Electrochemical oxygen sensor

Abstract

The present disclosure relates to a continuous oxygen sensor comprising a working electrode comprising carbon, and a redox mediator comprising a polymer, an electron transfer agent, and no enzyme; wherein the redox mediator is disposed on at least a portion of the working electrode. The present disclosure is further related to a dual analyte sensor which can sense oxygen and a second analyte of interest. The present disclosure is further related to a method of sensing oxygen, which includes exposing the continuous oxygen sensor, or dual sensor, to a biofluid comprising oxygen; applying a potential to the working electrode, wherein the potential is sufficient to induce an oxidation reduction cascade that reduces oxygen to water, hydrogen peroxide, or a combination thereof; obtaining a signal that is proportional to a concentration of oxygen in the biofluid; and correlating the signal to the concentration of oxygen in the biofluid.

Description

ELECTROCHEMICAL OXYGEN SENSOR BACKGROUND

[0001] Oxygen saturation is a crucial measure of how well the lungs are working. Lungs transmit oxygen into tiny blood vessels called capillaries when air is breathed in. In turn, the capillaries send oxygen-rich blood to the heart, which then pumps it through arteries to the rest of the body. Organs need a constant supply of oxygen to work properly. When the capacity of the lungs to transport oxygen into the blood is impaired, blood oxygen saturation declines, potentially putting the organs in danger. A non-invasive pulse oximeter can quickly detect this drop in oxygen saturation, alerting people of the need for medical intervention. However, a pulse oximeter does not provide a precise measurement with respect to the concentration of oxygen. In addition, pulse oximeters cannot detect other analytes. While arterial blood samples can be drawn to improve the accuracy of determining oxygen concentration, such measurements are painful and riskier for already unwell patients.

[0002] Thus, there is a need for a biosensor that can provide accurate oxygen monitoring (e.g. continuous oxygen monitoring) in vivo with minimal invasiveness.BRIEF SUMMARY

[0003] The present disclosure relates to an oxygen sensor comprising a working electrode, and a redox mediator. As explained herein, in some aspects the redox mediator comprises a polymer and an electron transfer agent. In some aspects, the redox mediator does not comprise an enzyme, although the presence of an enzyme is not excluded. In some aspects, the redox mediator is disposed on at least a portion of the working electrode.

[0004] The present disclosure further relates to a continuous oxygen sensor comprising a first portion configured to be positioned above a user’ s skin and a second portion configured to be transcutaneously positioned beneath the skin and in contact with an interstitial fluid to detect oxygen in vivo, the second portion comprising: a working electrode comprising carbon, and a redox mediator comprising a polymer, an electron transfer agent, and noenzyme; wherein the redox mediator is disposed on at least a portion of the working electrode.

[0005] In some aspects, the polymer comprises poly(vinylpyridine), poly(vinylimidazole), poly(thiophene), poly(aniline), poly(pyrrole), poly(acetylene), polyethylene, polyacrylate, polymethacrylate, polystyrene, polyurethane, polyurea, or any combination thereof. In some aspects, the polymer comprises a polymer or copolymer repeat unit comprising at least one pendant pyridinyl group, imidazolyl group, or both a pyridinyl and imidazolyl group.

[0006] In some aspects, the electron transfer agent comprises a transition metal complex.In some aspects, the transition metal complex comprises osmium, ruthenium, iron, cobalt, vanadium, or any combination thereof and at least one ligand coupled to the polymer backbone. In some aspects, the transition metal complex is an osmium transition metal complex comprising one or more ligands, wherein at least one ligand comprises a nitrogencontaining heterocycle coupled to the polymer backbone. In some aspects, the redox mediator comprises an osmium complex bonded to a poly(4-vinylpyridine)-based polymer.

[0007] In some aspects, the redox mediator further comprises a cross linking agent. In some aspects, the cross linking agent is a polyepoxide, cyanuric chloride, N- hydroxysuccinimide, an imidoester, epichlorohydrin, or any combination thereof. In some aspects, the cross linking agent is a polyethylene glycol diglycidyl ether (PEGDGE).

[0008] In some aspects, the continuous oxygen sensor further comprises an albumin and optionally a pH buffer.

[0009] In some aspects, the oxygen sensor or continuous oxygen sensor further comprises a membrane overcoating the redox mediator and at least a portion of the working electrode. In some aspects, the membrane comprises poly(4-vinylpyridine).

[0010] In some aspects, the oxygen sensor or continuous oxygen sensor further comprises a reference electrode, a counter electrode, or both a reference electrode and a counter electrode. In some aspects, the oxygen sensor or continuous oxygen sensor further comprises at least one insulating layer. In some aspects, the oxygen sensor or continuous oxygen sensor further comprises a substrate, wherein the working electrode is disposed on the substrate. In some aspects, the substrate is an implantable portion (e.g., insertion tip) configured for implantation into a tissue.

[0011] In some aspects, the redox mediator is continuously disposed on the working electrode. In some aspects, the redox mediator is discontinuously disposed on the working electrode. A redox mediator is considered to be “continuously disposed” on a working electrode when the redox mediator is applied in a fashion that is uninterrupted across the surface of the working electrode, i.e. a single spot or line, etc. A redox mediator is considered to be “discontinuously disposed” on a working electrode when the redox mediator is applied as at least two discrete shapes on the working electrode, such as two spots, two lines, a spot and a line, or a plurality (e.g., an array) of spots, lines, or combination thereof. The number of discontinuous applications of the redox mediator as a series of spots and / or lines is not considered to be particularly limited, but can range from 2 to about 10 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10, including about 3 to about 8, or from about 4 to about 6).

[0012] The present disclosure further relates to dual analyte sensor system comprising the oxygen sensor or continuous oxygen sensor described herein, and a second sensor that senses a second analyte. In some aspects, the second analyte is glucose, lactate, or a ketone. In some aspects, the second sensor comprises a working electrode for the second analyte, and a sensing region disposed on at least a portion of the working electrode for the second analyte, wherein the sensing region comprises an enzyme responsive to the second analyte.

[0013] The present disclosure also relates to a method of sensing oxygen comprising:a) exposing the continuous oxygen sensor or the dual analyte sensor, as described herein, to a biofluid comprising oxygen;b) applying a potential to the working electrode, wherein the potential is sufficient to induce an oxidation reduction cascade that reduces oxygen to water, hydrogen peroxide, or a combination thereof;c) obtaining a signal that is proportional to a concentration of oxygen in the biofluid; d) correlating the signal to the concentration of oxygen in the biofluid; and e) repeating steps a) through d) to provide the concentration of oxygen continuously for a period of time.

[0014] In some aspects, the applied potential is from about -500 mV to about +500 mV vs the reference electrode.

[0015] In some aspects, the period of time is from about 7 days to about 30 days.

[0016] Additional aspects and advantages of the disclosure will be set forth, in part, in the description that follows, and will flow from the description, or can be learned by practice of the disclosure.

[0017] It is to be understood that both the foregoing summary and the following detailed description are exemplary and explanatory only, and do not restrict the scope of the claims.BRIEF DESCRIPTION OF THE DRAWINGS / FIGURES

[0018] FIG. 1 shows a diagram of an illustrative sensing system that can incorporate an analyte sensor of the present disclosure.

[0019] FIGs. 2A-2C show cross-sectional diagrams of analyte sensors including a single sensing layer.

[0020] FIGs. 3A-3C show cross-sectional diagrams of analyte sensors including two sensing layers.

[0021] FIG. 4 shows a cross-sectional diagram of an analyte sensor including two sensing layers.

[0022] FIGs. 5A-5C show perspective views of analyte sensors including two sensing layers upon separate working electrodes.

[0023] FIG. 6 shows a redox mediator acting as a catalyst to reduce oxygen with an electron provided by the working electrode.

[0024] FIG. 7 shows products of oxygen reduction.

[0025] FIG. 8 shows a cyclic voltammogram of an osmium-containing poly(4- vinylpyridine)-based polymer on a carbon electrode at a scan rate of 10 mV / sec; the potential is shown vs an Ag / AgCl reference electrode.

[0026] FIG. 9 shows an average sensor current (nA) versus time (hours) plot of exemplary analyte sensors of the present disclosure comprising dip coated membranes at varying oxygen concentrations; data obtained at a potential of -200 mV vs Ag / AgCl.

[0027] FIG. 10 shows the average calibration curve of exemplary analyte sensors of the present disclosure comprising dip coated membranes at varying oxygen concentrations.

[0028] FIG. 11 shows the stability of four exemplary analyte sensors comprising dip coated membranes over a 7 day period.

[0029] FIG. 12 shows the stability of an enzyme-containing oxygen sensor and an enzyme free oxygen sensor over a period of 12 days.DETAILED DESCRIPTION

[0030] The headings provided herein are not limitations of the various aspects of the disclosure, which can be defined by reference to the specification as a whole. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.Definitions

[0031] 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 aspects, 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.

[0032] 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.

[0033] As used herein, the term “about” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. Typically, “about” means ±10% of a given value.

[0034] 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.

[0035] 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 aspect disclosed herein “comprises” certain elements, it should be understood that present disclosure also specifically contemplates and discloses aspects that “consist essentially of’ those elements and that “consist of’ those elements.

[0036] 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 ingredients which materially affect the basic and novel characteristics of an aspect are included.

[0037] As used herein, the terms “consists of,” “consisting of,” and the like are to be construed as closed terms, such that an aspect “consisting of’ a particular set of elements excludes any element, step, or ingredient not specified in the aspect.

[0038] As used herein, an “analyte” is an enzyme substrate that is subject to be measured or detected. The analyte can be from, for example, a biofluid and can be tested in vivo, ex vivo, or in vitro. In most aspects herein, the analyte is oxygen.

[0039] As used herein, an “analyte sensor” is a sensor as described herein for an analyte.When the analyte is oxygen, the analyte sensor is an oxygen sensor.

[0040] 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. In certain aspects, the biofluid is dermal fluid or interstitial fluid.

[0041] As used herein, the term “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 aspects 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.

[0042] As used herein, the term “crosslinking agent” 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 thantwo reactive groups can be capable of both intermolecular and intramolecular crosslinkings at the same time.

[0043] As used herein, the term “electrolysis” refers to the electrooxidation or electroreduction of a compound either directly at an electrode or via one or more electron transfer agents.

[0044] As used herein, the term “electron transfer agent” refers to 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.

[0045] As used herein, components are “immobilized” or “attached” to a polymer and / or a sensor, for example, when the components are entrapped on, entrapped within, covalently bound, ionically bound, electrostatically bound, or coordinatively bound to constituents of a polymer, a sol-gel matric, membrane, and / or sensor, which reduces or precludes mobility.

[0046] As used herein, the term “non-leachable” compound, or a compound that is “non- leachably disposed” is meant to define a compound that is affixed on the sensor such that it does not substantially diffuse away from the sensing layer of the working electrode for the period in which the sensor is used (e.g., the period in which the sensor is implanted in a patient or measuring a sample).

[0047] As used herein, 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.”

[0048] As used herein, the term “precursor polymer” refers to the starting polymer before the various modifier groups are attached to form a modified polymer.

[0049] As used herein, the term “reactive group” refers to a functional group of a molecule (e.g., a polymer, a crosslinking agent, an enzyme) that can react 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 byelectron-withdrawing groups such as sulfo, nitro, cyano, or halo groups; or carboxylic acids activated by carbodiimides.

[0050] As used herein, the term “redox mediator” refers to 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.”

[0051] As used herein, the term “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. An example of a reference electrode is an Ag / AgCl reference electrode.

[0052] As used herein, the term “sensing layer” refers to a component of the sensor including constituents that facilitate the electrolysis of the analyte. The sensing layer can include constituents such as a redox mediator (e.g., an electron transfer agent or a redox polymer), a catalyst (e.g., an analyte-specific 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 aspects of the present disclosure, a sensor includes a sensing layer that is non-leachably disposed in proximity to or on the working electrode.

[0053] As used herein, the term “sensor” refers to a device configured to detect the presence, absence and / or measure the level (e.g. concentration) 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 correlated to (e.g., is proportional to) an amount, concentration, or level of an analyte in the sample.

[0054] As used herein, the term “continuous” as it relates to a continuous analyte sensor (e.g. “a continuous oxygen sensor”) refers to a sensor that is configured to take one or more measurements of the analyte (e.g. oxygen) over a period of time. A continuous sensor may take sequential measurements according to its sampling frequency. For example, one or more measurements may be taken about every 1 ms, about every 10 ms, about every 100 ms, about every 1 s, about every 10 seconds, about every 30 seconds, about every minute, about every 5 minutes, about every 10 minutes, about every 30 minutes, or about every hour. The measurements may be taken continuously e.g. over a contiguous time period of at least 1 hour, 6 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2weeks, 3 weeks, 1 month or longer. A continuous oxygen sensor is typically continuously in contact with a sample, such as a biofluid. For example, a continuous oxygen sensor may comprise an implantable portion or member as defined herein which in use is in continuous contact with a biofluid such as dermal fluid or interstitial fluid, such that measurements can be taken continuously or periodically according to the sampling frequency of the sensor over the continuous time period.

[0055] As used herein, the term “substituted” functional group (e.g., substituted alkyl, alkenyl, alkoxy, aryl) includes at least one substituent (e.g., 1, 2, 3, 4, or 5) that can be, for example, halo, alkoxy, mercapto, aryl, alkoxycarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, hydroxy, amino, alkylamino, dialkylamino, trialkylammonium, alkanoylamino, aryl carb oxami do, hydrazino, alkylthio, alkenyl, and reactive groups.

[0056] As used herein, the term “working electrode” refers to an electrode at which the analyte or background interferent is electrooxidized or electroreduced with or without the agency of an electron transfer agent.

[0057] As used herein, the term “Ce-30 aryl” refers to an aromatic compound comprising a mono-, bi-, or tricyclic carbocyclic ring system having one, two, or three aromatic rings, for example, phenyl, naphthyl, anthracenyl, or biphenyl. The aromatic compound generally contains from, for example, 6 to 30 carbon atoms, from 6 to 18 carbon atoms, from 6 to 14 carbon atoms, or from 6 to 10 carbon atoms. It is understood that the term aryl includes carbocyclic moieties that are planar and comprise 4n+2 TI electrons, according to Hiickel’s Rule, wherein n=l, 2, or 3.

[0058] As used herein, the term “halo” refers to a radical of a halogen, i.e., F, Cl, Br, or I.

[0059] As used herein, the term “Ci-6 alkyl” refers to a straight-chain or branched alkyl substituent containing from, for example, from about 1 to about 6 carbon atoms, e.g., from about 1 to about 4 carbon atoms or about 1 to about 3 carbons. Examples of alkyl group include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, n-hexyl, and the like. This definition also applies wherever “alkyl” occurs as part of a group, such as, e.g., Ci-6 haloalkyl (e.g., - trifluoromethyl (-CF3)).

[0060] As used herein, the term “C2-6 alkenyl” refers to a linear alkenyl substituent containing from, for example, 2 to about 6 carbon atoms (branched alkenyls are about 3 to about 6 carbons atoms). In accordance with an aspect, the alkenyl group is a C2-4 alkenyl.Examples of alkenyl group include, but are not limited to, ethenyl, allyl, 2-propenyl, 1- butenyl, 2-butenyl, 1 -pentenyl, 2-pentenyl, 3 -pentenyl, 1 -hexenyl, and the like.

[0061] As used herein, the term “C2-6 alkynyl” refers to a linear alkynyl substituent containing from, for example, 2 to about 6 carbon atoms (branched alkynyls are about 3 to about 6 carbons atoms). In accordance with an aspect, the alkynyl group is a C2-4 alkynyl. Examples of alkynyl group include, but are not limited to, ethynyl, propynyl, 1-butynyl, 2- butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 1-hexynyl, and the like.

[0062] As used herein, the term “hydroxy” refers to -OH.

[0063] As used herein, the term “nitro” refers to -NO2.

[0064] As used herein, the term “cyano” refers to -CN.

[0065] As used herein, the term “amino” refers to -NH2. The terms mono- and di-Ci-6 alkylamino refer to a nitrogen bonded to one or two C1-6 alkyl groups, respectively, i.e., -NHR or -NRR', in which R and R' are the same or different C1-6 alkyl groups.

[0066] As used herein, the term “C1-6 alkoxy” refers to a C1-6 alkyl group bonded to an oxygen, i.e., -OR, in which R is a C1-6 alkyl group.

[0067] As used herein, the term “Ce-io aryloxy” refers to an aryl group bonded to an oxygen, i.e., -O(Ar), in which Ar is a Ce-io aryl group.

[0068] As used herein, the term “aralkoxy” refers to the group -OR(Ar), in which R is an C1-6 alkyl group and Ar is a Ce-io aryl group.

[0069] As used herein, the term “carboxy” refers to -C(O)OH.

[0070] As used herein, the term “C1-6 alkylcarboxy” refers to a carboxy group wherein the hydrogen bound to the carboxy group has been replaced with a C1-6 alkyl group, i.e., -C(O)OR, wherein R is an C1-6 alkyl group.

[0071] As used herein, the term “amido” refers to the structure -C(O)NH or -NHC(O). The term “C1-6 alkylamido” refers to -C(O)NR or -NRC(O), wherein R is C1-6 alkyl.

[0072] As used herein, the term “C1-6 haloalkylamido” refers to a C1-6 alkylamido group in which the C1-6 alkyl group is substituted with 1, 2, or 3 halo groups, as described herein.

[0073] As used herein, the term “heteroaryl” refers to an aromatic compound, as described herein, containing a 5 or 6 membered ring in which 1 or 2 carbons have been replaced with nitrogen, sulfur, and / or oxygen. Examples of heteroaryl include, but are not limited to, pyridinyl, furanyl, pyrrolyl, quinolinyl, thiophenyl, indolyl, oxazolyl, isoxazolyl, pyrazolyl,imidazolyl, thiazolyl, isothiazolyl, 1,3,4-thiadiazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, and triazinyl.

[0074] As used herein, the term “heterocycloalkyl” refers to a monocyclic, bicyclic, or spiro ring system containing 3 to 7 carbon atom ring members and 1, 2, or 3 other atoms selected from nitrogen, sulfur, and / or oxygen. Examples of such heterocycloalkyl rings include, but are not limited to, aziridinyl, oxiranyl, thiazolinyl, imidazolidinyl, piperazinyl, homopiperazinyl, pyrrolinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydrothiofuranyl, pyranyl, tetrahydropyranyl, piperidinyl, and morpholinyl.Methods, Sensors, and Compositions of the Disclosure

[0075] As will be apparent from the discussion herein, the present disclosure provides an oxygen sensor comprising a working electrode as described herein. The provided oxygen sensor is also referred to herein as an analyte sensor (e.g. wherein the analyte is oxygen). In some aspects the sensor further comprises a redox mediator as described herein. In some aspects the redox mediator is disposed on at least a portion of the working electrode. In some aspects the oxygen sensor is an implantable oxygen sensor. Thus, in some aspects the oxygen sensor comprises an implantable portion (e.g., insertion tip) configured for implantation into a tissue. In some aspects the oxygen sensor is configured for continuous oxygen sensing. Thus, in some aspects the oxygen sensor is a continuous oxygen sensor. Certain aspects described herein thus relate to aspects of a continuous oxygen sensor. However, unless implied otherwise by the context, the present disclosure also provides such features as aspects of an oxygen sensor as described herein which may be continuous or discontinuous.

[0076] In some aspects the oxygen sensor does not rely on an enzyme to generate a signal.Accordingly, in some aspects the oxygen sensor does not rely on enzymatic oxygen reduction to generate a detectable signal at the working electrode. In some aspects the sensor is configured to generate a detectable signal in the absence of any enzyme at the working electrode. In some aspects the working electrode does not comprise an enzyme. In some aspects the sensor comprises no enzyme. In some aspects the oxygen sensor is enzyme-free.

[0077] Before describing the analyte sensors (e.g. oxygen sensors) of the present disclosure and their components in further detail, a brief overview of suitable in vivo analyte sensorconfigurations and sensor systems employing the analyte sensors will be provided so that the aspects 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 certain aspects. 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 certain 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 can also 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 certain aspects, 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 certain aspects, 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 certain aspects.

[0078] 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 certain aspects.

[0079] 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. Thus, the sensor may be configured to penetrate the skin of a subject. The sensor may comprise a member capable of penetrating the skin of a subject. For example, the member may be an insertable tip, tail, probe or needle capable of penetrating the skin of a subject. The member may be an implantable portion. Thus, sensor 104 can include an implantable portion of sufficient length for insertion to a desired depth in a given tissue. The implantable portion can include at least one working electrode. In certain configurations, the implantable portion can include a sensing layer for detecting an analyte (e.g., oxygen). A counter electrode can be present in combination with the at least one working electrode. Particular electrode configurations upon the implantable portion are described in more detail below.

[0080] The sensing layer can be configured for detecting a particular analyte (e.g., oxygen).For example, but not by way of limitation, the disclosed analyte sensors include at least one sensing layer configured to detect an analyte (e.g., oxygen).

[0081] In certain aspects of the present disclosure, an analytes (e.g., oxygen) can be monitored in any fluid of interest. In certain aspects the fluid is a 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 certain particular aspects, 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 certain aspects, the biofluid is interstitial fluid.

[0082] 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 (e.g., oxygen concentration) can be communicated automatically and periodically, such as at a certain frequency as data is obtained or after a certain time period has passed, with the data beingstored in a memory until transmittal (e.g., every minute, five minutes, or other predetermined time period). In certain other aspects, 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 certain other aspects, 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.

[0083] An introducer can be present transiently to promote introduction of sensor 104 into a tissue. In certain illustrative aspects, 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 aspects. 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 aspects. After opening the access pathway, the needle or other introducer can be withdrawn so that it does not represent a sharps hazard. In certain aspects, suitable needles can be solid or hollow, beveled or nonbeveled, and / or circular or non-circular in cross-section. In more particular aspects, 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.

[0084] In certain aspects, 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 certain aspects, sensor 104 can reside within a lumen or groove 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.

[0085] Sensor configurations featuring a single sensing layer 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 layers 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 layers within the same implantable portion, since the signal contribution from each sensing layer can be determined more readily.

[0086] 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. 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.

[0087] 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. Typically, as explained in more detail here, the working electrode configured for sensing oxygen is enzyme-free. The working electrode for sensing a further analyte may be enzyme-free or may comprise one or more enzymes.

[0088] FIG. 2A shows a diagram of an illustrative two-electrode analyte sensor configuration, which is compatible for use in the disclosure herein. As shown, analytesensor 200 includes substrate 212 disposed between working electrode 214 and counter / 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 layer 218 is disposed as at least one layer upon at least a portion of working electrode 214. Sensing layer 218 can include multiple spots or a single spot configured for detection of an analyte (e.g., oxygen), as discussed further herein.

[0089] Referring still to FIG. 2 A, membrane 220 overcoats at least sensing layer 218. In certain aspects, 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 layer 218 (i.e., membrane 220 is a mass transport limiting membrane having some permeability for the analyte of interest). In some aspects, and further described below, membrane 220 is not crosslinked. Analyte sensor 200 can be operable for assaying an analyte (e.g., oxygen) by any of coulometric, amperometric, voltammetric, or potentiometric electrochemical detection techniques.

[0090] 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 sensor 200 in FIG.2 A, except for the inclusion of additional electrode 217 in analyte sensors 201 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 aspects, electrode 214 (working electrode) and electrode 216 (counter electrode) can be located upon opposite faces of substrate 212, with electrode 217 (referenceelectrode) being located upon one of electrodes 214 or 216 and 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. 2A, sensing layer 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.

[0091] Like analyte sensor 200, membrane 220 can also overcoat sensing layer 218, as well as other sensor components, in analyte sensors 201 and 202, thereby serving as a mass transport limiting membrane. In certain aspects, 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 certain aspects 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. 2 A), 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 aspects disclosed herein, with alternative electrode and / or layer configurations remaining within the scope of the present disclosure.

[0092] FIG. 3 A shows an illustrative configuration for sensor 203 having a single working electrode with two different sensing layers disposed thereon. FIG. 3A is similar to FIG.2 A, except for the presence of two sensing layers upon working electrode 214: first sensing layer 218a and second sensing layer 218b, which are responsive to different analytes and are laterally spaced apart from one another upon the surface of working electrode 214. Sensing layers 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 layers 218a and 218b. First sensing layer 218a and second sensing layer 218b can be configured to detect their corresponding analytes at working electrode potentials that differ from one another, as discussed further below.

[0093] FIGs. 3B and 3C show cross-sectional diagrams of illustrative three-electrode sensor configurations for sensors 204 and 205, respectively, each featuring a single workingelectrode having first sensing layer 218a and second sensing layer 218b disposed thereon. FIGs. 3B and 3C are otherwise similar to FIGs. 2B and 2C and can be better understood by reference thereto. As with FIG. 3A, the composition of membrane 220 can vary or be compositionally the same at sensing layers 218a and 218b.

[0094] 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.

[0095] 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 layer 310a is disposed upon the surface of working electrode 304, and second sensing layer 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 layers 310a and 310b, according to various aspects, with other components of analyte sensor 300 or the entirety of analyte sensor 300 optionally being overcoated with membrane 340.

[0096] Like analyte sensors 200, 201, and 202, analyte sensor 300 can be operable for assaying an analyte (e.g., oxygen) by any of coulometric, amperometric, voltammetric, or potentiometric electrochemical detection techniques.

[0097] Alternative sensor configurations having multiple working electrodes and differing from the configuration shown in FIG. 4 can feature a counter / reference electrode instead 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 bereversed 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.

[0098] 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 hereinbelow. 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.

[0099] FIG. 5A 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 implantable portion (e.g., 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 implantable portion 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 implantable portion 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.

[0100] Referring still to FIG. 5A, first sensing layers 414a and second sensing layers 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 layers 414a and 414b have been depicted as three discrete spots in FIG. 5 A, it is to be appreciated that fewer or greater thanthree spots, including a continuous layer of sensing layer, can be present in alternative sensor configurations.

[0101] In FIG. 5A, sensor 400 is partially coated with membrane 450 upon working electrodes 410 and 420 and sensing layers 414a and 414b disposed thereon. FIG. 5B shows an alternative sensor configuration in which the substantial entirety of sensor 401 is overcoated with membrane 450. Membrane 450 can be the same or vary compositionally at sensing layers 414a and 414b.

[0102] 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. 5A and 5B. Similarly, the positions of working electrodes 410 and 420 are not limited to those that are expressly depicted in FIGs. 5A and 5B. FIG. 5C shows an alternative sensor configuration to that shown in FIG. 5B, in which sensor 405 contains counter electrode 430 and reference electrode 440 that are located more proximal to implantable portion 404 and working electrodes 410 and 420 that are located more distal to implantable portion 404. Sensor configurations in which working electrodes 410 and 420 are located more distal to implantable portion 404 can be advantageous by providing a larger surface area for deposition of sensing layers 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.

[0103] Several parts of the sensor are further described below.

[0104] The present disclosure relates to an electrochemical oxygen sensor and associated methods of detecting oxygen (e.g. detecting the presence, absence, or level (e.g. concentration) of oxygen in a fluid such as a biofluid). In some embodiments, the sensor comprises a first portion configured to be positioned above a user’s skin and a second portion configured to be transcutaneously positioned beneath the skin and in contact with an interstitial fluid to detect the analyte (e.g., oxygen) in vivo. In some embodiments, the second portion comprises a working electrode and a redox mediator.

[0105] An advantage of the present disclosure is that in some aspects the redox mediator does not comprise an enzyme, i.e. is enzyme-free. This can simplify manufacture andstorage of the sensor and may in some aspects increase the usable lifetime of the sensor. Typically, an enzyme-free sensor may have a more stable response over time than an enzyme-based sensor. For example, an enzyme-free oxygen sensor may exhibit a more stable current response to given oxygen levels over time than a comparable enzymatic sensor e.g. comprising bilirubin oxidase for oxygen reduction. However, the use of a redox mediator comprising an enzyme is within the scope of the disclosure. Some suitable enzymes are provided herein. In some aspects incorporating an enzyme may provide for greater initial sensitivity of the electrode. In some aspects incorporating an enzyme can allow additional measurements to be taken.

[0106] The present disclosure relates to an electrochemical continuous oxygen sensor and methods of detecting oxygen. The use of a redox mediator as a catalyst to reduce oxygen without the need for an enzyme is shown diagrammatically in FIG. 6. Without wishing to be bound by theory, it is believed that electrons provided by the working electrode can reduce the redox material (e.g., Os3+) to form a reduced redox material (e.g., Os2+), which in turn, can reduce oxygen e.g. to hydrogen peroxide and / or water according to the pathway shown in FIG. 7.

[0107] It is envisioned that the electrochemical continuous oxygen sensor can be used on its own or in combination with other types of sensors. For example, the electrochemical continuous oxygen sensor can be used in conjunction with a lactate sensor, since lactate levels in biofluids are closely related to oxygen levels. Detecting both oxygen and lactate levels can provide more accurate measurements for lactate sensing by, for example, predicting a rise in lactate. In another example, the accuracy of an oxidase-based sensor can be improved by applying one or more correction factors based on their oxygen effect. In yet another example, the electrochemical continuous oxygen sensor can be used as an indicating factor for reducing early signal attenuation (ESA), early signal rise (ESR), late signal attenuation (LSA), and / or late signal rise (LSR). Suitable lactate sensors are described, for example, in U.S. Patent Publication No. 2019 / 0320947 (the contents of which are incorporated by reference herein in their entirety). In further examples, the oxygen sensor can be used in conjunction with a sensor for glucose, lactate or ketone.

[0108] Oxygen monitoring in an individual can occur periodically or continuously over a period of time. Periodic oxygen monitoring can take place by withdrawing a sample of bodily fluid, such as blood, at set time intervals and analyzing ex vivo. Continuous oxygenmonitoring can be conducted using one or more sensors that remain implanted within a tissue of an individual, such as dermally, subcutaneously, or intravenously, whereby analyses can take place in vivo. Implanted sensors can collect oxygen data continuously, at planned intervals, or sporadically, depending on an individual's particular health needs and / or previously determined oxygen levels. Continuous oxygen sensors can also transmit data from a sensor control device to a reader device continuously without prompting, according to a schedule or at time periods selected by the user through a software application.

[0109] As explained herein, the working electrode can comprise any suitable material.Suitable materials include but are not limited to carbon. The redox mediator can comprise a polymer and an electron transfer agent. In some aspects the electron transfer agent is coupled to the polymer. Examples are provided herein.

[0110] Thus, in one aspect, the present disclosure is directed to a continuous oxygen sensor comprising a working electrode comprising carbon, and a redox mediator comprising a polymer, an electron transfer agent, and no enzyme; wherein the redox mediator is disposed on at least a portion of the working electrode.Working Electrode[OHl] In some aspects, the continuous oxygen sensor comprises a working electrode and a redox mediator on a portion of a working electrode. As discussed above, an enzyme is not required as part of the redox mediator in order to sense oxygen. In aspects in which the continuous oxygen sensor acts a dual analyte sensor, a second working electrode is present to detect a second analyte (e.g., glucose, lactate, or ketone). Such a sensor can, and typically does comprise, one or more enzymes as is appreciated in the art (e.g. lactate oxidase for a lactate sensor, glucose oxidase for a glucose sensor, a ketoreductase for a ketone sensor, etc.). Further examples of enzymes that can be present on a working electrode in a sensor provided herein include bilirubin oxidase, laccase, lactate oxidase, glucose dehydrogenase, glucose oxidase, P-hydroxybutyrate dehydrogenase, creatinine amidohydrolase, and creatine amidinohydrolase.

[0112] In the continuous oxygen sensor, the working electrode (e.g., a first working electrode, a second working electrode) can be any suitable conductive material. Examples of suitable conductive materials include, e.g., aluminum, carbon (including graphite),cobalt, copper, gallium, gold, indium, iridium, iron, lead, magnesium, mercury (as an amalgam), nickel, niobium, osmium, palladium, platinum, rhenium, rhodium, selenium, silicon (e.g., doped polycrystalline silicon), silver, tantalum, tin, titanium, tungsten, uranium, vanadium, zinc, zirconium, mixtures thereof, and alloys, oxides, or metallic compounds of these elements. In some aspects, a working electrode (e.g., a first working electrode and / or a second working electrode) can comprise carbon.Sensing Layer of the Oxygen Sensor

[0113] The sensing layer of the continuous oxygen sensor comprises a redox mediator comprising a polymer and an electron transfer agent. As discussed elsewhere herein, the sensing layer can further optionally comprise a stabilizing agent such as an albumin, a pH buffer, or both. In some aspects, the sensing layer of the continuous oxygen sensor does not comprise an enzyme.

[0114] In some aspects, the polymer in the redox mediator can be any suitable polymer that allows the transfer of electrons between the electron transfer agent and the working electrode. For example, the polymer can be a polyvinylpyridine (e.g., poly(4- vinylpyridine; PVP)), a polyvinylimidazole (e.g., poly(l-vinylimidazole; PVI)), poly(aniline), poly(pyrrole), poly(acetylene), poly(acrylic acid), styrene / maleic anhydride copolymer, methylvinylether / maleic anhydride copolymer, poly(vinylbenzylchloride), poly(allylamine), poly(lysine), poly(acrylamide-co-l -vinyl imidazole), poly(4- vinylpyridine) quaternized with carboxypentyl groups, or poly(sodium 4-styrene sulfonate). These polymers can be considered precursor polymers in that the polymers are further modified to immobilize (e.g., attach) the electron transfer agent. In some aspects, the polymer can comprise a backbone comprising poly(4-vinylpyridine), poly(l- vinylimidazole), poly(styrene), poly(thiophene), poly(aniline), poly(pyrrole), poly(acetylene), or any combination thereof. In other aspects, the polymer can comprise a polymer or copolymer repeat unit that can comprise at least one (e.g., 1, 2, 3, 4, 5, or 6) pendant pyridinyl group, imidazolyl group, or both a pyridinyl and imidazolyl group. For example, a suitable polymer can include partially or fully quaternized poly(4-vinylpyridine) and poly(l-vinylimidazole), in which quaternized pyridine and imidazole groups, respectively, can be used to form spacers by reaction with (e.g., complexation with) an electron transfer agent.

[0115] In some aspects, the polymer comprises poly(vinylpyridine), poly(vinylimidazole), poly(thiophene), poly(aniline), poly(pyrrole), poly(acetylene), polyethylene, polyacrylate, polymethacrylate, polystyrene, polyurethane, polyurea, or any combination thereof. In some aspects, the polymer comprises a polymer or copolymer repeat unit comprising at least one pendant pyridinyl group, imidazolyl group, or both a pyridinyl and imidazolyl group.

[0116] In some aspects, the electron transfer agent in the redox mediator can comprise a transition metal complex. The transition metal in the transition metal complex can be any suitable transition metal that can be effectively reduced and oxidized in the method described herein. For example, the transition metal complex can comprise osmium, ruthenium, iron, cobalt, vanadium, or any combination thereof. In some aspects, the transition metal can be ruthenium or osmium. In some aspects, the transition metal can be osmium. According to some aspects, suitable electron transfer agents can include low- potential osmium complexes, such as those described in U.S. Pat. Nos. 6,134,461, 6,605,200, 6,736,957, 7,501,053, and 7,754,093, the disclosures of each of which are incorporated herein by reference in their entirety. Other suitable examples of electron transfer mediators and polymer-bound electron transfer mediators can include those described in U.S. Pat. Nos. 8,444,834, 8,268,143, and 6,605,201, the disclosures of which are incorporated herein by reference in their entirety.

[0117] The transition metal complex can further comprise at least one ligand, which can be monodentate or multidentate (e.g., bidentate, tridentate, tetradentate). Typically, the complex will include enough ligands to provide a full coordination sphere. In some aspects, at least one ligand (e.g., 1, 2, 3, 4, 5, or 6) can comprise a nitrogen-containing heterocycle.

[0118] Monodentate ligands include, for example, -F, -Cl, -Br, -I, -CN, -SCN, -OH, NH3, alkylamine, dialkylamine, trialkylamine, alkoxy, a heterocyclic compound, compounds containing such groups, a solvent molecule (e.g., H2O, EtOH), or a reactive group. For example, an alkyl (e.g., C1-12, C1-6, Ci-4, C1-3) or aryl (e.g., phenyl, benzyl, naphthyl) portions of a ligand can be optionally substituted by, e.g., F, Cl, Br, I, alkylamino, dialkylamino, trialkylammonium (except aryl portions), alkoxy, alkylthio, and aryl. Examples of suitable heterocyclic monodentate ligands include imidazole, pyrazole, oxazole, thiazole, pyridine, and pyrazine, each of which can be unsubstituted or substituted,as described herein (e.g., with at least one reactive group, such as 1, 2, 3, or 4 reactive groups).

[0119] Examples of suitable bidentate ligands include, for example, 1,10-phenanthroline, an amino acid, oxalic acid, acetyl acetone, a diaminoalkane, an or / Ao-diaminoarene, 2,2'- biimidazole, 2,2'-bioxazole, 2,2'-bithiazole, 2-(2-pyridyl)imidazole, and 2,2'-bipyridine, each of which can be unsubstituted or substituted, as described herein (e.g., substituted with at least one reactive group, such as 1, 2, 3, or 4 reactive groups). Particularly suitable bidentate ligands for the electron transfer agent include substituted and unsubstituted 2,2'- biimidazole, 2-(2-pyridyl)imidazole, and 2,2'-bipyridine. Examples of suitable terdentate ligands include, for example, diethylenetriamine, 2,2',2"-terpyridine, 2,6-bis(A- pyrazolyl)pyridine, each of which can substituted or unsubstituted (e.g., substituted with one more alkyl groups, such as methyl, or one or more reactive groups).

[0120] A suitable 2,2'-biimidazole ligand can be a ligand according to formula (I):R1R2(I).

[0121] In formula (I), R1and R2are the same or different and each is a substituted or unsubstituted alkyl, alkenyl, or aryl. Generally, R1and R2are the same or different and each is an unsubstituted C1-12 alkyl (e.g., Ci-4 alkyl). In some aspects, both R1and R2are methyl.

[0122] In formula (I), R3, R4, R5, and R6are the same or different and each is H, F, Cl, Br, I, NO2, CN, CO2H, SO3H, SH, alkoxycarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, hydroxy, alkoxy, amino, alkylamino, dialkylamino, alkanoylamino, aryl carb oxami do, hydrazino, alkylhydrazino, hydroxylamino, alkoxyamino, alkylthio, alkyl, alkenyl, or aryl. Alternatively, R3and R4, in combination, or R5and R6, in combination, independently form a saturated or unsaturated 5- or 6-membered ring (e.g., benzo). Typically, the alkyl and alkoxy portions are C1-12. The alkyl or aryl portions of any of the substituents can be optionally substituted by one or more substituents (e.g., 1, 2, 3, 4, 5, or 6), such as F, Cl, Br, I, amino, alkylamino, dialkylamino, trialkylammonium (excepton aryl portions), alkoxy, alkylthio, aryl, or a reactive group (e.g., CO2H). Generally, R3, R4, R5, and R6are the same or different and each is H or an unsubstituted C1-12 alkyl (e.g., Ci-4 alkyl). In some aspects, R3, R4, R5, and R6are all H.

[0123] A suitable 2-(2-pyridyl)imidazole ligand can be a ligand according to formula (II):(II).

[0124] In formula (II), R1is a substituted or unsubstituted alkyl, alkenyl, or aryl. Generally, R1is an unsubstituted C1-12 alkyl (e.g., Ci-4 alkyl) or a C1-12 alkyl that is optionally substituted with a reactive group. In some aspects, R1is methyl.

[0125] In formula (II), R3, R4, Ra, Rb, Rc, and Rdare the same or different and each is H, F, Cl, Br, I, NO2, CN, CO2H, SO3H, SH, alkoxycarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, hydroxy, alkoxy, amino, alkylamino, dialkylamino, alkanoylamino, aryl carb oxami do, hydrazino, alkylhydrazino, hydroxylamino, alkoxyamino, alkylthio, alkyl, alkenyl, or aryl. Alternatively, R3and R4, in combination, or two adjacent substituents of Ra, Rb, Rc, and Rd(e.g., Raand Rb, Rband Rc, or Rcand Rd) in combination, independently form a saturated or unsaturated 5- or 6-membered ring (e.g., benzo). Typically, the alkyl and alkoxy portions are C1-12. The alkyl or aryl portions of any of the substituents can be optionally substituted by one or more substituents (e.g., 1, 2, 3, 4, 5, or 6), such as F, Cl, Br, I, amino, alkylamino, dialkylamino, trialkylammonium (except on aryl portions), alkoxy, alkylthio, aryl, or a reactive group (e.g., CO2H). Generally, R3, R4, Ra, Rb, Rc, and Rdare the same or different and each is H or an unsubstituted C1-12 alkyl (e.g., Ci-4 alkyl). In some aspects, R3, R4, Ra, Rb, Rc, and Rdare all H.

[0126] A suitable 2,2'-bipyridine ligand can be a ligand according to formula (III):R18R19R20R21(III).

[0127] In formula (III), R16, R17, R18, R19, R20, R21, R22, and R23are the same or different and each is H, F, Cl, Br, I, NO2, CN, CO2H, SO3H, SH, alkoxycarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, hydroxy, alkoxy, amino, alkylamino, dialkylamino, alkanoylamino, arylcarboxamido, hydrazino, alkylhydrazino, hydroxylamino, alkoxyamino, alkylthio, alkyl, alkenyl, or aryl. Typically, the alkyl and alkoxy portions are C1-12. The alkyl or aryl portions of any of the substituents can be optionally substituted by one or more substituents (e.g., 1, 2, 3, 4, 5, or 6), such as F, Cl, Br, I, amino, alkylamino, dialkylamino, trialkylammonium (except on aryl portions), alkoxy, alkylthio, aryl, or a reactive group (e.g., CO2H).

[0128] Specific examples of suitable combinations include R16and R23are both H or both methyl and / or R17and R23are both H or both methyl and / or R18and R21are both H or both methyl and / or R19and R20are both H or both methyl. An alternative combination is where one or more adjacent pairs of substituents (e.g., R16and R17, R17and R18, R18and R19, R23and R22, R22and R21, or R21and R20), in combination, form a saturated or unsaturated 5- or 6-membered ring (e.g., benzo).

[0129] In an aspect, the one or more ligand is 4,4'-dimethyl-2,2'-bipyridine, mono-, di-, or polyalkoxy-2, 2'-bipyridines (e.g., 4,4'-dimethoxy-2,2'-bipyridine), 4,7-dimethyl-l,10- phenanthroline, mono, di-, or polyalkoxy-l,10-phenanthrolines (e.g., 4,7-dimethoxy-l,10- phenanthroline), or a combination of any of these.

[0130] In some aspects, the transition metal complex can include a counterion (X) to balance the charge of the transition metal. Typically, there can be 1 to 5 (i.e., 1, 2, 3, 4, or 5) counterions. Multiple counterions in the complex are not necessarily all the same. Examples of suitable counterions include anions, such as halide (e.g., fluoride, chloride, bromide, or iodide), sulfate, phosphate, hexafluorophosphate, and tetrafluorob orate, and cations (e.g., a monovalent cation), such as lithium, sodium, potassium,tetralkylammonium, and ammonium. In some aspects, the counterion is a halide, such as chloride.

[0131] In an aspect, the transition metal complex can be an osmium transition metal complex that can comprise one or more ligands, wherein at least one (e.g., 1, 2, 3, 4, 5, or 6) ligand that can comprise a nitrogen-containing heterocycle (e.g., imidazole, pyrazole, oxazole, thiazole, pyridine, and pyrazine). In some aspects, the osmium transition metal complex can comprise one or more ligands selected from 4,4'-dimethyl-2,2'-bipyridine, mono-, di-, or polyalkoxy-2, 2'-bipyridines (e.g., 4, 4'-dimethoxy-2, 2' -bipyridine), 4,7- dimethyl-l,10-phenanthroline, mono, di-, or polyalkoxy-l,10-phenanthrolines (e.g., 4,7- dimethoxy-1, 10-phenanthroline).

[0132] In an aspect, the redox mediator can comprise an osmium complex bonded to a polymer or copolymer of poly(l-vinyl imidazole) or poly(4-vinylpyridine). The poly(4- vinylpyridine)-based polymer is a prepolymer that has been modified, as shown in the following structure, to attach an osmium complex (e.g., a poly(biimidizyl) osmium complex):wherein n can be 2, n' can be 17, and n" can be 1. Other reactive groups and / or spacer groups can be used.

[0133] In an aspect, the electron redox mediator can comprise an osmium-containing poly(4-vinylpyridine)-based polymer, referred to herein as “osmium-containing poly(4- vinylpyridine)-based polymer,” as shown below.osmium-containing poly(4-vinylpyridine)-based polymer, wherein n is 2, n' is 17, and n" is 1.

[0134] In some aspects, the transition metal complex comprises osmium, ruthenium, iron, cobalt, vanadium, or any combination thereof and at least one ligand coupled to the polymer backbone. In some aspects, the transition metal complex is an osmium transition metal complex comprising one or more ligands, wherein at least one ligand comprises a nitrogen-containing heterocycle coupled to the polymer backbone. In some aspects, the redox mediator comprises an osmium complex bonded to a poly(4-vinylpyridine)-based polymer.

[0135] In some aspects, the electron transfer agent can be attached (e.g., non-leachably and / or covalently bonded) to the polymer in the redox mediator. For example, covalent bonding of the electron transfer agent to the polymer can take place by polymerizing a monomer unit bearing a covalently bound electron transfer agent, or the electron transfer agent can be reacted with the polymer separately after the polymer has already been synthesized.

[0136] According to some aspects, a bifunctional spacer can be used to attach (e.g., covalently bond) the electron transfer agent to the polymer in the redox mediator, with a first reactive group being reactive with the polymer (e.g., a functional group capable of quaternizing a pyridine nitrogen atom or an imidazole nitrogen atom) and a second reactive group being reactive with the electron transfer agent (e.g., a functional group that is reactive with a ligand coordinating a metal ion). Typically, covalent bonds are formed between the two reactive groups to generate a linkage. Suitable reactive groups include, for example, activated ester (e.g., succinimidyl, benzotriazolyl, or an aryl substituted with one more electron withdrawing groups, such as sulfo, nitro, cyano, or halo), acrylamido, acyl azido, acyl halide, carboxy (-COO- or -CO2H), aldehyde, ketone, alkyl halide, alkyl sulfonato, anhydride, aziridino, epoxy, halotriazinyl, imido ester, isocyanato, isothiocyanato, maleimido, sulfonyl halide, amino, thiol (-SH), hydroxy, pyridinyl, imidazolyl, and hydroxyamino. The reaction between two reactive groups can form a covalent linkage between the transition metal complex and the polymer that is a carboxamido, thioether, hydrazonyl, oximyl, alkylamino, ester, carboxylic ester, imidazolium, pyridinium, ether, thioether, aminotriazinyl, triazinyl ether, amidinyl, urea, urethanyl, thiourea, thioether, sulfonamide, or any combination. In addition to the reactive groups, the bifunctional spacer typically can further comprise an alkylenyl (i.e., -(CH2)n-) and / or ethylenyloxy (i.e., -(CH2CH2O)m-, in which n and m are each independently an integer from 1 to 12 (e.g., 1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2).

[0137] In some aspects, the redox mediator can further comprise a cross linking agent. In general, the cross linking agent is any suitable multifunctional (e.g., bifunctional) short chain molecule that enables the electron transfer agent to attach (e.g., covalently bond) to the polymer of the redox mediator. For example, the cross linking agent can include apolyepoxide (e.g., a polyethylene glycol diglycidylether (PEGDGE), ethylene glycol diglycidyl ether (EGDGE), resorcinol diglycidyl ether, 1,2, 7, 8-diepoxy octane, Gly3), cyanuric chloride, / ' / -hydroxysuccinimide, an imidoester, epichlorohydrin, or any combination thereof. In some aspects, the redox mediator further comprises a cross linking agent. In some aspects, the cross linking agent is a polyepoxide, cyanuric chloride, N- hydroxysuccinimide, an imidoester, epichlorohydrin, or any combination thereof. In some aspects, the cross linking agent is a polyethylene glycol diglycidyl ether (PEGDGE). In an aspect, the cross linking agent is a PEGDGE of the following formula:wherein n is an integer from 1 to about 50 (e.g., 1 to about 45, 1 to about 40, 1 to about 35, 1 to about 30, 1 to about 25, about 5 to about 50, about 5 to about 45, about 5 to about 40, about 5 to about 35, or about 5 to about 30).

[0138] In a particular example, the PEGDGE can be PEGDGE200, PEGDGE400 (n is 10), PEGDGE500, PEGDGE600, PEGDGE1000, or PEGDGE2000, in which the number denotes the average molecular weight (Mn). In an aspect, the crosslinking agent can be PEGDGE400.

[0139] In some aspects, the sensing layer can further comprise a stabilizing agent such as a protein, e.g. an albumin. In an aspect, the albumin can be a serum albumin, such as bovine serum albumin (BSA) or human serum albumin (HSA). In certain aspects, the sensing layer can comprise human serum albumin. In certain aspects, the sensing layer can comprise bovine serum albumin (BSA). In some aspects the stabilizing agent is present in an amount of from about 1 to about 100 mg / mL such as from about 5 to about 20 mg / mL e.g. about 10 mg / mL.

[0140] In some aspects, the sensing layer can comprise a pH buffer. The buffer can be any suitable composition that is water soluble and controls (i.e., maintains) the pH of the sensing composition within a pH of about 5 to about 8 (e.g., maintains a pH of about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, or about 8). In some aspects, the pH can be controlled to be within a range of about 6 to about 8. For example, the buffer can comprise a phosphate (e.g., monobasic and dibasic sodium phosphate), 4-(2- hydroxyethyl)piperazine-l -ethanesulfonic acid (HEPES), 2-(A-morpholino)ethanesulfonicacid (MES), 3-(7V-morpholino) propanesulfonic acid (MOPS), 2-amino-2- (hydroxymethyl)- 1,3 -propanediol (TRIS), a carbonate (e.g., carbonic acid and a carbonate salt, such as sodium carbonate; sodium carbonate and sodium bicarbonate), or a citrate (e.g., citric acid and a citrate salt, such as trisodium citrate). The buffer can optionally comprise one or more (e.g., 1, 2, 3, or 4) additional salts (e.g., Group I or Group II halide salts, e.g., sodium chloride, potassium chloride, magnesium chloride). In an aspect, the buffer can be phosphate-buffered saline (PBS), which comprises disodium hydrogen phosphate, sodium chloride, and optionally potassium chloride and potassium dihydrogen phosphate. In another aspect, the buffer can be MES or a phosphate buffer, which can comprise phosphate, sodium chloride, potassium chloride, and / or magnesium chloride. For example, in one aspect the buffer is MES.

[0141] In some aspects, the buffer comprises buffer salts at a concentration of from about 1 mM to about 500 mM, such as from about 5 mM to about 100 mM, e.g., from about 10 mM to about 50 mM.

[0142] In some aspects, the buffer typically can be an aqueous buffer. In other aspects, nonaqueous solvents can be present, such as an alcohol (e.g., ethanol). In some aspects, the buffer can comprise water as the only solvent. In other aspects, the buffer can comprise water and at least one (e.g., 1, 2, or 3) non-aqueous solvents in any suitable ratio, such as a non-aqueous solvent to water volume ratio ranging from 99.9:0.1 to 0.1:99.9. In some aspects, the non-aqueous solvent to water volume ratio can be about 1 :99, about 5 :95, about 10:90, about 15:85, about 20:80, about 25:75, about 30:70, about 35:65, about 40:60, about 45:55, about 50:50, about 55:45, about 60:40, about 65:35, about 70:30, about 75:25, about 80:20, about 85:15, about 90:10, about 95:5, or about 99:1, etc.). In a specific example, ethanol (EtOH) and water can be used in a volume ratio ranging from 50:50 to 90:10 EtOELEhO (e.g., 70:30, about 75:25, about 80:20, about 85:15, or about 90:10, etc.).

[0143] In some aspects the buffer comprises one or more buffer components as described herein (e.g. one or more buffer salts) at a concentration of from about 1 mM to about 100 mM, e.g. about 2 mM to about 80 mM, e.g. about 5 mM to about 50 mM, such as from about 10 mM to about 20 mM, e.g. about 10 mM. For example, in some aspects the buffer is an aqueous buffer comprising from about 1 mM to about 100 mM (e.g. about 10 mM) MES.

[0144] In some aspects, the continuous oxygen sensor further comprises a stabilizing agent such as a protein, e.g. an albumin; and optionally a pH buffer. In some aspects, the continuous oxygen sensor further comprises an albumin. In some aspects, the continuous oxygen sensor further comprises a stabilizing agent such as a protein, e.g. an albumin and a pH buffer.

[0145] The oxygen sensing layer, the second analyte sensing layer, or both sensing layers can be continuously or discontinuously disposed on at least a portion of the respective working electrode. A discontinuous application means that the sensing layer can form a discrete shape on the working electrode, such as a spot, a line, or a plurality (e.g., an array) of spots and / or lines. The number of spots or lines is not considered to be particularly limited, but can range from 2 to about 10 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10, including about 3 to about 8, or from about 4 to about 6). In any of the aspects herein, the oxygen sensing layer, the second analyte sensing layer, or both can be continuously disposed on the respective working electrode. In other aspects, the oxygen sensing layer, the second analyte sensing layer, or both can be discontinuously disposed on the respective working electrode.

[0146] The redox mediator can be applied to a working electrode using any suitable technique, such as spray coating, painting, inkjet printing, stenciling, roller coating, dip coating, or any combination thereof. In some aspects, the redox mediator can be applied by dip coating at least a portion of the working electrode into a solution of the redox mediator. One application or multiple applications can be applied (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 applications). In some aspects, the redox mediator can be applied in 1, 2, 3, or 4 applications (e.g., passes). In some aspects, the redox mediator can be applied in 1 or 2 applications (e.g., passes).

[0147] In some aspects, the redox mediator in the continuous oxygen sensor is continuously disposed on the working electrode. In some aspects, the redox mediator in the continuous oxygen sensor is discontinuously disposed on the working electrode.

[0148] The total size of the sensing layer or layers (e.g., combined area of all spots, layers, or active areas) can be at least about 0.05 mm2and can be up to about 100 mm2. In some aspects, the total size can be about 0.05 mm2to about 100 mm2, about 0.05 mm2to about 75 mm2, about 0.05 mm2to about 50 mm2, about 0.05 mm2to about 40 mm2, about 0.05 mm2to about 30 mm2, about 0.05 mm2to about 25 mm2, about 0.05 mm2to about 15 mm2, about 0.05 mm2to about 10 mm2, about 0.05 mm2to about 5 mm2, about 0.05 mm2to about1 mm2, or about 0.05 mm2to about 0.1 mm2. In a particular aspect, the total size of the sensing layer or layers ranges from about 0.05 to about 0.1 mm2, about 0.05 to about 100 mm2, about 0.1 to about 50 mm2, about 0.5 to about 30 mm2, about 1 to about 20 mm2, or about 1 to about 15 mm2.

[0149] The oxygen sensing layer typically has a thickness that ranges from about 0.1-10 pm. For example, the oxygen sensing layer can be 0.1 pm thick or more (e.g., 0.2 pm or more, 0.3 pm or more, 0.5 pm or more, 0.8 pm or more, 1 pm or more, 2 pm or more, 3 pm or more, 5 pm or more, or 8 pm or more) and typically will have a thickness of 10 pm or less (e.g., 8 pm or less, 5 pm or less, 3 pm or less, 2 pm or less, 1 pm or less, 0.8 pm or less, 0.5 pm or less, 0.3 pm or less, or 0.2 pm or less). In an example, the oxygen sensing layer can have a thickness of about 0.1 to about 10 pm, about 0.2 to about 8 pm, about 0.5 to about 5 pm, about 1 to about 4 pm, or about 2 pm.

[0150] In some aspects, a conductive material such as, for example, carbon nanotubes, graphene, or metal nanoparticles, can be combined within the oxygen sensing layer 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 each sensing layer (e.g., about 1 to about 50 pbw, about 1 to about 10 pbw, or about 0.1 to about 10 pbw).

[0151] In some aspects, the sensing layer is allowed to cure on the electrode before a membrane is applied thereto. In some aspects the sensing layer is allowed to cure for from about 1 hour to about 48 hours (e.g. from about 6 hours to about 24 hours, e.g. about 12 hours). In some aspects the curing takes place at about 10 to about 30 °C e.g. from about 15 to about 25 °C such as at about 20 °C. In some aspects the curing takes place under an atmosphere having a humidity level of from about 50% to about 80% such as about 60% to about 70%.

[0152] In some aspects, therefore, a working electrode comprised in an analyte sensor as described herein (e.g. an oxygen sensor, e.g. a continuous oxygen sensor) comprises: a working electrode comprising carbon,a sensing layer comprising a redox mediator comprising a polymer and an electron transfer agent disposed on at least a portion of the working electrode;- the working electrode comprised in an implantable portion of the sensor;- the redox mediator comprising an osmium complex bonded to a polymer or copolymer of poly(l -vinyl imidazole) or poly(4-vinylpyridine);- the sensing layer optionally further comprising a stabilizing agent (e.g. a protein, e.g. an albumin); and / or a pH buffer (e.g. a buffer that maintains the pH of the sensing layer to a pH of about pH 5 to about pH 8);- wherein the working electrode does not comprise an enzyme (i.e. is enzyme free).

[0153] In some aspects, the working electrode comprised in an analyte sensor as described herein (e.g. an oxygen sensor, e.g. a continuous oxygen sensor) comprises:a working electrode comprising carbon, e.g. a screen-printed carbon electrode, a sensing layer comprising a redox mediator comprising a polymer and an electron transfer agent disposed on at least a portion of the working electrode;- the working electrode comprised in an implantable portion of the sensor;- the redox mediator comprising an osmium-containing poly(4-vinylpyridine)-based polymer as defined herein; and optionally comprising a cross-linking agent (e.g. a polyepoxide (e.g., a polyethylene glycol diglycidylether (PEGDGE), ethylene glycol diglycidyl ether (EGDGE), resorcinol diglycidyl ether, 1,2, 7, 8-di epoxy octane, Gly3), cyanuric chloride, / f-hydroxysuccinimide, an imidoester, epichlorohydrin, or any combination thereof);- the sensing layer comprising an aqueous buffer that maintains the pH of the sending layer to a pH of about pH 5 to about pH 8); wherein the buffer comprises from about 1 mM to about 100 mM of one or more buffer components (e.g. about 10 mM MES); - the sensing layer optionally further comprising a stabilizing agent (e.g. a protein, e.g.an albumin);- wherein the working electrode does not comprise an enzyme (i.e. is enzyme free).Membrane

[0154] In an embodiment, the sensing electrode can further comprise a membrane that overcoats at least the oxygen sensing layer and optionally other components. The overcoating forms an outer membrane that provides stability to the sensing reagents (e.g., the redox mediator), mass-transport limitations, biocompatibility, and / or prevents electrode fouling. The membrane can optionally coat all or part of the working electrode and optionally any counter or reference electrode that can be present. In an embodiment, the membrane coats (e.g., encapsulates) the entire system, including the sensing electrode with its sensing layer(s), and any counter electrodes, reference electrodes, and / or substrates thatcan be present. As explained here, in most aspects disclosed herein the sensing reagents do not comprise an enzyme (i.e. the working electrode is enzyme-free). However, in aspects wherein an enzyme is present, the outer membrane typically also provides stability to the enzyme.

[0155] The membrane can comprise one or more polymeric membrane materials with a physical structure that allows analyte flux to the sensing layer (i.e., the membrane is a mass transport limiting membrane). The composition of the membrane can vary (e.g., the degree of hydrophobicity and / or the degree of crosslinking) to promote a desired flux of oxygen (and any other additional analytes) to the sensing electrode, thereby providing a desired signal intensity and stability as described further herein. In an embodiment, the membrane is permeable to oxygen.

[0156] The coating of the membrane over at least the oxygen sensing layer can be performed by any suitable technique. Typically, the membrane will be coated by spray coating, painting, inkjet printing, roller coating, dip coating, or any combination thereof. The coating step can be performed once or multiple times (e.g., 2, 3, 4, or 5 times), which will affect the thickness of the membrane coating. In an embodiment, the coating step can be performed twice to form a bilayer.

[0157] In some aspects, the coating of the membrane is performed by dip coating. Any suitable dip coating speed can be used. In some aspects the dip coating speed is at least about 1 mm / s, such as from about 1 mm / s to about 100 mm / s, e.g. from about 1 mm / s to about 10 mm / s. In some aspects, the dip coating speed is from about 1 mm / s to about 100 mm / s, from about 1 mm / s to about 90 mm / s, from about 1 mm / s to about 80 mm / s, from about 1 mm / s to about 70 mm / s, from about 1 mm / s to about 60 mm / s, from about 1 mm / s to about 50 mm / s, from about 1 mm / s to about 40 mm / s, from about 1 mm / s to about 30 mm / s, from about 1 mm / s to about 20 mm / s, from about 1 mm / s to about 10 mm / s. In some aspects, the dip coating speed is about 1 mm / s, about 2 mm / s, about 3 mm / s, about 4 mm / s, about 5 mm / s, about 6 mm / s, about 7 mm / s, about 8 mm / s, about 9 mm / s, about 10 mm / s, about 25 mm / s, about 50 mm / s, about 75 mm / s, about 100 mm / s.

[0158] In general, if multiple coats are applied, the first coat will be dried prior to applying the subsequent coat(s). The amount of time between coating steps will vary depending on the types of membrane, working electrode, and sensing layer, and the atmospheric conditions. In general, the drying time will be 1 minute or longer (e.g., 2 min or more, 3min or more, 5 min or more, 10 min or more, 15 min or more, or 20 min or more). Once the membrane coating has been applied, the coating can be cured. In an embodiment, the coating can be cured for 12 hours or more (e.g., 18 hours or more, 24 hours or more, 30 hours or more, 36 hours or more, 42 hours or more, or 48 hours or more). The curing can be at room temperature (i.e., about 20 °C) or at a slightly elevated temperature (e.g., 100 °C or less, 80 °C or less, 70 °C or less, 60 °C or less, 50 °C or less, 40 °C or less, 30 °C or less, or 25 °C or less). In general, the curing will not occur at less than about 20 °C.

[0159] The membrane typically has a thickness that ranges from about 1 pm to about 100 pm. For example, in some aspects, the membrane can have a thickness of about 1 pm or more (e.g., about 5 pm or more, about 10 pm or more, about 15 pm or more, about 20 pm or more, about 25 pm or more, about 30 pm or more, about 35 pm or more, about 40 pm or more, about 50 pm or more, about 60 pm or more, about 70 pm or more, about 80 pm or more, or about 90 pm or more) and typically will have a thickness of about 100 pm or less (e.g., about 90 pm or less, about 80 pm or less, about 70 pm or less, about 60 pm or less, about 50 pm or less, about 45 pm or less, about 40 pm or less, about 35 pm or less, about 30 pm or less, about 25 pm or less, about 20 pm or less, about 15 pm or less, about 10 pm or less, or about 5 pm or less). In an example, the membrane can have a thickness of about 5 to about 80 pm, about 10 to about 80 pm, about 10 to about 60 pm, about 15 to about 60 pm, about 20 to about 50 pm, about 20 to about 40 pm, about 25 to about 35 pm, or a thickness of about 30 pm. In an example, the membrane can have a thickness of about 15 to about 35 pm. In an example, the membrane can have a thickness of about 20 to about 35 pm. In an example, the membrane can have a thickness of about 20 to about 30 pm. In an example, the membrane can have a thickness of about 25 pm. In another example, a membrane can have a thickness of from about 5 to about 15 pm, such as about 8 to about 10 pm. In another example, a membrane can have a thickness of from about 5 to about 30 pm, such as from about 8 to about 25 pm.

[0160] In an embodiment, the membrane can comprise optionally crosslinked poly(4- vinylpyridine), poly(vinyl alcohol), poly(acrylic acid), poly(methacrylic acid), or a combination thereof. In an example, the membrane can comprise at least a poly(4- vinylpyridine) homopolymer or copolymer, in which the poly(4-vinylpyridine) can be optionally cross linked. Particularly, the membrane can comprise poly(4-vinylpyridine)crosslinked with a crosslinking agent, as described herein (e.g., high molecular weight (such as molecular weight 400 g / mol) poly(ethylene glycol) diglycidyl ether).

[0161] Suitable poly(4-vinylpyridine) copolymers for inclusion in the membrane can comprise up to about 25% comonomers (based on the total amount of monomers in the copolymer), such as from about 0.1% to about 5% comonomers, or about 5% to about 15% comonomers, or about 15% to about 25% comonomers, or about 1% to about 10% comonomers. Suitable comonomers are not particularly limited, provided that the membrane affords sufficient oxygen permeability.

[0162] In some aspects, the membrane can comprise multiple layers in which each layer has a different composition and / or degree of crosslinking. For example, the membrane coating can be a bilayer membrane that can comprise a first layer formed from a poly(4- vinylpyridine) homopolymer or copolymer and a second layer formed from a crosslinked poly(4-vinylpyridine) homopolymer or copolymer (e.g., crosslinked with PEGDGE). The variation in composition between layers allows tuning the permeability of the membrane to both oxygen and a second analyte of interest.

[0163] In some aspects, the membrane can comprise a polyurethane. For example, but not by way of limitation, the membrane can be a single-component membrane or a multicomponent membrane comprising a polyurethane. In certain aspects, a polymer for use in the present disclosure, e.g., a polyurethane, is capable of absorbing from about 30% to about 95% of its weight in water, e.g., from about 30% to about 70%. In certain aspects, the polyurethane is capable of absorbing at least about 30% of its weight in water. In certain aspects, the polyurethane is capable of absorbing at least about 40% of its weight in water. In certain aspects, the polyurethane is capable of absorbing at least about 50% of its weight in water. In certain aspects, the polyurethane is capable of absorbing at least about 60% of its weight in water. In certain aspects, the polyurethane is capable of absorbing at least about 70% of its weight in water. In certain aspects, a polyurethane for use in the present disclosure is low heat curable. For example, but not by way of limitation, a polymer for use in the present disclosure, e.g., a polyurethane, is curable at a temperature from about 20 °C to about 90 °C, e.g., about 25 °C to about 85 °C, about 30 °C to about 80 °C, about 35 °C to about 75 °C, about 40 °C to about 70 °C, about 45 °C to about 65 °C, about 20 °C to about 70 °C, about 20 °C to about 60 °C, about 20 °C to about 50 °C, about 30 °C to about 90 °C, about 40 °C to about 90 °C, about 50 °C to about 90 °C, about 60 °C to about90 °C or about 70 °C to about 90 °C. In certain aspects, the polymer for use in the present disclosure, e.g., a polyurethane, has a molecular weight from about 50 to about 500 kDa.

[0164] In certain aspects, the polyurethane can be a commercially available hydrophilic polyurethane. In certain aspects, the hydrophilic polyurethane can be a polyurethane of the HydroMed™ Series from AdvanSource biomaterials (Wilmington, MA). For example, but not by way of limitation, the commercially available hydrophilic polyurethane can be HydroMed™ DI, HydroMed™ D2, HydroMed™ D3, HydroMed™ D4, HydroMed™ D6, HydroMed™ D640, HydroMed™ D7, HydroMed™ Hydroslip C, or a combination thereof. In certain aspects, the polyurethane can comprise HydroMed™ D7. In certain aspects, the polyurethane can comprise HydroMed™ DI.

[0165] In certain aspects, the membrane is deposited from an aqueous solution. In certain aspects, the membrane is deposited from a solution comprising one or more non-aqueous solvents, such as an alcohol (e.g., ethanol). In some aspects the membrane is deposited from a solution comprising water and at least one (e.g., 1, 2, or 3) non-aqueous solvents in any suitable ratio, such as a non-aqueous solvent to water volume ratio ranging from 99.9:0.1 to 0.1:99.9. In some aspects, the non-aqueous solvent to water volume ratio can be about 1:99, about 5:95, about 10:90, about 15:85, about 20:80, about 25:75, about 30:70, about 35:65, about 40:60, about 45:55, about 50:50, about 55:45, about 60:40, about 65:35, about 70:30, about 75:25, about 80:20, about 85:15, about 90:10, about 95:5, or about 99:1, etc.). In a specific example, ethanol (EtOH) and water can be used in a volume ratio ranging from 50:50 to 90:10 EtOH:H2O (e.g., 70:30, about 75:25, about 80:20, about 85:15, or about 90:10, etc.).

[0166] In some aspects the aqueous solution comprises one or more buffer components.Any suitable buffer components as described herein can be used. In some aspects the buffer comprises a phosphate (e.g., monobasic and dibasic sodium phosphate), 4-(2- hydroxyethyl)piperazine-l -ethanesulfonic acid (HEPES), 2-(A-morpholino)ethanesulfonic acid (MES), 3-(A-morpholino) propanesulfonic acid (MOPS), 2-amino-2- (hydroxymethyl)- 1,3 -propanediol (TRIS), a carbonate (e.g., carbonic acid and a carbonate salt, such as sodium carbonate; sodium carbonate and sodium bicarbonate), or a citrate (e.g., citric acid and a citrate salt, such as trisodium citrate). The buffer can optionally comprise one or more (e.g., 1, 2, 3, or 4) additional salts (e.g., Group I or Group II halidesalts, e.g., sodium chloride, potassium chloride, magnesium chloride). In an aspect, the buffer comprises HEPES.

[0167] In some aspects, therefore:- the membrane comprises optionally crosslinked poly(4-vinylpyridine), poly(vinyl alcohol), poly(acrylic acid), poly(methacrylic acid), or a combination thereof; and - the membrane has a thickness of from about 1 pm to about 100 pm.

[0168] In some aspects:- the membrane is coated by spray coating, painting, inkjet printing, roller coating, dip coating, or any combination thereof; and the coating step is performed once, 2, 3, 4 or 5 times;the membrane comprises poly(4-vinylpyridine) thereof crosslinked with PEGDGE; - the membrane has a thickness of from about 5 pm to about 30 pm;- the membrane is optionally deposited on the electrode from an aqueous solution optionally comprising one or more non-aqueous solvents (e.g. ethanol) and / or one or more buffer components (e.g. HEPES).

[0169] In some aspects an electrode in accordance with the present disclosure is stable under operational conditions. In some aspects an electrode in accordance with the present disclosure exhibits a stable current response under constant oxygen levels (e.g. in the presence of about 21% oxygen) for at least 7 days, such as at least 10 days, at least 14 days, at least 21 days, or at least 1 month. In some aspects the current response alters (e.g. decreases) by less than 10%, more typically less than 5%, e.g. less than 4%, less than 3%, less than 2% or less than 1% over at least 7 days, such as at least 10 days, at least 14 days, at least 21 days, or at least 1 month.

[0170] Also provided herein is a method of producing an electrode for sensing oxygen, comprising disposing on the surface of an electrode a sensing composition as described in more detail herein and disposing on the sensing composition a membrane as described herein. In some aspects the method further comprises incorporating the electrode into a sensor or other apparatus as described herein.Second Sensors

[0171] In certain aspects, the sensor described here can include a at least a second sensor for sensing a second analyte. As noted previously, such sensor typically comprises a secondsensing layer comprising one or more enzymes suitable for detecting a particular analyte of interest. The enzyme catalyzes a reaction that consumes the analyte of interest or produces a product that is detectable by the sensor. The analyte-responsive enzyme will be selected based on the analyte that is to be detected (e.g., glucose, lactate, ketone, glutamate, pyruvate, creatinine, sarcosine, and / or alcohol (e.g., ethanol)). In some aspects, the enzyme is an oxidase enzyme or a dehydrogenase enzyme. Suitable examples of analyte-responsive enzymes include, but are not limited to, glucose oxidase, glucose dehydrogenase, glutamate oxidase, lactate oxidase, lactate dehydrogenase, pyruvate oxidase, alcohol oxidase, xanthine oxidase, [3-hydroxybutyrate dehydrogenase, 1 l[3-hydroxysteroid dehydrogenase type 2 (11P-HSD-2), creatine amidohydrolase, sarcosine oxidase, nicotinamide adenine dinucleotide (NADH)-dependent oxidase, NADPH dehydrogenase, a flavin adenine dinucleotide (FAD)-dependent oxidase, a flavin mononucleotide (FMN)-dependent oxidase, diaphorase, catalase, and combinations thereof.

[0172] In some aspects, the enzyme is glucose oxidase and / or glucose dehydrogenase to detect glucose. In some aspects, the enzyme is glutamate oxidase to detect glutamate. In some aspects, the enzyme is lactate oxidase and / or lactate dehydrogenase to detect lactate. In some aspects, the enzyme is pyruvate oxidase to detect pyruvate. In some aspects, the enzymes are alcohol oxidase and xanthine oxidase to detect ethanol or other alcohols. In some aspects, the enzyme is [3-hydroxybutyrate dehydrogenase to detect a ketone. In some aspects, the enzymes are creatine amidinohydrolase, creatinine amidohydrolayse, and sarcosine oxidase to detect creatinine. If necessary, one or more cofactors can be included with the enzyme, which serves as a catalyst for the electron transfer. Suitable cofactors include, e.g., pyrroloquinoline quinone (PQQ), thiamine pyrophosphate (TPP), flavin adenine dinucleotide (FAD), nicotinamide adenine dinucleotide (NAD), and any combination thereof such as those described in U.S. Ser. No. 18 / 440,379 and U.S. Pat. Nos.6,736,957 and 8,268,143, the disclosures of each of which are incorporated herein by reference in their entirety.

[0173] For example, but not by way of limitation, a glutamate sensing layer can include a glutamate oxidase. In certain aspects, a glutamate-responsive sensing layer contains a glutamate oxidase that converts L-glutamate into a-ketoglutarate (also referred to as “a- ketoglutaric acid”) and reduces glutamate oxidase. The reduced form of the glutamate oxidase can then transfer electron(s) to a redox mediator, which in turn can then be oxidizedat an anode, i.e., the working electrode. The electrons transferred during this reaction provide the basis for glutamate detection at the working electrode. The electrochemical signal obtained can then be correlated to the amount of glutamate that was initially present in the sample.

[0174] In certain aspects, the enzyme can be an oxidoreductase. In certain aspects, the oxidoreductase can be an enzyme belonging to enzyme class 1. For example, but not by way of limitation, the enzyme can belong to enzyme class 1.1 (e.g., 1.1.1 or 1.1.3), enzyme class 1.4 (e.g., 1.4.3), or enzyme class 1.5. In certain aspects, the enzyme can be a NAD(P)+-dependent dehydrogenase. In certain aspects, the enzyme can be a flavin adenine dinucleotide (FAD)-dependent oxidoreductase. In certain aspects, the enzyme can be a hydrolase. In certain aspects, the hydrolase can be an enzyme belonging to enzyme class 3. For example, but not by way of limitation, the enzyme can belong to enzyme class 3.5, e.g., 3.5.2 or 3.5.3.

[0175] In certain aspects, the one or more enzymes can be present in the sensing layer of the second sensor in various amounts. For example, but not by way of limitation, the enzyme can be present in the sensing layer of the second sensor in amount from about 0.005 pg to about 20 pg, e.g., from about 0.005 pg to about 15 pg, from about 0.005 pg to about 10 pg, from about 0.005 pg to about 5 pg, from about 0.01 pg to about 20 pg, from about 0.01 pg to about 15 pg, from about 0.01 pg to about 10 pg, from about 0.01 pg to about 5 pg, 0.01 pg to about 3 pg, from about 0.1 pg to about 15 pg, from about 0.1 pg to about 10 pg, from about 0.1 pg to about 5 pg, from about 1 pg to about 20 pg, from about 1 pg to about 15 pg, from about 1 pg to about 10 pg, from about 1 pg to about 5 pg or from about 5 pg to about 20 pg. In certain aspects, the enzyme is present in the sensing layer of the second sensor in an amount from about 1 pg to about 20 pg. In certain aspects, the enzyme is present in the sensing layer of the second sensor in an amount from about 0.01% to about 100% by weight of the sensing layer composition, such as, but not limited to, from about 0.1% to about 95% by weight, from about 0.1% to about 90% by weight, from about 0.1% to about 85% by weight, from about 0.1% to about 80% by weight, from about 0.1% to about 75% by weight, from about 0.1% to about 70% by weight, from about 0.1% to about 65% by weight, from about 0.1% to about 60% by weight, from about 0.1% to about 55% by weight, from about 0.1% to about 50% by weight, from about 0.1% to about 45% by weight, from about 0.1% to about 40% by weight, from about 0.1% to about 35% by weight,from about 0.1% to about 30% by weight, from about 0.1% to about 25% by weight, from about 0.1% to about 20% by weight, from about 0.1% to about 15% by weight or from about 0.1% to about 10% by weight or any values in between based on the weight of the total sensing layer composition. In certain aspects, the enzyme is present in the sensing layer of the second sensor in an amount from about 5% to about 20% by weight of the sensing layer composition.

[0176] In certain aspects, the sensing layer of the second analyte sensor can include one or more enzymes that can be used to detect glucose. For example, but not by way of limitation, an analyte sensor of the present disclosure can include at least a second sensor comprising a glucose-responsive sensing layer comprising one or more enzymes for detecting glucose, e.g., disposed on a first working electrode. In certain aspects, the glucose-responsive sensing layer of the second sensor can include one or more enzymes, as described in U.S. Patent 8,268,143 (the contents of which are incorporated by reference herein in their entirety). In certain aspects, the second analyte sensor can include a sensing layer comprising a glucose oxidase and / or a glucose dehydrogenase for detecting glucose. In certain aspects, the glucose dehydrogenase can be a pyrroloquinoline quinone (PQQ) or a cofactor-dependent glucose dehydrogenase, e.g., flavin adenine dinucleotide (FAD)- dependent glucose dehydrogenase or nicotinamide adenine dinucleotide (NAD)-dependent glucose dehydrogenase. In certain aspects, the sensing layer of the second sensor can further include diaphorase. In certain aspects, the enzyme for detecting glucose in the second sensor is an FAD-dependent glucose oxidase.

[0177] In certain aspects, the sensing layer of the second analyte sensor can include one or more enzymes that can be used to detect ketones. For example, but not by way of limitation, an analyte sensor of the present disclosure can include at least a second sensor comprising a ketone-responsive sensing layer comprising one or more enzymes, e.g., an enzyme system, for detecting ketones, e.g., disposed on a first working electrode. In certain aspects, the ketones-responsive sensing layer of the second sensor can include an enzyme system comprising multiple enzymes that are capable of acting in concert to facilitate detection of ketones, as described in U.S. Patent Publication No. 2020 / 0237275 (the contents of which are incorporated by reference herein in their entirety). In certain aspects, the second analyte sensor can include a sensing layer comprising P-hydroxybutyrate dehydrogenase fordetecting ketones. In certain aspects, the sensing layer of the second sensor can further include diaphorase.

[0178] In certain aspects, the sensing layer of the second analyte sensor can include one or more enzymes that can be used to detect lactate. For example, but not by way of limitation, an analyte sensor of the present disclosure can include at least a second sensor comprising a lactate-responsive sensing layer comprising one or more enzymes, e.g., an enzyme system, for detecting lactate, e.g., disposed on a first working electrode. In certain aspects, the lactate-responsive sensing layer of the second sensor can include an enzyme system comprising multiple enzymes that are capable of acting in concert to facilitate detection of lactate, as described in U.S. Publication No. 2019 / 0320947 (the contents of which are incorporated by reference herein in their entirety). In certain aspects, the second analyte sensor can include a sensing layer comprising a lactate dehydrogenase and / or a lactate oxidase for detecting lactate. In certain aspects, the sensing layer of the second sensor can further include diaphorase.

[0179] In certain aspects, the sensing layer of the second analyte sensor can include one or more enzymes that can be used to detect alcohol, e.g., ethanol. For example, but not by way of limitation, an analyte sensor of the present disclosure can include at least a second sensor comprising an alcohol-responsive sensing layer comprising one or more enzymes, e.g., an enzyme system, for detecting alcohol, e.g., disposed on a first working electrode. In certain aspects, the ethanol-responsive sensing layer of the second sensor can include an enzyme system comprising multiple enzymes that are capable of acting in concert to facilitate detection of ethanol, as in U.S. Patent Publication No. 2020 / 0237277 (the contents of which are incorporated by reference herein in their entirety). In certain aspects, the second analyte sensor can include a sensing layer comprising an alcohol dehydrogenase or a ketoreductase for detecting alcohol.

[0180] In certain aspects, the sensing layer of the second analyte sensor can include one or more enzymes that can be used to detect creatinine. For example, but not by way of limitation, an analyte sensor of the present disclosure can include at least a second sensor comprising a creatinine-responsive sensing layer comprising one or more enzymes, e.g., an enzyme system, for detecting creatinine, e.g., disposed on a first working electrode. In certain aspects, the creatinine-responsive sensing layer of the second sensor can include an enzyme system comprising multiple enzymes that are capable of acting in concert tofacilitate detection of creatinine, e.g., as described in U.S. Patent Publication No.2020 / 0241015 (the contents of which are incorporated by reference herein in their entirety). In certain aspects, the second analyte sensor can include a sensing layer comprising an creatinine amidohydrolase, creatine amidinohydrolase and sarcosine oxidase for detecting creatinine.

[0181] In certain aspects, the sensing layer of the second analyte sensor can include one or more enzymes that can be used to detect glutamate. For example, but not by way of limitation, an analyte sensor of the present disclosure can include at least a second sensor comprising a glutamate-responsive sensing layer comprising one or more enzymes, e.g., an enzyme system, for detecting glutamate, e.g., disposed on a first working electrode. In certain aspects, the second analyte sensor can include a sensing layer comprising a glutamate dehydrogenase or a glutamate oxidase for detecting glutamate.

[0182] In certain aspects, the sensing layer of the second analyte sensor can include one or more enzymes that can be used to detect pyruvate. For example, but not by way of limitation, an analyte sensor of the present disclosure can include at least a second sensor comprising a pyruvate-responsive sensing layer comprising one or more enzymes, e.g., an enzyme system, for detecting pyruvate, e.g., disposed on a first working electrode. In certain aspects, the second analyte sensor can include a sensing layer comprising a pyruvate oxidase for detecting pyruvate.

[0183] In some aspects, the sensing layer of the second analyte sensor further comprises one or more additional cofactors for the analyte-sensing enzyme. In some aspects, the one or more additional cofactors comprise flavin adenine dinucleotide (FAD) and / or nicotinamide adenine dinucleotide (NAD). In some aspects the cofactor(s) is present in an amount of from about 1 to about 100 mg / mL such as from about 5 to about 20 mg / mL e.g. about 10 mg / mL.

[0184] In some aspects, the sensing layer of the second analyte sensor can further comprise a stabilizing agent such as a protein, e.g. an albumin. In an aspect, the albumin can be a serum albumin, such as bovine serum albumin (BSA) or human serum albumin (HSA). In certain aspects, the sensing layer of the second analyte sensor can comprise human serum albumin. In certain aspects, the sensing layer of the second analyte sensor can comprise bovine serum albumin (BSA). In some aspects the stabilizing agent is present in an amount of from about 1 to about 100 mg / mL such as from about 5 to about 20 mg / mL e.g. about10 mg / mL. The sensing layer of the optional second analyte sensor can generally be produced as described above in more detail for the first analyte sensor. For example, the sensing layer of the optional second analyte sensor typically has a thickness that ranges from about 0.1-10 pm. For example, each sensing layer can be 0.1 pm thick or more (e.g., 0.2 pm or more, 0.3 pm or more, 0.5 pm or more, 0.8 pm or more, 1 pm or more, 2 pm or more, 3 pm or more, 5 pm or more, or 8 pm or more) and typically will have a thickness of 10 pm or less (e.g., 8 pm or less, 5 pm or less, 3 pm or less, 2 pm or less, 1 pm or less, 0.8 pm or less, 0.5 pm or less, 0.3 pm or less, or 0.2 pm or less). In an example, the sensing layer of the second analyte sensor can have a thickness of about 0.1 to about 10 pm, about 0.2 to about 8 pm, about 0.5 to about 5 pm, about 1 to about 4 pm, or about 2 pm.

[0185] In some aspects, a conductive material such as, for example, carbon nanotubes, graphene, or metal nanoparticles, can be combined within the sensing layer of the second analyte sensor to promote rapid attainment of a steady state current. Any of the conductive materials described above for the first analyte sensor can be used, and when a second analyte sensor is present the conductive materials used for the first and second analyte sensors may be the same or different. Conductive material can be included in a range from about 0.1% to about 50% by weight (pbw) of the sensing layer of the second analyte sensor (e.g., about 1 to about 50 pbw, about 1 to about 10 pbw, or about 0.1 to about 10 pbw).

[0186] Both the first sensing electrode (e.g., oxygen sensing layer) and the optional second sensing electrode (e.g., second analyte sensing layer) comprise a redox mediator. Any of the redox mediators described above for the first analyte sensor can be used. Each redox mediator can be the same or different. In some aspects, the redox mediator can be the same for both the first and second sensing electrodes.

[0187] In some aspects, the sensing layer of the second analyte sensor can further comprise a buffer. Any of the buffers described above for the first analyte sensor can be used, and when a second analyte sensor is present the buffer used for the first and second analyte sensors may be the same or different.

[0188] A buffer and / or a stabilizing agent may be present in the second analyte sensor.Any of the buffers described above for the first analyte sensor can be used, and when a second analyte sensor is present the buffer used for the first and second analyte sensors may be the same or different. Any of the stabilizing agents described above for the first analytesensor can be used, and when a second analyte sensor is present the stabilizing agents used for the first and second analyte sensors may be the same or different.

[0189] The redox mediator of the second analyte sensing layer can be applied to a working electrode using any suitable technique, such as spray coating, painting, inkjet printing, stenciling, roller coating, dip coating, or any combination thereof. When a second analyte sensor is present the application method used for the first and second analyte sensors may be the same or different. In some aspects, the redox mediator can be applied by dip coating at least a portion of the working electrode into a solution of the redox mediator. One application or multiple applications can be applied (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 applications). In some aspects, the redox mediator can be applied in 1, 2, 3, or 4 applications (e.g., passes). In some aspects, the redox mediator can be applied in 1 or 2 applications (e.g., passes).

[0190] Accordingly, in some aspects provided herein is an oxygen sensor (e.g. a continuous oxygen sensor) comprising:a working electrode comprising carbon, the working electrode comprised in an implantable portion of the sensor; wherein the working electrode does not comprise an enzyme (i.e. is enzyme free).a sensing layer comprising a redox mediator comprising a polymer and an electron transfer agent disposed on at least a portion of the working electrode;- the redox mediator comprising an osmium complex bonded to a polymer or copolymer of poly(l -vinyl imidazole) or poly(4-vinylpyridine);a membrane coating the sensing layer,- the membrane comprising optionally crosslinked poly(4-vinylpyridine), poly(vinyl alcohol), poly(acrylic acid), poly(methacrylic acid), or a combination thereof; and having a thickness of from about 1 pm to about 100 pm.

[0191] In some aspects, provided herein is an oxygen sensor (e.g. a continuous oxygen sensor) comprising:a working electrode comprising carbon, e.g. a screen-printed carbon electrode, - the working electrode comprised in an implantable portion of the sensor; wherein the working electrode does not comprise an enzyme (i.e. is enzyme free).a sensing layer comprising a redox mediator comprising a polymer and an electron transfer agent disposed on at least a portion of the working electrode;- the redox mediator comprising an osmium-containing poly(4-vinylpyridine)-based polymer as defined herein; and optionally comprising a cross-linking agent (e.g. a polyepoxide (e.g., a polyethylene glycol diglycidylether (PEGDGE), ethylene glycol diglycidyl ether (EGDGE), resorcinol diglycidyl ether, 1,2, 7, 8-di epoxy octane, Gly3), cyanuric chloride, A-hydroxysuccinimide, an imidoester, epichlorohydrin, or any combination thereof);a membrane coating the sensing layerthe membrane comprising poly(4-vinylpyridine) thereof crosslinked with PEGDGE and having a thickness of from about 5 pm to about 30 pm.

[0192] In some aspects, provided herein is a dual analyte sensor (e.g. a continuous oxygen / dual analyte sensor) comprising:a first sensor which is an oxygen sensor or continuous oxygen sensor as described herein;a second sensor which is a sensor for a second analyte, e.g. glucose, lactate, ketone, glutamate, pyruvate, creatinine, sarcosine, and / or alcohol;- the second sensor comprising a working electrode comprising a sensing layer comprising a redox mediator comprising a polymer and an electron transfer agent disposed on at least a portion of the working electrode;- the redox mediator comprising an osmium complex bonded to a polymer or copolymer of poly(l -vinyl imidazole) or poly(4-vinylpyridine);- the sensing layer further comprising an enzyme for detecting the second analyte;- the sensing layer optionally further comprising a stabilizing agent (e.g. a protein, e.g.an albumin); and / or a pH buffer (e.g. a buffer that maintains the pH of the sensing layer to a pH of about pH 5 to about pH 8);a membrane coating the sensing layer, and- the membrane comprising optionally crosslinked poly(4-vinylpyridine), poly(vinyl alcohol), poly(acrylic acid), poly(methacrylic acid), or a combination thereof; and having a thickness of from about 1 pm to about 100 pm.Methods of Detecting Oxygen and, Optionally, Other Analytes

[0193] The present disclosure is also directed to a method for sensing oxygen.Accordingly, in some aspects provided herein is a method for detecting the presence,absence, or level (e.g. concentration) of oxygen in a sample (e.g. in a biofluid), the method comprising contacting the sample with a sensor as described herein; and taking one or more measurements characteristic of oxygen reduction by the sensor. In some aspects the one or more measurements are continuous measurements and the method is a method for determining the level (e.g. concentration) of oxygen in the sample (e.g. the biofluid) over time.

[0194] The present disclosure is also directed to a method for sensing (e.g. continuously sensing) oxygen that can comprise: a) exposing the continuous oxygen sensor or dual analyte sensor as described herein to a biofluid comprising oxygen; b) applying a potential to the working electrode, wherein the potential is sufficient to induce an oxidation reduction cascade that reduces oxygen (e.g. to water, hydrogen peroxide, or a combination thereof); c) obtaining a signal (e.g. a signal that is proportional to a concentration of oxygen in the biofluid); d) correlating the signal to the presence, absence, or level (e.g. concentration) of oxygen in the biofluid; and e) repeating steps a) through d) to provide the concentration of oxygen continuously for a period of time.

[0195] As described herein, the applied potential is from about -500 mV to about +500 mV vs the reference electrode. In some aspects, the potential applied to the continuous oxygen sensor can be from about -500 mV to about +500 mV, including ranges from about -500 mV to about +475 mV, from about -500 mV to about +450 mV, from about -500 mV to about +425 mV, from about -500 mV to about +400 mV, from about -500 mV to about +375 mV, from about -500 mV to about +350 mV, from about -500 mV to about +325 mV, from about -500 mV to about +300 mV, from about -500 mV to about +275 mV, from about -500 mV to about +250 mV, from about -500 mV to about +225 mV, from about -500 mV to about +200 mV, from about -500 mV to about +175 mV, from about - 500 mV to about +150 mV, from about -500 mV to about +125 mV, from about -500 mV to about +100 mV, from about -475 mV to about +100 mV, from about -450 mV to about +100 mV, from about -425 mV to about +100 mV, from about -400 mV to about +100 mV, from about -375 mV to about +100 mV, from about -350 mV to about +100 mV, from about -325 mV to about +100 mV, from about -300 mV to about +100 mV, from about -275 mV to about +100 mV, from about -250 mV to about +100 mV, from about - 225 mV to about +100 mV, from about -200 mV to about +100 mV, from about -175 mV to about +100 mV, from about -150 mV to about +100 mV, from about -100 mV to about+100 mV, or any range or value between any two preceding values, relative to the reference electrode. In some aspects, the applied potential can be about -500 mV, about -450 mV, about -400 mV, about -350 mV, about -300 mV, about -250 mV, about -200 mV, about - 150 mV, about -100 mV, about -50 mV, about +50 mV, about +100 mV, about +150 mV, about +200 mV, about +250 mV, about +300 mV, about +350 mV, about +400 mV, about +450 mV, about +500 mV, or any range or value between any two preceding values, vs the reference electrode. In some aspects, the applied potential is about -200 mV vs the reference electrode. Typically, a reference electrode as used herein is an Ag / AgCl reference electrode.

[0196] In some aspects, the method of continuously sensing oxygen provides the concentration of oxygen continuously for a period of time from about 1 day to about 30 days. In some aspects, the method of continuously sensing oxygen provides the concentration of oxygen continuously for a period of time from about 1 day to about 29 days, from about 2 days to about 28 days, from about 3 days to about 27 days, from about 4 days to about 26 days, from about 5 days to about 24 days, from about 6 days to about 22 days, from about 7 days to about 20 days, from about 8 days to about 18 days, from about 9 days to about 16 days, from about 10 days to about 14 days, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 21 days, about 22 days, about 23 days, about 24 days, about 25 days, about 26 days, about 27 days, about 28 days, about 29 days, or about 30 days. In some aspects, the method of continuously sensing oxygen provides the concentration of oxygen continuously for a period of time from about 7 days to about 30 days.

[0197] In some aspects, the steps (a) through (d) above are repeated at a sampling rate (sampling frequency). In some aspects a measurement is taken about every 1 ms, about every 10 ms, about every 100 ms, about every 1 s, about every 10 seconds, about every 30 seconds, about every minute, about every 5 minutes, about every 10 minutes, about every 30 minutes, or about every hour. In some aspects the potential is applied to the electrode constantly and the signal is obtained about every 1 ms, about every 10 ms, about every 100 ms, about every 1 s, about every 10 seconds, about every 30 seconds, about every minute, about every 5 minutes, about every 10 minutes, about every 30 minutes, or about everyhour. In some aspects the potential is applied about every 1 ms, about every 10 ms, about every 100 ms, about every 1 s, about every 10 seconds, about every 30 seconds, about every minute, about every 5 minutes, about every 10 minutes, about every 30 minutes, or about every hour and the signal is obtained when the potential is applied to the electrode.

[0198] 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 low degree of variability, e.g. change in detection signal. In some aspects, the continuous oxygen sensor or dual analyte sensor as described herein is stable for at least about 5 days, for at least about 6 days, for at least about 7 days, for at least about 8 days, for at least about 9 days, for at least about 10 days, for at least about 11 days, for at least about 12 days, for at least about 13 days, for at least about 14 days. In some aspects, the continuous oxygen sensor or dual analyte sensor as described herein is stable for at least about 7 days.

[0199] In some aspects, the continuous oxygen sensor or dual analyte sensor as described herein demonstrates an average signal variation of less than about 5% after 7 days. In some aspects, the continuous oxygen sensor or dual analyte sensor as described herein demonstrates an average change in detection signal after 7 days of about 10% or less, about 9.5% or less, about 9% or less, about 8.5% or less, about 8% or less, about 7.5% or less, about 7% or less, about 6.5% or less, about 6% or less, about 5.5% or less, about 5% or less, about 4.5% or less, 4% or less, about 3.5% or less, 3% or less, about 2.5% or less, 2% or less, about 1.5% or less, 1% or less, about 0.5% or less, about 0.4% or less, about 0.35% or less, about 0.3% or less, about 0.25% or less, about 0.2% or less, about 0.15% or less, about 0.1% or less, or any range or value between any two preceding values, e.g. about 0.1% to about 1% variability over 7 days, about 0.1% to about 0.5% variability over 7 days, or, for example, about 0.3 to about 0.4% variability over 7 days. In some aspects, the continuous oxygen sensor or dual analyte sensor as described herein demonstrates an average variability after 7 days ranging from about 0.01% to about 5%. In some aspects, the continuous oxygen sensor or dual analyte sensor as described herein demonstrates an average variability of about 3% or less after 7 days.

[0200] In general, the method uses a system (e.g., a sensor), as disclosed herein, for measuring concentrations of oxygen and can be used in an in vivo monitoring system, which while positioned in vivo in a user (e.g., a patient, such as a human) makes contact with thebiofluid of the user and senses one or more oxygen levels contained therein. An in vivo monitoring system can include one or more reader devices that receives sensed analyte data from a sensor control device. The reader device can process and / or display the sensed oxygen data or sensor data in any number of forms to the user. In some aspects, the reader device can be a mobile communication device, such as a dedicated reader device (configured for communication with a sensor control device) optionally in conjunction with a computer system, a mobile telephone (e.g., a WiFi or internet-enabled smart phone), a tablet, a personal digital assistant (PDA), or a mobile smart wearable electronics assembly (e.g., a smart glass, smart glasses, watch, bracelet, or necklace). Configuring a reader device to an in vivo monitoring system is described, for example, in U.S. Patent 11,371,957, the disclosure of which is incorporated herein by reference in its entirety.

[0201] The reader device typically includes an input component, a display, and processing circuitry, which can include one or more processors, microprocessors, controllers, and / or microcontrollers, each of which can be a discrete chip or distributed amongst (and a portion of) a number of different chips. The processing circuitry can include a communications processor having on-board memory and an applications processor having on-board memory. The reader device can further include radio frequency (RF) communication circuitry coupled with an RF antenna, a memory, multi-functional circuitry with one or more associated antennas, a power supply, power management circuitry, and / or a clock. It will be recognized that other hardware and functionality can be included in the reader device.

[0202] The present disclosure is further illustrated by the following embodiments.

[0203] (1) An oxygen sensor comprising a first portion configured to be positioned above a user’s skin and a second portion configured to be transcutaneously positioned beneath the skin and in contact with an interstitial fluid to detect oxygen in vivo, the second portion comprising a working electrode, and a redox mediator, wherein the redox mediator is disposed on at least a portion of the working electrode.

[0204] (2) The oxygen sensor of embodiment 1, wherein the redox mediator does not comprise an enzyme.

[0205] (3) The oxygen sensor of embodiment 1 or 2, wherein the redox mediator comprises a polymer and an electron transfer agent.

[0206] (4) The oxygen sensor of any one of the preceding embodiments, which is a continuous oxygen sensor.

[0207] (5) The oxygen sensor or continuous oxygen sensor of any one of embodiments 3- 4, wherein the polymer comprises poly(vinylpyridine), poly(vinylimidazole), poly(thiophene), poly(aniline), poly(pyrrole), poly(acetylene), polyethylene, polyacrylate, polymethacrylate, polystyrene, polyurethane, polyurea, or any combination thereof.

[0208] (6) The oxygen sensor or continuous oxygen sensor of any one of embodiments 3- 5, wherein the polymer comprises a polymer or copolymer repeat unit comprising at least one pendant pyridinyl group, imidazolyl group, or both a pyridinyl and imidazolyl group.

[0209] (7) The oxygen sensor or continuous oxygen sensor of any one of embodiments 3- 6, wherein the electron transfer agent comprises a transition metal complex.

[0210] (8) The oxygen sensor or continuous oxygen sensor of embodiment 7, wherein the transition metal complex comprises osmium, ruthenium, iron, cobalt, vanadium, or any combination thereof and at least one ligand coupled to the polymer backbone.

[0211] (9) The oxygen sensor or continuous oxygen sensor of embodiment 7 or 8, wherein the transition metal complex is an osmium transition metal complex comprising one or more ligands, wherein at least one ligand comprises a nitrogen-containing heterocycle coupled to the polymer backbone.

[0212] (10) The oxygen sensor or continuous oxygen sensor of any one of embodiments 1- 9, wherein the redox mediator comprises an osmium complex bonded to a poly(4- vinylpyridine)-based polymer.

[0213] (11) The oxygen sensor or continuous oxygen sensor of any one of embodiments 1- 10, wherein the redox mediator further comprises a cross linking agent.

[0214] (12) The oxygen sensor or continuous oxygen sensor of embodiment 11, wherein the cross linking agent is a polyepoxide, cyanuric chloride, N-hydroxysuccinimide, an imidoester, epichlorohydrin, or any combination thereof.

[0215] (13) The oxygen sensor or continuous oxygen sensor of embodiment 11 or 9, wherein the cross linking agent is a polyethylene glycol diglycidyl ether (PEGDGE).

[0216] (14) The oxygen sensor or continuous oxygen sensor of any one of embodiments 1- 13, wherein the oxygen sensor or continuous oxygen sensor further comprises an albumin and optionally a pH buffer.

[0217] (15) The oxygen sensor or continuous oxygen sensor of any one of embodiments 1- 14, further comprising a membrane overcoating the redox mediator and at least a portion of the working electrode.

[0218] (16) The oxygen sensor or continuous oxygen sensor of embodiment 15, wherein the membrane comprises poly(4-vinylpyridine).

[0219] (17) The oxygen sensor or continuous oxygen sensor of any one of embodiments 1- 16, further comprising a reference electrode, a counter electrode, or both a reference electrode and a counter electrode.

[0220] (18) The oxygen sensor or continuous oxygen sensor of any one of embodiments 1- 17, further comprising at least one insulating layer.

[0221] (19) The oxygen sensor or continuous oxygen sensor of any one of embodiments 1- 18, further comprising a substrate, wherein the working electrode is disposed on the substrate.

[0222] (20) The oxygen sensor or continuous oxygen sensor of embodiment 19, wherein the substrate comprises an implantable portion configured for implantation into a tissue.

[0223] (21) The oxygen sensor or continuous oxygen sensor of any one of embodiments 1- 20, wherein the redox mediator is continuously disposed on the working electrode.

[0224] (22) The oxygen sensor or continuous oxygen sensor of any one of embodiments 1- 20, wherein the redox mediator is discontinuously disposed on the working electrode.

[0225] (23) A dual analyte sensor system comprising the oxygen sensor or continuous oxygen sensor of any one of embodiments 1-22, and a second sensor that senses a second analyte.

[0226] (24) The dual analyte sensor system of embodiment 23, wherein the second analyte is glucose, lactate, or ketone.

[0227] (25) The dual analyte sensor system of embodiment 23 or 24, wherein the second sensor comprises a working electrode for the second analyte, and a sensing region disposed on at least a portion of the working electrode for the second analyte, wherein the sensing region comprises an enzyme responsive to the second analyte.

[0228] (26) A method of detecting the presence, absence, or level (e.g. concentration) of oxygen in a sample (e.g. in a biofluid), the method comprising contacting the sample with the oxygen sensor or continuous oxygen sensor of any one of embodiments 1-25; and taking one or more measurements characteristic of oxygen reduction by the sensor.

[0229] (27) A method of continuously sensing oxygen comprising:a) exposing the continuous oxygen sensor of any one of embodiments 4-22 or the dual analyte sensor of any one of embodiments 23-25 to a biofluid comprising oxygen; b) applying a potential to the working electrode, wherein the potential is sufficient to induce an oxidation reduction cascade that reduces oxygen to water, hydrogen peroxide, or a combination thereof;c) obtaining a signal that is proportional to a concentration of oxygen in the biofluid; d) correlating the signal to the concentration of oxygen in the biofluid; and e) repeating steps a) through d) to provide the concentration of oxygen continuously for a period of time.

[0230] (28) The method of embodiment 27, wherein the applied potential is from about - 500 mV to about +500 mV vs the reference electrode, wherein the reference electrode is an Ag / AgCl reference electrode.

[0231] (29) The method of embodiment 27, wherein the period of time is from about 7 days to about 30 days.EXAMPLES

[0232] The examples presented below are provided for the purpose of illustration only and the aspects described herein should in no way be construed as being limited to these examples. Rather, the aspects should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.Example 1

[0233] Enzyme free oxygen sensing layers were deposited using a solution formulated as specified in Table 1 below. The solution comprised a MES buffer, the osmium-based redox material known as osmium-containing poly(4-vinylpyridine)-based polymer, and crosslinking agent PEGDGE400. 24 nL of the solution mixture was deposited onto each of four screen-printed carbon electrodes to form a sensing layer on each electrode, the sensing layers each having an area of about 0.19 mm2. The resulting electrodes having a sensing layer were then cured overnight at 25 °C and at 60% humidity to provide a dried sensor.

[0234] Each dried electrode was then dip coated four times at an exit speed of 1 mm / s with the membrane formulation provided in Table 2 to form a membrane over each sensing layer, resulting in four dip coated electrodes having a membrane thickness of about 8 pm. Following the completion of dip coating, each of the dip coated electrodes was cured overnight at 25 °C and at 60% humidity. Spray coating, screen printing, or similar processes can also be used to deposit the membrane over at least the sensing layer.Table 1: Sensing LayerReagents Final (mg / mL)Os-PVP polymer 16.55Pegdge400 5.51Buffer (10 mM MES)Os-PVP polymer: osmium-containing poly(4-vinylpyridine)-based polymerPEGDGE400: polyethylene glycol diglycidylether with an average molecularweight (Mn) of about 400 g / molMES: 2-(N-morpholino)ethanesulfonic acidTable 2: Membrane CompositionMembrane Details100 mg / mLPVPin 80:20 EtOH:HEPES5 mg / mLPEGDGE400in 80:20 EtOH:HEPESPVP: poly(4-vinylpyridine) (PVP)EtOH: ethanolHEPES: 4-(2-hydroxyethyl)piperazine-l-ethanesulfonic acid (10 mM, pH 8)Example 2

[0235] The Test Electrodes prepared in Example 1 were each tested in a system comprising reference and counter electrodes to measure (a) an oxygen detection signal of each electrode (nA) at various oxygen concentrations over time; and (b) to assess whether the sensors were responsive to both increasing and decreasing oxygen concentrations. The sensors were tested in pH 7.4 PBS buffer at 33 °C. A potentiostat (CH Instrument modelCH1040C) was used to measure the detection signal (nA) of each sensor at varying oxygen concentrations. The reference and counter electrodes were Ag / AgCl and carbon, respectively. A constant potential of -200 mV was applied to the working electrode and currents were recorded as a sensor output signal. Argon and oxygen gas were mixed through a gas flow meter at different ratios to purge and vary the oxygen concentration in the buffer. The oxygen concentration started under air with about 21% oxygen and was subsequently decreased to 5%, 2.5%, 1.6%, 0.8%, and 0% by purging with the argon and oxygen gas mixture. The sensor’s responsiveness to increasing oxygen concentrations was then tested by increasing the oxygen concentration back to 0.8%, 1.6%, 2.5%, 5%, and finally back to 21% under air. Data was collected for each sensor and the resulting data was averaged. The averaged sensor response is shown in FIG. 9. The results shown in FIG. 9 demonstrate that the sensors were responsive to both increasing and decreasing oxygen concentrations. With these data, an average calibration curve shown in FIG. 10 was prepared. The average detection signal (nA) for the Test Electrodes is shown in Table 3. The Test Electrodes showed a sensitivity to change in oxygen concentration with an oxygen response of 0.253 nA / Ch%.

[0236] The stability of the four Test Electrodes is shown in FIG. 11 with the average change (%) in detection signal (nA) over a period of 7 days shown in Table 3. Stability was monitored by measuring current continuously for a period of about 7 days at the highest oxygen concentration (21%). Sensors were calibrated again with changing oxygen concentration by repeating the argon and oxygen purging procedure as described above and as shown in FIG. 9. The average sensor response after 7 days was substantially the same as the result at day 0, as shown in FIG. 11. These results demonstrate that the Test Electrodes remained stable over a period of 7 days. See e.g., Table 3.Table 3: Average of Test Electrodes 1-4 responsesAverage Oxygen StabilityElectrode Current CV* Response (variability(nA)* (nA / O2%) over 7 days)Test Electrodes-6.28 0.67 0.253 0.37%1-4* collected at 21% oxygen concentration at 17 hoursExample 3

[0237] Bilirubin oxidase is known to be able to catalyze the reduction of oxygen. To compare the effects of the presence of an enzyme in the oxygen sensor, an enzyme containing oxygen sensor and an enzyme free oxygen sensor were prepared using the oxygen sensing layer formulations as specified in Table 4, shown below. Each sensing layer was applied to the electrode using the process described in Example 1. After application of the sensing layers, the membrane formulation as specified in Table 2 was then dip coated over each electrode using the process described in Example 1.

[0238] A constant potential of -200 mV was applied to the working electrodes and the currents were recorded as the sensor output signal. Stability was monitored by measuring the signal (current) continuously for a period of about 12 days, as shown in FIG. 12. The results show that although the initial sensitivity of the enzyme-containing electrode was higher, the sensor signal started decaying after about 2 days while the enzyme-free oxygen sensor maintained stability over 12 days.Table 4.Final (mg / mL) Final (mg / mL)Reagentswith enzyme without enzymeBilirubin oxidase 16.55 -BSA 13.8 13.8Os-PVP polymer 16.55 16.55Pegdge400 5.51 5.51Buffer (lO mMMES) (lO mMMES)BSA: bovine serum albuminOs-PVP polymer: Osmium-containing poly(4-vinylpyridine)-based polymerPEGDGE400: polyethylene glycol diglycidylether with an average molecular weight(Mn) of about 400 g / molMES: 2-(N-morpholino)ethanesulfonic acidExample 4

[0239] A dual oxygen and lactate sensor is prepared using the oxygen sensing layer as specified in Table 1 shown above, and the lactate sensing layer formulation as specified inTable 5 below. The oxygen sensing layer is applied to a first electrode and the lactate sensing layer is applied to a second electrode as described in Example 1. A membrane is dip coated over the electrodes with the oxygen and lactate sensing layers using an alcohol- buffer solution comprising the materials specified in Table 2. The membrane solution is deposited using 5 dips at a dipping exit speed of 2 mm / s, resulting in a membrane thickness of about 23 pm.Table 5.in 10 mM MESComponent(pH5.5) Volumemg / mL mLBufferLOX 80 0.32HSA 80 0.32Os-PVPpolymer 40 0.24PEGDGE400 40 0.16LOX: lactate oxidaseHSA: human serum albuminOs-PVP polymer: Osmium-containing poly(4-vinylpyridine)-based polymerPEGDGE400: polyethylene glycol diglycidylether with an average molecular weight (Mn)of about 400 g / molExample 5

[0240] A dual oxygen and glucose sensor is prepared using the oxygen sensing layer as specified in Table 1 shown above, and the glucose sensing layer formulation as specified in Table 6 or Table 7 below. The oxygen sensing layer is applied to a first electrode and the glucose sensing layer is applied to a second electrode as described in Example 1. A membrane is dip coated over the electrodes with the oxygen and glucose sensing layers using an alcohol-buffer solution comprising the materials specified in Table 2. The membrane solution is deposited using 5 dips at a dipping exit speed of 2 mm / s, resulting in a membrane thickness of about 23 pm.Table 6.in lO mMHEPESComponent(pH8) Volumemg / mL RatioBufferFADGDH 20 0.12Os-PVP polymer 20 0.05PEGDGE400 20 0.044FADGDH: flavin adenine dinucleotide dependent glucose dehydrogenaseOs-PVP polymer: osmium-containing poly(4-vinylpyridine)-based polymerPEGDGE400: polyethylene glycol diglycidylether with an average molecular weight (Mn)of about 400 g / molTable 7.in lO mMHEPES FinalComponent(PH8) Concentrationmg / mL mg / mLBufferGOX 60 24.6Os-PVP polymer 60 20.4PEGDGE400 60 15GOX: glucose oxidaseOs-PVP polymer: osmium-containing poly(4-vinylpyridine)-based polymerPEGDGE400: polyethylene glycol diglycidylether with an average molecularweight (Mn) of about 400 g / molExample 6

[0241] A dual oxygen and ketone sensor is prepared using the oxygen sensing layer as specified in Table 1 shown above, and the ketone sensing layer formulation as specified in Table 8 below. The oxygen sensing layer is applied to a first electrode and the ketone sensing layer is applied to a second electrode as described in Example 1. A membrane is dip coated over the electrodes with the oxygen and ketone sensing layers using an alcohol- buffer solution comprising the materials specified in Table 2. The membrane solution is deposited using 5 dips at a dipping exit speed of 2 mm / s, resulting in a membrane thickness of about 23 pm.Table 8.in lO mMMESComponent(pH5.5)mg / mLBufferHBDH 8Diaphorase 4Albumin 8NAD+ 8Os-PVP polymer 8PEGDGE400 4HBDH: p-hydroxybutyrate dehydrogenaseNAD+: nicotinamide adenine dinucleotideOs-PVP polymer: osmium-containing poly(4-vinylpyridine)-based polymerPEGDGE400: polyethylene glycol diglycidylether with an average molecular weight (Mn)of about 400 g / molExample 7

[0242] A dual oxygen and creatinine sensor is prepared using the oxygen sensing layer as specified in Table 1 shown above, and the creatinine sensing layer formulation as specified in Table 9 below. The oxygen sensing layer is applied to a first electrode and the creatinine sensing layer is applied to a second electrode as described in Example 1. A membrane is dip coated over the electrodes with the oxygen and creatinine sensing layers using an alcohol-buffer solution comprising the materials specified in Table 2. The membrane solution is deposited using 5 dips at a dipping exit speed of 2 mm / s, resulting in a membrane thickness of about 23 pm.Table 9.Concentration (mg / mL)Componentin 10 mM MESCNH 20CRH 40SOX 5Os-PVP polymer 8.5PEGDGE400 6.5CNH: creatinine amidohydrolaseCRH: creatine amidinohydrolaseSOX: sarcosine oxidaseOs-PVP polymer: osmium-containing poly(4-vinylpyridine)-based polymerPEGDGE400: polyethylene glycol diglycidylether with an average molecular weight (Mn)of about 400 g / mol

[0243] It is to be appreciated that the Detailed Description section, and not the Summary and Abstract sections, is intended to be used to interpret the claims. The Summary and Abstract sections may set forth one or more but not all exemplary embodiments of thepresent invention as contemplated by the inventor(s), and thus, are not intended to limit the present invention and the appended claims in any way.

[0244] The present disclosure has been described above with the aid of functional building blocks illustrating the implementation of specified functions and relationships thereof. The boundaries of these functional building blocks have been arbitrarily defined herein for the convenience of the description. Alternate boundaries can be defined so long as the specified functions and relationships thereof are appropriately performed.

[0245] The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying knowledge within the skill of the art, readily modify and / or adapt for various applications such specific embodiments, without undue experimentation, without departing from the general concept of the present invention. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance.

[0246] The breadth and scope of the present invention should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the following claims and their equivalents.

Claims

WHAT IS CLAIMED IS:

1. A continuous oxygen sensor comprisinga first portion configured to be positioned above a user’s skin; and a second portion configured to be transcutaneously positioned beneath the skin and in contact with an interstitial fluid to detect oxygen in vivo, the second portion comprising:a working electrode comprising carbon, anda redox mediator comprising a polymer, an electron transfer agent, and no enzyme; wherein the redox mediator is disposed on at least a portion of the working electrode.The continuous oxygen sensor of claim 1, wherein the polymer comprises poly(vinylpyridine), poly(vinylimidazole), poly(thiophene), poly(aniline), poly(pyrrole), poly(acetylene), polyethylene, polyacrylate, polymethacrylate, polystyrene, polyurethane, polyurea, or any combination thereof.3 The continuous oxygen sensor of claim 1 or 2, wherein the polymer comprises a polymer or copolymer repeat unit comprising at least one pendant pyridinyl group, imidazolyl group, or both a pyridinyl and imidazolyl group.The continuous oxygen sensor of any one of claims 1-3, wherein the electron transfer agent comprises a transition metal complex.5 The continuous oxygen sensor of claim 4, wherein the transition metal complex comprises osmium, ruthenium, iron, cobalt, vanadium, or any combination thereof and at least one ligand coupled to the polymer backbone.6 The continuous oxygen sensor of claim 4 or 5, wherein the transition metal complex is an osmium transition metal complex comprising one or more ligands, wherein at least one ligand comprises a nitrogen-containing heterocycle coupled to the polymer backbone.7 The continuous oxygen sensor of any one of claims 1-6, wherein the redox mediator comprises an osmium complex bonded to a poly(4-vinylpyridine)-based polymer.8 The continuous oxygen sensor of any one of claims 1-7, wherein the redox mediator further comprises a cross linking agent.

9. The continuous oxygen sensor of claim 8, wherein the cross linking agent is a polyepoxide, cyanuric chloride, N-hydroxysuccinimide, an imidoester, epichlorohydrin, or any combination thereof.

10. The continuous oxygen sensor of claim 8 or 9, wherein the cross linking agent is a polyethylene glycol diglycidyl ether (PEGDGE).

11. The continuous oxygen sensor of any one of claims 1-10, wherein the continuous oxygen sensor further comprises an albumin and optionally a pH buffer.

12. The continuous oxygen sensor of any one of claims 1-11, further comprising a membrane overcoating the redox mediator and at least a portion of the working electrode.

13. The continuous oxygen sensor of claim 12, wherein the membrane comprises poly(4- vinylpyridine).

14. The continuous oxygen sensor of any one of claims 1-13, further comprising a reference electrode, a counter electrode, or both a reference electrode and a counter electrode.

15. The continuous oxygen sensor of any one of claims 1-14, further comprising at least one insulating layer.

16. The continuous oxygen sensor of any one of claims 1-15, further comprising a substrate, wherein the working electrode is disposed on the substrate.

17. The continuous oxygen sensor of claim 16, wherein the substrate is a implantable portion configured for implantation into a tissue.

18. The continuous oxygen sensor of any one of claims 1-17, wherein the redox mediator is continuously disposed on the working electrode.

19. The continuous oxygen sensor of any one of claims 1-17, wherein the redox mediator is discontinuously disposed on the working electrode.

20. A dual analyte sensor system comprising the continuous oxygen sensor of any one of claims 1-19, and a second sensor that senses a second analyte.

21. The dual analyte sensor system of claim 20, wherein the second analyte is glucose, lactate, or ketone.

22. The dual analyte sensor system of claim 20 or 21, wherein the second sensor comprises:a working electrode for the second analyte, anda sensing region disposed on at least a portion of the working electrode for the second analyte, wherein the sensing region comprises an enzyme responsive to the second analyte.

23. A method of continuously sensing oxygen comprising:a) exposing the continuous oxygen sensor of any one of claims 1-19 or the dual analyte sensor of any one of claims 20-22 to a biofluid comprising oxygen;b) applying a potential to the working electrode, wherein the potential is sufficient to induce an oxidation reduction cascade that reduces oxygen to water, hydrogen peroxide, or a combination thereof;c) obtaining a signal that is proportional to a concentration of oxygen in the biofluid; d) correlating the signal to the concentration of oxygen in the biofluid; and e) repeating steps a) through d) to provide the concentration of oxygen continuously for a period of time.

24. The method of claim 23, wherein the applied potential is from about -500 mV to about +500 mV vs the reference electrode.

25. The method of claim 23, wherein the period of time is from about 7 days to about 30 days.

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