), creatine, DNA, RNA, growth factors, growth hormones, hormones (e.g., thyroid-stimulating hormone), steroids, vitamins (e.g., ascorbic acid), uric acid, neurochemicals (e.g., acetylcholine, norepinephrine, and dopa), or the like. The analyte may be an analyte of interest, such as ATP, ATP, ATP-dependent agonists, ATP-dependent agonists, ATP-dependent agonists (ATP-dependent agonists), ...

[0112] In certain embodiments, a method for detecting an analyte can include (i) preparing an analyte sensor for detecting an analyte, such as glucose. In certain embodiments, the analyte sensor includes (a) a sensor tail including at least a first working electrode, (b) an analyte-responsive active area including a first enzyme system and a second enzyme system and disposed on the surface of the first working electrode, and (c) a mass transport limiting membrane overcoating the analyte-responsive active area and allowing the analyte to permeate, such as a membrane described herein. In certain embodiments, the method further includes (ii) applying a potential to the first working electrode, (iii) acquiring a first signal at or above the redox potential of the analyte-responsive active area, the first signal being proportional to the concentration of the first analyte in a fluid in contact with the analyte-responsive active area, and (iv) correlating the first signal with the concentration of the first analyte in the fluid. In certain embodiments, the mass transport limiting membrane is a membrane with low temperature dependence, such as a membrane disclosed herein, such that analyte detection is not adversely affected by temperature changes. Non-limiting examples of mass transport limiting membranes that can be included in the analyte sensors are disclosed in Section II.5 herein.

[0113] In certain embodiments, a method of the present disclosure may include (i) exposing an analyte sensor to a fluid containing an analyte of interest, the analyte sensor including (a) a sensor tail including at least a first working electrode, (b) an analyte-responsive active area including at least an enzyme for detecting the analyte and optionally a polymer, the analyte-responsive active area being disposed on the surface of the first working electrode, and (c) a mass transport limiting membrane overcoating the analyte-responsive active area and allowing the analyte to permeate, e.g., a membrane disclosed herein. In certain embodiments, the method may further include (ii) applying a potential to the first working electrode, (iii) acquiring a first signal at or above the redox potential of the first analyte-responsive active area, the first signal being proportional to the analyte concentration in the fluid, and (iv) correlating the first signal with the analyte concentration in the fluid. In certain embodiments, the mass transport limiting membrane is a membrane with low temperature dependence, e.g., a membrane disclosed herein, such that analyte detection is not adversely affected by temperature changes. Non-limiting examples of mass transport limiting membranes that can be included in the analyte sensors are disclosed in Section II.5 herein.

[0114] In certain embodiments, the disclosed methods may further include preparing an analyte sensor including a second active area and / or detecting another analyte by exposing the analyte sensor including a second active area to a fluid containing the analyte. In certain embodiments, an analyte sensor for use in the disclosed methods may include a second working electrode and a second active area disposed on the surface of the second working electrode, the second active area including a second polymer, at least one enzyme responsive to the analyte to be detected, and optionally a redox mediator, and a portion of the mass transport limiting membrane, e.g., the second portion, overcoating the second active area. Alternatively, the second active area may be covered by a second mass transport limiting membrane that is separate and / or different from the mass transport limiting membrane overcoating the first analyte-responsive active area. In certain embodiments, the disclosed methods may further include (ii) applying a potential to the second working electrode, (iii) acquiring a second signal at or above the redox potential of the second analyte-responsive active area that is proportional to the concentration of the second analyte in the fluid, and (iv) correlating the second signal with the concentration of the second analyte in the fluid. Non-limiting examples of mass transport limiting membranes that can be included in the analyte sensor are disclosed in Section II.5 herein.

[0115] IV. Method of Manufacturing The present disclosure further provides methods for fabricating the analyte sensors disclosed herein. In certain embodiments, the analyte sensors of the present disclosure include one or more active areas (for detecting one or more analytes) and one or more working electrodes. For example, but not by way of limitation, the present disclosure provides methods for fabricating an analyte sensor including a first active area disposed on a first working electrode. In certain embodiments, a second active area can be disposed on a second working electrode or on the first working electrode.

[0116] In certain embodiments, the method includes creating a first working electrode, for example, by screen printing. In certain embodiments, creating the first working electrode can include printing with a carbon ink. In certain embodiments, creating the first working electrode can include printing with a carbon ink onto a substrate, for example, a non-conductive substrate. In certain embodiments, the method may further include applying a composition comprising one or more enzymes to the surface of the working electrode to generate an analyte-responsive active area on the working electrode. In certain embodiments, the enzyme composition may comprise one or more enzymes for detecting an analyte described herein. In certain embodiments, the composition comprising one or more enzymes may further comprise an electron transfer agent and / or a cross-linking agent.

[0117] In certain embodiments, the method may further include depositing a membrane directly on top of the first and / or second active areas. In certain embodiments, the membrane composition may include a copolymer described herein. Non-limiting examples of copolymers that can be included in the membrane composition are disclosed in Section II.5 herein. In certain embodiments, the membrane is a membrane disclosed herein that has low temperature dependency, such that analyte detection is not adversely affected by temperature changes. For example, but not by way of limitation, the membrane may include a copolymer of at least a first monomer and a second monomer, wherein the first monomer includes acrylamide. In certain embodiments, the copolymer is a poly(4-vinylpyridine-co-N-isopropylacrylamide) polymer. In certain embodiments, the membrane is applied by dip coating. In certain embodiments, the dip coating process may include depositing multiple layers. In certain embodiments, a single layer deposited by a single dip into a polymer-solvent solution can be used to produce a functional sensor. Alternatively, the membrane can be produced by a multiple-dip process. In certain embodiments, the dip-coating process may include (1) dipping the sensor tail into a membrane solution and (2) drying the membrane solution on the sensor tail. In certain embodiments, steps (1) and (2) may be repeated until the membrane has a desired thickness. For example, but not by way of limitation, this dipping process may be repeated at least two times, at least three times, at least four times, or at least five times.

[0118] In certain embodiments, the method can include creating a second working electrode, e.g., by screen printing, on the same substrate as the first working electrode. In certain embodiments, the method can further include applying an enzyme system, e.g., a composition including a second enzyme system, to the surface of the second working electrode to create a second analyte-responsive active area on the second working electrode. In certain embodiments, the membrane of the present disclosure overcoats the second analyte-responsive active area.

[0119] V. Illustrative Embodiments A. The present disclosure provides a membrane structure comprising an enzyme layer and a membrane disposed proximate to the enzyme layer, the membrane comprising a copolymer of at least a first monomer and a second monomer, the first monomer comprising acrylamide. A1. The membrane structure of A, wherein the second monomer comprises a heterocycle-containing moiety. A2. The membrane structure of A or A1, wherein the acrylamide is an N-alkylacrylamide. A2-1. The membrane structure according to A2, wherein the alkyl of the N-alkylacrylamide is a C1-C6 linear or branched alkyl group or a C3-C6 cycloalkyl group. A2-2. The membrane structure according to A2-1, wherein the alkyl of the N-alkylacrylamide is a branched alkyl group. A2-3. The membrane structure according to A2-2, wherein the branched alkyl group is selected from the group consisting of isopropyl, isobutyl, sec-butyl, tert-butyl, tert-pentyl, neopentyl, isopentyl, sec-pentyl, 3-pentyl, sec-isopentyl, active pentyl, isohexyl, tert-hexyl, neohexyl, and sec-hexyl. A2-4. The membrane structure according to A2-3, wherein the branched alkyl group is an isopropyl group. A2-5. The membrane structure according to any one of A2 to A2-4, wherein the N-alkylacrylamide is N-isopropylacrylamide. A3. The film structure according to any one of A1 to A2-5, wherein the heterocycle-containing component is selected from the group consisting of furan, thiophene, pyrrole, pyridine, pyrimidine, imidazole, oxadiazole, isoxazole, oxazole, pyrazole, isothiazole, thiazole, pyrazine, isoquinoline, quinoline, benzofuran, and benzimidazole.

[0120] A4. The membrane structure according to any one of A1 to A3, wherein the heterocycle is pyridine. A5. The membrane structure of A4, wherein the pyridine is vinylpyridine. A6. The membrane structure of A5, wherein the vinylpyridine is 2-vinylpyridine, 4-vinylpyridine, or a combination thereof. A7. The membrane structure of A6, wherein the vinylpyridine is 4-vinylpyridine. A8. The membrane structure of A6, wherein the vinylpyridine is 2-vinylpyridine. A9. The membrane structure according to any one of A1 to A3, wherein the heterocycle is imidazole. A10. The membrane structure according to A9, wherein the imidazole is vinylimidazole. A11. The membrane structure according to A10, wherein the vinylimidazole is 1-vinylimidazole, 2-vinylimidazole, 4-vinylimidazole, or a combination thereof. A12. The membrane structure according to A11, wherein the vinylimidazole is 1-vinylimidazole. A13. The membrane structure according to A11, wherein the vinylimidazole is 2-vinylimidazole. A14. The membrane structure according to any one of A1 to A3, wherein the acrylamide is an N-alkylacrylamide and the heterocycle is pyridine. A15. The membrane structure according to A14, wherein the N-alkylacrylamide is N-isopropylacrylamide. A16. The membrane structure according to A14 or A15, wherein the pyridine is vinylpyridine. A17. The membrane structure according to A16, wherein the vinylpyridine is 4-vinylpyridine.

[0121] A18. The membrane structure according to any one of A14 to A17, wherein the acrylamide is N-alkylacrylamide and the pyridine is 4-vinylpyridine. A18-1. The membrane structure according to any one of A to A18, wherein the copolymer comprises about 20 mer% to about 70 mer% of the first monomer. A18-2. The membrane structure according to any one of A to A18-1, wherein the copolymer comprises about 40 mer% to about 60 mer% of the first monomer. A18-3. The membrane structure according to A18-1, wherein the copolymer comprises about 30 mer% to about 60 mer% of the first monomer. A18-4. The membrane structure according to any one of A to A18-3, wherein the copolymer comprises about 30 mer% to about 80 mer% of the second monomer. A18-5. The membrane structure according to any one of A to A18-4, wherein the copolymer comprises about 30 mer% to about 65 mer% of the second monomer. A18-6. The membrane structure according to any one of A to A18-5, wherein the copolymer comprises about 30 mer% to about 50 mer% of the second monomer. A18-7. The membrane structure according to A18-4, wherein the copolymer comprises about 40 mer% to about 70 mer% of the second monomer. A18-8. The membrane structure of A17, wherein the copolymer comprises at least about 40 mer% of the first monomer. A18-9. The membrane structure of A17, wherein the copolymer comprises at least about 60 mer% of the first monomer. A18-10. The membrane structure of A17, wherein the copolymer comprises at least about 60 mer% of a second monomer. A18-11. The membrane structure of A17, wherein the copolymer comprises at least about 40 mer% of a second monomer. A18-12. The membrane structure according to any one of A14 to A18-11, wherein the copolymer is poly(4-vinylpyridine-co-N-isopropylacrylamide).

[0122] A19. The copolymer has formula I: [ka] The membrane structure according to any one of A to A3, having a structure represented by the formula: wherein m and n are each a positive integer. A20. The membrane structure according to A19, wherein the ratio of m to n is about 1:1 to about 1:100. A21. The membrane structure according to A19, wherein the ratio of m to n is from about 1:1 to about 100:1. A21-1. The membrane structure according to A19, wherein the ratio of m to n is about 4:1 to about 1:4. A21-2. The membrane structure according to A21-1, wherein the ratio of m to n is about 1:1 to about 1:4. A21-3. The membrane structure according to A21-2, wherein the ratio of m to n is about 1:1 to about 1:3. A21-4. The membrane structure according to A21-3, wherein the ratio of m to n is about 1:1 to about 1:2. A22. The membrane structure according to A19, wherein m is in the range of about 1 to about 90 and n is in the range of about 1 to about 90. A22-1. The membrane structure according to A19, wherein m is in the range of about 30 to about 50, and n is in the range of about 50 to about 70. A23. The membrane structure according to A22, wherein m is about 80 and n is about 22, or m is about 65 and n is about 35, or m is about 50 and n is about 50. A23-1. The membrane structure according to A22-1, wherein m is about 40 and n is about 60.

[0123] A24. The membrane structure according to any one of A to A23-1, wherein the membrane comprises one or more crosslinking agents. A25. The membrane structure according to A24, wherein the one or more cross-linking agents are selected from the group consisting of polyethylene glycol diglycidyl ether, polyethylene glycol tetraglycidyl ether, and polyetheramines. A26. The membrane structure according to A25, wherein the cross-linking agent is polyethylene glycol diglycidyl ether. A27. The membrane structure according to any one of A to A26, wherein the enzyme layer comprises one or more enzymes that respond to the first analyte. A28. The membrane structure according to any one of A to A27, wherein the enzyme layer comprises an electron transfer agent. A29. The membrane structure of A27 and A28, wherein the first analyte is selected from the group consisting of glucose, glutamate, ketone, lactate, creatinine, potassium, sarcosine, and ascorbate. A30. The membrane structure of A29, wherein the first analyte is glucose. A31. The membrane structure of A29, wherein the first analyte is lactate. A32. The membrane structure of any one of A-A31, wherein the change in diffusion rate of the analyte through the membrane structure in response to a change in temperature is less than about 5%. A33. The membrane structure of any one of A-A32, wherein the change in the diffusion rate of the analyte through the membrane structure in response to a change in temperature is less than about 1%.

[0124] B. This disclosure: (i) a sensor tail including at least a first working electrode; (ii) a first active area disposed on a surface of a first working electrode; (iii) a first mass transport limiting membrane overcoating at least the first active area and permeable to the first analyte; an analyte sensor comprising: The first mass transport limiting membrane comprises a copolymer of a first monomer and a second monomer, the first monomer comprising at least acrylamide. B1. The analyte sensor of B, wherein the second monomer comprises a heterocycle-containing moiety.

[0125] B2. The analyte sensor of B or B1, wherein the acrylamide is an N-alkylacrylamide. B2-1. The analytical substance sensor according to B2, wherein the alkyl of the N-alkylacrylamide is a C1-C6 linear or branched alkyl group or a C3-C6 cycloalkyl group. B2-2. The analytical substance sensor according to B2-1, wherein the alkyl of the N-alkylacrylamide is a branched alkyl group. B2-3. The analytical substance sensor according to B2-2, wherein the branched alkyl group is selected from the group consisting of isopropyl, isobutyl, sec-butyl, tert-butyl, tert-pentyl, neopentyl, isopentyl, sec-pentyl, 3-pentyl, sec-isopentyl, active pentyl, isohexyl, tert-hexyl, neohexyl, and sec-hexyl. B2-4. The analytical substance sensor according to any one of B2 to B2-3, wherein the N-alkylacrylamide is N-isopropylacrylamide. B3. The analyte sensor of any one of B1 to B2-4, wherein the heterocycle-containing component is selected from the group consisting of furan, thiophene, pyrrole, pyridine, pyrimidine, imidazole, oxadiazole, isoxazole, oxazole, pyrazole, isothiazole, thiazole, pyrazine, isoquinoline, quinoline, benzofuran, and benzimidazole. B4. The analyte sensor of any one of B1 to B3, wherein the heterocycle is pyridine.

[0126] B5. The analyte sensor of B4, wherein the pyridine is vinylpyridine. B6. The analyte sensor of B5, wherein the vinylpyridine is 2-vinylpyridine, 4-vinylpyridine, or a combination thereof. B7. The analyte sensor of B6, wherein the vinylpyridine is 4-vinylpyridine. B8. The analyte sensor of B6, wherein the vinylpyridine is 2-vinylpyridine. B9. The analyte sensor of any one of B1 to B3, wherein the heterocycle is imidazole. B10. The analyte sensor of B9, wherein the imidazole is vinylimidazole. B11. The analyte sensor of B10, wherein the vinylimidazole is 1-vinylimidazole, 2-vinylimidazole, 4-vinylimidazole, or a combination thereof. B12. The analyte sensor of B11, wherein the vinylimidazole is 1-vinylimidazole. B13. The analyte sensor of B11, wherein the vinylimidazole is 2-vinylimidazole. B14. The analyte sensor of any one of B1 to B3, wherein the acrylamide is an N-alkylacrylamide and the heterocycle is pyridine.

[0127] B15. The analyte sensor of B14, wherein the N-alkylacrylamide is N-isopropylacrylamide. B16. The analyte sensor of B14 or B15, wherein the pyridine is vinylpyridine. B17. The analyte sensor of B16, wherein the vinylpyridine is 4-vinylpyridine. B18. The analyte sensor according to any one of B14 to B17, wherein the acrylamide is N-alkylacrylamide and the pyridine is 4-vinylpyridine. B18-1. The analyte sensor of any one of B-B18, wherein the copolymer comprises about 20 mer% to about 70 mer% of the first monomer. B18-2. The analyte sensor of any one of B to B18-1, wherein the copolymer comprises about 40 mer% to about 60 mer% of the first monomer. B18-3. The analyte sensor of any one of B to B18-2, wherein the copolymer comprises about 30 mer% to about 60 mer% of the first monomer. B18-4. The analyte sensor of any one of B to B18-3, wherein the copolymer comprises about 30 mer% to about 80 mer% of the second monomer. B18-5. The analyte sensor of any one of B to B18-4, wherein the copolymer comprises about 30 mer% to about 65 mer% of the second monomer.

[0128] B19. The analyte sensor of any one of B-B18-5, wherein the copolymer comprises about 30 mer% to about 50 mer% of the second monomer. B19-1. The analyte sensor of any one of B to B18-4, wherein the copolymer comprises about 40 mer% to about 70 mer% of the second monomer. B19-2. The analyte sensor of any one of B-B18, wherein the copolymer comprises at least about 40 mer% of the first monomer. B19-3. The analyte sensor of any one of B-B18, wherein the copolymer comprises at least about 60 mer% of the first monomer. B19-4. The analyte sensor of any one of B-B18, wherein the copolymer comprises at least about 60 mer% of the second monomer. B19-5. The analyte sensor of any one of B-B18, wherein the copolymer comprises at least about 40 mer% of a second monomer. B19-6. The analyte sensor of any one of B14 to B19-5, wherein the copolymer is poly(4-vinylpyridine-co-N-isopropylacrylamide).

[0129] B20. A copolymer having Formula I: [ka] The analyte sensor according to any one of B to B8, having the structure: wherein m and n are each a positive integer. B21. The analyte sensor of B20, wherein the ratio of m to n is from about 1:1 to about 1:100. B22. The analyte sensor of B20, wherein the ratio of m to n is from about 1:1 to about 100:1. B22. The analyte sensor of B19 or B20, wherein the ratio of m to n is from about 4:1 to about 1:4.

[0130] B23. The analyte sensor of B19 or B20, wherein the ratio of m to n is from about 4:1 to about 1:1. B24. The analyte sensor according to B19 or B20, wherein the ratio of m to n is from about 1:1 to about 1:4. B25. The analyte sensor of B24, wherein the ratio of m to n is about 1:1 to about 1:3. B26. The analyte sensor according to B25, wherein the ratio of m to n is about 1:1 to about 1:2. B27. The analyte sensor of B19, wherein m is in the range of about 1 to about 90 and n is in the range of about 1 to about 90. B27-1. The analyte sensor according to B27, wherein m is in the range of about 30 to about 50 and n is in the range of about 50 to about 70. B27-2. The analyte sensor of B27-1, wherein m is about 40 and n is about 60. B28. The analyte sensor of B27, wherein m is about 80 and n is about 22, or m is about 65 and n is about 35, or m is about 50 and n is about 50. B29. The analyte sensor of any one of B-B28, wherein the membrane comprises one or more cross-linking agents.

[0131] B30. The analyte sensor of B29, wherein the one or more cross-linking agents are selected from the group consisting of polyethylene glycol diglycidyl ether, polyethylene glycol tetraglycidyl ether, and polyether amines. B31. The analyte sensor of B30, wherein the cross-linking agent is polyethylene glycol diglycidyl ether. B32. The analyte sensor of any one of B-B31, wherein the first active area comprises one or more enzymes responsive to a first analyte. B33. The analyte sensor of any one of B-B32, wherein the first active area comprises an electron transfer agent. B34. The analyte sensor of any one of B-B33, wherein the first analyte is selected from the group consisting of glucose, glutamate, ketone, lactate, creatinine, potassium, sarcosine, and ascorbate. B35.(iv) a second working electrode, and (v) a second active area disposed on the surface of the second working electrode, the second active area responding to a second analyte different from the first analyte or for detecting a background signal; further comprising The analyte sensor of any one of paragraphs B-B34, wherein a second portion of the first mass transport limiting membrane overcoats the second active area, or a second, separate mass transport limiting membrane overcoats the second active area.

[0132] B36. The analyte sensor of B35, wherein the concentration of the first analyte in the fluid can be obtained by subtracting the background signal from the first signal obtained from the first working electrode. B37. The analyte sensor of B35 or B36, wherein the second active area comprises at least one enzyme responsive to a second analyte. B38. The analyte sensor of any one of B35-B37, wherein the second distinct mass transport limiting membrane comprises a different polymer than the first distinct mass transport limiting membrane. B39. The analyte sensor of any one of B35-B37, wherein the second, distinct mass transport limiting membrane comprises the same copolymer as the first, distinct mass transport limiting membrane. B40. The analyte sensor of any one of B-B39, wherein the change in diffusion rate of the analyte through the mass transport limiting membrane in response to a change in temperature is less than about 5%. B41. The analyte sensor of any one of B-B40, wherein the change in diffusion rate of the analyte through the mass transport limiting membrane in response to a change in temperature is less than about 1%. B42. The analyte sensor of any one of B-B41, wherein the change in sensitivity of the analyte sensor in response to a change in temperature is less than about 5%. B43. The analyte sensor of any one of B-B42, wherein the change in sensitivity of the analyte sensor in response to a change in temperature is less than about 1%.

[0133] C. This disclosure (i) providing an analyte sensor according to any one of B to B43; (ii) obtaining a first signal proportional to the concentration of a first analyte in a fluid in contact with the first active area at or above the redox potential of the active area; and (iii) correlating the first signal with the concentration of the first analyte in the fluid. The present invention provides a method for detecting an analyte, comprising: D. This disclosure: (i) providing an analyte sensor according to any one of B to B39; (ii) obtaining a first signal at or above the redox potential of the first active area, the first signal being proportional to the concentration of the first analyte in the fluid in contact with the first active area; (iii) obtaining a second signal at or above the redox potential of the second active area, the second signal being proportional to the concentration of a second analyte in the fluid in contact with the second active area; (iv) correlating the first signal with the concentration of the first analyte in the fluid; and (v) correlating the second signal with the concentration of a second analyte in the fluid. The present invention provides a method for detecting an analyte, comprising:

[0134] E. The present disclosure provides a copolymer comprising at least a first monomer and a second monomer, wherein the first monomer comprises an acrylamide and the second monomer comprises a heterocycle-containing moiety. E1. The copolymer of E, wherein the acrylamide is an N-alkyl acrylamide. E2. A copolymer according to E1, wherein the alkyl of the N-alkylacrylamide is a C1-C6 linear or branched alkyl group or a C3-C6 cycloalkyl group. E3. A copolymer according to E2, wherein the alkyl of the N-alkylacrylamide is a branched alkyl group. E4. The copolymer according to E3, wherein the branched alkyl group is selected from the group consisting of isopropyl, isobutyl, sec-butyl, tert-butyl, tert-pentyl, neopentyl, isopentyl, sec-pentyl, 3-pentyl, sec-isopentyl, active pentyl, isohexyl, tert-hexyl, neohexyl, and sec-hexyl.

[0135] E5. The copolymer of any one of E-E4, wherein the N-alkylacrylamide is N-isopropylacrylamide. E6. The copolymer of any one of E-E5, wherein the heterocycle-containing component is selected from the group consisting of furan, thiophene, pyrrole, pyridine, pyrimidine, imidazole, oxadiazole, isoxazole, oxazole, pyrazole, isothiazole, thiazole, pyrazine, isoquinoline, quinoline, benzofuran, and benzimidazole. E7. The copolymer of any one of E-E6, wherein the heterocycle is pyridine. E8. The copolymer according to E7, wherein the pyridine is vinylpyridine. E9. The copolymer of E8, wherein the vinylpyridine is 2-vinylpyridine, 4-vinylpyridine, or a combination thereof. E10. The copolymer according to E8 or E9, wherein the vinylpyridine is 4-vinylpyridine. E11. The copolymer according to E8 or E9, wherein the vinylpyridine is 2-vinylpyridine.

[0136] E12. The copolymer of any one of E-E6, wherein the heterocycle is imidazole. E13. The copolymer according to E12, wherein the imidazole is vinylimidazole. E14. The copolymer according to E13, wherein the vinylimidazole is 1-vinylimidazole, 2-vinylimidazole, 4-vinylimidazole, or a combination thereof. E15. The copolymer according to E14, wherein the vinylimidazole is 1-vinylimidazole. E16. The copolymer according to E14, wherein the vinylimidazole is 2-vinylimidazole. E17. The copolymer of any one of E-E16, wherein the acrylamide is an N-alkylacrylamide and the heterocycle is pyridine. E18. The copolymer according to E17, wherein the N-alkylacrylamide is N-isopropylacrylamide. E19. A copolymer according to E17 or E18, wherein the pyridine is vinylpyridine.

[0137] E20. The copolymer according to E19, wherein the vinylpyridine is 4-vinylpyridine. E21. The copolymer according to any one of E17 to E20, wherein the acrylamide is an N-alkylacrylamide and the pyridine is 4-vinylpyridine. E22. The copolymer of any one of E-E21, comprising from about 20 mer% to about 70 mer% of the first monomer. E22-1. A copolymer according to any one of E-E22, comprising from about 40 mer% to about 60 mer% of the first monomer. E22-2. A copolymer according to any one of E to E22-1, comprising about 30 mer% to about 60 mer% of the first monomer.

[0138] E23. The copolymer of any one of E-E22-2, comprising about 30 mer% to about 80 mer% of a second monomer. E23-1. The copolymer of any one of E-E23, comprising from about 30 mer% to about 65 mer% of a second monomer. E23-2. The copolymer according to any one of E to E23-1, comprising about 30 mer% to about 50 mer% of a second monomer. E23-3. The copolymer of any one of E-E23, comprising about 40 mer% to about 70 mer% of a second monomer. E23-4. The copolymer of any one of E-E21, comprising at least about 40 mer% of the first monomer. E23-5. The copolymer of any one of E-E21, comprising at least about 60 mer% of the first monomer. E23-6. The copolymer of any one of E-E21, comprising at least about 60 mer% of a second monomer. E23-7. The copolymer of any one of E-E21, comprising at least about 40 mer% of a second monomer. E23-8. The copolymer according to any one of E17 to E23-7, which is poly(4-vinylpyridine-co-N-isopropylacrylamide).

[0139] E24.Formula I: [ka] The copolymer of any one of E-E10, having the structure: wherein m and n are each positive integers. E25. A copolymer according to E24, wherein the ratio of m to n is from about 1:1 to about 1:100. E26. A copolymer according to E24, wherein the ratio of m to n is from about 1:1 to about 100:1. E27. A copolymer according to E24, wherein the ratio of m to n is from about 4:1 to about 1:4. E28. A copolymer according to E27, wherein the ratio of m to n is from about 1:1 to about 1:4. E29. A copolymer according to E28, wherein the ratio of m to n is from about 1:1 to about 1:3. E30. The copolymer according to E29, wherein the ratio of m to n is from about 1:1 to about 1:2.

[0140] E31. A copolymer according to E24, wherein m is in the range of about 1 to about 90 and n is in the range of about 1 to about 90. E31-1. The copolymer according to E31, wherein m is in the range of about 30 to about 50 and n is in the range of about 50 to about 70. E31-2. A copolymer according to E31-1, wherein m is about 40 and n is about 60. E32. The copolymer according to E31, wherein m is about 80 and n is about 22, or m is about 65 and n is about 35, or m is about 50 and n is about 50. E33. The copolymer of any one of E-E32, wherein the rate of diffusion of the analyte through the copolymer changes by less than about 5% in response to a change in temperature. E34. The copolymer of any one of E-E33, wherein the rate of diffusion of the analyte through the copolymer changes by less than about 1% in response to a change in temperature.

[0141] F. The present disclosure further comprises: (i) a sensor tail including at least a first working electrode; (ii) a first active area disposed on a surface of a first working electrode; (iii) a first mass transport limiting membrane overcoating at least the first active area and permeable to the first analyte; an analyte sensor comprising: The first mass transport limiting membrane comprises a copolymer according to any one of paragraphs E-E32. F1. The analyte sensor of F, wherein the first mass transport limiting membrane further comprises a second polymer. F2. The analyte sensor of F1, wherein the second polymer comprises a silicone. G. The present disclosure further provides an analyte sensor comprising the membrane structure of any one of A-A33. [Example]

[0142] The presently disclosed subject matter may be better understood with reference to the following examples, which are offered by way of illustration, but not limitation, of the presently disclosed subject matter. Example 1 Preparation of poly(4-vinylpyridine-co-N-isopropylacrylamide) This example provides the synthesis of poly(4-vinylpyridine-co-N-isopropylacrylamide) which was used to prepare a mass transport limiting membrane with low temperature dependence. Poly(4-vinylpyridine-co-N-isopropylacrylamide) was prepared by coupling a vinylpyridine, such as 4-vinylpyridine, with N-isopropylacrylamide (NIPAAM), as shown in Scheme I below. [ka] Scheme I

[0143] Example 2 Glucose sensor testing This example provides a test of the temperature dependence of a glucose sensor using a membrane of the present disclosure. In particular, this example provides a test of a glucose sensor having various amounts of poly(4-vinylpyridine-co-N-isopropylacrylamide) at various temperatures to evaluate the temperature dependence of the sensor. The poly(4-vinylpyridine-co-N-isopropylacrylamide) polymer used in the membrane of this example was prepared as described in Example 1. A glucose sensor with a working electrode containing glucose oxidase (GOX) and flavin adenine dinucleotide (FAD) in the enzyme layer was coated with poly(4-vinylpyridine-co-N-isopropylacrylamide) polymer containing different percentages of N-isopropylacrylamide (NIPAAM) and the crosslinker PEG-DGE1000. The film thickness was approximately 20–30 μm. A control sensor was prepared by coating a poly(4-vinylpyridine-co-styrene) polymer film onto a working electrode having a GOX enzyme layer. Examples of poly(4-vinylpyridine-co-styrene) polymer films used as controls include those described in U.S. Patent No. 6,932,894, the disclosure of which is incorporated herein by reference. The poly(4-vinylpyridine-co-styrene) polymer film used as a control may include a poly(4-vinylpyridine-co-styrene) copolymer derivatized with propyl sulfonate and poly(ethylene oxide) moieties.

[0144] The analyte sensors were tested in 0.1 M phosphate buffered saline (PBS) containing 0.1 M NaCl (pH 7.4) and 10 mM glucose at temperatures ranging from 22° C. to 42° C. The temperature was controlled by a circulating water system with a digital temperature controller. Figure 6 provides the response curve of a glucose sensor (control) with a poly(4-vinylpyridine-co-styrene) polymer membrane on a working electrode with a GOX enzyme layer. As shown in Figure 6, increasing the temperature increases the membrane's permeability. With increasing temperature, more water molecules form hydrogen bonds with the polar groups of the polymer, causing the membrane to expand. As a result, more glucose can diffuse, thereby increasing the sensitivity of the sensor.

[0145] Table 1 provides membrane compositions with varying amounts of NIPAAm in poly(4-vinylpyridine-co-N-isopropylacrylamide) copolymer. Membranes were prepared with 0%, 20%, 35%, and 50% NIPAAm by molar monomer ratio. The amount of crosslinker was kept constant. [Table 1]

[0146] Figure 7 provides normalized response curves for glucose sensors with the membrane compositions provided in Table 1. Table 2 provides the percent change in sensor response over the temperature range of 22°C to 42°C. As shown, the working electrode contains 6% glucose oxidase (GOX) alone or a combination of 6% GOX and 3% flavin adenine dinucleotide (FAD). As shown in Figure 7 and Table 2, when the membrane polymer contains a higher percentage of NIPAAm, the temperature dependence of the sensor response is less. In particular, 50% NIPAAm results in a membrane that is significantly less temperature sensitive than membrane polymers with lower NIPAAm content. [Table 2]

[0147] The experiment was repeated varying the amount of cross-linker as shown in Table 3. Figure 8 provides the normalized response curves of glucose sensors having the membrane compositions provided in Table 3. [Table 3]

[0148] Table 4 provides the percent change in sensor response over a temperature range of 22°C to 42°C. As indicated, the working electrode contained 6% glucose oxidase (GOX) alone or a combination of 6% GOX and 3% flavin adenine dinucleotide (FAD). The analyte sensors were tested in 0.1 M phosphate buffered saline (PBS) containing 10 mM glucose at temperatures ranging from 22°C to 42°C. Temperature was controlled by a circulating water system with a digital temperature controller. As shown in Table 4 and Figure 8, different levels of crosslinker do not affect sensor response at varying temperatures. [Table 4]

[0149] The results disclosed in this example demonstrate that using a membrane containing a polymer containing NIPAAM allows for the preparation of sensors with only a 1% change in sensitivity over a temperature range of 22°C to 42°C, compared to a control membrane that exhibited a 7% change in sensitivity. It was also observed that a higher amount of NIPAAM in the polymer reduced the change in sensitivity over the temperature range. Furthermore, this example demonstrates that different levels of crosslinker do not affect the sensor response at varying temperatures.

[0150] Example 3 Membranes containing increasing amounts of N-isopropylacrylamide (NIPAAM) This example provides a test of the temperature dependence of a glucose sensor using a membrane of the present disclosure having increasing amounts of NIPAAM. The poly(4-vinylpyridine-co-N-isopropylacrylamide) polymer used in the membrane of this example was prepared as described in Example 1. This example provides a test of a glucose sensor at various temperatures to evaluate the temperature dependence of the sensor with a membrane containing poly(4-vinylpyridine-co-N-isopropylacrylamide) containing 50% or 60% mole percent of NIPAAM compared to a control sensor with a membrane without poly(4-vinylpyridine-co-N-isopropylacrylamide). Table 5 provides membrane compositions with varying amounts of NIPAAM in poly(4-vinylpyridine-co-N-isopropylacrylamide) copolymer. Membranes with 50% and 60% NIPAAM (molar ratio) were prepared and designated SM50 and SM60, respectively, and placed on top of an enzyme layer containing GOX or FADGDH (FAD-glucose dehydrogenase). The amount of crosslinker was kept constant.

[0151] [Table 5]

[0152] Table 6 provides the percent change in sensor response over a temperature range of 22°C to 42°C. As indicated, the working electrode contains GOX. The analyte sensors were tested in 0.1 M phosphate buffered saline (PBS) containing 10 mM glucose at temperatures ranging from 22°C to 42°C. As shown in Table 6 and Figure 9, the temperature dependence of the sensor response is less when the membrane polymer contains a higher percentage of NIPAAM. In particular, 60% NIPAAM is less temperature sensitive than membrane polymers with lower NIPAAM content, resulting in a membrane that is significantly less temperature sensitive than a control membrane without NIPAAM. [Table 6]

[0153] Table 7 provides the percent change in sensor response over a temperature range of 22°C to 42°C. As indicated, the working electrode contained GOX and FADGDH. GOX was used with a control membrane, and FADGDH was used with a temperature-insensitive membrane. The analyte sensors were tested in 0.1 M phosphate buffered saline (PBS) containing 10 mM glucose at temperatures ranging from 22°C to 42°C. As shown in Table 7 and Figure 10, the temperature dependence of the sensor response is less when the membrane polymer contains a higher percentage of NIPAAM. In particular, 60% NIPAAM is less temperature sensitive than membrane polymers with lower NIPAAM content, resulting in a membrane that is significantly less temperature sensitive than a control membrane without NIPAAM.

[0154] [Table 7] The results disclosed in this example demonstrate that using membranes containing copolymers with 50% or 60% NIPAAM by molar ratio yields sensors with only about a 1% change in sensitivity over the temperature range of 22°C to 42°C compared to control membranes. It was also observed that a higher amount of NIPAAM in the polymer reduces the change in sensitivity over the temperature range. For example, a copolymer with 60% NIPAAM by molar ratio exhibits significantly less temperature sensitivity.

[0155] Example 4 Lactate sensors containing membranes containing increasing amounts of N-isopropylacrylamide (NIPAAM) This example provides a study of the temperature dependence of lactate sensors using membranes of the present disclosure with increasing amounts of NIPAAM. The poly(4-vinylpyridine-co-N-isopropylacrylamide) polymer used in the membranes of this example was prepared as described in Example 1. This example provides testing of lactate sensors at various temperatures to evaluate the temperature dependence of the sensors with membranes containing poly(4-vinylpyridine-co-N-isopropylacrylamide) with 50% or 60% mole percent NIPAAM compared to control sensors with membranes that do not contain poly(4-vinylpyridine-co-N-isopropylacrylamide), such as a control sensor with a polymer containing only poly(4-vinylpyridine).

[0156] Table 8 provides membrane compositions with varying amounts of NIPAAM in poly(4-vinylpyridine-co-N-isopropylacrylamide) copolymer. Membranes with 50% and 60% NIPAAM by molar ratio were prepared and designated SM50 and SM60, respectively, and placed on top of an enzyme layer containing lactate oxidase. The amount of crosslinker was kept constant. The sensor was immersed in the copolymer four times at a withdrawal speed of 5 mm / s and once at a withdrawal speed of 1 mm / s (referred to as 4x5 and 1x1 in Table 8). [Table 8]

[0157] Table 9 and Figure 11 provide the percent change in sensor response over a temperature range of 22°C to 42°C. As shown, the working electrode contains lactate oxidase (LOX). The analyte sensors were tested at temperatures ranging from 22°C to 42°C. As shown in Table 9 and Figure 11, when the membrane polymer contains a higher percentage of NIPAAM, the temperature dependence of the sensor response is less. In particular, 60% NIPAAM is less temperature sensitive than membrane polymers with lower NIPAAM content, resulting in a membrane that is significantly less temperature sensitive than a control membrane without NIPAAM. [Table 9]

[0158] The results disclosed in this example demonstrate that using membranes containing copolymers with 50% or 60% NIPAAM by molar ratio yields lactate sensors with only about a 1-2% change in sensitivity over the temperature range of 22°C to 42°C compared to control membranes. It was also observed that a higher amount of NIPAAM in the polymer reduces the change in sensitivity over the temperature range. For example, copolymers with 60% NIPAAM by molar ratio exhibit significantly less temperature sensitivity.

[0159] While the presently disclosed subject matter and its advantages have been described in detail, it should be understood that various changes, substitutions, and alterations can be made therein without departing from the spirit and scope of the disclosed subject matter. Furthermore, the scope of this application is not intended to be limited to the particular embodiments of the processes, machines, manufacture, and compositions, methods, and processes described in the specification. As will be readily apparent to those skilled in the art from the disclosed subject matter of the presently disclosed subject matter, any now-existing or later-developed process, machine, manufacture, composition, method, or step that performs substantially the same function or achieves substantially the same result as the corresponding embodiment described herein can be utilized in accordance with the presently disclosed subject matter. Accordingly, it is intended that the appended claims include within their scope such processes, machines, manufacture, compositions, methods, or steps. Throughout this application, various patents, patent applications, publications, product descriptions, protocols, and sequence accession numbers are cited, the inventions of which are incorporated herein by reference in their entirety for all purposes.

Claims

1. an enzyme layer, and a membrane positioned adjacent to the enzyme layer A membrane structure comprising: the membrane comprises a copolymer of at least a first monomer and a second monomer; the first monomer comprises acrylamide; The membrane structure wherein the second monomer comprises a heterocycle-containing component.

2. The membrane structure of claim 1, wherein the acrylamide is an N-alkyl acrylamide.

3. The alkyl of the N-alkylacrylamide is a C1-C6 linear or branched alkyl group or a C 3 -C 6 The membrane structure of claim 2, wherein the alkyl group is a cycloalkyl group.

4. The membrane structure of claim 3, wherein the alkyl of the N-alkylacrylamide is a branched alkyl group.

5. The membrane structure according to any one of claims 2 to 4, wherein the N-alkylacrylamide is N-isopropylacrylamide.

6. The film structure according to any one of claims 1 to 5, wherein the heterocycle of the heterocycle-containing component is selected from the group consisting of furan, thiophene, pyrrole, pyridine, pyrimidine, imidazole, oxadiazole, isoxazole, oxazole, pyrazole, isothiazole, thiazole, pyrazine, isoquinoline, quinoline, benzofuran, and benzimidazole.

7. The membrane structure according to any one of claims 1 to 6, wherein the heterocycle is pyridine.

8. 7. The membrane structure according to claim 5, wherein the heterocycle is vinylpyridine.

9. 9. The membrane structure of claim 8, wherein the vinylpyridine is selected from the group consisting of 2-vinylpyridine, 4-vinylpyridine, and combinations thereof.

10. 10. The membrane structure according to claim 8, wherein the vinylpyridine is 4-vinylpyridine.

11. 11. The membrane structure of claim 1, wherein the copolymer comprises (i) from about 20 mer% to about 70 mer% of the first monomer, and / or (ii) from about 30 mer% to about 80 mer% of the second monomer.

12. The copolymer comprises (i) from about 40 mer% to about 60 mer% of the first monomer, and / or (ii) from about 30 12. The membrane structure of claim 1, comprising from 0.5% to about 60% of said second monomer.

13. The membrane structure according to any one of claims 1 to 12, wherein the acrylamide is N-isopropylacrylamide and the heterocycle is 4-vinylpyridine.

14. The copolymer has formula I: 【Chemistry 1】 14. The membrane structure according to claim 1, having a structure of: wherein m and n are each a positive integer.

15. 15. The membrane structure of claim 14, wherein the ratio of m to n is from about 1:1 to about 1:

100.

16. 15. The membrane structure of claim 14, wherein the ratio of m to n is from about 1:1 to about 100:

1.

17. 15. The membrane structure of claim 14, wherein the ratio of m to n is from about 4:1 to about 1:

4.

18. 18. The membrane structure of claim 17, wherein the ratio of m to n is from about 1:1 to about 1:

4.

19. 15. The membrane structure of claim 14, wherein m is in the range of about 1 to about 90 and n is in the range of about 1 to about 90.

20. 20. The membrane structure of claim 19, wherein m is about 80 and n is about 22, or m is about 65 and n is about 35, or m is about 50 and n is about 50, or m is about 4 and n is about 6.

21. The membrane structure of any one of claims 1 to 20, comprising one or more cross-linking agents.

22. 22. The membrane structure of claim 21, wherein the one or more cross-linking agents are selected from the group consisting of polyethylene glycol diglycidyl ether, polyethylene glycol tetraglycidyl ether, and polyetheramine.

23. (i) a sensor tail including at least a first working electrode; (ii) a first active area disposed on a surface of the first working electrode; (iii) a first mass transport limiting membrane overcoating at least the first active area and permeable to a first analyte; 1. An analyte sensor comprising: An analyte sensor, wherein the first mass transport limiting membrane comprises a copolymer of at least a first monomer comprising an acrylamide and a second monomer comprising a heterocycle-containing moiety.

24. 24. The analyte sensor of claim 23, wherein the first active area comprises one or more enzymes responsive to a first analyte.

25. 25. The analyte sensor of claim 23 or 24, wherein the first active area comprises an electron transfer agent.

26. 26. The analyte sensor of any one of claims 23 to 25, wherein the first analyte is selected from the group consisting of glucose, glutamate, ketone, lactate, creatine, creatinine, potassium, sarcosine, and ascorbate.

27. 27. The analytical substance sensor of any one of claims 23 to 26, wherein the acrylamide is an N-alkylacrylamide.

28. The alkyl of the N-alkylacrylamide is a C1-C6 linear or branched alkyl group or a C 3 -C 6 28. The analyte sensor of claim 27, wherein the group is a cycloalkyl group.

29. 29. The analyte sensor of claim 28, wherein the alkyl of the N-alkylacrylamide is a branched alkyl group.

30. 30. The analytical substance sensor of any one of claims 27 to 29, wherein the N-alkylacrylamide is N-isopropylacrylamide.

31. 31. The analytical substance sensor of claim 23, wherein the heterocycle of the heterocycle-containing component is selected from the group consisting of furan, thiophene, pyrrole, pyridine, pyrimidine, imidazole, oxadiazole, isoxazole, oxazole, pyrazole, isothiazole, thiazole, pyrazine, isoquinoline, quinoline, benzofuran, and benzimidazole.

32. 32. The analyte sensor of claim 31 , wherein the heterocycle is pyridine.

33. 33. The analyte sensor of claim 31 or 32, wherein the heterocycle is vinylpyridine.

34. 34. The analyte sensor of claim 33, wherein the vinylpyridine is selected from the group consisting of 2-vinylpyridine, 4-vinylpyridine, and combinations thereof.

35. 35. The analyte sensor of claim 33 or 34, wherein the vinylpyridine is 4-vinylpyridine.

36. 36. The analyte sensor of any one of claims 23 to 35, wherein the copolymer comprises (i) from about 20 mer% to about 70 mer% of the first monomer, and / or (ii) from about 30 mer% to about 80 mer% of the second monomer.

37. The copolymer comprises (i) from about 40 mer% to about 60 mer% of the first monomer, and / or (ii) from about 30 37. The analyte sensor of any one of claims 23 to 36, comprising from 1 mer% to about 60 mer% of the second monomer.

38. 38. The analytical substance sensor of any one of claims 23 to 37, wherein the acrylamide is N-isopropylacrylamide and the heterocycle is 4-vinylpyridine.

39. The copolymer has formula I: 【Chemistry 2】 39. The analyte sensor of any one of claims 23 to 38, having the structure: wherein m and n are each positive integers.

40. 40. The analyte sensor of claim 39, wherein the ratio of m to n is from about 1:1 to about 1:

100.

41. 40. The analyte sensor of claim 39, wherein the ratio of m to n is from about 1:1 to about 100:

1.

42. 40. The analyte sensor of claim 39, wherein the ratio of m to n is from about 4:1 to about 1:

4.

43. 43. The analyte sensor of claim 42, wherein the ratio of m to n is from about 1:1 to about 1:

4.

44. 40. The analyte sensor of claim 39, wherein m is in the range of about 1 to about 90 and n is in the range of about 1 to about 90.

45. 45. The analyte sensor of claim 44, wherein m is about 80 and n is about 22, or m is about 65 and n is about 35, or m is about 50 and n is about 50, or m is about 4 and n is about 6.

46. (iv) a second working electrode, and (v) a second active area disposed on the surface of the second working electrode, the second active area responding to a second analyte different from the first analyte or for detecting a background signal; further comprising 46. ​​The analyte sensor of any one of claims 23-45, wherein a second portion of the first mass transport limiting membrane overcoats the second active area, or a second, separate mass transport limiting membrane overcoats the second active area.

47. 47. The analyte sensor of claim 46, wherein the concentration of the first analyte in a fluid can be obtained by subtracting the background signal from a first signal obtained from the first working electrode.

48. 48. The analyte sensor of claim 46 or 47, wherein the second active area comprises at least one enzyme responsive to the second analyte.

49. 49. The analyte sensor of any one of claims 46-48, wherein the second distinct mass transport limiting membrane comprises a different polymer than the first distinct mass transport limiting membrane.

50. 49. The analyte sensor of any one of claims 46-48, wherein the second distinct mass transport limiting membrane comprises the same copolymer as the first distinct mass transport limiting membrane.

51. (i) providing an analyte sensor according to any one of claims 23 to 50; (ii) obtaining a first signal proportional to the concentration of the first analyte in a fluid in contact with the first active area at or above the redox potential of the active area; and (iii) correlating the first signal with the concentration of the first analyte in the fluid.

1. A method for detecting an analyte, comprising:

52. (i) providing an analyte sensor according to any one of claims 23 to 50; (ii) obtaining a first signal at or above the redox potential of the first active area, the first signal being proportional to the concentration of the first analyte in a fluid in contact with the first active area; (iii) obtaining a second signal at or above the redox potential of the second active area, the second signal being proportional to the concentration of the second analyte in the fluid in contact with the second active area; (iv) correlating the first signal with the concentration of the first analyte in the fluid; and (v) correlating the second signal with the concentration of the second analyte in the fluid.

1. A method for detecting an analyte, comprising:

53. A copolymer comprising at least a first monomer and a second monomer, wherein the first monomer comprises an acrylamide and the second monomer comprises a heterocycle-containing component.

54. 54. The copolymer of claim 53, wherein the acrylamide is an N-alkyl acrylamide.

55. The alkyl of the N-alkylacrylamide is a C1-C6 linear or branched alkyl group or a C 3 -C 6 55. The copolymer of claim 54, which is a cycloalkyl group.

56. 56. The copolymer of claim 55, wherein the alkyl of the N-alkylacrylamide is a branched alkyl group.

57. 57. The copolymer of any one of claims 54 to 56, wherein the N-alkylacrylamide is N-isopropylacrylamide.

58. 58. The copolymer of any one of claims 53 to 57, wherein the heterocycle-containing component is selected from the group consisting of furan, thiophene, pyrrole, pyridine, pyrimidine, imidazole, oxadiazole, isoxazole, oxazole, pyrazole, isothiazole, thiazole, pyrazine, isoquinoline, quinoline, benzofuran, and benzimidazole.

59. 59. The copolymer of claim 58, wherein the heterocycle is pyridine.

60. 60. The copolymer of claim 59, wherein the pyridine is vinylpyridine.

61. 61. The copolymer of claim 60, wherein the vinylpyridine is 2-vinylpyridine, 4-vinylpyridine, or a combination thereof.

62. 62. The copolymer of claim 60 or 61, wherein the vinylpyridine is 4-vinylpyridine.

63. 63. The copolymer of any one of claims 53 to 62, wherein the acrylamide is N-isopropylacrylamide and the heterocycle is 4-vinylpyridine.

64. 64. The copolymer of any one of claims 53-63, comprising: (i) from about 20 mer% to about 70 mer% of said first monomer; and / or (ii) from about 30 mer% to about 80 mer% of said second monomer.

65. The copolymer comprises (i) from about 40 mer% to about 60 mer% of the first monomer, and / or (ii) from about 30 65. The analyte sensor of claim 64, comprising from 1 mer % to about 60 mer % of the second monomer.

66. Formula I: 【Transformation 3】 66. The copolymer of any one of claims 53 to 65, having the structure:

67. 67. The copolymer of claim 66, wherein the ratio of m to n is from about 1:1 to about 1:

100.

68. 67. The copolymer of claim 66, wherein the ratio of m to n is from about 1:1 to about 100:

1.

69. 67. The copolymer of claim 66, wherein the ratio of m to n is from about 4:1 to about 1:

4.

70. 70. The copolymer of claim 69, wherein the ratio of m to n is from about 1:1 to about 1:

4.

71. 67. The copolymer of claim 66, wherein m is in the range of about 1 to about 90 and n is in the range of about 1 to about 90.

72. 72. The copolymer of claim 71, wherein m is about 80 and n is about 22, or m is about 65 and n is about 35, or m is about 50 and n is about 50, or m is about 4 and n is about 6.

73. An analyte sensor comprising a membrane structure according to any one of claims 1 to 22.

74. Use of an analyte sensor according to any one of claims 23 to 50 for detecting an analyte.

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