Dispensed area and drop solid content tunability

Abstract

The present disclosure provides analyte sensors comprising a first working electrode, a sensing area disposed upon a surface of the first working electrode, and a membrane that overcoats the sensing area, wherein the first sensing area comprises from about 0.19% to about 16% by weight of a surfactant. The present disclosure also provides methods of using such analyte sensors for detecting one or more analytes present in a biological sample and methods of manufacturing the analyte sensors.

Description

DISPENSED AREA AND DROP SOLID CONTENT TUNABILITYFIELD

[0001] The present disclosure provides analyte sensors comprising a first working electrode, a first sensing area disposed upon a surface of the first working electrode, and a membrane that overcoats at least a part of the first sensing area, wherein the first sensing area comprises from about 0.19% to about 16% by weight of a surfactant. The present disclosure also provides methods of using such analyte sensors for detecting one or more analytes present in a biological sample and methods of manufacturing the analyte sensors.BACKGROUND

[0002] The detection of various analytes within an individual can sometimes be vital for monitoring the condition of their health, as deviations from normal analyte levels can be indicative of a physiological condition. For example, monitoring glucose levels can enable people suffering from diabetes to take appropriate corrective action including administering medicine or consuming a particular food or beverage product to avoid significant physiological harm. Other analytes can be desirable to monitor for other physiological conditions. In some instances, it can be desirable to monitor more than one analyte when monitoring single or multiple physiological conditions, particularly if a person is suffering from comorbid conditions that result in simultaneous dysregulation of two or more analytes in combination with one another.

[0003] Analyte monitoring in an individual can take place periodically or continuously over a period of time. Periodic analyte monitoring can take place by withdrawing a sample of bodily fluid, such as blood or urine, at set time intervals and analyzing the same ex vivo. Periodic, ex vivo analyte monitoring can be sufficient to determine the physiological condition of many individuals. However, ex vivo analyte monitoring can be inconvenient or painful in some instances. Moreover, there is no way to recover lost data if an analyte measurement is not obtained at an appropriate time.

[0004] Continuous analyte monitoring can be conducted using one or more sensors that remain at least partially implanted within a tissue of an individual, such as dermally, subcutaneously, or intravenously, so that analyses can be conducted in vivo. Implantedsensors can collect analyte data on-demand, at a set schedule, or continuously, depending on an individual’s particular health needs and / or previously measured analyte levels.Analyte monitoring with an in vivo implanted sensor can be a more desirable approach for individuals having severe analyte dysregulation and / or rapidly fluctuating analyte levels, although it can also be beneficial for other individuals as well. Since implanted analyte sensors often remain within a tissue of an individual for an extended period of time, it can be highly desirable for such analyte sensors to be made from stable materials exhibiting a high degree of biocompatibility.

[0005] While continuous monitoring is desirable, there are several challenges associated with optimizing manufacturing protocols to reduce the cost and time associated with manufacturing analyte sensors constructed for in vivo and / or in vitro use. During the manufacturing process for an analyte sensor, an aqueous solution (e.g., a sensing formulation) contacts a surface of a substrate. When a liquid contacts a solid surface in air, in general, a droplet of the liquid forms. The shape of the droplet depends upon the interaction between the liquid and the solid surface. If the interaction is strong, i.e., if the liquid “likes” the surface, the liquid drop will be “flat.” On the contrary, if the interaction is weak, i.e., the liquid “dislikes” the surface, it will “bead up.”

[0006] Currently used aqueous solutions deposited on analyte sensor substrates tend to bead up on the surface of the substrate. If each individual droplet of solution beads up on the surface of a substrate, it results in a droplet covering a reduced surface area compared to the surface area that could be covered by a droplet that does not bead up on the surface. Thus, a solution that beads up on the surface of a substrate requires depositing more droplets to cover a substrate resulting in an increase in the time and cost associated with manufacturing the analyte sensor.SUMMARY

[0007] The purpose and advantages of the disclosed subject matter will be set forth in and are apparent from the description that follows, as well as will be learned by practice of the disclosed subject matter. Additional advantages of the disclosed subject matter will be realized and attained by the devices particularly pointed out in the detailed description and claims hereof, as well as from the appended drawings.

[0008] The present disclosure provides an analyte sensor comprising:

[0009] a proximal portion configured to be positioned above a user’s skin; and

[0010] a distal portion configured to be transcutaneously positioned through the user’s skin and in contact with the user’s bodily fluid to detect or monitor an analyte in the bodily fluid in vivo, the distal portion comprising:

[0011] a first working electrode;

[0012] a first sensing area disposed upon a surface of the first working electrode, the first sensing area being responsive to a first analyte; and

[0013] a membrane that overcoats at least a part of the first sensing area;

[0014] wherein the first sensing area comprises from about 0.19% to about 16% by weight of a surfactant.

[0015] The present disclosure provides an analyte sensor comprising:

[0016] a first working electrode;

[0017] a first sensing area disposed upon a surface of the first working electrode, the first sensing area being responsive to a first analyte; and

[0018] a membrane that overcoats at least a part of the first sensing area;

[0019] wherein the first sensing area comprises from about 0.19% to about 16% by weight of a surfactant.

[0020] In some embodiments, the surfactant in the first sensing area is biocompatible.

[0021] In some embodiments, the surfactant in the first sensing areais selected from the group consisting of an anionic surfactant, a nonionic surfactant, a cationic surfactant, an amphoteric surfactant, a zwitterionic surfactant, and combinations thereof.

[0022] In some embodiments, the surfactant in the first sensing area is a nonionic surfactant.

[0023] In some embodiments, the surfactant is an ethoxylated nonionic surfactant.

[0024] In some embodiments, the surfactant is an ethoxylated nonionic surfactant selected from the group consisting of polyethylene glycol sorbitan monolaurate, polyoxyethylene sorbitan monopalmitate, polyethylene glycol sorbitan monostearate, polyethylene glycol sorbitan monooleate, polyethylene glycol tert-octylphenyl ether, polyethylene glycol 4-tert-octylphenyl ether, and combinations thereof.

[0025] In some embodiments, the sensing area comprises from about 1% to about 10% by weight of a surfactant.

[0026] In some embodiments, the sensing area further comprises a polymer and at least one redox mediator.

[0027] In some embodiments, the redox mediator in the sensing areaarea comprises a transition metal complex.

[0028] In some embodiments, the transition metal complex is an osmium-containing transition metal complex.

[0029] In some embodiments, the polymer in the sensing area comprises poly(vinylpyridine), poly(thiophene), poly(aniline), poly(pyrrole), or poly(acetylene).

[0030] In some embodiments, the sensing area comprises an osmium-containing transition metal complex bonded to a poly(vinylpyridine)-based polymer.

[0031] In some embodiments, the polymer in the sensing area is crosslinked with a crosslinking agent.

[0032] In some embodiments, the crosslinking agent is a polyepoxide, cyanuric chloride, N-hydroxysuccinamide, an imidoester, epichlorohydrin, or any combination thereof.

[0033] In some embodiments, the crosslinking agent is a polyethylene glycol diglycidylether (PEGDGE).

[0034] In some embodiments, the sensing area further comprises an analyte-responsive enzyme.

[0035] In some embodiments, the analyte sensor comprising the surfactant has an analyte sensitivity that is greater than the analyte sensitivity of an otherwise identical sensor lacking a surfactant.

[0036] In some embodiments, the analyte sensor comprising the surfactant has an analyte sensitivity that is at least 75% greater than the analyte sensitivity of an otherwise identical sensor lacking the surfactant.

[0037] In some embodiments, the analyte sensor comprises a proximal portion configured to be positioned above a user’s skin and a distal portion configured to be positioned through the user’s skin and in contact with a user’s bodily fluid to detect or monitor an analyte in the bodily fluid in vivo.

[0038] In some embodiments, the analyte sensor further comprises sensor electronics coupled to the analyte sensor, the sensor electronics comprising a processor and a memory.

[0039] In some embodiments, the analyte sensor further comprises:

[0040] a second working electrode; and

[0041] a second sensing area disposed upon a surface of the second working electrode, the second sensing area being responsive to a second analyte differing from the first analyte;

[0042] wherein the second sensing area comprises at least one enzyme responsive to the second analyte.

[0043] The present disclosure comprises a method of manufacturing an analyte sensor comprising:

[0044] depositing a sensing area formulation upon a surface of a working electrode, the sensing area formulation comprising at least one surfactant in an amount from about 0.01% to about 1% by weight.

[0045] In some embodiments, the depositing is performed by a non-impact printing method.

[0046] In some embodiments, the non-impact printing method comprises piezoelectric pulse-jet deposition.

[0047] In some embodiments, the non-impact printing method comprises thermoelectric pulse-jet deposition.

[0048] In some embodiments, a droplet deposited during a single activation event of the non-impact printing method has a volume from about 0.01 pL to 1000 pL.

[0049] In some embodiments, a droplet deposited during a single activation event of the non-impact printing method covers an area from about 0.005 mm2to about 0.10 mm2.

[0050] In some embodiments, the depositing is performed by dip coating.

[0051] In some embodiments, the sensing area formulation comprises the surfactant in an amount from about 0.25% to about 0.75% by weight.

[0052] In some embodiments, the sensing area formulation comprising at least one surfactant has a concentration (mass by volume) of solid material that is greater than the concentration (mass by volume) of solid material of an otherwise identical sensing area formulation lacking the surfactant.

[0053] In some embodiments, the sensing area formulation comprising at least one surfactant has a concentration of solid material that is at least 25% greater than the concentration of an otherwise identical sensing area formulation lacking the surfactant.

[0054] In some embodiments, the sensing area formulation comprising at least one surfactant has a concentration of solid material that is at least 75% greater than the concentration of an otherwise identical sensing area formulation lacking the surfactant.

[0055] The present disclosure also provides a method of manufacturing an analyte sensor, the method comprising:

[0056] depositing a sensing area upon a surface of an electrode;

[0057] applying a voltage to the electrode; and

[0058] at least partially removing a liquid from the sensing area while the voltage is applied to provide a contact area between the sensing area and the electrode of from about 0.075 mm2to about 0.25 mm2.

[0059] In some embodiments, the contact area between the sensing area and the electrode is from about 0.075 mm2to about 0.10 mm2.

[0060] In some embodiments, at least partially removing the liquid from the sensing area while the voltage is applied provides a contact angle between the surface of the electrode and the sensing area from about 5° to about 85°.

[0061] In some embodiments, the contact angle between the sensing area and electrode is from about 5° to about 45°.

[0062] In some embodiments, the electrode is porous and at least partially removing the liquid from the sensing area while the voltage is applied provides a penetration depth of the sensing area into the electrode of from about 1 pm to about 25 pm.

[0063] In some embodiments, the penetration depth of the sensing area into the electrode is from about 1 pm to about 10 pm.

[0064] In some embodiments, at least partially removing the liquid from the sensing area is provided by adjusting a temperature of the sensing area, the electrode, or a combination thereof.

[0065] In some embodiments, the temperature of the sensing area, the electrode, or a combination thereof is adjusted to a temperature sufficient to cause the liquid in the sensing area to evaporate.

[0066] In some embodiments, the contact angle between the sensing area and the electrode is determined using cyclic voltammetry (CV).

[0067] The present disclosure also provides a method of manufacturing an analyte sensor, the method comprising:

[0068] depositing a sensing area on a surface of an electrode using a gantry configured to move relative to an analyte sensor;

[0069] applying a voltage to the electrode; and

[0070] at least partially removing a liquid from the sensing area while the voltage is applied to provide a contact area between the sensing area and the electrode of from about 0.075 mm2to about 0.25 mm2.

[0071] The present disclosure also provides an analyte sensor comprising:

[0072] a proximal portion configured to be positioned above a user’s skin; and

[0073] a distal portion configured to be transcutaneously positioned through the user's skin and in contact with a user’s bodily fluid to detect or monitor an analyte in the bodily fluid in vivo, the distal portion comprising:

[0074] a first working electrode;

[0075] a first sensing area disposed upon a surface of the first working electrode, the first sensing area being responsive to a first analyte; and

[0076] a membrane that overcoats at least a part of the first sensing area;

[0077] wherein a contact area between the first sensing area and the first working electrode is at least 0.075 mm2.

[0078] The present disclosure also provides an analyte sensor comprising:

[0079] a first working electrode;

[0080] a first sensing area disposed upon a surface of the first working electrode, the first sensing area being responsive to a first analyte; and

[0081] a membrane that overcoats at least a part of the first sensing area;

[0082] wherein a contact area between the first sensing area and the first working electrode is at least 0.075 mm2.

[0083] In some embodiments, the first sensing area comprises a water-soluble solution of one or more enzymes and one or more electroactive mediators.

[0084] In some embodiments, the first working electrode comprises a carbon-based electrode.

[0085] In some embodiments, the first working electrode comprises carbon ink.

[0086] In some embodiments, the first working electrode has a porosity in a range from about 20% to about 80%.

[0087] In some embodiments, a penetration depth of the first sensing area into the first working electrode is at least 1 pm.BRIEF DESCRIPTION OF THE DRAWINGS

[0088] The following figures are included to illustrate some aspects of the present disclosure and should not be viewed as exclusive embodiments. The subject matter disclosed is capable of considerable modifications, alterations, combinations, and equivalents in form and function, without departing from the scope of this disclosure.

[0089] FIG. 1 shows a diagram of an illustrative sensing system that can incorporate an analyte sensor of the present disclosure.

[0090] FIGS. 2A-2C show cross-sectional diagrams of analyte sensors including a single sensing area.

[0091] FIGS. 3A-3C show cross-sectional diagrams of analyte sensors including two sensing areas.

[0092] FIG. 4 shows a cross-sectional diagram of an analyte sensor including two sensing areas.

[0093] FIGS. 5A-5C show perspective views of analyte sensors including two sensing areas upon separate working electrodes.

[0094] FIG. 6 is a line graph showing sensor current (nA) versus concentration (mM) of exemplary glucose sensors comprising a working electrode coated with six spots of a sensing area formulation dispensed in four passes, where the sensing area formulation comprises an osmium-containing poly(4-vinylpyridine)-based polymer, glucose oxidase, PEGDGE 400, and no polyethylene glycol tert-octylphenyl ether (♦), 0.1% polyethylene glycol tert-octylphenyl ether(a), 0.5% polyethylene glycol tert-octylphenyl ether (A), and 1% polyethylene glycol tert-octylphenyl ether (X).

[0095] FIG. 7 is a bar graph showing the average response time (hours:minutes:seconds) of exemplary glucose sensors comprising a working electrode coated with six spots of a sensing area formulation dispensed in four passes, where the sensing area formulation comprises osmium-containing poly(4-vinylpyridine)-based polymer, glucose oxidase, PEGDGE 400 overcoated with a layer of a derivatized polyvinylpyridine-co-styrene copolymer, and 1% polyethylene glycol tert-octylphenyl ether, 0.5% polyethylene glycoltert-octylphenyl ether, 0.1% polyethylene glycol tert-octylphenyl ether, and no polyethylene glycol tert-octylphenyl ether.DETAILED DESCRIPTION

[0096] The present disclosure provides analyte sensors comprising a first working electrode, a first sensing area disposed upon a surface of the first working electrode, and a membrane that overcoats at least a part of the first sensing area, wherein the first sensing area comprises from about 0.19% to about 16% by weight of a surfactant. By adding a surfactant to the sensing area formulation, the sensing area formulation has increased wettability which allows a single drop of the sensing area formulation to cover an increased surface area of a substrate. Increasing the surface area covered by a single drop of the sensing area formulation reduces the number of drops needed to cover the substrate which results in the reduction of manufacturing time and cost.

[0097] In some embodiments, the present disclosure is directed to methods and devices for improving the distribution of one or more components of an aqueous solution (e.g., a sensing area formulation) by including a surfactant in the sensing area formulation, wherein the components are disposed proximate to a working electrode of an analyte sensor. Currently used aqueous solutions (e.g., sensing area formulations) deposited on analyte sensor substrates tend to bead up on the surface of the substrate. The present disclosures provides for a surfactant to be included in an aqueous solution (e.g., a sensing area formulation), resulting in an increase in the wettability of the solution on a surface, thereby providing coverage of a greater surface area than a solution without a surfactant.

[0098] During the manufacturing process for an analyte sensor, an aqueous solution (e.g., a sensing area formulation) contacts a surface of a substrate. When a liquid contacts a solid surface in air, in general, a droplet of the liquid forms. The shape of the droplet depends upon the interaction between the liquid and the solid surface. If the interaction is strong, i.e., if the liquid “likes” the surface, the liquid drop will be “flat.” On the contrary, if the interaction is weak, i.e., the liquid “dislikes” the surface, it will “bead up.” This property is described quantitatively by the concept of contact angle, i.e. the angle (theta) at which the air-liquid and solid-liquid interfaces meet, also referred to as the “three-phase contact point.” The contact angle is the tangent of the drop profile at the three-phase contact point and it provides an inverse measurement of wettability. See, e.g,Ponomar, M., et al., Membranes 12(8):765 (2022). A contact angle less than 90° indicates that wetting of the surface with the liquid is favorable, and if theta is less than 10° then the liquid will spread over a large area of the surface. If theta is greater than 90°, it means that wetting of the surface is unfavorable.

[0099] In some embodiments, the addition of a surfactant to an aqueous solution (e.g., a sensing area formulation) used in the manufacturing in vivo and / or in vitro analyte sensors greatly improves uniformity and / or distribution of one or more components of the sensor (e.g., an enzyme-containing sensing area). Surfactants are additives that reduce the contact angle of the solution, thereby lowering the interfacial tension between the solution and the surface. The interfacial tension is the adhesive force between the liquid phase of one substance and either a solid, liquid, or gas phase of another substance. Here, the interfacial tension controls the contact angle between the solution and the surface. The contact angle is specific for any given system. On many highly hydrophilic surfaces, water droplets will exhibit contact angles of 0° to 30°. If the solid surface is hydrophobic, the contact angle can be larger than 90°. On highly hydrophobic surfaces, the surfaces can have a water contact angle of 150° or even as high as nearly 180°. On these highly hydrophobic surfaces, water droplets simply rest on the surface, without actually wetting to any significant extent.

[0100] In some embodiments, the surfactant reduces the contact angle of the solution by modifying the aqueous solution’s surface tension. This results in an increase in the uniformity of distribution of the constituents of a solution deposited on a substrate upon drying and curing, as well as an increase in the surface area of the solution upon drying and curing as compared to a solution lacking the surfactant. Increasing the surface area covered by each individual droplet allows for a reduction of the number of droplets dispensed and reduces the time required to cover the same surface area. This, in turn, improves the overall manufacturing process of the analyte sensor.

[0101] The present disclosure provides solutions that exhibit a desirable combination of reduction in the number of droplets dispensed and reduction in the time required to cover a given surface area — allowing for a reduction in manufacturing time and cost.

[0102] The present disclosure further provides methods of detecting an analyte using the disclosed sensors and methods of manufacturing the disclosed analyte sensors.I. DEFINITIONS

[0103] For convenience, the meaning of some terms and phrases used in the specification, examples, and appended claims are provided below. Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed technology, because the scope of the technology is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs. If there is an apparent discrepancy between the usage of a term in the art and its definition provided herein, the definition provided within the specification will control.

[0104] The articles “a,” “an,” and “the” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0105] As used herein, the term “about” means ± 10% of the specified value, unless otherwise indicated.

[0106] The term “at least” prior to a number or series of numbers is understood to include the number associated with the term “at least,” and all subsequent numbers or integers that could logically be included, as clear from context. When “at least” is present before a series of numbers or a range, it is understood that “at least” can modify each of the numbers in the series or range. For example, “at least 3” means at least 3, at least 4, at least 5, etc. When “at least” is present before a component in a method step, then that component is included in the step, whereas additional components are optional.

[0107] As used herein, the terms “comprises,” “comprising,” “having,” “including,” “containing,” and the like are open-ended terms meaning “including, but not limited to.” To the extent a given embodiment disclosed herein “comprises” certain elements, it should be understood that present disclosure also specifically contemplates and discloses embodiments that “consist essentially of’ those elements and that “consist of’ those elements.

[0108] As used herein the terms “consists essentially of,” “consisting essentially of,” and the like are to be construed as a semi-closed terms, meaning that no other ingredientswhich materially affect the basic and novel characteristics of an embodiment are included.

[0109] As used herein, the terms “consists of,” “consisting of,” and the like are to be construed as closed terms, such that an embodiment “consisting of’ a particular set of elements excludes any element, step, or ingredient not specified in the embodiment.

[0110] As used herein, the terms “measure,” “measuring,” and “measured” can encompass the meaning of a respective one or more of the terms “determine,” “determining,” “determined,” “calculate,” “calculating,” and “calculated.”[OHl] As used herein, an “analyte” is a substance that is measured or detected. The analyte can be from, for example, a biofluid and can be tested in vivo, ex vivo, or in vitro. Glucose, glutamate, ketones, alcohols, lactate, creatinine, and combinations thereof are exemplary analytes in the present disclosure. Typically, the analytes are measured in vivo.

[0112] As used herein, a “sensor” is a device configured to detect the presence and / or measure the level of an analyte in a sample via electrochemical oxidation and reduction reactions on the sensor. These reactions are transduced to an electrical signal that can be indicative of presence and be correlated to an amount, concentration, or level of an analyte in the sample.

[0113] As used herein, a “working electrode” is an electrode at which the analyte (or additional compound(s) whose level depends on the level of the analyte) is electrooxidized or electroreduced with or without the agency of an electron transfer agent.

[0114] As used herein, a “counter electrode” refers to an electrode paired with the working electrode, through which passes a current equal in magnitude and opposite in sign to the current passing through the working electrode. In the context of embodiments of the present disclosure, the term “counter electrode” includes both a) counter electrodes and b) counter electrodes that also function as reference electrodes (i.e., counter / reference electrodes), unless otherwise indicated.

[0115] As used herein, a “reference electrode” includes both a) reference electrodes and b) reference electrodes that also function as counter electrodes (i.e., counter / reference electrodes), unless otherwise indicated.

[0116] As used herein, “electrolysis” is the electrooxidation or electroreduction of a compound either directly at an electrode or via one or more electron transfer agents.

[0117] As used herein, an “electron transfer agent” is a compound that carries electrons between the analyte and the working electrode, either directly, or in cooperation with other electron transfer agents. One example of an electron transfer agent is a redox mediator.

[0118] As used herein, a “redox mediator” is an electron-transfer agent for carrying electrons between an analyte, an analyte-reduced or analyte-oxidized, enzyme, and an electrode, either directly, or via one or more additional electron-transfer agents. A redox mediator that includes a polymeric backbone can also be referred to as a “redox polymer.”

[0119] A “reactive group” is a functional group of a molecule (e.g., a polymer, a crosslinking agent, an enzyme) that is capable of reacting with another compound to couple at least a portion (e.g., another reactive group) of that other compound to the molecule. 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 photo-reactive azido aryl groups. Activated esters, as understood in the art, generally include esters of succinimidyl, benzotri azolyl, or aryl substituted by electron-withdrawing groups such as sulfo, nitro, cyano, or halo groups; or carboxylic acids activated by carbodiimides.

[0120] As used herein, a “sensing area” is a component of the sensor including constituents that facilitate the electrolysis of the analyte. The sensing area can include constituents such as a redox mediator (e.g., an electron transfer agent or a redox polymer), a catalyst (e.g., an analyte-responsive enzyme), which catalyzes a reaction of the analyte to produce a response at the working electrode, or both an electron transfer agent and a catalyst. In some embodiments of the present disclosure, a sensor includes a sensing area that is non-leachably disposed in proximity to or on the working electrode. As used herein, the terms “sensing area” and “sensing layer” can be used interchangeably.

[0121] As used herein, a “sensing element” is an application or region of an analytespecific reactant disposed with the sensing area. As such, a sensing element is capable of interacting with the analyte. A sensing area can have more than one sensing element making up the analyte detection area disposed on the working electrode. In someembodiments, the sensing element includes an analyte-specific reactant and an electron transfer agent (e.g., electron transfer agent). In some embodiments, the sensing element includes an analyte-specific reactant, a redox mediator, and a crosslinker.

[0122] As used herein, “crosslinking agent” or “crosslinker” is a molecule that contains at least two (e.g., 2, 3, or 4) reactive groups (e.g., terminal functional groups) that can link at least two molecules together (intermolecular crosslinking) or at least two portions of the same molecule together (intramolecular crosslinking). A crosslinking agent having more than two reactive groups can be capable of both intermolecular and intramolecular crosslinkings at the same time.

[0123] A “membrane solution” is a solution that contains the components for crosslinking and forming the membrane, including, e.g., polymer (e.g., a modified polymer containing heterocyclic nitrogen groups), a crosslinking agent, and a solvent (e.g., a buffer or an alcohol -buffer mixed solvent).

[0124] As used herein, a “biofluid” is any bodily fluid or bodily fluid derivative in which the analyte can be measured. Examples of biofluid include, for example, dermal fluid, subcutaneous fluid, interstitial fluid, plasma, blood (e.g., from a vein or blood vessel), lymph, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage, amniotic fluid, sweat, or tears.

[0125] The term “patient” refers to a living animal, and thus encompasses a living mammal and a living human, for example. The term “user” can be used herein as a term that encompasses the term “patient.”

[0126] All ranges disclosed herein are inclusive of the endpoints, and the endpoints are independently combinable with each other. Each range disclosed herein constitutes a disclosure of any point or sub-range lying within the disclosed range. For example, the range from X to Y, is inclusive of X and Y. And, the range between X and Y, is inclusive of X and Y.

[0127] The phrase “enzyme composition” refers to a composition that includes one or more enzymes for detecting and / or measuring an analyte. In some non-limiting embodiments, the enzyme compositions can include one or more enzymes, polymers, redox mediators, and / or crosslinkers.

[0128] As used herein, the phrase “multi-component membrane” refers to a membrane comprising two or more types of membrane polymers.

[0129] As used herein, the phrase “multilayered membrane” refers to a membrane system comprising two of more layers of membrane polymer. The two or more layers of membrane polymer can comprise multiple layers of the same membrane polymer as long as there is at least one different membrane polymer layer between the two membrane polymer layers comprising the same membrane polymer.

[0130] As used herein, the term “NAD(P)” refers to the nicotinamide adenine dinucleotides NAD+(and its reduced form NADH) and / or NADP+(and its reduced form NADPH). NAD+and NADP+are electron acceptors and NADH and NADPH are electron donors.

[0131] As used herein, the phrase “NAD(P)-dependent enzyme” refers to an enzyme that uses NAD+(and its reduced form NADH) and / or NADP+(and its reduced form NADPH) as a cofactor in a redox reaction.II. ANALYTE SENSORSSensors, Compositions, and Methods of the Disclosure

[0132] Before describing the analyte sensors of the present disclosure and their components in further detail, a brief overview of suitable in vivo analyte sensor configurations and sensor systems employing the analyte sensors will be provided so that the embodiments of the present disclosure can be better understood. FIG. 1 shows a diagram of an illustrative sensing system that can incorporate an analyte sensor of the present disclosure. As shown, sensing system 100 includes sensor control device 102 and reader device 120 that are configured to communicate with one another over a local communication path or link 140, which can be wired or wireless, uni- or bi-directional, and encrypted or non-encrypted. Reader device 120 can constitute an output medium for viewing analyte concentrations and alerts or notifications determined by sensor 104 or a processor associated therewith, as well as allowing for one or more user inputs, according to some embodiments. Reader device 120 can be a multi-purpose smartphone or a dedicated electronic reader instrument. While only one reader device 120 is shown, multiple reader devices 120 can be present in some instances. Reader device 120 can also be in communication with remote terminal 170 and / or trusted computer system 180 via communication path(s) / link(s) 141 and / or 142, respectively, which also can be wired or wireless, uni- or bi-directional, and encrypted or non-encrypted. Reader device 120 canalso or alternately be in communication with network 150 (e.g., a mobile telephone network, the internet, or a cloud server) via communication path / link 151. Network 150 can be further communicatively coupled to remote terminal 170 via communication path / link 152 and / or trusted computer system 180 via communication path / link 153. Alternately, sensor 104 can communicate directly with remote terminal 170 and / or trusted computer system 180 without an intervening reader device 120 being present. For example, but not by the way of limitation, sensor 104 can communicate with remote terminal 170 and / or trusted computer system 180 through a direct communication link to network 150, according to some embodiments, as described in U.S. Patent Application Publication 2011 / 0213225 and incorporated herein by reference in its entirety. Any suitable electronic communication protocol can be used for each of the communication paths or links, such as near field communication (NFC), radio frequency identification (RFID), BLUETOOTH® or BLUETOOTH® Low Energy protocols, WiFi, or the like. Remote terminal 170 and / or trusted computer system 180 can be accessible, according to some embodiments, by individuals other than a primary user who have an interest in the user’s analyte levels. Reader device 120 can include display 122 and optional input component 121. Display 122 can include a touch-screen interface, according to some embodiments.

[0133] Sensor control device 102 includes sensor housing 103, which can house circuitry and a power source for operating sensor 104. Optionally, the power source and / or active circuitry can be omitted. A processor (not shown) can be communicatively coupled to sensor 104, with the processor being physically located within sensor housing 103 or reader device 120. Sensor 104 protrudes from the underside of sensor housing 103 and extends through adhesive layer 105, which is adapted for adhering sensor housing 103 to a tissue surface, such as skin, according to some embodiments.

[0134] Sensor 104 is adapted to be at least partially inserted into a tissue of interest, such as within the dermal or subcutaneous layer of the skin, wherein sensor 104 can comprise a proximal portion and a distal portion. In some embodiments, for example, the distal portion of the sensor can be configured for in vivo placement (e.g., for transcutaneous positioning through the skin of a subject). In some embodiments, an introducer (e.g., a needle or a sharp) can create an insertion path through the subject’s skin during the transcutaneous positioning of the distal portion of the sensor). In some embodiments, thesensor can comprise a member capable of penetrating the skin of a subject. In some embodiments, the member can comprises an insertable tip, tail, probe, or needle capable of penetrating the skin of a subject. In some embodiments, the distal portion of sensor 104 can comprise an implantable portion (e.g. sensor tail) of sufficient length for insertion to a desired depth in a given tissue. The implantable portion (e.g., sensor tail) can include at least one working electrode. In some configurations, the implantable portion (e.g. sensor tail) can include a sensing area for detecting an analyte. A counter electrode can be present in combination with the at least one working electrode. Particular electrode configurations upon the implantable portion (e.g., sensor tail) are described in more detail below. In another embodiment, the proximal portion of the sensor can be configured to remain above the skin (ex vivo) and can be configured to be electrically coupled with the circuitry disposed in the sensor housing 103 of sensor control device 102.

[0135] The sensing area can be configured for detecting a particular analyte. For example, but not by way of limitation, the disclosed analyte sensors include at least one sensing area configured to detect an analyte (e.g., glucose, ketone). In some embodiments, a sensor of the present disclosure includes two sensing areas, where each sensing area is configured to detect a different analyte. Alternatively, the two sensing areas can be configured to detect the same analyte. In some embodiments, a first sensing area can be configured to detect an analyte (e.g., glucose) and a second sensing area can be configured to detect the first (i.e., same) analyte or a second analyte different from the first analyte (e.g., ketone, creatinine).

[0136] In some embodiments of the present disclosure, one or more analytes can be monitored in any biological fluid of interest such as dermal fluid, interstitial fluid, plasma, blood, lymph, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage, amniotic fluid, or the like. In some embodiments, analyte sensors of the present disclosure can be adapted for assaying dermal fluid or interstitial fluid to determine a concentration of one or more analytes in vivo. In some embodiments, the biological fluid is interstitial fluid.

[0137] Referring still to FIG. 1, sensor 104 can automatically forward data to reader device 120. For example but not by the way of limitation, analyte concentration data (i.e., glucose concentration) can be communicated automatically and periodically, such as at a some frequency as data is obtained or after a time period has passed, with the data being stored in a memory until transmittal (e.g., every minute, five minutes, or otherpredetermined time period). In some embodiments, sensor 104 can communicate with reader device 120 in a non-automatic manner and not according to a set schedule. For example, but not by the way of limitation, data can be communicated from sensor 104 using RFID technology when the sensor electronics are brought into communication range of reader device 120. Until communicated to reader device 120, data can remain stored in a memory of sensor 104. Thus, a user does not have to maintain close proximity to reader device 120 at all times, and can instead upload data at a convenient time. In some embodiments, a combination of automatic and non-automatic data transfer can be implemented. For example, and not by the way of limitation, data transfer can continue on an automatic basis until reader device 120 is no longer in communication range of sensor 104.

[0138] The introducer, a component of an applicator, can be transiently deployed to facilitate the introduction of sensor 104 into a tissue.. In some illustrative embodiments, the introducer can include a needle or similar sharp. As would be readily recognized by a person skilled in the art, other types of introducers, such as sheaths or blades, can be present in alternative embodiments. More specifically, the needle or other introducer can transiently reside in proximity to sensor 104 prior to tissue insertion and then be withdrawn afterward. While present, the needle or other introducer can facilitate insertion of sensor 104 into a tissue by opening an access pathway for sensor 104 to follow. For example, and not by the way of limitation, the needle can facilitate penetration of the epidermis as an access pathway to the dermis to allow implantation of sensor 104 to take place, according to one or more embodiments. After opening the access pathway, the needle or other introducer can be withdrawn so that it does not represent a sharps hazard. In some embodiments, suitable needles can be solid or hollow, beveled or non-beveled, and / or circular or non-circular in cross-section. In some embodiments, suitable needles can be comparable in cross-sectional diameter and / or tip design to an acupuncture needle, which can have a cross-sectional diameter of about 250 microns. However, suitable needles can have a larger or smaller cross-sectional diameter if needed for certain particular applications.

[0139] In some embodiments, a tip of the needle (while present) can be angled over the terminus of sensor 104, such that the needle penetrates a tissue first and opens an access pathway for sensor 104. In some embodiments, sensor 104 can reside within a lumen orgroove of the needle, with the needle similarly opening an access pathway for sensor 104. In either case, the needle is subsequently withdrawn after facilitating sensor insertion.

[0140] Sensor configurations featuring a single sensing area that is configured for the detection of a corresponding single analyte can employ two-electrode or three-electrode detection motifs, as described further herein in reference to FIGS. 2A-2C. Sensor configurations featuring two different sensing areas for detection of separate analytes, either upon separate working electrodes or upon the same working electrode, are described separately thereafter in reference to FIGS. 3A-5C. Sensor configurations having multiple working electrodes can be particularly advantageous for incorporating two different sensing areas within the same implantable portion (e.g., sensor tail), since the signal contribution from each sensing area can be determined more readily.

[0141] When a single working electrode is present in an analyte sensor, three-electrode sensor configurations can include a working electrode, a counter electrode, and a reference electrode. Related two-electrode sensor configurations can include a working electrode and a second electrode, in which the second electrode can function as both a counter electrode and a reference electrode (i.e., a counter / reference electrode). The various electrodes can be at least partially stacked (layered) upon one another and / or laterally spaced apart from one another upon the implantable portion (e.g., sensor tail). Suitable sensor configurations can be substantially flat in shape, substantially cylindrical in shape or any other suitable shape. In any of the sensor configurations disclosed herein, the various electrodes can be electrically isolated from one another by a dielectric material or similar insulator.

[0142] Analyte sensors featuring multiple working electrodes can similarly include at least one additional electrode. When one additional electrode is present, the one additional electrode can function as a counter / reference electrode for each of the multiple working electrodes. When 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.

[0143] FIG. 2A shows a diagram of an illustrative two-electrode analyte sensor configuration, which is compatible for use in the disclosure herein. As shown, analyte sensor 200 includes substrate 212 disposed between working electrode 214 andcounter / reference electrode 216. Alternately, working electrode 214 and counter / reference electrode 216 can be located upon the same side of substrate 212 with a dielectric material interposed in between (configuration not shown). Sensing area 218 is disposed as at least one layer upon at least a portion of working electrode 214. Sensing area 218 can include multiple spots or a single spot configured for detection of an analyte, as discussed further herein.

[0144] Referring still to FIG. 2A, membrane 220 overcoats at least sensing area 218. In some embodiments, membrane 220 can also overcoat some or all of working electrode 214 and / or counter / reference electrode 216, or the entirety of analyte sensor 200. One or both faces of analyte sensor 200 can be overcoated with membrane 220. Membrane 220 can include one or more polymeric membrane materials having capabilities of limiting analyte flux to sensing area 218 (i.e., membrane 220 is a mass transport limiting membrane having some permeability for the analyte of interest). In some embodiments, and further described below, membrane 220 is not crosslinked. Analyte sensor 200 can be operable for assaying an analyte by any of coulometric, amperometric, voltammetric, or potentiometric electrochemical detection techniques.

[0145] FIGS. 2B and 2C show diagrams of illustrative three-electrode analyte sensor configurations, which are also compatible for use in the disclosure herein. Three- electrode analyte sensor configurations can be similar to that shown for analyte sensor200 in FIG. 2 A, except for the inclusion of additional electrode 217 in analyte sensors201 and 202 (FIGS. 2B and 2C). With additional electrode 217, counter / reference electrode 216 can then function as either a counter electrode or a reference electrode, and additional electrode 217 fulfills the other electrode function not otherwise accounted for. Working electrode 214 continues to fulfill its original function. Additional electrode 217 can be disposed upon either working electrode 214 or electrode 216, with a separating layer of dielectric material in between. For example, and not by the way of limitation, as depicted in FIG. 2B, dielectric layers 219a, 219b and 219c separate electrodes 214, 216 and 217 from one another and provide electrical isolation. Alternatively, at least one of electrodes 214, 216 and 217 can be located upon opposite faces of substrate 212, as shown in FIG. 2C. Thus, in certain embodiments, electrode 214 (working electrode) and electrode 216 (counter electrode) can be located upon opposite faces of substrate 212, with electrode 217 (reference electrode) being located upon one of electrodes 214 or 216and spaced apart therefrom with a dielectric material. Reference material layer 230 (e.g., Ag / AgCl) can be present upon electrode 217, with the location of reference material layer 230 not being limited to that depicted in FIGS. 2B and 2C. As with sensor 200 shown in FIG. 2 A, sensing area 218 in analyte sensors 201 and 202 can include multiple spots or a single spot. Additionally, analyte sensors 201 and 202 can be operable for assaying an analyte by any of coulometric, amperometric, voltammetric, or potentiometric electrochemical detection techniques.

[0146] Like analyte sensor 200, membrane 220 can also overcoat sensing area 218, as well as other sensor components, in analyte sensors 201 and 202, thereby serving as a mass transport limiting membrane. In some embodiments, the additional electrode 217 can be overcoated with membrane 220. Although FIGS. 2B and 2C have depicted electrodes 214, 216, and 217 as being overcoated with membrane 220, it is to be recognized that in some embodiments only working electrode 214 is overcoated. Moreover, the thickness of membrane 220 at each of electrodes 214, 216, and 217 can be the same or different. As in two-electrode analyte sensor configurations (FIG. 2A), one or both faces of analyte sensors 201 and 202 can be overcoated with membrane 220 in the sensor configurations of FIGS. 2B and 2C, or the entirety of analyte sensors 201 and 202 can be overcoated. Accordingly, the three-electrode sensor configurations shown in FIGS. 2B and 2C should be understood as being non-limiting of the embodiments disclosed herein, with alternative electrode and / or layer configurations remaining within the scope of the present disclosure.

[0147] FIG. 3 A shows an illustrative configuration for sensor 203 having a single working electrode with two different sensing areas disposed thereon. FIG. 3 A is similar to FIG. 2 A, except for the presence of two sensing areas upon working electrode 214: first sensing area 218a and second sensing area 218b, which are responsive to different analytes and are laterally spaced apart from one another upon the surface of working electrode 214. Sensing areas 218a and 218b can include multiple spots or a single spot configured for detection of each analyte. The composition of membrane 220 can vary or be compositionally the same at sensing areas 218a and 218b. First sensing area 218a and second sensing area 218b can be configured to detect their corresponding analytes at working electrode potentials that differ from one another, as discussed further below.

[0148] FIGS. 3B and 3C show cross-sectional diagrams of illustrative three-electrode sensor configurations for sensors 204 and 205, respectively, each featuring a single working electrode having first sensing area 218a and second sensing 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. 3 A, the composition of membrane 220 can vary or be compositionally the same at sensing areas 218a and 218b.

[0149] Illustrative sensor configurations having multiple working electrodes, specifically two working electrodes, are described in further detail in reference to FIGS. 4-5C. Although the following description is primarily directed to sensor configurations having two working electrodes, it is to be appreciated that more than two working electrodes can be incorporated through extension of the disclosure herein. Additional working electrodes can be used to impart additional sensing capabilities to the analyte sensors beyond just a first analyte and a second analyte, e.g., for the detection of a third and / or fourth analyte.

[0150] FIG. 4 shows a cross-sectional diagram of an illustrative analyte sensor configuration having two working electrodes, a reference electrode and a counter electrode, which is compatible for use in the disclosure herein. As shown, analyte sensor 300 includes working electrodes 304 and 306 disposed upon opposite faces of substrate 302. First sensing area 310a is disposed upon the surface of working electrode 304, and second sensing area 310b is disposed upon the surface of working electrode 306. Counter electrode 320 is electrically isolated from working electrode 304 by dielectric layer 322, and reference electrode 321 is electrically isolated from working electrode 306 by dielectric layer 323. Outer dielectric layers 330 and 332 are positioned upon reference electrode 321 and counter electrode 320, respectively. Membrane 340 can overcoat at least sensing areas 310a and 310b, according to various embodiments, with other components of analyte sensor 300 or the entirety of analyte sensor 300 optionally being overcoated with membrane 340.

[0151] Like analyte sensors 200, 201, and 202, analyte sensor 300 can be operable for assaying an analyte by any of coulometric, amperometric, voltammetric, or potentiometric electrochemical detection techniques.

[0152] Alternative sensor configurations having multiple working electrodes and differing from the configuration shown in FIG. 4 can feature a counter / reference electrodeinstead of separate counter and reference electrodes 320, 321, and / or feature layer and / or membrane arrangements varying from those expressly depicted. For example, and not by the way of limitation the positioning of counter electrode 320 and reference electrode 321 can be reversed from that depicted in FIG. 4. In addition, working electrodes 304 and 306 need not necessarily reside upon opposing faces of substrate 302 in the manner shown in FIG. 4.

[0153] Although suitable sensor configurations can feature electrodes that are substantially planar in character, it is to be appreciated that sensor configurations featuring non-planar electrodes can be advantageous and particularly suitable for use in the disclosure herein. In particular, substantially cylindrical electrodes that are disposed concentrically with respect to one another can facilitate deposition of a mass transport limiting membrane, as described herein below. FIGs. 5A-5C show perspective views of analyte sensors featuring two working electrodes that are disposed concentrically with respect to one another. It is to be appreciated that sensor configurations having a concentric electrode disposition but lacking a second working electrode are also possible in the present disclosure.

[0154] FIG. 5 A shows a perspective view of an illustrative sensor configuration in which multiple electrodes are substantially cylindrical and are disposed concentrically with respect to one another about a central substrate. As shown, analyte sensor 400 includes central substrate 402 about which all electrodes and dielectric layers are disposed concentrically with respect to one another. In particular, working electrode 410 is disposed upon the surface of central substrate 402, and dielectric layer 412 is disposed upon a portion of working electrode 410 distal to sensor tip 404. Working electrode 420 is disposed upon dielectric layer 412, and dielectric layer 422 is disposed upon a portion of working electrode 420 distal to sensor tip 404. Counter electrode 430 is disposed upon dielectric layer 422, and dielectric layer 432 is disposed upon a portion of counter electrode 430 distal to sensor tip 404. Reference electrode 440 is disposed upon dielectric layer 432, and dielectric layer 442 is disposed upon a portion of reference electrode 440 distal to sensor tip 404. As such, exposed surfaces of working electrode 410, working electrode 420, counter electrode 430, and reference electrode 440 are spaced apart from one another along longitudinal axis B of analyte sensor 400.

[0155] Referring still to FIG. 5A, first sensing areas 414a and second sensing areas 414b, which are responsive to different analytes or the same analyte, are disposed upon the exposed surfaces of working electrodes 410 and 420, respectively, thereby allowing contact with a fluid to take place for sensing. Although sensing areas 414a and 414b have been depicted as three discrete spots in FIG. 5A, it is to be appreciated that fewer or greater than three spots, including a continuous layer of sensing area, can be present in alternative sensor configurations (e.g., a sensing area that overcoats an entire surface of the working electrode, like a membrane).

[0156] In FIG. 5A, analyte sensor 400 is partially coated with membrane 450 upon working electrodes 410 and 420 and sensing areas 414a and 414b disposed thereon. FIG. 5B shows an alternative sensor configuration in which the substantial entirety of analyte sensor 401 is overcoated with membrane 450. Membrane 450 can be the same or vary compositionally at sensing areas 414a and 414b.

[0157] It is to be further appreciated that the positioning of the various electrodes in FIGs. 5 A and 5B can differ from that expressly depicted. For example, the positions of counter electrode 430 and reference electrode 440 can be reversed from the depicted configurations in FIGs. 5 A and 5B. Similarly, the positions of working electrodes 410 and 420 are not limited to those that are expressly depicted in FIGs. 5 A and 5B. FIG. 5C shows an alternative sensor configuration to that shown in FIG. 5B, in which analyte sensor 405 contains counter electrode 430 and reference electrode 440 that are located more proximal to sensor tip 404 and working electrodes 410 and 420 that are located more distal to sensor tip 404. Sensor configurations in which working electrodes 410 and 420 are located more distal to sensor tip 404 can be advantageous by providing a larger surface area for deposition of sensing areas 414a and 414b (five discrete sensing spots illustratively shown in FIG. 5C), thereby facilitating an increased signal strength in some cases. Similarly, central substrate 402 can be omitted in any concentric sensor configuration disclosed herein, wherein the innermost electrode can instead support subsequently deposited layers.

[0158] Several parts of the sensor are further described below.III. GENERAL STRUCTURE OF THE ANALYTE SENSOR SYSTEM

[0159] In some embodiments, the present disclosure is directed to an analyte sensor comprising:

[0160] a proximal portion configured to be positioned above a user’s skin; and

[0161] a distal portion configured to be transcutaneously positioned through the user’s skin and in contact with the user’s bodily fluid to detect or monitor an analyte in the bodily fluid in vivo; the distal portion comprising:

[0162] a first working electrode;

[0163] a first sensing area disposed upon a surface of the first working electrode, the first sensing area being responsive to a first analyte; and

[0164] a membrane that overcoats at least a part of the first sensing area;

[0165] wherein the first sensing area comprises from about 0.19% to about 16% by weight of a surfactant.

[0166] In some embodiments, the present disclosure is directed to an analyte sensor comprising:

[0167] a first working electrode;

[0168] a first sensing area disposed upon a surface of the first working electrode, the first sensing area being responsive to a first analyte; and

[0169] a membrane that overcoats at least a part of the first sensing area;

[0170] wherein the first sensing area comprises from about 0.19% to about 16% by weight of a surfactant.

[0171] In some embodiments, the present disclosure is directed to a analyte sensor comprising:

[0172] a proximal portion configured to be positioned above a user’s skin; and

[0173] a distal portion configured to be transcutaneously positioned through the user’s skin and in contact with the user’s bodily fluid to detect or monitor an analyte in the bodily fluid in vivo; the distal portion comprising:

[0174] a first working electrode;

[0175] a first sensing area disposed upon a surface of the first working electrode, the first sensing area being responsive to a first analyte;

[0176] a membrane that overcoats at least a part of the first sensing area;

[0177] wherein the first sensing area comprises from about 1% to about 16% by weight of a surfactant;

[0178] a second working electrode; and

[0179] a second sensing area disposed upon a surface of the second working electrode, the second sensing area being responsive to a second analyte differing from the first analyte; wherein the second sensing area comprises at least one enzyme responsive to the second analyte.

[0180] In some embodiments, the present disclosure is directed to a analyte sensor comprising:

[0181] a first working electrode;

[0182] a first sensing area disposed upon a surface of the first working electrode, the first sensing area being responsive to a first analyte;

[0183] a membrane that overcoats at least a part of the first sensing area;

[0184] wherein the first sensing area comprises from about 1% to about 16% by weight of a surfactant;

[0185] a second working electrode; and

[0186] a second sensing area disposed upon a surface of the second working electrode, the second sensing area being responsive to a second analyte differing from the first analyte; wherein the second sensing area comprises at least one enzyme responsive to the second analyte.

[0187] Embodiments of the present disclosure relate to a sensing area composition for improving the manufacturing process of one or more components of an analyte sensor by including a surfactant in the solution of a sensing area that reduces the contact angle of the solution by modifying its surface tension. It has been surprisingly discovered that adding the surfactant results in an increase in the uniformity of distribution of the constituents of a solution deposited on a substrate upon drying and curing, as well as an increase in the surface area of the solution upon drying and curing as compared to a solution lacking the surfactant. Increasing the surface area covered by each individual droplet allows for a reduction of the number of droplets required to be dispensed and reduces the time required to cover the same surface area. This, in turn, improves the overall manufacturing process of the analyte sensor.1. Working Electrode

[0188] In the analyte sensor, the working electrode can be any suitable conductive material, such as carbon, gold, palladium, or platinum. In some embodiments, the working electrode can be a carbon working electrode. The sensing area is responsive to adesired analyte (e.g., glucose) and can be continuously or discontinuously disposed on at least a portion of the working electrode. A discontinuous application means that the sensing area forms a discrete shape on the working electrode, such as a spot, a line, or a plurality (i.e., an array) of spots and / or lines. The number of spots is not considered to be particularly limited, but can range from about 1 to about 1000, from about 3 to about 100, or from about 4 to about 10. In some embodiments, the number of spots can be 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the sensing area can form a shape, e.g., a circle or a rectangle. In some embodiments, the sensing area can be continuous on the working electrode. In some embodiments, the sensing area can be discontinuous on the working electrode.2. Sensing area

[0189] In some embodiments, the working electrode can comprise at least one sensing area. In some embodiments, the working electrode can comprise one sensing area. In some embodiments, the working electrode can comprise two sensing areas. In some embodiments, the working electrode can comprise a first sensing area and a second sensing area, wherein the analyte for the first sensing area is different from the analyte for the second sensing area. In this instance, the first sensing area and second sensing area can form an array of multiple spots of each sensing area, in which some spots sense a first analyte (e.g., glucose) and other spots sense a second analyte different from the first analyte (e.g., ketone, creatinine). Each spot can range in size from about 0.01 mm2to about 1 mm2in diameter.

[0190] In some embodiments, each sensing area comprises multiple spots or features (e.g., sensing elements) that form an array. As used herein, the term “array” refers to any one-dimensional, two-dimensional, or substantially two-dimensional arrangement of regions that comprise a particular composition (e.g., sensing area formulation) associated with that region. For example, an array can contain multiple sensing elements of the same formulation, such as multiple sensing elements of the same sensing area formulation. In some embodiments, an array comprises individual sensing elements, where each sensing element in the array comprises the same sensing area formulation.

[0191] In some embodiments, a sensing area can comprise two or more, five or more, ten or more, 100 or more, or 1000 or more spots. In some embodiments, a sensing area can comprise from about 1 to about 1000 spots. In some embodiments, a sensing area cancomprise from about 1 to about 1000, from about 1 to about 50, from about 1 to about 25, from about 1 to about 10, from about 1 to about 5, from about 5 to about 1000, from about 5 to about 100, from about 5 to about 50, from about 5 to about 25, from about 5 to about 10, from about 10 to about 1000, from about 10 to about 100, from about 10 to about 50, from about 10 to about 25, from about 25 to about 1000, from about 25 to about 100, from about 25 to about 50, from about 50 to about 1000, from about 50 to about 100, or from about 100 to about 1000 spots. In some embodiments, a sensing area can comprise about 1000, about 100, about 50, about 25, about 10, about 5, or about 1 spots. In some embodiments, a sensing area comprises 6 spots.

[0192] In some embodiments, the total size of a spot of the sensing area can be from about 0.01 mm2to about 10 mm2. In some embodiments, the total size of a spot of the sensing area can be about 10 mm2or less, about 5 mm2or less, about 1 mm2or less, about 0.1 mm2or less, or about 0.01 mm2or less. In some embodiments, the total size of a spot of the sensing area can range from about 0.01 mm2to about 10 mm2, from about 0.01 mm2to about 5 mm2, from about 0.01 mm2to about 1 mm2, from about 0.01 mm2to about 0.1 mm2, from about 0.1 mm2to about 10 mm2, from about 0.1 mm2to about 5 mm2, from about 0.1 mm2to about 1 mm2, from about 1 mm2to about 10 mm2, from about 1 mm2to about 5 mm2, or from about 5 mm2to about 10 mm2.

[0193] In some embodiments, the total size of a sensing element of the sensing area can be from about 0.01 mm2to about 25 mm2. In some embodiments, the total size of a sensing element of the sensing area can be about 25 mm2or less, about 20 mm2or less, about 15 mm2or less, about 10 mm2or less, about 5 mm2or less, about 1 mm2or less, about 0.1 mm2or less, or about 0.01 mm2or less. In some embodiments, the total size of the sensing area can range from about 0.01 mm2to about 25 mm2, from about 0.01 mm2to about 20 mm2, from about 0.01 mm2to about 15 mm2, from about 0.01 mm2to about 10 mm2, from about 0.01 mm2to about 5 mm2, from about 0.01 mm2to about 1 mm2, from about 0.01 mm2to about 0.1 mm2, from about 0.1 mm2to about 25 mm2, from about 0.1 mm2to about 20 mm2, from about 0.1 mm2to about 15 mm2, from about 0.1 mm2to about 10 mm2, from about 0.1 mm2to about 5 mm2, from about 0.1 mm2to about 1 mm2, from about 1 mm2to about 25 mm2, from about 1 mm2to about 20 mm2, from about 1 mm2to about 15 mm2, from about 1 mm2to about 10 mm2, from about 1 mm2to about 5 mm2, from about 5 mm2to about 25 mm2, from about 5 mm2to about 20 mm2, fromabout 5 mm2to about 15 mm2, from about 5 mm2to about 10 mm2, from about 10 mm2to about 25 mm2, from about 10 mm2to about 20 mm2, from about 10 mm2to about 15 mm2, from about 15 mm2to about 25 mm2, from about 15 mm2to about 20 mm2, or from about 20 mm2to about 25 mm2.

[0194] In some embodiments, the total size of the sensing area (combined area of all sensing elements) can be from about 0.075 mm2to about 100 mm2. In some embodiments, the total size of the sensing area can be about 100 mm2or less, about 75 mm2or less, about 50 mm2or less, about 40 mm2or less, about 30 mm2or less, about 25 mm2or less, about 15 mm2or less, about 10 mm2or less, about 5 mm2or less, about 1 mm2or less, or about 0.1 mm2or less. In some embodiments, the total size of the sensing area can range from about 0.075 mm2to about 100 mm2, from about 0.075 mm2to about 0.1 mm2, from about 0.1 mm2to about 50 mm2, from about 0.5 mm2to about 30 mm2, from about 1 mm2to about 20 mm2, or from about 1 mm2to about 15 mm2.

[0195] In some embodiments, the sensing area can have a thickness that ranges from about 0.1 gm to about 10 gm. For example, each sensing area can be about 0.1 pm thick or more (e.g., about 0.2 pm or more, about 0.3 pm or more, about 0.5 pm or more, about 0.8 pm or more, about 1 pm or more, about 2 pm or more, about 3 pm or more, about 5 pm or more, or about 8 pm or more) and typically can have a thickness of 10 pm or less (e.g., about 8 pm or less, about 5 pm or less, about 3 pm or less, about 2 pm or less, about 1 pm or less, about 0.8 pm or less, about 0.5 pm or less, about 0.3 pm or less, or about 0.2 pm or less). In some embodiments, each sensing area can have a thickness from about 0.1 pm to about 10 pm, from about 0.2 pm to about 8 pm, from about 0.5 pm to about 5 pm, from about 1 pm to about 4 pm, or from about 1 pm about 2 pm.

[0196] In some embodiments, the sensing surface can include inter-feature areas. As used herein, the phrase “inter-feature area” refers to an area that does not include (e.g., is substantially free of) an analyte-responsive enzyme. In some embodiments, the interfeature areas surround the sensing elements. In some embodiments, the sensing elements can have an inter-feature distance (i.e., the distance between areas of a sensing surface that are substantially free of an analyte-responsive enzyme) ranging from about 1 pm to about 500 pm. In some embodiments, the sensing elements can have an inter-feature distance ranging from about 1 pm to about 500 pm, from about 1 pm to about 250 pm, from about 1 pm to about 100 pm, from about 1 pm to about 50 pm, from about 1 pm toabout 10 pm, from about 1 pm to about 5 pm, from about 5 pm to about 500 pm, from about 5 pm to about 250 pm, from about 5 pm to about 100 pm, from about 5 pm to about 50 pm, from about 5 pm to about 10 pm, from about 10 pm to about 500 pm, from about 10 pm to about 250 pm, from about 10 pm to about 100 pm, from about 10 pm to about 50 pm, from about 50 pm to about 500 pm, from about 50 pm to about 250 pm, from about 50 pm to about 100 pm, from about 100 pm to about 500 pm, from about 100 pm to about 250 pm, or from about 250 pm to about 500 pm.

[0197] In some embodiments, a sensing area array can be fabricated using drop deposition of a sensing area formulation onto a surface of a working electrode. The sensing area formulation can be deposited by any non-impact or impact printing method. In some embodiments, the sensing area formulation can be deposited using a pulse-jet device. A “pulse-jet” device is a device that can dispense drops in the formation of an array. Pulse jet devices operate by delivering a pulse of pressure to liquid adjacent to an outlet or orifice such that a drop will be dispensed therefrom (for example, by a piezoelectric or thermoelectric element positioned in the same chamber as the orifice). In some embodiments, the drops can be dispensed using a dispenser device configured to operate similar to an inkjet printing device. In some embodiments, the pulse-jet device includes a dispensing head configured to dispense drops, such as, but not limited to, of a sensing area formulation, in the formation of an array. The dispensing head can be of a type commonly used in a inkjet type of printer and can, for example, include one or more deposition chambers for containing the formulation(s) to be deposited. The amount of fluid that is deposited in a single activation event of a pulse jet can be controlled by changing one or more of a number of parameters, including the size of the orifice in the dispensing head (e.g., the orifice diameter), the size of the deposition chamber, or the size of the piezoelectric or thermoelectric element.

[0198] In some embodiments, the amount of sensing area formulation deposited during a single activation event can range from about 0.01 pL to about 1000 pL. In some embodiments, the amount of sensing area formulation deposited during a single activation event can range from about 0.01 pL to about 1000 pL, from about 0.01 pL to about 500 pL, from about 0.01 pL to about 100 pL, from about 0.01 pL to about 50 pL, from about 0.01 pL to about 25 pL, from about 0.01 pL to about 10 pL, from about 0.01 pL to about 5 pL, from about 0.01 pL to about 1 pL, from about 0.01 pL to about 0.1 pL, from about0.1 pL to about 1000 pL, from about 0.1 pL to about 500 pL, from about 0.1 pL to about 100 pL, from about 0.1 pL to about 50 pL, from about 0.1 pL to about 25 pL, from about 0.1 pL to about 10 pL, from about 0.1 pL to about 5 pL, from about 0.1 pL to about 1 pL, from about 1 pL to about 1000 pL, from about 1 pL to about 500 pL, from about 1 pL to about 100 pL, from about 1 pL to about 50 pL, from about 1 pL to about 25 pL, from about 1 pL to about 10 pL, from about 1 pL to about 5 pL, from about 5 pL to about 1000 pL, from about 5 pL to about 500 pL, from about 5 pL to about 100 pL, from about 5 pL to about 50 pL, from about 5 pL to about 25 pL, from about 5 pL to about 10 pL, from about 10 pL to about 1000 pL, from about 10 pL to about 500 pL, from about 10 pL to about 100 pL, from about 10 pL to about 50 pL, from about 10 pL to about 25 pL, from about 25 pL to about 1000 pL, from about 25 pL to about 500 pL, from about 25 pL to about 100 pL, from about 25 pL to about 50 pL, from about 50 pL to about 1000 pL, from about 50 pL to about 500 pL, from about 50 pL to about 100 pL, from about 100 pL to about 1000 pL, from about 100 pL to about 500 pL, or from about 500 pL to about 1000 pL.

[0199] In some embodiments, a droplet of sensing area formulation deposited during a single activation event can cover an area from about 0.005 mm2to about 0.10 mm2. In some embodiments, a droplet of sensing area formulation deposited during a single activation event can cover an area from about 0.005 mm2to about 0.10 mm2, from about 0.005 mm2to about 0.075 mm2, from about 0.005 mm2to about 0.01 mm2, from about 0.01 mm2to about 0.10 mm2, from about 0.01 mm2to about 0.075 mm2, or from about 0.075 mm2to about 0.10 mm2.

[0200] In some embodiments, each sensing element (e.g., each spot or feature on the array) can have a volume ranging from about 0.01 pL to about 1000 pL. In some embodiments, each sensing element can have a volume ranging from about 0.01 pL to about 1000 pL, from about 0.01 pL to about 500 pL, from about 0.01 pL to about 100 pL, from about 0.01 pL to about 50 pL, from about 0.01 pL to about 25 pL, from about 0.01 pL to about 10 pL, from about 0.01 pL to about 5 pL, from about 0.01 pL to about 1 pL, from about 0.01 pL to about 0.1 pL, from about 0.1 pL to about 1000 pL, from about 0.1 pL to about 500 pL, from about 0.1 pL to about 100 pL, from about 0.1 pL to about 50 pL, from about 0.1 pL to about 25 pL, from about 0.1 pL to about 10 pL, from about 0.1 pL to about 5 pL, from about 0.1 pL to about 1 pL, from about 1 pL to about 1000 pL,from about 1 pL to about 500 pL, from about 1 pL to about 100 pL, from about 1 pL to about 50 pL, from about 1 pL to about 25 pL, from about 1 pL to about 10 pL, from about 1 pL to about 5 pL, from about 5 pL to about 1000 pL, from about 5 pL to about 500 pL, from about 5 pL to about 100 pL, from about 5 pL to about 50 pL, from about 5 pL to about 25 pL, from about 5 pL to about 10 pL, from about 10 pL to about 1000 pL, from about 10 pL to about 500 pL, from about 10 pL to about 100 pL, from about 10 pL to about 50 pL, from about 10 pL to about 25 pL, from about 25 pL to about 1000 pL, from about 25 pL to about 500 pL, from about 25 pL to about 100 pL, from about 25 pL to about 50 pL, from about 50 pL to about 1000 pL, from about 50 pL to about 500 pL, from about 50 pL to about 100 pL, from about 100 pL to about 1000 pL, from about 100 pL to about 500 pL, or from about 500 pL to about 1000 pL.

[0201] In some embodiments, a solution (e.g., a sensing area formulation) is contacted with a surface of a substrate (e.g., the surface of a working electrode), forming a droplet on the surface of a substrate. In some embodiments, the droplet is allowed to dry and cure. In some embodiments, the constituents of the droplet can migrate toward the outer edges of the droplet due to a faster rate of evaporation at the thinner peripheral edges of the droplet. This can result in a greater concentration of the constituents of the solution at the peripheral edges of the site of deposition.

[0202] In some embodiments, the sensing area can be dried at a temperature above room temperature. In some embodiments, the sensing area can be dried at a temperature from about 25 °C to about 100 °C. In some embodiments, the sensing area can be dried at a temperature from about 25 °C to about 100 °C, from about 25 °C to about 80 °C, from about 25 °C to about 60 °C, from about 25 °C to about 40 °C, from about 25 °C to about 30 °C, from about 30 °C to about 100 °C, from about 30 °C to about 80 °C, from about 30 °C to about 60 °C, from about 30 °C to about 40 °C, from about 40 °C to about 100 °C, from about 40 °C to about 80 °C, from about 40 °C to about 60 °C, from about 60 °C to about 100 °C, from about 60 °C to about 80 °C, or from about 80 °C to about 100 °C.

[0203] In some embodiments, the sensing area can comprise a surfactant in a range from about 0.19% by weight to about 16% by weight. In some embodiments, the sensing area can comprise a surfactant in a range from about 0.19% by weight to about 16% by weight, from about 0.19% by weight to about 10% by weight, from about 0.19% by weight to about 8% by weight, from about 0.19% by weight to about 6% by weight, fromabout 0.19% by weight to about 4% by weight, from about 0.19% by weight to about 2% by weight, from about 0.19% by weight to about 1% by weight, from about 1% by weight to about 16% by weight, from about 1% by weight to about 10% by weight, from about 1% by weight to about 8% by weight, from about 1% by weight to about 6% by weight, from about 1% by weight to about 4% by weight, from about 1% by weight to about 2% by weight, from about 2% by weight to about 16% by weight, from about 2% by weight to about 10% by weight, from about 2% by weight to about 8% by weight, from about 2% by weight to about 6% by weight, from about 2% by weight to about 4% by weight, from about 4% by weight to about 16% by weight, from about 4% by weight to about 10% by weight, from about 4% by weight to about 8% by weight, from about 4% by weight to about 6% by weight, from about 6% by weight to about 16% by weight, from about 6% by weight to about 10% by weight, from about 6% by weight to about 8% by weight, from about 8% by weight to about 16% by weight, from about 8% by weight to about 10% by weight, or from about 10% by weight to about 16% by weight. In some embodiments, the sensing area can comprise a surfactant in an amount of about 0.19% by weight, about 1% by weight, about 2% by weight, about 4% by weight, about 6% by weight, about 8% by weight, about 10% by weight, or about 16% by weight.

[0204] In some embodiments, the sensing area can comprise a nonionic surfactant (e.g. an ethoxylated nonionic surfactant) in a range from about 0.19% by weight to about 16% by weight. In some embodiments, the sensing area can comprise a nonionic surfactant in a range from about 0.19% by weight to about 16% by weight, from about 0.19% by weight to about 10% by weight, from about 0.19% by weight to about 8% by weight, from about 0.19% by weight to about 6% by weight, from about 0.19% by weight to about 4% by weight, from about 0.19% by weight to about 2% by weight, from about 0.19% by weight to about 1% by weight, from about 1% by weight to about 16% by weight, from about 1% by weight to about 10% by weight, from about 1% by weight to about 8% by weight, from about 1% by weight to about 6% by weight, from about 1% by weight to about 4% by weight, from about 1% by weight to about 2% by weight, from about 2% by weight to about 16% by weight, from about 2% by weight to about 10% by weight, from about 2% by weight to about 8% by weight, from about 2% by weight to about 6% by weight, from about 2% by weight to about 4% by weight, from about 4% by weight to about 16% by weight, from about 4% by weight to about 10% by weight, fromabout 4% by weight to about 8% by weight, from about 4% by weight to about 6% by weight, from about 6% by weight to about 16% by weight, from about 6% by weight to about 10% by weight, from about 6% by weight to about 8% by weight, from about 8% by weight to about 16% by weight, from about 8% by weight to about 10% by weight, or from about 10% by weight to about 16% by weight. In some embodiments, the sensing area can comprise a nonionic surfactant in an amount of about 0.19% by weight, about 1% by weight, about 2% by weight, about 4% by weight, about 6% by weight, about 8% by weight, about 10% by weight, or about 16% by weight.

[0205] In some embodiments, the sensing area can comprise a polyethylene glycol tertoctylphenyl ether in a range from about 0.19% by weight to about 16% by weight. In some embodiments, the sensing area can comprise a polyethylene glycol tert-octylphenyl ether in a range from about 0.19% by weight to about 16% by weight, from about 0.19% by weight to about 10% by weight, from about 0.19% by weight to about 8% by weight, from about 0.19% by weight to about 6% by weight, from about 0.19% by weight to about 4% by weight, from about 0.19% by weight to about 2% by weight, from about 0.19% by weight to about 1% by weight, from about 1% by weight to about 16% by weight, from about 1% by weight to about 10% by weight, from about 1% by weight to about 8% by weight, from about 1% by weight to about 6% by weight, from about 1% by weight to about 4% by weight, from about 1% by weight to about 2% by weight, from about 2% by weight to about 16% by weight, from about 2% by weight to about 10% by weight, from about 2% by weight to about 8% by weight, from about 2% by weight to about 6% by weight, from about 2% by weight to about 4% by weight, from about 4% by weight to about 16% by weight, from about 4% by weight to about 10% by weight, from about 4% by weight to about 8% by weight, from about 4% by weight to about 6% by weight, from about 6% by weight to about 16% by weight, from about 6% by weight to about 10% by weight, from about 6% by weight to about 8% by weight, from about 8% by weight to about 16% by weight, from about 8% by weight to about 10% by weight, or from about 10% by weight to about 16% by weight. In some embodiments, the sensing area can comprise a polyethylene glycol tert-octylphenyl ether in an amount of about 0.19% by weight, about 1% by weight, about 2% by weight, about 4% by weight, about 6% by weight, about 8% by weight, about 10% by weight, or about 16% by weight.

[0206] In some embodiments, a conductive material such as, for example, carbon nanotubes, graphene, or metal nanoparticles, can be combined within the sensing area or layers to promote rapid attainment of a steady state current. Conductive material can be included in a range from about 0.1% to about 50% by weight (pbw) of the sensing area (e.g., from about 1 pbw to about 50 pbw, from about 1 pbw to about 10 pbw, or from about 0.1 pbw to about 10 pbw).3. Surfactants

[0207] The present disclosure is based on the surprising discovery that the addition of a surfactant to sensing area formulations used in the manufacture of in vivo and / or in vitro analyte sensors improves the distribution of one or more components of the sensing area.

[0208] In some embodiments, at least one sensing area can comprise a surfactant. In some embodiments, one, two, or three sensing areas can comprise a surfactant. In some embodiments, one sensing area comprises a surfactant. In some embodiments, each sensing area comprises one, two, or three surfactants. In some embodiments, each sensing area comprises one surfactant.

[0209] As used herein, the term “surfactant” refers to a substance that when added to a liquid, reduces its surface tension, thereby increasing its spreading and wetting properties. In some embodiments, the surfactant can be selected from the group consisting of an anionic surfactant, a non-ionic surfactant, a cationic surfactant, an amphoteric surfactant, a zwitterionic surfactant, and combinations thereof. In some embodiments, the surfactant can be a non-ionic surfactant.

[0210] In some embodiments, the surfactant can be biocompatible.

[0211] In some embodiments, the surfactant can be an ethoxylated nonionic surfactant.In some embodiments, the ethoxylated nonionic surfactant can be polyethylene glycol sorbitan monolaurate, polyoxyethylene sorbitan monopalmitate, polyethylene glycol sorbitan monostearate, polyethylene glycol sorbitan monooleate, polyethylene glycol tertoctylphenyl ether, or polyethylene glycol 4-tert-octylphenyl ether. In some embodiments, the surfactant can be polyethylene glycol tert-octylphenyl ether.

[0212] In some embodiments, a co-solvent can be used with a surfactant to achieve low surface tension. In some embodiments, the co-solvent can be an alcohol, a glycol, or a ketone. In some embodiments, the co-solvent can be an alcohol. In some embodiments, the co-solvent can be a short-chained linear alcohol. In some embodiments, the co-solvent can be an alcohol such as ethanol, isopropanol, or methanol. In some embodiments, the co-solvent can be a glycol. In some embodiments, the co-solvent can be a glycol such as ethylene glycol or propylene glycol. In some embodiments, the cosolvent can be a ketone. In some embodiments, the co-solvent can be a ketone such as acetone. In some embodiments, the co-solvent can be selected from the group consisting of ethanol, isopropanol, methanol, ethylene glycol, propylene glycol, acetone, and combinations thereof.

[0213] The sensing area can include, in addition to the surfactant and among other constituents, a supply of a cofactor, enzymes, a redox mediator, and combinations thereof. In some embodiments, the sensing area can also include optional components such as a polymer and a crosslinker.

[0214] In some embodiments, the sensing area formulation can comprise a surfactant in a range from about 0.19% by weight to about 16% by weight. In some embodiments, the sensing area formulation can comprise a surfactant in a range from about 0.19% by weight to about 16% by weight, from about 0.19% by weight to about 10% by weight, from about 0.19% by weight to about 8% by weight, from about 0.19% by weight to about 6% by weight, from about 0.19% by weight to about 4% by weight, from about 0.19% by weight to about 2% by weight, from about 0.19% by weight to about 1% by weight, from about 1% by weight to about 16% by weight, from about 1% by weight to about 10% by weight, from about 1% by weight to about 8% by weight, from about 1% by weight to about 6% by weight, from about 1% by weight to about 4% by weight, from about 1% by weight to about 2% by weight, from about 2% by weight to about 16% by weight, from about 2% by weight to about 10% by weight, from about 2% by weight to about 8% by weight, from about 2% by weight to about 6% by weight, from about 2% by weight to about 4% by weight, from about 4% by weight to about 16% by weight, from about 4% by weight to about 10% by weight, from about 4% by weight to about 8% by weight, from about 4% by weight to about 6% by weight, from about 6% by weight to about 16% by weight, from about 6% by weight to about 10% by weight, from about 6% by weight to about 8% by weight, from about 8% by weight to about 16% by weight, from about 8% by weight to about 10% by weight, or from about 10% by weight to about 16% by weight. In some embodiments, the sensing area formulation can comprise a surfactant in an amount of about 0.19% by weight, about 1% by weight, about 2% byweight, about 4% by weight, about 6% by weight, about 8% by weight, about 10% by weight, or about 16% by weight.

[0215] In some embodiments, the sensing area formulation can comprise a nonionic surfactant (e.g. an ethoxylated nonionic surfactant) in a range from about 0.19% by weight to about 16% by weight. In some embodiments, the sensing area formulation can comprise a nonionic surfactant (e.g. an ethoxylated nonionic surfactant) in a range from about 0.19% by weight to about 16% by weight. In some embodiments, the sensing area formulation can comprise a nonionic surfactant in a range from about 0.19% by weight to about 16% by weight, from about 0.19% by weight to about 10% by weight, from about 0.19% by weight to about 8% by weight, from about 0.19% by weight to about 6% by weight, from about 0.19% by weight to about 4% by weight, from about 0.19% by weight to about 2% by weight, from about 0.19% by weight to about 1% by weight, from about 1% by weight to about 16% by weight, from about 1% by weight to about 10% by weight, from about 1% by weight to about 8% by weight, from about 1% by weight to about 6% by weight, from about 1% by weight to about 4% by weight, from about 1% by weight to about 2% by weight, from about 2% by weight to about 16% by weight, from about 2% by weight to about 10% by weight, from about 2% by weight to about 8% by weight, from about 2% by weight to about 6% by weight, from about 2% by weight to about 4% by weight, from about 4% by weight to about 16% by weight, from about 4% by weight to about 10% by weight, from about 4% by weight to about 8% by weight, from about 4% by weight to about 6% by weight, from about 6% by weight to about 16% by weight, from about 6% by weight to about 10% by weight, from about 6% by weight to about 8% by weight, from about 8% by weight to about 16% by weight, from about 8% by weight to about 10% by weight, or from about 10% by weight to about 16% by weight. In some embodiments, the sensing area formulation can comprise a nonionic surfactant (e.g. an ethoxylated nonionic surfactant) in an amount of about 0.19% by weight, about 1% by weight, about 2% by weight, about 4% by weight, about 6% by weight, about 8% by weight, about 10% by weight, or about 16% by weight.

[0216] In some embodiments, the sensing area formulation can comprise a polyethylene glycol tert-octylphenyl ether in a range from about 0.19% by weight to about 16% by weight. In some embodiments, the sensing area formulation can comprise a polyethylene glycol tert-octylphenyl ether in a range from about 0.19% by weight to about 16% byweight. In some embodiments, the sensing area formulation can comprise a polyethylene glycol tert-octylphenyl ether in a range from about 0.19% by weight to about 16% by weight, from about 0.19% by weight to about 10% by weight, from about 0.19% by weight to about 8% by weight, from about 0.19% by weight to about 6% by weight, from about 0.19% by weight to about 4% by weight, from about 0.19% by weight to about 2% by weight, from about 0.19% by weight to about 1% by weight, from about 1% by weight to about 16% by weight, from about 1% by weight to about 10% by weight, from about 1% by weight to about 8% by weight, from about 1% by weight to about 6% by weight, from about 1% by weight to about 4% by weight, from about 1% by weight to about 2% by weight, from about 2% by weight to about 16% by weight, from about 2% by weight to about 10% by weight, from about 2% by weight to about 8% by weight, from about 2% by weight to about 6% by weight, from about 2% by weight to about 4% by weight, from about 4% by weight to about 16% by weight, from about 4% by weight to about 10% by weight, from about 4% by weight to about 8% by weight, from about 4% by weight to about 6% by weight, from about 6% by weight to about 16% by weight, from about 6% by weight to about 10% by weight, from about 6% by weight to about 8% by weight, from about 8% by weight to about 16% by weight, from about 8% by weight to about 10% by weight, or from about 10% by weight to about 16% by weight. In some embodiments, the sensing area formulation can comprise a polygylcol tert-octylphenyl ether in an amount of about 0.19% by weight, about 1% by weight, about 2% by weight, about 4% by weight, about 6% by weight, about 8% by weight, about 10% by weight, or about 16% by weight.

[0217] In some embodiments, the surfactant reduces the contact angle of the solution by modifying the interfacial tension between the solution and the substrate surface. This results in an increase in the uniformity of distribution of the constituents of a solution deposited on a substrate upon drying and curing, as well as an increase in the surface area of the solution upon drying and curing as compared to a solution lacking the surfactant. Increasing the surface area covered by each individual droplet allows for a reduction of the number of droplets dispensed and reduces the time required to cover the same surface area. This, in turn, improves the overall manufacturing process of the analyte sensor.

[0218] In some embodiments, the contact angle of the solution (e.g,. sensing area formulation) comprising the surfactant on the substrate surface is from about 10° to about90°, from about 10° to about 60°, from about 10° to about 30°, from about 10° to about20°, from about 20° to about 90°, from about 20° to about 60°, from about 20° to about30°, from about 30° to about 90°, from about 30° to about 60°, or from about 60° to about90°.

[0219] In some embodiments, the contact angle of the solution (e.g., sensing area formulation) comprising the surfactant on the substrate surface is reduced from about 1% to about 90%, from about 1% to about 70%, from about 1% to about 50%, from about 1% to about 40%, from about 1% to about 20%, from about 1% to about 10%, from about 10% to about 90%, from about 1% to about 70%, from about 10% to about 50%, from about 10% to about 40%, from about 10% to about 20%, from about 20% to about 90%, from about 20% to about 70%, from about 20% to about 50%, from about 20% to about 40%, from about 40% to about 90%, from about 40% to about 70%, from about 40% to about 50%, from about 50% to about 90%, from about 50% to about 70%, or from about 70% to about 90% compared to the contact angle of the same solution lacking the surfactant.4. Cofactor Supply

[0220] In some embodiments, the analyte sensors of the present disclosure can include a supply of a cofactor in the sensing area. For example, but not by way of limitation, the present disclosure provides analyte sensors that can include a supply of a cofactor that allows the controlled release of the cofactor over an extended period of the time.

[0221] The exact amount of the cofactor supply present within an analyte sensor can vary based on the particular application of the analyte sensor, e.g., which analyte is being detected, the duration of analyte detection, and the conditions under which the detection of the analyte occurs.

[0222] In some embodiments, the cofactor is NAD(P) or a derivative thereof. Nonlimiting examples of NAD(P) derivatives are disclosed in WO 2007 / 012494 and WO 1998 / 033936, the contents of each which are incorporated herein by reference in their entireties. In some embodiments, the present disclosure provides analyte sensors that can include a supply of NAD(P) or a derivative thereof in the sensing area that allows the controlled release of NAD(P) or derivative thereof over an extended period of the time. In some embodiments, the supply of NAD(P) or derivative thereof can be an internalsupply of NAD(P), e.g., an NAD(P) depot, as disclosed in US 2022 / 0186277, the contents of which are incorporated herein by reference in its entirety.

[0223] In some embodiments, the amount of NAD(P) present within a sensing area or an NAD(P) depot can vary depending on the duration of use of the analyte sensor. In some embodiments, NAD(P) can be present in a sensing area or an NAD(P) depot in an amount from about 0.1 pg to about 900 pg. In some embodiments, from about 0.1 pg to about 900 pg, from about 0.1 pg to about 600 pg, from about 0.1 pg to about 300 pg, from about 0.1 pg to about 100 pg, from about 0.1 pg to about 50 pg, from about 0.1 pg to about 25 pg, from about 0.1 pg to about 10 pg, from about 0.1 pg to about 1 pg, from about 0.1 pg to about 0.5 pg, from about 0.5 pg to about 900 pg, from about 0.5 pg to about 600 pg, from about 0.5 pg to about 300 pg, from about 0.5 pg to about 100 pg, from about 0.5 pg to about 50 pg, from about 0.5 pg to about 25 pg, from about 0.5 pg to about 10 pg, from about 0.5 pg to about 1 pg, from about 1 pg to about 900 pg, from about 1 pg to about 600 pg, from about 1 pg to about 300 pg, from about 1 pg to about 100 pg, from about 1 pg to about 50 pg, from about 1 pg to about 25 pg, from about 1 pg to about 10 pg, from about 10 pg to about 900 pg, from about 10 pg to about 600 pg, from about 10 pg to about 300 pg, from about 10 pg to about 100 pg, from about 10 pg to about 50 pg, from about 10 pg to about 25 pg, from about 25 pg to about 900 pg, from about 25 pg to about 600 pg, from about 25 pg to about 300 pg, from about 25 pg to about 100 pg, from about 25 pg to about 50 pg, from about 50 pg to about 900 pg, from about 50 pg to about 600 pg, from about 50 pg to about 300 pg, from about 50 pg to about 100 pg, from about 100 pg to about 900 pg, from about 100 pg to about 600 pg, from about 100 pg to about 300 pg, from about 300 pg to about 900 pg, from about 300 pg to about 600 pg, or from about 600 pg to about 900 pg of NAD(P) can be present in a sensing area or an NAD(P) depot. In some embodiments, NAD(P) can be present in a sensing area or an NAD(P) depot in an amount from about 0.1 pg to about 100 pg.

[0224] In some embodiments, the amount of NAD(P) present in the sensing area or NAD(P) depot can vary depending on the lifetime of the analyte sensor. In some embodiments, the amount of NAD(P) in the sensing area or NAD(P) depot can allow the analyte sensor to detect an analyte using an NAD(P)-dependent enzyme for at least 7 days, for at least 8 days, for at least 9 days, for at least 10 days, for at least 11 days, for at least 12 days, for at least 13 days, for at least 14 days, for at least 15 days, for at least 16days, for at least 17 days, for at least 18 days, for at least 19 days, for at least 20 days, for at least 25 days, for at least 30 days, for at least 35 days, or for at least 40 days. In some embodiments, the amount of NAD(P) in the sensing area or NAD(P) depot can allow the analyte sensor to detect an analyte using an NAD(P)-dependent enzyme for at least 14 days. In some embodiments, the amount of NAD(P) in the sensing area or NAD(P) depot can allow the analyte sensor to detect an analyte using an NAD(P)-dependent enzyme for greater than about two weeks, for greater than about three weeks, for greater than about four weeks, for greater than about five weeks, for greater than about six weeks, for greater than about seven weeks, or for greater than about eight weeks.5. Multilayered Membrane

[0225] In some embodiments, the sensing area is overcoated with a multilayered membrane in at least one membrane layer. In some embodiments, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% of the sensing area can be overcoated with a multilayered membrane. In some embodiments, the sensing area can be entirely overcoated with a multilayered membrane. In some embodiments, the multilayered membrane limits NAD(P) release. The composition of the multilayered membrane can vary depending on the desired release kinetics of the NAD(P), e.g., rate of NAD(P) release, from the sensing area.

[0226] In some embodiments, the cofactor, e.g., NAD(P), can be physically retained within the sensing area. For example, but not by way of limitation, a multilayered membrane overcoating the sensing area can aid in retaining the cofactor within the sensing area while still permitting sufficient inward diffusion of the analyte to permit detection thereof.

[0227] The multilayered membrane can be applied over the sensing area(s) by placing a droplet or droplets of membrane solution on at least the one or more sensing areas of an analyte sensor, such as by dipping the implantable portion (e.g., sensor tail) into a membrane solution, by spraying the membrane solution on the implantable portion (sensor tail), by heat pressing or melting the membrane solution, vapor depositing the membrane solution, powder coating the membrane solution, or combinations thereof.

[0228] Generally, the thickness of the multilayered membrane can be controlled by the number of different membrane solutions, the concentration of the membrane solution(s),by the number of droplets of the membrane solution(s) applied, by the number of times the implantable portion (e.g. sensor tail) is dipped in the membrane solution(s), by the volume of membrane solution(s) sprayed on the implantable portion (sensor tail), or by any combination thereof. In some embodiments, the multilayered membrane can have a thickness of less than about 100 pm. In some embodiments, the multilayered membrane can have a thickness of less than about 50 pm. In some embodiments, the multilayered membrane can have a thickness ranging from about 0.1 pm to about 100 pm, from about 0.1 pm to about 50 pm, from about 0.1 pm to about 40 pm, from about 0.1 pm to about 20 pm, from about 0.1 pm to about 10 pm, from about 0.1 pm to about 5 pm, from about 0.1 pm to about 1 pm, from about 1 pm to about 100 pm, from about 1 pm to about 50 pm, from about 1 pm to about 40 pm, from about 1 pm to about 20 pm, from about 1 pm to about 10 pm, from about 1 pm to about 5 pm, from about 5 pm to about 100 pm, from about 5 pm to about 50 pm, from about 5 pm to about 40 pm, from about 5 pm to about 20 pm, from about 5 pm to about 10 pm, from about 10 pm to about 100 pm, from about 10 pm to about 50 pm, from about 10 pm to about 40 pm, from about 10 pm to about 20 pm, from about 20 pm to about 100 pm, from about 20 pm to about 50 pm, from about 20 pm to about 40 pm, from about 40 pm to about 100 pm, from about 40 pm to about 50 pm, or from about 50 pm to about 100 pm. In some embodiments, the multilayered membrane can have a thickness of less than about 100 pm, less than about 50 pm, less than about 40 pm, less than about 20 pm, less than about 10 pm, less than about 5 pm, or less than about 1 pm. For example, but not by way of limitation, a sensor (or working electrode) of the present disclosure can be dipped in a membrane solution, or in each different membrane solution if multiple membrane solutions are used, at least once, at least twice, at least three times, at least four times, or at least five times to obtain the desired multilayered membrane thickness.

[0229] In some embodiments, the multilayered membrane can be single-component (i.e. can comprise a single membrane polymer). In some embodiments, the multilayered membrane can be multi-component (i.e. can comprise two or more different membrane polymers). In some embodiments, the multilayered membrane can comprise 1, 2, 3, 4, 5, 6, 7, or 8 different membrane polymers. In some embodiments, the multilayered membrane can comprise three different membrane polymers.

[0230] In some embodiments, the multilayered membrane can include two or more layers. In some embodiments, each layer can be formed by depositing a membrane solution upon a surface, for example by dip coating, and allowing the membrane solution to dry. Thus, a first layer can be formed on a sensing area by dipping a implantable portion (e.g. sensor tail) into a membrane solution followed by allowing the membrane solution to dry. Then, a second layer can be formed on the first layer by dipping the implantable portion (e.g. sensor tail) comprising the first layer into a second membrane solution, which is different than the first membrane solution, followed by allowing the second membrane solution to dry. Subsequent layers can be formed after each membrane solution is allowed to dry. The two or more layers of membrane polymer can comprise multiple layers of the same membrane polymer as long as there is at least one different membrane polymer layer between the two membrane polymer layers comprising the same membrane polymer.

[0231] In some embodiments, the multilayered membrane can comprise crosslinked polymers containing heterocyclic nitrogen groups. In some embodiments, a multilayered membrane can comprise a polyvinylpyridine-based polymer. Non-limiting examples of polyvinylpyridine-based polymers are disclosed in U.S. Patent Publication No. 2003 / 0042137 (e.g., Formula 2b therein), the entirety of which is incorporated herein by reference.

[0232] In some embodiments, the multilayered membrane can comprise a polyvinylpyridine (e.g., poly(4-vinylpyridine) or poly(2-vinylpyridine)), a polyvinylimidazole, a polyvinylpyridine copolymer (e.g., a copolymer of vinylpyridine and styrene), a polyacrylate, a polyurethane, a polyether urethane, a silicone, a polytetrafluoroethylene, a polyethylene-co-tetrafluoroethylene, a polyolefin, a polyester, a polycarbonate, a biostable polytetrafluoroethylene, homopolymers, copolymers or terpolymers of polyurethanes, a polypropylene, a polyvinylchloride, a polyvinylidene difluoride, a polybutylene terephthalate, a polymethylmethacrylate, a polyether ether ketone, cellulosic polymers, polysulfones and block copolymers thereof including, for example, di-block, tri-block, alternating, random and graft copolymers or a chemically related material and the like.

[0233] In some embodiments, the multilayered membrane can comprise a copolymer of vinylpyridine and styrene. In some embodiments, the multilayered membrane cancomprise a polyvinylpyridine-co-styrene copolymer. For example, but not by way of limitation, a polyvinylpyridine-co-styrene copolymer for use in the present disclosure can include a derivatized polyvinylpyridine-co-styrene copolymer in which a portion of the pyridine nitrogen atoms are functionalized with a non-crosslinked polyethylene glycol tail and a portion of the pyridine nitrogen atoms are functionalized with an alkylsulfonic acid group. In some embodiments, the derivatized polyvinylpyridine-co-styrene copolymer for use as a membrane polymer can be the polymer shown below:wherein x=0.85, y=0.1, z=0.05, n=9, m=l, and p=about 10.

[0234] In some embodiments, the multilayered membrane can comprise polymers such as, but not limited to, poly(styrene co-maleic anhydride), dodecylamine and polypropylene glycol)-block-polyethylene glycol)-block-poly(propylene glycol) (2- aminopropyl ether) crosslinked with polypropylene glycol)-block-poly(ethylene glycol)- block-polypropylene glycol) bis(2-aminopropyl ether); poly(N-isopropyl acrylamide); a copolymer of polypthylene oxide) and polypropylene oxide); or a combination thereof.

[0235] In some embodiments, the multilayered membrane can comprise a polyurethane membrane that includes both hydrophilic and hydrophobic regions. In some embodiments, a hydrophobic polymer component can be a polyurethane, a polyurethane urea or poly(ether-urethane-urea). In some embodiments, a polyurethane can be a polymer produced by the condensation reaction of a diisocyanate and a difunctional hydroxyl-containing material. In some embodiments, the polyurethane urea can be a polymer produced by the condensation reaction of a diisocyanate and a difunctional amine-containing material. In some embodiments, diisocyanates for use herein include aliphatic diisocyanates, e.g., diisocyanates comprising from about 4 to about 8 methylene units, or diisocyanates comprising cycloaliphatic moieties. Additional non-limitingexamples of polymers that can be used for the generation of a multilayered membrane comprising a calibrant, as described below, in at least one membrane layer of a presently disclosed sensor include vinyl polymers, polyethers, polyesters, polyamides, inorganic polymers (e.g., polysiloxanes and polycarbosiloxanes), natural polymers (e.g., cellulosic and protein based materials) and mixtures (e.g., admixtures or layered structures) or combinations thereof. In some embodiments, the hydrophilic polymer component can be polyethylene oxide and / or polyethylene glycol. In some embodiments, the hydrophilic polymer component can be a polyurethane copolymer. For example, but not by way of limitation, a hydrophobic-hydrophilic copolymer component for use in the present disclosure can be a polyurethane polymer that comprises about 10% to about 50%, e.g., 20%, hydrophilic polyethylene oxide.

[0236] In some embodiments, the multilayered membrane can comprise a silicone polymer / hydrophobic-hydrophilic polymer blend. In some embodiments, the hydrophobic-hydrophilic polymer for use in the blend can be any suitable hydrophobic- hydrophilic polymer such as, but not limited to, polyvinylpyrrolidone, polyhydroxyethyl methacrylate, polyvinylalcohol, polyacrylic acid, polyethers such as polyethylene glycol or polypropylene oxide, and copolymers thereof, including, for example, di-block, triblock, alternating, random, comb, star, dendritic, and graft copolymers. In some embodiments, the hydrophobic-hydrophilic polymer is a copolymer of poly(ethylene oxide) (PEO) and polypropylene oxide) (PPO). Non-limiting examples of PEO and PPO copolymers include PEO-PPO diblock copolymers, PPO-PEO-PPO triblock copolymers, PEO-PPO-PEO triblock copolymers, alternating block copolymers of PEO-PPO, random copolymers of ethylene oxide and propylene oxide and blends thereof. In some embodiments, the copolymers can be substituted with hydroxy substituents.

[0237] In some embodiments, hydrophilic or hydrophobic modifiers can be used to “finetune” the permeability of the resulting multilayered membrane comprising a calibrant in at least one membrane layer to an analyte of interest. In some embodiments, hydrophilic modifiers such as poly(ethylene) glycol, hydroxyl or polyhydroxyl modifiers and the like, and any combinations thereof, can be used to enhance the biocompatibility of the multilayered membrane.

[0238] In some embodiments, the multilayered membrane can comprise a membrane polymer crosslinked with a crosslinking agent disclosed herein.

[0239] In some embodiments, the multilayered membrane can comprise from 1 layer to 10 layers of poly(4-vinylpyridine). In some embodiments, the multilayered membrane can comprise from 1 layer to 10 layers, from 1 layer to 8 layers, from 1 layer to 6 layers, from 1 layer to 4 layers, from 1 layer to 2 layers, from 2 layers to 10 layers, from 2 layers to 8 layers, from 2 layers to 6 layers, from 2 layers to 4 layers, from 4 layers to 10 layers, from 4 layers to 8 layers, from 4 layers to 6 layers, from 6 layers to 10 layers, from 6 layers to 8 layers, or from 8 layers to 10 layers of poly(4-vinylpyridine). In some embodiments, the multilayered membrane can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 layers of poly(4-vinylpyridine). In some embodiments, the multilayered membrane can comprise 6 layers of poly(4-vinylpyridine).

[0240] In some embodiments, the multilayered membrane can comprise from 1 layer to 10 layers of a derivatized polyvinylpyridine-co-styrene copolymer. In some embodiments, the multilayered membrane can comprise from 1 layer to 10 layers, from 1 layer to 8 layers, from 1 layer to 6 layers, from 1 layer to 4 layers, from 1 layer to 2 layers, from 2 layers to 10 layers, from 2 layers to 8 layers, from 2 layers to 6 layers, from 2 layers to 4 layers, from 4 layers to 10 layers, from 4 layers to 8 layers, from 4 layers to 6 layers, from 6 layers to 10 layers, from 6 layers to 8 layers, or from 8 layers to 10 layers of a derivatized polyvinylpyridine-co-styrene copolymer. In some embodiments, the multilayered membrane can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 layers of the derivatized polyvinylpyridine-co-styrene copolymer. In some embodiments, the multilayered membrane can comprise 2 layers of the derivatized polyvinylpyridine-co- styrene copolymer.6. Enzymes

[0241] The sensors of the present disclosure include one or more enzymes for detecting one or more analytes in at least one sensing area. Suitable enzymes for use in a sensor of the present disclosure can include a NAD(P)-dependent enzyme or an NAD(P)- independent enzyme. For example, an NAD(P)-dependent enzyme for use in the present disclosure can be used for detecting glucose, ketones, lactate, oxygen, hemoglobin A1C, albumin, alcohol, alkaline phosphatase, alanine transaminase, aspartate aminotransferase, bilirubin, blood, urea, nitrogen, calcium, carbon dioxide, chloride, creatinine, hematocrit, lactate, magnesium, oxygen, pH, phosphorus, potassium, sodium, total protein, uric acid, etc. In some embodiments, the analyte to be detected using an NAD(P)-dependentenzyme can be glucose, lactate, ketones, creatinine, alcohol, e.g., ethanol, or the like. In some embodiments, a sensing area can include multiple enzymes, e.g., an enzyme system, that are collectively responsive to the analyte.

[0242] In some embodiments, the enzymes for use in a sensor of the present disclosure can include an enzyme that is NAD(P)-independent. In some embodiments, the NAD(P)- independent enzyme for use in present disclosure can be used for detecting glucose or glutamate.

[0243] In some embodiments, the sensing area of a presently disclosed analyte sensor can include at least one NAD(P)-dependent enzyme. In some embodiments, the sensing area of a presently disclosed analyte sensor can include two or more NAD(P)-dependent enzymes. In some embodiments, the analyte sensor of the present disclosure can include two sensing areas that each include at least one NAD(P)-dependent enzyme. Alternatively, an analyte sensor of the present disclosure in some embodiments can include two sensing areas, where only one sensing area includes an NAD(P)-dependent enzyme. Non-limiting examples of NAD(P)-dependent enzymes are disclosed in Vidal et al., Biochimica et Biophysica Acta - Proteins and Proteomics 1866(2):327-347 (2018) (see Tables 1-2), the contents of which are incorporated by reference in its entirety.

[0244] In some embodiments, an analyte sensor of the present disclosure can include one or more internal supplies of NAD(P) for an NAD(P)-dependent enzyme included in one or more sensing areas of the analyte sensor.

[0245] In some embodiments, a sensing area can include an NAD(P)-dependent dehydrogenase. Non-limiting examples of NAD(P)-dependent dehydrogenases include glucose dehydrogenase (EC.1.1.1.47), lactate dehydrogenase (EC 1.1.1.27 and EC1.1.1.28), malate dehydrogenase (EC1.1.1.37), glycerol dehydrogenase (EC1.1.1.6), alcohol dehydrogenase (EC 1.1.1.1), alpha-hydroxybutyrate dehydrogenase, sorbitol dehydrogenase, amino acid dehydrogenase such as L-amino acid dehydrogenase (EC1.4.1.5), diaphorase (EC1.8.1.4), and combinations thereof.

[0246] In some embodiments, the NAD(P)-dependent dehydrogenase can include diaphorase, glucose dehydrogenase, alcohol dehydrogenase, lactate dehydrogenase, and P-hydroxybutyrate dehydrogenase. In some embodiments, the enzyme system can include two or more NAD(P)-dependent dehydrogenases, e.g., a first NAD(P)-dependent dehydrogenase and diaphorase. For example, but not by way of limitation, the NAD(P)-dependent dehydrogenase can convert the analyte and oxidized nicotinamide adenine dinucleotide (NAD+) into an oxidized analyte and reduced nicotinamide adenine dinucleotide (NADH), respectively. The enzyme cofactors NAD+and NADH aid in promoting the concerted enzymatic reactions disclosed herein. The NADH can then undergo reduction under diaphorase mediation, with the electrons transferred during this process providing the basis for analyte detection at the working electrode.

[0247] In some embodiments, an analyte sensor of the present disclosure can include a glucose-responsive sensing area, a ketones-responsive sensing area, a lactate-responsive sensing area, a creatinine-responsive sensing area, an alcohol-responsive sensing area, or any combination thereof. In some embodiments, a glucose-responsive sensing area can include one or more NAD(P)-dependent enzymes for detecting glucose. In some embodiments, a ketones-responsive sensing area can include one or more NAD(P)- dependent enzymes for detecting ketones. In some embodiments, a lactate-responsive sensing area can include one or more NAD(P)-dependent enzymes for detecting lactate. In some embodiments, a creatinine-responsive sensing area can include one or more NAD(P)-dependent enzymes for detecting creatinine. In some embodiments, an alcoholresponsive sensing area can include one or more NAD(P)-dependent enzymes for detecting alcohol. In some embodiments, a sensing area can include an enzyme system comprising two or more enzymes that are collectively responsive to the analyte. For example, but not by way of limitation, a ketones-responsive sensing area can include an enzyme system comprising at least one NAD(P)-dependent enzyme.

[0248] In some embodiments, an analyte sensor disclosed herein can include at least one sensing area that includes one or more NAD(P)-dependent enzymes, as disclosed herein, for detecting an analyte. Alternatively, an analyte sensor disclosed herein can include two or more sensing areas, with each sensing area containing one or more enzymes, e.g., where one of the sensing areas includes one or more NAD(P)-dependent enzymes. For example, but not by way of limitation, an analyte sensor of the present disclosure can include a first sensing area that comprises a first enzyme (or enzyme system) for use in detecting a first analyte and a second sensing area that includes a second enzyme (or second enzyme system) for detecting a second analyte, where one of the first sensing area or second sensing area can include an NAD(P)-dependent enzyme.

[0249] In some embodiments, the sensing area can include by weight from about 10% to about 80%, e.g., from about 15% to about 75%, from about 20% to about 70%, from about 25% to about 65%, or from about 30% to about 60%, of one or more enzymes disclosed herein.

[0250] In some embodiments, the sensing area can further include a stabilizer, e.g., for stabilizing the enzyme. For example, but not by way of limitation, the stabilizer can be an albumin, e.g., a serum albumin. Non-limiting examples of serum albumins include bovine serum albumin and human serum albumin. In some embodiments, the stabilizer can be a human serum albumin. In some embodiments, the stabilizer can be a bovine serum albumin. In some embodiments, the stabilizer can be catalase. In some embodiments, the sensing area can include a ratio of stabilizer to the one or more enzymes present in the sensing area, from about 40: 1 to about 1 :40, e.g., from about 35: 1 to about 1 :35, from about 30: 1 to about 1 :30, from about 25: 1 to about 1 :25, from about 20: 1 to about 1 :20, from about 15: 1 to about 1 : 15, from about 10: 1 to about 1 : 10, from about 9:1 to about 1 :9, from about 8: 1 to about 1 :8, from about 7: 1 to about 1 :7, from about 6: 1 to about 1 :6, from about 5: 1 to about 1 :5, from about 4: 1 to about 1 :4, from about 3 : 1 to about 1 :3, from about 2: 1 to about 1 :2 or about 1 : 1. In some embodiments, the sensing area can include a ratio of stabilizer to the one or more enzymes present in the sensing area, from about 2: 1 to about 1 :2. In some embodiments, the sensing area can include by weight from about 10% to about 50%, e.g., from about 15% to about 45%, from about 20% to about 40%, from about 20% to about 35%, or from about 20% to about 30% of the stabilizer. In some embodiments, the sensing area can include from about 15% to about 35% of the stabilizer by weight.

[0251] In some embodiments, the sensing area can further include a cofactor for one or more enzymes present in the sensing area. In some embodiments, the cofactor can be NAD(P). In some embodiments, the cofactor can be a cofactor different from NAD(P). In some embodiments, the sensing area can include a ratio of cofactor to enzyme from about 40: 1 to about 1 :40, e.g., from about 35: 1 to about 1 :35, from about 30:1 to about 1 :30, from about 25: 1 to about 1 :25, from about 20: 1 to about 1 :20, from about 15: 1 to about 1 : 15, from about 10: 1 to about 1 : 10, from about 9: 1 to about 1 :9, from about 8: 1 to about 1 :8, from about 7: 1 to about 1 :7, from about 6: 1 to about 1 :6, from about 5: 1 to about 1 :5, from about 4: 1 to about 1 :4, from about 3 : 1 to about 1 :3, from about 2: 1 toabout 1 :2, or from about 2: 1 to about 1 :2. In some embodiments, the sensing area can include a ratio of cofactor to enzyme from about 2: 1 to about 1 :2. In some embodiments, the sensing area can include by weight from about 10% to about 50%, e.g., from about 15% to about 45%, from about 20% to about 40%, from about 20% to about 35%, or from about 20% to about 30% of the cofactor. In some embodiments, the sensing area can include from about 15% to about 35% by weight of the cofactor.

[0252] In some embodiments, an analyte sensor of the present disclosure can include an implantable portion (e.g. sensor tail) comprising at least one working electrode, and a sensing area disposed upon the surface of the working electrode, where the sensing area includes at least one NAD(P)-independent enzyme. In some embodiments, an analyte sensor of the present disclosure can include an implantable portion (e.g. sensor tail) comprising a substrate, at least one working electrode, and a sensing area disposed upon the surface of the working electrode, where the sensing area includes at least one NAD(P)-independent enzyme. In some embodiments, the NAD(P)-independent enzyme can be glucose oxidase or glutamate oxidase. For example, but not by way of limitation, a sensor of the present disclosure can include an implantable portion (e.g. sensor tail) comprising at least one working electrode and a sensing area disposed upon the surface of the working electrode, where the sensing area includes an enzyme system comprising glucose oxidase or glutamate oxidase.

[0253] In some embodiments, an analyte sensor of the present disclosure can include an implantable portion (e.g. sensor tail) comprising at least one working electrode and a glucose-responsive sensing area disposed upon the surface of the working electrode, where the glucose-responsive sensing area includes an enzyme system comprising an NAD(P)-independent oxidase, e.g., glucose oxidase.

[0254] In some embodiments, an analyte sensor of the present disclosure can include an implantable portion (e.g. sensor tail) comprising at least one working electrode and a glutamate-responsive sensing area disposed upon the surface of the working electrode, where the glutamate-responsive sensing area includes an enzyme system comprising an NAD(P)-independent oxidase, e.g., glutamate oxidase.

[0255] In some embodiments, an analyte sensor of the present disclosure can include a second sensing area, e.g., for detecting an analyte different from the analyte detected by the first sensing area. In some embodiments, the second sensing area can be disposedupon the same working electrode as the first sensing area or on a second working electrode. In some embodiments, the second sensing area can be a ketones-responsive sensing area, a lactate-responsive sensing area, a creatinine-responsive sensing area, or an alcohol-responsive sensing area.

[0256] In some embodiments, an analyte sensor can include two working electrodes, e.g., a first sensing area disposed on a first working electrode and a second sensing area disposed on a second working electrode. For example, but not by way of limitation, an analyte sensor disclosed herein can feature a first sensing area disposed on a first working electrode and a second sensing area disposed upon the surface of a different working electrode, e.g., second working electrode, where one of the sensing areas can include an NAD(P)-independent enzyme. In some embodiments, the second sensing area can be configured to detect a different analyte or the same analyte detected by first sensing area. In some embodiments, such analyte sensors can include an implantable portion (e.g. sensor tail) with a first working electrode and a second working electrode, a first sensing area disposed upon a surface of the first working electrode and a second sensing area disposed upon a surface of the second working electrode, where one of the sensing areas can include an NAD(P)-independent enzyme.

[0257] In some embodiments, when the sensor is configured to detect two or more analytes using two working electrodes, detection of each analyte can include applying a potential to each working electrode separately, such that separate signals are obtained from each analyte. The signal obtained from each analyte can then be correlated to an analyte concentration through use of a calibration curve or function, or by employing a lookup table. In some embodiments, correlation of the analyte signal to an analyte concentration can be conducted through use of a processor.

[0258] In some analyte sensor configurations, the first sensing area and the second sensing area can be disposed upon a single working electrode. For example, but not by way of limitation, an analyte sensor disclosed herein can feature a first sensing area and a second sensing area disposed upon the surface of a single working electrode, where one of the sensing areas includes an NAD(P)-independent enzyme. In some embodiments, a first signal can be obtained from the first sensing area, e.g., at a low potential, and a second signal containing a signal contribution from both sensing areas can be obtained at a higher potential. Subtraction of the first signal from the second signal can then allowthe signal contribution arising from the second analyte to be determined. The signal contribution from each analyte can then be correlated to an analyte concentration in a similar manner to that described for sensor configurations having multiple working electrodes. In some embodiments, when a glutamate-responsive sensing area and a second sensing area configured to detect a different analyte, e.g., a glucose-responsive sensing area, are arranged upon a single working electrode in this manner, one of the sensing areas can be configured such that it can be interrogated separately to facilitate detection of each analyte. For example, either the glutamate-responsive sensing area or glucose-responsive sensing area can produce a signal independently of the other sensing area.

[0259] It is also to be appreciated that the sensitivity (output current) of the analyte sensors toward each analyte can be varied by changing the coverage (area or size) of the sensing areas, the area ratio of the sensing areas with respect to one another, or the identity, thickness and / or composition of a mass transport limiting membrane overcoating the sensing areas. Variation of these parameters can be conducted readily by one having ordinary skill in the art once granted the benefit of the disclosure herein.7. Redox Mediators

[0260] In some embodiments, an analyte sensor of the present disclosure can include an electron transfer agent. For example, but not by way of limitation, one or more sensing areas of an analyte sensor can include an electron transfer agent. In some embodiments, an analyte sensor can include one sensing area that includes an electron transfer agent and a second sensing area that does not include an electron transfer agent. In some embodiments, the presence of an electron transfer agent in a sensing area can depend on the enzyme or enzyme system used to detect the analyte and / or the composition of the working electrode. Alternatively, an analyte sensor can include two sensing areas, where both sensing areas include an electron transfer agent.

[0261] Suitable electron transfer agents can facilitate conveyance of electrons to the adjacent working electrode after an analyte undergoes an enzymatic oxidation-reduction reaction within the corresponding sensing area, thereby generating a current that is indicative of the presence of that particular analyte. The amount of current generated is proportional to the quantity of analyte that is present. In some embodiments, suitable electron transfer agents can include electroreducible and electrooxidizable ions,complexes or molecules (e.g., quinones) having oxidation-reduction potentials that are a few hundred millivolts above or below the oxidation-reduction potential of the standard calomel electrode (SCE). In some embodiments, the redox mediators can include osmium complexes and other transition metal complexes, such as those described in U.S. Patent Nos. 6,134,461 and 6,605,200, which are incorporated herein by reference in their entirety. Additional examples of suitable redox mediators include those described in U.S. Patent 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 entirety. Other examples of suitable redox mediators include metal compounds or complexes of ruthenium, osmium, iron (e.g., polyvinylferrocene or hexacyanoferrate), or cobalt, including metallocene compounds thereof, for example. Suitable ligands for the metal complexes can also include, for example, bidentate or higher denticity ligands such as, for example, bipyridine, biimidazole, phenanthroline, or pyridyl(imidazole). Other suitable bidentate ligands can include, for example, amino acids, oxalic acid, acetylacetone, diaminoalkanes, or o- diaminoarenes. Any combination of monodentate, bidentate, tridentate, tetradentate, or higher denticity ligands can be present in a metal complex, e.g., osmium complex, to achieve a full coordination sphere.

[0262] In some embodiment, the electron redox mediator can comprise an osmium- containing poly(4-vinylpyridine)-based polymer as shown below.wherein n is 2, n' is 17, and n" is 1.

[0263] In some embodiments, electron transfer agents disclosed herein can comprise suitable functionality to promote covalent bonding to a polymer (also referred to herein as a polymeric backbone) within the sensing areas as discussed further below. For example, but not by way of limitation, an electron transfer agent for use in the present disclosure can include a polymer-bound electron transfer agent. Suitable non-limiting examples of polymer-bound electron transfer agents include those described in U.S. Patent Nos. 8,444,834, 8,268,143 and 6,605,201, the disclosures of which are incorporated herein byreference in their entirety. In some embodiments, the electron transfer agent is a bidentate osmium complex bound to a polymer described herein, e.g., a polymeric backbone described below. In some embodiments, the polymer-bound electron transfer agent shown in FIG. 3 of U.S. Patent No. 8,444,834 can be used in a sensor of the present disclosure.

[0264] In some embodiments of the present disclosure, an analyte sensor can include at least one working electrode and at least one sensing area disposed upon the surface of the working electrode, where the sensing area can be overcoated with a multi-layered membrane, and at least one redox mediator, e.g., an osmium complex. In some embodiments, the sensing area includes an enzyme system comprising glutamate oxidase or glucose oxidase and a redox mediator, e.g., an osmium complex.8. Polymeric Backbone

[0265] In some embodiments, one or more sensing areas for promoting analyte detection can include a polymer to which an enzyme and / or redox mediator is covalently bound. Any suitable polymeric backbone can be present in the sensing area for facilitating detection of an analyte through covalent bonding of the enzyme and / or redox mediator thereto. Non-limiting examples of suitable polymers within the sensing area include polyvinylpyridines, e.g., poly(4-vinylpyridine) and / or poly(2-vinylpyridine), and polyvinylimidazoles, e.g., poly(N-vinylimidazole) and poly(l-vinylimidazole), or a copolymer thereof, for example, in which quatemized pyridine groups serve as a point of attachment for the redox mediator or enzyme thereto. Illustrative copolymers that can be suitable for inclusion in the sensing areas include those containing monomer units such as styrene, acrylamide, methacrylamide, or acrylonitrile, for example. In some embodiments, polymers that can be present in a sensing area include a polyurethane or a copolymer thereof, and / or polyvinylpyrrolidone. In some embodiments, polymers that can be present in the sensing area include, but are not limited to, those described in U.S. Patent 6,605,200, the contents of which are incorporated herein by reference in their entirety, such as poly(acrylic acid), styrene / maleic anhydride copolymer, methylvinylether / maleic anhydride copolymer (GANTREZ polymer), poly(vinylbenzylchloride), poly(allylamine), polylysine, poly(4-vinylpyridine) quaternized with carboxypentyl groups, and poly(sodium 4-styrene sulfonate). In someembodiments where the analyte sensor includes two sensing areas, the polymer within each sensing area can be the same or different.

[0266] In some embodiments, the polymer can be polyvinylpyridine or a copolymer thereof. In some embodiments, the polymer can be a co-polymer of vinylpyridine and styrene.

[0267] In some embodiments, when an enzyme system with multiple enzymes is present in a given sensing area, all of the multiple enzymes can be covalently bonded to the polymer. In some embodiments, only a subset of the multiple enzymes are covalently bonded to the polymer. For example, and not by the way of limitation, one or more enzymes within an enzyme system can be covalently bonded to the polymer and at least one enzyme can be non-covalently associated with the polymer, such that the non- covalently bonded enzyme is physically retained within the polymer. In some embodiments, the NAD(P)-dependent enzyme can be covalently bonded to the polymer. Alternatively, the NAD(P)-dependent enzyme can be non-covalently associated with the polymer. In some embodiments, the NAD(P)-dependent dehydrogenase and the diaphorase can be covalently bonded to a polymer within a sensing area of the disclosed analyte sensors. In some embodiments, the NAD(P)-dependent dehydrogenase can be covalently bonded to the polymer and diaphorase can be non-covalently associated with the polymer. Alternatively, diaphorase can be covalently bonded to the polymer and the NAD(P)-dependent dehydrogenase can be non-covalently associated with the polymer.

[0268] In some embodiments, when a stabilizer is present in a sensing area, one or more enzymes within the area can be covalently bonded to the stabilizer. For example, and not by the way of limitation, one or more enzymes within an enzyme system, e.g., one or more NAD(P)-dependent enzymes, can be covalently bonded to the stabilizer, e.g., albumin, present in the sensing area.

[0269] In some particular embodiments, covalent bonding of the one or more enzymes and / or redox mediators to the polymer and / or stabilizer in a given sensing area can take place via crosslinking introduced by a suitable crosslinking agent. In some embodiments, crosslinking of the polymer to the one or more enzymes and / or redox mediators can reduce the occurrence of delamination of the enzyme compositions from the electrode. Suitable crosslinking agents can include one or more crosslinkable functionalities such as, but not limited to, vinyl, alkoxy, acetoxy, enoxy, oxime, amino, hydroxyl, cyano, halo,acrylate, epoxide, and isocyanato groups. In some embodiments, the crosslinking agent can comprise one or more, two or more, three or more, or four or more epoxide groups. For example, but not by way of limitation, a crosslinker for use in the present disclosure can include mono-, di-, tri- and tetra-ethylene oxides. In some embodiments, crosslinking agents for reaction with free amino groups in the enzyme (e.g., with the free side chain amine in lysine) can include crosslinking agents such as, for example, polyethylene glycol dibutyl ethers, polypropylene glycol dimethyl ethers, polyalkylene glycol allyl methyl ethers, polyethylene glycol diglycidyl ether (PEGDGE), or other polyepoxides, cyanuric chloride, N-hydroxysuccinimide, imidoesters, epichlorohydrin, or derivatized variants thereof. In some embodiments, the crosslinking agent can be PEGDGE, e.g., having an average molecular weight (Mn) from about 200 to 1,000, e.g., about 400. In some embodiments, the crosslinking agent can be PEGDGE 400. In some embodiments, the crosslinking agent can be glutaraldehyde. Suitable crosslinking agents for reaction with free carboxylic acid groups in the enzyme can include, for example, carbodiimides. In some embodiments, the crosslinking agent can be polyethylene glycol diglycidyl ether. In some embodiments, the crosslinking of the enzyme to the polymer can generally be intermolecular. In some embodiments, the crosslinking of the enzyme to the polymer can generally be intramolecular.9. Mass Transport Limiting Membrane

[0270] In some embodiments, an analyte sensor of the present disclosure can further comprise a mass transport limiting membrane permeable to an analyte that overcoats at least one sensing area, e.g., a first sensing area and / or a second sensing area. In some embodiments, the mass transport limiting membrane overcoats one or more of the sensing areas of an analyte sensor.

[0271] In some embodiments, a mass transport limiting membrane overcoating a sensing area can improve biocompatibility. A mass transport limiting membrane can act as a diffusion-limiting barrier to reduce the rate of mass transport of the analyte, e.g., glucose, an alcohol, a ketone, lactate or P-hydroxybutyrate, when the sensor is in use. For example, but not by way of limitation, limiting access of an analyte, e.g., an alcohol, to the sensing area with a mass transport limiting membrane can aid in avoiding sensor overload (saturation), thereby improving detection performance and accuracy. In some embodiments, the mass transport limiting membrane can limit the flux of an analyte to theelectrode in an electrochemical sensor so that the sensor is linearly responsive over a large range of analyte concentrations.

[0272] In some embodiments, the mass transport limiting membrane can have a thickness, e.g., dry thickness, ranging from about 0.1 pm to about 1,000 pm, e.g., from about 1 pm to about 500 pm, from about 1 pm to about 100 pm, or from about 10 pm to about 100 pm. In some embodiments, the mass transport limiting membrane can have a thickness from about 0.1 pm to about 10 pm, e.g., from about 0.5 pm to about 10 pm, from about 1 pm to about 10 pm, from about 1 pm to about 5 pm, or from about 0.1 pm to about 5 pm.

[0273] In some embodiments, the mass transport limiting membrane can be formed by depositing a mass transport limiting membrane solution upon a surface, for example by dipping, and allowing the membrane solution to dry. In some embodiments, the sensor can be dipped in the mass transport limiting membrane solution more than once. For example, but not by way of limitation, a sensor (or working electrode) of the present disclosure can be dipped in an mass transport limiting membrane solution at least twice, at least three times, at least four times, or at least five times to obtain the desired mass transport limiting membrane thickness.

[0274] In some embodiments, the mass transport limiting membrane can be singlecomponent (contain a single membrane polymer). Alternatively, the mass transport limiting membrane can be multi-component (contain two or more different membrane polymers). In some embodiments, the mass transport limiting membrane can include two or more layers, e.g., a bilayer or trilayer membrane. In some embodiments, each layer can comprise a different polymer or the same polymer at different concentrations or thicknesses.

[0275] In some embodiments, a mass transport limiting membrane can include a polyvinylpyridine (e.g., poly(4-vinylpyridine) or poly(4-vinylpyridine)), a polyvinylimidazole, a polyvinylpyridine copolymer (e.g., a copolymer of vinylpyridine and styrene), a polyacrylate, a polyurethane, a polyether urethane, a silicone, a polytetrafluoroethylene, a polyethylene-co-tetrafluoroethylene, a polyolefin, a polyester, a polycarbonate, a biostable polytetrafluoroethylene, homopolymers, copolymers or terpolymers of polyurethanes, a polypropylene, a polyvinylchloride, a polyvinylidene difluoride, a polybutylene terephthalate, a polymethylmethacrylate, a polyether etherketone, cellulosic polymers, polysulfones and block copolymers thereof including, for example, di-block, tri-block, alternating, random and graft copolymers or a chemically related material and the like.

[0276] In some embodiments, the mass transport limiting membrane for use in the present disclosure, e.g., a single-component membrane, can include a polyvinylpyridine (e.g., poly(4-vinylpyridine) and / or poly(2-vinylpyridine)). In some embodiments, a mass transport limiting membrane for use in the present disclosure, e.g., a single-component membrane, can include poly(4-vinylpyridine). In some embodiments, a mass transport limiting membrane for use in the present disclosure, e.g., a single-component membrane, can include a copolymer of vinylpyridine and styrene. In some embodiments, the mass transport limiting membrane can comprise a polyvinylpyridine-co-styrene copolymer. For example, but not by way of limitation, a polyvinylpyridine-co-styrene copolymer for use in the present disclosure can include a polyvinylpyridine-co-styrene copolymer in which a portion of the pyridine nitrogen atoms were functionalized with a noncrosslinked polyethylene glycol tail and a portion of the pyridine nitrogen atoms were functionalized with an alkylsulfonic acid group. In some embodiments, a derivatized polyvinylpyridine-co-styrene copolymer for use as a membrane polymer can be the polymer as described in U.S. Patent No. 8,761,857, the contents of which are incorporated by reference in their entirety. In some embodiments, the polyvinylpyridine- based polymer can have a molecular weight from about 50 Da to about 500 kDa.

[0277] In some embodiments, the mass transport limiting membrane can comprise polymers such as, but not limited to, poly(styrene co-maleic anhydride), dodecylamine and polypropylene glycol)-block-polyethylene glycol)-block-poly(propylene glycol) (2- aminopropyl ether) crosslinked with polypropylene glycol)-block-polypthylene glycol)- block-polypropylene glycol) bis(2-aminopropyl ether); poly(N-isopropyl acrylamide); a copolymer of polypthylene oxide) and polypropylene oxide); or a combination thereof.

[0278] In some embodiments, the mass transport limiting membrane can include a polyurethane membrane that includes both hydrophilic and hydrophobic regions. In some embodiments, a hydrophobic polymer component can be a polyurethane, a polyurethane urea or poly ther-urethane-urea). In some embodiments, a polyurethane is a polymer produced by the condensation reaction of a diisocyanate and a difunctional hydroxylcontaining material. In some embodiments, a polyurethane urea is a polymer producedby the condensation reaction of a diisocyanate and a difunctional amine-containing material. In some embodiments, diisocyanates for use herein can include aliphatic diisocyanates, e.g., containing from about 4 to about 8 methylene units, or diisocyanates containing cycloaliphatic moieties. Additional non-limiting examples of polymers that can be used for the generation of a mass transport limiting membrane of the presently disclosed sensor include vinyl polymers, polyethers, polyesters, polyamides, inorganic polymers (e.g., polysiloxanes and polycarbosiloxanes), natural polymers (e.g., cellulosic and protein based materials) and mixtures (e.g., admixtures or layered structures) or combinations thereof. In some embodiments, the hydrophilic polymer component can be polyethylene oxide and / or polyethylene glycol. In some embodiments, the hydrophilic polymer component can be a polyurethane copolymer. For example, but not by way of limitation, a hydrophobic-hydrophilic copolymer component for use in the present disclosure can be a polyurethane polymer that comprises about 10% to about 50%, e.g., 20%, hydrophilic polyethylene oxide.

[0279] In some embodiments, the mass transport limiting membrane can include a silicone polymer / hydrophobic-hydrophilic polymer blend. In some embodiments, the hydrophobic-hydrophilic polymer for use in the blend can be any suitable hydrophobic- hydrophilic polymer such as, but not limited to, polyvinylpyrrolidone, polyhydroxyethyl methacrylate, polyvinylalcohol, polyacrylic acid, polyethers such as polyethylene glycol or polypropylene oxide, and copolymers thereof, including, for example, di-block, triblock, alternating, random, comb, star, dendritic, and graft copolymers. In some embodiments, the hydrophobic-hydrophilic polymer can be a copolymer of poly(ethylene oxide) (PEO) and polypropylene oxide) (PPO). Non-limiting examples of PEO and PPO copolymers include PEO-PPO diblock copolymers, PPO-PEO-PPO triblock copolymers, PEO-PPO-PEO triblock copolymers, alternating block copolymers of PEO-PPO, random copolymers of ethylene oxide and propylene oxide and blends thereof. In some embodiments, the copolymers can be substituted with hydroxy substituents.

[0280] In some embodiments, hydrophilic or hydrophobic modifiers can be used to “finetune” the permeability of the resulting membrane to an analyte of interest. In some embodiments, hydrophilic modifiers such as poly(ethylene) glycol, hydroxyl or polyhydroxyl modifiers and the like, and any combinations thereof, can be used toenhance the biocompatibility of the polymer or the resulting mass transport limiting membrane.

[0281] In some embodiments where multiple sensing areas are present, the mass transport limiting membrane can overcoat each sensing area. For example, but not by way of limitation, a mass transport limiting membrane can be disposed on the first sensing area, e.g., an alcohol-responsive sensing area, and a separate, second mass transport limiting membrane can overcoat the second sensing area, e.g., a glucose-responsive sensing area. In some embodiments, the two mass transport limiting membranes can be spatially separated and do not overlap each other. In some embodiments, the first mass transport limiting membrane does not overlap the second mass transport limiting membrane and the second mass transport limiting membrane does not overlap the first mass transport limiting membrane. In some embodiments, the first mass transport limiting membrane comprises different polymers than the second mass transport limiting membrane. Alternatively, the first mass transport limiting membrane comprises the same polymers as the second mass transport limiting membrane. In some embodiments, the first mass transport limiting membrane comprises the same polymers as the second mass transport limiting membrane but comprises different crosslinking agents.

[0282] In some embodiments, polydimethylsiloxane (PDMS) can be incorporated in any of the mass transport limiting membranes disclosed herein.

[0283] In some embodiments when a first sensing area and a second sensing area configured for assaying different analytes are disposed on separate working electrodes, the mass transport limiting membrane can have differing permeability values for the first analyte and the second analyte. For example, but not by way of limitation, the mass transport limiting membrane overcoating at least one of the sensing areas can include an admixture of a first membrane polymer and a second membrane polymer or a bilayer of the first membrane polymer and the second membrane polymer. A homogeneous membrane can overcoat the sensing area not overcoated with the admixture or the bilayer, wherein the homogeneous membrane includes only one of the first membrane polymer or the second membrane polymer. Advantageously, the architectures of the analyte sensors disclosed herein readily allow a continuous membrane having a homogenous membrane portion to be disposed upon a first sensing area and a multi-component membrane portion to be disposed upon a second sensing area of the analyte sensors, thereby equalizing thepermeability values for each analyte concurrently to afford improved sensitivity and detection accuracy. Continuous membrane deposition can take place through sequential dip coating operations in particular embodiments.

[0284] In some embodiments, the mass transport limiting membrane can comprise a membrane polymer crosslinked with a crosslinking agent disclosed herein. In some embodiments where there are two mass transport limiting membranes, e.g., a first mass transport limiting membrane and a second mass transport limiting membrane, each membrane can be crosslinked with a different crosslinking agent. For example, but not by way of limitation, the crosslinking agent can result in a membrane that is more restrictive to diffusion of certain compounds, e.g., analytes within the membrane, or less restrictive to diffusion of certain compounds, e.g., by affecting the size of the pores within the membrane. For example, but not by way of limitation, in a sensor that is configured to detect alcohol and glucose, the mass transport limiting membrane overcoating the alcohol-responsive area can have a pore size that restricts the diffusion of compounds larger than alcohol, e.g., glucose, through the membrane.

[0285] In some embodiments, crosslinking agents for use in the present disclosure can include polyepoxides, carbodiimide, cyanuric chloride, triglycidyl glycerol, N- hydroxysuccinimide, imidoesters, epichlorohydrin or derivatized variants thereof. In some embodiments, a membrane polymer overcoating one or more sensing areas can be crosslinked with a branched crosslinker, e.g., which can decrease the amount of extractables obtainable from the mass transport limiting membrane. Non-limiting examples of a branched crosslinker include branched glycidyl ether crosslinkers, e.g., including branched glycidyl ether crosslinkers that include two or three or more crosslinkable groups. In some embodiments, the branched crosslinker can include two or more crosslinkable groups, such as polyethylene glycol diglycidyl ether. In some embodiments, the branched crosslinker can include three or more crosslinkable groups, such as polyethylene glycol tetraglycidyl ether. In some embodiments, the membrane polymer can include polyvinylpyridine or a copolymer of vinylpyridine and styrene crosslinked with a branched glycidyl ether crosslinker including two or three crosslinkable groups, such as polyethylene glycol tetraglycidyl ether or polyethylene glycol diglycidyl ether. In some embodiments, the epoxide groups of a polyepoxides, e.g., polyethylene glycol tetraglycidyl ether or polyethylene glycol diglycidyl ether, canform a covalent bond with pyridine or an imidazole via epoxide ring opening resulting in a hydroxyalkyl group bridging a body of the crosslinker to the heterocycle of the membrane polymer.

[0286] In some embodiments, the crosslinking agent can be polyethylene glycol diglycidyl ether (PEGDGE). In some embodiments, the PEGDGE used to promote crosslinking (e.g., intermolecular crosslinking) between two or more membrane polymer backbones can exhibit a broad range of suitable molecular weights. In some embodiments, the molecular weight of the PEGDGE can range from about 100 g / mol to about 5,000 g / mol. The number of ethylene glycol repeat units in each arm of the PEGDGE can be the same or different, and can typically vary over a range within a given sample to afford an average molecular weight. In some embodiments, the PEGDGE for use in the present disclosure has an average molecular weight (Mn) from about 200 to 1,000, e.g., about 400. In some embodiments, the crosslinking agent can be PEGDGE 400.

[0287] In some embodiments, the polyethylene glycol tetraglycidyl ether used to promote crosslinking (e.g., intermolecular crosslinking) between two or more membrane polymer backbones can exhibit a broad range of suitable molecular weights. Up to four polymer backbones can be crosslinked with a single molecule of the polyethylene glycol tetraglycidyl ether crosslinker. The number of ethylene glycol repeat units in each arm of the polyethylene glycol tetraglycidyl ether can be the same or different, and can typically vary over a range within a given sample to afford an average molecular weight.10. Interference Domain

[0288] In some embodiments, the sensor of the present disclosure, e.g., implantable portion (e.g. sensor tail), can further comprise an interference domain. In some embodiments, the interference domain can include a polymer domain that restricts the flow of one or more interferents, e.g., to the surface of the working electrode. In some embodiments, the interference domain can function as a molecular sieve that allows analytes and other substances that are to be measured by the working electrode to pass through, while preventing passage of other substances such as interferents. In some embodiments, the interferents can 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.

[0289] In some embodiments, the interference domain can be located between the working electrode and one or more sensing areas, e.g., alcohol-responsive sensing area. In some embodiments, non-limiting examples of polymers that can be used in the interference domain include polyurethanes, polymers having pendant ionic groups, and polymers having controlled pore size. In some embodiments, the interference domain is formed from one or more cellulosic derivatives. Non-limiting examples of cellulosic derivatives include polymers such as cellulose acetate, cellulose acetate butyrate, 2- hydroxyethyl cellulose, cellulose acetate phthalate, cellulose acetate propionate, cellulose acetate trimellitate, and the like.

[0290] In some embodiments, the interference domain can be part of the mass transport limiting membrane and not a separate membrane.

[0291] In some embodiments, the interference domain can include a thin, hydrophobic membrane that is non-swellable and restricts diffusion of high molecular weight species. For example, but not by way of limitation, the interference domain can be permeable to relatively low molecular weight substances, such as hydrogen peroxide, while restricting the passage of higher molecular weight substances, such as ketones, glucose, acetaminophen, and / or ascorbic acid.

[0292] In some embodiments, the interference domain can be deposited directly onto the working electrode, e.g., onto the surface of the working electrode. In some embodiments, the interference domain has a thickness, e.g., dry thickness, ranging from about 0.1 pm to about 1,000 pm, e.g., from about 1 pm to about 500 pm, from about 1 pm to about 100 pm, or from about 10 pm to about 100 pm. In some embodiments, the interference domain can have a thickness from about 0.1 pm to about 10 pm, e.g., from about 0.5 pm to about 10 pm, from about 1 pm to about 10 pm, from about 1 pm to about 5 pm, or from about 0.1 pm to about 5 pm. In some embodiments, the sensor can be dipped in the interference domain solution more than once. For example, but not by way of limitation, a sensor (or working electrode) of the present disclosure can be dipped in an interference domain solution at least once, at least twice, at least three times, at least four times, or at least five times to obtain the desired interference domain thickness.11. Manufacturing

[0293] The present disclosure further provides methods for manufacturing the presently disclosed analyte sensors that includes one or more sensing areas and one or more working electrodes.

[0294] In some embodiments, the method can include depositing one or more enzymes on a working electrode. In some embodiments, an enzyme composition can include one or more enzymes, a crosslinking agent, e.g., polyethylene glycol diglycidyl ether, and / or a redox mediator. In some embodiments, the enzyme composition can be deposited onto the surface of a working electrode as one large application which covers the desired portion of the working electrode or in the form of an array of a plurality of enzyme compositions, e.g., spaced apart from each other, to generate one or more sensing areas for detecting one or more analytes. In some embodiments, the method can further include curing the enzyme composition.

[0295] In some embodiments, the enzyme composition (i.e., sensing area formulation) can be deposited onto the surface of a working electrode by any non-impact or impact printing method. In some embodiments, the enzyme composition can be deposited using a pulse-jet device. In some embodiments, the enzyme composition can be deposited using thermoelectric pulse-jet deposition.

[0296] In some embodiments, the method includes depositing one or more NAD(P)- dependent enzymes, e.g., an NAD(P)-dependent dehydrogenase, on a working electrode. In some embodiments, the enzyme composition can include one or more additional enzymes, e.g., diaphorase, a crosslinking agent, e.g., polyethylene glycol diglycidyl ether, and / or a redox mediator. In some embodiments, the enzyme composition can be deposited onto the surface of a working electrode as one large application which covers the desired portion of the working electrode or in the form of an array of a plurality of enzyme compositions, e.g., spaced apart from each other, to generate one or more sensing areas for detecting one or more analytes. In some embodiments, the method can further include curing the enzyme composition.

[0297] In some embodiments, the sensing area composition can be prepared as a solution that dries or cures to solidify after deposition. Therefore, in some embodiments, all layers can be deposited in an automated fashion using small-volume liquid handling or similar techniques for high-throughput sensor fabrication.

[0298] In some embodiments, the method can further include adding a membrane composition on top of the cured sensing area and / or around the entire sensor. In some embodiments, the membrane composition is a mass transport limiting membrane, or a combination. In some embodiments, the method can include curing the membrane composition.IV. ANALYTE MONITORING

[0299] The present disclosure further provides methods of using the analyte sensors disclosed herein to detect an analyte in vivo. In some embodiments, the present disclosure provides methods for detecting one or more analytes, e.g., one analyte or two analytes. For example, but not by way of limitation, the present disclosure provides methods for detecting one or more analytes including glucose, ketones, lactate, oxygen, hemoglobin A1C, albumin, alcohol, alkaline phosphatase, alanine transaminase, aspartate aminotransferase, bilirubin, blood, urea, nitrogen, calcium, carbon dioxide, chloride, creatinine, hematocrit, lactate, magnesium, oxygen, pH, phosphorus, potassium, sodium, total protein, and / or uric acid using one or more NAD(P)-dependent or NAD(P)- independent enzymes. In some embodiments, the analyte can be ketones, alcohol, glucose, and / or lactate using one or more NAD(P)-dependent enzymes. In some embodiments, the analyte can be glucose or glutamate using one or more NAD(P)- independent enzymes. For example, but not by way of limitation, the present disclosure provides methods for detecting one or more ketones. In some embodiments, the present disclosure provides methods for detecting glucose. In some embodiments, the present disclosure provides methods for detecting creatinine. In some embodiments, the present disclosure provides methods for detecting lactate. In some embodiments, the present disclosure provides methods for detecting alcohol. In some embodiments, the present disclosure provides methods for detecting glutamate.

[0300] In some embodiments, the present disclosure provides methods for monitoring in vivo levels of an analyte over time with analyte sensors that include one or more NAD(P)- dependent enzymes or NAD(P)-independent enzymes. Generally, monitoring the in vivo concentration of an analyte in a fluid of the body of a subject includes inserting at least partially under a skin surface an in vivo analyte sensor as disclosed herein, contacting the monitored fluid (interstitial, blood, dermal, and the like) with the inserted sensor and generating a sensor signal at the working electrode. The presence and / or concentration ofthe analyte detected by the analyte sensor can be displayed, stored, forwarded, and / or otherwise processed. A variety of approaches can be employed to determine the concentration of analyte (e.g., glucose, an alcohol, a ketone, and / or lactate) with the disclosed sensors. In some embodiments, monitoring the concentration of analyte using the sensor signal can be performed by coulometric, amperometric, voltammetric, potentiometric, or any other convenient electrochemical detection technique.

[0301] In some embodiments, the analyte sensors comprising a surfactant in the sensing area exhibit increased stability. In some embodiments, the analyte sensors comprising a surfactant in the sensing area exhibit less than a 20% decrease (signal drop) in current over a period of 14 days. In some embodiments, the analyte sensors comprising a surfactant in the sensing area exhibit less than a 20% decrease, less than a 15% decrease, less than a 10% decrease, or less than a 5% decrease in current over a period of 14 days. In some embodiments, the analyte sensors comprising a surfactant in the sensing area exhibit less than a 15% decrease in current over a period of 7 days, over a period of 14 days, over a period of 16 days, over a period of 18 days, over a period of 20 days, over a period of 22 days, over a period of 24 days, over a period of 26 days, over a period of 28 days, or over a period of 30 days. In some embodiments, the analyte sensors comprising a surfactant in the sensing area exhibit less than a 10% decrease in current over a period of 7 days, over a period of 14 days, over a period of 16 days, over a period of 18 days, over a period of 20 days, over a period of 22 days, over a period of 24 days, over a period of 26 days, over a period of 28 days, or over a period of 30 days. In some embodiments, the analyte sensors comprising a surfactant in the sensing area exhibit less than a 20% decrease in current over a period of 7 days, over a period of 14 days, over a period of 16 days, over a period of 18 days, over a period of 20 days, over a period of 22 days, over a period of 24 days, over a period of 26 days, over a period of 28 days, or over a period of 30 days.

[0302] In some embodiments, the analyte sensor comprising a surfactant in the sensing area can have an analyte sensitivity that is greater than the analyte sensitivity of an otherwise identical sensor lacking the surfactant. In some embodiments, the analyte sensor comprising a surfactant in the sensing area can have an analyte sensitivity that is at least 95% greater than the analyte sensitivity of an otherwise identical sensor lacking the surfactant. In some embodiments, the analyte sensor comprising a surfactant in thesensing area can have an analyte sensitivity that is at least 85% greater than the analyte sensitivity of an otherwise identical sensor lacking the surfactant. In some embodiments, the analyte sensor comprising a surfactant in the sensing area can have an analyte sensitivity that is at least 75% greater than the analyte sensitivity of an otherwise identical sensor lacking the surfactant. In some embodiments, the analyte sensor comprising a surfactant in the sensing area can have an analyte sensitivity that is at least 50% greater than the analyte sensitivity of an otherwise identical sensor lacking the surfactant. In some embodiments, the analyte sensor comprising a surfactant in the sensing area can have an analyte sensitivity that is at least 25% greater than the analyte sensitivity of an otherwise identical sensor lacking the surfactant. In some embodiments, the analyte sensor comprising a surfactant in the sensing area can have an analyte sensitivity that is at least 10% greater than the analyte sensitivity of an otherwise identical sensor lacking the surfactant.

[0303] In some embodiments, the sensing area formulation comprising at least one surfactant can have a concentration (mass by volume) of solid material that is greater than the concentration (mass by volume) of solid material of an otherwise identical sensing area formulation lacking the surfactant. In some embodiments, the sensing area formulation comprising at least one surfactant can have a concentration (mass by volume) of solid material that is at least 5% greater than the concentration (mass by volume) of solid material of an otherwise identical sensing area formulation lacking the surfactant. In some embodiments, the sensing area formulation comprising at least one surfactant can have a concentration (mass by volume) of solid material that is at least 10% greater than the concentration (mass by volume) of solid material of an otherwise identical sensing area formulation lacking the surfactant. In some embodiments, the sensing area formulation comprising at least one surfactant can have a concentration (mass by volume) of solid material that is at least 15% greater than the concentration (mass by volume) of solid material of an otherwise identical sensing area formulation lacking the surfactant. In some embodiments, the sensing area formulation comprising at least one surfactant can have a concentration (mass by volume) of solid material that is at least 20% greater than the concentration (mass by volume) of solid material of an otherwise identical sensing area formulation lacking the surfactant. In some embodiments, the sensing area formulation comprising at least one surfactant can have a concentration (mass by volume)of solid material that is at least 25% greater than the concentration (mass by volume) of solid material of an otherwise identical sensing area formulation lacking the surfactant. In some embodiments, the sensing area formulation comprising at least one surfactant can have a concentration (mass by volume) of solid material that is at least 50% greater than the concentration (mass by volume) of solid material of an otherwise identical sensing area formulation lacking the surfactant. In some embodiments, the sensing area formulation comprising at least one surfactant can have a concentration (mass by volume) of solid material that is at least 75% greater than the concentration (mass by volume) of solid material of an otherwise identical sensing area formulation lacking the surfactant.

[0304] In some embodiments, a method for detecting an analyte includes:

[0305] (i) applying a potential to a first working electrode of an analyte sensor, wherein the analyte sensor comprises:

[0306] (a) a first working electrode;

[0307] (b) a first sensing area disposed upon a surface of the first working electrode, the first sensing area being responsive to a first analyte; and

[0308] (c) a membrane that overcoats at least a part of the first sensing area;

[0309] wherein the first sensing area comprises from about 0.19% to about 16% by weight of surfactant;

[0310] (ii) obtaining a first signal at or above an oxidation-reduction potential of the first active area, the first signal being proportional to a concentration of a first analyte in a fluid contacting the first active area; and

[0311] (iii) correlating the first signal to the concentration of the first analyte in the fluid.

[0312] In some embodiments, a method for detecting glucose includes:

[0313] (i) applying a potential to a first working electrode of an analyte sensor, wherein the analyte sensor comprises:

[0314] (a) a first working electrode;

[0315] (b) a glucose-responsive sensing area disposed upon a surface of the first working electrode, the glucose-responsive sensing area being responsive to a first analyte; and

[0316] (c) a membrane that overcoats at least a part of the glucose-responsive sensing area;

[0317] wherein the first sensing area comprises from about 1% to about 10% by weight of surfactant;

[0318] (ii) obtaining a first signal at or above an oxidation-reduction potential of the first active area, the first signal being proportional to a concentration of glucose in a fluid contacting the first active area; and

[0319] (iii) correlating the first signal to the concentration of glucose in the fluid.

[0320] In some embodiments, a method for detecting glutamate includes:

[0321] (i) applying a potential to a first working electrode of an analyte sensor, wherein the analyte sensor comprises:

[0322] (a) a first working electrode;

[0323] (b) a glutamate-responsive sensing area disposed upon a surface of the first working electrode, the glutamate-responsive sensing area being responsive to a first analyte; and

[0324] (c) a membrane that overcoats at least a part of the glutamateresponsive sensing area;

[0325] wherein the glutamate-responsive sensing area comprises from about 0.19% to about 16% by weight of surfactant;

[0326] (ii) obtaining a first signal at or above an oxidation-reduction potential of the first active area, the first signal being proportional to a concentration of glutamate in a fluid contacting the first active area; and

[0327] (iii) correlating the first signal to the concentration of glutamate in the fluid.

[0328] In some embodiments, the method of the present disclosure can further include detecting a second analyte by providing an analyte sensor that includes a second analyte- responsive active area and / or exposing an analyte sensor that includes a second analyte- responsive active area to a fluid comprising the first analyte and the second analyte. In some embodiments, the analyte sensor for use in a method for detecting a first analyte and a second analyte can further include a second working electrode; and a second analyte- responsive active area disposed upon a surface of the second working electrode and responsive to a second analyte differing from the first analyte, where the second analyte- responsive active area comprises a second polymer, at least one enzyme responsive to the second analyte covalently bonded to the second polymer and, optionally, a redox mediator covalently bonded to the second polymer; wherein a portion, e.g., secondportion, of the mass transport limiting membrane overcoats the second analyte-responsive active area. Alternatively, the second analyte-responsive active site can be covered by a second mass transport limiting membrane that is separate and / or different than a mass transport limiting membrane that overcoats the first analyte-responsive active area. In some embodiments, at least one enzyme responsive to the second analyte can comprise an enzyme system comprising multiple enzymes that are collectively responsive to the second analyte.

[0329] In some embodiments, the method further includes attaching an electronics unit to the skin of the patient, coupling conductive contacts of the electronics unit to contacts of the sensor, collecting data using the electronics unit regarding a level of analyte from signals generated by the sensor, and forwarding the collected data from electronics unit to a receiver unit, e.g., by RF. In some embodiments, the receiver unit is a mobile telephone. In some embodiments, the mobile telephone includes an application related to the monitored analyte. In some embodiments, analyte information is forwarded by RFID protocol, such as BLUETOOTH®, and the like.

[0330] In some embodiments, the analyte sensor can be positioned in a user for automatic analyte sensing, e.g., continuously or periodically. In some embodiments, the level of the analyte can be monitored over a time period ranging from seconds to minutes, hours, days, weeks or months. In some embodiments, the methods disclosed herein can be used to predict future levels of the analyte, based on the obtained information, such as but not limited to current analyte level at time zero, as well as the rate of change of the analyte concentration or amount.V. ANALYTE SENSORS MANUFACTURED BY ELECTROWETTING

[0331] Provided herein are system, apparatus, device, method, and / or process embodiments, and / or combinations and sub-combinations thereof, for manufacturing an analyte sensor by electrowetting. A method as described below can deposit a sensing area on an electrode, apply a voltage to the electrode, and control the interfacial tension between a surface of the electrode and the sensing area. An analyte sensor as described below can include a sensing area and an electrode directly contacting the sensing area. Although the method and the analyte sensor are described below as a stand-alone apparatus, system, and / or method, embodiments of this disclosure can be used with otherapparatuses, systems, and / or methods, for example, analyte sensors 200-205, 300, 400, 401, and 405.

[0332] The term “electrowetting” or “electrowef ’ as used herein indicates controlling the interfacial tension between a surface and a liquid using an applied electric field, for example, modifying the ability of a liquid (e.g., sensing area) to maintain contact with a solid surface (e.g., electrode). In some embodiments, electrowetting can occur when a voltage is applied to and / or through an electrode, thereby forming an electric field between the electrode and a sensing area in contact with the electrode, for example, pulling (attracting) oppositely charged counter ions of the sensing area towards and / or within the electrode via electrostatic interactions.

[0333] In some embodiments, a method of manufacturing an analyte sensor (e.g., similar to analyte sensors 200-205, 300, 400, 401, 405 as described herein) can include depositing a sensing area (e.g., similar to sensing area 218 as described herein) on an electrode (e.g., similar to working electrode 214 as described herein), and applying a voltage to the electrode thereby controlling the interfacial tension between a surface of the electrode and the sensing area. In some embodiments, the electrowetting can increase a contact area between the sensing area and the electrode. In some embodiments, the contact area between the sensing area and the electrode can be from about 0.075 mm2to about 0.25 mm2. In some embodiments, the contact area between the sensing area and the electrode can be from about 0.075 mm2to about 0.25 mm2, from about 0.075 mm2to about 0.15 mm2, from about 0.075 mm2to about 0.10 mm2, from about 0.075 mm2to about 0.08 mm2, from about 0.08 mm2to about 0.25 mm2, from about 0.08 mm2to about 0.15 mm2, from about 0.08 mm2to about 0.10 mm2, from about 0.10 mm2to about 0.25 mm2, from about 0.10 mm2to about 0.15 mm2, or from about 0.15 mm2to about 0.25 mm2. In some embodiments, the electrowetting can increase the contact area to at least 0.075 mm2. In some embodiments, the electrowetting can increase the contact area to at least 0.08 mm2. In some embodiments, a voltage can be applied to the electrode prior to depositing the sensing area on the electrode such that electrowetting occurs as soon as the sensing area contacts the charged electrode.

[0334] In some embodiments, the electrowetting (e.g., via electrostatic interactions between the sensing area and the electrode) can decrease a contact angle between the sensing area and the electrode. The contact angle between the sensing area and theelectrode (e.g., between the liquid-vapor interface and the solid-liquid interface) can be determined by a balance between adhesive and cohesive forces of the sensing area on the electrode. The contact angle is inversely proportional to the wettability of the electrode such that as the contact angle decreases (lowers), the wettability of the electrode increases (e.g., a contact angle of 0° would be perfect wetting). In some embodiments, the electrowetting can decrease the contact angle between the sensing area and the electrode to a range of about 5° to about 85°. In some embodiments, the contact angle (e.g., during electrowetting) can be less than or equal to 90°. In some embodiments, the contact angle (e.g., during electrowetting) can be less than or equal to 45°. In some embodiments, the contact angle (e.g., during electrowetting) can be in a range of about 0° to about 45°. In some embodiments, the sensing area can include one or more surfactants configured to increasethe interfacial tension between the sensing area and the electrode surface.

[0335] In some embodiments, the contact angle between the liquid (e.g., the sensing area) and the substrate surface (e.g., the electrode) during electrowetting is from about 5° to about 90°, from about ° to about 60°, from about 5° to about 30°, from about 5° to about 10°, from about 10° to about 85°, from about 10° to about 60°, from about 10° to about 30°, from about 30° to about 85°, from about 30° to about 60°, or from about 60° to about 85°.

[0336] In some embodiments, the contact angle between the liquid (e.g., the sensing area) and the substrate surface (e.g., the electrode) during electrowetting is reduced by from about 1% to about 90%, from about 1% to about 70%, from about 1% to about 50%, from about 1% to about 40%, from about 1% to about 20%, from about 1% to about 10%, from about 10% to about 90%, from about 1% to about 70%, from about 10% to about 50%, from about 10% to about 40%, from about 10% to about 20%, from about 20% to about 90%, from about 20% to about 70%, from about 20% to about 50%, from about 20% to about 40%, from about 40% to about 90%, from about 40% to about 70%, from about 40% to about 50%, from about 50% to about 90%, from about 50% to about 70%, or from about 70% to about 90% compared to the contact angle between the liquid and the substrate surface before electrowetting.

[0337] In some embodiments, the electrode can be porous and include one or more pores. In some embodiments, the electrowetting can increase a penetration depth of the sensing area into the porous electrode. In some embodiments, the electrode can have a porosity ina range of about 20% to about 95% or from about 20% to about 80%. In some embodiments, for example, the porosity can be in a range of about 50% to about 95%. In some embodiments, a porosity of the electrode can depend upon the thickness of the electrode. In some embodiments, the electrowetting can increase the penetration depth of the sensing area into the porous electrode to at least 1 pm. In some embodiments, the penetration depth can be at least 5 pm. In some embodiments, the penetration depth can be in a range from about 1 pm to about 1,000 pm, from about 1 pm to about 100 pm, from about 1 pm to about 50 pm, from about 1 pm to about 25 pm, from about 1 pm to about 10 pm, from about 1 pm to about 5 pm, from about 5 pm to about 1,000 pm, from about 5 pm to about 100 pm, from about 5 pm to about 50 pm, from about 5 pm to about 25 pm, from about 5 pm to about 10 pm, from about 10 pm to about 1,000 pm, from about 10 pm to about 100 pm, from about 10 pm to about 50 pm, from about 10 pm to about 25 pm, from about 25 pm to about 1,000 pm, from about 25 pm to about 100 pm, from about 25 pm to about 50 pm, from about 50 pm to about 1,000 pm, from about 50 pm to about 100 pm, or from about 100 pm to about 1,000 pm. In some embodiments, the penetration depth can be at least 1 pm. In some embodiments, the penetration depth can be at least 5 pm. In some embodiments, the penetration depth can be in a range of about 15% to about 100% of a thickness of the electrode. In some embodiments, the penetration depth can be at least 50% of a thickness of the electrode. In some embodiments, the electrowetting can include absorbing and / or adsorbing a portion of the sensing area (e.g., 20% to 80%) into one or more pores of the electrode. In some embodiments, the electrowetting can include absorbing and / or adsorbing about 100% of the sensing area into one or more pores of the electrode.

[0338] In some embodiments, the method can further include adjusting a temperature of the sensing area, the electrode, or a combination thereof. In some embodiments, for example, the temperature of the sensing area can be adjusted (e.g., during the electrowetting) to a temperature sufficient to cause liquid in the sensing area to evaporate (e.g., about 100 °C), thereby resulting in a dried sensing area on the electrode. In some embodiments, for example, the temperature of the electrode can be adjusted (e.g., during the electrowetting) to a temperature sufficient to cause liquid in the sensing area to evaporate (e.g., about 100 °C), thereby resulting in a dried sensing area on the electrode. In some embodiments, the temperature of the sensing area, the electrode, or acombination thereof can be adjusted in a range of about 20 °C to about 150 °C. In some embodiments, the temperature of the sensing area, the electrode, or a combination thereof can be adjusted (e.g., during the electrowetting) based on one or more parameters. For example, the one or more parameters can include a thickness of the electrode, a surface area of the electrode, a conductivity of the electrode, a permittivity of the electrode, a porosity of the electrode, a surface roughness of the electrode, a volume of the sensing area, a density of the sensing area, a conductivity of the sensing area, a permittivity of the sensing area, or a combination thereof.

[0339] In some embodiments, the method can further include annealing the electrode prior to depositing the sensing area. In some embodiments, for example, the annealing can include flash lamp annealing (FLA), rapid thermal annealing (RTA), high temperature annealing, a combination thereof, or any other suitable form of annealing. In some embodiments, the electrode can be annealed prior to depositing the sensing area at a temperature in a range of about 100 °C to about 1500 °C.

[0340] In some embodiments, the method can further include etching the electrode prior to depositing the sensing area. In some embodiments, for example, the etching can include plasma surface etching, chemical mechanical polishing (CMP), chemical etching (e.g., HF etch, buffered HF etch, etc.), dry etching (e.g., reactive ion etching (RIE), etc.), thermal etching (e.g., thermal atomic layer etching (ALE)), preferential oxidation, or a combination thereof. In some embodiments, the electrode can be etched prior to depositing the sensing area to remove a native oxide and / or any passivation layer on a surface of the electrode.

[0341] In some embodiments, the method can further include conducting cyclic voltammetry (CV) on the electrode. In some embodiments, for example, CV can be performed (e.g., during and / or after the electro wetting) to determine the contact area between the sensing area and the electrode. In some embodiments, CV can be performed in situ with the electrowetting process. In some embodiments, the applied voltage to the electrode can be ramped over time (e.g., 0 V to 2 V for 10 sec, 2 V to 0 V for 10 sec, etc.) to determine a cyclic voltammogram trace of the sensing area absorbed and / or adsorbed within the electrode. In some embodiments, for example, the determined cyclic voltammogram trace can be used to extract a double-layer capacitance (Cai) between the sensing area-electrode interface, along with a specific capacitance (Cs) of the electrode, toestimate an electrochemically active surface area (ECSA) (e.g., contact area between the sensing area and the electrode), where ECSA = Cdi / Cs. In some embodiments, the ECSA can be calculated based on the determined cyclic voltammogram trace and the Randles- Sevcik equation, relating the peak current measurement to the ECSA. In some embodiments, the method can further include calibrating the analyte sensor based at least in part on the determined contact area (e.g., about 0.08 mm2).

[0342] In some embodiments, the electrode can be part of an electrode assembly. In some embodiments, the electrode assembly can include the electrode (e.g., a working electrode, similar to working electrode 214 as described herein) and a second electrode (e.g., a counter / reference electrode, similar to counter / reference electrode 216 as described herein). In some embodiments, the electrode assembly can include the electrode (e.g., a working electrode, similar to working electrode 214 as described herein), a second electrode (e.g., a counter / reference electrode, similar to counter / reference electrode 216 as described herein), and a third electrode (e.g., a dummy electrode, similar to additional electrode 217 as described herein).

[0343] In some embodiments, the method can further include heating the electrode after the electrowetting process. In some embodiments, for example, the heating can include annealing, thermal baking (e.g., hot plate, oven, etc.), thermal curing, resistive heating, radiative heating, convective heating, optical heating (e.g., IR, UV, etc.), a combination thereof, or any other suitable form of heating. In some embodiments, the electrode is heated after deposition of the sensing area to a temperature sufficient to cause the liquid in the sensing area to evaporate (e.g., about 100 °C), thereby resulting in a dried sensing area on the electrode.

[0344] In some embodiments, the method can further include performing the electrowetting process for two or more cycles. In some embodiments, for example, the electrowetting process can be performed for a number of cycles until all of the sensing area has been deposited and dried (e.g., by evaporation, heating, etc.) on and / or within the electrode. In some embodiments, the electrowetting can be performed for a single cycle. In some embodiments, the electrowetting can be performed for two cycles.

[0345] In some embodiments, applying a voltage to the electrode can include the application of a range of voltages to the electrode. In some embodiments, for example, the applied voltage to the electrode can be in a range of -50 V to about +50 V. In someembodiments, applying the voltage to the electrode can include applying a voltage based on one or more parameters. For example, the one or more parameters can include a thickness of the electrode, a surface area of the electrode, a conductivity of the electrode, a permittivity of the electrode, a temperature of the electrode, a porosity of the electrode, a surface roughness of the electrode, a volume of the sensing area, a density of the sensing area, a conductivity of the sensing area, a permittivity of the sensing area, or a combination thereof.

[0346] In some embodiments, the method can further include manufacturing an analyte sensor. In some embodiments, for example, the analyte sensor can be manufactured with a gantry configured to move relative to an analyte sensor. In some embodiments, manufacturing an analyte sensor can include depositing a sensing area on an electrode with the gantry and applying a voltage to the electrode with the gantry thereby controlling the interfacial tension between a surface of the electrode and the sensing area. In some embodiments, the gantry can deposit the sensing area on the electrode. In some embodiments, a separate machine (e.g., dispensing machine) can deposit the sensing area on the electrode. In some embodiments, the gantry can apply a voltage to the electrode and / or the sensing area. In some embodiments, the gantry can deposit the sensing area on the electrode, the gantry can apply a voltage to the electrode and / or the sensing area, or a combination thereof.

[0347] In some embodiments, an analyte sensor can include a sensing area configured to respond to one or more analytes where the sensing area is disposed upon a surface of an electrode. In some embodiments, the sensing area directly contacts the electrode. In some embodiments, a contact area between the sensing area and the electrode can be at least 0.08 mm2. In some embodiments, the sensing area can include a water-soluble solution of one or more enzymes and / or one or more electroactive mediators. In some embodiments, the sensing area can include sensing area 218 as described herein.

[0348] In some embodiments, the electrode can include a carbon-based electrode. In some embodiments, for example, the electrode can include carbon ink (e.g., hydrophobic carbon ink). In some embodiments, the electrode can include working electrode 214 as described herein.

[0349] In some embodiments, the electrode can include one or more grooves (e.g., trenches, cuts, depressions, etched surface, patterns, etc.) configured to increase thecontact area between the sensing area and the electrode. In some embodiments, the surface of the electrode can include a rough surface. In some embodiments, for example, the surface of the electrode can have a surface roughness of at least 100 nm.VI. EXEMPLARY EMBODIMENTS

[0350] (1) In some non-limiting embodiments, the presently disclosed subject matter provides for analyte sensors comprising:

[0351] a first working electrode;

[0352] a first sensing area disposed upon a surface of the first working electrode, the first sensing area being responsive to a first analyte; and

[0353] a membrane that overcoats at least a part of the first sensing area;

[0354] wherein the first sensing area comprises from about 0.19% to about 16% by weight of a surfactant.

[0355] (2) The analyte sensor of (1), wherein the surfactant is biocompatible.

[0356] (3) The analyte sensor of (1) or (2), wherein the surfactant is selected from the group consisting of an anionic surfactant, a nonionic surfactant, a cationic surfactant, an amphoteric surfactant, a zwitterionic surfactant, and combinations thereof.

[0357] (4) The analyte sensor of any one of ( 1 )-(3), wherein the surfactant is a nonionic surfactant.

[0358] (5) The analyte sensor of (4), wherein the nonionic surfactant is an ethoxylated nonionic surfactant.

[0359] (6) The analyte sensor of (5), wherein the ethoxylated nonionic surfactant is polyethylene glycol sorbitan monolaurate, polyoxyethylene sorbitan monopalmitate, polyethylene glycol sorbitan monostearate, polyethylene glycol sorbitan monooleate, polyethylene glycol tert-octylphenyl ether, or polyethylene glycol 4-tert-octylphenyl ether.

[0360] (7) The analyte sensor of any one of ( 1 )-(6), wherein the sensing area comprises from about 1% to about 10% by weight of a surfactant.

[0361] (8) The analyte sensor of any one of (l)-(7), wherein the sensing area further comprises a polymer and at least one redox mediator.

[0362] (9) The analyte sensor of (8), wherein the redox mediator comprises a transition metal complex.

[0363] (10) The analyte sensor of (9), wherein the transition metal complex is an osmium-containing transition metal complex.

[0364] (11) The analyte sensor of any one of (8)-(l 0), wherein the polymer comprises poly(vinylpyridine), poly(thiophene), poly(aniline), poly(pyrrole), or poly(acetylene).

[0365] (12) The analyte sensor of any one of (8)-(l 1), wherein the sensing area comprises an osmium-containing transition metal complex bonded to a poly(vinylpyridine)-based polymer.

[0366] (13) The analyte sensor of any one of (8)-( 12), wherein the polymer is crosslinked with a crosslinking agent.

[0367] (14) The analyte sensor of (13), wherein the crosslinking agent is a polyepoxide, cyanuric chloride, N-hydroxysuccinamide, an imidoester, epichlorohydrin, or combinations thereof.

[0368] (15) The analyte sensor of (13) or (14), wherein the crosslinking agent is a polyethylene glycol diglycidylether (PEGDGE).

[0369] (16) The analyte sensor of any one of (l)-(l 5), wherein the sensing area further comprises an analyte-responsive enzyme.

[0370] (17) The analyte sensor of any one of (l)-(l 6), wherein the sensing area further comprises a co-solvent.

[0371] (18) The analyte sensor of any one of (l)-(l 7), wherein the analyte sensor comprising the surfactant can have an analyte sensitivity that is greater than the analyte sensitivity of an otherwise identical sensor lacking a surfactant.

[0372] (19) The analyte sensor of (18), wherein the analyte sensor comprising the surfactant has an analyte sensitivity that is at least 75% greater than the analyte sensitivity of an otherwise identical sensor lacking the surfactant.

[0373] (20) The analyte sensor of any one of (l)-(l 9), wherein the analyte sensor comprises a first portion positionable above a surface of a skin and a second portion positionable below a surface of the skin.

[0374] (21) The analyte sensor of any one of (l)-(20), further comprising sensor electronics coupled to the analyte sensor, the sensor electronics comprising a processor and a memory.

[0375] (22) The analyte sensor of any one of (l)-(21), further comprising:

[0376] a second working electrode; and

[0377] a second sensing area disposed upon a surface of the second working electrode, the second sensing area being responsive to a second analyte differing from the first analyte;

[0378] wherein the second sensing area comprises at least one enzyme responsive to the second analyte.

[0379] (23) A method of manufacturing an analyte sensor comprising:

[0380] providing a working electrode; and

[0381] depositing a sensing area formulation upon a surface of the working electrode, the sensing area formulation comprising at least one surfactant in an amount from about 0.01% to about 1% by weight.

[0382] (24) The method of (23), wherein the depositing is performed by a non-impact printing method.

[0383] (25) The method of (24), wherein the non-impact printing method comprises piezoelectric pulse-jet deposition.

[0384] (26) The method of (24), wherein the non-impact printing method comprises thermoelectric pulse-jet deposition.

[0385] (27) The method of (24), wherein a droplet deposited during a single activation event of the non-impact printing method has a volume from about 0.01 pL to 1000 pL.

[0386] (28) The method of (24), wherein a droplet deposited during a single activation event of the non-impact printing method covers an area from about 0.005 mm2to about 0.10 mm2.

[0387] (29) The method of (23), wherein the depositing is performed by dip coating.

[0388] (30) The method of any one of (23)-(29), wherein the sensing area formulation comprises the surfactant in an amount from about 0.25% to about 0.75% by weight.

[0389] (31) The method of any one of (23)-(31), wherein the sensing area formulation further comprises a polymer and at least one redox mediator.

[0390] (32) The method of (31), wherein the redox mediator comprises a transition metal complex.

[0391] (33) The method of (32), wherein the transition metal complex is an osmium- containing transition metal.

[0392] (34) The method of any one of (31 )-(33), wherein the polymer comprises poly(vinylpyridine), poly(thiophene), poly(aniline), poly(pyrrole), or poly(acetylene).

[0393] (35) The method of any one of (31 )-(34), wherein the polymer and at least one redox mediator comprises an osmium complex bonded to a poly(vinylpyridine)-based polymer.

[0394] (36) The method of any one of (31 )-(35), wherein the polymer is crosslinked with a crosslinking agent.

[0395] (37) The method of (36), wherein the crosslinking agent is a polyepoxide, cyanuric chloride, N-hydroxysuccinamide, an imidoester, epichlorohydrin, or any combination thereof.

[0396] (38) The method of (36) or (37), wherein the crosslinking agent is a polyethylene glycol diglycidylether (PEGDGE).

[0397] (39) The method of any one of (23)-(38), wherein the analyte sensor comprising the surfactant has an analyte sensitivity that is greater than the analyte sensitivity of an otherwise identical sensor lacking the surfactant.

[0398] (40) The method of (39) wherein the analyte sensor comprising the surfactant has an analyte sensitivity that is at least 75% greater than the analyte sensitivity of an otherwise identical sensor lacking the surfactant.

[0399] (41) The method of any one of (23)-(40), wherein the sensing area formulation comprising at least one surfactant has a concentration (mass by volume) of solid material that is greater than the concentration (mass by volume) of solid material of an otherwise identical sensing area formulation lacking the surfactant.

[0400] (42) The method of (41), wherein the sensing area formulation comprising at least one surfactant has a concentration of solid material that is at least 25% greater than the concentration of an otherwise identical sensing area formulation lacking the surfactant.

[0401] (43) The method of (41), wherein the sensing area formulation comprising at least one surfactant has a concentration of solid material that is at least 75% greater than the concentration of an otherwise identical sensing area formulation lacking the surfactant.

[0402] (44) A method of manufacturing an analyte sensor, the method comprising: depositing a sensing area upon a surface of an electrode; applying a voltage to the electrode; and at least partially removing a liquid from the sensing area while the voltage is applied to provide a contact area between the sensing area and the electrode of from about 0.075 mm2to about 0.25 mm2.

[0403] (45) The method of (44), wherein the contact area between the sensing area and the electrode is from about 0.075 mm2to about 0.10 mm2.

[0404] (46) The method of (44) or (45), wherein at least partially removing a liquid from the sensing area while the voltage is applied provides a contact angle between the surface of the electrode and the sensing area from about 5° to about 85°.

[0405] (47) The method of (46), wherein the contact angle is from about 5° to about 45°.

[0406] (48) The method of any one of (44)-(47), wherein the electrode is porous and at least partially removing the liquid from the sensing area while the voltage is applied provides a penetration depth of the sensing area into the electrode of from about 1 pm to about 25 pm.

[0407] (49) The method of (48), wherein the penetration depth is from about 1 pm to about 10 pm.

[0408] (50) The method of any one of (44)-(49), wherein at least partially removing the liquid comprises adjusting a temperature of the sensing area, the electrode, or a combination thereof.

[0409] (51) The method of (50), wherein the temperature of the sensing area, the electrode, or a combination thereof is adjusted to a temperature sufficient to cause the liquid in the sensing area to evaporate.

[0410] (52) The method of any one of (44)-(51), wherein the contact angle between the sensing area and the electrode is determined using cyclic voltammetry (CV).

[0411] (53) A method of manufacturing an analyte sensor, the method comprising: depositing a sensing area on a surface of an electrode using a gantry configured to move relative to an analyte sensor; applying a voltage to the electrode; and at least partially removing a liquid from the sensing area while the voltage is applied to provide a contact area between the sensing area and the electrode of from about 0.075 mm2to about 0.25 mm2.

[0412] (54) An analyte sensor comprising: a first working electrode; a first sensing area disposed upon a surface of the first working electrode, the first sensing area being responsive to a first analyte; and a membrane that overcoats at least a part of the first sensing area;wherein a contact area between the first sensing area and the first working electrode is at least 0.075 mm2.

[0413] (55) The analyte sensor of (54), wherein the first sensing area comprises a water- soluble solution of one or more enzymes and one or more electroactive mediators.

[0414] (56) The analyte sensor of (54) or (55), wherein the first working electrode comprises a carbon-based electrode.

[0415] (57) The analyte sensor of any one of (54)-(56), wherein the first working electrode comprises carbon ink.

[0416] (58) The analyte sensor of any one of (54)-(57), wherein the first working electrode has a porosity in a range from about 20% to about 80%.

[0417] (59) The analyte sensor of any one of (54)-(58), wherein a penetration depth of the first sensing area into the first working electrode is at least 1 pm.

[0418] (60) In some non-limiting embodiments, the presently disclosed subject matter provides for analyte sensors comprising:

[0419] a proximal portion configured to be positioned above a user’s skin; and

[0420] a distal portion configured to be transcutaneously positioned through the user’s skin and in contact with a user’s bodily fluid to detect or monitor an analyte in the bodily fluid in vivo, the distal portion comprising:

[0421] a first working electrode;

[0422] a first sensing area disposed upon a surface of the first working electrode, the first sensing area being responsive to a first analyte; and

[0423] a membrane that overcoats at least a part of the first sensing area;

[0424] wherein the first sensing area comprises from about 0.19% to about 16% by weight of a surfactant.EXAMPLES

[0425] The presently disclosed subject matter will be better understood by reference to the following Examples, which are provided as exemplary of the presently disclosed subject matter, and not by way of limitation.Example 1: Stock Solution for Comparative Sensing area Formulation

[0426] A stock solution of the comparative formulation was prepared by mixing 60 mg / mL of an osmium-containing poly(4-vinylpyridine)-based polymer, 60 mg / mL of glucose oxidase, and 30 mg / mL of polyethylene glycol diglycidyl ether (PEGDGE) 400. The stock solution of the comparative formulation was added to a N-(2- hydroxyethyl)piperazine-N’-(2-ethanesulfonic acid) (HEPES) buffer resulting in a total amount of solid materials in the comparative sensing area formulation of 52.5 mg / mL.Example 2: Stock Solutions of Sensing area Comprising Surfactants

[0427] 0.1% polyethylene glycol tert-octylphenyl ether stock solution: A stock solution of 0.1% polyethylene glycol tert-octylphenyl ether in HEPES buffer was prepared by mixing 1 g / mL polyethylene glycol tert-octylphenyl ether in 10 mM HEPES buffer. This solution was mixed with the stock solution of Example 1 (60 mg / mL of an osmium- containing poly(4-vinylpyridine)-based polymer, 60 mg / mL of glucose oxidase, and 30 mg / mL of PEGDGE 400) to provide a solution comprising solid materials.

[0428] 0.5% polyethylene glycol tert-octylphenyl ether stock solution: A stock solution of 0.5% polyethylene glycol tert-octylphenyl ether in HEPES buffer was prepared by mixing 5 g / mL polyethylene glycol tert-octylphenyl ether in 10 mM HEPES buffer. This solution was mixed with the stock solution of Example 1 (60 mg / mL of an osmium- containing poly(4-vinylpyridine)-based polymer, 60 mg / mL of glucose oxidase, and 30 mg / mL of PEGDGE 400) to provide a solution comprising solid materials.

[0429] 1.0% polyethylene glycol tert-octylphenyl ether stock solution: A stock solution of 0.5% polyethylene glycol tert-octylphenyl ether in HEPES buffer was prepared by mixing 10 g / mL polyethylene glycol tert-octylphenyl ether in 10 mM HEPES buffer. This solution was mixed with the stock solution of Example 1 (60 mg / mL of an osmium- containing poly(4-vinylpyridine)-based polymer, 60 mg / mL of glucose oxidase, and 30 mg / mL of PEGDGE 400) to provide a solution comprising solid materials.Example 3: Preparation of Glucose Sensors with Comparative Sensing area Formulation

[0430] The sensing area of a glucose sensor was prepared by dispensing four passes (each pass comprising six spots) of the comparative formulation of Example 1 onto a working electrode surface.

[0431] A derivatized polyvinylpyridine-co-styrene copolymer membrane solution was prepared by mixing 4 mL of a derivatized polyvinylpyridine-co-styrene copolymer at 100 mg / mL in a 80:20 ratio (volume by volume) of ethanol: 10 mM HEPES (N-2- hydroxyethylpiperazine-N’-2-ethanesulfonic acid) buffer at a pH of 8.0, 1 mL of glycerol triglycidyl ether (Gly3) at 12.5 mg / mL in a 80:20 ratio (volume by volume) of ethanol:HEPES buffer (at a pH of 8.0), and 13.2 pL of PDMS at 100 mg / mL in 100% ethanol.

[0432] All dipping steps were conducted at 55% relative humidity and at a temperature of 21 °C. After drying, the glucose sensor was dipped five times with a 2 mm / sec entry speed and 15 mm / sec exit speed into the derivatized polyvinylpyridine-co-styrene copolymer membrane solution. The glucose sensor was allowed to dry for 10 minutes between dips with a 20 minute dry time after the last dip. After dipping was complete, the glucose sensor was stored at 60% relative humidity and at a temperature of 25 °C for 24 hours to cure. After 24 hours, the glucose sensor was transferred to a 56 °C oven for 72 hours. The resulting membrane had a thickness of about 30 pm.Example 4: Preparation of Glucose Sensors with Stock Solutions Comprising Surfactants

[0433] The sensing area of a glucose sensor was prepared by dispensing four passes (each pass comprising six spots) of the sensing area formulations of Example 2 onto the surface of three separate working electrodes.

[0434] A derivatized polyvinylpyridine-co-styrene copolymer membrane solution was prepared by mixing 4 mL of a derivatized polyvinylpyridine-co-styrene copolymer at 100 mg / mL in a 80:20 ratio (volume by volume) of ethanol: 10 mM HEPES (N-2- hydroxyethylpiperazine-N’-2-ethanesulfonic acid) buffer at a pH of 8.0, 1 mL of glycerol triglycidyl ether (Gly3) at 12.5 mg / mL in a 80:20 ratio (volume by volume) of ethanokHEPES buffer (at a pH of 8.0), and 13.2 pL of PDMS at 100 mg / mL in 100% ethanol.

[0435] All dipping steps were conducted at 55% relative humidity and at a temperature of 21 °C. After drying, the glucose sensors were dipped three times with a 10 mm / sec (entry and exit) speed into a derivatized polyvinylpyridine-co-styrene copolymer membrane solution. The glucose sensor was allowed to dry for 10 minutes after the first dip, 10 minutes after the second dip, and 20 minutes after the third dip. After dipping wascomplete, the glucose sensors were stored at 60% relative humidity and at a temperature of 25 °C for 24 hours to cure. After 24 hours, the glucose sensors were transferred to a desiccated vial and aged at 56 °C for 24 hours. The resulting membranes had a thickness of 35 pm.Example 5: Sensitivity of the Glucose Sensors

[0436] The comparative glucose sensor of Example 3 and the glucose sensors prepared with stock solutions comprising surfactants of Example 4 were each cut to form a single sensor.

[0437] The sensitivity of the glucose sensor prepared with the comparative sensing area solution, the glucose sensor prepared with the 0.1% polyethylene glycol tert-octylphenyl ether sensing area solution, the glucose sensor prepared with the 0.5% polyethylene glycol tert-octylphenyl ether sensing area solution, and the glucose sensor prepared with the 1% polyethylene glycol tert-octylphenyl ether sensing area solution were measured using increasing concentrations of glucose. The results, shown in FIG. 6, demonstrate that the glucose sensor prepared with the polyethylene glycol tert-octylphenyl ether sensing area solution were more sensitive to an increase in glucose concentration than the comparative glutamate sensor. The measured sensitivities are shown in Tables 1 and 2.

[0438] TABLE 1 : Sensitivity of Glucose Sensor

[0439] TABLE 2: Coefficients of Variation (CV) of Glucose Sensors (Percentage)

[0440] The response time can be measured by calculating the time between the 10% to 90% signal jump / change during the sensor read in the 5 mM glucose to 7 mM glucose step. The response time of the glucose sensor prepared with the comparative sensing area solution, the glucose sensor prepared with the 0.1% polyethylene glycol tert-octylphenyl ether sensing area solution, the glucose sensor prepared with the 0.5% polyethylene glycol tert-octylphenyl ether sensing area solution, and the glucose sensor prepared with the 1% polyethylene glycol tert-octylphenyl ether sensing area solution were measured using increasing concentrations of glucose. The results, shown in FIG. 7, demonstrate that the glucose sensors prepared with the polyethylene glycol tert-octylphenyl ether sensing area solutions provided a similar response time to the glucose sensor prepared with the comparative sensing area solution.Example 6: Stock Solution of Sensing area Comprising 0.1% Surfactant with an Additional 25% of Solid Materials

[0441] A stock solution of 0.1% polyethylene glycol tert-octylphenyl ether was prepared by mixing 75 mg / mL of an osmium-containing poly(4-vinylpyridine)-based polymer, 75 mg / mL of glucose oxidase, and 37.5 mg / mL of PEGDGE 400 to provide a solution comprising solid materials. To the solid materials solution was added 0.1% polyethylene glycol tert-octylphenyl ether and HEPES buffer resulting in a total amount of solid materials in the sensing area formulation of 65.6 mg / mL. This was an increase in the solid materials of 25%. The increase in solid materials allowed a reduction in the number of dispensing passes to three passes (each pass providing six spots). This resulted in a relative total solid amount of 93.8% compared to the solid material dispensed in four passes (each pass providing six spots) using the comparative stock solution of Example 1.Example 7: Stock Solution of Sensing area Comprising 0.5% Surfactant with an Additional 50% of Solid Materials

[0442] A stock solution of 0.5% polyethylene glycol tert-octylphenyl ether was prepared by mixing 90 mg / mL of an osmium-containing poly(4-vinylpyridine)-based polymer, 90 mg / mL of glucose oxidase, and 45 mg / mL of PEGDGE 400. To the stock solution was added 0.5% polyethylene glycol tert-octylphenyl ether and HEPES buffer resulting in a total amount of solid materials in the sensing area formulation of 78.8 mg / mL. This was an increase in the solid materials of 50%. The increase in solid materials allowed a reduction in the number of dispensing passes to three passes (each pass providing sixspots). This resulted in a relative total solid amount of 112.5% compared to the solid material dispensed in four passes (each pass providing six spots) using the comparative stock solution of Example 1.Example 8: Stock Solution of Sensing area Comprising 1 % Surfactant with an Additional 75% of Solid Materials

[0443] A stock solution of 1% polyethylene glycol tert-octylphenyl ether was prepared by mixing 105 mg / mL of an osmium-containing poly(4-vinylpyridine)-based polymer, 105 mg / mL of glucose oxidase, and 52.5 mg / mL of PEGDGE 400. To the stock solution was added 1% polyethylene glycol tert-octylphenyl etherand HEPES buffer resulting in a total amount of solid materials in the sensing area formulation of 91.9 mg / mL. This was an increase in the solid materials of 75%. The increase in solid materials allowed a reduction in the number of dispensing passes to three passes (each pass providing six spots). This resulted in a relative total solid amount of 131.3% compared to the solid material dispensed in four passes (each pass providing six spots) using the comparative stock solution of Example 1.

[0444] Although 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 herein without departing from the spirit and scope of the disclosed subject matter. Moreover, the scope of the present application is not intended to be limited to the particular embodiments of the process, machine, manufacture, and composition of matter, methods and processes described in the specification. As one of ordinary skill in the art will readily appreciate from the disclosed subject matter of the presently disclosed subject matter, processes, machines, manufacture, compositions of matter, methods, or steps, presently existing or later to be developed that perform substantially the same function or achieve substantially the same result as the corresponding embodiments described herein can be utilized according to the presently disclosed subject matter. Accordingly, the appended claims are intended to include within their scope such processes, machines, manufacture, compositions of matter, methods, or steps.

[0445] Various patents, patent applications, publications, product descriptions, protocols, and sequence accession numbers are cited throughout this application, the inventions of which are incorporated herein by reference in their entireties for all purposes.

Claims

WHAT IS CLAIMED IS:

1. An analyte sensor comprising: a first working electrode; a first sensing area disposed upon a surface of the first working electrode, the first sensing area being responsive to a first analyte; and a membrane that overcoats at least a part of the first sensing area; wherein the first sensing area comprises from about 0.19% to about 16% by weight of a surfactant.

2. The analyte sensor of claim 1, wherein the surfactant is biocompatible.

3. The analyte sensor of claim 1 or 2, wherein the surfactant is selected from the group consisting of an anionic surfactant, a nonionic surfactant, a cationic surfactant, an amphoteric surfactant, a zwitterionic surfactant, and combinations thereof.

4. The analyte sensor of any one of claims 1-3, wherein the surfactant is a nonionic surfactant.

5. The analyte sensor of claim 4, wherein the nonionic surfactant is an ethoxylated nonionic surfactant.

6. The analyte sensor of claim 5, wherein the ethoxylated nonionic surfactant is polyethylene glycol sorbitan monolaurate, polyoxyethylene sorbitan monopalmitate, polyethylene glycol sorbitan monostearate, polyethylene glycol sorbitan monooleate, polyethylene glycol tert-octylphenyl ether, or polyethylene glycol 4-tert-octylphenyl ether.

7. The analyte sensor of any one of claim 1-6, wherein the first sensing area comprises from about 1% to about 10% by weight of a surfactant.

8. The analyte sensor of any one of claims 1-7, wherein the sensing area further comprises a polymer and at least one redox mediator.

9. The analyte sensor of claim 8, wherein the redox mediator comprises a transition metal complex.

10. The analyte sensor of claim 9, wherein the transition metal complex is an osmium- containing transition metal complex.

11. The analyte sensor of any one of claims 8-10, wherein the polymer comprises poly(vinylpyridine), poly(thiophene), poly(aniline), poly(pyrrole), or poly(acetylene).

12. The analyte sensor of any one of claims 8-11, wherein the sensing area comprises an osmium-containing transition metal complex bonded to a poly(vinylpyridine)-based polymer.

13. The analyte sensor of any one of claims 8-12, wherein the polymer is crosslinked with a crosslinking agent.

14. The analyte sensor of claim 13, wherein the crosslinking agent is a polyepoxide, cyanuric chloride, N-hydroxysuccinamide, an imidoester, epichlorohydrin, or combinations thereof.

15. The analyte sensor of claim 13 or 14, wherein the crosslinking agent is a polyethylene glycol diglycidylether (PEGDGE).

16. The analyte sensor of any one of claims 1-15, wherein the sensing area further comprises an analyte-responsive enzyme.

17. The analyte sensor of any one of claims 1-16, wherein the analyte sensor comprising the surfactant has an analyte sensitivity that is greater than the analyte sensitivity of an otherwise identical sensor lacking a surfactant.

18. The analyte sensor of claim 17, wherein the analyte sensor comprising the surfactant has an analyte sensitivity that is at least 75% greater than the analyte sensitivity of an otherwise identical sensor lacking the surfactant.

19. The analyte sensor of any one of claims 1-18, wherein the analyte sensor comprises a proximal portion configured to be positioned above a user’s skin and a distal portion configured to be transcutaneously positioned through the user’s skin and in contact with the user’s bodily fluid to detect or monitor an analyte in the bodily fluid in vivo.

20. The analyte sensor of any one of claims 1-19, further comprising sensor electronics coupled to the analyte sensor, the sensor electronics comprising a processor and a memory.

21. The analyte sensor of any one of claims 1-20, further comprising: a second working electrode; and a second sensing area disposed upon a surface of the second working electrode, the second sensing area being responsive to a second analyte differing from the first analyte; wherein the second sensing area comprises at least one enzyme responsive to the second analyte.

22. A method of manufacturing an analyte sensor comprising: depositing a sensing area formulation upon a surface of a working electrode, the sensing area formulation comprising at least one surfactant in an amount from about 0.01% to about 1% by weight.

23. The method of claim 22, wherein the depositing is performed by a non-impact printing method.

24. The method of claim 23, wherein the non-impact printing method comprises piezoelectric pulse-jet deposition.

25. The method of claim 23, wherein the non-impact printing method comprises thermoelectric pulse-jet deposition.

26. The method of claim 23, wherein a droplet deposited during a single activation event of the non-impact printing method has a volume from about 0.01 pL to 1000 pL.

27. The method of claim 23, wherein a droplet deposited during a single activation event of the non-impact printing method covers an area from about 0.005 mm2to about 0.10 mm2.

28. The method of claim 22, wherein the depositing is performed by dip coating.

29. The method of any one of claims 22-28, wherein the sensing area formulation comprises the surfactant in an amount from about 0.25% to about 0.75% by weight.

30. The method of any one of claims 22-29, wherein the sensing area formulation further comprises a polymer and at least one redox mediator.

31. The method of claim 30, wherein the redox mediator comprises a transition metal complex.

32. The method of claim 31, wherein the transition metal complex is an osmium-containing transition metal complex.

33. The method of any one of claims 30-32, wherein the polymer comprises poly(vinylpyridine), poly(thiophene), poly(aniline), poly(pyrrole), or poly(acetylene).

34. The method of any one of claims 30-33, wherein the sensing area comprises an osmium- containing transition metal complex bonded to a poly(vinylpyridine)-based polymer.

35. The method of any one of claims 30-34, wherein the polymer is crosslinked with a crosslinking agent.

36. The method of claim 35, wherein the crosslinking agent is a polyepoxide, cyanuric chloride, N-hydroxysuccinamide, an imidoester, epichlorohydrin, or any combination thereof.

37. The method of claim 35 or 36, wherein the crosslinking agent is a polyethylene glycol diglycidylether (PEGDGE).

38. The method of any one of claims 22-37, wherein the analyte sensor comprising the surfactant has an analyte sensitivity that is greater than the analyte sensitivity of an otherwise identical sensor lacking the surfactant.

39. The method of claim 38 wherein the analyte sensor comprising the surfactant has an analyte sensitivity that is at least 75% greater than the analyte sensitivity of an otherwise identical sensor lacking the surfactant.

40. The method of any one of claims 22-39, wherein the sensing area formulation comprising at least one surfactant has a concentration (mass by volume) of solid material that is greater than the concentration (mass by volume) of solid material of an otherwise identical sensing area formulation lacking the surfactant.

41. The method of claim 40, wherein the sensing area formulation comprising at least one surfactant has a concentration of solid material that is at least 25% greater than the concentration of an otherwise identical sensing area formulation lacking the surfactant.

42. The method of claim 40, wherein the sensing area formulation comprising at least one surfactant has a concentration of solid material that is at least 75% greater than the concentration of an otherwise identical sensing area formulation lacking the surfactant.

43. A method of manufacturing an analyte sensor, the method comprising: depositing a sensing area upon a surface of an electrode; applying a voltage to the electrode; and at least partially removing a liquid from the sensing area while the voltage is applied to provide a contact area between the sensing area and the electrode of from about 0.075 mm2to about 0.25 mm2.

44. The method of claim 43, wherein the contact area between the sensing area and the electrode is from about 0.075 mm2to about 0.10 mm2.

45. The method of claim 43 or claim 44, wherein at least partially removing a liquid from the sensing area while the voltage is applied provides a contact angle between the surface of the electrode and the sensing area from about 5° to about 85°.

46. The method of claim 45, wherein the contact angle is from about 5° to about 45°.

47. The method of any one of claims 43-46, wherein the electrode is porous and at least partially removing a liquid from the sensing area while the voltage is applied provides a penetration depth of the sensing area into the electrode of from about 1 pm to about 25 pm.

48. The method of claim 47, wherein the penetration depth is from about 1 pm to about 10 pm.

49. The method of any one of claims 43-48, wherein at least partially removing a liquid is provided by adjusting a temperature of the sensing area, the electrode, or a combination thereof.

50. The method of claim 49, wherein the temperature of the sensing area, the electrode, or a combination thereof is adjusted to a temperature sufficient to cause the liquid in the sensing area to evaporate.

51. The method of any one of claims 43-50, wherein the contact angle between the sensing area and the electrode is determined using cyclic voltammetry (CV).

52. A method of manufacturing an analyte sensor, the method comprising: depositing a sensing area on a surface of an electrode using a gantry configured to move relative to an analyte sensor; applying a voltage to the electrode; and at least partially removing a liquid from the sensing area while the voltage is applied to provide a contact area between the sensing area and the electrode of from about 0.075 mm2to about 0.25 mm2.

53. An analyte sensor comprising: a first working electrode; a first sensing area disposed upon a surface of the first working electrode, the first sensing area being responsive to a first analyte; and a membrane that overcoats at least a part of the first sensing area; wherein a contact area between the first sensing area and the first working electrode is at least 0.075 mm2.

54. The analyte sensor of claim 53, wherein the first sensing area comprises a water-soluble solution of one or more enzymes and one or more electroactive mediators.

55. The analyte sensor of claim 53 or claim 54, wherein the first working electrode comprises a carbon-based electrode.

56. The analyte sensor of any one of claims 53-55, wherein the first working electrode comprises carbon ink.

57. The analyte sensor of any one of claims 53-56, wherein the first working electrode has a porosity in a range from about 20% to about 80%.

58. The analyte sensor of any one of claims 53-57, wherein a penetration depth of the first sensing area into the first working electrode is at least 1 pm.

59. The analyte sensor of any one of claims 1-21, wherein the sensing area further comprises a co-solvent.

60. The analyte sensor of claim 59, wherein the co-solvent is selected from the group consisting of an alcohol, a glycol, and a ketone.

61. The analyte sensor of claim 59, wherein the co-solvent is ethanol.

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