pH-INSENSITIVE APTAMERIC SENSING SYSTEM

Abstract

An analyte monitoring sensor configured for in vivo measurement of at least one analyte is provided, the sensor comprising An electrochemical aptamer biosensor comprising: at least one aptamer electrically associated with at least one working electrode, the at least one aptamer configured to undergo a reversible conformational change in the presence of at least one analyte; a redox moiety coupled to the one or more aptamer, wherein the redox moiety provides a signal corresponding to a concentration of the at least one analyte and / or the at least one aptamer is sensitive to changes in pH in about the at least one electrode; and at least one pH sensor electrically isolated from the at least one working electrode. Methods of extending in vivo performance of the analyte monitoring sensor are also described.

Description

Attorney Docket No.: 0978-PCT01-0243pH-INSENSITIVE APTAMERIC SENSING SYSTEMTechnical Field

[0001] This disclosure is directed to protective materials for an aptamer-based biosensor construct or device suitable for wholly or partially implantation in a subject for continuous monitoring of an analyte.BACKGROUND

[0002] Aptamer biosensors (AB) are a class of affinity biosensors in which the recognition element is the aptamer (single stranded DNA / RNA) which has a specific affinity to the analyte, and such aptamer-analyte interaction induces a measurable transduced signal (optical, electrical). Existing aptamer biosensors (AB's) are limited to the poor stability observed when placed in physiological relevant environments. Electrochemically -based Aptamer biosensors (EABs) are currently limited in useful end-of-life. Aptamer sensors with useable redox probes for signal transduction are prone to variation in signal due to many factors, including, but not limited to pH, temperature, and leaching metal ions from electrodes.SUMMARY

[0003] In examples, an electrochemical aptamer biosensor is provided, the electrochemical aptamer biosensor comprising: at least one aptamer electrically associated with at least one working electrode, the at least one aptamer configured to undergo a reversible conformational change in the presence of at least one analyte; a redox moiety coupled to the one or more aptamer, wherein the redox moiety provides a signal corresponding to a concentration of the at least one analyte wherein the at least one aptamer and / or the redox moiety is sensitive to changes in pH; and at least one pH sensor electrically isolated from the at least one working electrode.

[0004] In aspects, reduction and oxidation of the redox moiety is reversible. In aspects, alone or in combination with any previous aspect, the at least one working electrode comprises a first working electrode and a second working electrode, the second working electrode electrically isolated from the first working electrode. In aspects, alone or in combination with any previous aspect, the at least second working electrode is configured to generate a signal associated with a second analyte, the second analyte being chemically different from the first analyte.Attorney Docket No.: 0978-PCT01-0243

[0005] In aspects, alone or in combination with any previous aspect, the electrochemical aptamer biosensor further comprises a reference electrode and / or a counter electrode.

[0006] In aspects, alone or in combination with any previous aspect, the at least one aptamer is present on a wire substrate or a planar substrate. In aspects, alone or in combination with any previous aspect, the pH sensor is present on a wire substrate or a planar substrate.

[0007] In aspects, alone or in combination with any previous aspect, the at least one working electrode comprises a first working electrode and a second working electrode, the second working electrode being electrically isolated from the first working electrode. In aspects, alone or in combination with any previous aspect, the at least one aptamer is present on the first electrode wire substrate and the pH electrode is present on the second working electrode.

[0008] In aspects, alone or in combination with any previous aspect, the planar substrate comprises a first electrode and a second working electrode, the second working electrode being electrically isolated from the first working electrode. In aspects, alone or in combination with any previous aspect, the at least one aptamer is present on the first electrode of the planar substrate and the pH electrode is present on the second working electrode of the planar substrate.

[0009] In aspects, alone or in combination with any previous aspect, at least a portion of the working electrode is a conductive metal. In aspects, alone or in combination with any previous aspect, at least a portion of the working electrode is gold, carbon, conductive ink, graphene, or graphene oxide. In aspects, alone or in combination with any previous aspect, at least a portion of the pH sensor comprises an ion selective polymer coated wire or an ion selective polymer coated contact pad.

[0010] In aspects, alone or in combination with any previous aspect, at least a portion of the pH sensor comprises a field effect transistor. In aspects, alone or in combination with any previous aspect, at least a portion of the pH sensor comprises an ion-sensitive field effect transistor (ISFET). In aspects, alone or in combination with any previous aspect, at least a portion of the pH sensor comprises a non-silicone based ion-sensitive field effect transistor. In aspects, alone or in combination with any previous aspect, at least a portion of the pH sensor comprises a nano ion-sensitive field effect transistor.Attorney Docket No.: 0978-PCT01-0243

[0011] In aspects, alone or in combination with any previous aspect, at least a portion of the pH sensor comprises transition metal dichalcogenide, graphene, carbon nanotubes, zinc oxide, compound semiconductor or combinations thereof. In aspects, alone or in combination with any previous aspect, at least a portion of the pH sensor comprises a silicon-on insulator ion-sensitive field effect transistor. In aspects, alone or in combination with any previous aspect, at least a portion of the pH sensor comprises an extended gate field effect transistor.

[0012] In aspects, alone or in combination with any previous aspect, at least a portion of the pH sensor comprises metal oxide. In aspects, alone or in combination with any previous aspect, In aspects, alone or in combination with any previous aspect, at least a portion of the pH sensor comprises a thin / thick film metal oxide electrode. In aspects, alone or in combination with any previous aspect, at least a portion of the pH sensor comprises a complementary metal oxide semiconductor, at least a portion of the pH sensor comprises a complementary metal oxide semiconductor ISFET. In aspects, alone or in combination with any previous aspect, at least a portion of the pH sensor comprises a silicon-on insulator metal oxide semiconductor field-effect transistor.

[0013] In aspects, alone or in combination with any previous aspect, at least a portion of the pH sensor comprises a conductive polymer. In aspects, alone or in combination with any previous aspect, the electrochemical aptamer biosensor further comprises a temperature sensor.

[0014] In aspects, alone or in combination with any previous aspect, the sensor is configured for transcutaneous insertion. In aspects, alone or in combination with any previous aspect, the sensor further comprises one or more of a transmitter, receiver, controller, or power supply.

[0015] In aspects, alone or in combination with any previous aspect, the reversible redox moiety comprises iron, iridium, ruthenium, osmium, a thiazine dye, or derivative thereof. In aspects, alone or in combination with any previous aspect, the reversible redox moiety comprises a ferrocene, methylene blue, or derivative thereof.

[0016] In aspects, alone or in combination with any previous aspect, the at least one aptamer is physically associated to a portion of the working electrode. In aspects, alone or inAttorney Docket No.: 0978-PCT01-0243combination with any previous aspect, the at least one aptamer is covalently associated to a portion of the working electrode.

[0017] In aspects, alone or in combination with any previous aspect, further comprising at least one co-adsorbate. In aspects, alone or in combination with any previous aspect, the at least one co-adsorbate is physically associated to a portion of the working electrode. In aspects, alone or in combination with any previous aspect, the at least one co-adsorbate is covalently associated to a portion of the working electrode.

[0018] In aspects, alone or in combination with any previous aspect, the at least one aptamer comprises RNA or DNA nucleotide sequences. In aspects, alone or in combination with any previous aspect, the at least one aptamer comprises at least one of 2'-O-methyl modification of a nucleotide, disulfide bridges, a 3' cap with an inverted 2-deoxy thymidine, a 3'-3'-thymidine linkage at 3' terminus, a 2'-F modification, conjugation to biotin, or a double stranded section.

[0019] In aspects, alone or in combination with any previous aspect, the at least one aptamer comprises the RNA or the DNA sequences with a first linker moiety on a 5' end and the reversible redox moiety at a 3' end, or wherein the at least one aptamer comprises the RNA or the DNA sequences with a first linker moiety on a 3' end and the reversible redox moiety at a 5' end.

[0020] In aspects, alone or in combination with any previous aspect, the first linker moiety on the 5' end or 3' end comprises an amino group or a carboxyl group. In aspects, alone or in combination with any previous aspect, the first linker moiety is physically or chemically coupled to the substrate at the 5' end or 3' end. In aspects, alone or in combination with any previous aspect, the first linker moiety is physically or chemically coupled to the co-adsorbate at the 5' end or the 3' end.

[0021] In aspects, alone or in combination with any previous aspect, the at least one aptamer is a glycopeptide antibiotic binding aptamer. In aspects, alone or in combination with any previous aspect, the glycopeptide antibiotic is selected from vancomycin, teicoplanin, telavancin, ramoplanin, decaplanin, corbomycin, complestatin, or bleomycin.

[0022] In aspects, alone or in combination with any previous aspect, the at least one aptamer is a vancomycin binding aptamer. In aspects, alone or in combination with anyAttorney Docket No.: 0978-PCT01-0243previous aspect, the at least one aptamer is a neurotransmitter binding aptamer or a hormone binding aptamer.

[0023] In aspects, alone or in combination with any previous aspect, the at least one aptamer is a dopamine, L-DOPA, insulin, or glutamate binding aptamer. In aspects, alone or in combination with any previous aspect, the at least one aptamer is a carbohydrate, triglyceride or fatty acid binding aptamer. In aspects, alone or in combination with any previous aspect, the at least one aptamer is a glucose, a glycerol, or a beta-hydroxy butyrate binding aptamer.

[0024] In aspects, alone or in combination with any previous aspect, the at least one aptamer is physically or chemically coupled to a self-assembled monolayer (SAM). In aspects, alone or in combination with any previous aspect, the at least one aptamer is physically or chemically coupled to a mono-functional alkanethiol, a multi-functional alkanethiol, ora mercaptoalkanol.

[0025] In aspects, alone or in combination with any previous aspect, the at least one aptamer is physically or chemically coupled to an alkylthiol betaine. In aspects, alone or in combination with any previous aspect, the at least one aptamer is physically or chemically coupled to an aliphatic amine. In aspects, alone or in combination with any previous aspect, the at least one aptamer is physically or chemically coupled to an amino alkanoic acid.

[0026] In aspects, alone or in combination with any previous aspect, the reversible redox moiety comprises an organometallic compound comprising iron, iridium, ruthenium, or osmium. In aspects, alone or in combination with any previous aspect, the reversible redox moiety comprises a thiazine dye, or derivative thereof. In aspects, alone or in combination with any previous aspect, the reversible redox moiety comprises ferrocene, methylene blue, or a derivative thereof.

[0027] In aspects, alone or in combination with any previous aspect, the electrochemical aptamer biosensor is sterile.

[0028] In aspects, alone or in combination with any previous aspect, further comprising a reference electrode coating. In aspects, alone or in combination with any previous aspect, the reference electrode coating comprises a hydrophobic polymer or a hydrophilic polymer. In aspects, alone or in combination with any previous aspect, the reference electrode coating comprises a fluorine-containing polymer. In aspects, alone or in combination withAttorney Docket No.: 0978-PCT01-0243any previous aspect, the fluorine-containing polymer comprises Teflon or a fluorinated alkyl polymer.

[0029] In aspects, alone or in combination with any previous aspect, the reference electrode coating comprises a deposited film coating. In aspects, alone or in combination with any previous aspect, the deposited film coating comprises parylene C, parylene D, parylene N, or derivatives thereof.

[0030] In aspects, alone or in combination with any previous aspect, the reference electrode coating comprises a polymer chain having both hydrophilic and hydrophobic regions. In aspects, alone or in combination with any previous aspect, the reference electrode coating comprises a polymer chain having polyurethane and / or polyurea segments.

[0031] In aspects, alone or in combination with any previous aspect, the polymer comprises hard segments and soft segments. In aspects, alone or in combination with any previous aspect, the soft segments comprise poly(tetramethylene oxide) repeating units. In aspects, alone or in combination with any previous aspect, the soft segments comprise polydialkylsiloxane repeating units. In aspects, alone or in combination with any previous aspect, the soft segments comprise both poly(tetramethylene oxide) repeating units and polydialkylsiloxane repeating units.

[0032] In aspects, alone or in combination with any previous aspect, further comprising a contacting layer adjacent the at least one aptamer. In aspects, alone or in combination with any previous aspect, the contacting layer comprises a polymer having both hydrophilic and hydrophobic components.

[0033] In aspects, alone or in combination with any previous aspect, the contacting layer comprises a polyurethane and / or polyurea polymer. In aspects, alone or in combination with any previous aspect, the polyurethane and / or polyurea polymer comprises hard segments and soft segments. In aspects, alone or in combination with any previous aspect, the soft segments comprise poly(tetramethylene oxide) repeating units. In aspects, alone or in combination with any previous aspect, the soft segments comprise polydialkylsiloxane repeating units and / or polyalkylcarbonate repeating units. In aspects, alone or in combination with any previous aspect, the soft segments comprise polyftetramethylene oxide) repeating units and polydialkylsiloxane repeating units.Attorney Docket No.: 0978-PCT01-0243

[0034] In aspects, alone or in combination with any previous aspect, the contacting layer comprises a polyurethane and / or polyurea polymer blended with polyvinylpyrrolidone. In aspects, alone or in combination with any previous aspect, the contacting layer comprises a polyelectrolyte polymer or a polymerized monomer comprising a zwitterionic functional group.

[0035] In aspects, alone or in combination with any previous aspect, the contacting layer further comprises one or more co-adsorbates. In aspects, alone or in combination with any previous aspect, the one or more co-adsorbates comprises a self-assembled monolayer (SAM) associated with the working electrode. In aspects, alone or in combination with any previous aspect, each of the one or more co-adsorbates comprises a one or more functional groups. In aspects, alone or in combination with any previous aspect, the one or more co-adsorbates comprises a mono-functional or a multi-functional alkanethiol, mercaptoalkanol, alkylmercaptoalkanol, or arylmercaptoalkanol functional group.

[0036] In aspects, alone or in combination with any previous aspect, the functional group comprises one or more ammoniophosphonates, ammoniophosphinates, ammoniosulfonates, alkanethiol betaine, ammoniosulfates, ammoniocarboxylates,and ammoniocarboxylates.

[0037] In aspects, alone or in combination with any previous aspect, the contacting layer provides for one or more of a surface energy range, a pH range, a phase separation range, and an intermolecular interaction range.

[0038] In aspects, alone or in combination with any previous aspect, the contacting layer further comprises at least one mono-, di-, tri- or polysaccharide. In aspects, alone or in combination with any previous aspect, the at least one mono-, di-, tri- or polysaccharide is at least partially amorphous between a temperature range of 0 °C to 40 °C.

[0039] In aspects, alone or in combination with any previous aspect, further comprising at least one encapsulating layer adjacent the contacting layer. In aspects, alone or in combination with any previous aspect, the encapsulating layer is different than the contacting layer. In aspects, alone or in combination with any previous aspect, the encapsulating layer is different than the reference electrode coating.

[0040] In aspects, alone or in combination with any previous aspect, the encapsulating layer comprises a polymer having both hydrophilic and hydrophobic regions. In aspects,Attorney Docket No.: 0978-PCT01-0243alone or in combination with any previous aspect, the encapsulating layer comprises a polyurethane and / or polyurea polymer. In aspects, alone or in combination with any previous aspect, the polyurethane and / or polyurea polymer comprises hard segments and soft segments. In aspects, alone or in combination with any previous aspect, the soft segments comprise poly(tetramethylene oxide) repeating units. In aspects, alone or in combination with any previous aspect, the soft segments comprise polydialkylsiloxane repeating units and / or polyalkylcarbonate repeating units. In aspects, alone or in combination with any previous aspect, the soft segments comprise both poly(tetramethylene oxide) repeating units and / or polydialkylsiloxane repeating units and / or polyalkylcarbonate repeating units.

[0041] In aspects, alone or in combination with any previous aspect, the encapsulating layer comprises a polyurethane and / or polyurea polymer blended with polyvinylpyrrolidone. In aspects, alone or in combination with any previous aspect, the encapsulating layer comprises a polyelectrolyte polymer or a polymerized monomer comprising a zwitterionic functional group. In aspects, alone or in combination with any previous aspect, the encapsulating layer comprises a polymer with a styrene group. In aspects, alone or in combination with any previous aspect, the encapsulating layer comprises a polymer with a heterocyclic group.

[0042] In aspects, alone or in combination with any previous aspect, the encapsulating layer comprises a polymer chain having poly(l-vinyl imidazole), poly(4-vinyl pyridine), poly (2-vinyl pyridine), polyacrylonitrile, polyacrylamide, and / or copolymers or quaternized forms thereof.

[0043] In aspects, alone or in combination with any previous aspect, further comprising a topcoat adjacent the encapsulating layer, the topcoat comprising a polymer chain having poly(l-vinyl imidazole), poly(4-vinyl pyridine), poly(2-vinyl pyridine), acrylonitrile, acrylamide, and / or copolymers or quaternized forms thereof.

[0044] In aspects, alone or in combination with any previous aspect, the encapsulating layer and / or the topcoat is at least partially cross-linked.

[0045] In examples, a method of measuring a concentration of an analyte in vivo with an electrochemical aptamer biosensor system is provided, the method comprising: providing an electrochemical aptamer biosensor system configured for in vivo use, theAttorney Docket No.: 0978-PCT01-0243electrochemical aptamer biosensor system comprising: at least one aptamer associated with an electroactive surface configured for obtaining a first signal corresponding to a concentration of an analyte, the aptamer comprising a reversible redox moiety coupled thereto; and a pH sensor configured for obtaining a second signal corresponding to a pH value in proximity to the at least one aptamer.

[0046] In aspects, alone or in combination with any previous aspect, further comprising adjusting the first signal based on a parameter related to the second signal and improving accuracy of the electrochemical aptamer biosensor system.

[0047] In aspects, alone or in combination with any previous aspect, at least a portion of the pH sensor comprises an ion selective polymer or ion selective membrane coated wire or contact pad.

[0048] In aspects, alone or in combination with any previous aspect, at least a portion of the pH sensor comprises a field effect transistor (FET), an ion-sensitive field effect transistor (ISFET), a non-silicone based ion-sensitive field effect transistor a nano ion-sensitive field effect transistor (Nano-ISFET), a transition metal dichalcogenide (TMD), graphene, carbon nanotubes, zinc oxide, compound semiconductors, a silicon-on insulator ion-sensitive field effect transistor (SOI ISFET), an extended gate field effect transistor (EGFET). a metal oxide, a thin / thick film metal oxide electrode a complementary metal oxide semiconductor (CMOS), a complementary metal oxide semiconductor ISFET (CMOS ISFET), a silicon-on insulator metal oxide semiconductor field-effect transistor (SOI MOSFET), or combinations thereof.

[0049] In aspects, alone or in combination with any previous aspect, at least a portion of the pH sensor comprises a conductive polymer.

[0050] In aspects, alone or in combination with any previous aspect, the electrochemical aptamer biosensor system further comprises a temperature sensor.

[0051] In aspects, alone or in combination with any previous aspect, further comprising a reference and / or counter electrode. In aspects, alone or in combination with any previous aspect, the reference electrode comprises a reference electrode coating configured to reduce release of cations from the reference electrode.

[0052] In aspects, alone or in combination with any previous aspect, the reference electrode coating comprises a hydrophobic polymer or a hydrophilic polymer. In aspects,Attorney Docket No.: 0978-PCT01-0243alone or in combination with any previous aspect, the reference electrode coating comprises a polymer chain having both hydrophilic and hydrophobic regions.

[0053] In aspects, alone or in combination with any previous aspect, the reference electrode coating comprises a polymer chain having polyurethane and / or polyurea segments. In aspects, alone or in combination with any previous aspect, the polymer comprises hard segments and soft segments. In aspects, alone or in combination with any previous aspect, the soft segments comprise poly(tetramethylene oxide) repeating units.

[0054] In aspects, alone or in combination with any previous aspect, the soft segments comprise polydialkylsiloxane repeating units and / or polyalkylcarbonate repeating units. In aspects, alone or in combination with any previous aspect, the soft segments comprise both poly(tetramethylene oxide) repeating units and / or polydialkylsiloxane repeating units and / or polyalkylcarbonate repeating units.

[0055] In aspects, alone or in combination with any previous aspect, the reference electrode coating comprises a fluorine-containing polymer. In aspects, alone or in combination with any previous aspect, the fluorine-containing polymer comprises Teflon or a fluorinated alkyl polymer.

[0056] In aspects, alone or in combination with any previous aspect, the reference electrode coating comprises a deposited film coating. In aspects, alone or in combination with any previous aspect, the deposited film coating comprises parylene C, parylene D, parylene N, or derivatives thereof.

[0057] In examples, a method of operating an electrochemical aptamer analyte sensor for measuring a concentration of at least one analyte in a fluid is provided, the analyte sensor comprising: an analyte sensing portion disposed on a surface of a first working electrode, the analyte sensing portion comprising: at least one aptamer electrically associated with the first working electrode, the at least one aptamer configured to undergo a reversible conformational change in the presence of the at least one analyte; a reversible redox moiety coupled to the at least one aptamer; at least one pH electrode electrically isolated from the first working electrode; the analyte sensing portion configured for introduction to a subcutaneous space, the analyte sensing portion capable of at least measurin the concentration of the at least one analyte; the method comprising: applying a potential to the first working electrode at or above an oxidation-reduction potential of theAttorney Docket No.: 0978-PCT01-0243reversible redox moiety to generate a signal corresponding to the concentration in the subcutaneous space; applying a potential to the pH electrode to generate a second signal corresponding to the pH concentration in the subcutaneous space; and adjusting the first signal based on a parameter related to the second signal.BRIEF DESCRIPTION OF THE DRAWINGS

[0058] In order to understand and to see how the present disclosure may be carried out in practice, examples will now be described, by way of non-limiting examples only, with reference to the accompanying drawings, in which:

[0059] FIGs. 1A and IB are schematic diagrams illustrating an aptamer protective material employed in a biosensor in accordance with the broadest aspect of the present disclosure.

[0060] FIG. 1C is a representative schematic for a linear substrate aptamer construct with aptamer protective material in accordance with this disclosure.

[0061] FIG. ID is a hypothetical graph of potential vs. electric double layer, in accordance with this disclosure.

[0062] FIG. IE is a representative schematics for a microporous substrate aptamer construct with aptamer protective material in accordance with this disclosure.

[0063] FIG. IF is a hypothetical graph of potential vs. electric double layer in accordance with this disclosure.

[0064] FIGs. 2A and 2B, are a schematic representations of exemplary co-adsorbates in accordance with the broadest aspect of the present disclosure.

[0065] FIGs. 3A, 3B, 3C, and 3D are schematic diagrams illustrating alternative structures of a sensing region with exemplary aptamer protective layers of the exemplary aptamer biosensor as shown in FIG.3A.

[0066] FIGS. 4A-4E illustrate an exemplary double-sided, co-planar un-connected analyte sensor, in accordance with an example.

[0067] FIGS. 5A-5E illustrate an exemplary double-sided, co-planar connected analyte sensor, in accordance with an example.

[0068] FIG. 6A is a perspective view of the in vivo portion of an example of a multielectrode sensor system, in accordance with an example.

[0069] FIG. 6B is an enlarged view of the distal portion 6B of FIG. 6A.Attorney Docket No.: 0978-PCT01-0243

[0070] FIG. 7A is an illustration of an example analyte sensor in accordance with the broadest aspect of the present disclosure.

[0071] FIG. 7B is an enlarged view of an example analyte sensor of the analyte sensor system shown in FIG. 7A.

[0072] FIG. 7C is a cross-sectional view of the analyte sensor of FIG. 7B.

[0073] FIG. 8 is a diagram illustrating certain embodiments of an example continuous analyte monitoring sensor system communicating with at least one display device in accordance with various technologies described in the present disclosure.

[0074] FIG. 9 is a diagram showing one example of a medical device system described in the present disclosure.

[0075] FIG. 10 depicts peak current vs. peak voltage relationship of an EAB sensor.

[0076] FIGs. 11A, 11B depict experimental data demonstrating the effect of pH on peak voltage and peak current of a EAB with an exemplary redox moiety.

[0077] FIG. 12 depicts experimental results of peak current vs. peak voltage of a plurality of EAB sensors with redox moiety without analyte present, in vitro, demonstrating the effect of pH on EAB sensor performance.

[0078] FIG. 13 depicts peak current-peak voltage data of an in vivo EAB sensor with redox moiety and the effect of pH changes in the in vivo milieu.

[0079] FIG. 14 depicts peak current-peak voltage data of an in vivo EAB sensor over time, and the effect of pH changes on these parameters in the in vivo milieu.

[0080] FIGs. 15A, 15B depict experimental data demonstrating the effect of temperature on EAB peak current and peak voltage, respectively.DETAILED DESCRIPTION

[0081] Despite significant progress that has been made towards the implementation of AB and EAB devices in vivo, important challenges must be overcome in terms of aptamer stability to facilitate their continuous operation in complex samples such as blood or ISF. There is a need to develop novel EAB interfaces that resist degradation overtime as a result of continuous electrochemical interrogation in biological fluids for prolonged periods.

[0082] It has been observed that signals from electrochemical aptamer sensors (EAB) are prone to pH interference. While not to be held to any particular theory, it is believed that the pH interference is due to one or more of the following:Attorney Docket No.: 0978-PCT01-0243

[0083] i. reaction rate of the redox probe (used to transduce the aptamer-analyte binding event) is pH-sensitive. For example, with methylene blue (MB) redox moiety, the redox reaction of one MB molecule involves one proton, making it sensitive to hydrogen ion concentration (environmental pH variation) during use;

[0084] ii. DNA / RNA backbone of the Aptamer can be pH sensitive. For example, protonation / deprotonation of DNA / RNA bases results in different favored base pairing and thus may alter the secondary structure of aptamer. Variation in secondary structure will likely result in EAB signal variation during use; and

[0085] iii. subcutaneous interstitial fluid has varying pH (sensor insertion creates wound, and thus will cause variation of local pH about the aptameric sensor during use).

[0086] The presently disclosed systems and methods addresses the technical problems. In examples, alone or in combination, the presently disclosed systems and methods provide the following technical solutions:

[0087] altering both peak voltage and peak current during sensor interrogation (square wave voltammetry (SWV), differential pulse voltammetry (DPV), alternating current voltammetry (ACV), cyclic voltammetry (CV)); and / or

[0088] incorporating a pH sensing electrode (pH sensor) as a part of multi-analyte sensing strategy; and / or

[0089] chemically modifying the redox moiety.Definitions

[0090] The term "about" as used herein is a broad term, and is to be given its ordinary and customary meaning to a person of ordinary skill in the art (and is not be limited to a special or customized meaning), and refers without limitation to allowing for a degree of variability in a value or range, for example, within 10%, within 5%, or within 1% of a stated value or of a stated limit of a range, and includes the exact stated value or range. The term "substantially" as used herein refers to a majority of, or mostly, as in at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or at least about 99.999% or more, or 100%. The phrase "substantially free of" as used herein can mean having none or having a trivial amount of, such that the amount of material present does not affect the material properties of the composition including the material, such that about 0 wt. % to about 5 wt. % of the composition is the material, or about 0 wt. % to about 1 wt.Attorney Docket No.: 0978-PCT01-0243%, or about 5 wt. % or less, or less than or equal to about 4.5 wt. %, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.01, or about 0.001 wt. % or less, or about 0 wt. %.

[0091] The term "adhere" and "attach" as used herein are broad terms, and are to be given their ordinary and customary meaning to a person of ordinary skill in the art (and are not be limited to a special or customized meaning), and refer without limitation to hold, bind, or stick, for example, by gluing, bonding, grasping, interpenetrating, or fusing.

[0092] The terms "analyte" as used herein is a broad term, and is to be given its ordinary and customary meaning to a person of ordinary skill in the art (and is not to be limited to a special or customized meaning), and refers without limitation to a substance or chemical constituent in a biological fluid (e.g., blood, interstitial fluid, cerebral spinal fluid, lymph fluid, urine, sweat, saliva, etc.) that can be analyzed. Analytes can include naturally occurring substances, artificial substances, drugs, toxins, metabolites, and / or reaction products.

[0093] The phrases "analyte-measuring device," "analyte-monitoring device," "analytesensing device," "continuous analyte sensing device," "continuous analyte sensor device," and / or "multi-analyte sensor device" as used herein are broad phrases, and are to be given their ordinary and customary meaning to a person of ordinary skill in the art (and are not to be limited to a special or customized meaning), and refer without limitation to an apparatus and / or system responsible for the detection of, or transduction of a signal associated with, a particular analyte, or combination of analytes. For example, these phrases may refer without limitation to an instrument responsible for detection of a particular analyte or combination of analytes. In examples, the instrument includes a sensor coupled to circuitry disposed within a housing, and configure to process signals associated with analyte concentrations into information. In examples, such apparatuses and / or systems are capable of providing specific quantitative, semi-quantitative, qualitative, and / or semi qualitative analytical information using a biological recognition element combined with a transducing and / or detecting element.

[0094] The term "aptamer" as used herein is a broad term, and is to be given its ordinary and customary meaning to a person of ordinary skill in the art (and is not to be limited to a special or customized meaning), and refers without limitation to an oligonucleotide or a peptide that binds to a biological analyte. Aptamers can be ofAttorney Docket No.: 0978-PCT01-0243oligonucleotide or peptide origin. Oligonucleotide aptamers include nucleic acid species that have been engineered through repeated rounds of in vitro selection or equivalently, SELEX (systematic evolution of ligands by exponential enrichment) to bind to biological analytes such as small molecules, proteins, nucleic acids, and even cells, tissues and organisms. Peptide aptamers include polypeptides selected or engineered to bind an analyte. Peptide aptamers can comprise or consist of one or more peptide loops of variable sequence presented in a protein scaffold. Peptide aptamer selection can be made using different systems, including the yeast two-hybrid system, combinatorial peptide libraries constructed by phage display and other surface display technologies such as mRNA display, ribosome display, bacterial display and yeast display, collectively, "biopannings." Peptides aptamers can be chosen from the MimoDB database. Peptide aptamers can also be isolated from combinatorial libraries created by directed mutation or rounds of variable region mutagenesis and selection. Commercially available aptamers, including aptamers with a transducing element can be purchased, for example, from Biosearch Technologies (Hoddesdon, UK).

[0095] The phrases "aptamer protective material," "aptamer protective domain," "aptamer protective membrane," "aptamer protective region," "aptamer protective matrix," and "aptamer protective layer" as used herein and collectively referred to as "aptamer protective layer " or "APL", are broad phrases, and are to be given their ordinary and customary meaning to a person of ordinary skill in the art (and is not to be limited to a special or customized meaning), and refers without limitation to any substance, domain, membrane, region, polymer, matrix or layer that cooperatively functions with one or more aptamers configured to transduce a signal corresponding to a concentration of a biological analyte. For example, APL provides one or more of the following attributes: allows an aptamer to undergo conformational transformations within the APL; allows transport of one or more analytes; provides an electrochemical and / or physiochemical environment about the aptamer for stabilizing the aptamer itself, or its coupling to a substrate, or longevity of a redox moiety coupled to the aptamer; and reduces or eliminates drift of signal over time in vivo.

[0096] The phrase and term "bioactive agent" and "bioactive" as used herein is a broad phrase and a broad term, and are to be given their ordinary and customary meaning to aAttorney Docket No.: 0978-PCT01-0243person of ordinary skill in the art (and is not to be limited to a special or customized meaning), and refers without limitation to any substance that has an effect on or elicits a response from living tissue, for example, drugs, biologies, reactive oxygen scavenger (ROS), and metal ions.

[0097] The phrases "biointerface membrane," "biointerface domain," and "biointerface layer" as used interchangeably herein are broad phrases, and are to be given their ordinary and customary meaning to a person of ordinary skill in the art (and are not to be limited to a special or customized meaning), and refer without limitation to a permeable membrane (which can include multiple domains) or layer that functions as a bioprotective interface between host tissue and an implantable device. The terms "biointerface" and "bioprotective" are used interchangeably herein.

[0098] The terms "biosensor" and / or "sensor" as used herein are broad terms and are to be given their ordinary and customary meaning to a person of ordinary skill in the art (and are not to be limited to a special or customized meaning), and refer without limitation to a part of an analyte measuring device, analyte-monitoring device, analyte sensing device, continuous analyte sensing device, continuous analyte sensor device, and / or multi-analyte sensor device responsible for the detection of, or transduction of a signal associated with, a particular analyte or combination of analytes. In examples, the biosensor or sensor generally comprises a body, a working electrode, a reference electrode, and / or a counter electrode coupled to body and forming surfaces configured to provide signals during electrochemically reactions. One or more membranes can be affixed to the body and cover electrochemically reactive surfaces. In examples, such biosensors and / or sensors are capable of providing specific quantitative, semi-quantitative, qualitative, semi qualitative analytical signals using a biological recognition element combined with a detecting and / or transducing element.

[0099] The term "biostable" as used herein is a broad term, and is to be given its ordinary and customary meaning to a person of ordinary skill in the art (and is not to be limited to a special or customized meaning), and refers without limitation to materials that are relatively resistant to degradation by processes that are encountered in vivo.

[0100] The term "co-adsorbate" as used herein is a broad term, and is to be given its ordinary and customary meaning to a person of ordinary skill in the art (and is not to be limited to a special or customized meaning), and refers without limitation to materials thatAttorney Docket No.: 0978-PCT01-0243adsorb, associate, or couple via covalent, ionic, or molecular interaction to a substrate surface.

[0101] The term "comprising" as used herein is synonymous with "including," "containing," or "characterized by," and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps.

[0102] The term "conjugate" as used herein, is a broad term, and is to be given its ordinary and customary meaning to a person of ordinary skill in the art (and is not to be limited to a special or customized meaning), and refers without limitation to aptamers or bioactive agents covalently linked through a linker to a substrate, co-adsorbate, carrier, or nanocarrier, such as a metal surface, conductive surface, or polymer. The linker can be biologically inactive, as in resisting the separation of the aptamer from the substrate when exposed or presented to a biological environment over a period of time suitable for continuous monitoring, such as with a wearable, or in a subcutaneous or transcutaneous environment, with or without a protective layer. The linker can be biologically active, as in capable of allowing the separation of the bioactive agent (e.g., an anti-inflammatory) from the carrier when exposed or presented to a biological environment, such as a subcutaneous or transcutaneous environment.

[0103] The term "continuous" as used herein is a broad term, and is to be given its ordinary and customary meaning to a person of ordinary skill in the art (and is not to be limited to a special or customized meaning), and refers without limitation to an uninterrupted or unbroken portion, domain, coating, or layer.

[0104] The phrase "continuous analyte sensing" as used herein is a broad phrase, and is to be given its ordinary and customary meaning to a person of ordinary skill in the art (and is not to be limited to a special or customized meaning), and refers without limitation to the period in which monitoring of an analyte concentration is continuously, continually, and / or intermittently (but regularly) performed, for example, from about every 5 seconds or less to about 10 minutes or more. In further examples, continuous monitoring of analyte concentration is performed from about every 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 second to about 1.25, 1.50, 1.75, 2.00, 2.25, 2.50, 2.75, 3.00, 3.25, 3.50, 3.75, 4.00, 4.25, 4.50, 4.75, 5.00, 5.25, 5.50, 5.75, 6.00, 6.25, 6.50, 6.75, 7.00, 7.25, 7.50, 7.75, 8.00, 8.25,Attorney Docket No.: 0978-PCT01-02438.50, 8.75, 9.00, 9.25, 9.50 or 9.75 minutes. In further examples, continuous monitoring of analyte concentration is performed daily and can be performed for weeks.

[0105] The term "coupled" as used herein is a broad term, and is to be given its ordinary and customary meaning to a person of ordinary skill in the art (and is not to be limited to a special or customized meaning), and refers without limitation to two or more system elements or components that are configured to be at least one of electrically, mechanically, thermally, operably, chemically or otherwise attached. Similarly, the phrases "operably connected", "operably linked", and "operably coupled" as used herein may refer to one or more components linked to another component(s) in a manner that facilitates transmission of at least one signal between the components. In some examples, components are part of the same structure and / or integral with one another (i.e. "directly coupled"). In other examples, components are connected via remote means. For example, one or more electrodes can be used to detect an analyte in a sample and convert that information into a signal; the signal can then be transmitted to an electronic circuit. In this example, the electrode is "operably linked" to the electronic circuit. The phrase "removably coupled" as used herein may refer to two or more system elements or components that are configured to be or have been electrically, mechanically, thermally, operably, chemically, or otherwise attached and detached without damaging any of the coupled elements or components. The phrase "permanently coupled" as used herein may refer to two or more system elements or components that are configured to be or have been electrically, mechanically, thermally, operably, chemically, or otherwise attached but cannot be uncoupled without damaging at least one of the coupled elements or components.

[0106] The term "discontinuous" as used herein is a broad term, and is to be given its ordinary and customary meaning to a person of ordinary skill in the art (and is not to be limited to a special or customized meaning), and refers without limitation to disconnected, interrupted, or separated portions, layers, coatings, or domains.

[0107] The term "distal" as used herein is a broad term, and is to be given its ordinary and customary meaning to a person of ordinary skill in the art (and is not to be limited to a special or customized meaning), and refers without limitation to a region spaced relatively far from a point of reference, such as an origin or a point of attachment.Attorney Docket No.: 0978-PCT01-0243

[0108] The term "domain" as used herein is a broad term, and is to be given its ordinary and customary meaning to a person of ordinary skill in the art (and is not to be limited to a special or customized meaning), and refers without limitation to a region of the membrane system that can be a layer, a uniform or non-uniform gradient (for example, an anisotropic region of a membrane), or a portion of a membrane that is capable of sensing one, two, or more analytes. The domains discussed herein can be formed as a single layer, as two or more layers, as pairs of bi-layers, or as combinations thereof.

[0109] The term "drift" as used herein is a broad term, and is to be given its ordinary and customary meaning to a person of ordinary skill in the art (and is not to be limited to a special or customized meaning), and refers without limitation to a progressive increase or decrease in signal over time that is unrelated to changes in host systemic analyte concentrations. While not wishing to be bound by theory, it is believed that drift may be the result of a local decrease in analyte transport to the sensor, for example, due to a formation of a foreign body capsule (FBC). It is also believed that an insufficient amount of interstitial fluid surrounding the sensor may result in reduced transport to the sensor. In examples, an increase in local interstitial fluid may slow or reduce drift and thus improve sensor performance. Drift may also be the result of sensor electronics, or algorithmic models used to compensate for noise or other anomalies that can occur with electrical signals in ranges including the milliampere range, microampere range, picoampere range, nanoampere range, and femtoampere range, likewise with faradic, capacitance, and voltage measurements.

[0110] The phrases "bioactive releasing membrane" and "drug releasing layer" and "bioactive releasing domain" and "bioactive agent releasing membrane" are used interchangeably herein and are each a broad phrase, and each are to be given its ordinary and customary meaning to a person of ordinary skill in the art (and is not to be limited to a special or customized meaning), and refers without limitation to a permeable or semi-permeable membrane which is permeable to one or more bioactive agents. In examples, the "bioactive releasing membrane" and "drug releasing layer" and "bioactive releasing domain" and "bioactive agent releasing membrane" can be comprised of two or more domains and is typically of a few microns thickness or more. In examples the bioactive releasing membrane and / or bioactive releasing membrane and / or bioactive agent releasingAttorney Docket No.: 0978-PCT01-0243membrane and / or and bioactive agent releasing membrane are substantially the same as the biointerface layer and / or biointerface membrane. In another example, the bioactive releasing membrane and / or bioactive releasing membrane and / or bioactive agent releasing membrane and / or and bioactive agent releasing membrane are distinct from the biointerface layer and / or biointerface membrane.

[0111] The term "electrochemically reactive surface" as used herein is a broad term, and is to be given its ordinary and customary meaning to a person of ordinary skill in the art (and is not to be limited to a special or customized meaning), and refers without limitation to the surface of an electrode where an electrochemical reaction takes place. In another example, electron transfer is provided using a redox moiety associated with an aptamer, where the redox moiety is capable of undergoing reduction-oxidation (redox) that is related to a reversible binding interaction of the aptamer and an analyte proportional to the analyte concentration.

[0112] The term "gain" as used herein is a broad term, and is to be given its ordinary and customary meaning to a person of ordinary skill in the art (and is not to be limited to a special or customized meaning), and refers without limitation to a differential measure between signal OFF state and signal ON state. For example, a typical range of gain is 1-200% of a signal percentage change produced by analyte of certain concentration as compared to zero analyte concentration. Analyte concentration is typically quantified in micromolar (uM), nanomolar (nM), nanograms / milliliter (ng / mL) or picograms / milliliter (pg / mL).

[0113] The phrase "hard segment" as used herein is a broad phrase, and is to be given its ordinary and customary meaning to a person of ordinary skill in the art (and is not to be limited to a special or customized meaning), and refers without limitation to an element of a copolymer, for example, a polyurethane, a polycarbonate polyurethane, or a polyurethane urea copolymer, which imparts resistance properties, e.g., resistance to bending or twisting. The term "hard segment" can be further characterized as a crystalline, semi-crystalline, or glassy material with a glass transition temperature determined by dynamic scanning calorimetry ("Tg") typically above ambient temperature, and is typically made of diisocyanate with or without chain extender.

[0114] The term "host" as used herein is a broad term, and is to be given its ordinary and customary meaning to a person of ordinary skill in the art (and is not to be limited to aAttorney Docket No.: 0978-PCT01-0243special or customized meaning), and refers without limitation to mammals, for example humans.

[0115] The terms "implanted" or "implantable" as used herein are broad terms, and are to be given their ordinary and customary meaning to a person of ordinary skill in the art (and are not to be limited to a special or customized meaning), and refer without limitation to objects (e.g., sensors) that are inserted subcutaneously (i.e. in the layer of fat between the skin and the muscle) or transcutaneously (i.e. penetrating, entering, or passing through intact skin), which may result in a sensor that has an in vivo portion and an ex vivo portion.

[0116] The terms "interfe rants" and "interfering species" as used herein are broad terms, and are to be given their ordinary and customary meaning to a person of ordinary skill in the art (and are not to be limited to a special or customized meaning), and refer without limitation to effects and / or species that interfere with the measurement of an analyte of interest in a sensor to produce a signal that does not accurately represent the analyte measurement. In examples of an electrochemical aptamer sensor, interfering species are compounds with a redox (reduction-oxidation) potential that overlaps with the analyte to be measured or one or more redox moieties associated with one or more aptamers.

[0117] The term "in vivo’’ as used herein is a broad term, and is to be given its ordinary and customary meaning to a person of ordinary skill in the art (and is not to be limited to a special or customized meaning), and without limitation is inclusive of the portion of a device (for example, a sensor) adapted for insertion into and / or existence within a living body of a host.

[0118] The term "ex vivo" as used herein is a broad term, and is to be given its ordinary and customary meaning to a person of ordinary skill in the art (and is not to be limited to a special or customized meaning), and without limitation is inclusive of a portion of a device (for example, a sensor) adapted to remain and / or exist outside of a living body of a host.

[0119] The term "membrane" as used herein is a broad term, and is to be given its ordinary and customary meaning to a person of ordinary skill in the art (and is not to be limited to a special or customized meaning), and refers without limitation to a structure configured to perform functions including, but not limited to, protection of the exposed electrode surface from the biological environment, diffusion resistance (limitation) of theAttorney Docket No.: 0978-PCT01-0243analyte, service as a matrix for a catalyst for enabling an enzymatic reaction, limitation or blocking of interfering species, provision of hydrophilicity at the electrochemically reactive surfaces of the sensor interface, service as an interface between host tissue and the implantable device, modulation of host tissue response via drug (or other substance) release, and combinations thereof. When used herein, the terms "membrane" and "matrix" are meant to be interchangeable.

[0120] The phrase "membrane system" as used herein is a broad phrase, and is to be given its ordinary and customary meaning to a person of ordinary skill in the art (and is not to be limited to a special or customized meaning), and refers without limitation to a permeable or semi-permeable membrane that can be comprised of two or more domains, layers, or layers within a domain, and is typically constructed of materials of a few microns thickness or more, which is permeable to analyte. In examples, the membrane system comprises an immobilized or encapsulated aptamer, which enables transduction to occur between the aptamer and analyte whereby a concentration of analyte can be measured.

[0121] The term "micro," as used herein is a broad term, and is to be given its ordinary and customary meaning to a person of ordinary skill in the art (and is not to be limited to a special or customized meaning), and refers without limitation to a small object or scale of approximately 106m that is not visible without magnification. The term "micro" is in contrast to the term "macro," which refers to a large object that may be visible without magnification. Similarly, the term "nano" refers to a small object or scale of approximately 10‘9m.

[0122] The term "noise," as used herein, is a broad term and is used in its ordinary sense, including, without limitation, a signal detected by the sensor or sensor electronics that is unrelated to analyte concentration and can result in reduced sensor performance. One type of noise has been observed during the few hours (e.g., about 2 to about 24 hours) after sensor insertion. Afterthe first 24 hours, the noise may disappear or diminish, but in some hosts, the noise may last for about three to four days. In some cases, noise can be reduced using predictive modeling, artificial intelligence, and / or algorithmic means. In other cases, noise can be reduced by addressing immune response factors associated with the presence of the implanted sensor, such as using a bioactive releasing membrane with at least one bioactive agent. For example, noise of one or more exemplary biosensors asAttorney Docket No.: 0978-PCT01-0243presently disclosed can be determined and then compared qualitatively or quantitatively. By way of example, by obtaining a raw signal timeseries with a fixed sampling interval (in units of picoampere (pA)), a smoothed version of the raw signal timeseries can be obtained, e.g., by applying a 3rd order lowpass digital Chebyshev Type II filter. Others smoothing algorithms can be used. At each sampling interval, an absolute difference, in units of pA, can be calculated to provide a smoothed timeseries. This smoothed timeseries can be converted into units (the unit of "noise"), using, for example, an analyte sensitivity timeseries, where the analyte sensitivity timeseries is derived by using a mathematical model between the raw signal and reference blood analyte measurements. Optionally, the timeseries can be aggregated as desired, e.g., by hour or day. Comparison of corresponding timeseries between different exemplary biosensors with the presently disclosed bioactive releasing membrane and one or more bioactive agents provides for qualitative or quantitative determination of improvement of noise.

[0123] The term "optional" or "optionally" as used herein is a broad term, and is to be given its ordinary and customary meaning to a person of ordinary skill in the art (and is not to be limited to a special or customized meaning), and, without limitation, means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0124] The phrase "polymerization group" used herein is a broad phrase, and is to be given its ordinary and customary meaning to a person of ordinary skill in the art (and is not to be limited to a special or customized meaning), and refers without limitation to a functional group that permits polymerization of the monomer with itself to form a homopolymer or together with different monomers to form a copolymer. Depending on the type of polymerization methods employed, the polymerization group can be selected from alkene, alkyne, epoxide, lactone, amine, hydroxyl, isocyanate, carboxylic acid, anhydride, silane, halide, aldehyde, and carbodiimide.

[0125] The term "polyzwitterions" as used herein is a broad term, and is to be given its ordinary and customary meaning to a person of ordinary skill in the art (and is not to be limited to a special or customized meaning), and refers without limitation to polymers where a repeating unit of the polymer chain is a zwitterionic moiety. Polyzwitterions are also known as polybetaines. Since polyzwitterions have both cationic and anionic groups,Attorney Docket No.: 0978-PCT01-0243they are a type of polyampholytic polymer. They are unique, however, because the cationic and anionic groups are both part of the same repeating unit, which means a polyzwitterion has the same number of cationic groups and anionic groups whereas other polyampholytic polymers can have more of one ionic group than the other. Also, polyzwitterions have the cationic group and anionic group as part of a repeating unit. Polyampholytic polymers need not have cationic groups connected to anionic groups; they can be on different repeating units and thus may be distributed apart from one another at random intervals, or one ionic group may outnumber the other.

[0126] The term "proximal" as used herein is a broad term, and is to be given its ordinary and customary meaning to a person of ordinary skill in the art (and is not to be limited to a special or customized meaning), and refers without limitation to the spatial relationship between various elements in comparison to a particular point of reference. For example, some examples of a device include a membrane system having a biointerface layer and an enzyme layer. If the sensor is deemed to be the point of reference and the enzyme layer is positioned nearer to the sensor than the biointerface layer, then the enzyme layer is more proximal to the sensor than the biointerface layer.

[0127] The phrase and term "processor module" and "microprocessor" as used herein are each a broad phrase and term, and are to be given their ordinary and customary meaning to a person of ordinary skill in the art (and are not to be limited to a special or customized meaning), and refer without limitation to a computer system, state machine, processor, or the like designed to perform arithmetic or logic operations using logic circuitry that responds to and processes the basic instructions that drive a computer.

[0128] The term "semi-continuous" as used herein is a broad term, and is to be given its ordinary and customary meaning to a person of ordinary skill in the art (and is not to be limited to a special or customized meaning), and refers without limitation to a portion, coating, domain, or layer that includes one or more continuous and noncontinuous portions, coatings, domains, or layers. For example, a coating disposed around a sensing region but not about the sensing region is "semi-continuous."

[0129] The phrases "sensing portion," "sensing membrane," "sensing region," "sensing domain," and / or "sensing mechanism" as used herein are broad phrases, and are to be given their ordinary and customary meaning to a person of ordinary skill in the art (and areAttorney Docket No.: 0978-PCT01-0243not to be limited to a special or customized meaning), and refer without limitation to the part of a biosensor and / or a sensor responsible for the detection of, or transduction of a signal associated with, a particular analyte or combination of analytes. In examples, the sensing portion, sensing membrane, and / or sensing mechanism generally comprise an electrode configured to provide signals during electrochemically reactions with one or more membranes covering electrochemically reactive surface. In examples, such sensing portions, sensing membranes, and / or sensing mechanisms are capable of providing specific quantitative, semi-quantitative, qualitative, semi qualitative analytical signals using a biological recognition element combined with a detecting and / or transducing element.

[0130] During general operation of the analyte measuring device, biosensor, sensor, sensing region, sensing portion, or sensing mechanism, a biological sample, for example, blood or interstitial fluid, or a component thereof contacts, either directly, or after passage through one or more membranes, an aptamer, or RNA or DNA protein, orfor example, one or more periplasmic binding protein (PBP) or mutant or fusion protein thereof having one or more analyte binding regions, each region capable of specifically and reversibly binding to at least one analyte. The interaction of the biological sample or component thereof with the analyte measuring device, biosensor, sensor, sensing region, sensing portion, or sensing mechanism results in transduction of a signal that permits a qualitative, semi-qualitative, quantitative, or semi-qualitative determination of the analyte level in the biological sample.

[0131] In examples, the sensing region or sensing portion can comprise at least a portion of a conductive substrate or at least a portion of a conductive surface, for example, a wire or conductive trace or a substantially planar substrate including substantially planar trace(s), and a membrane. In examples, the sensing region or sensing portion can comprise a non-conductive body, a working electrode, a reference electrode, and a counter electrode (optional), forming an electrochemically reactive surface at one location on the body and an electronic connection at another location on the body, and a sensing membrane affixed to the body and covering the electrochemically reactive surface.

[0132] In examples, multiple working electrodes can be employed. For example, a second working electrode comprising a plurality of different analyte (e.g., analyte 1, analyte2, etc.) aptamer on the second working electrode to correct for sensor drift and / or interference. Likewise, a second working electrode comprising a non-selective aptamer to aAttorney Docket No.: 0978-PCT01-0243plurality of different analytes (e.g., analyte 1, analyte2, etc) on the second working electrode can be used to correct for sensor drift and / or interference.

[0133] In another example, the sensing region can comprise one or more periplasmic binding protein (P BP) or mutant or fusion protein thereof having one or more analyte binding regions, each region capable of specifically and reversibly binding to at least one analyte. Mutations of the PBP can contribute to or alter one or more of the binding constants, extended stability of the protein, including thermal stability, to bind the protein to a special encapsulation matrix, membrane or polymer, or to attach a detectable reporter group or "label" to indicate a change in the binding region. Specific examples of changes in the binding region include, but are not limited to, hydrophobic / hydrophilic environmental changes, three-dimensional conformational changes, changes in the orientation of amino acid side chains in the binding region of proteins, and redox states of the binding region. Such changes to the binding region provide for transduction of a detectable signal corresponding to the one or more analytes present in the biological fluid.

[0134] In examples, the sensing region determines the selectivity among one or more analytes, so that only the analyte which has to be measured leads to (transduces) a detectable signal. The selection may be based on any chemical or physical recognition of the analyte by the sensing region, where the chemical composition of the analyte is unchanged, or in which the sensing region causes or catalyzes a reaction of the analyte that changes the chemical composition of the analyte.

[0135] The sensing region transduces the recognition of analytes into a semi-quantitative or quantitative signal. Thus, "transducing" or "transduction" and their grammatical equivalents as are used herein encompasses optical, electrochemical, acoustical / mechanical, or colorimetrical technologies and methods. Electrochemical properties include current and / or voltage, capacitance, and potential. Optical properties include absorbance, fluorescence / phosphorescence, wavelength shift, phase modulation, bio / chemiluminescence, reflectance, light scattering, and refractive index.

[0136] The term "sensitivity" as used herein is a broad term, and is to be given its ordinary and customary meaning to a person of ordinary skill in the art (and is not to be limited to a special or customized meaning), and refers without limitation to an amount of signal (e.g., in the form of electrical current and / or voltage) produced by a predeterminedAttorney Docket No.: 0978-PCT01-0243amount (unit) of the measured analyte. For example, an amperometric sensor has a sensitivity (or slope) of from about 1 to about 100 picoAmps of current for every 1 mg / dL of analyte.

[0137] The phrases and terms "small diameter sensor," "small structured sensor," and "micro-sensor" as used herein are broad phrases and terms, and are to be given their ordinary and customary meaning to a person of ordinary skill in the art (and are not to be limited to a special or customized meaning), and refer without limitation to sensing mechanisms that are less than about 2 mm in at least one dimension. In further examples, the sensing mechanisms are less than about 1 mm in at least one dimension. In some examples, the sensing mechanism (sensor) is less than about 0.95, 0.9, 0.85, 0.8, 0.75, 0.7, 0.65, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 mm. In some examples, the maximum dimension of an independently measured length, width, diameter, thickness, or circumference of the sensing mechanism does not exceed about 2 mm. In some examples, the sensing mechanism is a needle-type sensor, wherein the diameter is less than about 1 mm, see, for example, U.S. Pat. No. 6,613,379 to Ward et al. and U.S. Pat. No. 7,497,827 to Brister et al., both of which are incorporated herein by reference in their entirety. In some alternate examples, the sensing mechanism includes electrodes deposited on a substantially planar substrate, wherein the thickness of the implantable portion is less than about 1 mm, see, for example U.S. Pat. No. 6,175,752 to Say et al. and U.S. Pat. No. 5,779,665 to Mastrototaro et. al., both of which are incorporated herein by reference in their entirety. Examples of methods of forming the sensors (sensor electrode layouts and membrane) and sensor systems discussed herein may be found in currently pending U.S. Pat. Pub. No. 2019-0307371, which is incorporated by reference in its entirety herein.

[0138] The phrase "soft segment" as used herein is a broad phrase, and is to be given its ordinary and customary meaning to a person of ordinary skill in the art (and is not to be limited to a special or customized meaning), and refers without limitation to an element of a copolymer, for example, a polyurethane, a polycarbonate polyurethane, or a polyurethane urea copolymer, which imparts flexibility to the chain. The phrase "soft segment" can be further characterized as an amorphous material with a low Tg, e.g., a Tg not typically higher than ambient temperature or normal mammalian body temperature.Attorney Docket No.: 0978-PCT01-0243

[0139] The phrase "solid portions" as used herein is a broad term, and is to be given its ordinary and customary meaning to a person of ordinary skill in the art (and is not to be limited to a special or customized meaning), and refers without limitation to portions of a membrane's material having a mechanical structure that demarcates cavities, voids, or other non-solid portions.

[0140] The term and phrases "zwitterion" and "zwitterionic compound" as used herein are each a broad term and phrase, and are to be given their ordinary and customary meaning to a person of ordinary skill in the art (and is not to be limited to a special or customized meaning), and refer without limitation to compounds in which a neutral molecule of the compound has a unit positive and unit negative electrical charge at different locations within the molecule. Such compounds are a type of dipolar compound, and are also sometimes referred to as "inner salts."

[0141] The phrases "zwitterion precursor" or "zwitterionic compound precursor" as used herein are broad phrases, and are to be given their ordinary and customary meaning to a person of ordinary skill in the art (and is not to be limited to a special or customized meaning), and refer without limitation to any compound that is not itself a zwitterion, but can become a zwitterion in a final or transition state through chemical reaction. In some examples described herein, devices comprise zwitterion precursors that can be converted to zwitterions prior to in vivo implantation of the device. Alternately, in some examples described herein, devices comprise zwitterion precursors that can be converted to zwitterions by some chemical reaction that occurs after in vivo implantation of the device. Such reactions are known to a person of ordinary skill in the art and include ring opening reaction, addition reaction such as Michael addition. This method is especially useful when the polymerization of betaine containing monomer is difficult due to technical challenges such as solubility of betaine monomer to achieve desired physical properties such as molecular weight and mechanical strength. Post-polymerization modification or conversion of betaine precursor can be a practical way to achieve desired polymer structure and composition. Examples of such as precursors include tertiary amines, quaternary amines, pyridines, and others detailed herein.

[0142] The phrases "zwitterion derivative" or "zwitterionic compound derivative" as used herein are broad phrases, and are to be given their ordinary and customary meaning toAttorney Docket No.: 0978-PCT01-0243a person of ordinary skill in the art (and is not to be limited to a special or customized meaning), and refer without limitation to any compound that is not itself a zwitterion, but rather is the product of a chemical reaction where a zwitterion is converted to a nonzwitterion. Such reactions can be reversible, such that under certain conditions zwitterion derivatives can act as zwitterion precursors. For example, hydrolysable betaine esters formed from zwitterionic betaines are cationic zwitterion derivatives that under the appropriate conditions are capable of undergoing hydrolysis to revert to zwitterionic betaines.

[0143] The phrases "zwitterionic repeating group" as used herein is a broad phrase, and is to be given their ordinary and customary meaning to a person of ordinary skill in the art (and is not to be limited to a special or customized meaning), and refer without limitation to, independently, two or more zwitterionic compounds, zwitterion derivatives or zwitterionic compound derivatives in the same compound or polymer.

[0144] Aptamer-based biosensors (AB's) and electrochemical aptamer-based biosensors (EAB's) are analytical platforms that can provide for continuous monitoring of specific molecular analytes, in vivo. EAB sensors typically present an architecture consisting of a selfassembled monolayer (SAM) of analyte-binding, alkanethiol-functionalized nucleic-acid aptamers or other bioreceptor comprising a redox moiety sensitive as signal transducing element to correlate analyte-binding events with measurable electrical energy changes, a SAM of electrode-blocking co-adsorbates of alkanethiols to prevent undesired electrochemical reactions and confer biocompatibility to the electrode surface.

[0145] The poor stability observed when the above AB or EAB's are placed in physiological relevant environments is at least attributed to desorption of the aptamer monolayer from a substrate surface or desorption of the underlying SAM monolayer used to immobilize the aptamer or the electrode-blocking SAM, as well as bioelectronic interface degradation (e.g., fouling, drift, etc.) upon continuous electrochemical interrogation— a process typically seen as a drop in faradaic and an increase in charging currents over time. As discussed in greater detail below, such performance deficits can be addressed with the presently disclosed aptamer protective layer (APL).

[0146] In examples, the present disclosure provides for AB or EAB's in an architecture consisting of a self-assembled monolayer (SAM) of analyte-binding, alkanethiol- or carboxyl-Attorney Docket No.: 0978-PCT01-0243functionalized nucleic-acid aptamers or other bioreceptor comprising a signal transducing element to correlate analyte-binding events with a measurable signal from the transducing element, SAM of electrode-blocking co-adsorbates of alkanethiols and / or functionalized alkanethiols and an aptamer protective layer (APL) adjacent or directly adjacent the above architecture to independently or collectively prevent undesired desorption, undesired reactions, reduce biofouling, / confer biocompatibility, aptamer stability, and device longevity.

[0147] With reference to FIGs. 1A and IB exemplary aptamer-based analyte monitoring sensor 100 configured for in vivo measurement of at least one analyte 99, is presented with schematic diagrams illustrating an contacting layer 105. Aptamer 102 with signal transducing element 104 is shown associated with optional monolayer 103 adjacent substrate 110. Monolayer 103 can be covalently or non-covalently coupled to substrate 110. In examples, aptamer 102 undergoes a reversible conformational change upon interaction with analyte 99, e.g., an analyte, metabolite, drug, etc., causing signal transducing element 104 to present in closer proximity to substrate 110 so as to provide a signal corresponding to the analyte 99 concentration or presence.

[0148] In examples, the signal transducing element 104 is a reversible redox moiety and the substrate 110 is electrically conductive and the reversible binding of aptamer 102 (and its subsequent reversible conformational change) upon interaction with analyte 99 causes a change in proximity of all or part of the signal transducing element 104 with that of the conductive substrate 110 such that the signal transducing element 104 is capable of undergoing detectable reversible reduction-oxidation reaction(s) via electron transfer with the conductive substrate 110 upon reversible binding of analyte 99 with aptamer 102. The detectable reversible reduction-oxidation reaction(s) via electron transfer with the conductive substrate 110 provide for correlation with analyte 99 concentration as further discussed below.

[0149] In another example, the reversible binding of aptamer 102 (and its subsequent reversible conformational change) upon interaction with analyte 99 can cause all or part of signal transducing element 104 to present in or to a different local environment, for example, from a hydrophobic local environment to a hydrophilic local environment (or visa-Attorney Docket No.: 0978-PCT01-0243versa) so as to provide a detectable signal corresponding to analyte 99 concentration or presence.

[0150] In examples, the signal transducing element 104 is an environmentally sensitive fluorescent dye or phosphorescence dye that upon exposure to electromagnetic radiation, e.g., light, is capable of undergoing a detectable change in emission wavelength or frequency and / or emission relaxation or emission decay rate, for example, upon reversible binding with analyte 99 and reversible conformation change from a hydrophobic local environment to a hydrophilic local environment (or visa-versa). The detectable change in emission wavelength or frequency and / or emission relaxation or emission decay rate so as to provide for correlation with analyte 99 concentration.

[0151] Signal transducing element 104 can be covalently or non-covalently coupled to aptamer 102, where the covalently or non-covalently coupling is such that it is sufficient for continuous signal transduction of signal over a time period commensurate with a transdermal, intradermal, subcutaneous, ocular, or skin based continuous analyte sensing devices. In examples, continuous signal transduction of signal over a time period of at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least one week, at least 2 weeks, at least 3 weeks, using the presently disclosed APLs is envisaged.

[0152] In examples, the signal transducing element 104 and aptamer 102 are conjugated or form a conjugate. In examples, the signal transducing element 104 and aptamer 102 conjugate are associated with monolayer 103. In examples, the signal transducing element 104 and aptamer 102 conjugate are covalently or non-covalently coupled to monolayer 103. In examples, the signal transducing element 104 and aptamer 102 conjugate are covalently or non-covalently coupled to substrate 110.

[0153] By way of example, any hereafter reference to a redox moiety as the signal transducing element 104 is for brevity and is not to limit the scope of signal transducing element 104. Thus, "redox moiety 104" and "signal transducing element 104" are hereinafter used interchangeably.

[0154] For example, the structural nature of the substrate can be determinative in the performance of an EAB, as shown in FIGs. 1C and ID, a planar substrate 110 with aptamer 102 and coupled redox moiety 104 present the shown potential vs electric double layer (EDL) relationship, a region contained within a Debye volume. In contrast, FIGs. IE showsAttorney Docket No.: 0978-PCT01-0243the same aptamer 102 and coupled redox moiety 104 construct with at least a portion of a nanometer and / or micrometer dimensioned pores 225 in substrate 222 surface, where as shown in FIG. IF, the potential vs electric double layer (EDL) relationship presents a smaller relative negative slope compared to the linear substrate. When used in combination with the presently disclosed APL, substrate 222 surfaces can provide increases in signal and limit of detection for continuous EAB devices.

[0155] FIG. 3A is a schematic diagram illustrating an exemplary aptamer biosensor 200 construct configured for continuous in vivo use in a subject. Thus biosensor 200 is shown as an elongated member having a sensing region 207, for example, created from a window in an electrically insulated coating 205 about a conductive wire. Alternatively, a window can be prepared in a jacket of an optical fiber for use with an optically based AB device. Additional electrodes 215 can be used. As shown in enlarged section views FIGs. 3B-3D, alternative structures 201, 202, and 203 of sensing region 207 are shown with substrate 110 surface, for example, structure 201 has substrate 110 surface with adjacent aptamer 102 and contacting layer 105. Structure 202 has substrate 110 surface with adjacent co-adsorbate 103, aptamer 102, contacting layer 105, and encapsulation layer 107. Structure 203 has substrate 110 surface adjacent co-adsorbate 103, aptamer 102, contacting layer 105, encapsulation layer 107, and drug-releasing membrane 113 most distal from substrate 110. Other configurations of the co-adsorbate 103, aptamer 102, contacting layer 105, encapsulation layer 107 and drug-releasing membrane 113 can be employed.Substrate

[0156] In examples, substrate 110 surface accepts the AB or EAB for use in a continuous sensing device. In examples, substrate 110 is or comprises a conductive material or is conductive. In examples, substrate 110 is an electrode that may be a wire, a planar structure or a substantially planar structure. In examples, substrate 110 can be configured to provide, independently, one or more of a working electrode, a reference electrode and optionally a counter electrode. In examples, substrate 110 comprises a wire formed from or coated with a conductive material, such as platinum, platinum-iridium, palladium, graphite, gold, carbon, graphene, graphene oxide, conductive polymer, alloys, or the like.

[0157] In examples, at least a portion of substrate 110 comprises pores having average pore diameters with nanometer and / or micrometer dimensions. Such pore diametersAttorney Docket No.: 0978-PCT01-0243dimensions in the aforementioned substrates can be formed with etching or plasma techniques, for example. Substrates with such nanometer and / or micrometer dimensions can be used in combination with the presently disclosed APL's.

[0158] In examples, at least a portion of substrate 110 surface comprises carbon, graphene, or graphene oxide. In examples at least a portion of substrate surface is comprised of nanomaterial. In examples, at least a portion of substrate 110 surface comprises carbon, graphene, or graphene oxide nanomaterial. In examples, the use of carbon, graphene, or graphene oxide nanomaterials improve aptamer loading onto substrate 110 surface to optimize aptamer 102 load and binding stability, among other things.

[0159] In examples, substrate 110 surface comprises gold and at least a portion of substrate 110 surface is configured to covalently couple or associate an alkyl thiol. In another example, at least a portion of substrate 110 surface is configured to covalently couple aliphatic amine or covalently couple amino alkanoic acid. In examples, at least a portion of substrate 110 surface is chemically modified with streptavidin, avidin, gold, biotin, or polymers such as dextrin and chitosan.

[0160] In examples, at least a portion of carbon, graphene, or graphene oxide nanomaterial substrate 110 surface comprises covalently coupled aliphatic amine or amino alkanoic acid. In examples, the amino alkanoic acid is also covalently coupled to aptamer 102 or aptamer-redox moiety 104 conjugates. For example, substrate 110 surface is modified with a carboxylated material to enable covalent immobilization with exposed COOH- groups of an amine-modified aptamer via EDC / NHS chemistry.

[0161] Among exemplary nanomaterials, graphene oxide (GO) shows significant advantages for use in EAB devices due to its large surface area with multiple exposed carboxylic groups (COOH) which can be used as anchor points to immobilize aptamer probes using a variety of different types of coupling chemistries. GO offers great versatility of functionalization and has demonstrated beneficial orientation effects in aptamer immobilization. Thus, in examples a partially or fully implantable-type sensor with a GO-functionalized substrate 110 surface (as a model carboxylated-surface) to serve as working electrode for the covalent immobilization of amine-functionalized aptamers. To covalently immobilize the aptamers to the GO surface, for example, activation of the GO - 'COOH'Attorney Docket No.: 0978-PCT01-0243moieties can be performed via N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide / Nhydroxysulfosuccinimide (EDC / NHS) chemistry, then forming the corresponding amide bond with the amine group present in the aptamer sequence. This immobilization strategy is envisaged to provide higher stability of the immobilized aptamer monolayer on the electrode surface, thus offering extended sensor lifetime and representing an alternative to thiol-based aptamer immobilization strategies.

[0162] In examples, substrate 110 surface is a carboxyl-functionalized substrate surface, a thiol functionalized substrate 110 surface, or a combination of a carboxyl -functionalized substrate 110 surface and a thiol functionalized substrate 110 surface.

[0163] In examples, substrate 110 surface is substantially a carboxyl-functionalized substrate surface. In examples, substrate 110 surface is essentially a carboxyl-functionalized substrate surface substantially without thiol functionalization.

[0164] In examples, substrate 110 surface is substantially a GO-functionalized substrate surface. In examples, substrate 110 surface is essentially a GO-functionalized substrate surface substantially without thiol functionalization.Co-Adsorbate Layer

[0165] In examples, aptamer-based biosensors (AB's) and electrochemical aptamerbased biosensors (EAB's) are analytical platforms that can provide for continuous monitoring of specific molecular analytes, in vivo. EAB sensors typically present an architecture consisting of a self-assembled monolayer (SAM) of analyte-binding, alkanethiol-functionalized nucleic-acid aptamers or other bioreceptor comprising a redox moiety sensitive as signal transducing element to correlate analyte-binding events with measurable electrical energy changes, a SAM of electrode-blocking co-adsorbates of alkanethiols to prevent undesired electrochemical reactions and confer biocompatibility to the electrode surface.

[0166] The poor stability observed when the above AB or EAB's are placed in physiological relevant environments is at least attributed to desorption of the aptamer monolayer from a substrate surface or desorption of the underlying SAM monolayer used to immobilize the aptamer or the electrode-blocking SAM, as well as bioelectronic interface degradation (e.g., fouling, drift, etc.) upon continuous electrochemical interrogation— a process typically seen as a drop in faradaic and an increase in charging currents over time.Attorney Docket No.: 0978-PCT01-0243As discussed in greater detail below, such performance deficits can be addressed with the presently disclosed aptamer protective layer (APL).

[0167] In examples, the presently disclosed AB or EAB device includes one or more coadsorbates. Co-adsorbates function to cover the substrate and alter the response to of the substrate to exposure of the surrounding environment such that undesired activity or reactions are eliminated or reduced.

[0168] In examples, the one or more co-adsorbates is present and provide, independently, an ionic strength and / or modulating or maintaining the ionic strength in proximity to the at least one aptamer conjugates and / or the substrate surface.

[0169] In examples, the one or more aptamer conjugates is physically or chemically coupled to a self-assembled monolayer (SAM). In examples, the one or more aptamer conjugates is physically or chemically coupled to a mono-functional or multi-functional alkanethiol or mercaptoalkanol. In examples, the one or more aptamer conjugates is physically or chemically coupled to an alkylthiol betaine. In examples, the one or more aptamer conjugates is physically or chemically coupled to an aliphatic amine.

[0170] In examples, the co-adsorbate comprises a self-assembled monolayer (SAM). In examples, the co-adsorbate comprises a mono-functional or a multi-functional alkanethiol.

[0171] In examples, a thiol functional group of the mono-functional alkanethiol or the multi-functional alkanethiol is covalently coupled to at least a portion of the substrate surface. In examples, a thiol functional group of the mono-functional alkanethiol or the multi-functional alkanethiol is covalently coupled to a gold substrate surface.

[0172] In examples, the co-adsorbate comprises a mono-functional or a multi-functional mercaptoalkanol. In examples, a thiol functional group of the mono-functional or the multifunctional mercaptoalkanol is covalently coupled to at least a portion of the substrate surface. In examples, a thiol functional group of the mono-functional or the multi-functional mercaptoalkanol is covalently coupled to at least a portion of a gold substrate surface.

[0173] In examples, the co-adsorbate comprises a zwitterionic repeating group associated with at least a portion of the substrate surface. In examples, the co-adsorbate comprises a zwitterionic repeating group coupled to at least a portion of the substrate surface. In examples, the co-adsorbate comprises a zwitterionic repeating group covalentlyAttorney Docket No.: 0978-PCT01-0243to at least a portion of the substrate surface. In examples, the zwitterionic repeating group comprises a betaine.

[0174] In examples, the zwitterionic repeating group comprises ammoniophosphates or lecithin analogs, ammoniophosphonates, ammoniophosphinates, ammoniosulfonates, ammoniosulfates, ammoniocarboxylates, or combinations thereof.

[0175] In examples, the zwitterionic repeating group comprises an alkanethiol betaine. In examples, the alkanethiol is linear and comprises a plurality of betaine groups along its chain. In examples, the alkanethiol is an end-terminated dithiol with at least one betaine group along its chain. In examples, the alkanethiol is linear and comprises an end-terminated betaine group. In examples, a thiol group of the end-terminated dithiol alkanethiol is covalently coupled to the substrate surface.

[0176] In examples, the zwitterionic repeating group comprises an mercaptoalkanol betaine. In examples, the mercaptoalkanol is linear and comprises a plurality of betaine groups along its chain. In examples, the mercaptoalkanol is linear and comprises an end-terminated betaine group. In examples, a thiol group of the mercaptoalkanol is covalently coupled to the substrate surface.

[0177] In examples, the co-adsorbate is one or more of the following structures:

[0178] wherein n, together with "' / vw'" js0-24; orAttorney Docket No.: 0978-PCT01-0243Attorney Docket No.: 0978-PCT01-0243

[0179] where W, Y, and Z are, independently, branched or straight chain alkyl, heteroalkyl, cycloalkyl, cycloheteroalkyl, aryl, or heteroaryl, any of which can be optionally substituted with O, OH, halogen, amido, oralkoxyl; Rl is H, alkyl, heteroalkyl, cycloalkyl, cycloheteroalkyl, aryl, or heteroaryl; and R2, R3, and R4, are independently chosen from alkyl, heteroalkyl, cycloalkyl, cycloheteroalkyl, aryl, or heteroaryl; n is an integer from 2-24. In examples, W is C1-C4 alkyl.

[0180] In examples, the co-adsorbate is one or more of the following ammoniosulfonates (sulfobetaines) or ammoniosulfates, structures:Rl\ - R2- -N- — Z - SO3®R3

[0181] where Z is branched or straight chain alkyl, heteroalkyl, cycloalkyl, cycloheteroalkyl, aryl, or heteroaryl; R1 is H, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; and R2 and R3, are independently chosen from alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; wherein one or more of Rl, R2, R3, and Z are substituted with a polymerization group; and ammoniocarboxylates having the structures:Attorney Docket No.: 0978-PCT01-0243

[0182] where Z is branched or straight chain alkyl, heteroalkyl, cycloalkyl, cycloheteroalkyl, aryl, or heteroaryl; R1 is H, alkyl, heteroalkyl, cycloa I kyl, heterocycloalkyl, aryl, or heteroaryl; and R2 and R3 are independently chosen from alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; wherein one or more of Rl, R2, R3, and Z are substituted with a polymerization group.

[0183] In each of these monomers, Z can have a length of from 1 to 12 atoms, e.g., from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 atoms, where any of these values can form an upper or lower endpoint of a range.

[0184] These compounds or monomers can be prepared by methods known to those of skilled in the art, e.g., as detailed in Laschewsky, "Structures and synthesis of zwitterionic polymers," Polymers 6:1544-1601, 2014. In certain examples, the disclosed polyzwitterions can have repeating zwitterionic units obtained from any of the zwitterionic compounds or monomers disclosed above.

[0185] In examples, the co-adsorbate is an end-terminated dithiol with at least one zwitterionic groups or zwitterionic repeating groups as disclosed herein. In examples, controlling ionic strength comprises configuring the APLs backbone or one or more appendages from its backbone with one or more zwitterionic betaine groups.Attorney Docket No.: 0978-PCT01-0243

[0186] In examples, the APLs comprises alkanethiol and one or more zwitterionic groups. In examples, controlling ionic strength comprises providing the APLs in combination with mercaptoalkanol having zwitterionic betaine groups.

[0187] In examples, the present disclosure provides for AB or EAB's in an architecture consisting of a self-assembled monolayer (SAM) of analyte-binding, alkanethiol- or carboxyl-functionalized nucleic-acid aptamers or other bioreceptor comprising a signal transducing element to correlate analyte-binding events with a measurable signal from the transducing element, SAM of electrode-blocking co-adsorbates of alkanethiols and / or functionalized alkanethiols and an aptamer protective layer (APL) adjacent or directly adjacent the above architecture to independently or collectively prevent undesired desorption, undesired reactions, reduce biofouling, / confer biocompatibility, aptamer stability, and device longevity.

[0188] In examples, the presently disclosed APLs alone in in combination with a SAM or a co-adsorbate, provides for a method to control or modulate the ionic strength about the aptamer-signal transducing element conjugate. For example, presently disclosed functionalized APLs, e.g., betaine functionalized APL's in combination with one or more coadsorbates can be present in an amount capable of modulating or maintaining the ionic strength about the aptamer-signal transducing element conjugate.

[0189] In examples, at least a portion of substrate 110 surface further comprises one or more co-adsorbates. In examples, the one or more co-adsorbates independently comprises a plurality of functional groups. FIGs. 2A and 2B depict schematic representations of exemplary co-adsorbates 402a, and 402b, respectively, in accordance with the broadest aspect of the present disclosure. Thus, FIG. 2A, shows an enlarged cross-sectional schematic of substrate 110 WE surface (as working electrode) of an implantable-type EAB with an exemplary architecture of co-adsorbate 402a having a linear segment 406a and end-group segments 404a. FIG. 2B shows an enlarged cross-sectional schematic of substrate 110 WE surface (as working electrode) of an implantable-type EAB with an exemplary architecture of co-adsorbate 402a having a linear segment 406b and backbone segments 404b. Linear segment 406a, 406b can be alkyl, alkylthiol, mercaptoakanol for example, as disclosed herein. End-group segments 404a or backbone segments 404b can be zwitterionic or repeating zwitterionic groups as disclosed herein. Substrate 110 can comprise aAttorney Docket No.: 0978-PCT01-0243combination of co-adsorbates 402a and 402b at various substrate surface area ratios. Coadsorbate 402a, 402b can be coupled to substrate 110 surface (indicated by "X" in Figure 2A, 2B) in a variety of ways, for example, a thiol, amine, amino, carboxyl, carboxylamine, or carboxylamino, via EDC / NHS chemistry, for example, as discussed herein. Co-adsorbate can be coupled to substrate 110 surface in a variety of ways, for example, a thiol, amine, amino, carboxyl, carboxylamine, or carboxylamino, via EDC / NHS chemistry, for example, as discussed herein. Substrate 110 surface can comprise a combination of co-adsorbates 402a, 402b at various substrate surface area ratios.

[0190] In examples, the continuous monitoring AB or EAB of the present disclosure comprises one or more co-adsorbates associated with substrate 110 surface, the one or more co-adsorbates being chemically different.

[0191] In examples, the aforementioned functionalized APL's can in part, also function as a co-adsorbate. Thus, in examples, at least a portion of the substrate surface comprises the functionalized APL, at least a portion of the substrate surface comprises the one or more co-adsorbates, and at least a portion of a remainder of the substrate surface comprises the one or more aptamer conjugates, where the sum of the percent portions of the substrate surface and the remainder can be 100 percent or less than 100 percent.

[0192] In examples, at least a portion of the substrate surface the one or more co-adsorbates and a portion of a remainder of the substrate surface comprises the one or more aptamer conjugates. In examples, the remainder of the substrate surface is about 50% of the total surface area of the substrate. In examples, the remainder of the substrate surface is less than 50% but more than 0 % of the total surface area of the substrate. In examples, the remainder of the substrate surface is greater than 50% and less than 100 % of the total surface area of the substrate.

[0193] In examples, at least a portion of the substrate surface comprises the one or more co-adsorbates and a portion of a remainder of the substrate surface comprises the one or more aptamer conjugates physically or chemically coupled to the substrate. In examples, the one or more co-adsorbates are physically or chemically coupled to the substrate surface and a portion of a remainder of the substrate surface comprises the one or more aptamer conjugates physically or chemically coupled to at least a portion of the co-adsorbate.Attorney Docket No.: 0978-PCT01-0243Aptamer / Aptamer-Signal Transducing Element Conjugates

[0194] In examples, the one or more aptamer and / or aptamer conjugates are present, for example, at densities of 10'9, IO10, 101X, IO12, to 1013molecules / cm2of substrate surface. Other densities can be used. In examples, the aptamers present at the substrate surface have substantially similar architecture (less than 2 base pair deviation), identical transducing elements or redox moieties, identical conjugate coupling chemistries attaching the aptamer to the working electrode surface, SAM, or co-adsorbate, identical aptamer / co-adsorbate mass ratios and / or densities, and identical manufacturing history. In examples, the aptamers present at the substrate surface have different architecture (more than 2 base pair deviation), different or the same transducing elements or redox moieties, different or identical conjugate coupling chemistries attaching the aptamer to the working electrode surface, SAM, or co-adsorbate, different aptamer / co-adsorbate mass ratios and / or densities, and different or identical manufacturing history.

[0195] In examples, the one or more aptamer 102 of the presently disclosed AB or EAB comprises RNA or DNA nucleotide sequences. In examples, the one or more aptamer 102 comprises at least one of : 2'-O-methyl modification of a nucleotide; disulfide bridges; a 3' cap with an inverted 2-deoxy thymidine; a 3'-3'-thymidine linkage at 3' terminus; a 2'-F modification; and a double stranded section. In examples, the one or more aptamer 102 comprises RNA or DNA sequences with a first linker moiety on a 5' end and the reversible redox moiety at a 3' end.

[0196] In examples, the first linker moiety on the 5' end of aptamer 102 comprises an amino group or carboxyl group. In examples, the first linker moiety of aptamer 102 is physically or chemically coupled to the substrate at the 5' end. In examples, the first linker moiety of aptamer 102 is physically or chemically coupled to the co-adsorbate at the 5' end.

[0197] In examples, the one or more aptamer 102 is a neurotransmitter binding aptamer. In examples, the one or more aptamer 102 is a dopamine or glutamate binding aptamer. In examples, the one or more aptamer 102 is a carbohydrate, triglyceride or fatty acid binding aptamer. In examples, the one or more aptamer conjugates 102 is a glucose, glycerol, or beta-hydroxybutyrate binding aptamer. In examples, the one or more aptamer 102 is a glycopeptide antibiotic binding aptamer. In examples, the one or more aptamer 102 is a vancomycin binding aptamer. Combinations of different aptamer conjugates 102 on theAttorney Docket No.: 0978-PCT01-0243same or different WE surfaces can be employed for providing a multi-analyte monitoring EAB device.Aptamer Protective Layer (APL)

[0198] It has been observed that specific attributes of the APLs effect the suitability of continuous operation of an AB or EAB in vivo. For example, a continuous AB or EAB having an APLs with sufficient free volume for aptamer conformational changes, favorable ionic characteristics, sufficient porosity to analyte and blocking of proteins, peptides, macrophages and other immune response biologies positively effects the EAB performance. One or more of the above characteristics further provides for, directly or indirectly, stability of the aptamer coupling (reduced desorption from substrate or SAM), reduced signal or sensitivity drift over in vivo time, and extended in vivo performance compared to an AB or EAB without an APL.

[0199] In examples, the APLs is a coating, multi-coating, matrix, membrane, multimembrane, domain, multi-domain, layer or multi-layer. In another example, the APLs is a coating, multi-coating, matrix, membrane, multi-membrane, domain, multi-domain, layer or multi-layer of a polymeric material. The polymeric material that forms a basis of an APLs can include one or more polymers, oligomers, layers of coatings, membranes, or matrixes. In examples, the APLs provides sufficient permeability to allow relevant analyte compounds to pass through it, for example, to allow the analyte to pass through the membrane from the sample under examination in order to reach the aptamer and allow for transduction of a signal corresponding to the analyte concentration in the sample.

[0200] In examples, APLs comprise a contacting layer. In examples, APLs comprise an encapsulation layer. In examples, APLs comprise a contacting layer and an encapsulation layer. In examples, APLs comprise a contacting layer more proximal to the electrode / electroactive surface than the encapsulation layer. In examples, APLs comprise a waterborne contacting layer and a solvent based encapsulation layer. In examples, APLs comprise a waterborne contacting layer and a waterborne encapsulation layer.

[0201] In examples, APLs comprises at least one polymer segment. In examples, the APLs comprises at least one polymer segment selected from the group consisting of polyurethane, polyurea, poly(urethane urea), epoxide, polyolefin, polysiloxane, polyamide, polystyrene, polyacrylate, polyether, polypyridine, polyvinylpyrrolidone, polyester,Attorney Docket No.: 0978-PCT01-0243polycarbonate, and copolymers thereof. In examples, APLs comprise a contacting layer and an encapsulation layer having polymer segments with the same or different weight percent, chain length, molecular weight (average or mean) or polymer chemistry. In examples, APLs comprise a contacting layer having polymer segments with at least one different weight percent, chain length, molecular weight (average or mean) or polymer chemistry than the encapsulation layer.

[0202] The hydrophilicity of the contacting layer and / or the encapsulation layer of the APLs can be adjusted by the selection of soft segment used and soft segment ratio during conventional PU or PUU synthesis, for example, by adjusting the amount of soft segment components (hydrophilic and hydrophobic polyols). Hydrophobic soft segment can be PDMS, polycarbonates, polyester, polyether or polymers with hydrophobic functional groups such as fluorine or silicone. The hydrophobic segment can be provided in the aforementioned APLs to be between 1-50 wt.%, 2-50 wt.%, 5-50 wt.%, 10-50 wt.%, 15-50 wt.%, 20-50 wt.%, 25-50 wt.%, 30-50 wt.%, 10-20 wt.%, 15-25 wt.%.

[0203] Hydrophilic soft segments can be polyethylene glycols, oligo polyether, polyoxazoline(POX), polypeptide, or zwitterionic polymer. By tuning the chemical composition, of soft and hard segment in the PU or PUU, addition or exclusion of functional group, desirable APLs properties and functionality can be achieved, e.g., surface charge / density, antifouling properties against protein such as serum albumin (SA). The hydrophilic segment can be provided in the aforementioned APLs to be between 1-50 wt.%, 2-50 wt.%, 5-50 wt.%, 10-50 wt.%, 15-50 wt.%, 20-50 wt.%, 25-50 wt.%, 30-50 wt.%, 10-20 wt.%, 15-25 wt.%.

[0204] In examples, the contacting layer and / or the encapsulation layer of the APLs comprises a segmented multiblock polymer. For example, the segmented multiblock polymer comprises a soft segment and a hard segment. In examples, the soft segment is hydrophobic or hydrophilic. In examples, the soft segment is hydrophobic and hydrophilic. In examples, the soft segment comprises hydrophobic polyol and hydrophilic polyol. In examples, the contacting layer and / or the encapsulation layer of the APLs comprises a segmented multiblock polyurethane polymer. In examples, the contacting layer and / or the encapsulation layer of the APLs comprises a segmented multiblock polyurethane, polyurethane-urea, or polyether-urethane, or polyether-urethane-urea polymer, copolymerAttorney Docket No.: 0978-PCT01-0243or blends thereof. In examples, the hard segment comprises urethane groups, urea groups, or combinations thereof.

[0205] In examples, the soft segment is one or more segments comprising polydimethylsiloxane, polycarbonate, polyester, polyether, and blends or copolymers thereof. In examples, the soft segment is one or more segments comprising polyethylene glycol, oligo polyether, polyoxazoline(POX), polypeptide, polyvinylpyrrolidone, zwitterionic repeating group polymer, and blends or copolymers thereof. In examples, end group functionalized- polyurethane (EGFPU) or polyurethane urea (EGFPUU) polymers can be used. EGFU / EGFPUU's can be synthesized using reactive functional monomers / oligomers end capping the polyurethane reaction intermediates to form polyurethane with functional groups on one chain end or both chain ends. The functional groups could be further deprotected to form reactive thiol groups connect to substrate 110 surface, e.g., gold.

[0206] Polyurethane, polyurethane-urea polymer can be produced by the condensation reaction of a diisocyanate and a difunctional hydroxyl-containing material or difunctional amine containing material. A polyurethane-urea is a polymer produced by the condensation reaction of a diisocyanate and a difunctional amine-containing material. In some examples, diisocyanates include aliphatic diisocyanates containing from about 4 to about 8 methylene units. Diisocyanates containing cycloaliphatic moieties can also be useful in the preparation of the polymer and copolymer components of the membranes of the present disclosure.

[0207] In examples, end group functionalized- polyurethane (EGFPU) or polyurethane urea (EGFPUU) polymers can be used, for example, as disclosed in co-assigned U.S. Patent No. 10,413,227B2. EGFU / EGFPUU's can be synthesized using reactive functional monomers / oligomers end capping the polyurethane reaction intermediates to form polyurethane with functional groups on one chain end or both chain ends. The functional groups could be further deprotected to form reactive thiol groups connect to substrate 110 surface, e.g., gold.

[0208] For example, an exemplary contacting layer and / or the encapsulation layer of the APLs hydrophobic-hydrophilic segmented copolymer component is a polyurethane polymer that includes about 20% hydrophilic polyethylene oxide. The polyethylene oxide portions of the copolymer are thermodynamically driven to separate from the hydrophobic portions of the copolymer and the hydrophobic polymer component. In examples, it hasAttorney Docket No.: 0978-PCT01-0243been observed that about 20% polyethylene oxide-based soft segment portion of the copolymer used to form the APLs affects the water pick-up and subsequent analyte permeability of the APLs membrane. In examples, the aforementioned exemplary contacting layer and / or the encapsulation layer of the APL's are dispensed as water-borne dispersions for use with the aforementioned aptamer-signal transducing element conjugates. For example, a hydrophilic aliphatic polyurethane, betaine functionalized, can be prepared as an aqueous dispersion that can be combined with an aqueous solution of the aforementioned aptamer-signal transducing element conjugates.

[0209] In examples, the contacting layer and / or the encapsulation layer of the APLs is comprised of non-polyurethane polymer. Examples of materials which can be used to make non-polyurethane type APL's include vinyl polymers, polyethers, polyesters, polyamides, polysilicones poly(dialkylsiloxanes), poly(alkylarylsiloxanes), poly(diarylsiloxanes), epoxide, polyolefin, polyamide, polystyrene, polycarbosiloxanes, polyacrylate, polyether, polyvinylpyridine, polyvinylpyrrolidone, polyester, polycarbonate, and copolymers thereof, Nation (sulfonated tetrafluoroethylene) natural polymers such as cellulosic and proteinbased materials, and mixtures, copolymers, or combinations thereof with or without the aforementioned polyurethane, or polyether-urethane-urea polymers. In examples, the contacting layer and / or the encapsulation layer of the APLs is comprised of poly(l-vinyl imidazole), poly(4-vinyl pyridine), poly(2-vinyl pyridine), polyacrylonitrile, polyacrylamide, and / or copolymers or quaternized forms thereof. In examples, the contacting layer and / or the encapsulation layer of the APLs is comprised grafted polymer chain having poly(l-vinyl imidazole), poly(4-vinyl pyridine), poly(2-vinyl pyridine), polyacrylonitrile, polyacrylamide, and / or copolymers or quaternized forms thereof.

[0210] In examples, the contacting layer and / or the encapsulation layer of the APLs layer is at least partially cross-linked using an amount of cross-linking agent sufficient to crosslink the APLs without deactivation of the aptamer or substantial reduction in the ability of the aptamer present therein to undergo conformation change sufficient to provide for signal transduction. In examples, the aptamer protective layer is completely cross-linked using an amount of cross-linking agent sufficient to crosslink the APLs without substantial reduction in the aptamer signal transduction.Attorney Docket No.: 0978-PCT01-0243

[0211] Suitable cross-linking agents include isocyanate, carbodiimide, gluteraldehyde or other aldehydes, aziridine, silane, epoxy, acrylates, free-radical based agents, ethylene glycol diglycidyl ether (EGDE), poly(ethylene glycol) diglycidyl ether (PEGDE), dicumyl peroxide (DCP), PVP-PEGDE or PVP-PEG. In one embodiment, from about 0.1% to about 15% w / w of cross-linking agent is added relative to the total dry weights of cross-linking agent and polymers added when blending the ingredients (in examples, about 1% to about 10%). During the curing process, substantially all of the cross-linking agent is believed to react, leaving substantially no detectable unreacted cross-linking agent in the final layer.

[0212] In examples, the contacting layer and / or the encapsulation layer of the APLs is a conductive polymer. In examples, the contacting layer and / or the encapsulation layer of the APLs is a functionalized polymer. The contacting layer and / or the encapsulation layer of the APLs can be functionalized, for example, with between 1-50 wt.%, 2-50 wt.%, 5-50 wt.%, 10-50 wt.%, 15-50 wt.%, 20-50 wt.%, 25-50 wt.%, 30-50 wt.%, 10-20 wt.%, 15-25 wt.%, of functional moiety. The functionalized polymer can be configured to couple with the substrate, or a SAM, or another layer, membrane, matrix, region, or polymer.

[0213] In examples, the functionalized polymer comprises alkanethiol groups. In examples, the alkanethiol groups are present at the end of the functionalized polymer chain or the alkanethiol groups are present along the backbone of the functionalized polymer chain.

[0214] In examples, the functionalized polymer comprises mercaptoalkanol groups. In examples, the mercaptoalkanol groups are present at the end of the functionalized polymer chain or are present along the backbone of the functionalized polymer chain.

[0215] In examples, the contacting layer and / or the encapsulation layer of the APLs comprises a zwitterionic group compound or a zwitterionic repeating group compound. In examples the functionalized polymer is prepared with at least one of a polymerizable zwitterionic monomer structure as follows:Attorney Docket No.: 0978-PCT01-0243

[0216] where X is 0, NH, or NR4, Y and Z are, independently, branched or straight chain alkyl, heteroalkyl, cycloalkyl, cycloheteroalkyl, aryl, or heteroaryl, and of which can be optionally substituted with OH, halogen, or alkoxyl; Ri, R3, R4, and Rs are independently H, alkyl, heteroalkyl, cycloalkyl, cycloheteroalkyl, aryl, or heteroaryl.

[0217] For example, the contacting layer and / or the encapsulation layer of the APLs can comprise, alone or in combination with other polymer structure / backbone, zwitterionic monomers including N-(2-methacryloyloxy)ethyl-N,N-dimethylammonio propanesulfonate, N-(3-methacryloylimino)propyl-N,N-dimethylammonio propanesulfonate, 2-(methacryloyloxy)ethyl phosphatidylcholine, and 3-(2'-vinyl-pyridinio) propanesulfonate.

[0218] In examples, the presently disclosed APLs provides an amount of a zwitterionic repeating group compound capable of modulating or maintaining an ionic strength aboutAttorney Docket No.: 0978-PCT01-0243the aptamer and / or transducing element (e.g. redox moiety) and / or substrate. In examples, the one or more zwitterionic repeating groups comprise a betaine compound or derivative thereof. In examples, the zwitterionic repeating groups are present at the end of the functionalized polymer chain or are present along the backbone of the functionalized polymer chain.

[0219] In examples, the functionalized polymer comprises alkanethiol and zwitterionic repeating groups. In examples, the functionalized polymer comprises alkanethiol and betaine groups. In examples, the functionalized polymer comprises mercaptoalkanol and zwitterionic repeating groups. In examples, the functionalized polymer comprises mercaptoalkanol and betaine groups.

[0220] In examples, the contacting layer and / or the encapsulation layer of the APLs is physically or chemically coupled to at least a portion of the substrate surface. In examples, the contacting layer and / or the encapsulation layer of the APLs is physically or chemically coupled to at least a portion of the substrate surface, with the one or more aptamer conjugates physically or chemically coupled to at least a portion of the substrate surface. In examples, the contacting layer and / or the encapsulation layer of the APLs is physically or chemically coupled to at least a portion of the substrate surface, with the one or more aptamer conjugates physically or chemically coupled to at least a portion of the substrate surface, and a substantial remainder of the substrate surface further comprising a physically or chemically coupled co-adsorbate.

[0221] In examples, the contacting layer and / or the encapsulation layer of the APLs provides sufficient free volume so as to allow reversible conformational change of the one or more aptamer conjugates. In examples, at least one of the one or more aptamer conjugates is physically or chemically coupled to an amino alkanoic acid.Signal Transducing Element

[0222] In examples, the signal transducing element comprises a redox moiety. The redox moiety may comprise any compound that upon a change in its proximity to an electrode at a biased potential causes a change in electron transfer kinetics. Exemplary redox species include methylene blue, organometallic redox moieties, ferrocene, viologen, anthraquinone or any other quinones, ethidium bromide, daunomycin, metallic porphyrin complexes, crown ether metallic complexes, bis-pyridine metal complexes, bis-imidizole metalAttorney Docket No.: 0978-PCT01-0243complexes, tris-py rid i ne metal complexes, ethylenetetracetic acid (EDTA)-metal complexes, and cytochromes. In examples, the reversible redox moiety comprises iron, iridium, ruthenium, a thiazine dye, or derivative thereof. In examples, the reversible redox moiety comprises ferrocene or methylene blue.

[0223] In examples, to reduce or eliminate the effects of pH and / or temperature on peak current / peak voltage of the EAB-redox moiety configuration, chemical modification of the redox moiety is envisaged. In examples, the aniline nitrogen of methylene blue is methylated. In examples, the redox moiety is a metal centered redox moiety, e.g., osmium-based, coupled or tethered to the aptamer.Methods

[0224] The presently disclosed APLs-EAB constructs provide advantages over EAB's without an APL. For example, the presently disclosed APLs-EAB constructs can be used in a method of determining an in vivo concentration of an analyte. For example, the method can comprise the steps of contacting, in vivo, an biological fluid comprising an analyte with the presently disclosed APLs-EAB constructs, the EAB coupled to a conductive substrate, the EAB encapsulated in the APLs, the APLs being permeable to an analyte, and the EAB producing a signal upon interaction with the analyte.

[0225] The presently disclosed APLs-EAB constructs are configured to receive bias voltage that is varied to reversibly oxidize and reduce a redox probe associated with the aptamer, with the aptamer being associated with a conductive substrate surface. The presently disclosed APLs-EAB constructs can be used in a method that comprises interrogating the conductive substrate or the APL-aptamer-redox moiety conjugate. The method can further comprise detecting the signal generated by the aptamer-redox moiety conjugate in the presence of a concentration of analyte and correlating an in vivo concentration of the analyte based on the detected signal, change of signal, difference of signal, etc. In examples, signal transduction by the presently disclosed APL-EAB constructs is determined by electron transfer rate from the reversible redox probe, where the electron transfer rate difference correlates to analyte concentrations.

[0226] In examples, the interrogating is continuous, semi-continuous, sequential, or a random temporal detecting of the signal. In examples, the method can further comprise the step of adjusting the signal based on a background signal produced as a result of nonAttorney Docket No.: 0978-PCT01-0243specific binding of the aptamer biosensor so as to produce an adjusted signal. Two or more working electrodes, with or without the presently disclosed APL-EAB constructs can be used. The method can further comprise determining the in vivo concentration of the analyte during a period of time based on the adjusted signal.

[0227] In examples, interrogating comprises a differential measurement technique. Exemplary differential measurement techniques include, for example, interrogating with a first square wave voltammetry (SWV) frequency to obtain a first signal and a second SWV frequency to obtain a second signal, taking the difference between the two signals, and dividing by the average of the two signals to obtain an adjusted signal. In examples, the interrogating comprises chronoamperometry. In examples, the interrogating comprises cyclic voltametry.

[0228] In examples, the presently disclosed APL's can control or modulate intermolecular interactions between the aptamer conjugate and the APL. In another example, the presently disclosed APL's constructs, alone or in combination with the presently disclosed co-adsorbate(s), provides for reducing decoupling of the aptamer from the substrate surface. In another example, the presently disclosed APL's can control or modulate diffusion of the aptamer from proximity about the substrate surface. Thus, if reversible desorption / decoupling of the aptamer from the substrate occurs, the presently disclosed APLs can keep the aptamer in proximity to the substrate surface so as to increase re-adsorption / re-coupling of the aptamer. In examples, the presently disclosed APLs are partially cross-linked. The presently disclosed APL's can be crosslinked in the presence of aptamer-transducing moiety with little or no adverse effect on the APL-EAB performance as discussed below.

[0229] In examples, the presently disclosed APLs can be used to extend the in vivo end-of-life for an EAB device. For example, the presently disclosed APLs have demonstrated extended end-of-life in bovine serum albumin for up to 20 hrs. It is envisaged that the presently disclosed APLs can provide EABs with up to one day, 2 days, one week, 2 weeks, 3 weeks, or one month in vivo end-of-life performance.

[0230] Methods of manufacturing the presently disclosed APL-EAB devices include presenting an aptamer comprising a reversible redox moiety to a surface of a conductive substrate and presenting the APLs to the portion of the surface of the conductive substrateAttorney Docket No.: 0978-PCT01-0243so as to encapsulate the aptamer conjugate in the APL. Alternatively, the presently disclosed APL-EABs are combined with the aptamer comprising a reversible redox moiety and presented to the substrate surface.Drug Releasing Layer

[0231] Devices and probes that are transcutaneously inserted or implanted into subcutaneous tissue conventionally elicit a foreign body response (FBR), which includes invasion of inflammatory cells that ultimately forms a foreign body capsule (FBC), as part of the body's response to the introduction of a foreign material. The continuous monitoring systems discussed herein include continuous analyte monitoring systems configured to monitor one, two, or more analytes concurrently, sequentially, and / or randomly (which is inclusive of events that can take place independently in picoseconds, nanoseconds, milliseconds, seconds, or minutes) to predict health-related events and health systems performance (e.g., the current and future performance of the human body's systems such as the circulatory, respiratory, digestive, or other systems or combinations of organs or systems). In examples, insertion or implantation of a device, for example, an EAB sensing device, can result in an acute inflammatory reaction resolving to chronic inflammation with concurrent building of fibrotic tissue, such as described in detail above. Eventually, over a period of time, a mature FBC, including primarily contractile fibrous tissue forms around the device. See Shanker and Greisler, Inflammation and Biomaterials in Greco RS, ed., "Implantation Biology: The Host Response and Biomedical Devices" pp 68-80, CRC Press (1994). The FBC surrounding conventional implanted devices has been shown to hinder or block the transport of analytes across the device-tissue interface. Thus, continuous extended life analyte transport (e.g., beyond the first few days) in vivo has been conventionally believed to be unreliable or impossible.

[0232] In some examples, certain aspects of the FBR in the first few days may play a role in noise. It has been observed that some sensors function more poorly during the first few hours after insertion than they do later. This is exemplified by noise and / or a suppression of the signal during the first few hours (e.g., about 2 to about 24 hours) after insertion. These anomalies often resolve spontaneously after which the sensors become less noisy, have improved sensitivity, and are more accurate than during the early period. It has been observed that some transcutaneous sensors and wholly implantable sensors are subject toAttorney Docket No.: 0978-PCT01-0243noise for a period of time after application to the host (i.e., inserted transcutaneously or wholly implanted below the skin).

[0233] Thus, with reference back to FIG. 3D, a drug releasing layer, membrane, gaseous sterilant scavenging matrix, or coating 113 can be positioned adjacent or directly adjacent the APLs 105 in examples the presently disclosed AB or EAB continuous sensor comprises an immune response attenuating layer or drug releasing layer configured to interact with the immune system of a host or release an active agent into the environment of the sensor. In examples, the immune response attenuating layer includes an active agent that is coupled to or entrapped in the layer, e.g., covalently coupled active agent (a dexamethasone derivative or analog) or a surface exposed, active agent (e.g., silver nanoparticles). In examples, the drug releasing layer comprises an active agent that is configured to release from the layer over time to mitigate or attenuate an immune response. Such drug releasing layers include, for example, segmented polyurethane polymers containing dexamethasone and / or dexamethasone acetate and / or other dexamethasone derivative or analog as disclosed in co-assigned U.S. Application No. 17 / 945,585, which is incorporated herein by reference.Manufacturing

[0234] The substrate can by formed by a variety of manufacturing techniques (bulk metal processing, deposition of metal onto the substrate, or the like). In examples, the substrate is plated wire (e.g., platinum on steel wire) or bulk metal (e.g., gold wire). It is believed that substrates for EAB's formed from bulk metal wire provide superior performance (e.g., in contrast to deposited electrodes), including increased stability of assay, simplified manufacturability, resistance to contamination (e.g., which can be introduced in deposition processes), and improved surface reaction (e.g., due to purity of material) without peeling or delamination. The substrate can be a metal wire with an outer insulator. The substrate can be a plurality of metal wires each with an outer insulator.

[0235] In examples wherein an outer insulator is disposed about a substrate, a portion of the coated assembly structure can be stripped or otherwise removed, for example, by hand, excimer lasing, chemical etching, laser ablation, grit-blasting (e.g., with sodium bicarbonate, solid carbon dioxide, or other suitable grit), or the like, to expose the electrochemically active surfaces. Alternatively, a portion of the electrode can be maskedAttorney Docket No.: 0978-PCT01-0243prior to depositing the insulator in order to maintain an exposed electrochemically active surface area. In one exemplary example, grit blasting is implemented to expose the electrochemically active surfaces, preferably utilizing a grit material that is sufficiently hard to ablate the polymer material, while being sufficiently soft so as to minimize or avoid damage to the underlying metal electrode (e.g., a platinum electrode). Although a variety of "grit" materials can be used (e.g., sand, talc, walnut shell, ground plastic, sea salt, solid carbon dioxide, and the like), in some one example, sodium bicarbonate is an advantageous grit-material because it is sufficiently hard to ablate, e.g., a parylene coating without damaging, e.g., an underlying platinum conductor. One additional advantage of sodium bicarbonate blasting includes its polishing action on the metal as it strips the polymer layer, thereby eliminating a cleaning step that might otherwise be necessary. Etching (chemical or plasma, for example) or other methods can be used to provide nanopores and / or micropores to the substrate surface.

[0236] In some examples, a radial window is formed through the insulating material to expose a circumferential electrochemically active surface of the working electrode.Additionally, sections of electrochemically active surface of the reference electrode are exposed. For example, the sections of electrochemically active surface can be masked during deposition of an outer insulating layer or etched after deposition of an outer insulating layer.

[0237] In examples, the APLs is deposited on the substrate comprising the aptamer conjugate to yield a domain thickness of from about 0.05 micron or less to about 40 microns or more, more preferably from about 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 1, 1.5, 2, 2.5, 3, or 3.5 to about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or more microns., In examples, a domain thickness of APLs is from about 20 microns to about 40 microns, including all ranges and subranges therebetween. In examples, the APLs is deposited together with the aptamer conjugate. In examples, the APLs (or APLs and aptamer conjugate) is deposited by spray coating or dip coating. The spraying process atomizes and mists the solution, and therefore most or all of the solvent is evaporated prior to the coating material settling on the underlying domain, thereby minimizing contact of the solvent with the aptamer. While not wishing to be bound by theory, it is believed that during the processAttorney Docket No.: 0978-PCT01-0243of depositing the APLs as described in the present disclosure, a structural morphology is formed about the aptamer that allows for substantially unimpeded conformational change by the aptamer with target analyte do to the hard-soft multi-segmented and / or functionalization of the APLs structure.

[0238] In examples, the APLs is deposited on the substrate / co-adsorbent by spraycoating a solution of from about 1 wt. % to about 5 wt. % polymer and from about 95 wt. % to about 99 wt. % solvent, including all ranges and subranges therebetween. In spraying a solution of APLs material, including a solvent, onto the substrate / co-adsorbent, it is desirable to mitigate or substantially reduce any contact with aptamer of any solvent in the spray solution that can deactivate the underlying aptamer, transducing element, or redox moiety. One or more solvents, including water, can be used, as is appreciated by one skilled in the art.

[0239] Although a variety of spraying or deposition techniques can be used, spraying the APLs material and rotating the sensor at least one time by 180° can provide adequate coverage by the APL. Spraying the APLs material and rotating the sensor at least two times by 120 degrees provides even greater coverage (one layer of 360° coverage), thereby ensuring protection to the AB or EAB, such as is described in more detail above.

[0240] In examples, the APLs is spray- or dip-coated and subsequently cured (e.g., if crosslinker is used) for a time of from about 15 to about 90 minutes at a temperature of from about 40 to about 60° C (and can be accomplished under vacuum (e.g., 20 to 30 mmHg)), including all ranges and subranges therebetween. A cure time of up to about 90 minutes or more can be advantageous to ensure complete drying of the APL. While not wishing to be bound by theory, it is believed that complete drying of the APLs aids in stabilizing the sensitivity of the AB or EAB sensor signal. It reduces drifting of the signal sensitivity over time, and complete drying is believed to stabilize performance of the AB or EAB sensor signal.

[0241] In examples, the APLs is formed by spray- or dip-coating one or more layers (e.g., rotating the sensor by 120° for 360° coverage) and optionally curing at 50° C under vacuum for 60 minutes. However, the APLs can be formed by dip-coating, depending upon the concentration of the solution, insertion rate, dwell time, withdrawal rate, and / or the desired thickness of the resulting APL.Attorney Docket No.: 0978-PCT01-0243

[0242] FIGS. 4A to 4B depict an exemplary planar sensor assembly 600, showing top-down drawings of a first side 602 and a second side 604 opposite the first side, in addition to a first end 612 and a second end 614. FIGS.4C to 4E depict schematic cross-section drawings of the full sensor assembly 600. The sensor assembly 600 can have The sensor assembly 600 can include substrate 610, conductive traces 620, 621, connector pads 622, 623 working electrodes 624, 625, counter electrode 626, insulating layers 630, 632, and reference electrode 640. In sensor assembly 600, a double-sided planar configuration is used. In the sensor assembly 600, a multiple-electrode sensor is shown, with two working electrodes (WE) 624, 625, a counter electrode (CE) 626 and a reference electrode (RE) 640. In sensor assembly 600, the electrodes are co-planar. The sensor assembly 600 is an unconnected variation. In examples, working electrodes (WE) 624, 625 are coated with the sensing membrane with multiple layers or domains as disclosed herein.

[0243] In sensor assembly 600, structures can be formed on both sides 602, 604, of the substrate 610. For example, the connector pads 622, 623, can be formed, respectively, on opposing sides 602, 604. This can allow for connection to the sensing electronics from both sides of the sensor assembly 600. Similarly, the conductive traces 620, 621, can be formed on both sides 602, 604, of the sensor assembly 600. On each individual side 602, 604 the conductive traces 620, 621, can be co-planar with each other.

[0244] The insulating layers 630, 632, such as a solder mask or other insulating material, can be deposited over the conductive layers including the conductive traces 620, 621. Openings can be formed in the insulating layers 630, 632, to form the working electrode 624 and pH electrode 625, and the counter electrode 626. An opening can be left for the reference electrode 640. A reference electrode material, such as silver / silver chloride, can be deposited on the designated sensing surface for the reference electrode 640. The insulating material can include epoxy, polyimide, polyurethane, polyethylene, or other materials or combinations of materials.

[0245] As illustrated in FIGS. 4A and 4B, the double-sided sensor assembly 600 can include a first working electrode 624, a pH sensitive electrode 625, a counter electrode 626, and a reference electrode 640. In some cases, such a double-sided sensor can contain more or less electrodes. For example, a double-sided sensor can include a single working electrode and a reference electrode. In examples, first working electrode 624, and pHAttorney Docket No.: 0978-PCT01-0243sensitive electrode 625 are electrically isolated from each other. In examples, additional working electrode(s) (not shown) can be employed for providing a multi-analyte, pH sensor configuration.

[0246] FIGS. 4C to 4E depict cross-sections of the sensor assembly 600. Shown in FIG. 4C is a cross section along line C-C, where the substrate 610 is situated between the two insulating layers 630, 632. The substrate 610 can be, for example, about 50 microns thick. Conductive traces 620, 621, can be seen. On the first side 602, three conductive traces 620 extend along the length of the sensor assembly 600, each connecting to a connector pad 622. The conductive traces 621 on the second side 604 can connect to the connector pad 623.

[0247] In examples, reference electrode 640 is coated with a material that reduces or eliminates release of ions from the reference electrode. In examples, reference electrode 640 is coated with reference electrode coating 629 that reduces or eliminates release of cations from the reference electrode. In examples, reference electrode 640 is coated with a reference electrode coating 629 that reduces or eliminates release of silver cations (Ag+). Exemplary materials for coating the reference electrode include parylene, fluoropolymers (Teflon), fluorinated alkyl polymer, orany one or combination of the contacting layeror encapsulating layer materials disclosed herein.

[0248] In FIG. 4D, the cross-section is taken along line D-D. The reference electrode 640 can be seen at this point. In FIG. 4E, the cross-section is taken along line E-E, working electrode 624 and pH electrode 625, are on opposing sides 602, 604, of the sensor assembly 600. In examples, at least a portion of pH electrode ("pH sensor") 625 comprises a field effect transistor (FET), an ion-sensitive field effect transistor (ISFET), a non-silicone based ion-sensitive field effect transistor a nano ion-sensitive field effect transistor (Nano-ISFET), a transition metal dichalcogenide (TMD), graphene, carbon nanotubes, zinc oxide, compound semiconductors, a silicon-on insulator ion-sensitive field effect transistor (SOI ISFET), an extended gate field effect transistor (EGFET). a metal oxide, a thin / th ick film metal oxide electrode a complementary metal oxide semiconductor (CMOS), a complementary metal oxide semiconductor ISFET (CMOS ISFET), a silicon-on insulator metal oxide semiconductor field-effect transistor (SOI MOSFET), or combinations thereof. In examples, at least a portionAttorney Docket No.: 0978-PCT01-0243of pH electrode ("pH sensor") 629 comprises a conductive polymer, e.g., polyaniline, a doped conductive polymer, and / or an ion selective polymer.

[0249] FIGS. 5A-5B illustrate a double-sided co-planar connected analyte sensor assembly 700, in accordance with an example. The sensor assembly 700 can include similar components to those of assembly 600 discussed above, except where otherwise noted.

[0250] FIGS. 5A to 5B depict schematic top-down drawings of opposing sides of the assembly 700. FIGS. 5C to 5E depict schematic cross-section drawings along cross-sections taken along C-C, D-D, and E-E, respectively, of the full sensor assembly 700. In some cases, the sensor assembly 700 can include a chamfer end, a rounded end, a flat end, or other appropriate shape. In some cases, the sensor assembly 700 can include a chamfer end, a rounded end, a flat end, or other appropriate shape.

[0251] The sensor assembly 700 can have a first side 702 and a second side 704 opposite the first side, in addition to a first end 712 and a second end 714. The sensor assembly 700 can include substrate 710, conductive traces 720, 721, connector pads 722, working electrode 724, and pH electrode 725, counter electrode 726, insulating layers 730, 732, and reference electrode 740. In sensor assembly 700, a double-sided planar configuration is used. In the sensor assembly 700, a multiple-electrode sensor is shown, with working electrode (WE) 724 and pH electrode 725, a counter electrode (CE) 726 and a reference electrode (RE) 740. In sensor assembly 700, the electrodes are co-planar. The assembly 700 is a co-planar, connected variation.

[0252] In sensor assembly 700, the substrate 710 is situated between two sides 702, 704, which can each host several co-planar components. For example, co-planar conductive traces 720 can be on the first side 702, and second conductive traces 721 can be on the second side 704. Each side 702, 704, can be covered by an insulating layer 730, 732. The insulating layers 730, 732, can define electrodes 724, 725, 726, and an area for the reference electrode 740.

[0253] In examples, reference electrode 740 is coated with a material that reduces or eliminates release of ions from the reference electrode. In examples, reference electrode 740 is coated with a reference electrode coating 729 that reduces or eliminates release of cations from the reference electrode. In examples, reference electrode 740 is coated with a reference electrode coating 729 that reduces or eliminates release of silver cations (Ag+).Attorney Docket No.: 0978-PCT01-0243Exemplary materials for coating the reference electrode include parylene, fluoropolymers (Teflon) fluorinated alkyl polymer fluorinated alkyl polymer, or any one or combination of the contacting layer or encapsulating layer materials disclosed herein.

[0254] The assembly 700 can also include vias, which can provide for an electrical connection between both sides 702, 704 of the sensor assembly 700. Including vias can allow for connection to the sensing electronics through the connector pads 722 on a single side 702 of the sensor, as well as routing traces to new locations, allowing flexible geometries to be used. The vias can be formed from various conductive materials discussed herein, including carbon, graphitic carbon, Pt, or combinations including Pt and C, Au and carbon. In some examples, the conductive material forming the vias between sides 702, 704 of the assembly or other assemblies as discussed herein may or may not further include conductive nanoparticles.

[0255] Shown in FIGS. 5A to 5E, the assembly 700 can include four connector pads 722 can be on a first side 702, electrically coupled to the electrodes 725, 740, on the second side 704 by vias and traces. In some cases, a WE, RE, and CE can be placed on the opposite side of the sensor assembly 700 to the connector pads 722. In some cases, as shown in assembly 700, a first working electrode 724 and counter electrode 726 can be located on the first side 702 of the sensor, while pH electrode 725 and a reference electrode 740 can be located on the other side 704 of the sensor. Vias can be used to establish electrical contact between traces and pads on both sides 702, 704 of the sensor assembly 700, since the connector pads 722 for connecting to the sensing electronics, in some examples, are located only on one side. In examples, working electrode (WE) 724 is coated with the sensing membrane with multiple layers or domains as disclosed herein.

[0256] In examples, at least a portion of pH electrode ("pH sensor") 729 comprises a field effect transistor (FET), an ion-sensitive field effect transistor (ISFET), a non-silicone based ion-sensitive field effect transistor a nano ion-sensitive field effect transistor (Nano-ISFET), a transition metal dichalcogenide (TMD), graphene, carbon nanotubes, zinc oxide, compound semiconductors, a silicon-on insulator ion-sensitive field effect transistor (SOI ISFET), an extended gate field effect transistor (EGFET). a metal oxide, a thin / thick film metal oxide electrode a complementary metal oxide semiconductor (CMOS), a complementary metal oxide semiconductor ISFET (CMOS ISFET), a silicon-on insulator metal oxideAttorney Docket No.: 0978-PCT01-0243semiconductor field-effect transistor (SOI MOSFET), or combinations thereof. In examples, at least a portion of pH electrode 729 comprises a conductive polymer, a doped conductive polymer, and / or an ion selective polymer.Dual Electrode / Dual Analyte Sensor

[0257] In examples, the presently disclosed continuous EAB analyte sensor is configured for the detection of multiple analytes or of one or more analyte and pH. In examples, signals corresponding to different analytes / pH are obtained intermittently, e.g., alternating between applied potentials. In examples, signals corresponding to pH and signals corresponding to one or more analytes are obtained in continuous time intervals, e.g., accumulating signals between changing the applied potential.

[0258] FIG. 6A is a perspective view of the in vivo portion of an example of a multielectrode sensor system 300 comprising working electrode 302, pH electrode 303, and at least one reference / counter electrode 320. The sensor 300 comprises first and second elongated bodies El, E2, each formed of a conductive core or of a core with a conductive layer deposited thereon. In this particular example, a wire-based sensor is shown, a planar arrangement being discussed above. In this particular example, an insulating layer 310, a conductive layer 320 e.g., a reference electrode, with reference electrode coating 329 as presently disclosed, and any one of the previously described membranes (not shown) are deposited on top of the elongated bodies El, E2. The insulating layer 310 separates the conductive layer 320 from the elongated body. Reference electrode coating 329 reduces or eliminates leaching from the reference electrode. The materials selected to form the insulating layer 310 may include any of the insulating materials described elsewhere herein, including polyurethane and polyimide.

[0259] The materials selected to form the pH electrode 303 can include any of the materials described elsewhere herein, including a field effect transistor (FET), an ionsensitive field effect transistor (ISFET), a non-silicone based ion-sensitive field effect transistor a nano ion-sensitive field effect transistor (Nano-ISFET), a transition metal dichalcogenide (TMD), graphene, carbon nanotubes, zinc oxide, compound semiconductors, a silicon-on insulator ion-sensitive field effect transistor (SOI ISFET), an extended gate field effect transistor (EGFET). a metal oxide, a thin / thick film metal oxide electrode a complementary metal oxide semiconductor (CMOS), a complementary metal oxideAttorney Docket No.: 0978-PCT01-0243semiconductor ISFET (CMOS ISFET), a silicon-on insulator metal oxide semiconductor fieldeffect transistor (SOI MOSFET), or combinations thereof, and / or a conductive polymer, a conductive polymer coating, and / or an ion selective polymer.

[0260] The materials selected to form the conductive layer 320 may include any of the conductive materials described elsewhere herein, including silver / silver chloride, platinum, gold, etc. Working electrode 302 and pH electrode 303 are formed by removing portions of the conductive layer 320, reference electrode coating 329, and the insulating layer 310, thereby exposing electroactive surface of the elongated bodies El, E2, respectively. FIG. 6B provides a close perspective view of the distal portion of the elongated bodies El, E2.

[0261] In examples, the two elongated bodies illustrated in FIG. 6A are fabricated to have substantially the same shape and dimensions. In examples, the two elongated bodies illustrated in FIG. 6A are fabricated to have substantially the same shape and dimensions but with one elongated body being a pH sensor, whereas the other elongated body includes one or more active aptamers with specificity towards one or more analytes.

[0262] In other examples, the two elongated bodies illustrated in FIG. 6A, but with one elongated body having no window and a drug releasing layer, e.g., an anti-inflammatory agent releasing layer, whereas the other elongated body includes a window 39 and sensing membrane with an active enzyme without a drug releasing layer to correct for any lag in signal corresponding to differences in blood and ISF analyte concentrations.

[0263] In some examples, the working electrodes of FIG. 6A are fabricated to have the same properties, thereby providing a sensor system capable of providing redundancy of signal measurements or providing unique signals representing two or more different analytes. In other examples, the working electrodes, associated with the elongated bodies El, E2, may each have one or more characteristics that distinguish each working electrode from the other. For example, In examples, each of the elongated bodies El, E2 may be different conductive surfaces, so that each working electrode has a different electrochemical property than the other working electrode. In addition, In examples, each of the elongated bodies El, E2 may be covered with different membrane(s), so that each working electrode has a different membrane property than the other working electrode.

[0264] Although not shown in FIGS. 6A-6B, in certain examples, the exposed distal ends 330, 331 of the core portions of the elongated bodies El, E2 may be covered with anAttorney Docket No.: 0978-PCT01-0243insulating material (e.g., polyurethane or polyimide). In alternative examples, the exposed distal ends 330, 331 of the core portions are covered with any of the previously described membrane system and / or serve as additional or "secondary" working electrode surface area.

[0265] Regarding fabrication of the sensor system illustrated in FIG. 6A-6B, In examples, the elongated bodies El, E2 may be formed as an elongated conductive core, or alternatively as a core (conductive or non-conductive) having at least one conductive material deposited thereon. Next, an insulating layer 310 is deposited onto each of the elongated bodies El, E2. Thereafter, a conductive layer 320 is deposited over the insulating layer 310. The conductive layer 320 with reference electrode coating 329, may serve as a reference / counter electrode and may be formed of silver / silver chloride, or any other material that may be used for a reference electrode. In alternative examples, the conductive layer 320 may be formed of a different conductive material, and may be used another working electrode. After these steps, a layer removal process is performed to remove portions of the deposited layers (i.e., the conductive layer 320 and / or the insulating layer 310) followed by coating with reference electrode coating 329.

[0266] Any of the techniques described elsewhere herein (e.g., laser ablation, chemical etching, grit blasting) may be used. In the example illustrated in FIGS. 6A-6B, layers of the conductive layer 320 and the insulating layer 310 are removed to form the working electrode 302 and pH electrode 303. Although in the example shown, layer removal is performed across the entire cross-sectional perimeter (e.g., circumference) of the deposited layer, it is contemplated that in other examples, layer removal may be performed across a preselected section of the cross-sectional perimeter, instead of across the entire cross-sectional perimeter.

[0267] In examples, the reference electrode coating 329, 629, 729 comprises a fluorine-containing polymer. In examples, the fluorine-containing polymer is a fluorinated alkyl polymer. In examples, the fluorine-containing polymer is Teflon or a derivative thereof. In examples, the reference electrode coating 329, 629, 729 comprises a deposited film coating. In examples, the deposited film coating comprises parylene C, parylene D, parylene N, or derivatives thereof.Attorney Docket No.: 0978-PCT01-0243

[0268] In examples, the reference electrode coating 329, 629, 729 comprises a hydrophobic polymer or a hydrophilic polymer. In examples, the reference electrode coating 329, 629, 729comprises a polymer having both hydrophilic and hydrophobic components. In examples, the reference electrode coating 329, 629, 729 comprises a polyurethane and / or polyurea polymer. In examples, the polyurethane and / or polyurea polymer comprises hard segments and soft segments.

[0269] In examples, the soft segments of the reference electrode coating 329, 629, 729 is different, chemically (composition) or structurally (molecular weight, segment length, etc.) than the APL, the contacting layer 105 and / or the encapsulation layer 107. In examples, the soft segments of the reference electrode coating 329, 629, 729 comprise poly(tetramethylene oxide) repeating units. In examples, the soft segments of the reference electrode coating 329, 629, 729 comprise polydialkylsiloxane repeating units and / or polyalkylcarbonate repeating units. In other examples, the soft segments of the reference electrode coating 329, 629, 729 comprise poly(tetramethylene oxide) repeating units and polydialkylsiloxane repeating units. In other examples, the reference electrode coating 329, 629, 729 comprises a fluorine-containing polymer, for example, Teflon or a fluorinated alkyl polymer. In examples, the reference electrode coating 329, 629, 729 comprises a deposited film coating, for example, parylene C, parylene D, parylene N, or derivatives thereof.

[0270] Contacts 304 are used to provide electrical connection between the working electrodes and other components of the sensor system may be formed in a similar manner. As shown, contacts 304 are separated from each other to prevent an electrical connection therebetween. Because the layer removal process is performed on each individual elongated body El, E2, instead of a single geometrically complicated elongated body, this particular sensor design (i.e., two elongated bodies placed side by side) may provide ease of manufacturing, as compared to the manufacturing processes involved with other multielectrode systems having other geometries.

[0271] After the conductive and insulating layers are deposited onto the elongated body, and after selected portions of the deposited layers have been removed, one or more aptamers, co-adsorbates, APLs are applied onto at least a portion of the elongated bodies using the apparatuses and method disclosed herein, either alone or in combination with the apparatuses and method disclosed herein or with other coating apparatuses and methods.Attorney Docket No.: 0978-PCT01-0243In certain examples, any of the aforementioned membrane systems are applied only to the working electrodes, but in other examples any of the aforementioned aptamers, coadsorbates, APLs are applied to the entire elongated body. In examples, any of the aforementioned aptamers, co-adsorbates, APLs are deposited onto the two working electrodes simultaneously while they are placed together (e.g., by bundling), but in other examples, any of the aforementioned aptamers, co-adsorbates, APLs are deposited onto each individual working electrode first, and the two working electrodes are then placed together.

[0272] In examples, the two elongated bodies illustrated in FIG. 6A are fabricated to have substantially the same shape and dimensions but with one elongated body having at least one enzyme with specificity towards a first analyte, whereas the other elongated body is a second analyte sensor. In this configuration, the applied potential can be alternated during a suitable frequency to detect the first and / or second analyte.

[0273] FIG. 7A is a side view of an analyte sensor system, the system mounted on the skin of a host, illustrating an analyte sensor 34 implanted into the host. A mounting unit 14 may be adhered to the host's skin using an adhesive pad 8. The adhesive pad 8 may be formed from an extensible material, which may be removably attached to the skin using an adhesive. The sensor electronics 106 may mechanically couple to the adhesive pad 8. In examples, the adhesive pad comprises conductive material (ink, or metal) configured to function as an external electrode, with or without the reference electrode coating.

[0274] FIG. 7B is an enlarged view of a distal portion of the analyte sensor 34. The analyte sensor 34 may be adapted for insertion under the host's skin and may be mechanically coupled to the mounting unit 14 and electrically coupled to the sensor electronics (not shown). The example analyte sensor 34 shown in FIG. 7B includes an elongated conductive body 41. The elongated conductive body 41 can include a core with various layers positioned thereon. A first layer 38 that at least partially surrounds the core and includes a working electrode, for example located in window 39. In some examples, the core and the first layer 38 are made of a single material. In some examples, the elongated conductive body 41 is a composite of two conductive materials, or a composite of at least one conductive material and at least one non-conductive material. A membrane system 32Attorney Docket No.: 0978-PCT01-0243is located over the working electrode and may cover other layers and / or electrodes of the sensor 34, as described herein.

[0275] The first layer 38 may be formed of a conductive material. The working electrode (at window 39) is an exposed portion of the surface of the first layer 38. Accordingly, the first layer 38 is formed of a material configured to provide a suitable electroactive surface for the working electrode. Examples of suitable materials include, but are not limited to, platinum, platinum-iridium, gold, palladium, iridium, nitinol, stainless steel, graphite, carbon, a ternary metal oxide composite, a conductive polymer, an alloy, and / or the like.

[0276] A second layer 40 surrounds at least a portion of the first layer 38, thereby defining boundaries of the working electrode. In some examples, the second layer 40 serves as an insulator and is formed of an insulating material, such as polyimide, polyurethane, parylene, or any other suitable insulating materials or materials. In some examples, the second layer 40 is configured such that the working electrode of the layer 38 is exposed via the window 39.

[0277] In some examples, the sensor 34 further includes a third layer 43 comprising a conductive material. The third layer 43 may comprise a reference electrode. In some examples, the third layer 43, including the reference electrode, is formed of a silver-containing material that is applied onto the second layer 40 e.g., an insulator. The silver-containing material may include various materials and be in various forms such as, for example, Ag / AgCl-polymer pastes, paints, polymer-based conducting mixtures, inks, etc. In examples, the reference electrode is coated with a material to reduce or eliminate release of ions therefrom, as discussed herein.

[0278] The analyte sensor 34 may include two or more electrodes, e.g., a working electrode at the layer 38 and exposed at window 39 and at least one additional electrode, such as a reference electrode of the layer 43. In the example arrangement of FIG. 7B, the reference electrode also functions as a counter electrode, although other arrangements can include a separate counter electrode. While the analyte sensor 34 may be used with a mounting unit in some examples, in other examples, the analyte sensor 34 may be used with other types of sensor systems. For example, the analyte sensor 34 may be part of a system that includes a battery and sensor in a single package, and may optionally include, for example, a near-field communication (NFC) circuit and / or drug delivery unit.Attorney Docket No.: 0978-PCT01-0243

[0279] FIG. 7C is a cross-sectional view through the sensor 34 of FIG. 7B on plane 7B-7B illustrating an exemplary membrane system 32. In examples, the membrane system 32 includes a number of domains e.g., layers. In examples, the membrane system 32 may include an aptamer domain 42, a diffusion resistance domain 44, and a bioprotective domain 46 located around the working electrode. In some examples, a diffusion resistance domain and bioprotective domain is included in the membrane system 32, e.g., wherein the functionality of both the diffusion resistance domain and bioprotective domain are incorporated into one domain.

[0280] The membrane system 32, in some examples, optionally includes an electrode layer 47. The electrode layer 47 may be arranged to provide an environment between the surfaces of the working electrode and the reference electrode that facilitates the electrochemical reaction between the electrodes. For example, the electrode layer 47 may include a coating that maintains a layer of water at the electrochemically reactive surfaces of the sensor 34.

[0281] In some examples, the sensor 34 may be configured for short-term implantation (e.g., from about 1 to 30 days). However, it is understood that the membrane system 32 can be modified for use in other devices, for example, by including only one or more of the domains, or additional domains. For example, a membrane system may include a plurality of resistance layers, or a plurality of enzyme layers. In some examples, the resistance domain 44 may include a plurality of resistance layers.

[0282] The diffusion resistance domain 44 may include a semipermeable membrane that controls the flux of interferents to the underlying aptamer domain 42.

[0283] In some examples, the membrane system 32 may include a bioprotective domain 46, also referred to as a domain or biointerface domain, comprising a base polymer as described in more detail elsewhere herein. However, the membrane system 32 of some examples can also include a plurality of domains or layers including, for example, an electrode domain, an interference domain, or a cell disruptive domain, such as described in more detail elsewhere herein and in U.S. Pat. Nos. 7,494,465, 8,682,408, and 9,44,199, which are incorporated herein by reference in their entirety.Attorney Docket No.: 0978-PCT01-0243Electronics

[0284] In examples, the presently disclosed continuous AB or EAB sensor further comprises one or more of a transmitter, receiver, controller, or power supply. Any electronics associated with continuous analyte sensors, such as non-invasive, minimally invasive, and / or invasive (e.g., transcutaneous and wholly implantable) sensors is applicable. For example, the sensor electronics and data processing as well as transceiver electronics, Wi-Fi, Bluetooth, RF and data processing known in the art can be incorporated into the presently disclosed AB or EAB sensor.

[0285] FIG. 8 is a diagram depicting an example continuous AB or EAB system 150 configured to measure one or more analytes alone or in combination with electrophysiological indicators (e.g., blood pressure, heart rate, core temperature, etc.) as discussed herein. The continuous AB or EAB system 150 includes exemplary continuous AB or EAB device 100, 200 operatively connected to a host 120 and a plurality of display devices 134 a-e according to certain aspects of the present disclosure. It should be noted that display device 134e alternatively or in addition to being a display device, may be a medicament delivery device that can act cooperatively with the continuous AB or EAB system 150 to deliver medicaments to host 120. In examples, the continuous AB or EAB system 150 is an EAB system, where a sensor electronics module 126 and a continuous EAB sensor 100 associated with the sensor electronics module 126. The sensor electronics module 126 may be in direct wireless communication with one or more of the plurality of the display devices 134a-e via wireless communications signals. In examples, display devices 134a-e may also communicate amongst each other and / or through each other to continuous AB or EAB system 150. For ease of reference, wireless communications signals from analyte sensor 100 to display devices 134a-e can be referred to as "uplink" signals 128. Wireless communications signals from, e.g., display devices 134a-e to continuous AB or EAB system 150 can be referred to as "downlink" signals 130. Wireless communication signals between two or more of display devices 134a-e may be referred to as "crosslink" signals 132. Additionally, wireless communication signals can include data transmitted by one or more of display devices 134a-d via "long-range" uplink signals 136 (e.g., cellular signals) to one or more remote servers 140 or network entities, such as cloud-based servers or databases, and receive long-range downlink signals 138 transmitted by remote servers 140.Attorney Docket No.: 0978-PCT01-0243

[0286] The sensor electronics module 126 includes sensor electronics that are configured to process sensor information and generate transformed sensor information. In certain examples, the sensor electronics module 126 includes electronic circuitry associated with measuring and processing data from continuous EAB sensor 100, including prospective algorithms associated with processing and calibration of the continuous analyte sensor data. The sensor electronics module 126 can be integral with (non-releasably attached to) or releasably attachable to the continuous EAB sensor 100 achieving a physical connection therebetween. The sensor electronics module 126 may include hardware, firmware, and / or software that enables analyte level measurement. For example, the sensor electronics module 126 can include a potentiostat, a power source for providing power to continuous EAB device 100, other components useful for signal processing and data storage, and a telemetry module for transmitting data from itself to one or more display devices 134a-e. Electronics can be affixed to a printed circuit board (PCB), or the like, and can take a variety of forms. For example, the electronics can take the form of an integrated circuit (IC), such as an Application-Specific Integrated Circuit (ASIC), an electrochemical analog front end, a microcontroller, and / or a processor. In examples, the electrochemical analog front end is configured with a sequencer or waveform synthesizer to create the appropriate waveforms to transduce the signal from the EAB. Exemplary waveforms include squarewave voltammetry, linear sweep voltammetry, cyclic voltammetry, differential pulse voltammetry, AC voltammetry, pulse voltammetry, staircase voltammetry, normal pulse voltammetry, chronoamperometry, and chronocoulometry. Examples of systems and methods for processing sensor analyte data are described in more detail in U.S. Pat. Nos. 7,310,544 and 6,931,327 and U.S. Patent Publication Nos. 2005 / 0043598, 2007 / 0032706, 2007 / 0016381, 2008 / 0033254, 2005 / 0203360, 2005 / 0154271, 2005 / 0192557, 2006 / 0222566, 2007 / 0203966 and 2007 / 0208245, each of which are incorporated herein by reference in their entirety for all purposes.

[0287] Display devices 134a-e are configured for displaying, alarming, and / or basing medicament delivery on the sensor information that has been transmitted by the sensor electronics module 126 (e.g., in a customized data package that is transmitted to one or more of display devices 134a-e based on their respective preferences). Each of the display devices 134a-e can include a display such as a touchscreen display for displaying sensorAttorney Docket No.: 0978-PCT01-0243information to a user (most often host 120 or a care taker / medical professional) and / or receiving inputs from the user. In some examples, the display devices 134a-e may include other types of user interfaces such as a voice user interface instead of or in addition to a touchscreen display for communicating sensor information to the user of the display device 134a-e and / or receiving user inputs. In some examples, one, some or all of the display devices 134a-e are configured to display or otherwise communicate the sensor information as it is communicated from the sensor electronics module 126 (e.g., in a data package that is transmitted to respective display devices 134a-e), without any additional prospective processing required for calibration and real-time display of the sensor information.

[0288] In the example of FIG. 8, one of the plurality of display devices 134a-e may be a custom display device 134a specially designed for displaying certain types of displayable sensor information associated with analyte values received from the sensor electronics module 126 (e.g., a numerical value and an arrow, in some examples). In some examples, one of the plurality of display devices 134a-e may be a handheld device 134c, such as a mobile phone based on the Android, iOS operating system or other operating system, a palm-top computer and the like, where handheld device 134c may have a relatively larger display and be configured to display a graphical representation of the continuous sensor data (e.g., including current and historic data). Other display devices can include other handheld devices, such as a tablet 134d, a smart watch 134b, a medicament delivery device 134e, a blood glucose meter, and / or a desktop or laptop computers.

[0289] As alluded to above, because the different display devices 134a-e provide different user interfaces, content of the data packages (e.g., amount, format, and / or type of data to be displayed, alarms, and the like) can be customized (e.g., programmed differently by the manufacture and / or by an end user) for each particular display device and / or display device type. Accordingly, in the example of FIG. 6, one or more of display devices 134a-e can be in direct or indirect wireless communication with the sensor electronics module 126 to enable a plurality of different types and / or levels of display and / or functionality associated with the sensor information, which is described in more detail elsewhere herein.

[0290] The analyte sensor system 150 also includes sensor electronics 106. In some examples, the analyte sensor 100 and sensor electronics 106 are provided as an integrated package. In other examples, the analyte sensor 100 and sensor electronics 106 are providedAttorney Docket No.: 0978-PCT01-0243as separate components or modules. For example, in one embodiment the analyte sensor system 150 includes a disposable (e.g., single-use) base that may include the analyte sensor 100, a component for attaching the sensor 100 to a host (e.g., an adhesive pad), or a mounting structure configured to receive another component. The system 102 also includes a sensor electronics package, which includes some or all of the sensor electronics 106 shown in FIG. 9 discussed below. The sensor electronics package may be reusable.

[0291] An analyte sensor 100 may use any known method, including invasive, minimally-invasive, or non-invasive sensing techniques (e.g., optically excited fluorescence, microneedle, transdermal monitoring of analytes), to provide a data stream indicative of the concentration of the analyte in a host 101. In some examples, the data stream is a raw data signal, which is converted into a calibrated and / or filtered data stream that is used to provide a useful value of the analyte (e.g., estimated blood analyte concentration level) to a user, such as a patient or a caretaker (e.g., a parent, a relative, a guardian, a teacher, a doctor, a nurse, or any other individual that has an interest in the wellbeing of the host 101).

[0292] Analyte sensor 100 may, for example, be a continuous analyte sensor, which may, for example, include a subcutaneous, transdermal (e.g., transcutaneous), or intravascular device. In some embodiments, such a sensor or device may recurrently (e.g., periodically or intermittently) analyze sensor data. The presently disclosed EAB analyte sensor may be used in combination with any method of analyte measurement, including enzymatic, chemical, physical, electrochemical, spectrophotometric, polarimetric, calorimetric, iontophoretic, radiometric, immunochemical, and the like. In various examples, the analyte sensor system 150 is or includes a continuous analyte monitor sensor.

[0293] In some examples, the system 100 also includes a second medical device 108, which may, for example, be a drug delivery device (e.g., pump). In some examples, the medical device 108 includes a sensor, such as another analyte sensor 100, a heart rate sensor, a respiration sensor, a motion sensor (e.g. accelerometer), posture sensor (e.g. 3-axis accelerometer), acoustic sensor (e.g. to capture ambient sound or sounds inside the body). In some examples, medical device 108 may be wearable, e.g., on a watch, glasses, contact lens, patch, wristband, ankle band, or other wearable item, or may be incorporated into a handheld device (e.g., a smartphone). In some examples, the medical device 108 mayAttorney Docket No.: 0978-PCT01-0243include a multi-sensor patch that may, for example, detect one or more of an analyte level (e.g., glycopeptide antibiotics, for example, vancomycin, teicoplanin, telavancin, ramoplanin and decaplanin, corbomycin, complestatin and the antitumor antibiotic bleomycin, L-DOPA, glucose, lactate, insulin or other substance), heart rate, respiration (e.g., using impedance), activity (e.g., using an accelerometer), posture (e.g., using an accelerometer), galvanic skin response, tissue fluid levels (e.g., using impedance or pressure).

[0294] The analyte sensor system 150 may communicate with the second medical device 108 via a wired connection, or via a wireless communication signal 110. For example, the analyte sensor system 150 may be configured to communicate using via radio frequency (e.g., Bluetooth, Medical Implant Communication System (MICS), Wi-Fi, NFC, RFID, Zigbee, Z-Wave or other communication protocols), optically (e.g., infrared), sonically (e.g., ultrasonic), or a cellular protocol (e.g., CDMA (Code Division Multiple Access) or GSM (Global System for Mobiles)), or via a wired connection (e.g., serial, parallel, etc.).

[0295] The system 150 may also include a wearable sensor 130, which may include a sensor circuit (e.g., a sensor circuit configured to detect an analyte concentration) and a communication circuit, which may, for example, be a near field communication (NFC) circuit. In some examples, information from the wearable sensor 130 may be retrieved from the wearable sensor 130 using a user device 132 such as a smart phone that is configured to communicate with the wearable sensor 130 via NFC when the user device 132 is placed near the wearable sensor 130 (e.g., swiping the user device 132 over the sensor 130 retrieves sensor data from the wearable sensor 130 using NFC). The use of NFC communication may reduce power consumption by the wearable sensor 130, which may reduce the size of a power source (e.g., battery or capacitor) in the wearable sensor 130 or extend the usable life of the power source. In some examples, the wearable sensor 130 may be wearable on an upper arm as shown. In some examples, a wearable sensor 130 may additionally or alternatively be on the upper torso of the patient (e.g., over the heart or over a lung), which may, for example, facilitate detecting heart rate, respiration, or posture. A wearable sensor 136 may also be on the lower body (e.g., on a leg).

[0296] In some examples, an array or network of sensors may be associated with the patient. For example, one or more of the analyte sensor system 150, medical device 108, wearable device 120 such as a watch, and an additional wearable sensor 130 mayAttorney Docket No.: 0978-PCT01-0243communicate with one another via wired or wireless (e.g., Bluetooth, MICS, NFC or any of the other options described above,) communication. The additional wearable sensor 130 may be any of the examples described above with respect to medical device 108. The analyte sensor system 150, medical device 108, and additional sensor 130 on the host 101 are provided for the purpose of illustration and description and are not necessarily drawn to scale.

[0297] The system 150 may also include one or more peripheral devices, such as a handheld smart device (e.g., smartphone) 112, tablet 114, smart pen 116 (e.g., drug delivery pen with processing and communication capability), computer 118, a wearable device 120 such as a watch, or peripheral medical device 122 (which may be a proprietary device such as a proprietary user device available from DexCom), any of which may communicate with the analyte sensor system 150 via a wireless communication signal 110, and may also communicate over a network 124 with a server system (e.g., remote data center) 126 or with a remote terminal 128 to facilitate communication with a remote user (not shown) such as a technical support staff member or a clinician.

[0298] The wearable device 120 may include an activity sensor, a heart rate monitor (e.g., light-based sensor or electrode-based sensor), a respiration sensor (e.g., acoustic- or electrode-based), a location sensor (e.g., GPS), or other sensors.

[0299] The system 150 may also include a wireless access point (WAP) 138 that may be used to communicatively couple one or more of analyte sensor system 150, network 124, server system 126, medical device 108 or any of the peripheral devices described above. For example, WAP 138 may provide Wi-Fi and / or cellular connectivity within system 150. Other communication protocols (e.g., Near Field Communication (NFC) or Bluetooth) may also be used among devices of the system 150. In some examples, the server system 126 may be used to collect analyte data from analyte sensor system 150 and / or the plurality of other devices, and to perform analytics on collected data, generate or apply universal or individualized models for analyte levels, and communicate such analytics, models, or information based thereon back to one or more of the devices in the system 100.

[0300] FIG. 9 is a schematic illustration of various example electronic components that may be part of system 150. In examples, the system 299 may include sensor electronics 106 and a base 290. While a specific example of division of components between the base 290Attorney Docket No.: 0978-PCT01-0243and sensor electronics 106 is shown, it is understood that some examples may include additional components in the base 290 or in the sensor electronics 106, and that some of the components (e.g., a battery or supercapacitor) that are shown in the sensor electronics 106 may be alternatively or additionally (e.g., redundantly) provided in the base 290.

[0301] In examples, the base 290 may include the analyte sensor 100 and a battery 292. In some examples, the base 290 may be replaceable, and the sensor electronics 106 may include a debouncing circuit (e.g., gate with hysteresis or delay) to avoid, for example, recurrent execution of a power-up or power down process when a battery is repeatedly connected and disconnected or avoid processing of noise signal associated with removal or replacement of a battery.

[0302] The sensor electronics 106 may include electronics components that are configured to process sensor information, such as sensor data, and generate transformed sensor data and displayable sensor information. The sensor electronics 106 may, for example, include electronic circuitry associated with measuring, processing, storing, or communicating continuous analyte sensor data, including prospective algorithms associated with processing and calibration of the sensor data. The sensor electronics 106 may include hardware, firmware, and / or software that enables measurement of levels of the analyte via an EAB sensor. Electronic components may be affixed to a printed circuit board (PCB), or the like, and can take a variety of forms. For example, the electronic components may take the form of an integrated circuit (IC), such as an Application-Specific Integrated Circuit (ASIC), a microcontroller, and / or a processor.

[0303] As shown in FIG. 9, the sensor electronics 106 may include a measurement circuit 202 (e.g., potentiostat), which may be coupled to the analyte sensor 100 and configured to recurrently obtain analyte sensor readings using the analyte sensor 100, for example by continuously or recurrently measuring a current flow indicative of concentration of an analyte. The sensor electronics 106 may include a gate circuit 294, which may be used to gate the connection between the measurement circuit 202 and the analyte sensor 100. In examples, the analyte sensor 100 accumulates charge over an accumulation period, and the gate circuit 294 is opened so that the measurement circuit 202 can measure the accumulated charge. Gating the analyte sensor 100 may improve the performance of the sensor system 102 by creating a larger signal to noise or interference ratio (e.g., becauseAttorney Docket No.: 0978-PCT01-0243charge accumulates from an analyte reaction, but sources of interference do not accumulate, or accumulate less than the charge from the analyte reaction). The sensor electronics 106 may also include a processor 204, which may retrieve instructions 206 from memory 208 and execute the instructions 206 to determine control application of bias potentials to the analyte sensor 100 via the potentiostat, interpret signals from the sensor 100, or compensate for environmental factors. The processor 204 may also save information in data storage memory 210 or retrieve information from data storage memory 210. In various examples, data storage memory 210 may be integrated with memory 208, or may be a separate memory circuit, such as a non-volatile memory circuit (e.g., flash RAM).Examples of systems and methods for processing sensor analyte data are described in more detail herein and in U.S. Pat. Nos. 7,310,544 and 6,931,327.

[0304] The sensor electronics 106 may also include a sensor 212, which may be coupled to the processor 204. The sensor 212 may be a temperature sensor, accelerometer, or another suitable sensor. The sensor electronics 106 may also include a power source such as a capacitor or battery 214, which may be integrated into the sensor electronics 106, or may be removable, or part of a separate electronics package. The battery 214 (or other power storage component, e.g., capacitor) may optionally be rechargeable via a wired or wireless (e.g., inductive or ultrasound) recharging system 216. The recharging system 216 may harvest energy or may receive energy from an external source or on-board source. In various examples, the recharge circuit may include a triboelectric charging circuit, a piezoelectric charging circuit, an RF charging circuit, a light charging circuit, an ultrasonic charging circuit, a heat charging circuit, a heat harvesting circuit, or a circuit that harvests energy from the communication circuit. In some examples, the recharging circuit may recharge the rechargeable battery using power supplied from a replaceable battery (e.g., a battery supplied with a base component).

[0305] The sensor electronics 106 may also include one or more supercapacitors in the sensor electronics package (as shown), or in the base 290. For example, the supercapacitor may allow energy to be drawn from the battery 214 in a highly consistent manner to extend the life of the battery 214. The battery 214 may recharge the supercapacitor after the supercapacitor delivers energy to the communication circuit or to the processor 204, so that the supercapacitor is prepared for delivery of energy during a subsequent high-load period.Attorney Docket No.: 0978-PCT01-0243In some examples, the supercapacitor may be configured in parallel with the battery 214. A device may be configured to preferentially draw energy from the supercapacitor, as opposed to the battery 214. In some examples, a supercapacitor may be configured to receive energy from a rechargeable battery for short-term storage and transfer energy to the rechargeable battery for long-term storage.

[0306] The supercapacitor may extend an operational life of the battery 214 by reducing the strain on the battery 214 during the high-load period. In some examples, a supercapacitor removes at least 10% of the strain off the battery during high-load events. In some examples, a supercapacitor removes at least 20% of the strain off the battery during high-load events. In some examples, a supercapacitor removes at least 30% of the strain off the battery during high-load events. In some examples, a supercapacitor removes at least 50% of the strain off the battery during high-load events.

[0307] The sensor electronics 106 may also include a wireless communication circuit 218, which may for example include a wireless transceiver operatively coupled to an antenna. The wireless communication circuit 218 may be operatively coupled to the processor 204 and may be configured to wirelessly communicate with one or more peripheral devices or other medical devices.

[0308] A peripheral device 250 may, for example, be a wearable device (e.g., activity monitor), such as a wearable device 120. In other examples, the peripheral device 250 may be a hand-held smart device 112 (e.g., smartphone or other device such as a proprietary handheld device available from Dexcom), a tablet 114, a smart pen 116, or special-purpose computer 118 shown in FIG. 8.

[0309] The peripheral device 250 may include a user interface 252, a memory circuit 254, a processor 256, a wireless communication circuit 258, a sensor 260, or any combination thereof. The peripheral device 250 may also include a power source, such as a battery. The peripheral device 250 may not necessarily include all of the components shown in FIG. 9. The user interface 252 may, for example, include a touch-screen interface, a microphone (e.g., to receive voice commands), or a speaker, a vibration circuit, or any combination thereof, which may receive information from a user or deliver information to the user such as analyte values, analyte trends (e.g., an arrow, graph, or chart), or analyte level alerts. The processor 256 may be configured to present information to a user, orAttorney Docket No.: 0978-PCT01-0243receive input from a user, via the user interface 252. The processor 256 may also be configured to store and retrieve information, such as communication information (e.g., pairing information or data center access information), user information, sensor data or trends, or other information in the memory circuit 254. The wireless communication circuit 258 may include a transceiver and antenna configured to communicate via a wireless protocol, such as Bluetooth, MICS, or any of the other options described above. The sensor 260 may, for example, include an accelerometer, a temperature sensor, a location sensor, biometric sensor, or blood glucose sensor, blood pressure sensor, heart rate sensor, respiration sensor, or other physiologic sensor. The peripheral device 250 may, for example, be a hand-held smart device 112 (e.g., smartphone or other device such as a proprietary handheld device available from Dexcom), tablet 114, smart pen 116, watch or other wearable device 120, or computer 118 shown in FIG. 8.

[0310] The peripheral device 250 may be configured to receive and display sensor information that may be transmitted by sensor electronics 106 (e.g., in a customized data package that is transmitted to the display devices based on their respective preferences). Sensor information (e.g., analyte concentration level) or an alert or notification (e.g., "high analyte level", "low analyte level" or "fall rate alert" may be communicated via the user interface 252 (e.g., via visual display, sound, or vibration). In some examples, the peripheral device 250 may be configured to display or otherwise communicate the sensor information as it is communicated from the sensor electronics 106 (e.g., in a data package that is transmitted to respective display devices). For example, the peripheral device 250 may transmit data that has been processed (e.g., an estimated concentration of an analyte level that may be determined by processing raw sensor data), so that a device that receives the data may not be required to further process the data to determine usable information (such as the estimated concentration of an analyte level). In other examples, the peripheral device 250 may process or interpret the received information (e.g., to declare an alert based on analyte values or a analyte trend). In various examples, the peripheral device 250 may receive information directly from sensor electronics 106, or over a network (e.g., via a cellular or Wi-Fi network that receives information from the sensor electronics 106 or from a device that is communicatively coupled to the sensor electronics 106).Attorney Docket No.: 0978-PCT01-0243

[0311] Referring again to FIG. 9, the medical device 270 may include a user interface 272, a memory circuit 274, a processor 276, a wireless communication circuit 278, a sensor 280, a therapy circuit 282, or any combination thereof. The user interface 272 may, for example, include a touch-screen interface, a microphone, or a speaker, a vibration circuit, or any combination thereof, which may receive information from a user (e.g., analyte values, alert preferences, calibration coding) or deliver information to the user, such as e.g., analyte values, analyte trends (e.g., an arrow, graph, or chart), or analyte alerts. The processor 276 may be configured to present information to a user, or receive input from a user, via the user interface 272. The processor 276 may also be configured to store and retrieve information, such as communication information (e.g., pairing information or data center access information), user information, sensor data or trends, or other information in the memory circuit 274. The wireless communication circuit 278 may include a transceiver and antenna configured communicate via a wireless protocol, such as Bluetooth, Medical Implant Communication System (MICS), Wi-Fi, Zigbee, or a cellular protocol (e.g., CDMA (Code Division Multiple Access) or GSM (Global System for Mobiles)). The sensor 280 may, for example, include an accelerometer, a temperature sensor, a location sensor, biometric sensor, blood pressure sensor, heart rate sensor, respiration sensor, or other physiologic sensor. The medical device 270 may include two or more sensors (or memories or other components), even though only one sensor 280 is shown in the example in FIG. 9. In various examples, the medical device 270 may be a smart handheld analyte sensor (e.g., meter), drug pump, or other physiologic sensor device, therapy device, or combination thereof. In various examples, the medical device 270 may be the medical device 108, peripheral medical device 122, wearable device 120, wearable sensor 130, or wearable sensor 136 shown in FIG. 7A.

[0312] In examples where the peripheral medical device 122 or medical device 270 is an drug pump, the pump and analyte sensor system 150 may be in two-way communication (e.g., so the pump can request a change to an analyte transmission protocol, e.g., request a data point or request data on a more frequent schedule), or the pump and analyte sensor system 150 may communicate using one-way communication (e.g., the pump may receive concentration of an analyte level information from the analyte sensor system). In one-way communication, an analyte value may be incorporated in an advertisement message, whichAttorney Docket No.: 0978-PCT01-0243may be encrypted with a previously-shared key. In a two-way communication, a pump may request a value, which the analyte sensor system 150 may share, or obtain and share, in response to the request from the pump, and any or all of these communications may be encrypted using one or more previously-shared keys. An drug pump may receive and track analyte values transmitted from analyte sensor system 150 using one-way communication to the pump for one or more of a variety of reasons. For example, a drug pump may suspend or activate administration based on an analyte value being below or above a threshold value.

[0313] In some examples, the system 150 shown in FIG. 9 may include two or more peripheral devices that each receives information directly or indirectly from the analyte sensor system 150. Because different display devices provide many different user interfaces, the content of the data packages (e.g., amount, format, and / or type of data to be displayed, alarms, and the like) may be customized (e.g., programmed differently by the manufacturer and / or by an end user) for each particular device. For example, in the example of FIG. 8, a plurality of different peripheral devices may be in direct wireless communication with a sensor electronics module (e.g., such as an on-skin sensor electronics 106 that is physically connected to the continuous analyte sensor 100) during a sensor session to enable a plurality of different types and / or levels of display and / or functionality associated with the displayable sensor information, or, to save battery power in the sensor system 102, one or more specified devices may communicate with the analyte sensor system 150 and relay (i.e., share) information to other devices directly or through a server system (e.g., a network-connected data center) 126.Experimental Results

[0314] A series of exemplary APL's were developed and tested with an aptamer-redox moiety conjugate EAB constructs and the effectiveness of the APL's in providing improvement in one or more attributes of the constructs evaluated. Characteristics of a representative sampling of the APL's are summarized in Table 1.Attorney Docket No.: 0978-PCT01-0243Tab e 1. Exemplary APLs. PUU = aliphatic polyurethane urea segmented block copolymer. PU = aliphatic polyurethane segmented block copolymer. Hydrophilic segment = polyethylene glycol and polycarbonate. Hydrophobic segment = poly dimethyl siloxane. Functional content = betaine. Crosslinker = polyglycol polyglycidyl (PEG-PG).

[0315] FIG. 10 demonstrates the current-voltage relationship of an exemplary EAB sensor construct and its peak current value 1005. FIGs. 11A, 11B depict the effect of pH on the reduction-oxidation peak voltage and peak current, respectively, of an exemplary redox moiety, methylene blue.

[0316] FIG. 12 depicts the effect on the current-voltage relationship with varying pH concentration. As shown, the current-voltage relationship changes with changing pH (data represents a pH range of 6.5 to 7.4 pH). Likewise, FIG. 13 shows the current-voltage relationship of a plurality of in vivo EAB sensors (without analyte present) where theoretically, the current-voltage relationship should be linear as depicted by line 1010, but deviates with the changing pH of the in vivo milieu. FIG. 14 depicts raw data of peak current 1012 and peak voltage 1014 over time of an in vivo EAB sensor, demonstrating the greater sensitivity to pH changes on peak current than peak voltage. FIGs. 15A and 15B depict the effect of temperature variation (25-35 °C) of a sampling of EAB sensors, demonstrating a greater effect of temperature on peak current.

[0317] While certain embodiments of the present disclosure have been illustrated with reference to specific combinations of elements, various other combinations may also be provided without departing from the teachings of the present disclosure. Thus, the presentAttorney Docket No.: 0978-PCT01-0243disclosure should not be construed as being limited to the particular exemplary embodiments described herein and illustrated in the Figures, but may also encompass combinations of elements of the various illustrated embodiments and aspects thereof.

Claims

1. Attorney Docket No.: 0978-PCT01-0243We CLAIM:

1. An electrochemical aptamer biosensor comprising:at least one aptamer electrically associated with at least one working electrode, the at least one aptamer configured to undergo a reversible conformational change in the presence of at least one analyte;a redox moiety coupled to the one or more aptamer, wherein the redox moiety provides a signal corresponding to a concentration of the at least one analyte wherein the at least one aptamer and / or the redox moiety is sensitive to changes in pH; andat least one pH sensor electrically isolated from the at least one working electrode.

2. The electrochemical aptamer biosensor of claim 1, where reduction and oxidation of the redox moiety is reversible.

3. The electrochemical aptamer biosensor of any one of the previous claims, wherein the at least one working electrode comprises a first working electrode and a second working electrode, the second working electrode electrically isolated from the first working electrode.

4. The electrochemical aptamer biosensor of any one of the previous claims, wherein the at least second working electrode is configured to generate a signal associated with a second analyte, the second analyte being chemically different from the first analyte.

5. The electrochemical aptamer biosensor of any one of the previous claims, further comprising a reference electrode and / or a counter electrode.

6. The electrochemical aptamer biosensor of any one of the previous claims, wherein the at least one aptamer is present on a wire substrate or a planar substrate.

7. The electrochemical aptamer biosensor of any one of the previous claims, wherein the pH sensor is present on a wire substrate or a planar substrate.

8. The electrochemical aptamer biosensor of any one of the previous claims, wherein the at least one working electrode comprises a first working electrode and a second working electrode, the second working electrode being electrically isolated from the first working electrode.

9. The electrochemical aptamer biosensor of claim 8, wherein the at least one aptamer is present on the first electrode wire substrate and the pH electrode is present on the second working electrode.Attorney Docket No.: 0978-PCT01-024310. The electrochemical aptamer biosensor of any one of the previous claims, wherein the planar substrate comprises a first electrode and a second working electrode, the second working electrode being electrically isolated from the first working electrode.

11. The electrochemical aptamer biosensor of claim 10, wherein the at least one aptamer is present on the first electrode of the planar substrate and the pH electrode is present on the second working electrode of the planar substrate.

12. The electrochemical aptamer biosensor of any one of the previous claims, wherein at least a portion of the working electrode is a conductive metal.

13. The electrochemical aptamer biosensor of any one of the previous claims, wherein at least a portion of the working electrode is gold, carbon, conductive ink, graphene, or graphene oxide.

14. The electrochemical aptamer biosensor of any one of the previous claims, wherein at least a portion of the pH sensor comprises an ion selective polymer coated wire or an ion selective polymer coated contact pad.

15. The electrochemical aptamer biosensor of any one of the previous claims, wherein at least a portion of the pH sensor comprises a field effect transistor.

16. The electrochemical aptamer biosensor of any one of the previous claims, wherein at least a portion of the pH sensor comprises an ion-sensitive field effect transistor (ISFET).

17. The electrochemical aptamer biosensor of any one of the previous claims, wherein at least a portion of the pH sensor comprises a non-silicone based ion-sensitive field effect transistor.

18. The electrochemical aptamer biosensor of any one of the previous claims, wherein at least a portion of the pH sensor comprises a nano ion-sensitive field effect transistor.

19. The electrochemical aptamer biosensor of any one of the previous claims, wherein at least a portion of the pH sensor comprises transition metal dichalcogenide, graphene, carbon nanotubes, zinc oxide, compound semiconductor or combinations thereof.

20. The electrochemical aptamer biosensor of any one of the previous claims, wherein at least a portion of the pH sensor comprises a silicon-on insulator ion-sensitive field effect transistor.

21. The electrochemical aptamer biosensor of any one of the previous claims, wherein at least a portion of the pH sensor comprises an extended gate field effect transistor.Attorney Docket No.: 0978-PCT01-024322. The electrochemical aptamer biosensor of any one of the previous claims, wherein at least a portion of the pH sensor comprises metal oxide.

23. The electrochemical aptamer biosensor of any one of the previous claims, wherein at least a portion of the pH sensor comprises a thin / thick film metal oxide electrode.

24. The electrochemical aptamer biosensor of any one of the previous claims, wherein at least a portion of the pH sensor comprises a complementary metal oxide semiconductor.

25. The electrochemical aptamer biosensor of any one of the previous claims, wherein at least a portion of the pH sensor comprises a complementary metal oxide semiconductor ISFET.

26. The electrochemical aptamer biosensor of any one of the previous claims, wherein at least a portion of the pH sensor comprises a silicon-on insulator metal oxide semiconductor field-effect transistor.

27. The electrochemical aptamer biosensor of any one of the previous claims, wherein at least a portion of the pH sensor comprises a conductive polymer.

28. The electrochemical aptamer biosensor of any one of the previous claims, wherein the electrochemical aptamer biosensor further comprises a temperature sensor.

29. The electrochemical aptamer biosensor of any one of the previous claims, wherein the sensor is configured for transcutaneous insertion.

30. The electrochemical aptamer biosensor of any one of the previous claims, wherein the sensor further comprises one or more of a transmitter, receiver, controller, or power supply.

31. The electrochemical aptamer biosensor of any one of the previous claims, wherein the reversible redox moiety comprises iron, iridium, ruthenium, osmium, a thiazine dye, or derivative thereof.

32. The electrochemical aptamer biosensor of any one of the previous claims, wherein the reversible redox moiety comprises a ferrocene, methylene blue, or derivative thereof.

33. The electrochemical aptamer biosensor of any one of the previous claims, wherein the at least one aptamer is physically associated to a portion of the working electrode.

34. The electrochemical aptamer biosensor of any one of the previous claims, wherein the at least one aptamer is covalently associated to a portion of the working electrode.Attorney Docket No.: 0978-PCT01-024335. The electrochemical aptamer biosensor of any one of the previous claims, further comprising at least one co-adsorbate.

36. The electrochemical aptamer biosensor of any one of the previous claims, wherein the at least one co-adsorbate is physically associated to a portion of the working electrode.

37. The electrochemical aptamer biosensor of any one of the previous claims, wherein the at least one co-adsorbate is covalently associated to a portion of the working electrode.

38. The electrochemical aptamer biosensor of any one of the previous claims, wherein the at least one aptamer comprises RNA or DNA nucleotide sequences.

39. The electrochemical aptamer biosensor of any one of the previous claims, wherein the at least one aptamer comprises at least one of 2'-O-methyl modification of a nucleotide, disulfide bridges, a 3' cap with an inverted 2-deoxy thymidine, a 3'-3'-thymidine linkage at 3' terminus, a 2'-F modification, conjugation to biotin, or a double stranded section.

40. The electrochemical aptamer biosensor of any one of the previous claims, wherein the at least one aptamer comprises the RNA or the DNA sequences with a first linker moiety on a 5' end and the reversible redox moiety at a 3' end, or wherein the at least one aptamer comprises the RNA or the DNA sequences with a first linker moiety on a 3' end and the reversible redox moiety at a 5' end.

41. The electrochemical aptamer biosensor of any one of the previous claims, wherein the first linker moiety on the 5' end or 3' end comprises an amino group or a carboxyl group.

42. The electrochemical aptamer biosensor of any one of the previous claims, wherein the first linker moiety is physically or chemically coupled to the substrate at the 5' end or 3' end.

43. The electrochemical aptamer biosensor of any one of the previous claims, wherein the first linker moiety is physically or chemically coupled to the co-adsorbate at the 5' end or the 3' end.

44. The electrochemical aptamer biosensor of any one of the previous claims, wherein the at least one aptamer is a glycopeptide antibiotic binding aptamer.

45. The electrochemical aptamer biosensor of claim 44, wherein the glycopeptide antibiotic is selected from vancomycin, teicoplanin, telavancin, ramoplanin, decaplanin, corbomycin, complestatin, or bleomycin.Attorney Docket No.: 0978-PCT01-024346. The electrochemical aptamer biosensor of any one of the previous claims, wherein the at least one aptamer is a vancomycin binding aptamer.

47. The electrochemical aptamer biosensor of any one of the previous claims, wherein the at least one aptamer is a neurotransmitter binding aptamer or a hormone binding aptamer.

48. The electrochemical aptamer biosensor of any one of the previous claims, wherein the at least one aptamer is a dopamine, L-DOPA, insulin, or glutamate binding aptamer.

49. The electrochemical aptamer biosensor of any one of the previous claims, wherein the at least one aptamer is a carbohydrate, triglyceride or fatty acid binding aptamer.

50. The electrochemical aptamer biosensor of any one of the previous claims, wherein the at least one aptamer is a glucose, a glycerol, or a beta-hydroxy butyrate binding aptamer.

51. The electrochemical aptamer biosensor of any one of the previous claims, wherein the at least one aptamer is physically or chemically coupled to a self-assembled monolayer (SAM).

52. The electrochemical aptamer biosensor of any one of the previous claims, wherein the at least one aptamer is physically or chemically coupled to a mono-functional, a multifunctional alkanethiol, or a mercaptoalkanol.

53. The electrochemical aptamer biosensor of any one of the previous claims, wherein the at least one aptamer is physically or chemically coupled to an alkylthiol betaine.

54. The electrochemical aptamer biosensor of any one of the previous claims, wherein the at least one aptamer is physically or chemically coupled to an aliphatic amine.

55. The electrochemical aptamer biosensor of any one of the previous claims, wherein the at least one aptamer is physically or chemically coupled to an amino alkanoic acid.

56. The electrochemical aptamer biosensor of any one of the previous claims, wherein the reversible redox moiety comprises an organometallic compound comprising iron, iridium, ruthenium, or osmium.

57. The electrochemical aptamer biosensor of any one of the previous claims, wherein the reversible redox moiety comprises a thiazine dye, or derivative thereof.

58. The electrochemical aptamer biosensor of any one of the previous claims, wherein the reversible redox moiety comprises ferrocene, methylene blue, or a derivative thereof.Attorney Docket No.: 0978-PCT01-024359. The electrochemical aptamer biosensor of any one of the previous claims, wherein the electrochemical aptamer biosensor is sterile.

60. The electrochemical aptamer biosensor of any one of the previous claims, further comprising a reference electrode coating.

61. The electrochemical aptamer biosensor of any one of the previous claims, wherein the reference electrode coating comprises a hydrophobic polymer or a hydrophilic polymer.

62. The electrochemical aptamer biosensor of any one of the previous claims, wherein the reference electrode coating comprises a fluorine-containing polymer.

63. The electrochemical aptamer biosensor of any one of the previous claims, wherein the fluorine-containing polymer comprises Teflon or a fluorinated alkyl polymer.

64. The electrochemical aptamer biosensor of any one of the previous claims, wherein the reference electrode coating comprises a deposited film coating.

65. The electrochemical aptamer biosensor of any one of the previous claims, wherein the deposited film coating comprises parylene C, parylene D, parylene N, or derivatives thereof.

66. The electrochemical aptamer biosensor of any one of the previous claims, wherein the reference electrode coating comprises a polymer chain having both hydrophilic and hydrophobic regions.

67. The electrochemical aptamer biosensor of claim 65, wherein the reference electrode coating comprises a polymer chain having polyurethane and / or polyurea segments.

68. The electrochemical aptamer biosensor of claim 66, wherein the polymer comprises hard segments and soft segments.

69. The electrochemical aptamer biosensor of claim 68, wherein the soft segments comprise poly(tetramethylene oxide) repeating units.

70. The electrochemical aptamer biosensor of any one of claims 68-69, wherein the soft segments comprise polydialkylsiloxane repeating units.

71. The electrochemical aptamer biosensorof any one of claims 68-70, wherein the soft segments comprise both poly(tetramethylene oxide) repeating units and polydialkylsiloxane repeating units.

72. The electrochemical aptamer biosensor of any one of the previous claims, further comprising a contacting layer adjacent the at least one aptamer.Attorney Docket No.: 0978-PCT01-024373. The electrochemical aptamer biosensor of claim 72, wherein the contacting layer comprises a polymer having both hydrophilic and hydrophobic components.

74. The electrochemical aptamer biosensor of any one of claims 72-73, wherein the contacting layer comprises a polyurethane and / or polyurea polymer.

75. The electrochemical aptamer biosensor of claim 74, wherein the polyurethane and / or polyurea polymer comprises hard segments and soft segments.

76. The electrochemical aptamer biosensorof of any one of claims 74-75, wherein the soft segments comprise poly(tetramethylene oxide) repeating units.

77. The electrochemical aptamer biosensor of any one of claims 74-76, wherein the soft segments comprise polydialkylsiloxane repeating units and / or polyalkylcarbonate repeating units.

78. The electrochemical aptamer biosensor of any one of claims 74-77, wherein the soft segments comprise poly(tetramethylene oxide) repeating units and polydialkylsiloxane repeating units.

79. The electrochemical aptamer biosensor of any one of claims 74-78, wherein the contacting layer comprises a polyurethane and / or polyurea polymer blended with polyvinylpyrrolidone.

80. The electrochemical aptamer biosensor of any one of claims 72-79, wherein the contacting layer comprises a polyelectrolyte polymer or a polymerized monomer comprising a zwitterionic functional group.

81. The electrochemical aptamer biosensor of any one of claims 72-80, wherein the contacting layer further comprises one or more co-adsorbates.

82. The electrochemical aptamer biosensor of claim 81, wherein the one or more coadsorbates comprises a self-assembled monolayer (SAM) associated with the working electrode.

83. The electrochemical aptamer biosensor of any one of claims 81-82, wherein each of the one or more co-adsorbates comprises a one or more functional groups.

84. The electrochemical aptamer biosensor of any one of claims 81-83, wherein the one or more co-adsorbates comprises a mono-functional or a multi-functional alkanethiol, mercaptoalkanol, alkylmercaptoalkanol, or arylmercaptoalkanol functional group.Attorney Docket No.: 0978-PCT01-024385. The electrochemical aptamer biosensor of any one of claims 81-84, wherein the functional group comprises one or more ammoniophosphonates, ammoniophosphinates, ammoniosulfonates, alkanethiol betaine, ammoniosulfates, ammoniocarboxylates,and ammoniocarboxylates.

86. The electrochemical aptamer biosensor of any one of claims 81-85, wherein the contacting layer provides for one or more of a surface energy range, a pH range, a phase separation range, and an intermolecular interaction range.

87. The electrochemical aptamer biosensor of any one of claims 81-86, wherein the contacting layer further comprises at least one mono-, di-, tri- or polysaccharide.

88. The electrochemical aptamer biosensor of claim 87, wherein the at least one mono-, di-, tri- or polysaccharide is at least partially amorphous between a temperature range of 0 °C to 40 °C.

89. The electrochemical aptamer biosensor of any one of claims 72-88, further comprising at least one encapsulating layer adjacent the contacting layer.

90. The electrochemical aptamer biosensor of any one of claims 72-89, wherein the encapsulating layer is different than the contacting layer.

91. The electrochemical aptamer biosensor of any one of claims 72-90, wherein the encapsulating layer is different than the reference electrode coating.

92. The electrochemical aptamer biosensor of any one of claims 69-71, wherein the encapsulating layer comprises a polymer having both hydrophilic and hydrophobic regions.

93. The electrochemical aptamer biosensor of any one of claims 69-72, wherein the encapsulating layer comprises a polyurethane and / or polyurea polymer.

94. The electrochemical aptamer biosensor of claim 93, wherein the polyurethane and / or polyurea polymer comprises hard segments and soft segments.

95. The electrochemical aptamer biosensor of any one of claims 93-94, wherein the soft segments comprise poly(tetramethylene oxide) repeating units.

96. The electrochemical aptamer biosensor of any one of claims 93-95, wherein the soft segments comprise polydialkylsiloxane repeating units and / or polyalkylcarbonate repeating units.Attorney Docket No.: 0978-PCT01-024397. The electrochemical aptamer biosensor of any one of claims 93-96, wherein the soft segments comprise both poly(tetramethylene oxide) repeating units and / or polydialkylsiloxane repeating units and / or polyalkylcarbonate repeating units.

98. The electrochemical aptamer biosensor of any one of claims 93-97, wherein the encapsulating layer comprises a polyurethane and / or polyurea polymer blended with polyvinylpyrrolidone.

99. The electrochemical aptamer biosensor of any one of claims 93-98, wherein the encapsulating layer comprises a polyelectrolyte polymer or a polymerized monomer comprising a zwitterionic functional group.

100. The electrochemical aptamer biosensor of any one of claims 93-100, wherein the encapsulating layer comprises a polymer with a styrene group.

101. The electrochemical aptamer biosensor of any one of claims 93-101, wherein the encapsulating layer comprises a polymer with a heterocyclic group.

102. The electrochemical aptamer biosensor of any one of claims 93-102, wherein the encapsulating layer comprises a polymer chain having poly(l-vinyl imidazole), poly(4-vinyl pyridine), poly(2-vinyl pyridine), polyacrylonitrile, polyacrylamide, and / or copolymers or quaternized forms thereof.

103. The electrochemical aptamer biosensor of any one of claims 93-103, further comprising a topcoat adjacent the encapsulating layer, the topcoat comprising a polymer chain having poly(l-vinyl imidazole), poly(4-vinyl pyridine), poly(2-vinyl pyridine), acrylonitrile, acrylamide, and / or copolymers or quaternized forms thereof.

104. The electrochemical aptamer biosensorof claim 103, wherein the encapsulating layer and / or the topcoat is at least partially cross-linked.

105. A method of measuring a concentration of an analyte in vivo with an electrochemical aptamer biosensor system, the method comprising:providing an electrochemical aptamer biosensor system configured for in vivo use, the electrochemical aptamer biosensor system comprising:at least one aptamer associated with an electroactive surface configured for obtaining a first signal corresponding to a concentration of an analyte, the aptamer comprising a reversible redox moiety coupled thereto; andAttorney Docket No.: 0978-PCT01-0243a pH sensor configured for obtaining a second signal corresponding to a pH value in proximity to the at least one aptamer.

106. The method of claim 105, further comprising adjusting the first signal based on a parameter related to the second signal and improving accuracy of the electrochemical aptamer biosensor system.

107. The method of any one of claims 105-106, wherein at least a portion of the pH sensor comprises an ion selective polymer or ion selective membrane coated wire or contact pad.

108. The method of any one of claims 105-107, wherein at least a portion of the pH sensor comprises a field effect transistor (FET), an ion-sensitive field effect transistor (ISFET), a nonsilicone based ion-sensitive field effect transistor a nano ion-sensitive field effect transistor (Nano-ISFET), a transition metal dichalcogenide (TMD), graphene, carbon nanotubes, zinc oxide, compound semiconductors, a silicon-on insulator ion-sensitive field effect transistor (SOI ISFET), an extended gate field effect transistor (EGFET). a metal oxide, a thin / thick film metal oxide electrode a complementary metal oxide semiconductor (CMOS), a complementary metal oxide semiconductor ISFET (CMOS ISFET), a silicon-on insulator metal oxide semiconductor field-effect transistor (SOI MOSFET), or combinations thereof.

109. The method of any one of claims 105-108, wherein at least a portion of the pH sensor comprises a conductive polymer.

110. The method of any one of claims 105-109, wherein the electrochemical aptamer biosensor system further comprises a temperature sensor.

111. The method of any one of claims 105-110, further comprising a reference and / or counter electrode.

112. The method of claim 111, wherein the reference electrode comprises a reference electrode coating configured to reduce release of cations from the reference electrode.

113. The method of any one of claims 111-112, wherein the reference electrode coating comprises a hydrophobic polymer or a hydrophilic polymer.

114. The method of any one of claims 111-113, wherein the reference electrode coating comprises a polymer chain having both hydrophilic and hydrophobic regions.

115. The method of any one of claims 111-113, wherein the reference electrode coating comprises a polymer chain having polyurethane and / or polyurea segments.Attorney Docket No.: 0978-PCT01-0243116. The method of claim 115, wherein the polymer comprises hard segments and soft segments.

117. The method of any one of claims 115-116, wherein the soft segments comprise poly(tetramethylene oxide) repeating units.

118. The method of any one of claims 115-117, wherein the soft segments comprise polydialkylsiloxane repeating units and / or polyalkylcarbonate repeating units.

119. The method of any one of claims 115-118, wherein the soft segments comprise both poly(tetramethylene oxide) repeating units and / or polydialkylsiloxane repeating units and / or polyalkylcarbonate repeating units.

120. The method of claim 112, wherein the reference electrode coating comprises a fluorine-containing polymer.

121. The method of claim 120, wherein the fluorine-containing polymer comprises Teflon or a fluorinated alkyl polymer.

122. The method of claim 112, wherein the reference electrode coating comprises a deposited film coating.

123. The method of claim 122, wherein the deposited film coating comprises parylene C, parylene D, parylene N, or derivatives thereof.

124. A method of operating an electrochemical aptamer analyte sensor for measuring a concentration of at least one analyte in a fluid, the analyte sensor comprising:an analyte sensing portion disposed on a surface of a first working electrode, the analyte sensing portion comprising:at least one aptamer electrically associated with the first working electrode, the at least one aptamer configured to undergo a reversible conformational change in the presence of the at least one analyte;a reversible redox moiety coupled to the at least one aptamer;at least one pH electrode electrically isolated from the first working electrode; the analyte sensing portion configured for introduction to a subcutaneous space, the analyte sensing portion capable of at least measuring the concentration of the at least one analyte;the method comprising:Attorney Docket No.: 0978-PCT01-0243applying a potential to the first working electrode at or above an oxidation-reduction potential of the reversible redox moiety to generate a signal corresponding to the concentration in the subcutaneous space;applying a potential to the pH electrode to generate a second signal corresponding to the pH concentration in the subcutaneous space; andadjusting the first signal based on a parameter related to the second signal.

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