Aptamer protection material and biosensor

Abstract

There is provided an analyte monitoring sensor configured for in vivo measurement of at least one analyte, the sensor comprising: a substrate having a substrate surface; an aptamer protective layer encapsulating at least a portion of the surface of the substrate, the aptamer protective layer being permeable to the at least one analyte; one or more aptamer conjugates associated with at least a portion of the surface of the substrate and located between the aptamer protective layer and the substrate for obtaining a measurement relating to the at least one analyte in vivo. Methods of extending the in vivo performance of the analyte monitoring sensor are also described.

Description

Technical Field

[0001] The present disclosure relates to protective materials for aptamer-based biosensor constructs or devices adapted to be fully or partially implanted into a subject for continuous monitoring of an analyte. Background Art

[0002] An aptamer biosensor (AB) is a class of affinity biosensors where the recognition element is an aptamer (single-stranded DNA / RNA) that has specific affinity for an analyte, and this aptamer-analyte interaction induces a measurable transduction signal (optical, electrical). Existing aptamer biosensors (ABs) are limited by the poor stability observed when placed in a physiologically relevant environment, which is at least partially attributed to the desorption of the aptamer monolayer from the substrate surface or the desorption of the underlying monolayer used to immobilize the aptamer. These desorption events limit the application of ABs for continuous monitoring of analytes in physiological environments. Another weakness of AB sensors, particularly electrochemical aptamer biosensors (EABs), is that their bioelectronic interfaces degrade during continuous electrochemical interrogation and / or biological fouling, a process commonly seen as a Faradaic decay and an increase in charging current over time. This progressive degradation limits the in vivo operational lifetime of EABs to 12 hours or less, a time much shorter than the elimination half-life of the vast majority of drugs in humans. Summary of the Invention

[0003] In a first example, there is provided an analyte monitoring sensor configured for in vivo measurement of at least one analyte, the analyte monitoring sensor comprising: a substrate having a substrate surface; an aptamer protective layer encapsulating at least a portion of the substrate surface, the aptamer protective layer being permeable to the at least one analyte; one or more aptamer conjugates associated with at least a portion of the substrate surface and located between the aptamer protective layer and the substrate for obtaining in vivo measurement results related to the at least one analyte; and a reversible redox moiety coupled to the one or more aptamer conjugates.

[0004] In one aspect, alone or in combination with any of the foregoing aspects, at least a portion of the substrate is a conductive metal. In one aspect, alone or in combination with any of the foregoing aspects, at least a portion of the substrate is gold, carbon, graphene, or graphene oxide. In one aspect, alone or in combination with any of the foregoing aspects, at least a portion of the substrate includes pores having an average pore diameter of nano- and / or micro-scale dimensions.

[0005] In one aspect, alone or in combination with any of the foregoing aspects, at least a portion of the substrate surface further comprises one or more co-adsorbents. In one aspect, alone or in combination with any of the foregoing aspects, the one or more co-adsorbents independently comprise a plurality of functional groups.

[0006] In one aspect, alone or in combination with any of the foregoing aspects, the one or more co-adsorbents independently provide one or more of a surface energy range, a phase separation range, and an intermolecular interaction range between one or more aptamers and an aptamer protection layer. In one aspect, alone or in combination with any of the foregoing aspects, the one or more co-adsorbents independently provide an ionic strength, a pH range, or a pH buffer, and the amount of the one or more co-adsorbents present is capable of regulating or maintaining the ionic strength, pH range, or pH buffer in the vicinity of at least one aptamer conjugate.

[0007] In one aspect, alone or in combination with any of the foregoing aspects, at least a portion of the substrate surface, the one or more co-adsorbents, and a portion of the remainder of the substrate surface comprise the one or more aptamer conjugates. In one aspect, alone or in combination with any of the foregoing aspects, at least a portion of the substrate surface comprises one or more co-adsorbents, and a portion of the remainder of the substrate surface comprises one or more aptamer conjugates physically or chemically coupled thereto. In one aspect, alone or in combination with any of the foregoing aspects, the one or more co-adsorbents are physically or chemically coupled to the substrate surface, and a portion of the remainder of the substrate surface comprises one or more aptamer conjugates physically or chemically coupled thereto.

[0008] In one aspect, alone or in combination with any of the foregoing aspects, the co-adsorbate comprises a self-assembled monolayer (SAM). In one aspect, alone or in combination with any of the foregoing aspects, the co-adsorbate coupled or tethered to the substrate is represented as follows:

[0009]

[0010] wherein X is -OH, -NHR1, -NH2, or -SH; wherein R1 is an acyclic alkyl, a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted aryl, a substituted or unsubstituted benzyl, a substituted or unsubstituted heteroalkyl, or a substituted or unsubstituted heterocycle; and a is 1 - 3.

[0011] In one aspect, alone or in combination with any of the foregoing aspects, the co-adsorbate comprises a mono-functional or multi-functional alkyl mercaptan, hydroxyalkyl mercaptan, alkoxy mercaptan, alkylaryl mercaptan, hydroxyalkylaryl mercaptan, hydroxyalkylaryl mercaptan, alkylarylmercaptoalkanol, alkylmercaptophenol, alkylmercaptocatechol, arylmercaptophenol, arylmercaptocatechol, alkoxyaryl mercaptan, alkoxyaryl mercaptan (collectively referred to hereinafter as "thiol co-adsorbents"). In one aspect, alone or in combination with any of the foregoing aspects, the thiol functional group of the mono-functional or multi-functional alkyl mercaptan is covalently coupled to at least a portion of the substrate surface.

[0012] In one aspect, alone or in combination with any of the foregoing aspects, the thiol co-adsorbent is covalently coupled to the surface of the gold substrate. In one aspect, alone or in combination with any of the foregoing aspects, the co-adsorbate comprises a mono-functional or multi-functional mercaptoalkanol, benzylmercaptoalkanol or arylmercaptoalkanol (collectively referred to hereinafter as "(aryl) mercaptoalkanol"). In one aspect, alone or in combination with any of the foregoing aspects, the thiol functional group of the mono-functional or multi-functional (aryl) mercaptoalkanol is covalently coupled to at least a portion of the substrate surface. In one aspect, alone or in combination with any of the foregoing aspects, the thiol functional group of the mono-functional or multi-functional (aryl) mercaptoalkanol is covalently coupled to at least a portion of the gold substrate surface.

[0013] In one aspect, alone or in combination with any of the foregoing aspects, at least a portion of the substrate surface comprises zwitterionic repeating groups. In one aspect, alone or in combination with any of the foregoing aspects, the zwitterionic repeating groups comprise betaine groups. In one aspect, alone or in combination with any of the foregoing aspects, the zwitterionic repeating groups comprise ammonium phosphates or lecithin analogs.

[0014] In one aspect, alone or in combination with any of the foregoing aspects, the zwitterionic repeating groups comprise ammonium phosphonates. In one aspect, alone or in combination with any of the foregoing aspects, the zwitterionic repeating groups comprise ammonium phosphites. In one aspect, alone or in combination with any of the foregoing aspects, the zwitterionic repeating groups comprise ammonium sulfonates. In one aspect, alone or in combination with any of the foregoing aspects, the zwitterionic repeating groups comprise ammonium sulfates. In one aspect, alone or in combination with any of the foregoing aspects, the zwitterionic repeating groups comprise ammonium carboxylates.

[0015] In one aspect, alone or in combination with any of the foregoing aspects, the zwitterionic repeating group includes an alkylthiol betaine, a phenylthiol betaine, or a benzylthiol betaine. In one aspect, alone or in combination with any of the foregoing aspects, the alkylthiol, phenylthiol, or benzylthiol is straight-chain and contains a plurality of betaine groups along its chain. In one aspect, alone or in combination with any of the foregoing aspects, the alkylthiol, phenylthiol, or benzylthiol is a mono-thiol or di-thiol that is end-capped with at least one betaine group along its chain or aromatic ring. In one aspect, alone or in combination with any of the foregoing aspects, the zwitterionic repeating group includes an n-mercaptoalkanol betaine. In one aspect, alone or in combination with any of the foregoing aspects, the mercaptoalkanol is straight-chain and contains a plurality of betaine groups along its chain. In one aspect, alone or in combination with any of the foregoing aspects, the mercaptophenol contains one or more betaine groups attached to the aromatic ring. In one aspect, alone or in combination with any of the foregoing aspects, the mercaptoalkanol or mercaptophenol is a 1,2-dithiol, 1,3-dithiol, or 1,4-dithiol of an alkyl or aromatic hydrocarbon compound.

[0016] In one aspect, alone or in combination with any of the foregoing aspects, the thiol groups of the end-capped dithiol alkylthiol are covalently coupled to the substrate surface. In one aspect, alone or in combination with any of the foregoing aspects, the thiol groups of the mercaptoalkanol are covalently coupled to the substrate surface.

[0017] In one aspect, alone or in combination with any of the foregoing aspects, the co-adsorbent is selected from one or more of the following structures:

[0018]

[0019] where represents a hydrocarbon chain; where R1 and R2 are independently a branched or unbranched acyclic alkyl, a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted aryl, a substituted or unsubstituted benzyl, a substituted or unsubstituted heteroalkyl, a substituted or unsubstituted heterocycle; where X is -OH, -NHR1, -NH2, or -SH; where n is an integer from 2 to about 1000; or

[0020]

[0021] Wherein X is -OH, -NHR1, -NH2 or -SH; wherein W, Y and Z are independently branched or straight-chain alkyl, heteroalkyl, cycloalkyl, cycloheteroalkyl, aryl or heteroaryl, any of which may optionally be substituted with O, OH, halogen, amide or alkoxy; R1 is H, branched or unbranched acyclic alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloheteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted benzyl, or substituted or unsubstituted heteroaryl; and R3, R4 and R5 are independently selected from acyclic alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloheteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted benzyl, or substituted or unsubstituted heteroaryl. In one example, one or more of R1, R2, R3, R4, R5 and Z are covalently or ionically coupled to the APL.

[0022] In one aspect, alone or in combination with any of the foregoing aspects, at least a portion of the substrate surface comprises covalently coupled aliphatic amine. In one aspect, alone or in combination with any of the foregoing aspects, at least a portion of the substrate surface comprises covalently coupled aminoalkanoic acid.

[0023] In one aspect, alone or in combination with any of the foregoing aspects, at least a portion of the carbon, graphene or graphene oxide substrate surface comprises covalently coupled aminoalkanoic acid. In one aspect, alone or in combination with any of the foregoing aspects, at least a portion of the carbon, graphene or graphene oxide substrate surface comprises covalently coupled aminoalkanoic acid, and the aminoalkanoic acid is also covalently coupled to the one or more aptamer conjugates.

[0024] In one aspect, alone or in combination with any of the foregoing aspects, a certain amount of crosslinking agent is used to at least partially crosslink the aptamer protective layer. In one aspect, alone or in combination with any of the foregoing aspects, the aptamer protective layer comprises a conductive polymer. In one aspect, alone or in combination with any of the foregoing aspects, the aptamer protective layer comprises a zwitterionic group compound. In one aspect, alone or in combination with any of the foregoing aspects, the aptamer protective layer comprises a zwitterionic repeating group compound.

[0025] In one aspect, alone or in combination with any of the foregoing aspects, the aptamer protective layer provides an ionic strength, a pH range, or a pH buffer. In one aspect, alone or in combination with any of the foregoing aspects, the aptamer protective layer provides an amount of zwitterionic repeating group compound, and the amount of the zwitterionic repeating group compound present is capable of regulating or maintaining the ionic strength, the pH range, or the pH buffer. In one aspect, alone or in combination with any of the foregoing aspects, the aptamer protective layer provides a free volume that allows reversible conformational changes of one or more aptamer conjugates present therein, and the free volume is sufficient to provide a signal in the presence of the at least one analyte.

[0026] In one aspect, alone or in combination with any of the foregoing aspects, the aptamer protective layer comprises a functionalized polymer. In one aspect, alone or in combination with any of the foregoing aspects, the functionalized polymer comprises an alkanethiol group. In one aspect, alone or in combination with any of the foregoing aspects, the alkanethiol group is present at the end of the functionalized polymer chain. In one aspect, alone or in combination with any of the foregoing aspects, the alkanethiol group is present along the backbone of the functionalized polymer chain.

[0027] In one aspect, alone or in combination with any of the foregoing aspects, the functionalized polymer comprises a mercaptoalkanol group. In one aspect, alone or in combination with any of the foregoing aspects, the mercaptoalkanol group is present at the end of the functionalized polymer chain. In one aspect, alone or in combination with any of the foregoing aspects, the mercaptoalkanol group is present along the backbone of the functionalized polymer chain.

[0028] In one aspect, alone or in combination with any of the foregoing aspects, the aptamer protective layer comprises a functionalized polymer that comprises one or more zwitterionic repeating groups. In one aspect, alone or in combination with any of the foregoing aspects, the one or more zwitterionic repeating groups include a betaine compound or a derivative thereof. In one aspect, alone or in combination with any of the foregoing aspects, the zwitterionic repeating group is present at the end of the functionalized polymer chain. In one aspect, alone or in combination with any of the foregoing aspects, the zwitterionic repeating group is present along the backbone of the functionalized polymer chain.

[0029] In one aspect, alone or in combination with any of the foregoing aspects, the functionalized polymer comprises an alkanethiol and a zwitterionic repeating group. In one aspect, alone or in combination with any of the foregoing aspects, the functionalized polymer comprises an alkanethiol and a betaine group. In one aspect, alone or in combination with any of the foregoing aspects, the functionalized polymer comprises a mercaptoalkanol and a zwitterionic repeating group.

[0030] In one aspect, alone or in combination with any of the foregoing aspects, the functionalized polymer comprises a mercaptoalkanol and a betaine group.

[0031] In one aspect, alone or in combination with any of the foregoing aspects, the aptamer protective layer is physically or chemically coupled to at least a portion of the substrate surface. In one aspect, alone or in combination with any of the foregoing aspects, the aptamer protective layer is physically or chemically coupled to at least a portion of the substrate surface, one or more aptamer conjugates are physically or chemically coupled to at least a portion of the substrate surface, and substantially the remaining portion of the substrate surface further comprises co-adsorbates that are physically or chemically coupled.

[0032] In one aspect, alone or in combination with any of the foregoing aspects, the aptamer protective layer comprises at least one polymer segment selected from the group consisting of polyurethanes, polyureas, poly(urethane ureas), epoxides, polyolefins, polysiloxanes, polyamides, polystyrenes, polyacrylates, polyethers, polyvinylpyridines, polyvinylpyrrolidones, polyesters, polycarbonates, and copolymers thereof.

[0033] In one aspect, alone or in combination with any of the foregoing aspects, the aptamer protective layer comprises a segmented multiblock polymer. In one aspect, alone or in combination with any of the foregoing aspects, the aptamer protective layer comprises a segmented multiblock polyurethane polymer. In one aspect, alone or in combination with any of the foregoing aspects, the aptamer protective layer comprises a segmented multiblock urea polymer.

[0034] In one aspect, alone or in combination with any of the foregoing aspects, the segmented multiblock polymer comprises soft segments and hard segments. In one aspect, alone or in combination with any of the foregoing aspects, the soft segments are hydrophobic or hydrophilic. In one aspect, alone or in combination with any of the foregoing aspects, the soft segments are hydrophobic and hydrophilic. In one aspect, alone or in combination with any of the foregoing aspects, the soft segments comprise hydrophobic polyols and hydrophilic polyols.

[0035] In one aspect, alone or in combination with any of the foregoing aspects, the soft segments are one or more segments comprising polydimethylsiloxane, polycarbonate, polyester, polyether, and blends or copolymers thereof. In one aspect, alone or in combination with any of the foregoing aspects, the soft segments are one or more segments comprising polyethylene glycol, oligo polyether, polyoxazoline (POX), polypeptides, polyvinylpyrrolidone, zwitterionic repeating group polymers, and blends or copolymers thereof.

[0036] In one aspect, alone or in combination with any of the foregoing aspects, the hard segments comprise polyurethane groups or urea groups.

[0037] In one aspect, alone or in combination with any of the foregoing aspects, one or more aptamer conjugates physically associate with a portion of the substrate surface. In one aspect, alone or in combination with any of the foregoing aspects, one or more aptamer conjugates covalently associate with a portion of the substrate surface.

[0038] In one aspect, alone or in combination with any of the foregoing aspects, one or more aptamer conjugates comprise an RNA or DNA nucleotide sequence. In one aspect, alone or in combination with any of the foregoing aspects, one or more aptamer conjugates comprise at least one of the following: 2'-O-methyl modification of nucleotides; disulfide bridges; 3' caps with inverted 2'-deoxythymidine; 3'-3'-thymidine linkages at the 3' terminus; 2'-F modification; and double-stranded segments. In one aspect, alone or in combination with any of the foregoing aspects, one or more aptamer conjugates comprise an RNA or DNA sequence having a first linker portion at the 5' terminus and a reversible redox moiety at the 3' terminus. In one aspect, alone or in combination with any of the foregoing aspects, one or more aptamer conjugates comprise an RNA or DNA sequence having a first linker portion at the 3' terminus and a reversible redox moiety at the 5' terminus.

[0039] In one aspect, alone or in combination with any of the foregoing aspects, the first linker portion at the 5' or 3' terminus of the aptamer comprises an amino group or a carboxyl group. In one aspect, alone or in combination with any of the foregoing aspects, the first linker portion is physically or chemically coupled to the substrate at the 5' terminus. In one aspect, alone or in combination with any of the foregoing aspects, the first linker portion is physically or chemically coupled to a co-adsorbate at the 5' terminus. In one aspect, alone or in combination with any of the foregoing aspects, the first linker portion is physically or chemically coupled to the substrate at the 3' terminus. In one aspect, alone or in combination with any of the foregoing aspects, the first linker portion is physically or chemically coupled to a co-adsorbate at the 3' terminus.

[0040] In one aspect, alone or in combination with any of the foregoing aspects, one or more aptamer conjugates are glycopeptide antibiotic-binding aptamers. In one aspect, alone or in combination with any of the foregoing aspects, one or more aptamer conjugates are vancomycin-binding aptamers. In one aspect, alone or in combination with any of the foregoing aspects, one or more aptamer conjugates are neurotransmitter-binding aptamers. In one aspect, alone or in combination with any of the foregoing aspects, one or more aptamer conjugates are dopamine- or glutamate-binding aptamers. In one aspect, alone or in combination with any of the foregoing aspects, one or more aptamer conjugates are carbohydrate-, triglyceride- or fatty acid-binding aptamers. In one aspect, alone or in combination with any of the foregoing aspects, one or more aptamer conjugates are glucose-, glycerol- or β-hydroxybutyrate-binding aptamers.

[0041] In one aspect, alone or in combination with any of the foregoing aspects, one or more aptamer conjugates are physically or chemically coupled to a self-assembled monolayer (SAM). In one aspect, alone or in combination with any of the foregoing aspects, one or more aptamer conjugates are physically or chemically coupled to a mono-functional or multi-functional alkanethiol or mercaptoalkanol. In one aspect, alone or in combination with any of the foregoing aspects, one or more aptamer conjugates are physically or chemically coupled to an alkylthiol betaine.

[0042] In one aspect, alone or in combination with any of the foregoing aspects, one or more aptamer conjugates are physically or chemically coupled to an aliphatic amine. In one aspect, alone or in combination with any of the foregoing aspects, one or more aptamer conjugates are physically or chemically coupled to an aminoalkanoic acid.

[0043] In one aspect, alone or in combination with any of the foregoing aspects, the reversible redox moiety comprises iron, iridium, ruthenium, osmium, thiazine dyes or derivatives thereof. In one aspect, alone or in combination with any of the foregoing aspects, the reversible redox moiety comprises ferrocene or methylene blue.

[0044] In one aspect, alone or in combination with any of the foregoing aspects, the sensor is configured for continuous, semi-continuous, sequential or random signal acquisition. In one aspect, alone or in combination with any of the foregoing aspects, the sensor further comprises one or more of a reference electrode, a working electrode and a counter electrode. In one aspect, alone or in combination with any of the foregoing aspects, the sensor further comprises one or more of a transmitter, a receiver, a controller or a power source. In one aspect, alone or in combination with any of the foregoing aspects, the sensor is configured for percutaneous insertion.

[0045] In a second example, a method of extending the end-of-life of an electrochemical aptamer biosensor (EAB) is provided, the method comprising: electroassociating at least one aptamer conjugate with a surface of a conductive substrate, the at least one aptamer conjugate comprising a reversible redox moiety; encapsulating the at least one aptamer conjugate in an aptamer protective layer, the at least one aptamer conjugate being configured to undergo a reversible conformational change within the aptamer protective layer in response to interaction with an analyte so as to generate a detectable signal; controlling one or more of: the ionic strength, pH range or pH buffering within the aptamer protective layer, the surface phase separation of the aptamer protective layer, and the intermolecular interaction between the at least one aptamer conjugate and the aptamer protective layer; and extending the end-of-life of the electrochemical aptamer sensor.

[0046] In one aspect, alone or in combination with any of the foregoing aspects, controlling the ionic strength, providing a pH range or pH buffering comprises introducing one or more co-adsorbents into the aptamer protective layer, the one or more co-adsorbents being present in an amount capable of regulating or maintaining the ionic strength, pH range or pH buffering.

[0047] In one aspect, alone or in combination with any of the foregoing aspects, the one or more co-adsorbents comprise zwitterionic betaine groups. In one aspect, alone or in combination with any of the foregoing aspects, the one or more co-adsorbents comprising zwitterionic betaine groups are selected from the following structures:

[0048]

[0049] wherein represents a hydrocarbon chain; wherein R1 and R2 are independently branched or unbranched acyclic alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted benzyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycle; wherein X is -OH, -NHR1, -NH2 or -SH; wherein n is an integer from 2 to about 1000; or

[0050]

[0051] Wherein X is -OH, -NHR1, -NH2 or -SH; wherein W, Y and Z are independently branched or straight-chain alkyl, heteroalkyl, cycloalkyl, cycloheteroalkyl, aryl or heteroaryl, any of which may optionally be substituted with O, OH, halogen, amide or alkoxy; R1 is H, branched or unbranched acyclic alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloheteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted benzyl, or substituted or unsubstituted heteroaryl; and R3, R4 and R5 are independently selected from acyclic alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloheteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted benzyl, or substituted or unsubstituted heteroaryl. In one example, one or more of R1, R2, R3, R4, R5 and Z are covalently or ionically coupled to the APL.

[0052] In one aspect, alone or in combination with any of the foregoing aspects, the co-adsorbate is a terminally-capped dithiol having at least one betaine group along its chain. In one aspect, alone or in combination with any of the foregoing aspects, the thiol groups of the terminally-capped dithiol alkanethiol are covalently coupled to the substrate surface. In one aspect, alone or in combination with any of the foregoing aspects, the zwitterionic betaine group includes mercaptoalkanol betaine.

[0053] In one aspect, alone or in combination with any of the foregoing aspects, the mercaptoalkanol is straight-chain and contains a plurality of betaine groups along its chain. In one aspect, alone or in combination with any of the foregoing aspects, the thiol groups of the mercaptoalkanol are covalently coupled to the substrate surface.

[0054] In one aspect, alone or in combination with any of the foregoing aspects, controlling the ionic strength, pH range or pH buffering includes providing an aptamer protection layer having one or more zwitterionic betaine groups. In one aspect, alone or in combination with any of the foregoing aspects, controlling the ionic strength includes providing an aptamer protection layer having mercaptoalkanol and zwitterionic betaine groups. In one aspect, alone or in combination with any of the foregoing aspects, the aptamer protection layer includes alkanethiol, mercaptoalkanol, benzylthiol, mercaptophenol and one or more zwitterionic groups.

[0055] In one aspect, alone or in combination with any of the foregoing aspects, controlling the ionic strength, pH range or pH buffering includes providing an aptamer protection layer having a pH regulating composition or a pH buffering composition.

[0056] In one aspect, alone or in combination with any of the foregoing aspects, controlling the intermolecular interaction between at least one aptamer conjugate and the aptamer protective layer includes providing an aptamer protective layer having a segmented multiblock polymer backbone. In one aspect, alone or in combination with any of the foregoing aspects, the segmented multiblock polymer backbone comprises a polyurethane polymer. In one aspect, alone or in combination with any of the foregoing aspects, the segmented multiblock polymer backbone comprises a polyurethane-urea polymer.

[0057] In one aspect, alone or in combination with any of the foregoing aspects, the segmented multiblock polymer comprises soft segments and hard segments. In one aspect, alone or in combination with any of the foregoing aspects, the soft segments are hydrophobic or hydrophilic. In one aspect, alone or in combination with any of the foregoing aspects, the soft segments are hydrophobic and hydrophilic. In one aspect, alone or in combination with any of the foregoing aspects, the soft segments comprise hydrophobic polyols and hydrophilic polyols. In one aspect, alone or in combination with any of the foregoing aspects, the soft segments are one or more segments comprising polydimethylsiloxane, polycarbonate, polyester, polyether, and blends or copolymers thereof.

[0058] In one aspect, alone or in combination with any of the foregoing aspects, the soft segments are one or more segments comprising polyethylene glycol, oligo polyether, polyoxazoline (POX), polypeptide, polyvinylpyrrolidone, polyvinylpyridine, zwitterionic repeating group polymer, and blends or copolymers thereof.

[0059] In one aspect, alone or in combination with any of the foregoing aspects, the segmented multiblock polymer comprises soft segments and hard segments. In one aspect, alone or in combination with any of the foregoing aspects, the hard segments comprise polyurethane groups or urea groups.

[0060] In one aspect, alone or in combination with any of the foregoing aspects, the soft segments are hydrophobic or hydrophilic. In one aspect, alone or in combination with any of the foregoing aspects, the soft segments are hydrophobic and hydrophilic. In one aspect, alone or in combination with any of the foregoing aspects, the soft segments comprise hydrophobic polyols and hydrophilic polyols. In one aspect, alone or in combination with any of the foregoing aspects, the soft segments are one or more segments comprising polydimethylsiloxane, polycarbonate, polyester, polyether, and blends or copolymers thereof. In one aspect, alone or in combination with any of the foregoing aspects, the soft segments are one or more segments comprising polyethylene glycol, oligo polyether, polyoxazoline (POX), polypeptide, polyvinylpyrrolidone, zwitterionic repeating group polymer, and blends or copolymers thereof.

[0061] In one aspect, alone or in combination with any of the foregoing aspects, reducing biological fouling includes providing an aptamer protection layer as defined in any of the foregoing aspects.

[0062] In one aspect, alone or in combination with any of the foregoing aspects, reducing the decoupling of at least one aptamer from the surface of a conductive substrate includes coupling at least one aptamer conjugate to the conductive substrate using a carbodiimide coupled to the conductive surface.

[0063] In one aspect, alone or in combination with any of the foregoing aspects, reducing the oxidation of an aptamer includes introducing one or more non-diffusible antioxidants into the aptamer protection layer.

[0064] In one aspect, alone or in combination with any of the foregoing aspects, controlling the diffusion of at least one aptamer includes at least partially crosslinking the aptamer protection layer.

[0065] In one aspect, alone or in combination with any of the foregoing aspects, the end-of-life is extended by up to one day, 2 days, one week, 2 weeks, 3 weeks or at least one month.

[0066] In another example, an aptamer protection layer configured for continuous in vivo online monitoring through the skin is provided, the aptamer protection layer comprising a polymer selected from: a functionalized polymer comprising at least one zwitterionic repeating group; a functionalized polymer derived from at least one of the following polymerizable zwitterionic monomer structures:

[0067]

[0068] wherein X is O, NH or NR4, Y and Z are independently acyclic alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloheteroalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, and which may optionally be substituted by OH, halogen or alkoxy; R1, R3, R4 and R5 are independently H, alkyl, heteroalkyl, cycloalkyl, cycloheteroalkyl, aryl or heteroaryl; a functionalized polymer comprising an alkylthiol group; a functionalized polymer comprising an alkylthiol group and a zwitterionic repeating group; a functionalized polymer comprising an (aryl) mercaptoalkanol group; a functionalized polymer comprising an (aryl) mercaptoalkanol group and a zwitterionic repeating group; or a segmented multiblock polymer.

[0069] In one aspect, alone or in combination with any of the foregoing aspects, the aptamer protection layer is at least partially crosslinked.

[0070] In one aspect, alone or in combination with any of the foregoing aspects, the aptamer protective layer provides an ionic strength, and the amount of the zwitterionic repeating group compound present is capable of regulating or maintaining the ionic strength. In one aspect, alone or in combination with any of the foregoing aspects, the aptamer protective layer provides a free volume that allows reversible conformational changes of one or more aptamer conjugates present to be sufficient to provide a detectable signal in the presence of an analyte.

[0071] In one aspect, alone or in combination with any of the foregoing aspects, an alkylthiol or arylthiol group is present at the end of the functionalized polymer chain. In one aspect, alone or in combination with any of the foregoing aspects, an alkylthiol or arylthiol group is present along the backbone of the functionalized polymer chain. In one aspect, alone or in combination with any of the foregoing aspects, an (aryl) mercaptoalkanol group is present at the end of the functionalized polymer chain. In one aspect, alone or in combination with any of the foregoing aspects, an (aryl) mercaptoalkanol group is present along the backbone of the functionalized polymer chain. In one aspect, alone or in combination with any of the foregoing aspects, a zwitterionic repeating group is present at the end of the functionalized polymer chain. In one aspect, alone or in combination with any of the foregoing aspects, a zwitterionic repeating group is present along the backbone of the functionalized polymer chain.

[0072] In one aspect, alone or in combination with any of the foregoing aspects, one or more zwitterionic repeating groups include a betaine compound or a derivative thereof. In one aspect, alone or in combination with any of the foregoing aspects, the aptamer protective layer is configured to physically or chemically couple to at least a portion of the substrate surface.

[0073] In one aspect, alone or in combination with any of the foregoing aspects, the aptamer protective layer includes a segmented multiblock polyurethane polymer.

[0074] In one aspect, alone or in combination with any of the foregoing aspects, the segmented multiblock polymer includes at least one of polyurethane, polyurea, poly(urethane urea), epoxide, polyolefin, polysiloxane, polyamide, polystyrene, polyacrylate, polyether, polyol, polyvinylpyridine, polyvinylpyrrolidone, polyester, polycarbonate, and copolymers thereof.

[0075] In one aspect, alone or in combination with any of the foregoing aspects, the aptamer protective layer includes a segmented multiblock urea polymer.

[0076] In one aspect, alone or in combination with any of the foregoing aspects, the segmented multi-block polymer comprises soft segments and hard segments. In one aspect, alone or in combination with any of the foregoing aspects, the soft segments are hydrophobic or hydrophilic. In one aspect, alone or in combination with any of the foregoing aspects, the soft segments are both hydrophobic and hydrophilic. In one aspect, alone or in combination with any of the foregoing aspects, the soft segments comprise hydrophobic polyols and hydrophilic polyols.

[0077] In one aspect, alone or in combination with any of the foregoing aspects, the soft segments are one or more segments comprising polydimethylsiloxane, polycarbonate, polyester, polyether, and blends or copolymers thereof. In one aspect, alone or in combination with any of the foregoing aspects, the soft segments are one or more segments comprising polyethylene glycol, oligo polyether, polyoxazoline (POX), polypeptide, polyvinylpyrrolidone, polyvinylpyridine, zwitterionic repeating group polymers, and blends or copolymers thereof.

[0078] In one aspect, alone or in combination with any of the foregoing aspects, the average molecular weight of the aptamer protective layer is from about 1 kDa to about 500 kDa.

[0079] In another example, a method for determining the in vivo concentration of an analyte is provided, the method comprising: contacting a biological fluid containing the analyte in vivo with an electrochemical aptamer biosensor coupled to a conductive substrate, the aptamer probe being encapsulated in an aptamer protective layer that is permeable to the analyte, the electrochemical aptamer biosensor generating a signal upon interaction with the analyte; and interrogating the conductive substrate or the electrochemical aptamer; and detecting a signal corresponding to the in vivo concentration of the analyte.

[0080] In one aspect, alone or in combination with any of the foregoing aspects, the interrogation is a continuous, semi-continuous, sequential, or random detection of the signal. In one aspect, alone or in combination with any of the foregoing aspects, it further comprises adjusting the signal based on a background signal generated due to non-specific binding of the aptamer biosensor so as to generate a regulated signal.

[0081] In one aspect, alone or in combination with any of the foregoing aspects, it further comprises determining the in vivo concentration of the analyte over a period of time based on the regulated signal. In one aspect, alone or in combination with any of the foregoing aspects, interrogating the conductive substrate comprises differential measurement techniques.

[0082] In one aspect, either alone or in combination with any of the foregoing aspects, a differential measurement technique includes: interrogating a conductive substrate with a first square wave voltammetry (SWV) frequency to obtain a first signal, and interrogating the conductive substrate with 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 a conditioned signal.

[0083] In one aspect, either alone or in combination with any of the foregoing aspects, the interrogation includes chronoamperometry. In one aspect, either alone or in combination with any of the foregoing aspects, the interrogation includes cyclic voltammetry.

[0084] In one aspect, either alone or in combination with any of the foregoing aspects, the conductive substrate is an electrode, a microporous or nanoporous conductive material.

[0085] In another example, a method of fabricating an electrochemical aptamer biosensor (EAB) is provided, the method including: presenting at least one aptamer to at least a portion of a surface of a conductive substrate, the at least one aptamer conjugate including a reversible redox moiety; and presenting an aptamer protective layer to a portion of the surface of the conductive substrate; and encapsulating at least a portion of the at least one aptamer conjugate within the aptamer protective layer.

[0086] In one aspect, either alone or in combination with any of the foregoing aspects, one or more co - adsorbents are further included into the aptamer protective layer. In one aspect, either alone or in combination with any of the foregoing aspects, the one or more co - adsorbents include zwitterionic betaine groups. In one aspect, either alone or in combination with any of the foregoing aspects, the one or more zwitterionic betaine groups are selected from the following structures:

[0087]

[0088] wherein represents a hydrocarbon chain; wherein R1 and R2 are independently a branched or unbranched acyclic alkyl, a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted aryl, a substituted or unsubstituted benzyl, a substituted or unsubstituted heteroalkyl, a substituted or unsubstituted heterocycle; wherein X is -OH, -NHR1, -NH2 or -SH; wherein n is an integer from 2 to about 1000; or

[0089]

[0090] wherein X is -OH, -NHR1, -NH2 or -SH; wherein W, Y and Z are independently a branched or straight-chain alkyl, heteroalkyl, cycloalkyl, cycloheteroalkyl, aryl or heteroaryl, any of which may optionally be substituted with O, OH, halogen, amide or alkoxy; R1 is H, a branched or unbranched acyclic alkyl, a substituted or unsubstituted heteroalkyl, a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted cycloheteroalkyl, a substituted or unsubstituted aryl, a substituted or unsubstituted benzyl, or a substituted or unsubstituted heteroaryl; and R3, R4 and R5 are independently selected from acyclic alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloheteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted benzyl, or substituted or unsubstituted heteroaryl. In one example, one or more of R1, R2, R3, R4, R5 and Z are covalently or ionically coupled to the APL.

[0091] In one aspect, alone or in combination with any of the foregoing aspects, the co-adsorbate is a terminally-capped dithiol having at least one betaine group along its chain. In one aspect, alone or in combination with any of the foregoing aspects, the thiol group of the terminally-capped dithiol alkanethiol is covalently coupled to the substrate surface.

[0092] In one aspect, alone or in combination with any of the foregoing aspects, the zwitterionic betaine group includes mercaptoalkanol betaine. In one aspect, alone or in combination with any of the foregoing aspects, the mercaptoalkanol is straight-chain and contains a plurality of betaine groups along its chain.

[0093] In one aspect, alone or in combination with any of the foregoing aspects, the thiol group of the mercaptoalkanol is covalently coupled to the substrate surface.

[0094] In one aspect, alone or in combination with any of the foregoing aspects, the aptamer protective layer includes an alkanethiol and one or more zwitterionic betaine groups.

[0095] In one aspect, alone or in combination with any of the foregoing aspects, the aptamer protective layer includes a segmented multi-block polymer backbone. In one aspect, alone or in combination with any of the foregoing aspects, the segmented multi-block polymer backbone comprises a polyurethane polymer. In one aspect, alone or in combination with any of the foregoing aspects, the segmented multi-block polymer backbone comprises a polyurethane urea polymer.

[0096] In one aspect, alone or in combination with any of the foregoing aspects, the segmented multi-block polymer comprises soft segments and hard segments. In one aspect, alone or in combination with any of the foregoing aspects, the soft segments are hydrophobic or hydrophilic. In one aspect, alone or in combination with any of the foregoing aspects, the soft segments are both hydrophobic and hydrophilic. In one aspect, alone or in combination with any of the foregoing aspects, the soft segments comprise hydrophobic polyols and hydrophilic polyols.

[0097] In one aspect, alone or in combination with any of the foregoing aspects, the soft segments are one or more segments comprising polydimethylsiloxane, polycarbonate, polyester, polyether, and blends or copolymers thereof. In one aspect, alone or in combination with any of the foregoing aspects, the soft segments are one or more segments comprising polyethylene glycol, oligo polyether, polyoxazoline (POX), polypeptide, polyvinylpyrrolidone, zwitterionic repeating group polymer, and blends or copolymers thereof.

[0098] In one aspect, alone or in combination with any of the foregoing aspects, the segmented multi-block polymer comprises soft segments and hard segments. In one aspect, alone or in combination with any of the foregoing aspects, the hard segments comprise polyurethane groups or urea groups.

[0099] In one aspect, alone or in combination with any of the foregoing aspects, the soft segments are one or more segments comprising polydimethylsiloxane, polycarbonate, polyester, polyether, and blends or copolymers thereof.

[0100] In one aspect, alone or in combination with any of the foregoing aspects, a certain amount of cross-linking agent is used to cross-link the aptamer protection layer. BRIEF DESCRIPTION OF THE DRAWINGS

[0101] To understand and appreciate how the present disclosure may be practiced in actuality, examples will now be described by way of non-limiting example only with reference to the accompanying drawings, in which:

[0102] Figure 1A and Figure 1B is a schematic diagram illustrating an aptamer protection material employed in a biosensor according to the broadest aspect of the present disclosure.

[0103] Figure 2A is a schematic diagram illustrating an exemplary aptamer biosensor construct.

[0104] Figure 2B , Figure 2C and Figure 2D are schematic diagrams showing alternative structures of the sensing region with aptamer protection material of the exemplary aptamer biosensor as shown in Figure 2A is shown.

[0105] Figure 2E and Figure 2F are a representative schematic diagram of a linear substrate aptamer construct with an aptamer protection material and a hypothetical diagram of the potential versus the electric double layer according to the present disclosure, respectively.

[0106] Figure 2G and Figure 2H are a representative schematic diagram of a microporous substrate aptamer construct with an aptamer protection material and a hypothetical diagram of the potential versus the electric double layer, respectively.

[0107] Figure 3 is a schematic diagram of an exemplary aptamer protection material according to the broadest aspect of the present disclosure.

[0108] Figure 4A and Figure 4B are schematic diagrams of exemplary co - adsorbents according to the broadest aspect of the present disclosure.

[0109] Figure 5A and Figure 5B are representative graphs of experimental charge - versus - frequency data for a control aptamer biosensor and an exemplary aptamer biosensor with an aptamer protection material according to the present disclosure, respectively.

[0110] Figure 6A and Figure 6B are representative graphs of experimental charge - versus - frequency data for protein fouling of a control aptamer biosensor and an exemplary aptamer biosensor with an aptamer protection material according to the present disclosure, respectively.

[0111] Figure 7A and Figure 7B are representative graphs of experimental charge - versus - frequency data for protein fouling of a control aptamer biosensor and an exemplary aptamer biosensor with an aptamer protection material according to the present disclosure, respectively.

[0112] Figure 8A and Figure 8B are representative graphs of experimental current - versus - frequency data for exemplary aminoglycoside aptamer biosensors without and with an aptamer protection material according to the present disclosure, respectively.

[0113] Figure 9A is a representative graph of experimental normalized read - out percentage versus time according to the present disclosure, which represents the drift of a control aptamer biosensor exposed to protein and exemplary aptamer biosensors with various aptamer protection materials.

[0114] Figure 9BIs a representative graph of the experimental normalized readout percentage versus time according to the present disclosure, which represents the drift of an exemplary aptamer biosensor with an aptamer protective layer exposed to bovine serum albumin.

[0115] Figure 9C Is a representative graph of the experimental normalized readout percentage versus time according to the present disclosure, which represents the stability of an exemplary aptamer biosensor with an aptamer protective layer.

[0116] Figure 10A And Figure 10B Are representative graphs of the experimental current versus frequency data over time for exemplary vancomycin aptamer biosensors without and with aptamer protection materials according to the present disclosure, respectively.

[0117] Figure 11 Is a representative graph of the experimental sensor response percentage versus analyte concentration according to the present disclosure, which represents exemplary aptamer biosensors with and without aptamer protection materials exposed to various analyte concentrations.

[0118] Figure 12A And Figure 12B Are representative graphs of exemplary vancomycin aptamer biosensors with different co - adsorbents according to the present disclosure, respectively.

[0119] Figure 13A And Figure 13B Are representative graphs of the shelf - life performance of uncoated EAB and exemplary APL - coated EAB after storage in the ambient environment for 5 hours, respectively.

[0120] Figure 13C And Figure 13D Is a representative graph of the calibration and drift performance of an exemplary vancomycin APL - coated EAB after storage in the ambient environment for one month.

[0121] Figure 13E And Figure 13F Is a representative graph of the calibration and drift performance of an exemplary vancomycin APL - coated EAB after storage in the ambient dark environment for two months.

[0122] Figure 14 Is a diagram showing certain embodiments of an example continuous analyte monitoring sensor system in communication with at least one display device according to various techniques as described in the present disclosure. Detailed Description

[0123] Although significant progress has been made in the in vivo implementation of AB and EAB devices, important challenges must be overcome in aptamer stability to facilitate their continuous operation in complex samples such as blood or ISF. Novel EAB interfaces need to be developed that resist degradation over time due to continuous electrochemical interrogation in biological fluids for extended periods, a process commonly regarded as Faradaic decay and an increase in charging current over time. This progressive degradation limits the in vivo operating lifetime of EABs to 12 hours or less, a time much shorter than the elimination half-life of the vast majority of drugs in humans. The present disclosure provides a technical solution to the above problems and uses aptamer protection materials alone or in combination with co-adsorbents to facilitate the continuous operation of AB and EAB devices in vivo.

[0124] Definition

[0125] As used herein, the term "about" is a broad term and will give its ordinary and customary meaning to a person of ordinary skill in the art (and is not limited to a special or customized meaning), and refers to (but is not limited to) a degree of variability of an allowable value or range, for example, within 10%, within 5%, or within 1% of the value or the limit of the range, and includes the exact value or range. As used herein, the term "substantially" means most or the majority, such as 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%. As used herein, the phrase "substantially free of" may mean not having or having a trace amount such that the amount of the material present does not affect the material properties of the composition including the material, such that the material accounts for from about 0 wt% to about 5 wt% or from about 0 wt% to about 1 wt% or about 5 wt% or less or less than or equal to about 4.5 wt%, 4 wt%, 3.5 wt%, 3 wt%, 2.5 wt%, 2 wt%, 1.5 wt%, 1 wt%, 0.9 wt%, 0.8 wt%, 0.7 wt%, 0.6 wt%, 0.5 wt%, 0.4 wt%, 0.3 wt%, 0.2 wt%, 0.1 wt%, 0.01 wt%, or about 0.001 wt% or less or about 0 wt%.

[0126] As used herein, the terms "adhere" and "attach" are broad terms and will give their ordinary and customary meaning to a person of ordinary skill in the art (and are not limited to a special or customized meaning), and refer to (but are not limited to) holding, binding, or sticking, for example, by adhesion, bonding, gripping, interpenetration, or fusion.

[0127] As used herein, the term "analyte" is a broad term and will give its ordinary and customary meaning to one of ordinary skill in the art (and is not limited to a special or customized meaning), and refers to (but is not limited to) a substance or chemical component that can be analyzed in a biological fluid (e.g., blood, interstitial fluid, cerebrospinal fluid, lymphatic fluid, urine, sweat, saliva, etc.). Analytes can include naturally occurring substances, man-made substances, drugs, toxins, metabolites, and / or reaction products. Exemplary analytes include troponin, BNP, insulin, GLP-1, dopamine, serotonin, L-DOPA, vancomycin, aminoglycosides, doxorubicin, cortisol, and luteinizing hormone.

[0128] As used herein, the phrases "analyte measurement device", "analyte monitoring device", "analyte sensing device", "continuous analyte sensing device", "continuous analyte sensor device", and / or "multi-analyte sensor device" are broad phrases and will give their ordinary and customary meaning to one of ordinary skill in the art (and is not limited to a special or customized meaning), and refer to (but are not limited to) devices and / or systems responsible for detecting or transducing signals associated with a specific analyte or analyte combination. For example, these phrases can refer to (but are not limited to) instruments responsible for detecting a specific analyte or analyte combination. In one example, the instrument includes a sensor coupled to a circuit disposed within a housing and configured to process a signal associated with an analyte concentration into information. In one example, such devices and / or systems are capable of using biorecognition elements in combination with transduction and / or detection elements to provide specific quantitative, semi-quantitative, qualitative, and / or semi-qualitative analysis information.

[0129] As used herein, the term "aptamer" is a broad term and will give its ordinary and customary meaning to one of ordinary skill in the art (and is not limited to a special or custom meaning), and refers to (but is not limited to) an oligonucleotide or peptide that binds to a bioanalyte. Aptamers can be of oligonucleotide or peptide origin. Oligonucleotide aptamers include nucleic acid substances that have been engineered by repeated rounds of in vitro selection or equivalently SELEX (systematic evolution of ligands by exponential enrichment) to bind bioanalytes such as small molecules, proteins, nucleic acids, and even cells, tissues, and organisms. Peptide aptamers include polypeptides that have been selected or engineered to bind an analyte. Peptide aptamers can comprise or consist of one or more peptide loops with variable sequences presented in a protein scaffold. Peptide aptamer selection can be performed using different systems, including yeast two-hybrid systems, combinatorial peptide libraries constructed by phage display and other surface display techniques such as mRNA display, ribosome display, bacterial display, and yeast display, collectively referred to as "biopanning". Peptide aptamers can be selected from the MimoDB database. Peptide aptamers can also be isolated from combinatorial libraries generated by directed mutagenesis or multiple rounds of variable region mutagenesis and selection. Commercially available aptamers, including aptamers with transduction elements, can be purchased, for example, from Biosearch Technologies (Hoddesdon, UK).

[0130] As used herein, the phrase "proximal" is a broad phrase and will give its ordinary and customary meaning to one of ordinary skill in the art (and is not limited to a special or custom meaning), and refers to (but is not limited to) an aptamer or bioactive agent covalently linked to a substrate, co-adsorbate, carrier, or nanocarrier (such as a metal surface, conductive surface, or polymer) via a linker. The linker can be biologically inactive, such as when exposed to or presented in a biological environment for a period of time, it resists the separation of the aptamer from the substrate, and the biological environment is suitable for continuous monitoring, such as with a wearable device, or in a subcutaneous or transdermal environment, whether or not a protective layer is present. The linker can be bioactive, such as when exposed to or presented in a biological environment (such as a subcutaneous or transdermal environment), it is capable of allowing a bioactive agent (such as an anti-inflammatory agent) to separate from the carrier. The phrase "aptamer conjugate" includes an aptamer comprising a linking moiety for coupling or tethering to a substrate, co-adsorbate, carrier, or nanocarrier, and an aptamer comprising a linking moiety and a redox moiety coupled thereto.

[0131] The phrases "aptamer protecting material", "aptamer protecting domain", "aptamer protecting film", "aptamer protecting region", "aptamer protecting matrix", and "aptamer protecting layer", which are used herein and collectively referred to as "aptamer protecting layer 105" or "APL", are broad phrases and will give their ordinary and customary meanings to those of ordinary skill in the art (and are not limited to special or custom meanings), and refer to (but are not limited to) any substance, domain, film, region, polymer, matrix, or layer that acts in concert with one or more aptamer conjugates that are configured to transduce a signal corresponding to the concentration of a bioanalyte. For example, the APL provides one or more of the following properties: allowing the aptamer conjugate to undergo a conformational transformation within the APL; allowing the transport of one or more analytes; providing an electrochemical and / or physiochemical environment for the aptamer to stabilize the aptamer itself, or its coupling to a substrate, or the lifetime of a redox moiety coupled to the aptamer; and reducing or eliminating signal drift in vivo over time.

[0132] As used herein, the phrases and terms "bioactive agent" and "bioactive substance" are broad phrases and broad terms and will give their ordinary and customary meanings to those of ordinary skill in the art (and are not limited to special or custom meanings), and refer to (but are not limited to) any substance that has an effect on or elicits a response from living tissue, such as drugs, biologics, reactive oxygen species scavengers (ROS), and metal ions.

[0133] As used interchangeably herein, the phrases "biointerface film", "biointerface domain", and "biointerface layer" are broad phrases and will give their ordinary and customary meanings to those of ordinary skill in the art (and are not limited to special or custom meanings), and refer to (but are not limited to) a permeable membrane (which may include multiple domains) or layer that serves as a bioprotective interface between recipient tissue and an implantable device. The terms "biointerface" and "bioprotective" are used interchangeably herein.

[0134] As used herein, the terms "biosensor" and / or "sensor" are broad terms and will give their ordinary and customary meaning to one of ordinary skill in the art (and are not limited to a special or customized meaning), and refer to (but are not limited to) a part of an analyte measurement device, an analyte monitoring device, an analyte sensing device, a continuous analyte sensing device, a continuous analyte sensor device, and / or a multi-analyte sensor device that is responsible for detecting or transducing a signal associated with a specific analyte or analyte combination. In an example, a biosensor or sensor typically includes a body, a working electrode, a reference electrode, and / or a counter electrode that are coupled to the body and form a surface configured to provide a signal during an electrochemical reaction. One or more membranes may be fixed to the body and cover the electrochemical reaction surface. In an example, such a biosensor and / or sensor is capable of providing a specific quantitative, semi-quantitative, qualitative, or semi-qualitative analysis signal using a biorecognition element in combination with a detection and / or transduction element.

[0135] As used herein, the term "biostable" is a broad term and will give its ordinary and customary meaning to one of ordinary skill in the art (and are not limited to a special or customized meaning), and refers to (but is not limited to) a material that is relatively resistant to degradation by processes encountered in the body.

[0136] As used herein, the term "co-adsorbate" is a broad term and will give its ordinary and customary meaning to one of ordinary skill in the art (and are not limited to a special or customized meaning), and refers to (but is not limited to) a material (adsorbent) that is absorbed, associated, or coupled to the surface of a substrate via covalent, ionic, or molecular interactions. Unless otherwise specified, the co-adsorbate is at least partially adsorbed onto the surface rather than into the surface.

[0137] As used herein, the term "comprising" is synonymous with "including", "containing", or "characterized by", and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps.

[0138] As used herein, the term "continuous" is a broad term and will give its ordinary and customary meaning to one of ordinary skill in the art (and are not limited to a special or customized meaning), and refers to (but is not limited to) a part, domain, coating, or layer that is uninterrupted or continuous.

[0139] As used herein, the phrase "continuous analyte sensing" is a broad term and will give its ordinary and customary meaning to one of ordinary skill in the art (and is not limited to a special or customized meaning), and refers to (but is not limited to) a period of time during which the monitoring of analyte concentration is performed continuously, continuously, or intermittently (but regularly) (e.g., about every 5 seconds or less to about 10 minutes or more). In another example, continuous monitoring of analyte concentration is performed about every 10 seconds, 15 seconds, 20 seconds, 25 seconds, 30 seconds, 35 seconds, 40 seconds, 45 seconds, 50 seconds, 55 seconds, or 60 seconds to about 1.25 minutes, 1.50 minutes, 1.75 minutes, 2.00 minutes, 2.25 minutes, 2.50 minutes, 2.75 minutes, 3.00 minutes, 3.25 minutes, 3.50 minutes, 3.75 minutes, 4.00 minutes, 4.25 minutes, 4.50 minutes, 4.75 minutes, 5.00 minutes, 5.25 minutes, 5.50 minutes, 5.75 minutes, 6.00 minutes, 6.25 minutes, 6.50 minutes, 6.75 minutes, 7.00 minutes, 7.25 minutes, 7.50 minutes, 7.75 minutes, 8.00 minutes, 8.25 minutes, 8.50 minutes, 8.75 minutes, 9.00 minutes, 9.25 minutes, 9.50 minutes, or 9.75 minutes. In another example, continuous monitoring of analyte concentration is performed daily and can be performed for several weeks.

[0140] As used herein, the term "coupled" is a broad term and will give to one of ordinary skill in the art its ordinary and customary meaning (and is not limited to a special or customized meaning), and means (but is not limited to this) that two or more system elements or components are configured to be attached electronically, mechanically, thermally, operatively, chemically, or otherwise attached in at least one of these ways. Similarly, phrases such as "operatively connected", "operatively linked", and "operatively coupled" as used herein can mean that one or more components are joined to another component in a way that facilitates the transmission of at least one signal between the components. In some examples, the components are part of the same structure and / or are integrated with each other (i.e., "directly coupled"). In other examples, the components are connected via a remote device. For example, one or more electrodes can be used to detect an analyte in a sample and convert that information into a signal; then the signal can be transmitted to a circuit. In this example, the electrode is "operatively linked" to the electronic circuit. The phrase "removably coupled" as used herein can mean that two or more system elements or components are configured to be or have been attached electronically, mechanically, thermally, operatively, chemically, or otherwise and can be separated without damaging any of the coupled elements or components. The phrase "permanently coupled" as used herein can mean that two or more system elements or components are configured to be or have been attached electronically, mechanically, thermally, operatively, chemically, or otherwise, but cannot be decoupled without damaging at least one of the coupled elements or components.

[0141] As used herein, the term "discontinuous" is a broad term and will give to one of ordinary skill in the art its ordinary and customary meaning (and is not limited to a special or customized meaning), and means (but is not limited to this) non - coherent, intermittent, or separated parts, layers, coatings, or domains.

[0142] As used herein, the term "distant" is a broad term and will give to one of ordinary skill in the art its ordinary and customary meaning (and is not limited to a special or customized meaning), and means (but is not limited to this) that a region is relatively far spaced from a reference point such as a starting point or an attachment point.

[0143] As used herein, the term "domain" is a broad term and will give to one of ordinary skill in the art its ordinary and customary meaning (and is not limited to a special or customized meaning), and means (but is not limited to this) a region of a membrane system, which can be a layer, a uniform or non - uniform gradient (e.g., an anisotropic region of a membrane), or a part of a membrane capable of sensing one, two, or more analytes. The domains discussed herein can be formed as a single layer, two or more layers, bilayer pairs, or combinations thereof.

[0144] As used herein, the term "drift" is a general term and will give its ordinary and customary meaning to one of ordinary skill in the art (and is not limited to a special or customized meaning), and refers to (but is not limited to) a gradual increase or decrease over time of a signal that is not related to changes in the analyte concentration of the host system. While not wishing to be bound by theory, it is believed that drift may be the result of a local decrease in the transport of the analyte to the sensor, for example, due to the formation of a foreign body capsule (FBC). It is also believed that insufficient amounts of interstitial fluid around the sensor may result in a decrease in transport to the sensor. In one example, an increase in local interstitial fluid can slow or reduce drift and thus improve sensor performance. Drift can also be the result of sensor electronics or an algorithmic model 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, and is equally applicable to Faraday, capacitance, and voltage measurements.

[0145] The phrases "bioactive agent release membrane" and "drug release layer" and "bioactive agent release domain" and "bioactive agent release membrane" are used interchangeably herein and are each general phrases and will each give their ordinary and customary meaning to one of ordinary skill in the art (and are not limited to a special or customized meaning), and refer to (but are not limited to) a permeable or semipermeable membrane that is permeable to one or more bioactive agents. In an example, the "bioactive agent release membrane" and "drug release layer" and "bioactive agent release domain" and "bioactive agent release membrane" can consist of two or more domains and typically have a thickness of several microns or greater. In an example, the bioactive agent release membrane and / or the bioactive agent release membrane and / or the bioactive agent release membrane and / or the bioactive agent release membrane is substantially the same as the biointerface layer and / or the biointerface membrane. In another example, the bioactive agent release membrane and / or the bioactive agent release membrane and / or the bioactive agent release membrane and / or the bioactive agent release membrane is different from the biointerface layer and / or the biointerface membrane.

[0146] As used herein, the term "electrochemical reaction surface" is a general term and will give its ordinary and customary meaning to one of ordinary skill in the art (and is not limited to a special or customized meaning), and refers to (but is not limited to) the surface of an electrode at which an electrochemical reaction occurs. In another example, an electron transfer is provided using a redox moiety associated with an aptamer conjugate, where the redox moiety is capable of undergoing reduction-oxidation (redox), which is related to the reversible binding interaction of the aptamer and the analyte and is proportional to the analyte concentration.

[0147] As used herein, the term "gain" is a broad term and will give its ordinary and customary meaning to one of ordinary skill in the art (and is not limited to a special or customized meaning), and refers to (but is not limited to) a differential measurement between a signal off state and a signal on state. For example, a typical gain range is 1% - 200% of the percent change in signal produced by a particular concentration of analyte compared to zero analyte concentration. Analyte concentration is typically quantified in micromoles (uM), nanomoles (nM), nanograms per milliliter (ng / mL), or picograms per milliliter (pg / mL).

[0148] As used herein, the phrase "hard segment" is a broad phrase and will give its ordinary and customary meaning to one of ordinary skill in the art (and is not limited to a special or customized meaning), and refers to (but is not limited to) an element of a copolymer, such as a polyurethane, a polycarbonate polyurethane, or a polyurethane-urea copolymer, that confers resistant properties, such as resistance to bending or twisting. The term "hard segment" can also be characterized as a crystalline, semi-crystalline, or glassy material that has a glass transition temperature ("Tg") that is typically above ambient temperature as determined by dynamic scanning calorimetry, and is typically made from a diisocyanate with or without a chain extender.

[0149] As used herein, the term "recipient" is a broad term and will give its ordinary and customary meaning to one of ordinary skill in the art (and is not limited to a special or customized meaning), and refers to (but is not limited to) a mammal, such as a human.

[0150] As used herein, the terms "implanted" or "implantable" are broad terms and will give their ordinary and customary meaning to one of ordinary skill in the art (and is not limited to a special or customized meaning), and refer to (but are not limited to) an object (e.g., a sensor) inserted subcutaneously (i.e., in the fat layer between the skin and muscle) or transcutaneously (i.e., penetrating, entering, or passing through intact skin), which can result in a sensor having an in vivo portion and an ex vivo portion.

[0151] As used herein, the terms "interferent" and "interfering substance" are broad terms and will give their ordinary and customary meaning to one of ordinary skill in the art (and is not limited to a special or customized meaning), and refer to (but are not limited to) an effect and / or substance that interferes with the measurement of an analyte of interest in a sensor to produce a signal that inaccurately represents the analyte measurement. In one example of an electrochemical aptamer sensor, an interfering substance is a compound having a redox (reduction-oxidation) potential that overlaps with that of the analyte to be measured, or with one or more redox moieties associated with one or more aptamers.

[0152] As used herein, the term "in vivo" is a broad term and will give its ordinary and customary meaning to one of ordinary skill in the art (and is not limited to a special or customized meaning), and includes (but is not limited to) portions of a device (e.g., a sensor) adapted to be inserted into and / or present within a recipient's living body.

[0153] As used herein, the term "ex vivo" is a broad term and will give its ordinary and customary meaning to one of ordinary skill in the art (and is not limited to a special or customized meaning), and includes (but is not limited to) portions of a device (e.g., a sensor) adapted to be retained and / or present outside of a recipient's living body.

[0154] As used herein, the term "linker" is a broad term and will give its ordinary and customary meaning to one of ordinary skill in the art (and is not limited to a special or customized meaning), and is not limited to including chemical groups or molecules that connect two molecules or portions, such as an aptamer and a substrate, a co-adsorbate, a carrier, or a nanocarrier. In one example, the linker is located between or flanks two groups, molecules, or other portions and is connected to each other via a covalent bond, thereby connecting the two. In one example, the linker is an oligonucleotide, biotin, maleimide (NHS) ester, polyethylene glycol-NHS ester, or a "click" chemistry component. In one example, thymidine nucleotides of length 2-10 with or without a spacer group are used.

[0155] As used herein, the term "membrane" is a broad term and will give its ordinary and customary meaning to one of ordinary skill in the art (and is not limited to a special or customized meaning), and refers to (but is not limited to) a structure configured to perform the following functions, which include but are not limited to: protecting an exposed electrode surface from the biological environment, diffusion resistance (limitation) of an analyte, serving as a matrix for enabling an enzymatic reaction to occur, restricting or blocking interfering substances, providing hydrophilicity at an electrochemically reactive surface of a sensor interface, serving as an interface between host tissue and an implantable device, regulating host tissue response via drug (or other substance) release, and combinations thereof. When used herein, the terms "membrane" and "matrix" are intended to be used interchangeably.

[0156] As used herein, the phrase "membrane system" is a broad phrase and will give its ordinary and customary meaning to one of ordinary skill in the art (and is not limited to a special or customized meaning), and refers to (but is not limited to) a permeable or semi-permeable membrane that can be composed of two or more domains, two or more layers, or two or more layers within a domain and is typically composed of a material with a thickness of several microns or greater, which is permeable to an analyte. In an example, the membrane system includes immobilized or encapsulated aptamers such that transduction can occur between the aptamer and the analyte, thereby enabling the concentration of the analyte to be measured.

[0157] As used herein, the term "minute" is a broad term and will give its ordinary and customary meaning to a person of ordinary skill in the art (and is not limited to a special or customized meaning), and refers to (but is not limited to) small objects or dimensions of about 10 -6 m that are not visible without magnification. The term "minute" is the opposite of the term "large", which refers to large objects that are visible without magnification. Similarly, the term "nano" refers to small objects or dimensions of about 10 -9 m.

[0158] As used herein, the term "noise" is a broad term and is used in its ordinary meaning, including but not limited to signals detected by a sensor or sensor electronics that are unrelated to analyte concentration and that may cause a decrease in sensor performance. One type of noise has been observed during the first few hours after sensor insertion (e.g., from about 2 hours to about 24 hours). After the first 24 hours, the noise may disappear or decrease, but in some hosts, the noise may persist for about three to four days. In some cases, predictive modeling, artificial intelligence, and / or algorithmic means can be used to reduce the noise. In other cases, the noise can be reduced by addressing immune response factors associated with the presence of the implanted sensor, such as by using a bioactive substance release membrane having at least one bioactive agent. For example, the noise of one or more exemplary biosensors disclosed in the present invention can be determined and then qualitatively or quantitatively compared. By way of example, by obtaining a raw signal time series with a fixed sampling interval (in picoamperes (pA)), a smoothed version of the raw signal time series can be obtained (e.g.) by applying a 3rd order Chebyshev type II low-pass digital filter. Other smoothing algorithms can be used. At each sampling interval, the absolute difference in pA can be calculated to provide a smoothed time series. The smoothed time series can be converted to units (the units of "noise") using, for example, an analyte sensitivity time series, where the analyte sensitivity time series is derived by using a mathematical model between the raw signal and a reference blood analyte measurement. Optionally, the time series can be aggregated as needed, e.g., hourly or daily. Comparison of the corresponding time series between different exemplary biosensors having the bioactive substance release membrane and one or more bioactive agents disclosed in the present invention provides a qualitative or quantitative determination of noise improvement.

[0159] As used herein, the term "optional" or "optionally" is a broad term and will give its ordinary and customary meaning to a person of ordinary skill in the art (and is not limited to a special or customized meaning), and refers to (but is not limited to) the subsequent described event or situation that may or may not occur, and the description includes the case where the event or situation occurs and the case where the event or situation does not occur.

[0160] As used herein, the phrase "polymerizable group" is a broad term and will give its ordinary and customary meaning to one of ordinary skill in the art (and is not limited to a special or customized meaning), and refers to (but is not limited to) a functional group that allows a monomer to polymerize with itself to form a homopolymer or with a different monomer to form a copolymer. Depending on the type of polymerization method employed, the polymerizable group can be selected from olefins, alkynes, epoxides, lactones, amines, hydroxyls, isocyanates, carboxylic acids, acid anhydrides, silanes, halides, aldehydes, and carbodiimides.

[0161] As used herein, the term "polyampholyte" is a broad term and will give its ordinary and customary meaning to one of ordinary skill in the art (and is not limited to a special or customized meaning), and refers to (but is not limited to) a polymer in which the repeating units of the polymer chain are zwitterionic moieties. Polyampholytes are also referred to as polybetaines. Since polyampholytes have both cationic and anionic groups simultaneously, they are a class of polyampholytic polymers. However, they are unique because the cationic and anionic groups are both part of the same repeating unit, which means that polyampholytes have the same number of cationic and anionic groups, whereas other polyampholytic polymers can have more of one ionic group than the other. Moreover, polyampholytes have cationic and anionic groups as part of the repeating unit. Polymers of polyampholytes do not need to have cationic groups attached to anionic groups; they can be on different repeating units and can thus be distributed randomly spaced apart from each other, or the number of one ionic group can exceed the number of the other ionic group.

[0162] As used herein, the term "proximate" is a broad term and will give its ordinary and customary meaning to one of ordinary skill in the art (and is not limited to a special or customized meaning), and refers to (but is not limited to) the spatial relationship between various components as compared to a specific reference point. For example, some examples of devices include membrane systems having a biointerface layer and an enzyme layer. If the sensor is considered the reference point and the enzyme layer is positioned closer to the sensor than the biointerface layer, then the enzyme layer is more proximate to the sensor compared to the biointerface layer.

[0163] As used herein, the phrases and terms "processor module" and "microprocessor" are each broad phrases and terms and will give their ordinary and customary meaning to one of ordinary skill in the art (and is not limited to a special or customized meaning), and refer to (but is not limited to) a computer system, state machine, processor, etc. that is designed to perform arithmetic or logical operations using logic circuits that respond to and process the basic instructions that drive the computer.

[0164] As used herein, the term "semi - continuous" is a broad term and will give its ordinary and customary meaning to one of ordinary skill in the art (and is not limited to a special or customized meaning), and refers to (but is not limited to) a part, coating, domain or layer that includes one or more continuous and discontinuous portions, coatings, domains or layers. For example, a coating that is disposed around rather than with respect to a sensing region is "semi - continuous".

[0165] As used herein, the phrases "sensing part", "sensing film", "sensing region", "sensing domain" and / or "sensing mechanism" are broad phrases and will give their ordinary and customary meaning to one of ordinary skill in the art (and is not limited to a special or customized meaning), and refer to (but are not limited to) a part of a biosensor and / or a sensor that is responsible for detecting or transducing a signal associated with a specific analyte or analyte combination. In an example, the sensing part, sensing film and / or sensing mechanism typically includes an electrode that is configured to provide a signal during an electrochemical reaction with one or more membranes covering an electrochemically reactive surface. In an example, such sensing part, sensing film and / or sensing mechanism is capable of providing a specific quantitative, semi - quantitative, qualitative, semi - qualitative analysis signal using a biorecognition element in combination with a detection and / or transduction element.

[0166] During the general operation of an analyte measuring device, biosensor, sensor, sensing region, sensing part or sensing mechanism, a biological sample (such as blood or interstitial fluid) or its components contact an aptamer, or RNA or DNA protein, or one or more periplasmic binding proteins (PBPs) or mutants or fusion proteins thereof having one or more analyte - binding regions, each region capable of specifically and reversibly binding at least one analyte, either directly or after passing through one or more membranes. The interaction of the biological sample or its components with the analyte measuring device, biosensor, sensor, sensing region, sensing part or sensing mechanism results in signal transduction that allows for a qualitative, semi - qualitative, quantitative or semi - quantitative determination of the analyte level in the biological sample.

[0167] In an example, the sensing region or sensing part may include at least a portion of a conductive substrate or at least a portion of a conductive surface (such as a wire or conductive trace or a substantially planar substrate including substantially planar traces) and a membrane. In an example, the sensing region or sensing part may include a non - conductive body; an electrochemically reactive surface formed at one location on the body and a working electrode, a reference electrode and an optional counter electrode that are electronically connected formed at another location on the body; and a sensing membrane attached to the body and covering the electrochemically reactive surface.

[0168] In one example, multiple working electrodes can be employed. For example, the second working electrode includes multiple different analyte (e.g., analyte 1, analyte 2, etc.) aptamer conjugates on the second working electrode to correct for sensor drift and / or interference. Similarly, a second working electrode including non-selective aptamers conjugated to multiple different analytes (e.g., analyte 1, analyte 2, etc.) on the second working electrode can be used to correct for sensor drift and / or interference.

[0169] In one example, a combination of at least two sets of identical aptamers is used, but one set has a different redox moiety for correcting sensor drift and / or interference. In one example, a combination of at least two sets of non-identical aptamer conjugates (e.g., different linkers / linker lengths, coupling chemistries, different selectivities, and / or binding affinities) is used, each set having the same redox moiety, to correct for sensor drift and / or interference and / or provide detection over a large range of physiological analyte concentrations. In one example, a combination of at least two sets of non-identical aptamer conjugates (e.g., different linkers / linker lengths, coupling chemistries, different selectivities, and / or binding affinities) is used, each set having a unique redox moiety, to correct for sensor drift and / or interference and / or provide detection over a large range of physiological analyte concentrations. In one example, identical or different aptamers are conjugated to different redox moieties having separate formal potentials to reduce or eliminate signals from interfering substances.

[0170] In another example, the sensing region can comprise one or more periplasmic binding proteins (PBPs) or mutants or fusion proteins thereof having one or more analyte binding regions, each region capable of specifically and reversibly binding at least one analyte. Mutations in the PBP can contribute to or alter one or more binding constants, extended protein stability (including thermal stability), to bind the protein to a specific encapsulating matrix, membrane, or polymer or attach a detectable reporter group or "tag" to indicate a change in the binding region. Specific examples of changes in the binding region include, but are not limited to, changes in the hydrophobic / hydrophilic environment, three-dimensional conformational changes, changes in the orientation of amino acid side chains in the protein binding region, and redox states of the binding region. Such changes in the binding region provide transduction of a detectable signal corresponding to one or more analytes present in the biological fluid.

[0171] In an example, the sensing region determines the selectivity between one or more analytes such that only the analyte(s) that must be measured produce(s) (transduce) a detectable signal. The selection can be based on any chemical or physical recognition of the analyte by the sensing region, where the chemical composition of the analyte is not altered, or where the sensing region causes or catalyzes a reaction of the analyte that changes the chemical composition of the analyte.

[0172] The sensing region transduces the recognition of the analyte into a semi - quantitative or quantitative signal. Thus, "transducing" (or "transduction") and their grammatical equivalents as used herein encompass optical, electrochemical, acoustic / mechanical, or colorimetric techniques and methods. Electrochemical properties include current and / or voltage, capacitance, and potential. Optical properties include absorption, fluorescence / phosphorescence, wavelength shift, phase modulation, bioluminescence / chemiluminescence, reflectance, light scattering, and refractive index.

[0173] As used herein, the term "sensitivity" is a broad term and will give its ordinary and customary meaning to a person of ordinary skill in the art (and is not limited to a special or customized meaning), and refers to (but is not limited to) the amount of signal (e.g., in the form of current and / or voltage) produced by a predetermined amount (unit) of the measured analyte. For example, for every 1 mg / dL of analyte, an amperometric analytical sensor has a sensitivity (or slope) of about 1 picoampere to about 100 picoamperes of current.

[0174] The phrases and terms "small - diameter sensor", "small - structure sensor", and "microsensor" as used herein are broad phrases and terms and will give their ordinary and customary meaning to a person of ordinary skill in the art (and are not limited to a special or customized meaning), and refer to (but are not limited to) a sensing mechanism that is less than about 2 mm in at least one dimension. In additional examples, the sensing mechanism is less than about 1 mm in at least one dimension. In some examples, the sensing mechanism (sensor) is less than about 0.95 mm, 0.9 mm, 0.85 mm, 0.8 mm, 0.75 mm, 0.7 mm, 0.65 mm, 0.6 mm, 0.5 mm, 0.4 mm, 0.3 mm, 0.2 mm, or 0.1 mm. In some examples, the largest dimension of the independently measured length, width, diameter, thickness, or perimeter of the sensing mechanism does not exceed about 2 mm. In some examples, the sensing mechanism is a needle - type sensor, where the diameter is less than about 1 mm, see, for example, U.S. Patent No. 6,613,379 to Ward et al. and U.S. Patent No. 7,497,827 to Brister et al., both of which are incorporated herein by reference in their entirety. In some alternative examples, the sensing mechanism includes electrodes deposited on a substantially planar substrate, where the thickness of the implantable portion is less than about 1 mm, see, for example, U.S. Patent No. 6,175,752 to Say et al. and U.S. Patent No. 5,779,665 to Mastrototaro et al., both of which are incorporated herein by reference in their entirety. Examples of methods for forming sensors (sensor electrode layouts and membranes) and sensor systems discussed herein can be found in currently pending U.S. Patent Publication No. 2019 - 0307371, which is incorporated herein by reference in its entirety.

[0175] As used herein, the phrase "soft segment" is a broad phrase and will give its ordinary and customary meaning to one of ordinary skill in the art (and is not limited to a special or customized meaning), and refers to (but is not limited to) an element of a copolymer, such as a polyurethane, polycarbonate polyurethane, or polyurethane urea copolymer, that imparts flexibility to the chain. The phrase "soft segment" can also be characterized as an amorphous material having a low Tg (e.g., typically not higher than ambient temperature or normal mammalian body temperature).

[0176] As used herein, the phrase "solid portion" is a broad term and will give its ordinary and customary meaning to one of ordinary skill in the art (and is not limited to a special or customized meaning), and refers to (but is not limited to) a portion of a membrane material that has a mechanical structure defining a cavity, void, or other non-solid portion.

[0177] As used herein, the terms and phrases "zwitterion" and "zwitterionic compound" are each broad terms and phrases and will give their ordinary and customary meaning to one of ordinary skill in the art (and is not limited to a special or customized meaning), and refer to (but are not limited to) a compound in which the neutral molecules of the compound have a unit positive charge and a unit negative charge at different positions within the molecule. Such compounds are a class of dipolar compounds and are sometimes also referred to as "inner salts".

[0178] As used herein, the phrase "zwitterion precursor" or "zwitterionic compound precursor" is a broad phrase and will give its ordinary and customary meaning to one of ordinary skill in the art (and is not limited to a special or customized meaning), and refers to (but is not limited to) any compound that is not itself a zwitterion but can be converted into a zwitterion in a final or transitional state through a chemical reaction. In some of the examples described herein, the device contains a zwitterion precursor that can be converted into a zwitterion before implantation of the device in vivo. Alternatively, in some of the examples described herein, the device contains a zwitterion precursor that can be converted into a zwitterion through some chemical reactions that occur after implantation of the device in vivo. Such reactions are known to one of ordinary skill in the art and include ring-opening reactions, addition reactions such as Michael addition. This method is particularly useful when the polymerization of betaine monomers is difficult to achieve desired physical properties such as molecular weight and mechanical strength due to technical challenges such as the solubility of betaine monomers. Post-polymerization modification or conversion of betaine precursors can be a practical way to achieve the desired polymer structure and composition. Examples of such precursors include tertiary amines, quaternary amines, pyridines, and other substances detailed herein.

[0179] As used herein, the phrase "zwitterionic derivative" or "zwitterionic compound derivative" is a broad phrase and will give to one of ordinary skill in the art their ordinary and accustomed meanings (and is not limited to a special or customized meaning), and refers to (but is not limited to) any compound that is not itself zwitterionic but is the product of a chemical reaction in which a zwitterion is converted to a non-zwitterionic species. Such reactions can be reversible such that under certain conditions the zwitterionic derivative can act as a zwitterionic precursor. For example, a hydrolyzable betaine ester formed from a zwitterionic betaine is a cationic zwitterionic derivative that is capable of undergoing hydrolysis under appropriate conditions to revert to the zwitterionic betaine.

[0180] As used herein, the phrase "zwitterionic repeat unit" is a broad phrase and will give to one of ordinary skill in the art their ordinary and accustomed meanings (and is not limited to a special or customized meaning) and independently refers to but is not limited to two or more zwitterionic compounds, zwitterionic derivatives, or zwitterionic compound derivatives in the same compound or polymer.

[0181] Aptamer-based biosensors (AB) and electrochemistry-based aptamer biosensors (EAB) are analytical platforms that can provide continuous monitoring of specific molecular analytes in vivo. EAB sensors typically present an architecture consisting of an analyte-binding, self-assembled monolayer (SAM) of an alkanethiol-functionalized nucleic acid aptamer or other bioreceptor that includes a redox moiety that serves as a signal transduction element sensitive to correlating analyte-binding events with a measurable change in electrical energy; a SAM of an electrode-blocking co-adsorbent of alkanethiol for preventing unwanted electrochemical reactions and conferring biocompatibility to the electrode surface.

[0182] The poor stability observed when the above AB or EAB is placed in a physiologically relevant environment is at least attributable to desorption of the aptamer monolayer from the 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.) during continuous electrochemical interrogation, which is a process generally viewed as a decrease in Faradaic and an increase in charging current over time. As discussed in more detail below, such performance deficiencies can be addressed with the aptamer protection layer (APL) disclosed in the present invention.

[0183] In one example, the present disclosure provides an AB or EAB aptamer in an architecture that consists of: a self-assembled monolayer (SAM) of an analyte-binding, alkanethiol-functionalized or carboxyl-functionalized nucleic acid aptamer or other bioreceptor, the self-assembled monolayer including a signal transduction element to correlate the event of analyte binding with a measurable signal from the transduction element; a SAM of an electrode-blocking co-adsorbent of alkanethiol and / or functionalized alkanethiol; and an aptamer protection layer (APL) adjacent or directly adjacent to the above architecture to prevent, independently or jointly, undesired desorption, undesired reactions, reduce biofouling / impart biocompatibility, aptamer stability, and device lifetime.

[0184] In one example, the aptamer conjugate and the APL are temperature-controlled during use. For example, the wearable sensor is thermally insulated and / or configured with a micro Peltier cooler and / or a heat exchange device, such as fins, or a combination of the above. In another example, the aptamer is prepared under conditions that closely match the in vivo thermodynamic environment of the sensor (e.g., by systematic evolution of ligands by exponential enrichment (SELEX)), thereby providing or improving high affinity and / or thermal stability.

[0185] Reference Figure 1A and Figure 1B , an exemplary aptamer-based analyte monitoring sensor 100 configured for in vivo measurement of at least one analyte 99 is presented in a schematic illustration showing an aptamer protection material 105. An aptamer 102 with a signal transduction element 104 is shown associated with an optional monolayer 103 of an adjacent substrate 110. The monolayer 103 can be coupled to the substrate 110 covalently or non-covalently. In one example, the aptamer 102 undergoes a reversible conformational change when interacting with an analyte 99 (e.g., an analyte, metabolite, drug, etc.), resulting in the signal transduction element 104 being presented closer to the substrate 110 to provide a signal corresponding to the concentration or presence of the analyte 99.

[0186] In one example, the signal transduction element 104 is a reversible redox moiety, and the substrate 110 is conductive, and the reversible binding of the aptamer 102 (and its subsequent reversible conformational change) upon interaction with the analyte 99 causes a change in the proximity of all or part of the signal transduction element 104 to the conductive substrate 110 such that when the analyte 99 reversibly binds to the aptamer 102, the signal transduction element 104 is capable of undergoing a detectable reversible reduction-oxidation reaction via electron transfer with the conductive substrate 110. The detectable reversible reduction-oxidation reaction via electron transfer with the conductive substrate 110 provides a correlation with the concentration of the analyte 99, as discussed further below.

[0187] In another example, the reversible binding of the aptamer 102 upon interaction with the analyte 99 (and its subsequent reversible conformational change) can result in all or part of the signal transduction element 104 being presented in or to a different local environment, for example, from a hydrophobic local environment to a hydrophilic local environment (or vice versa), in order to provide a detectable signal corresponding to the concentration or presence of the analyte 99.

[0188] In one example, the signal transduction element 104 is an environmentally sensitive fluorescent or phosphorescent dye that is capable of undergoing a detectable change in emission wavelength or frequency and / or emission relaxation or emission decay rate upon exposure to electromagnetic radiation (e.g., light), for example, upon reversible binding to the analyte 99 and a reversible conformational change from a hydrophobic local environment to a hydrophilic local environment (or vice versa). The detectable change in emission wavelength or frequency and / or emission relaxation or emission decay rate is provided in order to provide a correlation with the concentration of the analyte 99.

[0189] The signal transduction element 104 can be covalently or non-covalently coupled to the aptamer 102, where the covalent or non-covalent coupling is sufficient to effect continuous signal transduction of the signal over a period of time comparable to that of a transdermal, intradermal, subcutaneous, ocular, or cutaneous continuous analyte sensing device. In one example, it is contemplated to use the aptamer protective material 105 disclosed herein to effect continuous signal transduction of the signal over a 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.

[0190] In one example, the signal transduction element 104 and the aptamer 102 are conjugated or form a conjugate. In one example, the signal transduction element 104 and aptamer 102 conjugate associates with the monolayer 103. In one example, the signal transduction element 104 and aptamer 102 conjugate is covalently or non-covalently coupled to the monolayer 103. In one example, the signal transduction element 104 and aptamer 102 conjugate is covalently or non-covalently coupled to the substrate 110.

[0191] For example, any reference hereinafter to a redox moiety as the signal transduction element 104 is for the sake of brevity and not to limit the scope of the signal transduction element 104. Thus, "redox moiety 104" and "signal transduction element 104" may be used interchangeably hereinafter.

[0192] Figure 2AFIG. 0 is a schematic diagram showing an exemplary aptamer biosensor 200 construct configured for continuous in vivo use in a subject. Thus, the biosensor 200 is shown as an elongate member having a sensing region 207, e.g., created by a window in an electrically insulating coating 205 surrounding a wire. Alternatively, a window can be prepared in the sheath of an optical fiber for use with an optically-based AB device. Additional electrodes 215 (reference and / or counter electrodes) can be used alone or provided, e.g., as adjacent coaxial elongate members. As Figures 2B to 2D shown in the enlarged cross-sectional view of Figures 2B to 2D , alternative structures 201, 202, and 203 of the sensing region 207 are shown as having a substrate 110 surface. For example, structure 201 has a substrate 110 surface that has an adjacent aptamer 102 and aptamer protection material 105. Structure 202 has a substrate 110 surface that has an adjacent co-adsorbate 103, aptamer 102, and aptamer protection material 105. Structure 203 has a substrate 110 surface, adjacent co-adsorbate 103, aptamer 102, aptamer protection material 105, and a drug release membrane 113 that is furthest from the substrate 110. Other configurations of the co-adsorbate 103, aptamer 102, aptamer protection material 105, and drug release membrane 113 can be employed.

[0193] Substrate / Electrode

[0194] In an example, the substrate 110 surface receives an AB or EAB for a continuous sensing device. In one example, the substrate 110 is or includes a conductive material. In one example, the substrate 110 is an electrode, which can be a wire, planar structure, or substantially planar structure. In one example, the substrate 110 can be configured to independently provide one or more of a working electrode, reference electrode, and optional counter electrode. In one example, one or more of the working electrode, reference electrode, and optional counter electrode are arranged in a linear or substantially linear configuration. In one example, the reference electrode includes silver (Ag) and / or silver chloride (AgCl). In one example, the reference electrode includes silver (Ag) and / or silver chloride (AgCl) encapsulated or otherwise covered with a protective layer. In one example, the reference electrode having a protective layer is configured to reduce or eliminate the diffusion of AgCl + , Ag, AgCl -2 , ions, or particles from the reference electrode and / or reduce or eliminate the interaction of the reference electrode or AgCl+ ions with the aptamer and / or thiol co-adsorbate or thiol aptamer protective layer. In one example, the protective layer of the silver reference electrode is configured to inhibit or reduce the transport of AgCl+ ions while allowing the transport of chloride ions. Examples of protective layers suitable for silver reference electrodes include, but are not limited to, amphiphilic polyurethanes or polyureas, Teflon, microporous Teflon, ion-selective membranes, semipermeable membranes, PVC, and plasticized PVC.

[0195] In one example, the substrate 110 includes wires formed of or coated with a conductive material such as platinum, platinum-iridium, palladium, graphite, gold, carbon, graphene, graphene oxide, a conductive polymer, an alloy, etc.

[0196] In one example, at least a portion of the substrate 110 includes pores having an average pore size in the nano- and / or micro-scale. Such pore size in the foregoing substrate can be formed, for example, by etching or plasma techniques. Substrates having such nano- and / or micro-scale sizes can be used in combination with the APL disclosed in the present invention.

[0197] For example, the structural properties of the substrate can be decisive in the performance of the EAB, as Figure 2E and Figure 2F shown, a planar substrate 110 having an aptamer 102 and a coupled redox moiety 104 exhibits the shown potential vs. electric double layer (EDL) relationship, which is the region contained within the Debye volume. In contrast, Figure 2G shows the same situation with a construct having an aptamer 102 and a coupled redox moiety 104, wherein at least a portion of the substrate 222 surface has nano- and / or micro-scale pores 225, where as Figure 2H shown, compared to a linear substrate, the potential vs. electric double layer (EDL) relationship exhibits a smaller relative negative slope. When used in combination with the APL disclosed in the present invention, the substrate 222 surface can provide an increase in signal and detection limit for a continuous EAB device. In addition, the foregoing construct can provide redox moiety embedding between closely spaced aptamers, which can provide two sites to absorb two analytes through (pi-pi) π-π interactions of a selected analyte-redox moiety (e.g., methylene blue and dopamine), rather than absorbing one analyte on the aptamer alone, thereby doubling the detection sensitivity.

[0198] In one example, the surface of the substrate 110 includes a conductive surface, and at least a portion of the surface of the substrate 110 is configured to covalently couple, tether, or conjugate an aptamer conjugate to couple or tether to the conductive surface. In one example, the aptamer conjugate is coupled or tethered to the conductive surface by providing suitable coupling or tethering functional groups on one or both of the conductive surface and the aptamer and using one or more coupling chemistries to couple or tether the aptamer conjugate to the conductive surface, such as click chemistry using N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide / N-hydroxysulfosuccinimide (EDC / NHS) chemistry, silane-based coupling using diazonium salt / thiol-ene click chemistry, phosphonate coupling, (strept)avidin-biotin coupling, silane coupling, π-π stacking, bicyclo[6.1.0]nonyne (BCN)-azide coupling, biotinylation, Cu(I)-catalyzed azide-alkyne click chemical reaction (CuAAC), ligation of tetrazine and alkene (e.g., using trans-cyclooctene) and biocompatible strain-promoted azide-alkyne click chemistry (SPAAC) reagents, dibenzocyclooctyne (DBCO)-azide or DBCO-NHS reagents, DBCO-PEG-amine coupling reagents, DBCO-PEG-maleimide coupling reagents, DBCO-PEG-alcohol reagents, amine-reactive trans-cyclooctene (TCO) reagents for tetrazine coupling (e.g., TCO-NHS ester), carboxyl / carbonyl-reactive TCO reagents (e.g., TCO amine or amine salt), TCO-PEG-DBCO reagents, etc. In other examples, a polyethylene glycol (PEG) linker is used.

[0199] In one example, at least a portion of the surface of substrate 110 comprises carbon, graphene, graphene oxide, or carbon ink. In one example, at least a portion of the substrate surface is composed of nanomaterials. In one example, at least a portion of the surface of substrate 110 comprises carbon, graphene, or graphene oxide nanomaterials. In one example, the use of carbon, graphene, or graphene oxide nanomaterials improves the loading of the aptamer on the surface of substrate 110 to optimize aptamer 102 loading and binding stability, etc. In one example, the aptamer conjugate is configured for electrografting onto the carbon-based electrode using diazonium salt / thiol-ene click chemistry, phosphonate coupling, (strept)avidin-biotin coupling, silane coupling, π-π stacking, bicyclo[6.1.0]nonyne (BCN)-azide coupling, biotinylation, Cu(I)-catalyzed azide-alkyne click chemical reaction (CuAAC), ligation of tetrazine and alkene (e.g., using trans-cyclooctene), and biocompatible strain-promoted azide-alkyne click chemistry (SPAAC) reagents, dibenzocyclooctyne (DBCO)-azide or DBCO-NHS reagents, DBCO-PEG-amine coupling reagents, DBCO-PEG-maleimide coupling reagents, DBCO-PEG-alcohol reagents, amine-reactive trans-cyclooctene (TCO) reagents for tetrazine coupling (e.g., TCO-NHS ester), carboxyl / carbonyl-reactive TCO reagents (e.g., TCO amine or amine salt), TCO-PEG-DBCO reagents, etc. In other examples, a polyethylene glycol (PEG) linker for the aptamer conjugate is used to increase resistance to nucleases, or lipid conjugation with the aptamer is used, or alternative nucleic acids for aptamer construction are used (e.g., L-DNA or L-RNA with increased -OH activity for coupling / tethering), and peptide nucleic acid (PNA) is used for aptamer construction and combinations thereof as described above.

[0200] In one example, the surface of substrate 110 comprises gold, and at least a portion of the surface of substrate 110 is configured to covalently couple, tether, or associate with an alkyl thiol or mercapto thiol. In another example, at least a portion of the surface of substrate 110 is configured to covalently couple a straight-chain or branched-chain aliphatic amine, a substituted or unsubstituted benzylamine, or a substituted or unsubstituted aniline, or covalently couple a straight-chain or branched-chain amino alkanoic acid, a substituted or unsubstituted aminobenzoic acid, or a substituted or unsubstituted aminophenyl carboxylic acid. In one example, at least a portion of the surface of substrate 110 is chemically modified with streptavidin, avidin, gold, biotin, or a polymer (such as dextrin and chitosan). In one example, a substrate comprising a gold surface is modified or treated with graphene oxide and / or zinc sulfide (ZnS2) to improve the coupling or tethering of the aptamer to the substrate.

[0201] In one example, at least a portion of the surface of the carbon, graphene, or graphene oxide nanomaterial substrate 110 comprises a covalently coupled straight-chain or branched-chain aliphatic amine, a substituted or unsubstituted benzylamine, or a substituted or unsubstituted aniline, or a covalently coupled straight-chain or branched-chain aminoalkanoic acid, a substituted or unsubstituted aminobenzoic acid, or a substituted or unsubstituted aminophenylcarboxylic acid. In one example, the straight-chain or branched-chain aliphatic amine, the substituted or unsubstituted benzylamine, or the substituted or unsubstituted aniline or the covalently coupled straight-chain or branched-chain aminoalkanoic acid, the substituted or unsubstituted aminobenzoic acid, or the substituted or unsubstituted aminophenylcarboxylic acid is also covalently coupled to the aptamer 102 or the aptamer-redox moiety 104 conjugate. For example, the substrate 110 is modified with a carboxylated material such that covalent immobilization via EDC / NHS chemistry to the exposed COOH-groups of the amine-modified aptamer is enabled.

[0202] In exemplary nanomaterials, graphene oxide (GO) exhibits significant advantages for EAB devices due to its large surface area with multiple exposed carboxyl (COOH) and alcohol (COH) groups, which can be used as anchors to immobilize aptamer conjugate probes using a variety of different types of coupling chemistries. GO provides great flexibility in functionalization and has demonstrated beneficial orientation effects in aptamer immobilization. Thus, in one example, a partially or fully implantable sensor has a GO-functionalized substrate 110 surface (as a model carboxylated surface) to serve as a working electrode for covalent immobilization of amine-functionalized aptamers. To covalently immobilize the aptamer conjugate to the GO surface, for example, activation of the GO-carboxyl (‘COOH’) moiety can be carried out via N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide / N-hydroxysulfosuccinimide (EDC / NHS) chemistry, followed by formation of corresponding amide bonds with the amine groups present in the aptamer sequence. It is envisioned that this immobilization strategy provides higher stability of the aptamer monolayer immobilized on the GO electrode surface, thus providing an extended sensor lifetime and representing an alternative to thiol-based aptamer immobilization strategies. In a similar manner, activation of the GO-‘COH’ moiety can be carried out with an aptamer conjugate modified with trialkoxysilane. In one example, the aptamer conjugate can be electrochemically grafted onto the GO electrode surface.

[0203] In one example, the substrate 110 surface is a carboxyl-functionalized substrate 110 surface, a thiol-functionalized substrate 110 surface, or a combination of a carboxyl-functionalized substrate 110 surface and a thiol-functionalized substrate 110 surface.

[0204] In one example, the substrate 110 surface is substantially a carboxyl-functionalized substrate 110 surface. In one example, the substrate 110 surface is substantially a carboxyl-functionalized substrate 110 surface that is substantially free of thiol functionalization.

[0205] In one example, the surface of substrate 110 is substantially a GO-functionalized substrate 110 surface. In one example, the surface of substrate 110 is substantially a GO-functionalized substrate 110 surface that is substantially free of thiol functionalization.

[0206] Aptamer / Aptamer-Signal Transduction Element Conjugate

[0207] In one example, one or more aptamer conjugates 102 of the AB or EAB disclosed by the present invention comprise an RNA or DNA nucleotide sequence. In one example, one or more aptamer conjugates 102 comprise at least one of the following: 2'-O-methyl modification of nucleotides; disulfide bridges; 3' caps with inverted 2'-deoxythymidine; 3'-3'-thymidine linkages at the 3' terminus; 2'-F modification; and double-stranded segments. In one example, one or more aptamer conjugates 102 comprise an RNA or DNA sequence having a first linker portion at the 5' terminus and a reversible redox moiety at the 3' terminus. In one example, one or more aptamer conjugates 102 comprise an RNA or DNA sequence having a first linker portion at the 3' terminus and a reversible redox moiety at the 5' terminus. In one example, the redox moiety (e.g., methylene blue) is attached to the oligomeric portion of the aptamer, which is within the sequence linked by thymidine bases, at the 5' terminus of the sequence, or at the 3' terminus via a modified thymidine or a 5-7-carbon spacer.

[0208] In one example, the first linker portion at the 5' terminus of aptamer 102 comprises an amino group, a carboxyl group, or a trialkoxysilyl group. In one example, the first linker portion of aptamer 102 is physically or chemically coupled to the substrate at the 5' terminus. In one example, the first linker portion of aptamer 102 is physically or chemically coupled to the co-adsorbate at the 5' terminus. Alternatively, the first linker portion at the 3' terminus of aptamer 102 comprises an amino group, a carboxyl group, or a trialkoxysilyl group, and the first linker portion of aptamer 102 is physically or chemically coupled to the substrate at the 3' terminus. In one example, the first linker portion of aptamer 102 is physically or chemically coupled to the co-adsorbate at the 3' terminus.

[0209] In one example, one or more aptamer conjugates 102 are neurotransmitter-binding aptamers. In one example, one or more aptamer conjugates 102 are dopamine- or glutamate-binding aptamers. In one example, one or more aptamer conjugates 102 are carbohydrate-, triglyceride-, or fatty acid-binding aptamers. In one example, one or more aptamer conjugates 102 are glucose-, glycerol-, or β-hydroxybutyrate-binding aptamers. In one example, one or more aptamer conjugates 102 are glycopeptide antibiotic-binding aptamers. In one example, one or more aptamer conjugates 102 are vancomycin-binding aptamers. Combinations of different aptamer conjugates 102 on the same or different WE surfaces can be used to provide a multi-analyte monitoring EAB device.

[0210] In one example, one or more aptamer conjugates are physically or chemically coupled to a self-assembled monolayer (SAM). In one example, one or more aptamer conjugates are physically or chemically coupled to a mono-functional or multi-functional alkanethiol or mercaptoalkanol. In one example, one or more aptamer conjugates are physically or chemically coupled to an alkylthiol betaine. In one example, one or more aptamer conjugates are physically or chemically coupled to an aliphatic amine.

[0211] Aptamer Protection Layer (APL)

[0212] It has been observed that specific properties of the APL affect the suitability for continuous operation in vivo of an AB or EAB. For example, a continuous AB or EAB with an APL has a positive impact on EAB performance, the APL having sufficient free volume for aptamer conformational changes, favorable ionic properties, sufficient analyte porosity, and blocking of proteins, peptides, macrophages, and other immune response biologic agents. Compared to an AB or EAB without an APL, one or more of the above characteristics further directly or indirectly provide stability of aptamer coupling or tethering (reduced desorption from the substrate or SAM), reduced signal or sensitivity drift over time in vivo, and extended in vivo performance.

[0213] In one example, the APL is a coating, matrix, membrane, domain, or layer. In another example, the APL is a coating, matrix, membrane, domain, or layer of a polymeric material. The polymeric material forming the basis of the APL can include one or more polymers, oligomers, coatings, membranes, or matrices. In one example, the APL provides sufficient permeability to allow relevant analyte compounds to pass through it, e.g., to allow an analyte to pass through a membrane from the sample being examined in order to reach the aptamer and allow transduction of a signal corresponding to the analyte concentration in the sample.

[0214] Figure 3Schematic diagram of an exemplary APL according to the broadest aspects of the present disclosure. Thus, in one example, APL 305 includes at least one polymer segment 302, 304. In one example, the APL includes at least one polymer segment selected from the group consisting of: polyurethane, polyurea, poly(urethane urea), epoxide, polyolefin, polysiloxane, polyamide, polystyrene, polyacrylate, polyether, polyvinylpyridine, polyvinylpyrrolidone, polyester, polycarbonate, and copolymers thereof.

[0215] The hydrophilicity of APL 305 can be adjusted by selecting the soft segments and the soft segment ratio used during conventional PU or PUU synthesis. For example, the soft segment components are shown in Figure 3 (hydrophilic and hydrophobic polyols). The hydrophobic soft segment can be PDMS, polycarbonate, polyester, polyether, or a polymer having a hydrophobic functional group such as fluorine or siloxane. The hydrophobic segment can be provided in the foregoing APL at 1 wt% - 50 wt%, 2 wt% - 50 wt%, 5 wt% - 50 wt%, 10 wt% - 50 wt%, 15 wt% - 50 wt%, 20 wt% - 50 wt%, 25 wt% - 50 wt%, 30 wt% - 50 wt%, 10 wt% - 20 wt%, 15 wt% - 25 wt%.

[0216] The hydrophilic soft segment can be polyethylene glycol, oligo polyether, polyoxazoline (POX), polypeptide, or zwitterionic polymer. By adjusting the chemical composition and / or molecular weight or the distribution of soft and hard segments in PU or PUU, and / or adding or excluding functional groups, the desired APL properties and functionality can be achieved, such as surface charge / density, anti-fouling properties against proteins such as serum albumin (SA). The hydrophilic segment can be provided in the foregoing APL at 1 wt% - 50 wt%, 2 wt% - 50 wt%, 5 wt% - 50 wt%, 10 wt% - 50 wt%, 15 wt% - 50 wt%, 20 wt% - 50 wt%, 25 wt% - 50 wt%, 30 wt% - 50 wt%, 10 wt% - 20 wt%, 15 wt% - 25 wt%.

[0217] In one example, the APL includes a segmented multi-block polymer. Referring again to Figure 3, for example, the segmented multi-block polymer comprises a soft segment 306 and a hard segment (one or more of 308, 310, 312). In one example, the soft segment is hydrophobic or hydrophilic. In one example, the soft segment is both hydrophobic and hydrophilic. In one example, the soft segment comprises a hydrophobic polyol and a hydrophilic polyol. In one example, the APL comprises a segmented multi-block polyurethane polymer. In one example, the APL comprises a segmented multi-block multi-block polyurethane, polyurethane-urea, or polyether-urethane, or polyether-urethane-urea polymer, copolymer, or blends thereof. In one example, the hard segment comprises a urethane group, a urea group, or a combination thereof.

[0218] In one example, the soft segment is one or more segments comprising polydimethylsiloxane, polycarbonate, polyester, polyether, and blends or copolymers thereof. In one example, the soft segment is one or more segments comprising polyethylene glycol, oligo polyether, polyoxazoline (POX), polypeptide, polyvinylpyrrolidone, polyvinylpyridine, a polymer having repeating zwitterionic groups in its main chain and / or at its ends (referred to herein as "zwitterionic repeating group polymer"), and blends or copolymers thereof. In one example, end-group functionalized polyurethane (EGFPU) or polyurethane-urea (EGFPUU) polymers can be used. The EGFU / EGFPUU can be synthesized using reactive functional monomers / oligomers that cap polyurethane reaction intermediates to form polyurethanes having functional groups at one or both chain ends. The functional groups can be further deprotected to form reactive thiol groups that are attached to the surface of the substrate 110 (such as gold).

[0219] Polyurethane, polyurethane-urea polymers can be produced by the condensation reaction of diisocyanate and bifunctional hydroxyl-containing materials or bifunctional amine-containing materials. Polyurethane-urea is a polymer produced by the condensation reaction of diisocyanate and bifunctional amine-containing materials. In some examples, the diisocyanate includes an aliphatic diisocyanate containing about 4 to about 8 methylene units. Diisocyanates containing an alicyclic moiety can also be used to prepare the polymer and copolymer components of the membranes of the present disclosure.

[0220] In one example, end-group functionalized polyurethane (EGFPU) or polyurethane-urea (EGFPUU) polymers can be used, for example, as disclosed in co-owned U.S. Patent No. 10,413,227 B2. The EGFU / EGFPUU can be synthesized using reactive functional monomers / oligomers that cap polyurethane reaction intermediates to form polyurethanes having functional groups at one or both chain ends. The functional groups can be further deprotected to form reactive thiol groups that are attached to the surface of the substrate 110 (such as gold).

[0221] For example, an exemplary hydrophobic-hydrophilic segmented copolymer component is a polyurethane polymer comprising about 20% hydrophilic polyethylene oxide. The polyethylene oxide portion of the copolymer is thermodynamically driven to separate from the hydrophobic portion of the copolymer and the hydrophobic polymer component. In one example, it has been observed that about 20% of the polyethylene oxide-based soft segment portion of the copolymer used to form the APL affects the water absorption rate of the APL film and subsequent analyte permeability. In one example, the foregoing exemplary APL is prepared as an aqueous dispersion for use with the foregoing aptamer-signal transduction element conjugate. For example, a betaine-functionalized hydrophilic aliphatic polyurethane can be prepared as an aqueous dispersion, which can be combined with an aqueous solution of the foregoing aptamer-signal transduction element conjugate.

[0222] Incorporation of zwitterionic repeat units into the foregoing polyurethanes, polyurethane-urea polymers can be achieved by using zwitterionic monomers having diols or diamines, or can be attached to diols or diamines. Examples of such zwitterionic monomers include:

[0223]

[0224] wherein X is one or two of -OH, -NHR1, -NH2 or -SH; wherein W, Y and Z are independently branched or straight-chain alkyl, heteroalkyl, cycloalkyl, cycloheteroalkyl, aryl or heteroaryl, any of which may optionally be substituted with O, OH, halogen, amide or alkoxy; R1 is H, branched or unbranched acyclic alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloheteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted benzyl, or substituted or unsubstituted heteroaryl; and R3, R4 and R5 are independently selected from acyclic alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloheteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted benzyl, or substituted or unsubstituted heteroaryl.

[0225] These compounds can react with diisocyanates to form polyurethanes or polyureas containing zwitterionic repeat units. Alternatively, the carboxylate, sulfonate, phosphinate or phosphonate moieties of the precursor zwitterionic repeat units can be protected and then the protecting groups can be removed after polymerization. In another alternative, the amine can be a tertiary amine, which is then quaternized by alkylation after polymerization. Additional examples of PU and PUU polymers having zwitterionic repeat units can be found in commonly assigned U.S. Published Application No. 20170188923, U.S. Patent No. 11112377 and U.S. Patent No. 11179079, the disclosures of which regarding such polymers and their synthesis are incorporated herein by reference.

[0226] In one example, the APL is composed of a non-polyurethane polymer. Examples of materials that can be used to prepare non-polyurethane APLs include vinyl polymers, polyethers, polyesters, polyamides, polyorganosiloxanes (polydialkylsiloxanes), poly(alkylarylsiloxanes), poly(diarylsiloxanes), polycarbosilanes, polycarbonates, Nafion (sulfonated tetrafluoroethylene), natural polymers such as cellulose and protein-based materials, and mixtures, copolymers or combinations thereof with or without the aforementioned polyurethanes, or polyether-polyurethane-urea polymers.

[0227] The APLs disclosed herein can be formulated into mixtures that can be drawn into films or applied to surfaces using any method known in the art (e.g., spraying, coating, dip coating, vapor deposition, molding, 3-D printing, lithographic techniques (e.g., photolithography), micro- and nano-pipette printing techniques, screen printing, etc.). The mixture can then be cured at an elevated temperature (e.g., 50 °C - 150 °C). Other suitable curing methods can include, for example, ultraviolet or gamma radiation.

[0228] In one example, an amount of crosslinker is used to at least partially crosslink the aptamer protective layer, the amount being sufficient to crosslink the APL without inactivating the aptamer or without significantly reducing the ability of the aptamer present therein to undergo conformational changes sufficient to provide signal transduction. In one example, the aptamer protective layer is fully crosslinked using an amount of crosslinker sufficient to crosslink the APL while substantially not reducing aptamer signal transduction.

[0229] Suitable crosslinkers include isocyanates, carbodiimides, glutaraldehyde or other aldehydes, aziridines, silanes, epoxies, acrylates, free-radical based reagents, ethylene glycol diglycidyl ether (EGDE), poly(ethylene glycol) diglycidyl ether (PEGDE), dicumyl peroxide (DCP), PVP-PEGDE or PVP-PEG. In one embodiment, about 0.1 wt% to about 15 wt% of the crosslinker is added (in one example, about 1 wt% to about 10 wt%) relative to the total dry weight of these components added when blending the crosslinker and the polymer. During the curing process, it is believed that substantially all of the crosslinker reacts and substantially no detectable unreacted crosslinker remains in the final layer.

[0230] In one example, the APL is a conductive polymer. In one example, the APL is a functionalized polymer. The APL can be functionalized, for example, with a functional moiety in an amount of 1 wt% - 50 wt%, 2 wt% - 50 wt%, 5 wt% - 50 wt%, 10 wt% - 50 wt%, 15 wt% - 50 wt%, 20 wt% - 50 wt%, 25 wt% - 50 wt%, 30 wt% - 50 wt%, 10 wt% - 20 wt%, 15 wt% - 25 wt%. The functionalized polymer can be configured to couple with a substrate or a SAM or another layer, film, matrix, region, or polymer.

[0231] In one example, the functionalized polymer contains an alkanethiol group. In one example, the alkanethiol group is present at the end of the functionalized polymer chain, or the alkanethiol group is present along the backbone of the functionalized polymer chain.

[0232] In one example, the functionalized polymer contains a mercaptoalkanol group. In one example, the mercaptoalkanol group is present at the end of the functionalized polymer chain or along the backbone of the functionalized polymer chain.

[0233] In one example, the APL contains a zwitterionic group compound or a zwitterionic repeating group compound. In one example, the functionalized polymer is prepared with at least one of the following polymerizable zwitterionic monomer structures:

[0234]

[0235] where X is O, NH, or NR4, Y and Z are independently a branched or straight-chain alkyl, heteroalkyl, cycloalkyl, cycloheteroalkyl, aryl, or heteroaryl, and which may optionally be substituted with OH, halogen, or alkoxy; R1, R3, R4, and R5 are independently H, alkyl, heteroalkyl, cycloalkyl, cycloheteroalkyl, aryl, or heteroaryl.

[0236] For example, the APL can contain zwitterionic monomers alone or in combination with other polymer structures / backbones, including N-(2-methacryloyloxy)ethyl-N,N-dimethylammonium propanesulfonate, N-(3-methacryloylimino)propyl-N,N-dimethylammonium propanesulfonate, 2-(methacryloyloxy)ethyl phosphatidylcholine, and 3-(2'-vinyl-pyridinio)propanesulfonate.

[0237] In one example, the APL disclosed in the present invention provides an amount of zwitterionic repeating group compound that is capable of regulating or maintaining the ionic strength or pH around the aptamer and / or transduction element (such as a redox moiety) and / or substrate. In one example, one or more zwitterionic repeating groups include a betaine compound or its derivatives. In one example, the zwitterionic repeating group is present at the end of the functionalized polymer chain or along the backbone of the functionalized polymer chain.

[0238] In one example, the functionalized polymer comprises an alkanethiol and zwitterionic repeat groups. In one example, the functionalized polymer comprises an alkanethiol and betaine groups. In one example, the functionalized polymer comprises a mercaptoalkanol and zwitterionic repeat groups. In one example, the functionalized polymer comprises a mercaptoalkanol and betaine groups. In one example, the functionalized polymer comprises an arylthiol and zwitterionic repeat groups. In one example, the functionalized polymer comprises an arylthiol and betaine groups. In one example, the functionalized polymer comprises an arylmercaptoalkanol and zwitterionic repeat groups. In one example, the functionalized polymer comprises an arylmercaptoalkanol and betaine groups. In one example, the functionalized polymer comprises a benzylthiol and zwitterionic repeat groups. In one example, the functionalized polymer comprises a benzylthiol and betaine groups. In one example, the functionalized polymer comprises a benzylmercaptoalkanol and zwitterionic repeat groups. In one example, the functionalized polymer comprises a benzylmercaptoalkanol and betaine groups.

[0239] In one example, the APL is physically or chemically coupled to at least a portion of the substrate surface. In one example, the APL is physically or chemically coupled to at least a portion of the substrate surface, wherein one or more aptamer conjugates are physically or chemically coupled to at least a portion of the substrate surface. In one example, the APL is physically or chemically coupled to at least a portion of the substrate surface, wherein one or more aptamer conjugates are physically or chemically coupled to at least a portion of the substrate surface, and substantially the remainder of the substrate surface further comprises co-adsorbates that are physically or chemically coupled.

[0240] In one example, the APL provides sufficient free volume to allow reversible conformational changes of one or more aptamer conjugates. In one example, at least one of the one or more aptamer conjugates is physically or chemically coupled to an aminoalkanoic acid.

[0241] In one example, one or more aptamer conjugates are, for example, at 10 -9 molecules / cm 2 、10 -10 molecules / cm 2 、10 -11 molecules / cm 2 、10 -12 molecules / cm 2 to 10 -13 molecules / cm 2There is a density on the substrate surface. Other densities can be used. In one example, the aptamers present at the substrate surface have substantially similar architectures (less than 2 base pair deviations), the same transduction element or redox moiety, the same conjugate coupling chemistry for attaching the aptamer to the working electrode surface, SAM, or co - adsorbate, the same aptamer / co - adsorbate mass ratio and / or density, and the same manufacturing history. In one example, the aptamers present at the substrate surface have different architectures (greater than 2 base pair deviations), different or the same transduction element or redox moiety, different or the same conjugate coupling chemistry for attaching the aptamer to the working electrode surface, SAM, or co - adsorbate, different aptamer / co - adsorbate mass ratio and / or density, and different or the same manufacturing history.

[0242] Signal Transduction Element

[0243] In one example, the signal transduction element comprises a redox moiety. The redox moiety can comprise any compound that causes a change in electron transfer kinetics when its proximity to an electrode at a biased potential changes. Exemplary redox substances include methylene blue, organometallic redox moieties, ferrocene, viologen, anthraquinone or any other quinone, ethidium bromide, daunomycin, metal porphyrin complexes, crown ether metal complexes, bipyridyl metal complexes, bisimidazole metal complexes, terpyridyl metal complexes, ethylenediaminetetraacetic acid (EDTA) - metal complexes, and cytochromes. In one example, the reversible redox moiety comprises iron, iridium, ruthenium, osmium, thiazine dyes, or derivatives thereof. In one example, the reversible redox moiety comprises ferrocene or methylene blue.

[0244] In one example, the sensor is configured for continuous, semi - continuous, sequential, or random signal acquisition. In one example, the sensor is configured for percutaneous insertion.

[0245] Co - adsorbent

[0246] In one example, the AB or EAB device disclosed by the present invention includes one or more co - adsorbents. The role of the co - adsorbent is to cover the substrate (adsorbent) and change the response of the substrate to the surrounding environment exposure, thereby eliminating or reducing unwanted activities or reactions. The effectiveness of the co - adsorbate can be measured experimentally, for example, by tracking the baseline current level when the substrate is potentiostatically biased. In one example, the co - adsorbent is configured to independently provide a surface energy modulation environment, a phase separation modulation environment, and / or an intermolecular interaction modulation environment between the co - adsorbing molecules and / or between the co - adsorbing molecules and the aptamer molecules, the aptamer 102 and the surface of the substrate 110, any monolayer 103, and / or the aptamer protection material 105.

[0247] In one example, one or more co-adsorbents independently provide ionic strength, and there is an amount of one or more co-adsorbents that is capable of regulating or maintaining the ionic strength near at least one aptamer conjugate and / or the substrate surface.

[0248] In one example, at least a portion of the surface of substrate 110 further comprises one or more co-adsorbents. In one example, one or more co-adsorbents independently comprise a plurality of functional groups. Figure 4A and Figure 4B Schematic diagrams of exemplary co-adsorbents 402a and 402b are respectively depicted in accordance with the broadest aspects of the present disclosure. Thus, Figure 4A An enlarged cross-sectional schematic view of the substrate 110WE surface (as a working electrode) of an implantable EAB having an exemplary architecture with co-adsorbate 402a is shown, the co-adsorbate having a linear segment 406a and a terminal segment 404a. Figure 4B An enlarged cross-sectional schematic view of the substrate 110WE surface (as a working electrode) of an implantable EAB having an exemplary architecture with co-adsorbate 402a is shown, the co-adsorbate having a linear segment 406b and a backbone segment 404b. The linear segments 406a, 406b may comprise, for example, alkyl, alkylthiol, phenylthiol, benzylthiol, arylthiol, mercaptoalkanol, alkylsilane, aromatic silane, or alkylaromatic silane as disclosed herein. In one example, the linear segments 406a, 406b comprise aromatic thiol, alkylaromatic thiol. In one example, the terminal segment 404a or the backbone segment 404b may be a zwitterion or a repeating zwitterionic group as disclosed herein. Substrate 110 may include a random and / or patterned combination of co-adsorbents 402a and 402b with various substrate surface area ratios. The co-adsorbates 402a, 402b can be coupled to the substrate 110 surface (at Figure 4A , Figure 4B indicated by "X" in) in a variety of ways (e.g., thiol, amine, amino, carboxyl, carboxylamine, or carboxylamino) via EDC / NHS chemistry (e.g., click chemistry), as discussed herein. FIG. 4C shows an enlarged cross-sectional schematic view of the substrate 110WE of an implantable EAB having an exemplary architecture with co-adsorbate 403, the co-adsorbate having a linear segment 406 and a linear segment 405, wherein the linear segment 406 may be, for example, alkyl, alkylthiol, mercaptoalkanol as disclosed herein, and the linear segment 405 may be a zwitterion or a repeating zwitterionic group as disclosed herein. The co-adsorbate 403 can be coupled to the substrate 110 surface in a variety of ways (e.g., thiol, amine, amino, carboxyl, carboxylamine, or carboxylamino) via EDC / NHS chemistry or click chemistry, such as discussed herein. The substrate 110 surface may include a random and / or patterned combination of co-adsorbents 402a, 402b, and 403 with various substrate surface area ratios.

[0249] In one example, the continuous monitoring of AB or EAB of the present disclosure includes one or more co - adsorbents associated with the surface of substrate 110, and the one or more co - adsorbents are chemically different.

[0250] In one example, the aforementioned functionalized APL can also partially act as a co - adsorbate. Thus, in one example, at least a portion of the substrate surface includes functionalized APL, at least a portion of the substrate surface includes one or more co - adsorbents, and at least a portion of the remaining portion of the substrate surface includes one or more aptamer conjugates, where the sum of the percentage portions of the substrate surface and the remaining portion can be 100% or less than 100%.

[0251] In one example, at least a portion of the substrate surface, one or more co - adsorbents, and a portion of the remaining portion of the substrate surface include one or more aptamer conjugates. In one example, the remaining portion of the substrate surface is about 50% of the total surface area of the substrate. In one example, the remaining portion of the substrate surface is less than 50% but greater than 0% of the total surface area of the substrate. In one example, the remaining portion of the substrate surface is greater than 50% and less than 100% of the total surface area of the substrate.

[0252] In one example, at least a portion of the substrate surface includes one or more co - adsorbents, and a portion of the remaining portion of the substrate surface includes one or more aptamer conjugates physically or chemically coupled to the substrate. In one example, one or more co - adsorbents are physically or chemically coupled to the substrate surface, and a portion of the remaining portion of the substrate surface includes one or more aptamer conjugates physically or chemically coupled to at least a portion of the co - adsorbate.

[0253] In one example, the co - adsorbate comprises a self - assembled monolayer (SAM). In one example, the co - adsorbate comprises a mono - functional or multi - functional alkanethiol.

[0254] In one example, the thiol functional group of the mono - functional alkanethiol or multi - functional alkanethiol is covalently coupled to at least a portion of the substrate surface. In one example, the thiol functional group of the mono - functional alkanethiol or multi - functional alkanethiol is covalently coupled to the gold substrate surface.

[0255] In one example, the co - adsorbate comprises a mono - functional or multi - functional mercaptoalkanol. In one example, the thiol functional group of the mono - functional or multi - functional mercaptoalkanol is covalently coupled to at least a portion of the substrate surface. In one example, the thiol functional group of the mono - functional or multi - functional mercaptoalkanol is covalently coupled to at least a portion of the gold substrate surface.

[0256] In one example, the co - adsorbate comprises zwitterionic repeating groups associated with at least a portion of the substrate surface. In one example, the co - adsorbate comprises zwitterionic repeating groups coupled to at least a portion of the substrate surface. In one example, the co - adsorbate comprises zwitterionic repeating groups covalently coupled to at least a portion of the substrate surface. In one example, the zwitterionic repeating groups include betaine groups, such as sulfobetaine or carboxybetaine groups.

[0257] In one example, the zwitterionic repeating groups include ammonium phosphates or lecithin analogs, ammonium phosphonates, ammonium hypophosphonates, ammonium sulfonates, ammonium sulfates, ammonium carboxylates, or combinations thereof.

[0258] In one example, the zwitterionic repeating groups include alkanethiol betaines. In one example, the alkanethiol is linear and contains multiple betaine groups along its chain. In one example, the alkanethiol is a terminally - capped mono - or dithiol having at least one betaine group along its chain. In one example, the alkanethiol is linear and contains terminally - capped betaine groups. In one example, the thiol groups of the terminally - capped dithiol alkanethiol are covalently coupled to the substrate surface.

[0259] In one example, the zwitterionic repeating groups include mercaptoalkanol betaines. In one example, the mercaptoalkanol is straight - chain and contains multiple betaine groups along its chain. In one example, the mercaptoalkanol is straight - chain and contains terminally - capped betaine groups. In one example, the thiol groups of the mercaptoalkanol are covalently coupled to the substrate surface.

[0260] In one example, the co - adsorbate is one or more of the following structures:

[0261]

[0262] where represents a hydrocarbon chain; where the zwitterionic unit is attached to the main chain and the charge is on a side group pendant from the main chain, or the zwitterionic unit places one or two charges on the main chain; where R1 and R2 are independently branched or unbranched acyclic alkyls, substituted or unsubstituted cycloalkyls, substituted or unsubstituted aryls, substituted or unsubstituted benzyls, substituted or unsubstituted heteroalkyls, substituted or unsubstituted heterocycles; where X is - OH, - NHR1, - NH2, or - SH; where n is an integer from 2 to about 1000; or

[0263]

[0264] Wherein X is -OH, -NHR1, -NH2 or -SH; wherein W, Y and Z are independently branched or straight-chain alkyl, heteroalkyl, cycloalkyl, cycloheteroalkyl, aryl or heteroaryl, any of which may optionally be substituted with O, OH, halogen, amide or alkoxy; R1 is H, branched or unbranched acyclic alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloheteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted benzyl, or substituted or unsubstituted heteroaryl; and R3, R4 and R5 are independently selected from acyclic alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloheteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted benzyl, or substituted or unsubstituted heteroaryl.

[0265] In one example, the co-adsorbate is one or more of the following structures:

[0266]

[0267] Wherein R1 is H, alkyl, heteroalkyl, cycloalkyl, cycloheteroalkyl, aryl or heteroaryl; and R2, R3 and R4 are independently selected from alkyl, heteroalkyl, cycloalkyl, cycloheteroalkyl, aryl or heteroaryl; n is an integer from 2 to 24.

[0268] In one example, the co-adsorbate is one or more of the following ammonium sulfonates (sulfobetaines) or ammonium sulfate structures:

[0269]

[0270] Wherein 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 selected from alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl; wherein one or more of R1, R2, R3 and Z are substituted with a polymeric group; and ammonium carboxylates having the following structure:

[0271]

[0272] Wherein 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 selected from alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl; wherein one or more of R1, R2, R3 and Z are substituted with a polymeric group.

[0273] In each of these monomers, Z can have a length of from 1 to 12 atoms, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 atoms, where any of these values can form the upper or lower limit of a range.

[0274] These compounds or monomers can be prepared by methods known to those skilled in the art, for example, as detailed in Laschewsky, “Structures and synthesis of zwitterionic polymers,” Polymers 6:1544 - 1601, 2014. In certain examples, the disclosed zwitterions can have repeating zwitterionic units obtained from any of the zwitterionic compounds or monomers disclosed above. Exemplary zwitterionic compounds include octamidopropyl betaine, oleamidopropyl betaine, octyl sulfobetaine, octanoyl sulfobetaine, lauryl sulfobetaine, myristyl sulfobetaine, palmityl sulfobetaine, stearyl sulfobetaine, betaine (trimethylglycine), octyl betaine, phosphatidylcholine, glycine betaine, poly(carboxybetaine), poly(sulfobetaine), and their derivatives. Exemplary monomers containing one or more side or end groups having a zwitterionic group contain or are derived from octamidopropyl betaine, oleamidopropyl betaine, octyl sulfobetaine, octanoyl sulfobetaine, lauryl sulfobetaine, myristyl sulfobetaine, palmityl sulfobetaine, stearyl sulfobetaine, betaine (trimethylglycine), octyl betaine, phosphatidylcholine, glycine betaine, poly(carboxybetaine), and poly(sulfobetaine).

[0275] In one example, the co - adsorbate is a mono - or dithiol end - capped with at least one zwitterionic group or zwitterionic repeating group as disclosed herein. In one example, controlling the ionic strength, pH, etc. includes configuring the APL backbone or one or more appendages from its backbone with one or more zwitterionic betaine groups.

[0276] In one example, the APL contains an alkanethiol or (aryl) mercaptoalkanol and one or more zwitterionic groups. In one example, controlling the ionic strength, pH, etc. includes providing an APL in combination with a mercaptoalkanol having a zwitterionic betaine group.

[0277] Method

[0278] The APL-EAB constructs disclosed herein offer advantages over EABs that do not have APL. For example, the APL-EAB constructs disclosed herein can be used in methods for determining the in vivo concentration of an analyte. For example, the method can include the steps of contacting a biological fluid containing the analyte in vivo with the APL-EAB constructs disclosed herein, where the EAB is coupled to a conductive substrate, the EAB is encapsulated in APL, the APL is permeable to the analyte, and the EAB generates a signal when interacting with the analyte.

[0279] The APL-EAB constructs disclosed herein are configured to receive a bias voltage that is varied to reversibly oxidize and reduce a redox probe associated with an aptamer, where the aptamer is associated with the surface of the conductive substrate. The APL-EAB constructs disclosed herein can be used in methods that include interrogating the conductive substrate or the APL-aptamer-redox moiety conjugate. The method can further include detecting a signal generated by the aptamer-redox moiety conjugate in the presence of a certain concentration of the analyte, and correlating the in vivo concentration of the analyte based on the detected signal, signal change, signal difference, etc. In one example, signal transduction through the APL-EAB constructs disclosed herein is determined by the electron transfer rate from the reversible redox probe, where the difference in electron transfer rates is related to the analyte concentration.

[0280] In one example, the interrogation is a continuous, semi-continuous, sequential, or random time detection of the signal. In one example, the method can further include the step of adjusting the signal based on a background signal resulting from non-specific binding of the aptamer biosensor to produce a regulated signal. Two or more working electrodes with or without the APL-EAB constructs disclosed herein can be used. The method can further include determining the in vivo concentration of the analyte over a period of time based on the regulated signal.

[0281] In one example, the interrogation includes differential measurement techniques. Exemplary differential measurement techniques include, for example, interrogating with a first square wave voltammetry (SWV) frequency to obtain a first signal and with 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 a regulated signal. In one example, the interrogation includes chronoamperometry. In one example, the interrogation includes cyclic voltammetry.

[0282] In one example, the APL disclosed by the present invention can control or regulate the intermolecular interaction between the aptamer conjugate and the APL. In another example, the APL construct disclosed by the present invention alone or in combination with the co-adsorbent disclosed by the present invention provides a reduction in the decoupling of the aptamer from the substrate surface. In another example, the APL disclosed by the present invention can control or regulate the diffusion of the aptamer near the substrate surface. Thus, if reversible desorption / decoupling of the aptamer from the substrate occurs, the APL disclosed by the present invention can keep the aptamer near the substrate surface to increase the reabsorption / recoupling of the aptamer. In one example, the APL disclosed by the present invention is partially cross-linked. The APL disclosed by the present invention can be cross-linked in the presence of the aptamer-transduction moiety with little or no adverse effect on the APL-EAB performance, as discussed below.

[0283] In one example, the APL disclosed by the present invention can be used to extend the in vivo lifetime termination of the EAB device. For example, the APL disclosed by the present invention has been shown to have an extended lifetime termination in bovine serum albumin for up to 20 hours. It is contemplated that the APL disclosed by the present invention can provide in vivo lifetime termination performance for the EAB for up to one day, 2 days, one week, 2 weeks, 3 weeks, or one month.

[0284] In one example, the APL disclosed by the present invention alone or in combination with SAM or a co-adsorbent provides a method for controlling or regulating the ionic strength around the aptamer-signal transduction element conjugate. For example, the functionalized APL disclosed by the present invention, such as a betaine-functionalized APL in combination with one or more co-adsorbents, can be present in an amount that can regulate or maintain the ionic strength around the aptamer-signal transduction element conjugate.

[0285] The method of fabricating the APL-EAB device disclosed by the present invention includes presenting an aptamer containing a reversible redox moiety to the surface of a conductive substrate and presenting the APL to a portion of the surface of the conductive substrate to encapsulate the aptamer conjugate in the APL. Alternatively, the APL-EAB disclosed by the present invention is combined with an aptamer containing a reversible redox moiety and presented to the substrate surface.

[0286] Experimental Results

[0287] A series of exemplary APLs were developed, tested with aptamer-redox moiety conjugate EAB constructs, and the effectiveness of the APLs in providing improvements in one or more properties of the constructs was evaluated. A summary of the characteristics of a representative sampling of the APLs is presented in Table 1.

[0288]

[0289] Table 1. Exemplary APL. PUU = Aliphatic Polyurethane Urea Segmented Block Copolymer. PU = Aliphatic Polyurethane Segmented Block Copolymer. Hydrophilic Segment = Polyethylene Glycol and Polycarbonate. Hydrophobic Segment = Polydimethylsiloxane. Functional Content= Sulfobetaine or Carboxybetaine. Cross - linker = Polyethylene Glycol Poly Glycidyl Ether (PEG - PG). Wt% values can vary + / - 10％ 。

[0290] Figure 5A and Figure 5B are representative plots of experimental voltammetric readouts of exemplary aptamer biosensors with and without APL versus electron transfer readout (charge versus frequency) data. In this example, the co-adsorbate is 6-mercapto-1-hexanol, the coupling chemistry is thiol association on a gold substrate, and the APL used is PUU-3.

[0291] The aptamers tested are specific for vancomycin and aminoglycosides. Figure 5A and Figure 5B show approximately 71% kinetic differential measurement (KDM) signal (“control EAB”) of a 50 μmol / L analyte spike in pre-serum PBS buffer without APL, while the APL-coated sample (“APL-EAB”) gives a 67% KDM signal under the same conditions.

[0292] Reference Figure 6A 、 Figure 6B , samples after exposure to biological fluid (50 μL serum incubation) for 20 h show that the APL sample PUU-9 maintains good KDM, while the control shows significant degradation of KDM. After 20 h, the APL sample shows approximately 4 times the signal of the control.

[0293] Figure 7A and Figure 7B are representative plots of experimental charge versus frequency data of protein fouling of the same aptamer conjugate with and without aptamer protection material. The data show the advantage of APL in maintaining the EAB response by resisting biological fouling compared to the uncoated control.

[0294] Figure 8A and Figure 8B are representative plots of experimental current versus frequency voltammogram data obtained at different time intervals for an exemplary aminoglycoside aptamer biosensor in a protein-doped buffer without aptamer protection material compared to an APL-aminoglycoside aptamer sample. Figure 8A The data in Figure 8B show that the uncoated EAB has a continuous signal output decay in a protein-containing environment. For example, the peak height gradually shrinks over time, while conversely,

[0295] Figure 9AIt is a representative graph of the experimental normalized readout percentage versus time, which shows the drift of the control EAB compared to the APL EAB without biofouling challenge. The samples were exposed to bovine serum albumin (BSA), and as shown, the control EAB without APL started to drift from biofouling, etc. after less than 1 hour, while the APL-EAB provided stable performance for at least 5 hours.

[0296] Figure 9B It is a representative graph of the experimental normalized readout percentage versus time, which shows the drift of the control EAB compared to the APL-EAB in a buffer solution containing biofouling proteins. The samples were exposed to bovine serum albumin (BSA), and as shown, the control EAB without APL started to drift from biofouling, etc. after less than 1 hour, while the APL-EAB, such as sample PUU-9, provided stable performance for at least 22 hours (without analyte challenge). Thus, the functional content of zwitterions present at at least 10 wt% in the PU or PUU provides one or more performance improvements of the EAB, such as calibration stability, storage stability, drift stability, local pH stability, and interference reduction.

[0297] Figure 9C It is a representative graph of the experimental normalized readout percentage versus time, which shows the stability of the exemplary APL EAB sensor PUU-3 in PBS at 37 °C. As shown, the APL-EAB samples exhibited stability for at least 6 days and retained at least 80% of the signal. This data demonstrates the enhanced stability characteristics of the APL disclosed in the present invention.

[0298] Figure 10A and Figure 10B are respectively representative graphs of the experimental current-versus-frequency voltammogram data of an exemplary vancomycin aptasensor without an aptamer protection material compared to an APL-vancomycin aptamer sample. Similarly, the data shows that the uncoated EAB had a continuous decay of signal output during potential cycling, while the APL vancomycin aptamer sample was significantly more robust.

[0299] Reference Figure 11 , shows representative graphs of the experimental sensor response percentage versus analyte concentration of exemplary EABs with and without an aptamer protection material exposed to various analyte concentrations. The vancomycin EAB (PUU-3) with APL provided a signal-concentration curve that was substantially equivalent to that of the vancomycin EAB without APL, although the sensor response was slightly lower at a given vancomycin concentration.

[0300] In the foregoing experiments, the substrate used was a standard disk gold electrode. Subsequent studies on other electrode shape factors have shown that the EAB-APL of the present invention is not substrate shape-dependent and can be replicated, for example, using a wire electrode shape factor. The electrode shape factor does affect the absolute signal level, at least the difference in surface area.

[0301] Figure 12A and Figure 12B are representative calibration curves of exemplary vancomycin aptamer biosensors with different co-adsorbents 6-mercapto-1-hexanol (MCH) and 8-mercapto-1-hexanol (MCO) respectively, challenged with analytes at 0 uM, 10 uM, and 30 uM concentrations. Data from Figure 12A and Figure 12B demonstrate good calibration and compatibility of various co-adsorbents with the APL disclosed in the present invention.

[0302] Figure 13A and Figure 13B are representative graphs of the shelf-life performance of uncoated EAB and APL-coated EAB after storage in the ambient environment for 5 h. The uncoated sensors showed a significant performance decline with a large background current, while minimal changes in performance were observed on APL-EAB.

[0303] Figure 13C and Figure 13D are representative graphs of the calibration and drift data of an exemplary APL-EAB (targeting vancomycin) after storage in an ambient air environment at room temperature and relative humidity in the dark for 1 month. Figure 13E and Figure 13F are representative graphs of the calibration and drift data of an exemplary APL-EAB (targeting vancomycin) after storage in an ambient air environment at room temperature and relative humidity in the dark for 2 months. Data from Figures 13C to 13F demonstrate good calibration and drift performance of the APLs disclosed in the present invention for at least two months, which APLs employ the APLs and co-adsorbents as disclosed in the present invention.

[0304] Drug Release Layer

[0305] Devices and probes inserted percutaneously or implanted subcutaneously typically elicit a foreign body response (FBR), which includes the invasion of inflammatory cells that ultimately form a foreign body capsule (FBC), as part of the body's response to the introduction of foreign substances. The continuous monitoring systems discussed herein include continuous analyte monitoring systems that are configured to monitor one, two, or more analytes (which include events that can occur independently on the picosecond, nanosecond, millisecond, second, or minute scale) simultaneously, sequentially, and / or randomly to predict health-related events and health system performance (e.g., the current and future performance of a human system such as the circulatory, respiratory, digestive, or other systems or organs or combinations of systems). In an example, the insertion or implantation of a device (e.g., an EAB sensing device) may result in an acute inflammatory response that resolves to chronic inflammation while fibrotic tissue is established, as described in detail above. Ultimately, over time, a mature FBC forms around the device, including predominantly contractile fibrous tissue. See Shanker and Greisler, Inflammation and Biomaterials in: Greco RS, editor, “Implantation Biology: The Host Response and Biomedical Devices” pages 68 - 80, CRC Press (1994). The FBC surrounding a conventional implanted device has been shown to impede or block analyte transport across the device - tissue interface. Thus, continuous analyte transport with an extended lifespan in vivo (e.g., beyond the first few days) is generally considered unreliable or impossible.

[0306] In some examples, certain aspects of the FBR may play a role in the noise during the first few days. It has been observed that some sensors function worse during the first few hours after insertion than they do later. This is exemplified by the noise and / or suppression of the signal during the first few hours after insertion (e.g., from about 2 hours to about 24 hours). These anomalies typically resolve spontaneously, after which the sensor becomes less noisy, has improved sensitivity, and is more accurate than during the initial period. It has been observed that some percutaneous sensors and fully implantable sensors experience noise for a period of time after being applied to a recipient (i.e., percutaneously inserted or fully implanted under the skin).

[0307] Thus, return reference Figure 2D, the drug release layer, membrane, matrix, or coating 113 can be positioned adjacent to or directly adjacent to the APL 105. In one example, the AB or EAB continuous sensors disclosed in the present invention include an immune response attenuation layer or a drug release layer, which is configured to interact with the host's immune system or release an active agent into the environment of the sensor. In one example, the immune response attenuation layer includes an active agent coupled to or embedded in the layer, such as a covalently coupled active agent (dexamethasone derivative or analog) or a surface-exposed active agent (e.g., silver nanoparticles). In one example, the drug release layer includes an active agent that is configured to be released from the layer over time to attenuate or dampen the immune response. Such drug release layers include, for example, segmented polyurethane polymers containing dexamethasone and / or dexamethasone acetate and / or other dexamethasone derivatives or analogs, as disclosed in co-pending U.S. Application No. 17 / 945,585, which is incorporated herein by reference.

[0308] Manufacture

[0309] The substrate can be formed by a variety of manufacturing techniques (bulk metal processing, depositing metal onto a substrate, etc.). In one example, the substrate is a plated wire (e.g., platinum on a steel wire) or a bulk metal (e.g., a gold wire). It is believed that substrates of EABs formed from bulk metal wires provide excellent performance (e.g., compared to deposited electrodes), including improved assay stability, simplified manufacturability, resistance to contamination (e.g., contamination that may be introduced during deposition), and improved surface reactivity (e.g., due to the purity of the material) without peeling or delamination. The substrate can be a metal wire with an external insulator. The substrate can be multiple metal wires, each with an external insulator.

[0310] In examples where an outer insulator is disposed around a substrate, a portion of the coated component structure may be stripped or otherwise removed, e.g., by hand, excimer laser, chemical etching, laser ablation, sandblasting (e.g., with sodium bicarbonate, solid carbon dioxide, or other suitable grit), etc., to expose the electrochemically active surface. Alternatively, a portion of the electrode may be masked prior to depositing the insulator in order to maintain the exposed electrochemically active surface area. In one exemplary example, a sandblasting process is implemented to expose the electrochemically active surface, preferably using a grit material that is hard enough to abrade the polymeric material while being soft enough to minimize or avoid damage to the underlying metal electrode (e.g., a platinum electrode). Although a variety of "grit" materials may be used (e.g., sand, talc, walnut shells, abrasive plastics, sea salt, solid carbon dioxide, etc.), in some examples, sodium bicarbonate is a favorable grit material because it is hard enough to abrade, e.g., a parylene coating, without damaging, e.g., the underlying platinum conductor. An additional advantage of sodium bicarbonate sandblasting includes a polishing effect on the metal as it strips the polymeric layer, thereby eliminating a cleaning step that may otherwise be necessary. Etching (e.g., chemical or plasma) or other methods may be used to provide nanopores and / or micropores to the substrate surface.

[0311] In some examples, radial windows are formed through the insulating material to expose the circumferential electrochemically active surface of the working electrode. Additionally, multiple segments of the electrochemically active surface of the reference electrode are exposed. For example, multiple segments of the electrochemically active surface may be masked during deposition of the outer insulating layer or etched after deposition of the outer insulating layer.

[0312] In an example, APL is deposited on a substrate comprising an aptamer conjugate to produce a domain thickness of from about 0.05 microns or less to about 40 microns or greater, more preferably from about 0.05 microns, 0.1 microns, 0.15 microns, 0.2 microns, 0.25 microns, 0.3 microns, 0.35 microns, 0.4 microns, 0.45 microns, 0.5 microns, 1 micron, 1.5 microns, 2 microns, 2.5 microns, 3 microns or 3.5 microns to about 4 microns, 5 microns, 6 microns, 7 microns, 8 microns, 9 microns, 10 microns, 11 microns, 12 microns, 13 microns, 14 microns, 15 microns, 16 microns, 17 microns, 18 microns, 19 microns, 20 microns, 21 microns, 22 microns, 23 microns, 24 microns, 25 microns, 26 microns, 27 microns, 28 microns, 29 microns, 30 microns, 31 microns, 32 microns, 33 microns, 34 microns, 35 microns, 36 microns, 37 microns, 38 microns, 39 microns, 40 microns or greater. In one example, the domain thickness of the APL is from about 20 microns to about 40 microns, including all ranges and sub-ranges therebetween. In one example, the APL is deposited with the aptamer conjugate. In one example, the APL (or APL and aptamer conjugate) is deposited by spraying or dip coating. The spraying process atomizes the solution and forms a mist, so that most or all of the solvent evaporates before the coating material settles onto the underlying domain, thereby minimizing contact of the solvent with the aptamer. Without wishing to be bound by theory, it is believed that during the process of depositing APL as described in the present disclosure, a structural morphology is formed around the aptamer that allows substantially unhindered conformational changes of the aptamer with respect to the target analyte for the hard-soft multi-segmentation and / or functionalization of the APL structure.

[0313] In an example, APL is deposited onto a substrate / coadsorbent by spraying a solution of from about 1 wt% to about 5 wt% of a polymer and from about 95 wt% to about 99 wt% of a solvent (including all ranges and sub-ranges therebetween). When spraying a solution of the APL material (including the solvent) onto the substrate / coadsorbent, it is desirable to mitigate or significantly reduce any contact of the solvent in the spray solution that could deactivate the underlying aptamer, transduction element, or redox moiety with the aptamer. As will be understood by those skilled in the art, one or more solvents may be used, including water.

[0314] Although a variety of spraying or deposition techniques may be used, spraying the APL material and rotating the sensor at least once by 180° can provide sufficient coverage of the APL. Spraying the APL material and rotating the sensor at least twice by 120 degrees provides even greater coverage (a single layer of 360° coverage), thereby ensuring protection of the AB or EAB, such as described in more detail above.

[0315] In an example, the APL is spray-coated or dip-coated and then cured at a temperature of about 40 °C to about 60 °C (e.g., if a cross-linking agent is used) for a time of about 15 minutes to about 90 minutes (and can be done under vacuum (e.g., 20 mmHg to 30 mmHg)), including all ranges and sub-ranges therebetween. A cure time of up to about 90 minutes or longer can advantageously ensure complete drying of the APL. While not wishing to be bound by theory, it is believed that complete drying of the APL helps to stabilize the sensitivity of the AB or EAB sensor signal. It reduces the drift of the signal sensitivity over time, and it is thought that complete drying stabilizes the performance of the AB or EAB sensor signal.

[0316] In an example, the APL is formed by spray-coating or dip-coating one or more layers (e.g., rotating the sensor 120° to achieve 360° coverage) and optionally curing at 50 °C for 60 minutes under vacuum. However, depending on the concentration of the solution, the insertion rate, the residence time, the withdrawal rate, and / or the desired thickness of the resulting APL, the APL can be formed by dip-coating. In one example, the APL and / or the aptamer conjugate is combined with one or more antioxidants. In one example, an antioxidant is incorporated into the aptamer protection layer (APL). In one example, the antioxidant is incorporated into the aptamer protection layer (APL) in an amount of about 0.01 wt% to about 5 wt%. In one example, the antioxidant incorporated into the aptamer protection layer (APL) reduces the oxidized thiol moiety or the thiol-gold bond of the aptamer conjugate, thereby increasing the operating life and / or shelf life of the EAB disclosed herein. In one example, the antioxidant is lipophilic, such as vitamin E or other tocopherols. In one example, the antioxidant is butylated hydroxytoluene (BHT). In one example, the antioxidant is directly added to the APL polymer solution (submitted provisional patent) and deposited onto the aptamer conjugate, e.g., using a multiple dipping process.

[0317] In another example, the antioxidant is hydrophilic, such as ascorbic acid, trehalose, or sodium bisulfite, which is coated as a separate layer or introduced between the aptamer conjugate and the APL. In one example, the antioxidant is grafted onto the APL or onto a polymer chain that is miscible or compatible with the APL.

[0318] Electronic Device

[0319] In one example, the continuous AB or EAB sensor disclosed by the present invention further includes one or more of a transmitter, a receiver, a controller, or a power source. Any electronic device associated with a continuous analyte sensor, such as non-invasive, minimally invasive, and / or invasive (e.g., transcutaneous and fully implantable) sensors, is applicable. For example, sensor electronics and data processing known in the art, as well as transceiver electronics, Wi-Fi, Bluetooth, RF, and data processing, can be incorporated into the AB or EAB sensor disclosed by the present invention.

[0320] Figure 14 FIG. depicting an exemplary continuous AB or EAB system 150 configured to separately measure one or more analytes or in combination with electrophysiological metrics (e.g., blood pressure, heart rate, core temperature, etc.) as discussed herein. According to certain aspects of the present disclosure, the continuous AB or EAB system 150 includes an exemplary continuous AB or EAB device 100, 200 operatively connected to a host 120 and a plurality of display devices 134a-e. It should be noted that the display device 134e, alternatively or in addition to being a display device, can be a drug delivery device that can cooperate with the continuous AB or EAB system 150 to deliver a drug to the host 120. In one example, the continuous AB or EAB system 150 is an EAB system, where the sensor electronics module 126 and the continuous EAB sensor 122 associated with the sensor electronics module 126. The sensor electronics module 126 can directly wirelessly communicate with one or more of the plurality of display devices 134a-e via a wireless communication signal. In one example, the display devices 134a-e can also communicate with each other and / or communicate with the continuous AB or EAB system 150 through each other. For ease of reference, the wireless communication signal from the analyte sensor system 124 to the display devices 134a-e can be referred to as the "uplink" signal 128. The wireless communication signal from, for example, the display devices 134a-e to the continuous AB or EAB system 150 can be referred to as the "downlink" signal 130. The wireless communication signal between two or more of the display devices 134a-e can be referred to as the "crosslink" signal 132. Additionally, the wireless communication signal can include data transmitted by one or more of the display devices 134a-d to one or more remote servers 140 or network entities (such as cloud-based servers or databases) via a "long-range" uplink signal 136 (e.g., a cellular signal), and receive a long-range downlink signal 138 transmitted by the remote server 140.

[0321] The sensor electronics module 126 includes sensor electronics configured to process sensor information and generate transformed sensor information. In some examples, the sensor electronics module 126 includes electronics circuitry associated with measuring and processing data from the continuous EAB sensor 122, including a look-ahead algorithm associated with processing and calibrating continuous analyte sensor data. The sensor electronics module 126 may be integral (non-releasably attached) with the continuous EAB sensor 122 or releasably attached to the continuous EAB sensor to effect a physical connection therebetween. The sensor electronics module 126 may include hardware, firmware, and / or software capable of performing analyte level measurements. For example, the sensor electronics module 126 may include a potentiostat, a power source for providing power to the continuous EAB device 122, other components for signal processing and data storage, and a telemetry module for transmitting data from itself to one or more display devices 134a-e. The electronics may be fixed to a printed circuit board (PCB) or the like and may take a variety of forms. For example, the electronics may be in 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 one example, the electrochemical analog front end is configured with a sequencer or waveform synthesizer to create an appropriate waveform to transduce signals from the EAB. Exemplary waveforms include square wave 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. Patent Nos. 7,310,544 and 6,931,327 and U.S. Patent Publications 2005 / 0043598, 2007 / 0032706, 2007 / 0016381, 2008 / 0033254, 2005 / 0203360, 2005 / 0154271, 2005 / 0192557, 2006 / 0222566, 2007 / 0203966, and 2007 / 0208245, each of which is incorporated herein by reference in its entirety for all purposes.

[0322] The display devices 134a-e are configured to display, alert, and / or perform drug delivery based on sensor information transmitted by the sensor electronics module 126 (e.g., in a customized data packet transmitted to the display device based on the respective preferences of one or more of the display devices 134a-e). Each of the display devices 134a-e may include a display such as a touchscreen display for displaying sensor information to a user (most commonly the recipient 120 or a caregiver / healthcare professional) and / or receiving input from the user. In some examples, as an alternative or supplement to the touchscreen display, the display devices 134a-e may include other types of user interfaces, such as a voice user interface, for communicating sensor information to the user of the display device 134a-e and / or receiving user input. In some examples, one, some, or all of the display devices 134a-e are configured to display or otherwise communicate sensor information communicated from the sensor electronics module 126 (e.g., in a data packet transmitted to the respective display devices 134a-e) without any additional forward processing required for calibration and real-time display of the sensor information.

[0323] In Figure 14 an example, one of the plurality of display devices 134a-e may be a customized display device 134a that is specifically designed to display certain types of displayable sensor information (e.g., numerical values and arrows in some examples) associated with an analyte value received from the sensor electronics module 126. In some examples, one of the plurality of display devices 134a-e may be a handheld device 134c, such as a mobile phone, a personal digital assistant, etc. based on the Android, iOS operating system, or other operating systems, where the handheld device 134c may have a relatively large display and is configured to display a graphical representation of continuous sensor data (e.g., including current and historical data). Other display devices may include other handheld devices, such as a tablet computer 134d, a smartwatch 134b, a drug delivery device 134e, a blood glucose meter, and / or a desktop or laptop computer.

[0324] As mentioned above, since the different display devices 134a-e provide different user interfaces, the content of the data packet (e.g., the amount, format, and / or type of data to be displayed, alerts, etc.) may be customized (e.g., programmed differently by the manufacturer and / or by the end user) for each specific display device and / or display device type. Thus, in Figure 14 an example, one or more of the display devices 134a-e may communicate directly or indirectly wirelessly with the sensor electronics module 126 to enable multiple different types and / or levels of display and / or functionality associated with the sensor information, which is described in more detail elsewhere herein.

[0325] Sterilization

[0326] The APL-EAB disclosed by the present invention is configured for full or partial sterilization, including aseptic manufacturing and / or packaging. Examples of sterilization methods applicable to the APL-EAB disclosed by the present invention include, for example, high-energy radiation (UV, electron beam, x-ray), chemical treatment (ethylene oxide, CIDEX OPA TM (0.55% ortho-phthalaldehyde)) or autoclaving.

[0327] Although 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. Accordingly, the present disclosure should not be construed as limited to the specific exemplary embodiments described herein and shown in the drawings, but may also cover combinations of elements of the various shown embodiments and aspects thereof.

Claims

1. An analyte monitoring sensor configured for in vivo measurement of at least one analyte, the analyte monitoring sensor comprising: A substrate having a substrate surface; An aptamer protective layer encapsulating at least a portion of the substrate surface, the aptamer protective layer being permeable to the at least one analyte; One or more aptamer conjugates associated with at least a portion of the substrate surface and located between the aptamer protective layer and the substrate for obtaining in vivo measurement results related to the at least one analyte; And A reversible redox moiety coupled to the one or more aptamer conjugates.

2. The analyte monitoring sensor according to any one of the preceding claims, wherein at least a portion of the substrate is a conductive metal.

3. The analyte monitoring sensor according to any one of the preceding claims, wherein at least a portion of the substrate is gold, carbon, graphene or graphene oxide.

4. The analyte monitoring sensor according to any one of the preceding claims, wherein at least a portion of the substrate comprises pores having an average pore diameter in the nano- and / or micro-size range.

5. The analyte monitoring sensor according to any one of the preceding claims, wherein at least a portion of the substrate surface further comprises one or more co-adsorbents.

6. The analyte monitoring sensor according to any one of the preceding claims, wherein each of the one or more co-adsorbents independently comprises a plurality of functional groups.

7. The analyte monitoring sensor according to any one of the preceding claims, wherein the one or more co-adsorbents independently provide one or more of a surface energy range, a pH range, a phase separation range, and an intermolecular interaction range between the one or more aptamers and the aptamer protective layer.

8. The analyte monitoring sensor according to any one of the preceding claims, wherein the one or more co-adsorbents independently provide an ionic strength, and an amount of the one or more co-adsorbents is present, the amount being capable of adjusting or maintaining the ionic strength in the vicinity of the at least one aptamer conjugate.

9. The analyte monitoring sensor according to any one of the preceding claims, wherein the co-adsorbent comprises a self-assembled monolayer (SAM) associated with the substrate surface.

10. The analyte monitoring sensor according to any one of the preceding claims, wherein: At least a portion of the substrate surface comprises the one or more co-adsorbents, and the remaining portion of the substrate surface, a portion of the remaining portion of the substrate surface comprises the one or more aptamer conjugates.

11. The analyte monitoring sensor according to any one of the preceding claims, wherein at least a portion of the substrate surface comprises the one or more co-adsorbents physically or chemically coupled thereto and the remaining portion of the substrate surface, the remaining portion of the substrate surface comprises the one or more aptamer conjugates physically or chemically coupled thereto.

12. The analyte monitoring sensor according to any one of the preceding claims, wherein at least a portion of the substrate surface comprises said one or more co-adsorbents physically or chemically thereto, and said one or more aptamer conjugates are physically or chemically coupled to said one or more co-adsorbents.

13. The analyte monitoring sensor according to any one of the preceding claims, wherein the co-adsorbent comprises a mono-functional or multi-functional alkanethiol, mercaptoalkanol, alkyl mercaptoalkanol or aryl mercaptoalkanol.

14. The analyte monitoring sensor according to any one of the preceding claims, wherein the 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.

15. The analyte monitoring sensor according to any one of the preceding claims, wherein the thiol functional group of the mono-functional alkanethiol or the multi-functional alkanethiol is covalently coupled to a gold substrate surface.

16. The analyte monitoring sensor according to any one of the preceding claims, wherein the co-adsorbent comprises a mono-functional or multi-functional mercaptoalkanol.

17. The analyte monitoring sensor according to any one of the preceding claims, wherein the thiol functional group of the mono-functional or multi-functional mercaptoalkanol is covalently coupled to at least a portion of the substrate surface.

18. The analyte monitoring sensor according to any one of the preceding claims, wherein the thiol functional group of the mono-functional or multi-functional mercaptoalkanol is covalently coupled to at least a portion of a gold substrate surface.

19. The analyte monitoring sensor according to any one of the preceding claims, wherein at least a portion of the substrate surface comprises zwitterionic repeating groups.

20. The analyte monitoring sensor according to any one of the preceding claims, wherein the zwitterionic repeating group comprises betaine.

21. The analyte monitoring sensor according to any one of the preceding claims, wherein the zwitterionic repeating group comprises ammonium phosphate or a lecithin analogue.

22. The analyte monitoring sensor according to any one of the preceding claims, wherein the zwitterionic repeating group comprises ammonium phosphonate.

23. The analyte monitoring sensor according to any one of the preceding claims, wherein the zwitterionic repeating group comprises ammonium hypophosphonate.

24. The analyte monitoring sensor according to any one of the preceding claims, wherein the zwitterionic repeating group comprises ammonium sulfonate.

25. The analyte monitoring sensor according to any one of the preceding claims, wherein the zwitterionic repeating group comprises ammonium sulfate.

26. The analyte monitoring sensor according to any one of the preceding claims, wherein the zwitterionic repeating group comprises ammonium carboxylate.

27. The analyte monitoring sensor according to any one of the preceding claims, wherein the zwitterionic repeating group comprises alkanethiol betaine.

28. The analyte monitoring sensor according to any one of the preceding claims, wherein the alkanethiol is straight-chain and comprises a plurality of betaine groups along the chain of the alkanethiol.

29. The analyte monitoring sensor according to any one of the preceding claims, wherein the alkanethiol is a terminally capped dithiol having at least one betaine group along the chain of the alkanethiol.

30. The analyte monitoring sensor according to any one of the preceding claims, wherein the thiol groups of the terminally capped dithiol alkanethiol are covalently coupled to the substrate surface.

31. The analyte monitoring sensor according to any one of the preceding claims, wherein the zwitterionic repeating group comprises mercaptoalkanol betaine.

32. The analyte monitoring sensor according to any one of the preceding claims, wherein the mercaptoalkanol is straight-chain and comprises a plurality of betaine groups along the chain of the mercaptoalkanol.

33. The analyte monitoring sensor according to any one of the preceding claims, wherein the thiol groups of the mercaptoalkanol are covalently coupled to the substrate surface.

34. The analyte monitoring sensor according to any one of the preceding claims, wherein the co-adsorbate coupled or tethered to the substrate is represented as follows: wherein X is -OH, -NHR1, -NH2 or -SH; wherein R1 is a branched or unbranched acyclic alkyl, a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted aryl, a substituted or unsubstituted benzyl, a substituted or unsubstituted heteroalkyl, or a substituted or unsubstituted heterocycle; and a is 1 - 3.

35. The analyte monitoring sensor according to any one of the preceding claims, wherein at least a portion of the substrate surface comprises covalently coupled amines.

36. The analyte monitoring sensor according to any one of the preceding claims, wherein at least a portion of the substrate surface comprises covalently coupled aminoalkanoic acids.

37. The analyte monitoring sensor according to any one of the preceding claims, wherein at least a portion of the carbon, graphene or graphene oxide substrate surface comprises covalently coupled aminoalkanoic acids.

38. The analyte monitoring sensor according to any one of the preceding claims, wherein at least a portion of the carbon, graphene or graphene oxide substrate surface comprises covalently coupled aminoalkanoic acids, and the aminoalkanoic acids are also covalently coupled to the one or more aptamer conjugates.

39. The analyte monitoring sensor according to any one of the preceding claims, wherein a certain amount of cross-linking agent is used to at least partially cross-link the aptamer protective layer.

40. The analyte monitoring sensor according to any one of the preceding claims, wherein the aptamer protective layer comprises a conductive polymer.

41. The analyte monitoring sensor according to any one of the preceding claims, wherein the aptamer protective layer comprises zwitterionic groups.

42. The analyte monitoring sensor according to any one of the preceding claims, wherein the aptamer protective layer comprises zwitterionic repeating groups.

43. The analyte monitoring sensor according to any one of the preceding claims, wherein the aptamer protective layer provides an ionic strength or a local pH range, and a certain amount of zwitterionic repeating group compound, the amount of the zwitterionic repeating group compound present being capable of regulating or maintaining the ionic strength or the local pH range.

44. The analyte monitoring sensor according to any one of the preceding claims, wherein the aptamer protective layer provides a free volume that allows reversible conformational changes of the one or more aptamer conjugates present therein, the free volume being sufficient to provide a signal in the presence of the at least one analyte.

45. The analyte monitoring sensor according to any one of the preceding claims, wherein the aptamer protective layer comprises a functionalized polymer.

46. The analyte monitoring sensor according to any one of the preceding claims, wherein the functionalized polymer comprises an alkanethiol group.

47. The analyte monitoring sensor according to any one of the preceding claims, wherein the alkanethiol group is present at the end of the functionalized polymer chain.

48. The analyte monitoring sensor according to any one of the preceding claims, wherein the alkanethiol group is present along the backbone of the functionalized polymer chain.

49. The analyte monitoring sensor according to any one of the preceding claims, wherein the functionalized polymer comprises a mercaptoalkanol group.

50. The analyte monitoring sensor according to any one of the preceding claims, wherein the mercaptoalkanol group is present at the end of the functionalized polymer chain.

51. The analyte monitoring sensor according to any one of the preceding claims, wherein the mercaptoalkanol group is present along the backbone of the functionalized polymer chain.

52. The analyte monitoring sensor according to any one of the preceding claims, wherein the aptamer protective layer comprises a functionalized polymer, the functionalized polymer comprising one or more zwitterionic repeat groups.

53. The analyte monitoring sensor according to any one of the preceding claims, wherein the one or more zwitterionic repeat groups comprise a betaine compound or a derivative thereof.

54. The analyte monitoring sensor according to any one of the preceding claims, wherein the zwitterionic repeat group is present at the end of the functionalized polymer chain.

55. The analyte monitoring sensor according to any one of the preceding claims, wherein the zwitterionic repeat group is present along the backbone of the functionalized polymer chain.

56. The analyte monitoring sensor according to any one of the preceding claims, wherein the functionalized polymer comprises an alkanethiol and a zwitterionic repeat group.

57. The analyte monitoring sensor according to any one of the preceding claims, wherein the functionalized polymer comprises an alkanethiol and a betaine group.

58. The analyte monitoring sensor according to any one of the preceding claims, wherein the functionalized polymer comprises a mercaptoalkanol and a zwitterionic repeat group.

59. The analyte monitoring sensor according to any one of the preceding claims, wherein the functionalized polymer comprises a mercaptoalkanol and a betaine group.

60. The analyte monitoring sensor according to any one of the preceding claims, wherein the aptamer protective layer is physically or chemically coupled to at least a portion of the substrate surface.

61. The analyte monitoring sensor according to any one of the preceding claims, wherein the aptamer protective layer is physically or chemically coupled to at least a portion of the surface of the substrate, the one or more aptamer conjugates are physically or chemically coupled to at least a portion of the surface of the substrate, and substantially the remaining portion of the surface of the substrate further comprises co-adsorbates that are physically or chemically coupled.

62. The analyte monitoring sensor according to any one of the preceding claims, wherein the aptamer protective layer comprises at least one polymer segment selected from the group consisting of: polyurethanes, polyureas, poly(urethane ureas), epoxides, polyolefins, polysiloxanes, polyamides, polystyrenes, polyacrylates, polyethers, polyvinylpyridines, polyvinylpyrrolidones, polyesters, polycarbonates, and copolymers thereof.

63. The analyte monitoring sensor according to any one of the preceding claims, wherein the aptamer protective layer comprises a segmented multi-block polymer.

64. The analyte monitoring sensor according to any one of the preceding claims, wherein the aptamer protective layer comprises a segmented multi-block polyurethane polymer.

65. The analyte monitoring sensor according to any one of the preceding claims, wherein the aptamer protective layer comprises a segmented multi-block polyurethane urea polymer.

66. The analyte monitoring sensor according to any one of the preceding claims, wherein the segmented multi-block polymer comprises soft segments and hard segments.

67. The analyte monitoring sensor according to any one of the preceding claims, wherein the soft segments are hydrophobic or hydrophilic.

68. The analyte monitoring sensor according to any one of the preceding claims, wherein the soft segments are hydrophobic and hydrophilic.

69. The analyte monitoring sensor according to any one of the preceding claims, wherein the soft segments comprise hydrophobic polyols and hydrophilic polyols.

70. The analyte monitoring sensor according to any one of the preceding claims, wherein the soft segments are one or more segments comprising polydimethylsiloxane, polycarbonate, polyester, polyether, and blends or copolymers thereof.

71. The analyte monitoring sensor according to any one of the preceding claims, wherein the soft segments are one or more segments comprising polyethylene glycol, oligo polyether, polyoxazoline (POX), polypeptides, polyvinylpyrrolidone, polyvinylpyridine, zwitterionic repeating group polymers, and blends or copolymers thereof.

72. The analyte monitoring sensor according to any one of the preceding claims, wherein the hard segments comprise urethane groups or urea groups.

73. The analyte monitoring sensor according to any one of the preceding claims, wherein the one or more aptamer conjugates are physically associated with a portion of the surface of the substrate.

74. The analyte monitoring sensor according to any one of the preceding claims, wherein the one or more aptamer conjugates are covalently associated with a portion of the surface of the substrate.

75. The analyte monitoring sensor according to any one of the preceding claims, wherein the one or more aptamer conjugates comprise an RNA or DNA nucleotide sequence.

76. The analyte monitoring sensor according to any one of the preceding claims, wherein the one or more aptamer conjugates comprise at least one of the following: 2'-O-methyl modification of nucleotides; disulfide bridges; 3' caps having reverse 2-deoxythymidine; 3'-3'-thymidine linkages at the 3' terminus; 2'-F modification; and double-stranded segments.

77. The analyte monitoring sensor according to any one of the preceding claims, wherein the one or more aptamer conjugates comprise an RNA or DNA sequence having a first linker portion at the 5' terminus and the reversible redox moiety at the 3' terminus; or wherein the one or more aptamer conjugates comprise an RNA or DNA sequence having a first linker portion at the 3' terminus and the reversible redox moiety at the 5' terminus.

78. The analyte monitoring sensor according to any one of the preceding claims, wherein the first linker portion at the 5' terminus or the 3' terminus comprises an amino group or a carboxyl group.

79. The analyte monitoring sensor according to any one of the preceding claims, wherein the first linker portion is physically or chemically coupled to the substrate at the 5' terminus or the 3' terminus.

80. The analyte monitoring sensor according to any one of the preceding claims, wherein the first linker portion is physically or chemically coupled to the co-adsorbate at the 5' terminus or the 3' terminus.

81. The analyte monitoring sensor according to any one of the preceding claims, wherein the one or more aptamer conjugates are glycopeptide antibiotic-binding aptamers.

82. The analyte monitoring sensor according to any one of the preceding claims, wherein the one or more aptamer conjugates are vancomycin-binding aptamers.

83. The analyte monitoring sensor according to any one of the preceding claims, wherein the one or more aptamer conjugates are neurotransmitter-binding aptamers.

84. The analyte monitoring sensor according to any one of the preceding claims, wherein the one or more aptamer conjugates are dopamine, L-DOPA, insulin, or glutamate-binding aptamers.

85. The analyte monitoring sensor according to any one of the preceding claims, wherein the one or more aptamer conjugates are carbohydrate, triglyceride, or fatty acid-binding aptamers.

86. The analyte monitoring sensor according to any one of the preceding claims, wherein the one or more aptamer conjugates are glucose, glycerol, or β-hydroxybutyrate-binding aptamers.

87. The analyte monitoring sensor according to any one of the preceding claims, wherein the one or more aptamer conjugates are physically or chemically coupled to a self-assembled monolayer (SAM).

88. The analyte monitoring sensor according to any one of the preceding claims, wherein the one or more aptamer conjugates are physically or chemically coupled to a mono-functional or multi-functional alkanethiol or mercaptoalkanol.

89. The analyte monitoring sensor according to any one of the preceding claims, wherein the one or more aptamer conjugates are physically or chemically coupled to an alkylthiol betaine.

90. The analyte monitoring sensor according to any one of the preceding claims, wherein the one or more aptamer conjugates are physically or chemically coupled to an aliphatic amine.

91. The analyte monitoring sensor according to any one of the preceding claims, wherein the one or more aptamer conjugates are physically or chemically coupled to an aminoalkanoic acid.

92. The analyte monitoring sensor according to any one of the preceding claims, wherein the reversible redox moiety comprises iron, iridium, ruthenium, osmium, a thiazine dye, or a derivative thereof.

93. The analyte monitoring sensor according to any one of the preceding claims, wherein the reversible redox moiety comprises ferrocene or methylene blue.

94. The analyte monitoring sensor according to any one of the preceding claims, wherein the sensor is configured for continuous, semi - continuous, sequential, or random signal acquisition.

95. The analyte monitoring sensor according to any one of the preceding claims, wherein the sensor is configured for percutaneous insertion.

96. The analyte monitoring sensor according to any one of the preceding claims, wherein the sensor further comprises one or more of a reference electrode, a working electrode, and a counter electrode.

97. The analyte monitoring sensor according to any one of the preceding claims, wherein the sensor further comprises one or more of a transmitter, a receiver, a controller, or a power source.

98. A method of extending the end - of - life of an electrochemical aptamer biosensor (EAB), the method comprising: electro - associating at least one aptamer conjugate with the surface of a conductive substrate, the at least one aptamer conjugate comprising a reversible redox moiety; encapsulating the at least one aptamer conjugate within an aptamer protective layer, the at least one aptamer conjugate being configured to undergo a reversible conformational change within the aptamer protective layer in response to interaction with an analyte so as to generate a detectable signal; controlling one or more of: the ionic strength within the aptamer protective layer, the pH within the aptamer protective layer, the surface phase separation of the aptamer protective layer, and the intermolecular interaction between the at least one aptamer conjugate and the aptamer protective layer; and extending the end - of - life of the electrochemical aptamer sensor.

99. The method according to claim 98, wherein controlling the ionic strength comprises introducing one or more co - adsorbents into the aptamer protective layer, the one or more co - adsorbents being present in an amount capable of regulating or maintaining the ionic strength.

100. The method according to any one of claims 98 - 99, wherein the one or more co - adsorbents comprise zwitterionic betaine groups.

101. The method according to claim 100, wherein the zwitterionic betaine group comprises one of the following structures: Wherein X is -OH, -NHR1, -NH2 or -SH; wherein W, Y and Z are independently branched or straight-chain alkyl, heteroalkyl, cycloalkyl, cycloheteroalkyl, aryl or heteroaryl, any one of which can optionally be substituted by O, OH, halogen, amide group or alkoxy group; R1 is H, branched or unbranched acyclic alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloheteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted benzyl, or substituted or unsubstituted heteroaryl; and R3, R4 and R5 are independently selected from acyclic alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloheteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted benzyl, or substituted or unsubstituted heteroaryl; wherein one or more of R3, R4, R5, W, X, Y and Z are coupled to the aptamer protective layer or the substrate.

102. The method according to any one of claims 99-101, wherein the co-adsorbent is a terminally capped dithiol having at least one betaine group along the chain of the co-adsorbent.

103. The method according to claim 102, wherein the thiol group of the terminally capped dithiol alkanethiol is covalently coupled to the substrate surface.

104. The method according to any one of claims 99-103, wherein the zwitterionic betaine group comprises mercaptoalkanol betaine or arylmercaptoalkanol.

105. The method according to claim 104, wherein the mercaptoalkanol is straight-chain and contains a plurality of betaine groups along the chain of the mercaptoalkanol.

106. The method according to claim 105, wherein the thiol group of the mercaptoalkanol or the arylmercaptoalkanol is covalently coupled to the substrate surface.

107. The method according to any one of claims 98-106, wherein controlling the ionic strength or pH comprises providing the aptamer protective layer having one or more zwitterionic betaine groups.

108. The method according to any one of claims 98-107, wherein the aptamer protective layer comprises alkanethiol and one or more zwitterionic groups.

109. The method according to any one of claims 98-108, wherein controlling the ionic strength or pH comprises providing the aptamer protective layer having mercaptoalkanol and zwitterionic betaine groups.

110. The method according to any one of claims 98-109, wherein controlling the intermolecular interaction between the at least one aptamer conjugate and the aptamer protective layer comprises providing the aptamer protective layer having a segmented multi-block polymer backbone.

111. The method according to any one of claims 98-110, wherein the segmented multi-block polymer backbone comprises a polyurethane polymer.

112. The method according to any one of claims 98-111, wherein the segmented multi-block polymer backbone comprises a polyurethane-urea polymer.

113. The method according to any one of claims 98 - 112, wherein the segmented multi - block polymer comprises a soft segment and a hard segment.

114. The method according to any one of claims 98 - 113, wherein the soft segment is hydrophobic or hydrophilic.

115. The method according to any one of claims 98 - 114, wherein the soft segment is both hydrophobic and hydrophilic.

116. The method according to any one of claims 98 - 115, wherein the soft segment comprises a hydrophobic polyol and a hydrophilic polyol.

117. The method according to any one of claims 98 - 116, wherein the soft segment is one or more segments comprising polydimethylsiloxane, polycarbonate, polyester, polyether, and blends or copolymers thereof.

118. The method according to any one of claims 98 - 117, wherein the soft segment is one or more segments comprising polyethylene glycol, oligomeric polyether, polyoxazoline (POX), polypeptide, polyvinylpyrrolidone, polyvinylpyridine, zwitterionic repeating group polymer, and blends or copolymers thereof.

119. The method according to any one of claims 98 - 118, wherein the segmented multi - block polymer comprises a soft segment and a hard segment.

120. The method according to any one of claims 98 - 119, wherein the hard segment comprises a urethane group or a urea group.

121. The method according to any one of claims 98 - 120, wherein the soft segment is hydrophobic or hydrophilic.

122. The method according to any one of claims 98 - 121, wherein the soft segment is both hydrophobic and hydrophilic.

123. The method according to any one of claims 98 - 122, wherein the soft segment comprises a hydrophobic polyol and a hydrophilic polyol.

124. The method according to any one of claims 98 - 123, wherein the soft segment is one or more segments comprising polydimethylsiloxane, polycarbonate, polyester, polyether, and blends or copolymers thereof.

125. The method according to any one of claims 98 - 124, wherein the soft segment is one or more segments comprising polyethylene glycol, oligomeric polyether, polyoxazoline (POX), polypeptide, polyvinylpyrrolidone, polyvinylpyridine, zwitterionic repeating group polymer, and blends or copolymers thereof.

126. The method according to claim 98, wherein reducing biofouling comprises providing an aptamer protection layer as defined in any one of claims 98 - 124.

127. The method according to any one of claims 98 - 126, wherein reducing the decoupling of the at least one aptamer from the surface of the conductive substrate comprises coupling the at least one aptamer conjugate to the conductive surface.

128. The method according to any one of claims 98 - 127, wherein reducing the oxidation of the aptamer comprises introducing one or more non - diffusible antioxidants into the aptamer protection layer.

129. The method according to any one of claims 98 - 128, wherein controlling the diffusion of the at least one aptamer comprises at least partially cross - linking the aptamer protective layer.

130. The method according to any one of claims 98 - 129, wherein the end - of - life is extended by up to one day, 2 days, one week, 2 weeks, 3 weeks or one month.

131. An aptamer protective layer configured for continuous in - vivo transdermal on - line monitoring, the aptamer protective layer comprising a polymer selected from: A functionalized polymer comprising at least one zwitterionic repeat group; A functionalized polymer of at least one polymerizable zwitterionic monomer structure selected from: wherein X is O, NH or NR4, Y and Z are independently acyclic alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloheteroalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, and which can optionally be substituted by OH, halogen or alkoxy; R1, R3, R4 and R5 are independently H, alkyl, heteroalkyl, cycloalkyl, cycloheteroalkyl, aryl or heteroaryl; A functionalized polymer comprising an alkylthiol, phenylthiol or benzylthiol group; A functionalized polymer comprising an alkylthiol group, phenylthiol group or benzylthiol group combined with a zwitterionic repeat group; A functionalized polymer comprising a mercaptoalkanol group, arylmercaptoalkanol group, benzylmercaptoalkanol group or a mixture thereof; A functionalized polymer comprising a mercaptoalkanol group, arylmercaptoalkanol group, benzylmercaptoalkanol group or a mixture thereof combined with a zwitterionic repeat group; or A segmented multiblock polymer.

132. The aptamer protective layer according to claim 131, wherein the aptamer protective layer is at least partially cross - linked.

133. The aptamer protective layer according to any one of claims 131 - 132, wherein the aptamer protective layer provides an ionic strength, and a certain amount of a zwitterionic repeat group compound, the amount of which present can regulate or maintain the ionic strength or pH near the aptamer conjugate.

134. The aptamer protective layer according to any one of claims 131 - 133, wherein the aptamer protective layer provides a free volume, the free volume allowing reversible conformational changes of the one or more aptamer conjugates present to be sufficient to provide a detectable signal in the presence of an analyte.

135. The aptamer protective layer according to any one of claims 131 - 134, wherein the alkylthiol group is present at the end of the functionalized polymer chain.

136. The aptamer protective layer according to any one of claims 131 - 135, wherein the alkylthiol group is present along the backbone of the functionalized polymer chain.

137. The aptamer protective layer according to any one of claims 131 - 136, wherein the mercaptoalkanol group is present at the end of the functionalized polymer chain.

138. The aptamer protective layer according to any one of claims 131-137, wherein the mercaptoalkanol group is present along the main chain of the functionalized polymer chain.

139. The aptamer protective layer according to any one of claims 131-138, wherein the zwitterionic repeating group is present at the end of the functionalized polymer chain.

140. The aptamer protective layer according to any one of claims 131-139, wherein the zwitterionic repeating group is present along the main chain of the functionalized polymer chain.

141. The aptamer protective layer according to any one of claims 131-140, wherein the one or more zwitterionic repeating groups include a betaine compound or a derivative thereof.

142. The aptamer protective layer according to any one of claims 131-141, wherein the aptamer protective layer is configured to be physically or chemically coupled to at least a portion of the substrate surface.

143. The aptamer protective layer according to any one of claims 131-142, wherein the segmented multiblock polymer comprises at least one of polyurethane, polyurea, poly(urethane urea), epoxide, polyolefin, polysiloxane, polyamide, polystyrene, polyacrylate, polyether, polyol, polyvinylpyridine, polyvinylpyrrolidone, polyester, polycarbonate, and copolymers thereof.

144. The aptamer protective layer according to any one of claims 131-143, wherein the aptamer protective layer comprises a segmented multiblock polyurethane polymer.

145. The aptamer protective layer according to any one of claims 131-144, wherein the aptamer protective layer comprises a segmented multiblock polyurethane urea polymer.

146. The aptamer protective layer according to any one of claims 131-145, wherein the segmented multiblock polymer comprises a soft segment and a hard segment.

147. The aptamer protective layer according to any one of claims 131-146, wherein the soft segment is hydrophobic or hydrophilic.

148. The aptamer protective layer according to any one of claims 131-147, wherein the soft segment is hydrophobic and hydrophilic.

149. The aptamer protective layer according to any one of claims 131-148, wherein the soft segment comprises a hydrophobic polyol and a hydrophilic polyol.

150. The aptamer protective layer according to any one of claims 131-149, wherein the soft segment is one or more segments comprising polydimethylsiloxane, polycarbonate, polyester, polyether, and blends or copolymers thereof.

151. The aptamer protective layer according to any one of claims 131-150, wherein the soft segment is one or more segments comprising polyethylene glycol, oligo polyether, polyoxazoline (POX), polypeptide, polyvinylpyrrolidone, polyvinylpyridine, zwitterionic repeating group polymer, and blends or copolymers thereof.

152. The aptamer protective layer according to any one of claims 131-151, wherein the average molecular weight of the aptamer protective layer is from about 10 kDa to about 500 kDa.

153. A method for determining the in vivo concentration of an analyte, the method comprising: contacting, in vivo, a biological fluid containing the analyte with an electrochemical aptamer biosensor coupled to a conductive substrate, the aptamer probe being encapsulated in an aptamer protective layer that is permeable to the analyte, the electrochemical aptamer biosensor generating a signal upon interaction with the analyte; and interrogating the conductive substrate or the electrochemical aptamer; and detecting the signal corresponding to the in vivo concentration of the analyte.

154. The method according to claim 153, wherein the interrogation is a continuous, semi-continuous, sequential or random detection of the signal.

155. The method according to claim 153, the method further comprising adjusting the signal based on a background signal resulting from non-specific binding of the aptamer biosensor to produce a adjusted signal.

156. The method according to any one of claims 153-155, the method further comprising determining the in vivo concentration of the analyte over a period of time based on the adjusted signal.

157. The method according to any one of claims 153-156, wherein interrogating the conductive substrate comprises a differential measurement technique.

158. The method according to claim 157, wherein the differential measurement technique comprises: interrogating the conductive substrate with a first square wave voltammetry (SWV) frequency to obtain a first signal, and interrogating the conductive substrate with 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.

159. The method according to any one of claims 153-156, wherein the interrogation comprises chronoamperometry.

160. The method according to any one of claims 153-156, wherein the interrogation comprises cyclic voltammetry.

161. The method according to any one of claims 153-160, wherein the conductive substrate is an electrode, a microporous or a nanoporous conductive material.

162. A method of manufacturing an electrochemical aptamer biosensor (EAB), the method comprising: presenting at least one aptamer to at least a portion of the surface of a conductive substrate, the at least one aptamer conjugate comprising a reversible redox moiety; and presenting an aptamer protective layer to the portion of the surface of the conductive substrate; and encapsulating at least a portion of the at least one aptamer conjugate in the aptamer protective layer.

163. The method according to claim 162, the method further comprising including one or more co-adsorbents into the aptamer protective layer.

164. The method according to any one of claims 162-163, wherein the one or more co-adsorbents comprise zwitterionic betaine groups.

165. The method according to claim 164, wherein the one or more zwitterionic betaine groups are selected from the following structures: Wherein W, Y, and Z are independently branched or straight-chain alkyl, heteroalkyl, cycloalkyl, cycloheteroalkyl, aryl, or heteroaryl, any of which can optionally be substituted by O, OH, halogen, amide, or alkoxy; R1 is H, alkyl, heteroalkyl, cycloalkyl, cycloheteroalkyl, aryl, or heteroaryl; and R2, R3, and R4 are independently selected from alkyl, heteroalkyl, cycloalkyl, cycloheteroalkyl, aryl, or heteroaryl; wherein one or more of R1, R2, R3, W, X, Y, and Z are coupled to the co-adsorbent.

166. The method according to any one of claims 162 - 165, wherein the co-adsorbate is a terminally-capped dithiol having at least one betaine group along the chain of the co-adsorbate.

167. The method according to claim 166, wherein the thiol groups of the terminally-capped dithiol alkanethiol are covalently coupled to the substrate surface.

168. The method according to any one of claims 162 - 167, wherein the zwitterionic betaine group comprises a mercaptoalkanol betaine.

169. The method according to claim 168, wherein the mercaptoalkanol is straight-chain and comprises a plurality of betaine groups along the chain of the mercaptoalkanol.

170. The method according to any one of claims 162 - 169, wherein the thiol groups of the mercaptoalkanol are covalently coupled to the substrate surface.

171. The method according to any one of claims 162 - 170, wherein the aptamer protective layer comprises an alkanethiol and one or more zwitterionic betaine groups.

172. The method according to any one of claims 162 - 171, wherein the aptamer protective layer comprises a segmented multi-block polymer backbone.

173. The method according to any one of claims 162 - 172, wherein the segmented multi-block polymer backbone comprises a polyurethane polymer.

174. The method according to any one of claims 162 - 173, wherein the segmented multi-block polymer backbone comprises a polyurethane-urea polymer.

175. The method according to any one of claims 162 - 174, wherein the segmented multi-block polymer comprises soft segments and hard segments.

176. The method according to any one of claims 162 - 175, wherein the soft segments are hydrophobic or hydrophilic.

177. The method according to any one of claims 162 - 176, wherein the soft segments are hydrophobic and hydrophilic.

178. The method according to any one of claims 162 - 177, wherein the soft segments comprise hydrophobic polyols and hydrophilic polyols.

179. The method according to any one of claims 162 - 178, wherein the soft segments are one or more segments comprising polydimethylsiloxane, polycarbonate, polyester, polyether, and blends or copolymers thereof.

180. The method according to any one of claims 162 - 179, wherein the soft segment is one or more segments comprising polyethylene glycol, oligo polyether, polyoxazoline (POX), polypeptide, polyvinylpyrrolidone, polyvinylpyridine, zwitterionic repeating group polymer, and blends or copolymers thereof.

181. The method according to any one of claims 162 - 180, wherein the segmented multi - block polymer comprises a soft segment and a hard segment.

182. The method according to any one of claims 162 - 181, wherein the hard segment comprises a urethane group or a urea group.

183. The method according to any one of claims 162 - 182, wherein a certain amount of cross - linker is used to cross - link the aptamer protective layer.

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