Continuous analyte sensor devices and methods

Abstract

A continuous (multi)analyte sensor device is provided comprising a (multi)analyte sensor operably coupled to a signal transducer, the (multi)analyte sensor comprising at least one membrane, the at least one membrane comprising at least one first transducer element and at least one of a mediator and / or a regenerative cofactor.

Description

[Technical Field]

[0001] FIELD OF THE DISCLOSURE The present disclosure relates to continuous analyte sensor devices and methods, and continuous multi-analyte sensor devices and methods. [Background technology]

[0002] In vivo analyte sensors can be configured to analyze a single analyte, typically using an enzyme to provide specificity for that single analyte. Determining the concentrations of multiple physiologically relevant analytes may be desirable in certain medical cases. Continuous quantification of circulating analytes remains a major challenge in clinical medicine. Summary of the Invention

[0003] In a first aspect, there is provided a continuous (multi)analyte sensor device comprising an indwelling (multi)analyte sensor operably coupled to a signal transducer, the (multi)analyte sensor comprising at least one membrane system adjacent to the signal transducer, the at least one membrane system independently comprising a first layer including at least one first transducer element, and a second layer adjacent to the first layer, the second layer being the same as or different from the first layer.

[0004] In one embodiment, the signal transducer comprises at least one electrode. In another embodiment, alone or in combination with any one of the preceding embodiments, the at least one electrode comprises a first working electrode surface and a second working electrode surface spatially separated vertically, horizontally, or circumferentially from the first working electrode surface.

[0005] In another embodiment, alone or in combination with any one of the preceding embodiments, the first layer is adjacent to the first working electrode surface. In another embodiment, alone or in combination with any one of the preceding embodiments, the second layer is adjacent to the second working electrode surface. In another embodiment, alone or in combination with any one of the preceding embodiments, at least a portion of the first layer is adjacent to at least a portion of the first working electrode surface, and at least a portion of the second layer is separated vertically, horizontally, or circumferentially more distally from at least a portion of the first working electrode surface than the portion of the first layer.

[0006] In another embodiment, alone or in combination with any one of the preceding embodiments, the second layer includes at least one second transduction element, the second transduction element being different from the first transduction element.

[0007] In another embodiment, alone or in combination with any one of the preceding embodiments, the device further comprises at least one of a mediator and a regenerative cofactor present in the first layer, the second layer, or both the first and second layers, wherein the at least one mediator is operably associated with the at least one first conversion element or the at least one second conversion element.

[0008] In another embodiment, alone or in combination with any one of the preceding embodiments, the first layer comprises at least one mediator and at least one first transducer element, and the second layer comprises at least one regenerative cofactor.

[0009] In another embodiment, alone or in combination with any one of the preceding embodiments, the second layer comprises at least one second transducer element, and at least a portion of the first layer is proximal to the first working electrode surface and at least a portion of the second layer is distal to the first working electrode surface.

[0010] In another embodiment, alone or in combination with any one of the preceding embodiments, the first layer comprises at least one regenerative cofactor and at least one first transducer element, and the second layer comprises at least one mediator.

[0011] In another embodiment, alone or in combination with any one of the preceding embodiments, the second layer comprises at least one second transducer element, and at least a portion of the first layer is proximal to the first working electrode surface and at least a portion of the second layer is distal to the first working electrode surface.

[0012] In another embodiment, alone or in combination with any one of the preceding embodiments, the first layer comprises at least one mediator, at least one regeneration cofactor, and at least one first conversion element; the second layer comprises at least one second conversion element; and at least a portion of the first layer is proximal to the first working electrode surface and at least a portion of the second layer is distal to the first working electrode surface.

[0013] In another embodiment, alone or in combination with any one of the preceding embodiments, the first layer comprises at least one first conversion element, the second layer comprises at least one regenerative cofactor, at least one mediator, and at least one second conversion element, and at least a portion of the first layer is proximal to the first working electrode surface and at least a portion of the second layer is distal to the first working electrode surface.

[0014] In another embodiment, alone or in combination with any one of the preceding embodiments, the first layer comprises at least one mediator and at least one first transducer element, and the second layer comprises at least one regenerative cofactor.

[0015] In another embodiment, alone or in combination with any one of the preceding embodiments, the first layer comprises at least one regenerative cofactor and at least one first transducer element, and the second layer comprises at least one mediator.

[0016] In another embodiment, alone or in combination with any one of the preceding embodiments, the first layer comprises at least one mediator, at least one regeneration cofactor, and at least one first conversion element; the second layer comprises at least one second conversion element; at least a portion of the first layer is proximate to the first working electrode surface; and at least a portion of the second layer is proximate to the second working electrode surface.

[0017] In another embodiment, alone or in combination with any one of the preceding embodiments, the first layer comprises at least one first conversion element, the second layer comprises at least one regenerative cofactor, at least one mediator, and at least one second conversion element, and at least a portion of the first layer is proximate to the first working electrode surface, and at least a portion of the second layer is proximate to the second working electrode surface.

[0018] In another embodiment, alone or in combination with any one of the preceding embodiments, the at least one regenerating cofactor is one or more of NAD, NADH, NAD(P)H, NAD(P)+, mutant NAD+(nox) (or NADH oxidase or NADHox), ATP, flavin adenine dinucleotide (FAD), magnesium (Mg++), pyrroloquinoline quinone (PQQ), pyrroloquinoline quinone (PQQ), and functionalized derivatives thereof.

[0019] In another embodiment, alone or in combination with any one of the preceding embodiments, the mediator or at least one regeneration cofactor is covalently, electrostatically, ionically associated with, or physically captured or absorbed by, one or more of the first layer, the second layer, or the third layer. In another embodiment, alone or in combination with any one of the preceding embodiments, the mediator or at least one regeneration cofactor is covalently, electrostatically, ionically associated with, or physically captured or absorbed by, one or more working electrode surfaces.

[0020] In another embodiment, alone or in combination with any one of the preceding embodiments, the second layer includes at least one second transducer element, and at least a portion of the first layer is proximate to the first working electrode surface and at least a portion of the second layer is proximate to the second working electrode surface.

[0021] In another embodiment, alone or in combination with any one of the preceding embodiments, the second layer includes at least one second transducer element, and at least a portion of the first layer is proximate to the first working electrode surface and at least a portion of the second layer is proximate to the second working electrode surface.

[0022] In another embodiment, alone or in combination with any one of the preceding embodiments, the first layer or the second layer comprises a polymer. In another embodiment, alone or in combination with any one of the preceding embodiments, the polymer is a polyolefin, polystyrene, polyoxymethylene, polysiloxane, polyether, polyacrylate, polymethacrylate, polyester, polycarbonate, polyamide, polypyridine, poly(pyridine-styrene) copolymer, poly(etherketone), poly(etherimide), polyurethane, polyurethaneurea, polycarbonate-polyurethane copolymer, or blends thereof.

[0023] In another embodiment, alone or in combination with any one of the preceding embodiments, the device further comprises at least one third conversion element, wherein the at least one third conversion element is present in at least one of the first layer, the second layer, and the third layer.

[0024] In another embodiment, alone or in combination with any one of the preceding embodiments, the first layer has a first diffusion resistance to one or more analytes or one or more substrate reaction products, and the second layer has a second diffusion resistance to one or more analytes or one or more enzyme-substrate reaction products, the second diffusion resistance being substantially the same as or different from the first diffusion resistance to one or more analytes or one or more enzyme-substrate reaction products.

[0025] In another embodiment, alone or in combination with any one of the preceding embodiments, the one or more analytes include glucose, lactose, glycerol, beta-hydroxybutyrate, creatinine, creatine, alcohol, urea, uric acid, cholesterol, bilirubin, glutathione, urea, sodium, potassium, or glutamic acid.

[0026] In another embodiment, alone or in combination with any one of the preceding embodiments, the at least one first conversion element and the at least one second conversion element are independently selected from a dehydrogenase enzyme, a reductase enzyme, a kinase enzyme, a peroxidase enzyme, an esterase enzyme, an (amide)hydrolase enzyme, and an oxidase enzyme.

[0027] In another embodiment, alone or in combination with any one of the preceding embodiments, the at least one first conversion element and the at least one second conversion element each have a substrate and generate a reaction product with the substrate to provide a clinical value correlating with the health status of the mammal. In another embodiment, alone or in combination with any one of the preceding embodiments, the substrate or reaction product is selected from hydrogen peroxide, creatine, acetoacetate, dihydroxyacetone, oxygen, or an aldehyde.

[0028] In another embodiment, alone or in combination with any one of the preceding embodiments, the at least one first conversion element and the at least one second conversion element are beta-hydroxybutyrate dehydrogenase, alcohol dehydrogenase, lipase, amidohydrolase, glycerol kinase, creatinine kinase, creatine amidohydrolase, alcohol oxidase, cholesterol oxidase, galactose oxidase, choline oxidase, glutamate oxidase, glycerol-3-phosphate oxidase, bilirubin oxidase, ascorbate oxidase, urate oxidase, urease, pyruvate oxidase, xanthine oxidase, glucose oxidase, lactate oxidase, sarcosine oxidase, malate dehydrogenase, formaldehyde dehydrogenase, glutathione reductase, glutathione peroxidase, 3-hydroxysteroid dehydrogenase, or horseradish peroxidase.

[0029] In another embodiment, alone or in combination with any one of the preceding embodiments, the mediator is one or more of 2,2'-bipyridine, poly-1,10-phenanthroline-5,6-dione, polyvinylferrocene, hexacyanoferrate, phthalocyanine or organometallic compounds thereof, organometallic compounds of osmium or ruthenium, and functionalized derivatives thereof, and complexes of one or more transition metals with one or more polymers or ligands, and salts thereof.

[0030] In another embodiment, alone or in combination with any one of the preceding embodiments, the device further comprises a transmitter configured to wirelessly transmit data to a paired display device or therapeutic agent delivery device such that multiple analyte parameters are assessed in parallel.

[0031] In a second aspect, a continuous analyte sensor device is provided comprising an indwelling mediated analyte sensor operably coupled to a signal transducer, the mediated analyte sensor comprising at least one membrane system adjacent to the signal transducer, the at least one membrane system independently comprising a first layer comprising at least one first transducer element, a second layer adjacent to the first layer, the second layer being the same as or different from the first layer, at least one mediator, and a mediator system interference domain comprising at least one oxidase enzyme, at least one peroxidase enzyme, catalase, or a combination thereof.

[0032] In one embodiment, the indwelling mediated analyte sensor is a (multi-)analyte sensor. In another embodiment, alone or in combination with any one of the preceding embodiments, the indwelling mediated analyte sensor is a glucose and ketone analyte sensor. In another embodiment, alone or in combination with any one of the preceding embodiments, the indwelling mediated analyte sensor is a glucose and creatinine analyte sensor. In another embodiment, alone or in combination with any one of the preceding embodiments, the indwelling mediated analyte sensor is a glucose and alcohol analyte sensor.

[0033] In another embodiment, alone or in combination with any one of the preceding embodiments, the signal converter comprises at least one electrode. In another embodiment, alone or in combination with any one of the preceding embodiments, the at least one electrode comprises a first working electrode surface and a second working electrode surface spatially separated vertically, horizontally, or circumferentially from the first working electrode surface.

[0034] In another embodiment, alone or in combination with any one of the preceding embodiments, the first layer is adjacent to the first working electrode surface. In another embodiment, alone or in combination with any one of the preceding embodiments, the second layer is adjacent to the second working electrode surface.

[0035] In another example, alone or in combination with any one of the preceding examples, at least a portion of the first layer is adjacent to at least a portion of the first working electrode surface, and at least a portion of the second layer is separated vertically, horizontally, or circumferentially more distally from at least a portion of the first working electrode surface than the portion of the first layer.

[0036] In another embodiment, alone or in combination with any one of the preceding embodiments, the second layer comprises at least one second transducer element, and at least a portion of the first layer is proximal to the first working electrode surface and at least a portion of the second layer is distal to the first working electrode surface.

[0037] In another embodiment, alone or in combination with any one of the preceding embodiments, the second layer comprises at least one second transducer element, and at least a portion of the first layer is proximal to the first working electrode surface and at least a portion of the second layer is distal to the first working electrode surface.

[0038] In another embodiment, alone or in combination with any one of the preceding embodiments, the first layer includes at least one first transducer element and the second layer includes at least one second transducer element, the second transducer element being different from the first transducer element.

[0039] In another embodiment, alone or in combination with any one of the preceding embodiments, each of the at least one first conversion element and the at least one second conversion element has a substrate and generates a reaction product with the substrate to provide a clinical value correlating with the health status of the mammal.

[0040] In another embodiment, alone or in combination with any one of the preceding embodiments, the device further comprises at least one regenerative cofactor present in the first layer, the second layer, or both the first and second layers.

[0041] In another embodiment, alone or in combination with any one of the preceding embodiments, the first layer comprises at least one mediator and at least one first transducer element, and the second layer comprises at least one regenerative cofactor.

[0042] In another embodiment, alone or in combination with any one of the preceding embodiments, the first layer comprises at least one regenerative cofactor and at least one first transducer element, and the second layer comprises at least one mediator.

[0043] In another embodiment, alone or in combination with any one of the preceding embodiments, the first layer comprises at least one mediator, at least one regeneration cofactor, and at least one first conversion element; the second layer comprises at least one second conversion element; and at least a portion of the first layer is proximal to the first working electrode surface and at least a portion of the second layer is distal to the first working electrode surface.

[0044] In another embodiment, alone or in combination with any one of the preceding embodiments, the first layer comprises at least one mediator, at least one regeneration cofactor, and at least one first conversion element; the second layer comprises at least one second conversion element; at least a portion of the first layer is proximate to the first working electrode surface; and at least a portion of the second layer is proximate to the second working electrode surface.

[0045] In another embodiment, alone or in combination with any one of the preceding embodiments, the first layer comprises at least one first conversion element, the second layer comprises at least one regenerative cofactor, at least one mediator, and at least one second conversion element, and at least a portion of the first layer is proximal to the first working electrode surface and at least a portion of the second layer is distal to the first working electrode surface.

[0046] In another embodiment, alone or in combination with any one of the preceding embodiments, the first layer comprises at least one first conversion element, the second layer comprises at least one regenerative cofactor, at least one mediator, and at least one second conversion element, and at least a portion of the first layer is proximate to the first working electrode surface, and at least a portion of the second layer is proximate to the second working electrode surface.

[0047] In another embodiment, alone or in combination with any one of the preceding embodiments, the second layer comprises at least one second transducer element, and at least a portion of the first layer is proximal to the first working electrode surface and at least a portion of the second layer is distal to the first working electrode surface.

[0048] In another embodiment, alone or in combination with any one of the preceding embodiments, the second layer includes at least one second transducer element, and at least a portion of the first layer is proximate to the first working electrode surface and at least a portion of the second layer is proximate to the second working electrode surface.

[0049] In another embodiment, alone or in combination with any one of the preceding embodiments, the second layer includes at least one second transducer element, and at least a portion of the first layer is proximate to the first working electrode surface and at least a portion of the second layer is proximate to the second working electrode surface.

[0050] In another embodiment, alone or in combination with any one of the preceding embodiments, the device further comprises a third layer adjacent to the second layer. In another embodiment, alone or in combination with any one of the preceding embodiments, the device further comprises at least one third transduction element, the at least one third transduction element being in at least one of the first layer, the second layer, and the third layer.

[0051] In another embodiment, alone or in combination with any one of the preceding embodiments, the first layer has a first diffusion resistance to one or more analytes or one or more substrate reaction products, and the second layer has a second diffusion resistance to one or more analytes or one or more enzyme substrate reaction products, and the second diffusion resistance is, in another embodiment, alone or in combination with any one of the preceding embodiments, substantially the same as or different from the first diffusion resistance to one or more analytes or one or more enzyme substrate reaction products.

[0052] In another embodiment, alone or in combination with any one of the preceding embodiments, the at least one first conversion element and the at least one second conversion element are independently a dehydrogenase enzyme, a reductase enzyme, a kinase enzyme, a peroxidase enzyme, an esterase enzyme, an (amide)hydrolase enzyme, an oxidase enzyme, or a combination thereof.

[0053] In another embodiment, alone or in combination with any one of the preceding embodiments, the at least one first conversion element and the at least one second conversion element are independently beta-hydroxybutyrate dehydrogenase, alcohol dehydrogenase, lipase, amidohydrolase, glycerol kinase, creatinine kinase, creatine amidohydrolase, alcohol oxidase, cholesterol oxidase, galactose oxidase, choline oxidase, glutamate oxidase, glycerol-3-phosphate oxidase, bilirubin oxidase, urease, pyruvate oxidase, xanthine oxidase, glucose oxidase, lactate oxidase, sarcosine oxidase, malate dehydrogenase, formaldehyde dehydrogenase, glutathione reductase, glutathione peroxidase, 3-hydroxysteroid dehydrogenase, or a combination thereof.

[0054] In another embodiment, alone or in combination with any one of the preceding embodiments, the mediator is one or more of 2,2'-bipyridine, poly-1,10-phenanthroline-5,6-dione, polyvinylferrocene, hexacyanoferrate, phthalocyanine or organometallic compounds thereof, organometallic compounds of osmium or ruthenium, and functionalized derivatives thereof, and complexes of one or more transition metals with one or more polymers or ligands, and salts thereof.

[0055] In another embodiment, alone or in combination with any one of the preceding embodiments, the at least one oxidase enzyme is ascorbic acid oxidase or uric acid oxidase.In another embodiment, alone or in combination with any one of the preceding embodiments, the at least one peroxidase enzyme is horseradish peroxidase.

[0056] In another embodiment, alone or in combination with any one of the preceding embodiments, the mediator system interference domain comprises at least one polymer. In another embodiment, alone or in combination with any one of the preceding embodiments, the at least one polymer is a polyolefin, polystyrene, polyoxymethylene, polysiloxane, polyether, polyacrylate, polymethacrylate, polyester, polycarbonate, polyamide, polypyridine, poly(pyridine-styrene) copolymer, poly(ether ketone), poly(ether imide), polyurethane, polyurethane urea, polycarbonate-polyurethane copolymer, polyethylene vinyl acetate, or a blend thereof.

[0057] In another embodiment, alone or in combination with any one of the preceding embodiments, the device further comprises at least one of an electrode domain, an enzyme domain, a resistance domain, and an interference membrane.

[0058] In another embodiment, alone or in combination with any one of the preceding embodiments, the mediator system interference domain is present in the electrode domain. In another embodiment, alone or in combination with any one of the preceding embodiments, the mediator system interference domain is immediately adjacent to the electrode domain. In another embodiment, alone or in combination with any one of the preceding embodiments, the mediator system interference domain is present in the enzyme domain. In another embodiment, alone or in combination with any one of the preceding embodiments, the mediator system interference domain is immediately adjacent to the enzyme domain. In another embodiment, alone or in combination with any one of the preceding embodiments, the mediator system interference domain is present in the resistance domain. In another embodiment, alone or in combination with any one of the preceding embodiments, the mediator system interference domain is immediately adjacent to the resistance domain. In another embodiment, alone or in combination with any one of the preceding embodiments, the mediator system interference domain is present in the interference membrane. In another embodiment, alone or in combination with any one of the preceding embodiments, the mediator system interference domain is immediately adjacent to the interference membrane.

[0059] In another embodiment, alone or in combination with any one of the preceding embodiments, the device further comprises a transmitter configured to wirelessly transmit data to a paired display device or therapeutic agent delivery device such that at least two analyte parameters are assessed in parallel.

[0060] In one example, a continuous analyte sensor device is provided, comprising an analyte sensor operably coupled to a signal transducer, the analyte sensor comprising at least one membrane system adjacent to the signal transducer, the at least one membrane system independently comprising a first domain including at least one first transducer element, a second domain adjacent to the first domain, the second domain being the same or different from the first domain, and at least one regeneration cofactor.

[0061] In one aspect, the analyte sensor is a multi-analyte sensor. In one aspect, alone or in combination with any of the preceding aspects, the multi-analyte analyte sensor is a glucose and ketone analyte sensor, or a glucose and creatinine analyte sensor, or a ketone and potassium ion analyte sensor.

[0062] In one aspect, alone or in combination with any of the preceding aspects, the signal transducer comprises at least one electrode.

[0063] In one aspect, alone or in combination with any of the preceding aspects, the first domain includes at least one first transduction element and the second domain includes at least one second transduction element, the second transduction element being different from the first transduction element.

[0064] In one aspect, alone or in combination with any of the preceding aspects, the at least one regenerating cofactor is one or more of NAD, NADH, NAD(P)H, NAD(P)+, ATP, flavin adenine dinucleotide (FAD), magnesium (Mg++), pyrroloquinoline quinone (PQQ), pyrroloquinoline quinone (PQQ), and functionalized derivatives thereof.

[0065] In one embodiment, alone or in combination with any of the preceding embodiments, the device further includes at least one mediator present in the first domain, the second domain, or both the first and second domains. In one embodiment, alone or in combination with any of the preceding embodiments, the mediator is one or more of 2,2'-bipyridine, poly-1,10-phenanthroline-5,6-dione, polyvinylferrocene, hexacyanoferrate, phthalocyanine or organometallic compounds thereof, organometallic compounds of osmium or ruthenium, and functionalized derivatives thereof, and complexes of one or more transition metals with one or more polymers or ligands, and salts thereof.

[0066] In one aspect, alone or in combination with any of the preceding aspects, the first domain comprises at least one mediator and at least one first transducer, and the second domain comprises at least one regenerating cofactor, or the first domain comprises at least one regenerating cofactor and at least one first transducer, and the second domain comprises at least one mediator.

[0067] In one aspect, alone or in combination with any of the preceding aspects, the at least one first conversion element and the at least one second conversion element are independently a dehydrogenase enzyme, a reductase enzyme, a kinase enzyme, a peroxidase enzyme, an esterase enzyme, an (amide)hydrolase enzyme, an oxidase enzyme, or a combination thereof.

[0068] In one aspect, alone or in combination with any of the preceding aspects, the at least one first conversion element and the at least one second conversion element are independently beta-hydroxybutyrate dehydrogenase, alcohol dehydrogenase, lipase, amidohydrolase, glycerol kinase, creatinine kinase, creatine amidohydrolase, alcohol oxidase, cholesterol oxidase, galactose oxidase, choline oxidase, glutamate oxidase, glycerol-3-phosphate oxidase, bilirubin oxidase, urease, pyruvate oxidase, xanthine oxidase, glucose oxidase, lactate oxidase, sarcosine oxidase, malate dehydrogenase, formaldehyde dehydrogenase, glutathione reductase, glutathione peroxidase, 3-hydroxysteroid dehydrogenase, NADH oxidase, or a combination thereof.

[0069] In one aspect, alone or in combination with any of the preceding aspects, the at least one first conversion element is beta-hydroxybutyrate dehydrogenase and the at least one second conversion element is NADH oxidase.

[0070] In one aspect, alone or in combination with any of the preceding aspects, at least one electrode comprises platinum or palladium, an interference domain is deposited on the at least one electrode, a first domain adjacent to the interference domain, the first domain comprising beta-hydroxybutyrate dehydrogenase, NADH oxidase, and a cofactor, and a second domain adjacent to the first domain, the second domain comprising a polyvinylpyridine polymer or copolymer. In one aspect, alone or in combination with any of the preceding aspects, the continuous analyte sensor device is configured to provide a continuous analyte signal without a transition metal-containing mediator.

[0071] In one embodiment, alone or in combination with any of the preceding embodiments, the at least one regenerative cofactor is NAD.

[0072] In one aspect, alone or in combination with any of the preceding aspects, the interference domain is configured to block the diffusion of at least one of acetaminophen, ascorbic acid, bilirubin, cholesterol, creatinine, dopamine, ephedrine, ibuprofen, L-dopa, methyldopa, salicylic acid, tetracycline, tolazamide, tolbutamide, triglycerides, and uric acid from the electrode.

[0073] In one aspect, alone or in combination with any of the preceding aspects, the interference domain comprises alternating layers of polyurethane, polyurethane-zwitterionic polymer, polymer with pendant ionic groups, NAFION™, chitosan, cellulose, polyallylamine, and polyacrylate acid, or combinations or blends thereof.

[0074] In one embodiment, alone or in combination with any of the preceding embodiments, the first domain or the second domain comprises an amphiphilic polymer or copolymer.

[0075] In one embodiment, alone or in combination with any of the preceding embodiments, the first domain or the second domain comprises a heterocyclic polymer or copolymer, or an at least partially quaternized heterocyclic polymer or copolymer.

[0076] In one aspect, alone or in combination with any of the preceding aspects, the device further comprises a transmitter configured to wirelessly transmit data to a paired display device or therapeutic agent delivery device. [Brief explanation of the drawings]

[0077] In order to understand and see how the present disclosure may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which: [Figure 1A] 1 shows an exemplary enzyme domain configuration for a continuous multi-analyte sensor as disclosed and described herein. [Figure 1B] 1 shows an exemplary enzyme domain configuration for a continuous multi-analyte sensor as disclosed and described herein. [Figure 1C] 1 shows an exemplary enzyme domain configuration for a continuous multi-analyte sensor as disclosed and described herein. [Figure 1D] 1 shows an exemplary enzyme domain configuration for a continuous multi-analyte sensor as disclosed and described herein. [Figure 1E] 1 shows an exemplary enzyme domain configuration for a continuous multi-analyte sensor as disclosed and described herein. [Figure 1F] 1 shows exemplary experimental data for the enzyme domain configuration of the continuous multi-analyte sensor disclosed and described herein. [Figure 1G] A linear regression of the experimental data in Figure 1E is shown. [Figure 1H] 1 shows exemplary experimental data for the cofactor / enzyme resistance domain configuration of the continuous analyte sensor disclosed and described herein. [Figure 1I] 1 shows exemplary experimental data for the cofactor / enzyme resistance domain configuration of the continuous analyte sensor disclosed and described herein. [Figure 1J] 1 shows exemplary experimental data for the cofactor / enzyme resistance domain configuration of the continuous analyte sensor disclosed and described herein. [Figure 2] 1 shows exemplary experimental data for the enzyme domain configuration of the continuous multi-analyte sensor disclosed and described herein. [Figure 3A] 1 shows exemplary experimental data for the enzyme domain configuration of the continuous multi-analyte sensor disclosed and described herein. [Figure 3B] 3B shows exemplary experimental data for the enzyme domain configuration of the continuous multi-analyte sensor of FIG. 3A. [Figure 3C] 1 shows a series of exemplary experimental data for enzyme domain configurations of continuous multi-analyte sensors using different electrode compositions as disclosed and described herein. [Figure 4] 1 shows exemplary experimental data for the enzyme domain configuration of the continuous multi-analyte sensor disclosed and described herein. [Figure 5] 1 shows a set of exemplary experimental data for the enzyme domain configuration of the continuous multi-analyte sensor disclosed and described herein. [Figure 6A] 1 shows an alternative view of an exemplary dual electrode enzyme domain configuration for a continuous multi-analyte sensor as disclosed and described herein. [Figure 6B] 1 shows an alternative view of an exemplary dual electrode enzyme domain configuration for a continuous multi-analyte sensor as disclosed and described herein. [Figure 6C] 1 shows an alternative view of an exemplary dual electrode enzyme domain configuration for a continuous multi-analyte sensor as disclosed and described herein. [Figure 6D] 1 shows an alternative view of an exemplary dual electrode enzyme domain configuration for a continuous multi-analyte sensor as disclosed and described herein. [Figure 6E] 1 illustrates an exemplary dual electrode configuration for a continuous multi-analyte sensor as disclosed and described herein. [Figure 6F] 6A-B show exemplary experimental data for the dual electrode configuration of the continuous multi-analyte sensor. [Figure 7A]1 shows an exemplary enzyme domain configuration for a continuous multi-analyte sensor as disclosed and described herein. [Figure 7B] 7A and 7B show exemplary experimental data for the enzyme domain configuration of FIG. 7A. [Figure 7C] 1 shows an alternative exemplary enzyme domain configuration for the continuous multi-analyte sensor disclosed and described herein. [Figure 7D] 1 shows an alternative exemplary enzyme domain configuration for the continuous multi-analyte sensor disclosed and described herein. [Figure 7E] 7A and 7B show exemplary experimental data for the enzyme domain configuration of FIG. 7D. [Figure 8A] 1 shows an exemplary enzyme domain configuration for a continuous multi-analyte sensor as disclosed and described herein. [Figure 8B] 8B shows exemplary experimental data for the enzyme domain configuration of FIG. 8A. [Figure 8C] 8B shows exemplary experimental data for the enzyme domain configuration of FIG. 8A. [Figure 9A] 1A-1C show alternative views of exemplary dual electrode enzyme domain configurations G1-G4 for the continuous multi-analyte sensors disclosed and described herein. [Figure 9B] 1A-1C show alternative views of exemplary dual electrode enzyme domain configurations G1-G4 for the continuous multi-analyte sensors disclosed and described herein. [Figure 9C] 1A-1C show alternative views of exemplary dual electrode enzyme domain configurations G1-G4 for the continuous multi-analyte sensors disclosed and described herein. [Figure 9D] 1A-1C show alternative views of exemplary dual electrode enzyme domain configurations G1-G4 for the continuous multi-analyte sensors disclosed and described herein. [Figure 9E] 9D shows a linear regression of experimental data for the continuous multi-analyte sensor shown in FIG. 9C. [Figure 10A] FIG. 1 is a cross-sectional / side schematic view showing the in vivo portion of an analyte sensor disclosed herein. [Figure 10B] FIG. 1 is a perspective schematic diagram illustrating the in vivo portion of a continuous multi-analyte sensor as disclosed and described herein. [Figure 10C] FIG. 1 is a side schematic view showing the in vivo portion of a continuous multi-analyte sensor as disclosed and described herein. [Figure 10D] FIG. 1 is a cross-sectional / side schematic view illustrating the in vivo portion of a continuous multi-analyte sensor as disclosed and described herein. [Figure 10E] 1 is a cross-sectional schematic diagram illustrating the in vivo portion of a continuous multi-analyte sensor as disclosed and described herein. [Figure 10F] FIG. 1 is a side schematic view showing the in vivo portion of a continuous multi-analyte sensor as disclosed and described herein. [Figure 10G] FIG. 1 is a side schematic view showing the in vivo portion of a continuous multi-analyte sensor as disclosed and described herein. [Figure 11] FIG. 11 is a cross-sectional schematic diagram taken along line 11-11 of FIG. 10A showing an exemplary continuous multi-analyte sensor membrane configuration as disclosed and described herein. [Figure 12A] FIG. 1 is a perspective schematic diagram illustrating the in vivo portion of a continuous analyte sensor as disclosed and described herein. [Figure 12B] FIG. 1 is a perspective schematic diagram illustrating the in vivo portion of a continuous analyte sensor as disclosed and described herein. [Figure 13A] FIG. 1 is a perspective schematic diagram showing the in vivo portion of a continuous multi-electrode multi-analyte sensor. [Figure 13B] FIG. 13B is an enlarged perspective view of the distal portion, section 13B, of the example sensor shown in FIG. 13A. [Figure 14] FIG. 1 shows a basic schematic diagram of the operating principle of the amperometric enzymatic multi-analyte sensor disclosed and described herein. [Figure 15] FIG. 1 illustrates one embodiment of an exemplary continuous multi-analyte sensor system in communication with at least one display device according to various technologies disclosed and described herein. [Figure 16] 1 shows exemplary experimental calibration data for the mediated continuous analyte sensor disclosed and described herein. [Figure 17] 17 shows exemplary experimental drift data for the continuous analyte sensor of FIG. 16 as disclosed and described herein. [Figure 18]17 shows exemplary experimental in vivo data for the continuous analyte sensor of FIG. 16 as disclosed and described herein. [Figure 19] 1 shows exemplary experimental calibration data for the non-mediated continuous analyte sensor disclosed and described herein. [Figure 20] 20 shows exemplary experimental drift data for the continuous analyte sensor of FIG. 19 as disclosed and described herein. [Figure 21A] 1 illustrates an exemplary continuous ketone sensor configuration pathway as disclosed and described herein. [Figure 21B] 1 illustrates an exemplary continuous ketone sensor configuration pathway as disclosed and described herein. [Figure 21C] 1 illustrates an exemplary continuous ketone sensor configuration pathway as disclosed and described herein. DETAILED DESCRIPTION OF THE INVENTION

[0078] Multi-analyte sensors designed for in vivo applications are provided. Multi-analyte sensing may be used to aid in the diagnosis and / or monitoring of various health conditions, including chronic conditions. In some embodiments, continuous multi-analyte sensors are configured to measure two or more analytes to enable early intervention for adverse health conditions, including metabolic disorders, as well as to treat health conditions. Multi-analyte sensors have advantages in certain instances in that a single analyte may not provide sufficient information to make decisions regarding overall health. By enabling sensing of two or more analytes, a more accurate measurement of the body's biological status is achieved. For example, glucose levels often indicate a specific set of conditions that can be further refined using other analytes. Examples include diabetic ketoacidosis (DKA), metabolic function, insulin sensitivity and clearance, liver function, etc. Diabetic ketoacidosis (DKA) is the leading cause of death among individuals with type 1 diabetes under the age of 20. Body-worn continuous glucose monitors have been commercially available for the past two decades for the assessment of interstitial glucose levels. However, in some cases, this single-analyte measurement alone may not be clinically sufficient to identify cases of euglycemic DKA, which has become of increasing concern with the recent off-label use of SGLT-2 inhibitors among patients receiving intensive insulin therapy, for example.

[0079] The systems and methods discussed herein use multi-analyte sensing to provide patients and healthcare providers with feedback regarding the patient's health.

[0080] In one embodiment, a single wire electrode implementation is provided that supports amperometric measurements of multiple analytes.

[0081] In another embodiment, a dual coaxial wire electrode implementation supporting amperometric measurements of two or more analytes according to the generalized n-dimensional Cottrell relation:

[0082]

number

[0083] In another example, the multi-analyte sensor device comprises a transmitter configured to interface with the multi-analyte sensor, and in one aspect, wireless transmission of data to a paired display device is provided so that multiple analytes can be assessed in parallel.

[0084] The following description and drawings illustrate in detail the embodiments of the present disclosure. Those skilled in the art will recognize that there are many variations and modifications of the present invention that are encompassed by the scope of the present disclosure. Therefore, the description of the embodiments herein should not be considered as limiting the scope of the present disclosure. To facilitate understanding of the embodiments disclosed herein, several terms are defined below.

[0085] As used herein, the term "about" is a broad term and is to be given its ordinary and accustomed meaning to one of ordinary skill in the art (and is not limited to any special or customized meaning), and refers to allowing for a degree of variability in a value or range, for example, within 10%, within 5%, or within 1% of the stated limits of the stated value or range, including, but not limited to, the exactly stated value or range.

[0086] As used herein, the terms "stick" and "adhere" are broad terms and are to be given their ordinary and customary meaning to those skilled in the art (and are not limited to any special or customized meaning), and refer to, but are not limited to, holding, joining, or fastening, for example, by adhering, bonding, grasping, interpenetrating, or fusing.

[0087] As used herein, the term "analyte" is a broad term and is to be given its ordinary and customary meaning to one of ordinary skill in the art (and is not limited to any special or customized meaning), and refers to, but is not limited to, a substance or chemical constituent in a biological fluid (e.g., blood, interstitial fluid, cerebrospinal fluid, lymphatic fluid, urine, sweat, saliva, etc.) that can be analyzed. Analytes can include naturally occurring substances, man-made substances, metabolites, and / or reaction products. In some examples, the analyte measured by the sensing region, devices, and methods is glucose. However, other analytes are contemplated as well, including acarboxyprothrombin; acylcarnitines; adenine phosphoribosyltransferase; adenosine deaminase; albumin; α-fetoprotein; amino acid profile (arginine (Krebs cycle), histidine / urocanic acid, homocysteine, phenylalanine / tyrosine, tryptophan); andrenostenedione; antipyrine; arabinitol enantiomers; arginase; benzoylecgonine (cocaine); bilirubin; biotinidase; biopterin; c-reactive protein; carnitine; carnosinase; CD4; ceruloplasmin; chenodeoxycholic acid; chloroquine; cholesterol; cholinesterase; conjugated 1-β-hydroxycholic acid; cortisol; creatine; creatine kinase; creatinine Enzyme MM isoenzymes; creatinine; cyclosporine A; d-penicillamine; deethylchloroquine; dehydroepiandrosterone sulfate; DNA (acetylation polymorphisms, alcohol dehydrogenase, alpha-1-antitrypsin, cystic fibrosis, Duchenne / Becker muscular dystrophy, glucose-6-phosphate dehydrogenase, hemoglobin A, hemoglobin S, hemoglobin C, hemoglobin D, hemoglobin E, hemoglobin F, D-Punjab, beta-thalassemia, hepatitis B virus, HCMV, HIV-1, HTLV-1, Leber's hereditary optic neuropathy, MCAD, RNA, PKU, Plasmodium vivax, 21-deoxycortisol); desbutylhalofantrine; dihydropteridine reductase; diphtheria / tetanus antitoxin; erythrocyte arginase; erythrocyte protoporphyrin; esterase D;Fatty acids / acylglycines; free beta-human chorionic gonadotropin; free erythrocyte porphyrins; free thyroxine (FT4); free triiodothyronine (FT3); fumarylacetoacetase; galactose / gal-1-phosphate; galactose-1-phosphate uridyltransferase; gentamicin; glucose-6-phosphate dehydrogenase; glutathione; glutathione peroxidase; glycerol; glycocholate; glycosylated hemoglobin; halofantrine; hemoglobin variants; hexosaminidase A; human erythrocyte carbonic anhydrase I; 17-alpha-hydroxyprogesterone; Hypoxanthine phosphoribosyltransferase; immunoreactive trypsin; beta-hydroxybutyrate; ketones; lactate; lead; lipoproteins ((a), B / A-1, β); lysozyme; mefloquine; netilmicin; oxygen; phenobarbitone; phenytoin; phytanic acid / pristanic acid; potassium, sodium, and / or other blood electrolytes; progesterone; prolactin; prolidase; purine nucleoside phosphorylase; quinine; reverse triiodothyronine tri-iodothyronine, rT3); selenium; serum pancreatic lipase; sisomicin; somatomedin C; specific antibodies (adenovirus, antinuclear antibody, anti-zeta antibody, arbovirus, pseudorabies virus, dengue virus, guinea worm, Echinococcus granulosus, Entamoeba histolytica, enterovirus, giardiasis, Helicobacter pylori, hepatitis B virus, herpes virus, HIV-1, IgE (atopic disease), influenza virus, Leishmania donovani, Leptospirosis, measles / mumps / Rubella, Mycobacterium leprae, Mycoplasma pneumoniae, Myoglobin, Onchocerca volvulus, Parainfluenza virus, Plasmodium, Poliovirus, Pseudomonas aeruginosa, Respiratory syncytial virus, Rickettsia (tsutsugamushi disease), Schistosoma mansoni, Toxoplasma gondii, Treponema pallidum, Trypanosoma cruzi / rangeli, Vesicular stomatitis virus, Wuchereria bancrofti, Yellow fever virus); Specific antigens (Hepatitis B virus, HIV-1); Succinylacetone; Sulfadoxine; Theophylline; Thyrotropin (TSH); Thyroxine (T4);Analytes may be naturally occurring in biological fluids or may be endogenous, e.g., metabolites, hormones, antigens, antibodies, etc. ... Alternatively, the analyte can be introduced into the body or can be exogenous, such as a contrast agent for imaging, a radioisotope, a chemical agent, a fluorocarbon-based synthetic blood, or a drug or pharmaceutical composition, including, but not limited to, insulin; ethanol; cannabis (marijuana, tetrahydrocannabinol, hashish); inhalants (nitrous oxide, amyl nitrite, butyl nitrite, chlorohydrocarbons, hydrocarbons); cocaine (crack cocaine); stimulants (amphetamine, methamphetamine, RITALIN®, CYLERT®, PRELUDIN®, DIDREX®, PRESTATE®, VORANIL®, SANDREX®, PLEGINE®); antidepressants ( barbiturates, methaqualone, valium®, librium®, miltown®, serax®, equinanil®, tranxene®, and other tranquilizers; hallucinogens (phencyclidine, lysergic acid, mescaline, peyote, psilocybin); narcotics (heroin, codeine, morphine, opium, meperidine, percocet®, percodan®, tussionex®, fentanyl, darvon®, talwin®, lomotil®); synthetic narcotics (fentanyl, meperidine, amphetamine, methamphetamine, and phencyclidine analogues, e.g., ecstasy); anabolic steroids;Analytes of interest include, but are not limited to, nicotine and nicotine. Metabolites of drugs and pharmaceutical compositions are also contemplated. Analytes such as neurochemicals and other chemicals produced in the body, such as ascorbic acid, uric acid, dopamine, noradrenaline, 3-methoxytyramine (3MT), 3,4-dihydroxyphenylacetic acid (DOPAC®), homovanillic acid (HVA), 5-hydroxytryptamine (5HT), 5-hydroxyindoleacetic acid (FHIAA), and histamine, can also be analyzed.

[0088] As used herein, the phrases “analyte measuring device,” “analyte monitoring device,” “analyte sensing device,” and / or “multi-analyte sensor device” are broad terms and are to be given their ordinary and customary meaning to one of ordinary skill in the art (and are not limited to any special or customized meaning), and refer to, but are not limited to, apparatus and / or systems responsible for detecting a specific analyte or combination of analytes or transducing a signal associated therewith. For example, but not limited to, these terms may refer to an instrument responsible for detecting a specific analyte or combination of analytes. In one example, the instrument includes a sensor coupled to a circuit arranged within a housing and configured to process a signal associated with the analyte concentration into information. In one example, such a device and / or system is capable of providing specific quantitative, semi-quantitative, qualitative, and / or semi-qualitative analytical information using a biological recognition element combined with a transduction (detection) element.

[0089] As used herein, the term "amphiphilic" is a broad term and is to be given its ordinary and customary meaning to those of ordinary skill in the art (and is not to be limited to any special or customized meaning), and refers to, but is not limited to, chemical compounds or polymers that have both hydrophilic and hydrophobic segments or properties.

[0090] As used herein, the terms "biosensor" and / or "sensor" are broad terms and are to be given their ordinary and customary meaning to those skilled in the art (and are not limited to any special or customized meaning), and refer to, but are not limited to, a portion of an analyte measuring device, analyte monitoring device, analyte sensing device, and / or multi-analyte sensor device responsible for detecting a specific analyte or combination of analytes or transducing a signal associated therewith. In one embodiment, a biosensor or sensor generally comprises a body and working, reference, and / or counter electrodes coupled to the body and forming a surface configured to provide a signal during an electrochemical reaction. One or more membranes may be affixed to the body and cover the electrochemically reactive surface. In one embodiment, such biosensors and / or sensors are capable of providing a specific quantitative, semi-quantitative, qualitative, or semi-qualitative analytical signal using a biological recognition element combined with a transduction (detection) element.

[0091] As used herein, the phrases "sensing moiety," "sensing membrane," and "sensing mechanism" are broad terms and are to be given their ordinary and customary meaning to one of ordinary skill in the art (and are not limited to any special or customized meaning), and refer to, but are not limited to, the portion of a biosensor and / or sensor responsible for detecting a particular analyte or combination of analytes or transducing a signal associated therewith. In one example, the sensing moiety, sensing membrane, and / or sensing mechanism generally comprises electrodes configured to provide a signal during an electrochemical reaction with one or more membranes covering an electrochemically reactive surface. In one example, such sensing moieties, sensing membranes, and / or sensing mechanisms can provide specific quantitative, semi-quantitative, qualitative, or semi-qualitative analytical information using a biological recognition element combined with a transduction (detection) element.

[0092] As used herein, the term "substantially" refers to a majority or 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%.

[0093] As used herein, the phrase "substantially free" can mean having no or an insignificant amount of the material, such that the amount of material present does not affect the material properties of the composition including the material, such as about 0% to about 5% by weight of the composition being the material, or about 0% to about 1%, or about 5% by weight or less, or about 4.5% by weight or less, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.01, or about 0.001% by weight or less, or about 0% by weight.

[0094] As used herein, the terms "stick" and "adhere" are broad terms and are to be given their ordinary and customary meaning to those skilled in the art (and are not limited to any special or customized meaning), and refer to, but are not limited to, holding, joining, or fastening, for example, by adhering, bonding, grasping, interpenetrating, or fusing.

[0095] As used herein, the phrase "barrier cell layer" is a broad phrase that is to be given its ordinary and customary meaning to those skilled in the art (and is not to be limited to any special or customized meaning), and refers, without limitation, to the portion of the foreign body response that forms a cohesive monolayer of cells (e.g., macrophages and foreign body giant cells) that substantially blocks the transport of molecules and other substances into the implantable device.

[0096] As used herein, the term "bioactive agent" is a broad term and is to be given its ordinary and customary meaning to those of skill in the art (and is not to be limited to any special or customized meaning), and refers to, but is not limited to, any substance that has an effect on or elicits a response from living tissue.

[0097] As used interchangeably herein, the phrases "biointerface membrane" and "biointerface layer" are broad terms that are to be given their ordinary and customary meaning to those skilled in the art (and are not limited to any special or customized meaning), and refer to, but are not limited to, a permeable membrane (which may include multiple domains) or layer that acts as a bioprotective interface between the host tissue and the implantable device. The terms "biointerface" and "bioprotective" are used interchangeably herein.

[0098] As used herein, the term "biostable" is a broad term that is to be given its ordinary and customary meaning to those of skill in the art (and is not to be limited to any special or customized meaning), and refers to, but is not limited to, materials that are relatively resistant to degradation by processes encountered in vivo.

[0099] As used herein, the phrase "cell protrusion" is a broad phrase and is to be given its ordinary and customary meaning to those skilled in the art (and is not to be limited to any special or customized meaning), and refers to, but is not limited to, the pseudopodia of cells.

[0100] As used herein, the phrase "cell attachment" is a broad phrase and is to be given its ordinary and customary meaning to those skilled in the art (and is not limited to any special or customized meaning), and refers to, but is not limited to, the adhesion of cells and / or cell processes to a material at a molecular level and / or the attachment of cells and / or cell processes to a microporous or macroporous material surface. One example of a material used in the prior art that promotes cell attachment to a porous surface is the BIOPORE™ cell culture support, as marketed by Millipore (Bedford, Mass.) and described in U.S. Patent No. 5,741,330 to Brauker et al.

[0101] As used herein, the term "cofactor" is a broad term that is given its ordinary and customary meaning to those of skill in the art (and is not limited to any special or customized meaning) and refers, without limitation, to one or more substances whose presence contributes to or is required for the analyte-related activity of an enzyme. The analyte-related activity can include, but is not limited to, any one or combination of binding, electron transfer, and chemical transformation. Cofactors include coenzymes, non-protein chemical compounds, metal ions, and / or metal-organic complexes. Coenzymes include prosthetic groups and cosubstrates.

[0102] As used herein, the term "continuous" is a broad term and is to be given its ordinary and customary meaning to those of ordinary skill in the art (and is not to be limited to any special or customized meaning), and refers to, but is not limited to, an uninterrupted or unbroken portion, domain, coating, or layer.

[0103] As used herein, the phrases "continuous analyte sensing" and "continuous multi-analyte sensing" are broad phrases that are to be given their ordinary and customary meaning to one of ordinary skill in the art (and are not limited to any special or customized meaning) and refer to, but are not limited to, continuous, intermittent, and / or intermittent (but periodic) monitoring of analyte concentrations, for example, for periods of time that range from about 1 second or less to about 1 week or more. In further embodiments, monitoring of the analyte concentration is performed between about every 2, 3, 5, 7, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 seconds to about every 1.25, 1.50, 1.75, 2.00, 2.25, 2.50, 2.75, 3.00, 3.25, 3.50, 3.75, 4.00, 4.25, 4.50, 4.75, 5.00, 5.25, 5.50, 5.75, 6.00, 6.25, 6.50, 6.75, 7.00, 7.25, 7.50, 7.75, 8.00, 8.25, 8.50, 8.75, 9.00, 9.25, 9.50, or 9.75 minutes. In further embodiments, the analyte concentration monitoring is performed between about every 10, 20, 30, 40, or 50 minutes and about every 1, 2, 3, 4, 5, 6, 7, or 8 hours. In further embodiments, the analyte concentration monitoring is performed between about every 8 hours and about every 12, 16, 20, or 24 hours. In further embodiments, the analyte concentration monitoring is performed between about every day and about every 1.5, 2, 3, 4, 5, 6, or 7 days. In further embodiments, the analyte concentration monitoring is performed between about every week and about every 1.5, 2, 3, or more weeks.

[0104] As used herein, the term "coaxial" should be interpreted broadly to include sensor architectures having elements aligned along a shared axis around a core that may be configured to have a circular, elliptical, triangular, polygonal, or other cross-section, and such elements may include electrodes, insulating layers, or other elements that may be positioned circumferentially around a core layer, such as a core electrode or core polymer wire.

[0105] As used herein, the term "coupled" is a broad term and is to be given its ordinary and customary meaning to those skilled in the art (and is not limited to any special or customized meaning), and refers to, but is not limited to, two or more system elements or components that are configured to be electrically, mechanically, thermally, operatively, chemically, or otherwise attached to at least one other element. For example, an element is "coupled" if it is covalently, communicatively, electrostatically, thermally connected, mechanically connected, magnetically connected, or ionically associated with, or physically captured, adsorbed, or absorbed by, another element. Similarly, as used herein, the phrases "operably connected," "operably linked," and "operably coupled" can refer to one or more components coupled to another component in a manner that facilitates transmission of at least one signal between the components. In some examples, components are part of the same structure and / or integrated with one another, such as when they are covalently, electrostatically, mechanically, thermally, magnetically, ionically associated, or physically trapped or absorbed (i.e., "directly coupled," as in the absence of intervening elements). In other examples, components are connected via remote means. For example, one or more electrodes can be used to detect analytes in a sample and convert that information into a signal, which can then be transmitted to an electronic circuit. In this example, the electrodes are "operably coupled" to the electronic circuit. As used herein, the phrase "removably coupled" can refer to two or more system elements or components that are configured or configured to be attached and detached electrically, mechanically, thermally, operably, chemically, or otherwise, without damaging any of the coupled elements or components.As used herein, the phrase "permanently coupled" may refer to two or more system elements or components that are configured to be or are attached electrically, mechanically, thermally, operatively, chemically, or otherwise, but cannot be separated without damaging at least one of the coupled elements or components, being covalently, electrostatically, ionically associated, or being physically trapped or absorbed.

[0106] As used herein, the phrase "defined edge" is a broad phrase and is to be given its ordinary and customary meaning to one of ordinary skill in the art (and is not to be limited to any special or customized meaning), and refers to, but is not limited to, an abrupt, distinct edge or boundary between layers, domains, coatings, or portions. A "defined edge" is in contrast to a gradual transition between layers, domains, coatings, or portions.

[0107] As used herein, the term "discontinuous" is a broad term and is to be given its ordinary and customary meaning to one of ordinary skill in the art (and is not to be limited to any special or customized meaning), and refers to, but is not limited to, cut, interrupted, or separated portions, layers, coatings, or domains.

[0108] As used herein, the term "distal" is a broad term and is to be given its ordinary and customary meaning to those skilled in the art (and is not to be limited to any special or customized meaning), and refers to, but is not limited to, an area that is relatively far away from a reference point such as an origin or attachment point.

[0109] As used herein, the term "domain" is a broad term and is to be given its ordinary and customary meaning to one of ordinary skill in the art (and is not limited to any special or customized meaning), and refers to, but is not limited to, a region of a membrane system that may be a layer, a uniform or non-uniform gradient (e.g., an anisotropic region of a membrane), or a portion of a membrane that is capable of sensing one, two, or more analytes. The domains discussed herein can be formed as a single layer, as two or more layers, as a pair of bilayers, or as combinations thereof.

[0110] As used herein, the term "drift" is a broad term and is to be given its ordinary and customary meaning to those skilled in the art (and is not limited to any special or customized meaning), referring to, but not limited to, a gradual increase or decrease in signal over time that is unrelated to changes in host systemic analyte concentration (e.g., the host's postprandial glucose concentration). Without being bound by theory, it is believed that drift may be the result of a local decrease in glucose transport to the sensor, for example, due to the formation of a foreign body capsule (FBC) or due to an insufficient amount of interstitial fluid surrounding the sensor, resulting in reduced oxygen and / or glucose transport to the sensor. In one example, an increase in local interstitial fluid may slow or reduce drift, thus improving sensor performance. Drift may also be the result of sensor electronics or algorithmic models used to compensate for noise or other anomalies that may occur with electrical signals, for example, in the picoampere range, femtoampere range, nanoampere range, microampere range, milliampere range, ampere range, etc.

[0111] As used interchangeably herein, the phrases "drug-release membrane" and "drug-release layer" are each broad phrases that are to be given their ordinary and customary meaning to one of ordinary skill in the art (and are not limited to any 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 one example, the "drug-release membrane" and "drug-release layer" are typically several microns or more thick and can be composed of two or more domains. In one example, the drug-release layer and / or drug-release membrane are substantially the same as the biointerface layer and / or biointerface membrane. In another example, the drug-release layer and / or drug-release membrane are separate from the biointerface layer and / or biointerface membrane.

[0112] As used herein, the term "electrochemically reactive surface" is a broad term and is to be given its ordinary and customary meaning to one of ordinary skill in the art (and is not to be limited to any special or customized meaning), and refers to, but is not limited to, the surface of an electrode on which an electrochemical reaction occurs. In one example, the reaction is faradaic, resulting in a charge transfer between the surface and its environment. In one example, hydrogen peroxide produced by an enzyme-catalyzed reaction of an analyte being oxidized on the surface results in a measurable electronic current. For example, in the detection of glucose, glucose oxidase produces hydrogen peroxide (HO) as a by-product. HO reacts with the surface of the working electrode to release two protons (2H + ), two electrons (2e - ), and one oxygen molecule (O), which produces an electronic current that is detected. At the counter electrode, a reducible species, e.g., O, is reduced at the electrode surface to balance the current produced by the working electrode.

[0113] As used herein, the term "electrolysis" is a broad term and is to be given its ordinary and customary meaning to one of ordinary skill in the art (and is not to be limited to any special or customized meaning), and refers, without limitation, to the electro-oxidation or electro-reduction (collectively "redox") of a compound, either directly or indirectly, by one or more enzymes, cofactors, or mediators.

[0114] As used herein, the phrase "hard segment" is a broad phrase and is to be given its ordinary and customary meaning to one of ordinary skill in the art (and is not limited to any 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 resistance properties, such as resistance to bending or twisting. The phrase "hard segment" may be further characterized as a crystalline, semi-crystalline, or glassy material having a glass transition temperature, typically determined by dynamic scanning calorimetry ("Tg"), above ambient temperature. Exemplary hard segment elements used to prepare polycarbonate polyurethane or polyurethane urea hard segments include norbornane diisocyanate (NBDI), isophorone diisocyanate (IPDI), tolylene diisocyanate (TDI), 1,3-phenylene diisocyanate (MPDI), trans-1,3-bis(isocyanatomethyl)cyclohexane (1,3-H6XDI), bicyclohexylmethane-4,4'-diisocyanate, and the like. cyanate (HMDI), 4,4'-diphenylmethane diisocyanate (MDI), trans-1,4-bis(isocyanatomethyl)cyclohexane (1,4-H6XDI), 1,4-cyclohexyl diisocyanate (CHDI), 1,4-phenylene diisocyanate (PPDI), 3,3'-dimethyl-4,4'-biphenyl diisocyanate (TODI), 1,6-hexamethylene diisocyanate (HDI), or combinations thereof.

[0115] As used herein, the term "host" is a broad term and is given its ordinary and customary meaning to those of skill in the art (and is not limited to any special or customized meaning), and refers to a mammal, such as, but not limited to, a human.

[0116] As used herein, the terms "indwelling," "indwelling," "implanted," or "implantable" are broad terms that are to be given their ordinary and customary meaning to those of ordinary skill in the art (and are not to be limited to any special or customized meaning) and refer to an object (e.g., a sensor) that is inserted or configured to be inserted subcutaneously (i.e., within the fatty layer between the skin and muscle), intradermally (i.e., penetrating the stratum corneum and located within the epidermal or dermal layer of the skin), or transcutaneously (i.e., penetrating, entering, or passing through intact skin), which may result in a sensor having an in vivo portion and an ex vivo portion. The term "indwelling" also encompasses an object configured to be inserted subcutaneously, intradermally, or percutaneously, whether or not it is itself inserted.

[0117] As used herein, the phrase "insertable surface area" is a broad phrase that is to be given its ordinary and customary meaning to one of ordinary skill in the art (and is not limited to any special or customized meaning) and refers to the geometric surface area utilized in the analyte sensors described herein, e.g., the surface area of ​​the insertable portion of the analyte sensor, including, but not limited to, the surface area of ​​a planar, flat, substantially planar, and / or coaxial substrate.

[0118] As used herein, the phrase "insertable volume" is a broad phrase and is to be given its ordinary and customary meaning to one of ordinary skill in the art (and is not limited to any special or customized meaning), and refers to, but is not limited to, the volume anterior and lateral to the insertion path of the insertable portion of the analyte sensor, as described herein, and the incision made in the skin for inserting the insertable portion of the analyte sensor. The insertable volume also includes up to 5 mm radially or perpendicularly to the volume anterior and lateral to the insertion path.

[0119] As used herein, the terms "interferent" and "interfering species" are broad terms and are to be given their ordinary and customary meaning to one of ordinary skill in the art (and are not limited to any special or customized meaning), and refer to, but are not limited to, effects and / or species that interfere with the measurement of an analyte of interest in a sensor, producing a signal that does not accurately represent the analyte measurement. In one example of an electrochemical sensor, an interfering species is a compound that produces a signal that is not analyte-specific due to a reaction on an electrochemically active surface.

[0120] As used herein, the term "in vivo" is a broad term and is to be given its ordinary and customary meaning to one of ordinary skill in the art (and is not to be limited to any special or customized meaning), encompassing, but not limited to, portions of a device (e.g., a sensor) adapted for insertion into and / or presence within the body of a host.

[0121] As used herein, the term "ex vivo" is a broad term and is to be given its ordinary and customary meaning to one of ordinary skill in the art (and is not to be limited to any special or customized meaning), encompassing, but not limited to, a portion of a device (e.g., a sensor) that is adapted to reside and / or exist outside the host organism.

[0122] As used herein, the terms “mechanical storage medium,” “device storage medium,” and “computer storage medium” (collectively referred to as “mechanical storage medium”) mean the same thing and may be used interchangeably in this disclosure. The term refers to single or multiple storage devices and / or media (e.g., centralized or distributed databases and / or associated caches and servers) that store executable instructions and / or data, as well as cloud-based storage systems or storage networks that include multiple storage devices or devices. Accordingly, the term is intended to include, but is not limited to, solid-state memory, optical media, and magnetic media (including memory that is internal or external to a processor). Specific examples of mechanical storage media, computer storage media, and / or device storage media include non-volatile memory, examples of which include semiconductor memory devices, e.g., erasable programmable read-only memory (EPROM), electrically erasable programmable devices; magnetic disks, such as internal hard disks and removable disks; magneto-optical disks; and CD-ROM and DVD-ROM disks. The terms mechanical storage media, computer storage media, and device storage media specifically exclude carrier waves, modulated data signals, and other such media, at least some of which are covered by the term "signal media" discussed below.

[0123] As used herein, the terms "mediator" and "redox mediator" are broad terms and phrases that are to be given their ordinary and customary meaning to one of ordinary skill in the art (and are not limited to any special or customized meaning) and refer to, but are not limited to, any chemical compound or collection of compounds capable of direct or indirect electron transfer between an analyte, analyte precursor, analyte surrogate, analyte reductase or analyte oxidase, or cofactor and an electrode surface held at an electric potential. In one example, the mediator accepts electrons from or transfers electrons to one or more enzymes or cofactors and / or exchanges electrons with a sensor system electrode. In one example, the mediator is a transition metal-coordinated organic molecule capable of reversible oxidation and reduction reactions. In other examples, the mediator can be an organic molecule or a metal capable of reversible oxidation and reduction reactions.

[0124] As used herein, the term "membrane" is a broad term and is to be given its ordinary and customary meaning to those skilled in the art (and is not limited to any special or customized meaning), and refers to a structure configured to perform functions including, but not limited to, protecting exposed electrode surfaces from the biological environment, resisting (limiting) the diffusion of analytes, serving as a matrix for catalysts (e.g., one or more enzymes) to enable enzymatic reactions, limiting or screening interfering species, providing hydrophilicity at electrochemically reactive surfaces of a sensor interface, serving as an interface between host tissue and an implantable device, modulating host tissue response via drug (or other substance) release, and combinations thereof. As used herein, the terms "membrane" and "matrix" are meant to be interchangeable.

[0125] As used herein, the phrase "membrane system" is a broad phrase and is to be given its ordinary and customary meaning to one of ordinary skill in the art (and is not limited to any special or customized meaning), and refers, without limitation, to a permeable or semi-permeable membrane that may be composed of two or more domains, layers, or layers within domains, is typically composed of materials several microns or more in thickness, and is permeable to oxygen and, optionally, for example, glucose or another analyte. In one example, the membrane system includes an enzyme that allows an analyte reaction to occur, thereby allowing the concentration of the analyte to be measured.

[0126] The phrases "machine-readable medium," "computer-readable medium," and "device-readable medium" mean the same thing and can be used interchangeably in this disclosure. These phrases include both mechanical storage media and signal media operably coupled to a sensor, biosensor, analyte sensing device, or analyte monitoring device. Thus, these phrases include both storage devices / media and carrier / modulated data signals operably coupled to a sensor, biosensor, analyte sensing device, or analyte monitoring device.

[0127] As used herein, the term "micro" is a broad term and is to be given its ordinary and customary meaning to those skilled in the art (and is not to be limited to any special or customized meaning), and refers to a size of approximately 10 microns that is not visible without magnification. -6 "Micro" refers to objects or scales that are small, but not limited to, those on the order of 10 microns. The term "micro" is in contrast to the term "macro," which refers to objects that are large enough to be seen without magnification. Similarly, the term "nano" refers to objects that are approximately 10 -9 Refers to a small object or scale of m.

[0128] As used herein, the term "noise" is a broad term and is used in its ordinary sense to include, but is not limited to, signals detected by a sensor or sensor electronics that are unrelated to analyte concentration and may result in degraded sensor performance. Some types of noise are observed for several hours (e.g., about 2 to about 24 hours) after sensor insertion. After the first 24 hours, noise may disappear or diminish, but in some hosts, noise may persist for about 3 to 4 days. In some cases, noise can be reduced using predictive modeling, artificial intelligence, and / or algorithmic means. In other cases, noise can be reduced by addressing immune response factors associated with the presence of an implanted sensor, such as by using a drug-releasing layer with at least one bioactive agent. For example, the noise of one or more exemplary biosensors, such as those disclosed herein, can be determined and then compared qualitatively or quantitatively. For example, by acquiring raw signal time series at a fixed sampling interval (in pA), a smoothed version of the raw signal time series can be obtained, for example, by applying a third-order low-pass digital Chebyshev Type II filter. Other smoothing algorithms can also be used. At each sampling interval, the absolute difference in pA can be calculated to provide a smoothed time series. This smoothed time series can be converted to mg / dL (units of "noise") using the glucose sensitivity time series in pA / mg / dL, where the glucose sensitivity time series is derived by using a mathematical model between the raw signal and reference blood glucose measurements (e.g., obtained from a blood glucose meter). Optionally, the time series can be aggregated, for example, by hour or day, as desired. Comparison of corresponding time series between different exemplary biosensors having a drug-releasing layer and one or more bioactive agents of the present disclosure provides a qualitative or quantitative determination of noise improvement.

[0129] As used herein, the terms "optional" or "optionally" are broad terms and are to be given their ordinary and customary meaning to one of ordinary skill in the art (and are not limited to any special or customized meaning), meaning, without limitation, that the subsequently described event or circumstance may or may not occur, and that the description includes instances when the event or circumstance occurs and instances when it does not occur.

[0130] As used herein, the term "planar surface" should be broadly interpreted to describe a sensor architecture having a substrate including at least a first surface and an opposing second surface, and including, for example, a plurality of elements disposed on one or more surfaces or edges of the substrate. The plurality of elements may include conductive or insulating layers or elements configured to operate as a circuit. The plurality of elements may or may not be electrically or otherwise coupled. In one example, the planar surface includes one or more edges separating the opposing surfaces.

[0131] As used herein, the term "proximal" is a broad term and is to be given its ordinary and customary meaning to one of ordinary skill in the art (and is not limited to any special or customized meaning), and refers to, but is not limited to, the spatial relationship between various elements compared to a particular reference point. For example, some examples of devices include a membrane system having a biological interface layer and an enzyme domain or enzyme layer. If the sensor is considered to be the reference point, and the enzyme domain is located closer to the sensor than the biological interface layer, then the enzyme domain is more proximal to the sensor than the biological interface layer.

[0132] As used herein, the phrases and terms "processor module" and "microprocessor" are each broad phrases and terms that are to be given their ordinary and customary meaning to those skilled in the art (and are not limited to any special or customized meaning) and refer to, but are not limited to, a computer system, state machine, processor, or the like, designed to perform arithmetic or logical operations using logic circuitry that responds to and processes the basic instructions that drive a computer.

[0133] As used herein, the term "semi-continuous" is a broad term and is to be given its ordinary and customary meaning to one of ordinary skill in the art (and is not to be limited to any special or customized meaning), and refers to, but is not limited to, a portion, 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 but not over a sensing area is "semi-continuous."

[0134] As used herein, the phrases "sensing moiety," "sensing membrane," and "sensing mechanism" are broad terms and are to be given their ordinary and customary meaning to one of ordinary skill in the art (and are not limited to any special or customized meaning), and refer to, but are not limited to, the portion of a biosensor and / or sensor responsible for detecting a particular analyte or combination of analytes or transducing a signal associated therewith. In one example, a sensing moiety, sensing membrane, and / or sensing mechanism generally comprises electrodes configured to provide a signal during an electrochemical reaction with one or more membranes covering an electrochemically reactive surface. In one example, such a sensing moiety, sensing membrane, and / or sensing mechanism is capable of providing specific quantitative, semi-quantitative, qualitative, or semi-qualitative analytical information using a biological recognition element combined with a transduction (detection) element.

[0135] During typical operation of an analyte measuring device, biosensor, sensor, sensing region, sensing moiety, or sensing mechanism, a biological sample, e.g., blood or interstitial fluid, or components thereof, contacts, either directly or after passing through one or more membranes, an enzyme, e.g., glucose oxidase, DNA, RNA, or a protein or aptamer, e.g., one or more periplasmic binding proteins (PBPs) or variants or fusion proteins thereof, having one or more analyte-binding regions, each region capable of specifically or reversibly binding and / or reacting with at least one analyte. The interaction of the biological sample or its components with the analyte measuring device, biosensor, sensor, sensing region, sensing moiety, or sensing mechanism results in the transduction of a signal that allows for the qualitative, semi-qualitative, quantitative, or semi-qualitative determination of analyte levels, e.g., glucose, ketone, lactate, potassium, etc., in the biological sample.

[0136] In one example, the sensing region or sensing portion can comprise at least a portion of a conductive substrate or at least a portion of a conductive surface, e.g., a substantially planar substrate including wires (coaxial) or conductive traces, or substantially planar traces, and a membrane. In one example, the sensing region or sensing portion can comprise a non-conductive body, a working electrode, a reference electrode, and a counter electrode (optional) forming an electrochemically reactive surface at one location on the body and forming electronic connections at another location on the body, and a sensing membrane affixed to the body and covering the electrochemically reactive surface. In some examples, the sensing membrane further comprises an enzyme domain, e.g., an enzyme domain, and an electrolyte phase, e.g., a free-flowing liquid phase comprising an electrolyte-containing fluid, as further described below. These terms are broad enough to include the entire device, or just the sensing portion thereof (or anything in between).

[0137] In another example, the sensing region can comprise one or more periplasmic binding proteins (PBPs), including mutants or fusion proteins thereof, or an aptamer having one or more analyte-binding regions, each capable of specifically and reversibly binding at least one analyte. Alterations in the aptamer or mutations in the PBP can contribute to or alter one or more of the binding constants, long-term stability of the protein, including thermal stability, to bind the protein to a specific encapsulation matrix, membrane, or polymer, or to attach a detectable reporter group or "label" to indicate changes in the binding region, or to transduce a signal corresponding to one or more analytes present in the biological fluid. Specific examples of alterations 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 / nucleic acid side chains in the protein's binding region, and the redox state of the binding region. Such changes to the binding region provide for the transduction of a detectable signal corresponding to one or more analytes present in the biological fluid.

[0138] In one embodiment, the sensing region determines selectivity between one or more analytes such that only the analyte that must be measured results in (transduces) a detectable signal. This 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 changed, or where the sensing region causes or catalyzes a reaction of the analyte that changes the chemical composition of the analyte.

[0139] As used herein, the term "sensitivity" is a broad term and is to be given its ordinary and customary meaning to one of ordinary skill in the art (and is not limited to any special or customized meaning), and refers, without limitation, to the amount of signal (e.g., in the form of current and / or voltage) produced by a given amount (unit) of analyte measured. For example, in one embodiment, the sensor has a sensitivity (or slope) of about 1 to about 100 picoamps of current per 1 mg / dL of analyte.

[0140] The phrases "signal media" or "transmission media" should be interpreted to include all forms of modulated data signals, carrier waves, etc. The phrase "modulated data signal" means a signal that has one or more of its characteristics set or changed in such a manner as to encode information in the signal.

[0141] As used herein, the phrases and terms "small diameter sensor," "miniature structured sensor," and "microsensor" are broad phrases and terms that are to be given their ordinary and customary meaning to those skilled in the art (and are not limited to any special or customized meaning), and refer to, but are not limited to, a sensing mechanism having at least one dimension that is less than about 2 mm. In further embodiments, the sensing mechanism has at least one dimension that is less than about 1 mm. In some embodiments, the sensing mechanism (sensor) is less than about 0.95, 0.9, 0.85, 0.8, 0.75, 0.7, 0.65, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 mm. In some embodiments, the largest independently measured dimension of length, width, diameter, thickness, or circumference of the sensing mechanism does not exceed about 2 mm. In some embodiments, the sensing mechanism is a coaxial sensor having a diameter of less than about 1 mm (see, e.g., 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 by reference in their entireties). In some alternative embodiments, the sensing mechanism includes electrodes deposited on a planar or substantially planar substrate, and the thickness of the implantable portion is less than about 1 mm (see, e.g., 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 by reference in their entireties).

[0142] As used herein, the phrase "soft segment" is a broad phrase and is to be given its ordinary and customary meaning to one of ordinary skill in the art (and is not limited to any special or customized meaning), and refers to, but is not limited to, an element of a copolymer, such as, for example, a polyurethane, a polycarbonate polyurethane, or a polyurethane urea copolymer, that imparts flexibility to the chain. The phrase "soft segment" can be further characterized as an amorphous material having a low Tg, e.g., a Tg that is typically no higher than ambient temperature or normal mammalian body temperature.

[0143] As used herein, the phrase "solid portion" is a broad phrase that is to be given its ordinary and customary meaning to one of ordinary skill in the art (and is not to be limited to any special or customized meaning), and refers to, but is not limited to, the portion of the material of the membrane that has a mechanical structure that defines a cavity, void, or other non-solid portion.

[0144] As used herein, the terms "transduce" or "transduction" and their grammatical equivalents are broad terms and are given their ordinary and customary meaning to those skilled in the art (and are not limited to any special or customized meaning), and refer to, but are not limited to, optical, electrical, electrochemical, acoustic / mechanical, or colorimetric techniques and methods. Electrochemical properties include current and / or voltage, inductance, capacitance, impedance, and electric potential. Optical properties include absorbance, fluorescence / phosphorescence, fluorescence / phosphorescence decay rate, wavelength shift, dual-wave phase modulation, bio / chemiluminescence, reflectance, light scattering, and refractive index. For example, a sensing region converts the recognition of an analyte into a semi-quantitative or quantitative signal.

[0145] As used herein, the phrase "transducing element" is a broad term and is to be given its ordinary and customary meaning to one of ordinary skill in the art (and is not limited to any special or customized meaning), and refers to, but is not limited to, an analyte recognition moiety capable of directly or indirectly facilitating detectable signal transmission corresponding to the presence and / or concentration of a recognized analyte. In one example, the transducing element is one or more enzymes, one or more aptamers, one or more ionophores, one or more capture antibodies, one or more proteins, one or more biological cells, one or more oligonucleotides, and / or one or more DNA or RNA moieties. Transdermal continuous multi-analyte sensors can be used in vivo for various lengths of time. The continuous multi-analyte sensor systems discussed herein can be transdermal devices in that a portion of the device can be inserted through the host's skin into the underlying soft tissue, while a portion of the device remains on the surface of the host's skin. In one aspect, to overcome problems associated with short-term noise or other sensor function, an embodiment uses a material that promotes the formation of a fluid pocket around the sensor, e.g., a structure such as a porous biointerface membrane or matrix that creates a space between the sensor and the surrounding tissue. In some embodiments, the sensor includes a spacer adapted to provide a fluid pocket between the sensor and the host tissue. This spacer, e.g., a biointerface material, matrix, structure, etc., as described in more detail elsewhere herein, is believed to provide oxygen and / or glucose transport to the sensor.

[0146] Membrane Systems The membrane systems disclosed herein are suitable for use in implantable devices that contact biological fluids. For example, the membrane systems can be utilized with implantable devices such as devices for monitoring and determining analyte levels in biological fluids, e.g., devices for monitoring glucose levels in individuals with diabetes. In some embodiments, the analyte measuring device is a continuous device. The analyte measuring device can employ any suitable sensing element to provide a raw signal, including, but not limited to, those involving enzymatic, chemical, physical, electrochemical, spectrophotometric, amperometric, potentiometric, polarimetric, calorimetric, radiometric, immunochemical, etc. elements.

[0147] Suitable membrane systems for the aforementioned multi-analyte systems and devices can include, for example, the membrane systems disclosed in U.S. Pat. No. 6,015,572, U.S. Pat. No. 5,964,745, and U.S. Pat. No. 6,083,523, which are incorporated herein by reference in their entireties for their teachings of membrane systems.

[0148] Generally, membrane systems include multiple domains, such as an electrode domain, an interference domain, an enzyme domain, a resistance domain, and a biointerface domain. The membrane system can be deposited on the exposed electroactive surface using known thin-film techniques (e.g., vapor deposition, spraying, electrodeposition, dipping, brush coating, film coating, droplet coating, etc.). Additional steps, such as drying, annealing, and curing (e.g., UV curing, thermal curing, moisture curing, radiation curing, etc.), can be applied following deposition of the membrane material to enhance specific properties, such as mechanical properties, signal stability, and selectivity. In a typical process, upon deposition of the resistance domain membrane, a biointerface / drug-release layer is formed having a "dry film" thickness of about 0.05 microns (μm) or less to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 μm. The "dry film" thickness refers to the thickness of a cured film cast from a coating formulation using standard coating techniques.

[0149] In certain examples, the biointerface / drug-release layer is formed from a biointerface polymer, which comprises one or more membrane domains containing polyurethane and / or polyurea segments and one or more zwitterionic repeat units. In some examples, the biointerface / drug-release layer coating is formed from a polyurethaneurea having carboxyl betaine groups and nonionic hydrophilic polyethylene oxide segments incorporated into the polymer. The polyurethaneurea polymer is dissolved in an organic or non-organic solvent system according to a predetermined coating formulation, crosslinked with an isocyanate crosslinker, and cured at a moderate temperature of about 50°C. The solvent system can be a single solvent or a mixture of solvents to aid in dissolving or dispersing the polymer. The solvent can be the one selected as the polymerization medium or can be added after polymerization is complete. The solvent is selected from those with lower boiling points to facilitate drying and to be less toxic for implant applications. Examples of these solvents include aliphatic ketones, esters, ethers, alcohols, hydrocarbons, etc. Depending on the final thickness of the biointerface / drug-release layer and the solution viscosity (related to the percent polymer solids), the coating can be applied in a single step or multiple repeated steps of a selected process, such as dipping, to build up the desired thickness. In yet another example, the bioprotective polymer is formed from a polyurethaneurea having carboxylic acid and carboxyl betaine groups incorporated into the polymer and non-ionic hydrophilic polyethylene oxide segments, where the polyurethaneurea polymer is dissolved in an organic or non-organic solvent system in the coating formulation, crosslinked with a carbodiimide (e.g., 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) or a polycarbodiimide crosslinker), and cured at a moderate temperature of about 50°C. In one example, a polycarbodiimide crosslinker is used.

[0150] In another example, the biointerface / drug-release layer coating is formed from a polyurethaneurea having sulfobetaine groups and nonionic hydrophilic polyethylene oxide segments incorporated into the polymer. The polyurethaneurea polymer is dissolved in an organic or non-organic solvent system according to a predetermined coating formulation, crosslinked with an isocyanate crosslinker, and cured at a moderate temperature of approximately 50°C. The solvent system can be a single solvent or a mixture of solvents to aid in dissolving or dispersing the polymer. The solvent can be the one selected as the polymerization medium or can be added after polymerization is complete. Solvents are selected from those with lower boiling points to facilitate drying and to be less toxic for implant applications. Examples of these solvents include aliphatic ketones, esters, ethers, alcohols, hydrocarbons, etc. Depending on the final thickness of the biointerface / drug-release layer and the solution viscosity (related to the percent polymer solids), the coating can be applied in a single step or multiple repeated steps of a selected process, such as dipping, to build up the desired thickness. In yet another embodiment, the biointerface polymer is formed from a polyurethaneurea having unsaturated hydrocarbon groups and sulfobetaine groups incorporated into the polymer, and non-ionic hydrophilic polyethylene oxide segments, where the polyurethaneurea polymer is dissolved in an organic or non-organic solvent system in a coating formulation and crosslinked in the presence of an initiator by heat or irradiation, including UV, LED light, electron beam, etc., and cured at a moderate temperature of about 50° C. Examples of unsaturated hydrocarbons include allyl groups, vinyl groups, acrylates, methacrylates, alkenes, alkynes, etc.

[0151] In some embodiments, tethers are used. Tethers are polymers or chemical moieties that do not participate in the (electro)chemical reactions involved in sensing but form chemical bonds with the (electro)chemically active components of the membrane. In some embodiments, these bonds are covalent bonds. In one embodiment, the tethers can be formed in solution before one or more intermediate layers of the membrane are formed, and the tethers directly connect two (electro)chemically active components to each other, or alternatively, the tethers connect the (electro)chemically active components to a polymer backbone structure. In another embodiment, the (electro)chemically active components are mixed with a crosslinker (and optionally a polymer) having a tunable length, and the tethering reaction occurs as an in situ crosslink. Tethering can be used to maintain a predetermined number of degrees of freedom of NAD(P)H for efficient enzyme catalysis, and "efficient" enzyme catalysis allows the analyte sensor to continuously monitor one or more analytes over a period of about 5 to about 15 days or longer.

[0152] membrane manufacturing Polymers can be processed by solution-based techniques, such as spraying, dipping, casting, electrospinning, vapor deposition, spin-coating, and coating. Water-based polymer emulsions can be made to form films by methods similar to those used for solvent-based materials. In both cases, evaporation of the volatile liquid (e.g., organic solvent or water) leaves a film of polymer. Crosslinking of the deposited film or layer can be carried out through the use of multifunctional reactive components by a number of methods. Liquid systems can be cured by heat, moisture, high-energy radiation, ultraviolet light, or by driving the reaction to completion, which produces the final polymer in the mold or on the substrate to be coated.

[0153] In some examples, the wetting properties of the membrane (and thus the degree of sensor drift exhibited by the sensor) can be tuned and / or controlled by creating covalent crosslinks between surface-active group-containing polymers, functional group-containing polymers, polymers with zwitterionic groups (or precursors or derivatives thereof), and combinations thereof. Crosslinking can have a substantial effect on the film structure, which in turn can affect the surface wetting properties of the film. Crosslinking can also affect the tensile strength, mechanical strength, water absorption rate, and other properties of the film.

[0154] The crosslinked polymers can have different crosslink densities. In certain embodiments, a crosslinking agent is used to promote crosslinking between layers. In other embodiments, instead of (or in addition to) the crosslinking techniques described above, heat is used to form crosslinks. For example, in some embodiments, imide and amide bonds can form between two polymers as a result of elevated temperatures. In some embodiments, photocrosslinking is performed to form covalent bonds between the polycation and polyanion layers. One major advantage of photocrosslinking is that it offers the possibility of patterning. In certain embodiments, patterning using photocrosslinking is performed to modify the film structure and therefore tailor the wetting properties of membranes and membrane systems, as discussed herein.

[0155] Polymers having domains or segments functionalized to enable crosslinking can be prepared by at least the methods discussed herein. For example, polyurethaneurea polymers having aromatic or aliphatic segments with electrophilic functional groups (e.g., carbonyl, aldehyde, anhydride, ester, amide, isocyano, epoxy, allyl, or halo groups) can be crosslinked with a crosslinker having multiple nucleophilic groups (e.g., hydroxyl, amine, urea, urethane, or thiol groups). In a further example, polyurethaneurea polymers having aromatic or aliphatic segments with nucleophilic functional groups can be crosslinked with a crosslinker having multiple electrophilic groups. In one example, a polycarbodiimide crosslinker is used. Furthermore, polyurethaneurea polymers having hydrophilic segments with nucleophilic or electrophilic functional groups can be crosslinked with a crosslinker having multiple electrophilic or nucleophilic groups. The unsaturated functional groups on the polyurethaneurea can also be used for crosslinking by reacting with a multivalent free radical agent. Non-limiting examples of suitable crosslinking agents include isocyanates, carbodiimides, glutaraldehyde, aziridines, silanes or other aldehydes, epoxies, acrylates, free radical-based agents, ethylene glycol diglycidyl ether (EGDE), poly(ethylene glycol) diglycidyl ether (PEG-DE), or dicumyl peroxide (DCP). In one embodiment, about 0.1% to about 15% w / w of crosslinking agent is added based on the total dry weight of crosslinking agent and polymer added when blending the components. In another embodiment, about 1% to about 10% w / w of crosslinking agent is added based on the total dry weight of crosslinking agent and polymer added when blending the components. In yet another embodiment, about 5% to about 15% w / w of crosslinking agent is added based on the total dry weight of crosslinking agent and polymer added when blending the components.During the curing process, it is believed that substantially all of the crosslinker reacts, leaving substantially no detectable unreacted crosslinker in the final film.

[0156] The polymers disclosed herein can be formulated into a mixture that can be drawn into a film or applied to a surface using methods such as spraying, self-assembling monolayer (SAM), painting, dip coating, vapor deposition, molding, 3D printing, slot die coating, picojet printing, piezo inkjet printing, lithography techniques (e.g., photolithography), micro- and nanopipetting printing techniques, silkscreen printing, etc. The mixture can then be cured under elevated temperatures (e.g., about 30°C to about 150°C). Other suitable curing methods can include, for example, ultraviolet light, electron beam, or gamma radiation.

[0157] In some situations, using a continuous multi-analyte monitoring system including a sensor configured with a bioprotective and / or drug-releasing membrane, it is believed that the foreign body response is a primary event surrounding the extended implantation of the implanted device and can be managed or engineered to support analyte transport rather than impede or block it. In another aspect, to extend the sensor's lifespan, one embodiment employs a material that promotes vascularized tissue ingrowth, for example, within a porous biointerface membrane. For example, tissue ingrowth into the porous biointerface material surrounding the sensor can promote sensor function over extended periods of time (e.g., weeks, months, or years). It has been observed that tissue ingrowth and formation of a tissue bed can take up to three weeks. Tissue ingrowth and tissue bed formation are believed to be part of the foreign body response. As discussed herein, the foreign body response can be engineered through the use of a porous bioprotective material that surrounds the sensor and promotes tissue and microvasculature ingrowth over time.

[0158] Thus, sensors such as those discussed in the Examples herein may include a biointerface layer. The biointerface layer may include, for example, but is not limited to, a porous biointerface material including a solid portion and interconnected cavities, as well as a drug-release layer, all of which are described in more detail elsewhere herein. The biointerface layer may be used to improve sensor function over the long term (e.g., after tissue ingrowth).

[0159] Thus, sensors such as those discussed in the examples herein can include a drug-releasing membrane that functions at least partially as or in combination with a biointerface membrane. The drug-releasing membrane can include, for example, a material including a hard-soft segment polymer having hydrophilic and optionally hydrophobic domains, all of which are described in more detail elsewhere herein and can be used to improve sensor function over time (e.g., after tissue ingrowth). In one example, a material including a hard-soft segment polymer having hydrophilic and optionally hydrophobic domains is configured to release dexamethasone or a combination of a derivative form of dexamethasone acetate and dexamethasone, such that one or more different release rates of the anti-inflammatory drug are achieved, extending the useful life of the sensor. Other suitable drug release membranes of the present disclosure include silicone polymers, polytetrafluoroethylene, expanded polytetrafluoroethylene, ethylene tetrafluoroethylene copolymers, polyolefins, polyesters, polycarbonates, biostable polytetrafluoroethylene, polyurethane homopolymers, copolymers, and terpolymers, polypropylene (PP), polyvinylchloride (PVC), polyvinylidene fluoride (PVDF), polyvinyl alcohol (PVA), polyvinyl acetate, ethylene vinyl acetate (EVA), polybutylene terephthalate (PBT), polymethylmethacrylate (PMMA), polyether ether ketone ether (polyether ether ketone ether), and the like.ketone, PEEK), polyamides, polyurethanes and copolymers and blends thereof, polyurethaneurea polymers and copolymers and blends thereof, cellulose polymers and copolymers and blends thereof, poly(ethylene oxide) and copolymers and blends thereof, poly(propylene oxide) and copolymers and blends thereof, polysulfones and block copolymers (e.g., including diblock, triblock, alternating, random, and graft cellulose copolymers), hydrogel polymers, poly(2-hydroxyethyl methacrylate, pHEMA) and copolymers and blends thereof, hydroxyethyl methacrylate (HEMA) and copolymers and blends thereof, polyacrylonitrile-polyvinyl chloride (PCC) chloride, PAN-PVC) and copolymers and blends thereof, acrylic copolymers and copolymers and blends thereof, nylon and copolymers and blends thereof, polyvinyl difluoride, polyanhydrides, poly(l-lysine), poly(L-lactic acid), pan and copolymers and blends thereof, hydroxyapeptite and copolymers and blends thereof.

[0160] Sensing mechanism Generally, analyte sensors of the present disclosure include a sensing mechanism having, at least in part, a miniature structure (e.g., a miniature structured sensor, a microsensor, or a small diameter sensor), e.g., a coaxial or planar sensor. As used herein, "miniature structure" refers to an architecture having at least one dimension less than about 1 mm. The miniature structured sensing mechanism may be coaxial-based, or substrate-based (a flat or substantially planar substrate that may be single-sided or double-sided and may include one or more sensor elements on either the side or surface), or any other architecture. In some alternative embodiments, the term "miniature structure" can also refer to slightly larger structures, such as those having a smallest dimension greater than about 1 mm, but where the architecture (e.g., mass or size) is designed to minimize foreign body response due to size and / or mass.

[0161] The present disclosure includes sensor systems including two or more sensors, each configured to sense a different analyte. The two or more sensors may be configured to function independently or simultaneously to sense the two or more analytes simultaneously, sequentially, and / or randomly (including events that may occur independently in picoseconds, nanoseconds, milliseconds, seconds, or minutes), or in an alternating or overlapping manner. The two or more sensors of a sensor system may be communicatively coupled to electronics, such as a single transmitter or receiver. The two or more sensors of a sensor system may be communicatively coupled to separate, independent electronics.

[0162] In one embodiment of a continuous analyte monitoring system, a first single sensor is configured to continuously monitor at least a first analyte (e.g., glucose, glycerol, lactate, bilirubin, oxygen concentration, etc.) and a second, different analyte. In this embodiment, the single sensor may include a single coaxial or planar sensor configured to monitor at least a first analyte and a second analyte. In another embodiment, the first sensor is configured to monitor a first analyte and the second sensor is configured to continuously monitor a second analyte (e.g., ketones). Each of the first and second sensors may be planar, substantially planar, or coaxial, or a combination of two or more top, side, or cross-sectional shapes. In one embodiment, each of the first and second sensors is communicatively coupled to the same sensor electronics and network elements to continuously monitor and provide feedback to a device, such as a mobile device, tablet, laptop, wearable technology (clothing, jewelry, other accessories), or other Internet of Things (IoT) device, or combination of devices. In another embodiment, the first sensor and the second sensor are communicatively coupled to independent sensor electronics and network elements. Each of the first sensor and the second sensor is positioned within the subject through a skin layer in a subcutaneous layer. In another embodiment, the sensor system is configured as a monolithic sensor body including both the first sensor and the second sensor with electrodes configured to detect two or more analytes. At least one of the plurality of electrodes of the sensor system is configured to detect a first analyte, and the second plurality of electrodes is configured to detect a second analyte. The sensor system is positioned within the subject through a skin layer in a subcutaneous layer. In yet another embodiment, the sensor system includes a first sensor and a second sensor, each sensor of the sensor system including one or more fiber elements. For example, two or more sensors, such as the first sensor and the second sensor, may be electrically, mechanically, or otherwise coupled together ex vivo, in vivo, or both. Each of the first sensor and the second sensor of the sensor system is positioned within the subject through a skin layer in a subcutaneous layer.

[0163] The multi-analyte sensor devices and systems discussed herein may include elements such as on-body wearable devices, wireless communication capabilities, electronics, software, GUI(s), or other elements configured to allow the analyte monitoring system to continuously monitor analyte levels in a host. In response to this monitoring, various alerts and actions may be taken. As discussed herein, an "on-body" or wearable device includes a device configured to couple to a host for at least a predetermined period of time via one or more coupling elements, including sensors and / or in vivo components such as adhesives, mechanical elements, electrical elements, magnetic elements, or other combinations of elements.

[0164] sensing membrane In some embodiments, as shown in FIG. 1 , a sensing membrane is disposed on the electroactive surface of the continuous multi-analyte sensor 100 and includes one or more domains or layers. Generally, the sensing membrane functions, for example, to control the flow of biological fluid therethrough and / or to protect the sensitive area of ​​the sensor from contamination by the biological fluid. Some electrochemical enzyme-based analyte sensors generally include a sensing membrane that, for example, controls the flow of the analyte to be measured, protects the electrodes from contamination by the biological fluid, and / or provides an enzyme that catalyzes the reaction of the analyte with a cofactor. See, for example, U.S. Patent Application Publication No. 2005 / 0245799 to Brauker et al. and U.S. Patent No. 7,497,827 to Brister et al., both of which are incorporated by reference in their entireties.

[0165] The sensing membranes of the present disclosure can include any membrane configuration suitable for use with any analyte sensor (as described in more detail above). Generally, the sensing membranes of the present disclosure include one or more domains, all or some of which can be adhered or deposited on the analyte sensor, as will be understood by those skilled in the art. In one example, the sensing membrane generally provides one or more of the following functions, as described in the above-referenced U.S. Patent No. 7,497,827 to Brister et al.: 1) protection of exposed electrode surfaces from the biological environment; 2) analyte diffusion resistance (limitation); 3) catalysis to enable enzymatic reactions; 4) limiting or blocking interfering species; and 5) hydrophilicity at the electrochemically reactive surface of the sensor interface. The sensing membranes discussed herein may also include one or more adhesive layers positioned between two adjacent membrane layers. In one example, the one or more adhesive layers can increase the robustness and adhesion, thus improving the integrity of the sensing membrane. In various embodiments, the adhesive layer may include silane groups, polyvinyl alcohol (PVA), glutaraldehyde, or silicone-based or silicone-containing materials, or other adhesives or combinations of adhesives.

[0166] Electrode Domain In some embodiments, the membrane system includes an optional electrode domain. The electrode domain is provided to ensure that the electrochemical reaction between the electroactive surface of the working electrode and the electroactive surface of the reference electrode is promoted and / or enhanced, and therefore the electrode domain is positioned closer to the electroactive surface than the enzyme domain. In some embodiments, the electrode domain includes a semi-permeable coating that maintains a layer of water on the electrochemically reactive surface of the sensor; for example, a wetting agent in a binder material can be used as the electrode domain, which allows for complete transport of ions in an aqueous environment. The electrode domain can also help stabilize sensor operation by overcoming electrode activation and drift problems caused by insufficient electrolyte. The material forming the electrode domain can also protect against pH-mediated damage that can result from the formation of large pH gradients due to the electrochemical activity of the electrodes.

[0167] In one embodiment, the electrode domain comprises a flexible, water-swellable hydrogel film having a "dry film" thickness of about 0.05 microns or less to about 20 microns or more. In some examples, the "dry film" thickness is about 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 1, 1.5, 2, 2.5, 3, or 3.5 microns to about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 19.5 microns. In further embodiments, the "dry film" thickness is about 2, 2.5, or 3 microns to about 3.5, 4, 4.5, or 5 microns. "Dry film" thickness refers to the thickness of a cured film cast from a coating formulation by standard coating techniques.

[0168] In certain embodiments, the electrode domains are formed from a curable mixture of a urethane polymer and a hydrophilic polymer. A particularly preferred coating is formed from a polyurethane polymer having carboxylate functional groups and nonionic hydrophilic polyether segments, where the polyurethane polymer is crosslinked with a water-soluble carbodiimide (e.g., 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) or a polycarbodiimide crosslinker) in the presence of polyvinylpyrrolidone and cured at a moderate temperature of about 50°C.

[0169] In some embodiments, the electrode domains are deposited by spray coating or dip coating the electroactive surface of the sensor. In further embodiments, the electrode domains are formed by dip coating the electroactive surface in an electrode solution and curing the domain for about 15 to about 30 minutes at a temperature of about 40 to about 55°C (and can be accomplished under vacuum, e.g., 20 to 30 mmHg). In embodiments where dip coating is used to deposit the electrode domains, an insertion speed of about 1 to about 3 inches / minute, a residence time of about 0.5 to about 2 minutes, and a withdrawal speed of about 0.25 to about 2 inches / minute provide a functional coating. However, values ​​other than those listed above may be acceptable or even desirable in certain embodiments, depending, for example, on viscosity and surface tension, as will be understood by those skilled in the art. In one embodiment, the electroactive surface of the electrode system is dip coated in one coat and cured under vacuum at 50°C for 20 minutes.

[0170] As discussed herein, the insertable portion coating composition applied to the insertable portion may have a viscosity of about 10 centipoise (cP) to about 350 cP. In another example, the insertable portion coating composition as applied to the insertable portion has a viscosity of about 20 cP to about 200 cP. In yet another example, the insertable portion coating composition as applied to the insertable portion has a viscosity of about 30 cP to about 300 cP.

[0171] Although separate electrode domains are described herein, in some examples, sufficient hydrophilicity may be provided in the interference domain and / or the enzyme domain (depending on which domain is adjacent to the electroactive surface) to provide complete transport of ions in an aqueous environment (e.g., without a separate electrode domain).

[0172] Unmediated System Interference Domain In some embodiments, an optional interference domain is provided for the non-mediated systems disclosed herein, and generally comprises a polymer domain that restricts the flow of one or more interferents to the working electrode. In some embodiments, the interference domain functions as a molecular sieve that allows the analyte and other substances measured by the electrode to pass through, but prevents the passage of other substances, including interferents such as ascorbate and urea (see U.S. Patent No. 6,001,067 to Shults). Some known interferents for glucose oxidase-based electrochemical sensors include acetaminophen, ascorbic acid, bilirubin, cholesterol, creatinine, dopamine, ephedrine, ibuprofen, L-dopa, methyldopa, salicylic acid, tetracycline, tolazamide, tolbutamide, triglycerides, and uric acid.

[0173] Some polymer types that can be utilized as base materials for the interference domain include, for example, polyurethanes, polymers with pendant ionic groups (e.g., polyurethane-zwitterionic), NAFION™, chitosan, cellulose, or alternating layers of polyallylamine and polyacrylate acid, as well as polymers with controlled pore sizes. In one example, the interference domain comprises a thin, hydrophobic membrane that is non-swelling and limits the diffusion of low molecular weight species. The interference domain is permeable to relatively low molecular weight substances such as hydrogen peroxide, but restricts the passage of higher molecular weight substances, including glucose and ascorbic acid. In one example, the interference domain comprises a charged species (e.g., a polymer with pendant charged groups as disclosed herein) that functions to interact with one or more species of the sensing system, such as a cofactor, to reduce or eliminate migration from the domain.

[0174] Other systems and methods for reducing or eliminating interfering species that can be applied to the membrane systems of the present disclosure are described in U.S. Patent No. 7,816,004 to Muradov et al., U.S. Patent Application Publication No. 2005 / 0176136 to Burd et al., U.S. Patent No. 7,081,195 to Simpson et al., and U.S. Patent No. 7,715,893 to Kamath et al. In alternative embodiments, no separate interference domains are included.

[0175] In one embodiment, the interference domain is deposited on the electrode domain (or directly on the electroactive surface if no separate electrode domain is included) with a dry film domain thickness of about 0.05 microns or less to about 20 microns or more. In other embodiments, the dry film domain thickness is about 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 1, 1.5, 2, 2.5, 3, or 3.5 microns to about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 19.5 microns. In further embodiments, the dry film domain thickness is about 2, 2.5, or 3 microns to about 3.5, 4, 4.5, or 5 microns. Thicker membranes may also be useful, but in some embodiments thinner membranes have less effect on the rate of diffusion of hydrogen peroxide from the enzyme domain to the electroactive species.

[0176] As discussed herein, when two or more sensors are employed in a sensor system, each sensor optionally includes an interference domain configured to prevent the same interferent(s) from permeating the membrane. In another example, when two or more sensors are employed in a sensor system, each sensor optionally includes an interference domain configured to prevent different or overlapping but distinct interferent(s) from permeating the membrane.

[0177] Mediated System Interference Domain Some second-generation electrochemical analyte sensor technologies (second generation) utilize immobilized redox mediators to reduce the overpotential required to detect an analyte. This reduction can be significant in contrast to the typical operating potential of first-generation electrochemical analyte sensors (e.g., sensors that operate based on the principle of hydrogen peroxide detection on a catalytic metal surface). As an example, second-generation analyte sensors can be biased between +0.0 V and +0.3 V, whereas first-generation sensors can be biased between +0.5 V and +0.8 V. However, despite this reduction in operating potential and reduced susceptibility to electroactive interference from endogenous and pharmacological agents, these second-generation sensors can still succumb to the excessive influence of residual interferences.

[0178] For example, an exemplary second-generation analyte sensor contains a polymer-bound covalent redox mediator (e.g., polyvinylimidazole (PVI)-Os(4,4'-dimethyl-2,2'-bipyridine) 2Cl) that reduces the overpotential required for enzymatic detection of the target analyte. + / 2+ ) are utilized. Examples of such mediator-based sensors include systems in which excessive signal contributions resulting from the presence of co-circulating endogenous electroactive species can occur, as evidenced by product label alerts for large amounts of ascorbic acid / ascorbate ion (i.e., vitamin C), resulting in false hyperglycemia alerts and the like. Charge-selective membranes or further reduction of overpotential can mitigate such interference effects, but can result in significant impacts on sensitivity and signal-to-noise figures of merit. Thus, currently, mediated electrochemical analyte sensing systems continue to exhibit excessive signal contributions from endogenous metabolites such as ascorbic acid.

[0179] Thus, the present disclosure includes mediator system interference domains developed for second-generation sensor systems, whether they are continuous glucose monitoring systems or multi-analyte monitoring systems, e.g., ketone-glucose monitoring, in which the mediator system interference domain includes one or more oxidase enzymes that induce the enzymatic decomposition of an interfering metabolite or ensemble of metabolites into a peroxide product, e.g., hydrogen peroxide. The present disclosure provides domains that include oxidase enzymes alone or in combination with any of the conventional membranes (electrodes, enzymes, resistive domains / layers) used with indwelling second-generation (e.g., mediator) analyte sensors. Exemplary oxidase enzymes include, for example, ascorbic acid oxidase or uric acid oxidase configured to catalytically convert undesired interfering species (e.g., ascorbic acid, uric acid) to hydrogen peroxide products, which exhibit significantly less sensitivity to bias voltages / overpotentials conventionally applied in second-generation sensing systems. This conversion provides a reduction in the overall concentration of the interfering species at the electrode surface (e.g., exchanging the flux of the interfering species for the flux of hydrogen peroxide) and provides a less detrimental effect on the sensed signal than would otherwise be possible in the presence of the interfering species. The interfering domain may be used alone or in combination with other interfering domains, membranes, or domains, which may comprise, for example, the same polymer(s) matrix without one or more oxidase enzymes, peroxidase, or catalase.

[0180] In some embodiments of the mediator system interference domain, the oxidase enzyme can be combined with one or more peroxidase or peroxidase-like enzymes (e.g., horseradish peroxidase, catalase) to further cleave the generated hydrogen peroxide product from the oxidase enzyme(s), thereby inactivating the peroxide electroactive material and rendering it unable to undergo redox reactions at the electrode surface. The present disclosure includes the placement of the mediator system interference domain in one or more of an electrode domain, an enzyme domain, a resistor domain, and an interference membrane. The present disclosure includes the placement of the mediator system interference domain in one or more of an electrode domain, an enzyme domain, a resistor domain, and an interference domain.

[0181] Thus, in one example, an exemplary ketone / glucose multi-analyte sensor system can be used to include a mediator system interference domain comprising at least one of ascorbate oxidase, urate oxidase, horseradish peroxidase, or catalase present in an enzyme domain comprising a dehydrogenase enzyme (e.g., beta-hydroxybutyrate dehydrogenase), an NADH-acting enzyme (e.g., diaphorase, NAD(P)H dehydrogenase), a redox polymer (e.g., PVI-Os(bpy)Cl), and optionally a cofactor (e.g., NAD, NADP, if required), optionally crosslinked using, for example, PEG-DGE, CDI, or a polycarbodiimide crosslinker. A resistance domain of a biocompatible material or a hydrophobic / hydrophilic polymer blend, for example, PVP / PEG-DGE, can be applied over the enzyme domain / mediator system interference domain.

[0182] In another example, using an exemplary ketone / glucose multi-analyte sensor system, a mediator system interference domain comprising at least one of ascorbate oxidase, urate oxidase, horseradish peroxidase, or catalase resides in a resistance domain comprising a biocompatible material or a hydrophobic / hydrophilic polymer blend (e.g., PVP / PEG-DGE). A separate enzyme domain may be positioned adjacent to the mediator system interference domain present in the resistance domain, proximal to the electrode, and the enzyme domain may comprise a dehydrogenase enzyme (e.g., beta-hydroxybutyrate dehydrogenase, an NADH-acting enzyme (e.g., diaphorase, NAD(P)H dehydrogenase), a redox polymer (e.g., PVI-Os(bpy)Cl), optionally a cofactor (e.g., NAD+, NADP+, if required), and may optionally be crosslinked, for example, using PEG-DGE or a polycarbodiimide crosslinker.

[0183] In another example, using an exemplary ketone / glucose multi-analyte sensor system, a mediator interference domain comprising at least one of ascorbate oxidase, urate oxidase, horseradish peroxidase, or catalase is present between the enzyme domain and at least one electrode surface. A resistive domain of a biocompatible material or a hydrophobic / hydrophilic polymer blend, for example, PVP / PEG-DGE, can be applied over the enzyme domain.

[0184] In other examples, as discussed further herein, an exemplary mediator-free ketone or ketone / glucose multi-analyte sensor system is provided. In one example, an exemplary ketone or ketone / glucose multi-analyte sensor system is provided that does not include a metal-based mediator, e.g., an osmium complex of a biimidazole and / or imidazole ligand. In one example, an exemplary ketone or ketone / glucose multi-analyte sensor system is provided that does not include a metal-based mediator, e.g., an osmium complex of a biimidazole and / or imidazole ligand, configured to provide an amperometric signal at applied voltages greater than +0.2 V, greater than or equal to +0.3 V, greater than or equal to +0.4 V, greater than or equal to +0.5 V, or greater than or equal to +0.6 V. In one example, an exemplary ketone or ketone / glucose multi-analyte sensor system is provided that does not include a metal-based mediator and includes an interference layer. In one embodiment, an exemplary ketone or ketone / glucose multi-analyte sensor system is provided that is configured to provide an amperometric signal at applied voltages greater than +0.2 V, greater than or equal to +0.3 V, greater than or equal to +0.4 V, greater than or equal to +0.5 V, or greater than or equal to +0.6 V, and that includes an interference layer and does not include a metal-based mediator, e.g., an osmium complex of a biimidazole and / or imidazole ligand.

[0185] Transducer domain In one embodiment, the membrane system further comprises a transducer domain, e.g., an enzyme, RNA, DNA, aptamer, binding protein, etc., located more distally from the electroactive surface than the interference domain (or the electrode domain when no separate interference is included). In some embodiments, the transducer domain is deposited directly on the electroactive surface (when neither the electrode nor the interference domain is included). In one embodiment, the transducer domain provides an enzyme that catalyzes the reaction of the analyte and its co-reactant, as described in more detail below. In some embodiments, the transducer domain comprises glucose oxidase. However, other oxidases, e.g., galactose oxidase or uricase oxidase, can also be used.

[0186] For an enzyme-based electrochemical glucose sensor to function effectively and accurately, the sensor response must be limited by neither enzyme activity nor co-reactant concentration. Because enzymes, including glucose oxidase, can undergo inactivation as a function of time even under ambient conditions, this behavior is compensated for by forming an enzyme domain. In some embodiments, the enzyme domain is preferably comprised of an aqueous dispersion of a colloidal polyurethane polymer containing the enzyme. However, in alternative embodiments, the enzyme domain is comprised of an oxygen-enhancing material, such as at least one of silicone or fluorocarbon, to provide an excess supply of oxygen to ensure that oxygen does not limit the sensing reaction. In some embodiments, the enzyme is immobilized within the enzyme domain. See U.S. Patent No. 7,379,765 to Petisce et al.

[0187] In one embodiment, the transduction element domain is deposited on the interference domain at a "dry film" domain thickness of about 0.05 microns or less to about 20 microns or more. In other embodiments, the dry film domain thickness is about 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 1, 1.5, 2, 2.5, 3, or 3.5 microns to about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 19.5 microns. In further embodiments, the dry film domain thickness is about 2, 2.5, or 3 microns to about 3.5, 4, 4.5, or 5 microns. "Dry film" thickness refers to the thickness of a cured film cast from a coating formulation by standard coating techniques, including post-cure of the film.

[0188] However, in some embodiments, the transducer domain is deposited directly onto the electrode domain or onto the electroactive surface. In some embodiments, the transducer domain is deposited by spray or dip coating, slot die coating, 3D printing, picojet printing, piezo inkjet printing, or the like. In further embodiments, the transducer domain is formed by dip coating the electrode domain into a transducer domain solution and curing the transducer domain at a temperature of about 40 to about 55°C for about 15 to about 30 minutes (and can be achieved under vacuum, e.g., 20 to 30 mmHg). In embodiments where dip coating is used to deposit the transducer domain at room temperature, an insertion speed of about 1 inch / min to about 3 inches / min, a dwell time of about 0.5 minutes to about 2 minutes, and a withdrawal speed of about 0.25 inches / min to about 2 inches / min provides a functional coating. However, values ​​other than those listed above may be acceptable or even desirable in certain embodiments, depending, for example, on viscosity and surface tension, as will be understood by those skilled in the art. In one example, the transducer domain is formed by dip-coating twice in a coating solution (i.e., forming two layers) and curing under vacuum at 50°C for 20 minutes. However, in some examples, the transducer domain can be formed by dip-coating and / or spray-coating one or more layers at a predetermined concentration, insertion rate, dwell time, withdrawal rate, and / or desired thickness of the coating solution. In yet another example, the transducer layer is formed from multiple intermediate layers deposited by self-assembled monolayers (SAMs), typically formed by immersion in a solution that promotes surface chemistry. The substrate may be placed in the solution for a period of about 30 minutes to about 24 hours to form the desired transducer layer to a predetermined thickness. In another example, the substrate may be placed in the solution for a period of about 1 hour to about 18 hours. In another example, the substrate may be placed in the solution for a period of about 3 hours to about 12 hours.

[0189] In yet other embodiments, the conversion element layer is formed from multiple intermediate layers, one or more of which may be varied in various aspects, such as chemistry (composition), thickness, or other mechanical, electrical, biological, or other material properties, either alone or in combination, to achieve a target electron mobility or range of electron mobilities through each intermediate layer.

[0190] Resistance Domain In one example, the membrane system includes a resistance domain located more distally from the electroactive surface than the enzyme domain. While the following description is directed to a resistance domain for a glucose sensor, the resistance domain can be modified to facilitate detection of other analyte and co-reactant concentration(s). In one example, the resistance domain is configured to control the flux of oxygen through the membrane. In another example, the resistance domain is configured to control the flux of an analyte or co-reactant other than oxygen through the membrane. In yet another example, the resistance domain is configured to control the flux of two or more analytes through the membrane.

[0191] Immobilized enzyme-based glucose sensors that use oxygen as a coreactant are supplied with a non-rate-limiting excess of oxygen so that the sensor responds linearly to changes in glucose concentration but not to changes in oxygen concentration. Specifically, when the glucose monitoring reaction is oxygen-limited, linearity is not achieved above a minimum concentration of glucose. Without a semipermeable membrane placed over the enzyme domain to control the flux of glucose and oxygen, a linear response to glucose levels can only be obtained for glucose concentrations up to about 40 mg / dL. However, in a clinical setting, a linear response to glucose levels up to at least about 400 mg / dL is desirable.

[0192] In one embodiment, the resistance domain includes a semipermeable membrane that controls the flux of oxygen and glucose to the underlying enzyme domain, making oxygen a non-rate-limiting excess. As a result, the upper linearity limit of glucose measurement is extended to values ​​much higher than that achieved without the resistance domain. In one embodiment, the resistance domain exhibits an oxygen-to-glucose permeability ratio of about 50:1 or less to about 400:1 or more. In a further embodiment, the oxygen-to-glucose permeability ratio is about 200:1.

[0193] In an alternative embodiment, a lower ratio of oxygen to glucose may be sufficient to provide excess oxygen by using a high oxygen solubility domain (e.g., a silicone or fluorocarbon-based material or domain) to enhance oxygen supply / transport to the conversion element domain. Also, if more oxygen is supplied to the enzyme, more glucose may be supplied to the conversion element without creating an oxygen-limiting excess. In an alternative embodiment, the resistance domain is formed from a silicone composition such as that described in U.S. Patent Application Publication No. 2005 / 0090607 to Tapsak et al.

[0194] In one embodiment, the resistance domain includes a polyurethane membrane having both hydrophilic and hydrophobic regions. The hydrophilic and hydrophobic regions may be used in combination to control the diffusion of one or more analytes (e.g., glucose, oxygen, ketones, lactate, uric acid, etc.) to the analyte sensor. A suitable hydrophobic polymer component is polyurethane or polyetherurethaneurea. Polyurethane is a polymer produced by the condensation reaction of a diisocyanate with a difunctional hydroxyl-containing material. Polyurethaneurea is a polymer produced by the condensation reaction of a diisocyanate with a difunctional amine-containing material. In polyurethane and polyurethaneurea polymers, either the hard or soft segments can include multiple different chemical structures; for example, the soft segment can include hydrophobic and hydrophilic segments.

[0195] Examples of diisocyanates useful as hard segment components of the polyurethane or polyurethaneurea polymers of the present disclosure include aliphatic diisocyanates containing about 4 to about 8 methylene units. Diisocyanates containing alicyclic moieties can also be useful in preparing the polymer and copolymer components of the membranes of the present disclosure. The material forming the base of the hydrophobic matrix of the resistance domain may be selected to be sufficiently permeable to allow relevant compounds to pass through it, for example, to allow oxygen molecules from the test sample to pass through the membrane to reach the active enzyme or electrochemical electrode. Examples of materials that can be used to prepare non-polyurethane-type membranes include vinyl polymers (including polyvinylimidazole and polyvinylpyridine), inorganic polymers such as polyethers, polyesters, polyamides, polysiloxanes, and polycarbosiloxanes, natural polymers such as cellulosic and protein-based materials, and mixtures or combinations thereof. In some examples, these non-polyurethane-type membranes contain a crosslinker in addition to the base polymer to improve mechanical properties and / or tailor mass transport of analytes or other species. In some embodiments, the resistive domain may be polyvinyl butyral (PVB). In other embodiments, the base polymer may be a segmented block copolymer. In another embodiment, the hard segment may be about 15% to about 75% by weight. In yet another embodiment, the hard segment may be about 25% to about 55% by weight. In yet another embodiment, the hard segment may be about 35% to about 45% by weight. For example, the base polymer may include polyurethane and / or polyurea segments and one or more of polycarbonate, polydimethylsiloxane (PDMS), polyether, fluorine-modified segments, perfluoropolyol, or polyester segments. In other embodiments, the base polymer may be a polyurethane copolymer selected from the group including polyether-urethane-urea, polycarbonate-urethane, polyether-urethane, polyester-urethane, and / or copolymers thereof.

[0196] In one embodiment, the hydrophilic polymer component of the resistance domain is polyethylene oxide (PEO). For example, one useful hydrophobic-hydrophilic copolymer component is a polyurethane polymer containing about 1% to about 50% by weight of polyethylene oxide. In one embodiment, the resistance domain contains 5% to about 30% by weight of polyethylene oxide (PEO). In another embodiment, the resistance domain contains about 10% to about 40% by weight of PEO. The polyethylene oxide portion of the copolymer is thermodynamically driven to separate from the hydrophobic portion of the copolymer and the hydrophobic polymer component. The polyethylene oxide-based soft segment portion of the copolymer used to form the final blend influences the water uptake and subsequent glucose permeability of the membrane.

[0197] In one embodiment, one or more of NBDI, IPDI, TDI, MPDI, HMDI, MDI, 1,3-H6XDI, 1,4-H6XDI, CHDI, PPDI, TODI, or HDI diisocyanate are used to form various polyurethanes and polyurethane-ureas for the resistor domain and / or other sensor domains. In one embodiment, the polyurethanes and polyurethane-ureas have a soft segment that is aliphatic or amphiphilic. In one embodiment, the soft segment comprises a diol, diamine, diester, or dicarbonate. In one embodiment, the soft segment comprises two or more of a diol, diamine, diester, or dicarbonate.

[0198] In one embodiment, one or more of NBDI, IPDI, TDI, MPDI, HMDI, MDI, 1,3-H6XDI, 1,4-H6XDI, CHDI, PPDI, TODI, and HDI are reacted with one or more dicarbonates, polyethers, polyesters, polyalkyl-diols, or polyalkyl-diamines.

[0199] In one embodiment, one or more of NBDI, IPDI, TDI, MPDI, HMDI, MDI, 1,3-H6XDI, 1,4-H6XDI, CHDI, PPDI, TODI, and HDI are reacted with a C5 or C6 dicarbonate, such as U90 OXYMER™, or polyhexamethylene carbonate glycol (PHA). In one embodiment, NBDI, IPDI, TDI, MPDI, HMDI, MDI, 1,3-H6XDI, 1,4-H6XDI, CHDI, PPDI, TODI, and HDI, or a mixture thereof, are reacted with a C5 or C6 dicarbonate, such as U90 OXYMER™, and one or more polyethers, polyesters, polyalkyl-diols, or polyalkyl-diamines. In one embodiment, the dicarbonate is sterically branched to increase the Tg of the soft segment, e.g., to provide a Tg near body temperature.

[0200] In one embodiment, one or more of the following hard segment diisocyanates are reacted with one or more of polyethers, such as polytetramethylene oxide (PTMO), polypropylene oxide (PPO), polyethylene glycol (PEG), and polybutadiene diol (PBU), either alone or in combination with polydimethylpolysiloxane (PDMS). In one embodiment, the same polyether with different molecular weights (Mw) is used. In one embodiment, two or more polyethers with the same or different Mw are used. In one embodiment, one or more polyethers with the same or different Mw are used in combination with one or more PDMS polymers with the same or different Mw. Without being bound by any particular theory, it has been observed that as the molecular weight of the soft segment decreases, phase mixing of the different soft segment components increases. In one embodiment, it has been observed that higher molecular weight soft segments result in the formation of a rich phase, likely due to, among other things, entropy contributions.

[0201] In one embodiment, one or more hard segment diisocyanates of NBDI, IPDI, TDI, MPDI, HMDI, MDI, 1,3-H6XDI, 1,4-H6XDI, CHDI, PPDI, TODI, HDI are reacted with one or more polyesters, such as polyethylene adipate glycol (PEA), polytetramethylene adipate glycol (PBA), alone or in combination with one or more polyethers, polyalkyl-diols, or polyalkyl-diamines.

[0202] In one embodiment, NBDI, IPDI, TDI, MPDI, HMDI, MDI, 1,3-H6XDI, 1,4-H6XDI, CHDI, PPDI, TODI, HDI, or mixtures thereof are reacted with one or more polyalkyl-diols, alone or in combination with one or more polycarbonates, polyethers, polyesters, or polyalkyl-diamines.

[0203] In one embodiment, the resistive domain is deposited directly onto the electrode surface or onto one or more layers of the enzyme domain to yield a resistive domain thickness of about 0.05 microns or less to about 20 microns or more. In another embodiment, the total resistive domain thickness is about 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 1, 1.5, 2, 2.5, 3, or 3.5 microns to about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 19.5 microns. In another embodiment, the total resistive domain thickness is about 2, 2.5, or 3 microns to about 3.5, 4, 4.5, or 5 microns. In some embodiments, the resistive domain is deposited onto the enzyme domain by spray or dip coating, slot die coating, 3D printing, picojet printing, or piezo inkjet printing. In certain embodiments, spray coating is a deposition technique. Because the spraying process atomizes and mistizes the solution, most or all of the solvent evaporates before the coating material settles on the underlying domain, thereby minimizing contact between the solvent and the enzyme. One additional advantage of spray coating the resistive domain as described in this disclosure includes the formation of a membrane system that substantially blocks or resists ascorbate, a known electrochemical interferent in hydrogen peroxide-measuring glucose sensors. Without wishing to be bound by theory, it is believed that a structural morphology characterized by substantial impermeability to ascorbate is formed during the process of depositing the resistive domain as described in this disclosure.

[0204] Heterocyclic Resistance Domain and Cofactor Immobilization or Retention Domain In one embodiment, the cofactor and enzyme are present in an enzyme and / or resistor domain comprising a domain, e.g., polyvinylpyridine, polyvinylpyridine-co-styrene, polyvinylpyridine copolymers with vinyl and (meth)acrylic monomers, poly(styrene-co-acrylonitrile), polyvinylimidazole, or polyvinylimidazole copolymers with vinyl and (meth)acrylic monomers, and / or provided as a layer adjacent to the electrode domain or electrode surface. As used herein, "polyvinylpyridine" encompasses poly(2-vinylpyridine), 3-vinylpyridine, 3-vinylpyridine, and alkyl-substituted derivatives thereof. Blends and / or graphs of the above polymers can be used. Blends and / or graphs of the above polymers with chitosan, amphiphilic or aliphatic polyurethanes or polyurethaneureas, polyols (e.g., PEG, PTMO), or zwitterionic polymers can be used. In some embodiments, polystyrene copolymers with vinyl monomers containing electron-withdrawing groups, such as nitriles, can be used. In some embodiments, vinyl polymers with benzene and nitrile functional groups can be used.

[0205] In one embodiment, the cofactor and enzyme are present within domains, e.g., enzyme and / or resistance domains comprising at least partially crosslinked poly(4-vinylpyridine), polyvinylpyridine-co-styrene, polyvinylpyridine copolymers with vinyl and (meth)acrylic monomers, poly(styrene-co-acrylonitrile), polyvinylimidazole, or polyvinylimidazole copolymers with vinyl and (meth)acrylic monomers are used as resistance domains and / or provided as layers adjacent to electrode domains or electrode surfaces. In one embodiment, poly(4-vinylpyridine), polyvinylpyridine-co-styrene, polyvinypyridine copolymers with vinyl and (meth)acrylic monomers, poly(styrene-co-acrylonitrile), polyvinylimidazole, or polyvinylimidazole copolymers with vinyl and (meth)acrylic monomers, with or without crosslinking, provide for immobilization or retention of one or more cofactors in the resistance domain. In one embodiment, the immobilization or retention of the cofactor is via covalent bonding with functional groups of the polymer. In another embodiment, the immobilization or retention of the cofactor is achieved through non-covalent interactions, for example, through equilibration with functional groups on the polymer.

[0206] Examples of cofactor immobilization via non-covalent interactions with polymers include NAD+ with cationic polymers (e.g., chitosan, quaternized PVPy, polyzwitterionic polymers, etc.) and / or polymers containing boronic acid functional groups. Thus, in one example, the cofactor and enzyme are present in a domain, e.g., an enzyme and / or resistance domain, comprising a polymer with pendant boronic acid groups that provide strong, dynamic covalent binding with the diol functional group on a cofactor, e.g., NAD, at a specific pH, allowing for immobilization or retention of NAD(H). Thus, in one example, the 1,2 diol-containing ribose ring structure of the NADH and NAD+ structures is used to associate and / or bind to one or more boronic acid functional groups through at least covalent interactions, as shown in Scheme 1a and Scheme 1b for the NADH form. Similar covalent interactions are envisioned for NAD+ forms.

[0207] [ka]

[0208] In one embodiment, the boronic acid polymer structure and coating solution pH are adjusted to provide sufficient association of the NAD / NADH structures to reduce or eliminate migration from the polymer membrane. In one embodiment, the boronic acid polymer includes styrene polymers, styrene copolymers (e.g., with acrylics, acrylates, acrylamides, olefins, cyclic olefins), naphthyl, anthracenyl polymers, and copolymers thereof. In one embodiment, the boronic acid polymer is at least partially crosslinked.

[0209] In some embodiments, the domains are configured to repel cofactors, for example, the RL functions to "repel" NAD(H) from passing through, thereby attenuating its movement from the EZL.

[0210] In another embodiment, NAD is tethered to a domain or to an electrode surface. In another embodiment, NAD is directly tethered to a domain or directly bound to an electrode surface. In another embodiment, NAD is bound to an electrode surface using an electron transfer agent. In one embodiment, the free amine of the adenine group of NAD(H) is extended with an alkyl chain bearing a primary amine to provide EDC or (sulfo-)NHS coupling chemistry with the -COOH group on the mediator, as shown in Scheme 3, which shows a modified NAD cofactor with an extended free -NH2 bound to one of the -COOH groups on a PQQ (pyrroloquinoline quinone) mediator.

[0211] In another embodiment, the free amine of the adenine group of NAD(H) is extended with an alkyl chain bearing a primary amine to provide EDC or (sulfo-)NHS coupling chemistry with the HBDH enzyme.

[0212] In one example, the modified NAD+ cofactor has an extended free -NH2 that can be easily crosslinked to one of the -COOH groups in a PQQ (pyrroloquinoline quinone) mediator, which has another -COOH group that can then be crosslinked to a polymer backbone, an enzyme, or directly onto an electrode surface.

[0213] In one embodiment, the cofactor and enzyme are present in a domain, e.g., an enzyme and / or resistance domain, comprising an amphiphilic polyurethane or polyurethaneurea polymer as disclosed above for the biointerface / drug-release layer, where the aliphatic polyurethane or polyurethaneurea has about 20-40 wt. % hard segment content, about 10-30 wt. % polysiloxane segments, and about 15-40 wt. % polyglycol segments. In one embodiment, the amphiphilic polyurethane or polyurethaneurea also contains 0-25 wt. % polyvinylpyrrolidone polymer. In one embodiment, the amphiphilic polyurethane or polyurethaneurea also contains 0-25 wt. % polyvinylpyridine or alkylated or polyol-substituted pyridine polymer. Such amphiphilic polyurethane or polyurethaneurea polymer resistance domains provided acceptable sensitivity and stability for greater than two weeks. In one embodiment, the amphiphilic polyurethane or polyurethaneurea polymer is at least partially crosslinked. Such amphiphilic polyurethane or polyurethaneurea polymer resistance domains provided sensitivity and stability comparable to the PVPy resistance domains described above.

[0214] In one embodiment, the cofactor and enzyme are present in a domain, e.g., an enzyme and / or resistance domain, comprising an aliphatic polyurethane or polyurethane urea polymer having a hard segment content of about 40-60 wt. %, about 15-50 wt. % polytetrahydrofuran (PTMO) segments, and about 5-30 wt. % polysiloxane segments. In one embodiment, the aliphatic polyurethane or polyurethane urea polymer is at least partially crosslinked. In one embodiment, a polycarbodiimide crosslinker is used. Such aliphatic polyurethane or polyurethane urea resistance domains have demonstrated lower sensitivity and stability than amphiphilic polyurethane or polyurethane urea-based resistance domains.

[0215] In one embodiment, the cofactor and enzyme are present in a domain, e.g., an enzyme and / or resistance domain, comprising a block copolymer obtained by polycondensation of a carboxylic acid polyamide (e.g., PA6, PA11, PA12) with a polyether (e.g., polytetramethylene glycol, polyethylene glycol, PEG, polytetrahydrofuran PTMO). In one embodiment, the block copolymer obtained by polycondensation of a carboxylic acid polyamide with a polyether can be at least partially crosslinked.

[0216] In one embodiment, the cofactor and enzyme are present in a domain, e.g., an enzyme and / or resistance domain, comprising a polyvinylpyridine-poly(ethylene glycol) diglycidyl ether (PVPy-PEG-DGE) matrix. In one embodiment, the mass ratio of PEG-DGE to PVPy is about 1-10% by weight. In one embodiment, the polyvinylpyridine-PEG-DGE matrix is ​​at least partially crosslinked.

[0217] In one example, the cofactor and enzyme are present in a domain, e.g., an enzyme and / or resistance domain, comprising a water-dispersible polyurethane-zwitterionic polymer crosslinked with a carbodiimide or polycarbodiimide. Examples of such domains include those disclosed in U.S. Patent Application Publication No. 2017 / 0191955, U.S. Patent Application Publication No. 2017 / 0188922, and U.S. Patent No. 11,112,377 B2, the disclosures of which are incorporated herein by reference. In one example, the domain comprises an enzyme and a polymer comprising a polyurethane and / or polyurea segment and one or more zwitterionic repeat units. In one example, the domain comprises an enzyme and a blend of a polyurethane-based polymer and polyvinylpyrrolidone. In some examples, the enzyme domain is formed from a polyurethaneurea having carboxyl betaine groups and nonionic hydrophilic polyethylene oxide segments incorporated into the polymer, and the polyurethaneurea polymer is dissolved in an organic or non-organic solvent system according to a predetermined coating formulation and, optionally, crosslinked and / or cured. The domains described above can be from 0.01 μm to about 250 μm thick.

[0218] Depending on the embodiment, the resistance domain(s) discussed herein may be formed by any number of methods, including, but not limited to, dip coating or spray coating of any layer or layers, depending on the concentration of the solution, insertion rate, residence time, withdrawal rate, and / or desired thickness of the resulting film, or other factor or combination of factors.

[0219] Advantageously, sensors having the membrane system of the present disclosure, including an electrode domain and / or interference domain, an enzyme domain, and a resistance domain, provide a stable signal response to increasing glucose levels of about 40 to about 400 mg / dL and provide sustained functionality (at least 90% signal intensity) even at low oxygen levels (e.g., about 0.6 mg / L02). Without wishing to be bound by theory, it is believed that the resistance domain provides sufficient resistivity, or the enzyme domain provides sufficient enzyme, such that oxygen limitation is observed at much lower oxygen concentrations compared to prior art sensors.

[0220] In one example, the sensor signal has a current in the picoampere range, which is described in more detail elsewhere herein. However, the ability to generate a signal with a current in the picoampere range may depend on a combination of factors, including the electronic circuit design (e.g., A / D converter, bit resolution, etc.), the membrane system (e.g., analyte permeability through the resistive domain, enzyme concentration, and / or electrolyte availability for the electrochemical reaction at the electrode), and the exposed surface area of ​​the working electrode. For example, the resistive domain can be designed to be more or less restrictive to the analyte, depending on the design of the electronic circuit, the membrane system, and / or the exposed electroactive surface area of ​​the working electrode.

[0221] Thus, in one embodiment, the membrane system is designed with a sensitivity of about 1 pA / mg / dL to about 100 pA / mg / dL. In another embodiment, the sensitivity is about 5 pA / mg / dL to about 25 pA / mg / dL. In a further embodiment, the sensitivity is about 4 to about 7 pA / mg / dL. Without wishing to be bound by any theory, it is believed that membrane systems designed with sensitivities in the above ranges enable measurement of analyte signals in low analyte and / or low oxygen conditions. That is, conventional analyte sensors have exhibited reduced measurement accuracy in the low analyte range due to low analyte availability to the sensor and / or increased signal noise in the high analyte range due to insufficient oxygen required to react with the amount of analyte being measured. Without wishing to be bound by theory, it is believed that the membrane systems of the present disclosure, in combination with the electronic circuitry design and exposed electrochemically reactive surface area design, support the measurement of analytes in the picoampere range, which allows for improved levels of resolution and accuracy in both the low and high analyte ranges not seen in the prior art.

[0222] Although some example sensors described herein include an optional interference domain to block or reduce one or more interferents, sensors having the disclosed membrane system including an electrode domain, an enzyme domain, and a resistance domain have been shown to inhibit ascorbate without an additional interference domain. That is, the disclosed membrane system including an electrode domain, an enzyme domain, and a resistance domain has been shown to be substantially unresponsive to ascorbate within a physiologically acceptable range. Without wishing to be bound by theory, it is believed that the process of depositing the resistance domain by spray coating described herein results in a structural morphology that is substantially resistant to ascorbate.

[0223] Interference-free membrane system In general, it is believed that appropriate solvents and / or deposition methods can be selected for one or more domains of the membrane system to form one or more transition domains such that they are substantially impermeable to interferents. Thus, sensors can be constructed without separate or deposited interference domains that are unresponsive to interferents. Without wishing to be bound by theory, it is believed that simplified multilayer membrane systems, more robust multilayer fabrication processes, and reduced variability caused by the thickness of deposited micron-thin interference domains and associated oxygen and glucose sensitivity can be provided. Additionally, optional polymer-based interference domains that typically inhibit hydrogen peroxide diffusion are eliminated, thereby enhancing the amount of hydrogen peroxide that passes through the membrane system. In other examples, the interference domains can be configured to block or reduce the diffusion of one or more interfering species, including HO, acetaminophen, or other interferents or combinations of interferents.

[0224] Oxygen delivery tube As mentioned above, some sensors employ a transducer element within a membrane system through which a host's bodily fluid passes and in which analytes (e.g., glucose, ketones) in the bodily fluid react in the presence of a co-reactant (e.g., oxygen) to produce a product. This product is then measured using electrochemical methods, and the output of the electrode system thus serves as a measure of the analyte. For example, when the sensor is a glucose oxidase-based glucose sensor, the species measured at the working electrode is HO. The enzyme glucose oxidase catalyzes the conversion of oxygen and glucose to hydrogen peroxide and gluconic acid according to the following reaction: Glucose + O2 → gluconic acid + H2O2.

[0225] For each glucose molecule reacted, there is a proportional change in the product, HO, so changes in HO can be monitored to determine glucose concentration. The oxidation of HO by the working electrode is balanced by the reduction of ambient oxygen, enzymatically generated HO, and other reducible species, for example, at the counter electrode. See Fraser, D.M., "An Introduction to In vivo Biosensing: Progress and Problems," in "Biosensors and the Body," D.M. Fraser, ed., 1997, pp. 1-56, John Wiley and Sons, New York.

[0226] In vivo, glucose concentrations are generally about 100 times higher than oxygen concentrations. As a result, oxygen is the limiting reactant in the electrochemical reaction, and when insufficient oxygen is supplied to the sensor, the sensor is unable to accurately measure glucose concentrations. Therefore, reduced sensor function or inaccuracy is believed to be the result of problems in the availability of oxygen to the enzyme and / or electroactive surface.

[0227] Thus, in an alternative embodiment, an oxygen conduit (e.g., a high oxygen solubility domain formed from silicone or a fluorochemical or perfluorocarbon compound) is provided that extends from the ex vivo portion of the sensor to the in vivo portion of the sensor to increase the availability of oxygen to the enzyme. The oxygen conduit can be formed as part of the coating (insulating) material or can be a separate conduit associated with the assembly of wire(s) that form the sensor.

[0228] In some embodiments, one or more domains of the sensing membrane are formed from materials such as silicone, polytetrafluoroethylene, ethylene tetrafluoroethylene copolymer, polyolefin, polyester, polycarbonate, biostable polytetrafluoroethylene, homopolymer, copolymer, terpolymer of polyurethane, polypropylene (PP), polyvinylchloride (PVC), polyvinylidene fluoride (PVDF), polybutylene terephthalate (PBT), polymethylmethacrylate (PMMA), polyether ether ketone (PEEK), polyurethane, cellulosic polymers, poly(ethylene oxide), poly(propylene oxide), and copolymers and blends thereof, polysulfone, and block copolymers thereof (e.g., diblock, triblock, alternating, random, and graft copolymers). US Patent Application Publication No. 2005 / 0245799 to Brauker et al., which is incorporated herein by reference in its entirety, describes biological interfaces and sensing membrane configurations and materials that may be applied to the sensors of the present disclosure.

[0229] The sensing membrane can be deposited on the electroactive surface of the electrode material using known thin-film or thick-film techniques (e.g., spraying, electrodeposition, dipping, etc.). Note that the sensing membrane surrounding the working electrode need not be of the same structure as the sensing membrane surrounding the reference electrode, etc. For example, the transducer domain deposited on the working electrode does not necessarily need to be deposited on the reference and / or counter electrodes.

[0230] In one example, as described in detail herein and understood by one of ordinary skill in the art, the sensor is an enzyme-based electrochemical sensor, where the working electrode measures hydrogen peroxide produced by an enzyme-catalyzed reaction of glucose to be detected, generating a measurable electronic current (e.g., glucose detection utilizing glucose oxidase produces hydrogen peroxide as a by-product, and HO reacts with the surface of the working electrode to release two protons (2H + ), two electrons (2e - ), and one molecule of oxygen (O), which generates an electronic current that is detected. In some embodiments, one or more potentiostats are used to monitor the electrochemical reaction at the electroactive surface of the working electrode. A potentiostat applies a constant potential to the working electrode and its associated reference electrode to determine the current produced at the working electrode. The current produced at the working electrode (and flows through the circuit to the counter electrode) is substantially proportional to the amount of HO that diffuses to the working electrode. The output signal is typically a raw data stream used to provide, for example, a host or physician with a useful value of the measured analyte concentration in the host, e.g., the raw signal processed by an algorithm before displaying the value.

[0231] Some alternative analyte sensors that can benefit from the systems and methods of the present disclosure are described, for example, in U.S. Pat. No. 5,711,861 to Ward et al., U.S. Pat. No. 6,642,155 to Vachon et al., U.S. Pat. No. 6,654,625 to Say et al., U.S. Pat. No. 6,565,509 to Say et al., U.S. Pat. No. 6,514,718 to Heller, U.S. Pat. No. 6,465,666 to Essenpreis et al., U.S. Pat. No. 6,465,666 to Offenbacher et al., and U.S. Pat. No. 6,214,185 to Cunningham et al., U.S. Pat. No. 5,310,469 to Shaffer et al., U.S. Pat. No. 5,683,562 to Bonnecaze et al., U.S. Pat. No. 6,579,690 to Bonnecaze et al., U.S. Pat. No. 6,484,46 to Say et al., U.S. Pat. No. 6,512,939 to Colvin et al., U.S. Pat. No. 6,424,847 to Mastrototaro et al., and U.S. Pat. No. 6,424,847 to Mastrototaro et al. Each of the above patents is incorporated herein by reference in its entirety and is not intended to be exhaustive of all applicable analyte sensors, although it should be understood that the disclosed embodiments are generally applicable to a variety of analyte sensor configurations. In other examples of sensor systems including a biological interface / drug-releasing layer(s), the sensor may be a planar or substantially planar sensor.

[0232] Exemplary Multi-Analyte Sensor Membrane Configurations Continuous multi-analyte sensors are provided having various membrane configurations suitable for facilitating simultaneous, intermittent, and / or sequential signal transduction corresponding to analyte concentrations. In one embodiment, such sensors may be configured using signal transducers comprising one or more transducing elements ("TL"). Such continuous multi-analyte sensors may employ various transduction means, such as amperometric, voltammetric, potentiometric, and impedimetric methods, among other techniques.

[0233] In one embodiment, the transduction element comprises one or more membranes that can include one or more layers and / or domains, each of which can independently include one or more signal transducers, such as enzymes, RNA, DNA, aptamers, binding proteins, etc. As used herein, transduction element includes and is used interchangeably with enzymes, ionophores, RNA, DNA, aptamers, and binding proteins.

[0234] In one embodiment, the transducer element resides in one or more membranes, layers, or domains formed over the sensing region. In one embodiment, such a sensor may be constructed using a membrane domain containing one or more enzyme domains, e.g., enzyme domains also referred to as EZ layers ("EZLs"), each of which may contain one or more enzymes. References below to an "enzyme layer" are intended to include all or a portion of the enzyme domain, any of which may be all or a portion of a membrane system as discussed herein, e.g., as a single layer, as two or more layers, as a bilayer pair, or combinations thereof.

[0235] In one embodiment, the continuous multi-analyte sensor uses one or more of the following analyte-substrate / enzyme pairs, e.g., sarcosine oxidase in combination with creatinine amidohydrolase, creatinine amidohydrolase used for sensing creatinine. Other examples of analyte / oxidase enzyme combinations that can be used in the sensing region include, e.g., alcohol / alcohol oxidase, cholesterol / cholesterol oxidase, galactose-galactose / galactose oxidase, choline / choline oxidase, glutamate / glutamate oxidase, glycerol / glycerol-3-phosphate oxidase (or glycerol oxidase), bilirubin / bilirubin oxidase, ascorbic acid / ascorbic acid oxidase, uric acid / uric acid oxidase, pyruvate / pyruvate oxidase, hypoxanthine-xanthine / xanthine oxidase, glucose / glucose oxidase, lactate / lactate oxidase, L-amino acid oxidase, and glycine / sarcosine oxidase. Other analyte-substrate / enzyme pairs may be used, including those containing recombinant enzymes, immobilized enzymes, mediator-wired enzymes, dimerized enzymes, and / or fusion enzymes.

[0236] NAD-based multi-analyte sensor platform Nicotinamide adenine dinucleotide (NAD(P) + NAD(P)H) is a coenzyme, e.g., a dinucleotide consisting of two nucleotides linked through their phosphate groups. One nucleotide contains an adenine nucleobase, and the other contains nicotinamide. NAD exists in two forms, e.g., oxidized (NAD(P)+) and reduced (NAD(P)H) (H=hydrogen). The reaction between NAD+ and NADH is reversible, and therefore the coenzyme is essentially not consumed, and NAD(P) + / and NAD(P)H forms can be continuously cycled between.

[0237] In one example, one or more enzyme domains in the sensing region of a continuous multi-analyte sensor device of the present disclosure include an amount of NAD+ or NADH to provide transduction of a detectable signal corresponding to the presence or concentration of one or more analytes. In one example, one or more enzyme domains in the sensing region of a continuous multi-analyte sensor device of the present disclosure include an excess amount of NAD+ or NADH to provide enhanced transduction of a detectable signal corresponding to the presence or concentration of one or more analytes.

[0238] In one embodiment, NAD, NADH, NAD+, NAD(P)+, ATP, flavin adenine dinucleotide (FAD), magnesium (Mg++), pyrroloquinoline quinone (PQQ), and their functionalized derivatives may be used in combination with one or more enzymes in a continuous multi-analyte sensor device. In one embodiment, NAD, NADH, NAD+, NAD(P)+, ATP, flavin adenine dinucleotide (FAD), magnesium (Mg++), pyrroloquinoline quinone (PQQ), and their functionalized derivatives are incorporated into a sensing region. In one embodiment, NAD, NADH, NAD+, NAD(P)+, ATP, flavin adenine dinucleotide (FAD), magnesium (Mg++), pyrroloquinoline quinone (PQQ), and their functionalized derivatives are dispersed or distributed in one or more membranes or domains of the sensing region.

[0239] In one aspect of the present disclosure, continuous sensing of one or more analytes using NAD+-dependent enzymes is provided in one or more membranes or domains of the sensing region. In one example, the membranes or domains provide a mechanism for the retention and stable recycling of NAD+ and the conversion of NADH oxidation or NAD+ reduction into an amperometrically measurable current. In one example described below, continuous sensing of multiple analytes is provided, at least one of which is either reversibly coupled to or oxidized or reduced by an NAD+-dependent enzyme, such as ketones (beta-hydroxybutyrate dehydrogenase), glycerol (glycerol dehydrogenase), cortisol (11β-hydroxysteroid dehydrogenase), glucose (glucose dehydrogenase), alcohol (alcohol dehydrogenase), aldehydes (aldehyde dehydrogenase), and lactate (lactate dehydrogenase). In other examples described below, membranes are provided that allow for the continuous on-body sensing of multiple analytes that utilize FAD-dependent dehydrogenases, such as fatty acid (acyl-CoA dehydrogenase).

[0240] Exemplary configurations of one or more membranes or portions thereof, configured to provide NAD+ retention and recycling, are provided. Thus, the electrode surface of a conductive wire (coaxial) or a planar conductive surface is coated with at least one layer containing at least one enzyme, as depicted in FIG. 1A. Referring to FIG. 1B, one or more optional layers may be positioned between the electrode surface and one or more enzyme domains. For example, one or more interference domains (also referred to as "interferent-blocking layers") may be used to reduce or eliminate signal contributions from undesired species present, or one or more electrodes (not shown) may be used to aid in wetting, system equilibration, and / or start-up. As shown in FIGS. 1A and 1B, one or more membranes provide an NAD+ reservoir domain, providing a reservoir for NAD+. In one example, one or more interferent-blocking membranes are used, and a potentiostat is utilized to measure HO production or 0 consumption of an enzyme such as or similar to NADH oxidase, where the NAD reservoir and enzyme domain locations can be switched to facilitate better consumption of excess NAD and slower outward diffusion. Exemplary sensor configurations can be found in U.S. Provisional Patent Application No. 63 / 321,340, entitled "CONTINUOUS ANALYTE MONITORING SENSOR SYSTEMS AND METHODS OF USING THE SAME," filed March 18, 2022, and U.S. Provisional Patent Application No. 63 / 291,726, entitled "MEDIATOR-TETHERED NAD(H) FOR KETONE SENSING," filed December 20, 2021, both of which are incorporated herein by reference in their entireties.

[0241] In one embodiment, one or more mediators suitable for NADH oxidation are incorporated into one or more electrode domains or enzyme domains. In one embodiment, organic mediators such as phenanthrolinedione or nitrosoaniline are used. In another embodiment, organometallic mediators such as ruthenium-phenanthrolinedione or osmium(bpy)2Cl, polymers containing covalently bound organic mediators or organometallic coordination mediator polymers, e.g., polyvinylimidizole-Os(bpy)2Cl, or polyvinylpyridine-organometallic coordination mediators (including ruthenium-phenanthrolinedione), are used. Other mediators may be used, as discussed further below.

[0242] In humans, serum levels of beta-hydroxybutyrate (BHB) are typically in the low micromolar range but can rise to approximately 6-8 mM. Serum levels of BHB can reach 1-2 mM after strenuous exercise, and consistent levels above 2 mM are achieved with a low-carbohydrate ketogenic diet. While other ketones, such as acetoacetate and acetone, are present in serum, the majority of the dynamic range in ketone levels is in the form of BHB. Therefore, monitoring BHB, for example, continuously, is useful for providing health information to users or healthcare providers.

[0243] Thus, an exemplary continuous ketone analyte detection method is provided using an electrode-associated mediator / NAD+ / dehydrogenase, e.g., beta-hydroxybutyrate dehydrogenase (HBDH), for continuous monitoring of BHB. In one example, a continuous ketone sensor configuration capable of monitoring BHB is shown in FIG. 21A, in which the mediator / NAD+ / dehydrogenase is present adjacent to the electrode surface 198. Alternatively, for example, multiple enzyme domains can be used in an enzyme layer, with a mediator / NAD+-containing layer being more proximal to the electrode surface than an adjacent enzyme domain containing a dehydrogenase enzyme. In one example, NAD+ and / or HBDH are present in the same or different enzyme domains, both of which can be immobilized using, for example, an amine-reactive crosslinker (e.g., glutaraldehyde, epoxide, NHS ester, imidoester). In one example, NAD+ is conjugated to a polymer and present in the same or different enzyme domain as HBDH. In one embodiment, the molecular weight of NAD+ is increased to prevent or eliminate migration from the sensing region; for example, NAD+ is dimerized using its C6-terminal amine with any amine-reactive crosslinker, or NAD+ is immobilized to a polymer from its C6-terminal amine. In one embodiment, a mediator polymer containing an organic mediator or an organometallic coordination mediator polymer is used, covalently or otherwise operably coupled to an electrode. In other embodiments, NAD+ may be electrografted to an electroactive surface (e.g., a working electrode). In one embodiment, the electrografted NAD+ is enzymatically active. In another embodiment, the electrografted NAD+ is not enzymatically active.

[0244] In some embodiments, the flux of reactants / co-reactants, such as oxygen, through the sensing region has little, if any, effect on the transduced signal. In the above configuration, there is no oxygen consumption or hydrogen peroxide production; rather, there is a direct transfer of electrons from the enzyme to the electrode surface for signal transduction. Therefore, despite endogenous electroactive species such as ascorbate and urate, the need to preferentially attenuate the flux of analytes relative to other reactants, such as oxygen and peroxide, is reduced or eliminated. For example, a homogeneous polymer with a controlled mesh size may be used. In other embodiments, the sensing region includes one or more enzymes that are oxygen-dependent, and oxygen flux is maximized, including, for example, silicone, polysiloxane, or copolymers. Other membranes may be used, such as those described above, positioned between or above the EZL or NAD+ reservoir, e.g., the drug release layer and / or the biointerface layer.

[0245] Another example of a continuous ketone analyte detection configuration using mediator-bound diaphorase / NAD+ / dehydrogenase associated with electrode surface 198 is shown in FIG. 21B.

[0246] In one embodiment, the diaphorase is electrically coupled to an electrode having an organometallic coordination mediator polymer. In another embodiment, the diaphorase is covalently coupled to an electrode having an organometallic coordination mediator polymer.

[0247] Alternatively, multiple enzyme domains can be used in the enzyme layer, for example, separating electrode-associated diaphorase (closest to the electrode surface) from more distal adjacent NAD+ or dehydrogenase enzymes to essentially separate NADH oxidation from analyte (ketone) oxidation. Alternatively, NAD+ can be closer to the electrode surface than adjacent enzyme domains containing dehydrogenase enzymes. In one example, NAD+ and / or HBDH are present in the same or different enzyme domains, both of which can be immobilized using, for example, amine-reactive crosslinkers (e.g., glutaraldehyde, epoxides, NHS esters, imidoesters). In one example, NAD+ is conjugated to a polymer and present in the same or different enzyme domain as HBDH. In one example, the molecular weight of NAD+ is increased to prevent or eliminate migration from the sensing region, for example, NAD+ is dimerized using its C6-terminal amine with any amine-reactive crosslinker. In one example, NAD+ can be covalently attached to an aspect of the enzyme domain that has a higher molecular weight than NAD+, which can improve the stability profile of NAD+ and improve the ability to retain and / or immobilize NAD+ within the enzyme domain, for example, dextran-NAD.

[0248] In yet another example, as shown in FIG. 21C, the transduction signal from the transduction element for a sequential ketone (and one or more other analytes) sensor configuration can be provided using the oxidation of NADH oxidase enzyme to form hydrogen peroxide at the electrode surface 198 as the signal transduction species. In this configuration, an electrode surface 198, membrane, layer, or domain can be used that selectively reduces the flux of analyte and NAD+ while allowing high flux of oxygen to the sensing region. Thus, one or more interference domains, such as NAFION™ or alternating layers of polyallylamine and polyacrylate acid, are used. In one example, NADH and one or more other analyte-specific oxidase enzymes can be present in the same or different enzyme domains, either of which can be immobilized.

[0249] In one embodiment, NAD+ may be bound to or physically trapped within a polymer and reside in the same or a different enzyme domain as HBDH. In one embodiment, the molecular weight of NAD+ is increased to prevent or eliminate migration from the sensing region; for example, NAD+ is dimerized using its C6-terminal amine with an optional amine-reactive crosslinker. In one embodiment, superoxide dismutase (SOD) may be included in the configuration, e.g., in the same enzyme domain as NADH, to scavenge free radicals generated by NADH oxidase, thereby improving signal stability and sensor performance. In the above configuration, the transduced signal is oxygen-dependent, and oxygen flux is maximized, e.g., by using a homogeneous polymer membrane with a controlled mesh size and / or by including silicone, polysiloxane, or copolymers in one or more enzyme domains. In one embodiment, if the signal is oxygen-independent, a resistance domain is used to attenuate the flux of analyte(s) into the EZL so that the sensor response remains linear throughout the physiological range of the target analyte(s). A "target" analyte, as discussed herein, is an analyte intended to be detected by a sensor system as discussed herein. One or more target analytes may be detected and analyzed using a sensor system as discussed herein.

[0250] In one embodiment, the sensing region comprises one or more NADH acceptor oxidoreductases and one or more NAD-dependent dehydrogenases. In one embodiment, the sensing region comprises one or more NADH acceptor oxidoreductases and one or more NAD(P)-dependent dehydrogenases having NAD(P)+ or NAD(P)H as a cofactor present in the sensing region. In one embodiment, the sensing region comprises a quantity of diaphorase.

[0251] In one embodiment, a ketone sensing configuration suitable for combination with another analyte sensing configuration is provided. Thus, an approximately 1-20 μm thick EZL layer is prepared by providing an EZL solution composition in 10 mM HEPES in water with about 20 μL 500 mg / mL HBDH, about 20 μL [500 mg / mL NAD(P)H, 200 mg / mL polyethylene glycol-diglycol ether (PEG-DGE) of about 400 MW], about 20 μL 500 mg / mL diaphorase, and about 40 μL 250 mg / mL polyvinylimidazole-osmium bis(2,2'-bipyridine) chloride (PVI-Os(bpy)2Cl) on a substrate such as a working electrode, so as to provide, after drying, about 15-40 wt% HBDH, about 5-30% diaphorase, about 5-30% NAD(P)H, about 10-50% PVI-Os(bpy)2Cl, and about 1-12% PEG-DGE (400 MW). Substrates discussed herein, which may include a working electrode, may be formed from gold, platinum, palladium, rhodium, iridium, titanium, tantalum, chromium, and / or alloys or combinations thereof, or carbon (e.g., graphite, glassy carbon, carbon nanotubes, graphene, or doped diamond, and combinations thereof).

[0252] The enzyme domain is contacted with a resistance domain, also referred to as a resistance layer ("RL"). In one embodiment, the RL comprises about 55-100% polyvinylpyrrolidone (PVP) and about 0.1-45% PEG-DGE. In another embodiment, the RL comprises about 75-100% PVP and about 0.3-25% PEG-DGE. In yet another embodiment, the RL comprises about 85-100% PVP and about 0.5-15% PEG-DGE. In yet another embodiment, the RL comprises essentially 100% PVP.

[0253] The exemplary continuous ketone sensor depicted in FIGS. 1A and 1B, which includes an NAD(P)H reservoir domain, is configured so that NAD(P)H is not rate-limiting in any of the enzyme domains in the sensing region. In one example, the NAD(P)H loading in the NAD(P)H reservoir domain is greater than about 20%, 30%, 40%, or 50% w / w. One or more membranes or portions of one or more membrane domains (hereinafter also referred to as "membranes") may also contain polymers or protein binders, such as zwitterionic polyurethane and / or albumin. Alternatively, in addition to NAD(P)H, the membrane may contain one or more analyte-specific enzymes (e.g., HBDH, glycerol dehydrogenase, etc.), so that, optionally, the NAD(P)H reservoir membrane also provides catalytic function. In one example, NAD(P)H is dispersed or distributed within or with the polymer (or protein) and may be crosslinked to an extent that still allows proper enzyme / cofactor function and / or reduced NAD(P)H flux within the domain.

[0254] In one embodiment, an NADH oxidase enzyme, alone or in combination with a superoxide dismutase (SOD), is used in one or more membranes of the sensing region. In one embodiment, an amount of superoxide dismutase (SOD) capable of scavenging a portion or a majority of one or more free radicals generated by the NADH oxidase is used. In one embodiment, the NADH oxidase enzyme, alone or in combination with the superoxide dismutase (SOD), is used in combination with NAD(P)H and / or a functionalized polymer having NAD(P)H immobilized on the polymer from the C6-terminal amine in one or more membranes of the sensing region.

[0255] In one example, NAD(P)H is immobilized to a degree that maintains NAD(P)H catalytic function. In one example, dimerized NAD(P)H is used to capture NAD(P)H within one or more membranes by crosslinking their respective C6-terminal amines with a suitable amine-reactive crosslinker, such as glutaraldehyde or PEG-DGE or a polycarbodiimide crosslinker.

[0256] The above-described continuous ketone sensor configurations may be adapted for other analytes or used in combination with other sensor configurations. For example, analyte-dehydrogenase enzyme combinations may be used in any of the membranes in the sensing region, including glycerol (glycerol dehydrogenase), cortisol (11β-hydroxysteroid dehydrogenase), glucose (glucose dehydrogenase), alcohol (alcohol dehydrogenase), aldehyde (aldehyde dehydrogenase), and lactate (lactate dehydrogenase).

[0257] In one embodiment, a semipermeable membrane is used in or adjacent to the sensing region, or adjacent to one or more membranes in the sensing region, to attenuate the flux of at least one analyte or chemical species. In one embodiment, the semipermeable membrane attenuates the flux of at least one analyte or chemical species to provide a linear response from the transduced signal. In another embodiment, the semipermeable membrane prevents or eliminates the efflux of NAD(P)H from the sensing region or any membrane or domain. In one embodiment, the semipermeable membrane can be an ion-selective membrane selective for an ionic analyte of interest, such as ammonium ion.

[0258] In another example, a continuous multi-analyte sensor configuration containing one or more enzymes and / or at least one cofactor was prepared. FIG. 1C depicts this exemplary configuration of an enzyme domain 150 containing an enzyme with a quantity of cofactor located proximate at least a portion of the working electrode ("WE") surface, where the WE comprises an electrochemically reactive surface. In one example, a second membrane 151 containing a quantity of cofactor is located adjacent to the first enzyme domain. The amount of cofactor in the second membrane can provide an excess amount for the enzyme, for example, to extend sensor lifetime. One or more resistor domains 152 ("RL") are located adjacent to (or can be between) the second membrane. The RL can be configured to block diffusion of the cofactor from the second membrane. Electron transfer from the cofactor to the WE transducees a signal that corresponds directly or indirectly to the analyte concentration.

[0259] 1D shows an alternative enzyme domain configuration that includes a first membrane 151 with a quantity of cofactor located more proximally for at least a portion of the WE surface. An enzyme domain 150 containing a quantity of enzyme is located adjacent to the first membrane.

[0260] In the membrane configurations depicted in Figures 1C and 1D, production of electrochemically active species in the enzyme domain diffuses to the WE surface and transduces a signal that directly or indirectly corresponds to the analyte concentration. In some embodiments, the electrochemically active species includes hydrogen peroxide. In sensor configurations that include a cofactor, the cofactor from the first layer can diffuse to the enzyme domain, e.g., to extend sensor lifetime by regenerating the cofactor. In other sensor configurations, a cofactor can optionally be included to improve performance attributes such as stability. For example, a continuous ketone sensor utilizes NAD(P)H and Mg +2The WE surface may include a divalent metal cation such as . One or more resistive domains RL may be located adjacent to the second membrane (or may be between layers). The RL may be configured to block the diffusion of cofactors and / or interferents from the second membrane from reaching the WE surface. Other configurations, such as electrodes, resistors, biointerfaces, and drug-releasing membranes, layers, or domains, may be used in the foregoing configurations. In other examples, the continuous analyte sensor includes one or more cofactors that contribute to sensor performance.

[0261] FIG. 1E depicts another continuous multi-analyte membrane configuration in which a {beta}-hydroxybutyrate dehydrogenase (BHBDH) in a first enzyme domain 153 is located proximal to the working electrode WE, and a second enzyme domain 154, e.g., comprising alcohol dehydrogenase (ADH) and NADH, is located adjacent to the first enzyme domain. One or more resistor domains RL152 may be deployed adjacent to the second enzyme domain 154. In this configuration, the combined presence of alcohol and ketone in serum collectively serves to provide a transduced signal corresponding to at least one of the analyte concentrations, e.g., ketone. Thus, as NADH present in the more distal second enzyme domain consumes alcohol present in the serum environment, NADH is oxidized to NAD(P)H, which diffuses to the first membrane layer, providing BHBDH-catalyzed electron transfer of acetoacetate ketone and transduction of a detection signal corresponding to the concentration of ketone. In one example, an enzyme can be configured for reverse catalysis, creating a substrate that can be used for catalysis of another enzyme present in either the same or a different layer or domain. Other configurations, such as electrodes, resistors, biointerfaces, and drug-releasing membranes, layers, or domains, can be used in the above configurations. Thus, a first enzyme domain that is more distal from the WE than a second enzyme domain can be configured to generate a cofactor or other element that acts as a reactant (and / or reactant substrate) for the second enzyme domain to detect one or more target analytes.

[0262] Combinations of the above configurations can be employed. Figures 1F and 1G show experimental data and linear regressions, respectively, for an exemplary continuous ketone sensor configuration spanning a ketone range of 0 mM to 8 mM, providing continuous linear response and signal stability ex vivo.

[0263] Figures 1H, 1I, and 1J show experimental data (0 to 5 mM ketone) demonstrating the sensitivity, calibration, and drift (20 hours at 5 mM) of an exemplary continuous ketone sensor configuration using NAD+ cofactor and NADH oxidase 170, 172, respectively, from different natural sources, demonstrating continuous linear response and signal stability. Data 199 shown in Figures 1H-1J represent a sample containing no cofactor (BHBDH only). In one example, a mutant NAD+ cofactor is used to improve cofactor retention in one or more membranes, providing improved covalent or non-covalent binding of the cofactor to one or more membranes of the continuous ketone sensor.

[0264] Alcohol sensor configuration In one embodiment, a continuous alcohol (e.g., ethanol) sensor device configuration is provided, in which one or more enzyme domains including alcohol oxidase (AOX) are provided, where the presence and / or amount of alcohol is converted by the creation of hydrogen peroxide, alone or in combination with an oxygen-consuming or another substrate-oxidase enzyme system, e.g., glucose-glucose oxidase, and the hydrogen peroxide and / or oxygen and / or glucose can be detected and / or measured qualitatively or quantitatively using amperometry.

[0265] In one example, the sensing region for the enzyme substrate-oxidase enzyme configuration comprises one or more enzyme domains with one or more electrodes. In one example, the sensing region for the enzyme substrate-oxidase enzyme configuration comprises one or more enzyme domains with or without one or more electrodes, and further comprises one or more interference-blocking membranes (e.g., permselective membranes, charge-exclusion membranes) to attenuate diffusion of one or more interferents through the membrane to the working electrode. In one example, the sensing region for the substrate-oxidase enzyme configuration comprises one or more enzyme domains with or without one or more electrodes, and further comprises one or more resistance domains with or without one or more interference-blocking membranes to attenuate diffusion of one or more analytes or enzyme substrates. In one example, the sensing region for the substrate-oxidase enzyme configuration comprises one or more enzyme domains with or without one or more electrodes, and one or more resistance domains with or without one or more interference-blocking membranes to attenuate diffusion of one or more analytes or enzyme substrates, and further comprises one or more biointerface membranes and / or drug-releasing membranes, independently, to attenuate a host immune response after insertion.

[0266] In one embodiment, one or more interference-blocking films are deposited adjacent to the working electrode and / or electrode surface. In one embodiment, one or more interference-blocking films are deposited directly adjacent to the working electrode and / or electrode surface. In one embodiment, one or more interference-blocking films are deposited between another layer, film, or domain adjacent to the working electrode or electrode surface to attenuate one or all analytes diffusing through the sensing region, except for oxygen. Such films can be used not only to attenuate alcohol itself, but also to attenuate other electrochemically active species or other analytes that may interfere by producing a signal if they diffuse to the working electrode.

[0267] Figure 2 shows experimental data from a non-limiting example of an alcohol sensor comprising alcohol oxidase in one or more layers coated with one or more interference films. In one example, the working electrode used comprised platinum, and the applied potential was approximately 0.5 volts. As shown in Figure 2, the signal response (in amperes) versus time (in seconds) for stepwise addition of alcohol at concentrations from 0 mg / dL to 200 mg / dL of ethanol is plotted. Figure 2 demonstrates substantial linearity of the current response from 1 to approximately 200 mg / dL, which represents at least a portion of the pharmacological range of alcohol for humans.

[0268] In one example, electrochemical sensing of oxygen level changes can be performed, for example, using a Clark-type electrode setup or in a different configuration, for example, by coating the electrodes with one or more membranes of one or more polymers, such as NAFION™. Based on the change in potential, the change in oxygen concentration can be recorded, which is directly or indirectly correlated with the concentration of alcohol. When properly designed to follow stoichiometric behavior, the presence of a specific concentration of alcohol should cause a corresponding reduction in local oxygen in a direct (linear) relationship with the concentration of alcohol. Thus, a multi-analyte sensor for both alcohol and oxygen can thus be provided.

[0269] In another embodiment, the alcohol-sensing configuration described above can include one or more secondary enzymes that react with a reaction product of alcohol / alcohol oxidase catalysis, e.g., hydrogen peroxide, to provide an oxidized form of the secondary enzyme that converts an alcohol-dependent signal to WE / RE at a lower potential than in the absence of the secondary enzyme. Thus, in one embodiment, the alcohol / alcohol oxidase is used in conjunction with a reduced form of a peroxidase, e.g., horseradish peroxidase. The alcohol / alcohol oxidase can be in the same or a different layer as the peroxidase, or they can be spatially separated distally from the electrode surface, e.g., the alcohol / alcohol oxidase being more distal from the electrode surface and the peroxidase being closer to the electrode surface, or alternatively, the alcohol / alcohol oxidase being more proximal from the electrode surface and the peroxidase being more distal from the electrode surface. In one embodiment, the alcohol / alcohol oxidase more distal to the electrode surface and peroxidase further comprises any combination of electrodes, interferences, resistors, and biointerface membranes to optimize signal, durability, reduce drift, or extend end-of-life duration.

[0270] In another embodiment, the alcohol sensing configuration described above can include one or more mediators. In one embodiment, the one or more mediators are present in, on, or around one or more electrodes or electrode surfaces and / or deposited on or otherwise associated with the surface of a working electrode (WE) or reference electrode (RE). In one embodiment, the one or more mediators eliminate or reduce direct oxidation of interfering species that may reach the WE or RE. In one embodiment, the one or more mediators reduce the operating potential of the WE / RE to, for example, about 0.6 V to about 0.3 V or less on a platinum electrode, thereby reducing or eliminating oxidation of endogenous interfering species. Examples of the one or more mediators are provided below. Other electrodes, such as counter electrodes, can be used.

[0271] In one example, other enzymes or additional components can be added to the polymer mixture comprising any portion of the sensing region to increase the stability of the aforementioned sensor and / or reduce or eliminate by-products of the alcohol / alcohol oxidase reaction. Increased stability includes storage or shelf life and / or operational stability (e.g., retention of enzyme activity during use). For example, by-products of the enzymatic reaction may be undesirable for increased shelf life and / or operational stability and, therefore, may be desirable to reduce or eliminate. In one example, xanthine oxidase can be used to remove by-products of one or more enzymatic reactions.

[0272] In another embodiment, a dehydrogenase enzyme is used in conjunction with an oxidase for the detection of alcohol alone or in combination with oxygen. Thus, in one embodiment, alcohol dehydrogenase is used to oxidize alcohol to an aldehyde in the presence of reduced nicotinamide adenine dinucleotide (NAD(P)H) or reduced nicotinamide adenine dinucleotide phosphate (NAD(P)+). To provide a continuous source of NAD(P)H or NAD(P)+, NADH oxidase or NADPH oxidase is used to oxidize NAD(P)H or NAD(P)+ while consuming oxygen. In another embodiment, diaphorase can be used in place of or in combination with NADH oxidase or NADPH oxidase. Alternatively, an excess amount of NAD(P)H can be incorporated into one or more enzyme domains and / or one or more electrodes in an amount to accommodate the intended duration of the sensor's planned lifespan.

[0273] In the dual-enzyme configuration described above, the signal can be sensed by either (1) electrically coupled (e.g., "wired") alcohol dehydrogenase (ADH), for example, using an electroactive hydrogel polymer containing one or more mediators, or (2) oxygen electrochemical sensing to measure oxygen consumption by NADH oxidase. In an alternative embodiment, the cofactor NAD(P)H or NAD(P)+ can be conjugated to a polymer, such as dextran, which is immobilized along with ADH in the enzyme domain. This provides for retention of the cofactor and its availability to the active site of ADH. In the above embodiments, any combination of electrodes, interferometers, resistors, and biointerface membranes can be used to optimize signal, durability, reduce drift, or extend end-of-life. In one embodiment, direct or indirect electrical coupling via covalent or ionic bonds to at least a portion of the transducer element, such as an aptamer, enzyme, or cofactor, and at least a portion of the electrode surface is provided. Any chemical moiety capable of assisting electron transfer from the enzyme or cofactor to the electrode surface can be used, including one or more mediators as described below.

[0274] In one embodiment, any one of the aforementioned continuous alcohol sensor configurations is combined with any one of the aforementioned continuous ketone monitoring configurations to provide a continuous multi-analyte sensor device as described further below. In one embodiment, a continuous glucose monitoring configuration is combined with any one of the aforementioned continuous alcohol sensor configurations and any one of the aforementioned continuous ketone monitoring configurations to provide a continuous multi-analyte sensor device as described further below.

[0275] Uric acid sensor configuration In another embodiment, a continuous uric acid sensor device configuration is provided. Thus, in one embodiment, uric acid oxidase (UOX) can be included in one or more enzyme domains and positioned adjacent to the working electrode surface. Catalysis of uric acid using UOX produces hydrogen peroxide, which can be detected using amperometric, voltammetric, and impedance measurement methods, among other techniques. In one embodiment, to reduce or eliminate interference from direct oxidation of uric acid on the electrode surface, one or more electrode, interference, and / or resistance domains can be deposited on at least a portion of the working electrode surface. Such a membrane can be used to attenuate diffusion of uric acid and other analytes to the working electrode, which could interfere with signal transmission.

[0276] Figures 3A and 3B show experimental data from a continuous uric acid sensor configuration. In this example, a platinum working electrode is coated with at least one interference film, on which at least one enzyme domain containing UOX is located. The enzyme domain is covered with one or more resistive domains to control or attenuate diffusion characteristics. As shown in Figure 3A, the exemplary sensor exhibits sensitivity in the physiological concentration range, stability against drift (8 mg / dL UA, 14 days at 37°C, Figure 3B), and high reproducibility.

[0277] In an alternative embodiment, the uric acid continuous sensing device configuration includes sensing oxygen level changes around the WE surface, as in a Clark-type electrode setup, or one or more electrodes may independently include one or more different polymers, such as NAFION™, zwitterionic polymers, or polymeric mediators adjacent to at least a portion of the electrode surface. In one embodiment, an electrode surface having one or more electrode domains provides operation at different or lower voltages to measure oxygen. Oxygen levels and their changes can be sensed, recorded, and correlated to uric acid concentrations, for example, based on using conventional calibration methods.

[0278] In one embodiment, one or more coatings can be deposited on the WE surface to lower the potential at the WE for uric acid signaling, either alone or in combination with any of the aforementioned uric acid sensor configurations. The one or more coatings can be deposited or otherwise formed on the WE surface and / or on other coatings formed thereon using various techniques, including, but not limited to, immersion, electrodeposition, vapor deposition, spray coating, etc. In one embodiment, the coated WE surface can provide for a redox reaction, for example, of hydrogen peroxide, at a lower potential (compared to 0.6 V on a platinum electrode surface without such a coating). Examples of materials that can be coated or annealed on the WE surface include, but are not limited to, Prussian blue, Medola blue, methylene blue, methylene green, methyl viologen, ferrocyanide, ferrocene, cobalt ions, and cobalt phthalocyanine.

[0279] Referring to Figure 3C, a graphical representation of uric acid signaling in the presence of added hydrogen peroxide using bare and coated electrodes is shown. In the graph, the addition of hydrogen peroxide is detected by all electrodes. However, the addition of uric acid is detected by the "bare" platinum electrode (d), as opposed to (a) platinum with electrodeposited Prussian blue, (b) gold with electrodeposited Prussian blue, and (c) carbon with electrodeposited Prussian blue, demonstrating the selectivity of this electrode / enzyme domain configuration. In the above examples, any combination of electrodes, interferometers, resistors, and biointerface membranes can be configured to further optimize signal, durability, reduce drift, or extend end-of-life duration.

[0280] In one embodiment, one or more secondary enzymes, cofactors, and / or mediators (electronically coupled or polymeric mediators) can be attached to the UOX-bearing enzyme domain to facilitate direct or indirect electron transfer to the WE. In such a configuration, for example, regeneration of the initial oxidized form of the secondary enzyme is reduced by the WE for signal transduction. In one embodiment, the secondary enzyme is horseradish peroxidase (HRP).

[0281] Choline Sensor Configuration In one embodiment, a continuous choline sensor device can be provided, for example, using a choline oxidase enzyme to generate hydrogen peroxide by oxidation of choline. Thus, in one embodiment, at least one enzyme domain includes choline oxidase (COX) adjacent to at least one WE surface, optionally with one or more electrodes and / or interference membranes positioned between the WE surface and the at least one enzyme domain. Catalysis of choline with COX results in the creation of hydrogen peroxide, which can be detectable using amperometric, voltammetric, and impedimetric methods, among other techniques.

[0282] Figure 4 shows experimental data for a functional choline sensor configuration. In this example, a first enzyme domain is formed across one or more interference membranes adjacent to a platinum WE. The first enzyme domain, containing choline oxidase, is covered with one or more resistive domains to control diffusion characteristics. The data demonstrate that the choline sensor can provide sensitivity in the physiological concentration range with thermal operational stability over drift (37°C) and high reproducibility.

[0283] In one example, the continuous choline sensor configuration described above is combined with any one of the continuous alcohol and uric acid sensor configurations described above to provide a continuous multi-analyte sensor device as described further below. This continuous multi-analyte sensor device may further include continuous glucose monitoring capabilities. Other membranes may be used in the continuous choline sensor configuration described above, such as electrodes, resistors, biointerfaces, and drug-releasing membranes.

[0284] Cholesterol sensor configuration In one example, a continuous cholesterol sensor configuration can be created using cholesterol oxidase (CHOX), similar to the sensors described above. Thus, one or more enzyme domains containing CHOX can be located adjacent to at least one WE surface. Catalysis of free cholesterol with CHOX results in the creation of hydrogen peroxide, which can be detectable using amperometric, voltammetric, and impedimetric methods, among other techniques.

[0285] An exemplary cholesterol sensor configuration using a platinum WE was prepared, in which at least one interference film was located adjacent to at least one WE surface, on which at least one enzyme domain containing CHOX was located, and on which at least one resistance domain for controlling diffusion characteristics was located. Figure 5 shows data demonstrating that the above cholesterol sensor configuration can provide sensitivity in the physiological concentration range with thermal operational stability over drift (37°C) and high reproducibility.

[0286] The method described above and the cholesterol sensor described above can measure free cholesterol, but with modifications, this configuration can measure more types of cholesterol and total cholesterol concentrations. Measuring different types of cholesterol and total cholesterol is important because a significant amount of cholesterol is in unmodified and esterified forms due to the low solubility of cholesterol in water. Thus, in one embodiment, a total cholesterol sample is provided in which a secondary enzyme has been introduced into at least one enzyme domain, for example, to provide a combination of cholesterol esterase and CHOX cholesteryl ester, which is converted from the cholesterol present and essentially represents the total cholesterol, which can be indirectly measured from the signal formed by the esterase.

[0287] In one example, the continuous (total) cholesterol sensor configuration described above is combined with any one of the continuous alcohol sensor configuration and / or continuous uric acid sensor configuration described above to provide a continuous multi-analyte sensor system as described further below. This continuous multi-analyte sensor device can further include continuous glucose monitoring capability. Other membrane configurations can be used in the continuous cholesterol sensor configuration described above, such as one or more electrode domains, resistive domains, biointerface domains, and drug-releasing membranes.

[0288] Configuration of bilirubin sensor and ascorbic acid sensor In one embodiment, continuous bilirubin and ascorbic acid sensors are provided. These sensors can use bilirubin oxidase and ascorbic acid oxidase, respectively. However, unlike some oxidoreductase enzymes, the end product of analyte catalysis by bilirubin oxidase and ascorbic acid oxidase is water instead of hydrogen peroxide. Therefore, hydrogen peroxide redox detection to correlate with bilirubin or ascorbic acid is not possible. However, these oxidase enzymes still consume oxygen for catalysis, and the level of oxygen consumption correlates with the level of target analyte present. Therefore, bilirubin and ascorbic acid levels can be measured indirectly by electrochemically sensing changes in oxygen levels, for example, in a Clark-type electrode setup.

[0289] Alternatively, different configurations for sensing bilirubin and ascorbic acid can be used. For example, an electrode domain containing one or more electrode domains containing an electron transfer agent, such as NAFION™, a zwitterionic polymer, or a polymeric mediator, can be coated on the electrode. Measured oxygen levels converted from such enzyme domain configurations can be correlated with bilirubin concentrations and ascorbic acid levels. In one example, an electrode domain containing one or more mediators electrically coupled to the working electrode can be used and correlated with bilirubin and ascorbic acid levels.

[0290] In one example, the continuous bilirubin and ascorbic acid sensor configuration described above can be combined with any one of the continuous alcohol, uric acid, and cholesterol sensor configurations described above to provide a continuous multi-analyte sensor device as described further below. This continuous multi-analyte sensor device can further include continuous glucose monitoring capabilities. Other membranes can be used in the continuous bilirubin and ascorbic acid sensor configuration described above, such as electrodes, resistors, biointerfaces, and drug-releasing membranes.

[0291] One working electrode configuration for dual-analyte detection In one embodiment, at least a dual enzyme domain configuration is provided, with each layer containing one or more specific enzymes and, optionally, one or more cofactors. Broadly speaking, one example of a continuous multi-analyte sensor configuration is depicted in FIG. 6A , in which a first membrane 155 (EZL1) containing at least one enzyme (Enzyme 1) of at least two enzyme domain configurations is proximal to at least one surface of a WE. One or more analyte-substrate enzyme pairs with Enzyme 1 transduce at least one detectable signal to the WE surface by direct or mediated electron transfer, which corresponds directly or indirectly to the analyte concentration. A second membrane 156 (EZL2) containing at least one second enzyme (Enzyme 2) is located adjacent to 155EZL1 and is generally more distal from the WE than EZL1. One or more resistance domains (RL) 152 may be provided adjacent to EZL2 156 and / or between EZL1 155 and EZL2 156. Although the different enzymes catalyze the conversion of the same analyte, at least one enzyme in EZL2 156 provides hydrogen peroxide and at least one other enzyme in EZL1 155 does not provide hydrogen peroxide. Thus, each measurable species (e.g., hydrogen peroxide and other measurable species that are not hydrogen peroxide) generates a signal associated with its concentration.

[0292] For example, in the configuration shown in Figure 6A, a first analyte diffuses through RL 152 into EZL2 156 and, through interaction with Enzyme 2, produces peroxide. The peroxide diffuses through at least EZL1 155 to the WE, transducing a signal that corresponds directly or indirectly to the first analyte concentration. A second analyte, different from the first analyte, diffuses through RL 152 and EZL2 156 and interacts with Enzyme 1, which results in electron transfer to the WE, transducing a signal that corresponds directly or indirectly to the second analyte concentration.

[0293] As shown in Figure 6B, the above configuration can be adapted to a conductive wire electrode construction in which at least two different enzyme-containing layers are constructed on the same WE with a single active surface. In one example, the single WE is a wire, with the active surface located around the longitudinal axis of the wire. In another example, the single WE is a conductive trace on a substrate, with the active surface located around the longitudinal axis of the trace. In one example, the active surface is substantially continuous around the longitudinal axis or radius.

[0294] In the above configuration, at least two different enzymes are used that can catalyze the conversion of different analytes, with at least one enzyme in EZL2 156 providing hydrogen peroxide and at least one other enzyme in EZL1 155 not providing hydrogen peroxide, e.g., providing electron transfer to the WE surface that corresponds directly or indirectly to the concentration of the analyte.

[0295] In one embodiment, the inner layer of at least two enzyme domains EZL1, EZL2 155, 156 includes at least one immobilized enzyme combined with at least one mediator that can facilitate lower bias voltage operation of the WE than without the mediator. In one embodiment, a potential P1 is used for such direct electron conversion. In one embodiment, at least a portion of the inner layer EZL1 155 may have one or more intervening electrode domains and / or overlying interfering and / or biological interfaces and / or drug-releasing membranes, provided that they are closer to the WE surface and at least one mediator can facilitate lower bias voltage operation with the WE surface. In another embodiment, at least a portion of the inner layer EZL1 155 is directly adjacent to the WE.

[0296] The second layer (outer layer EZL2 156) of at least the dual enzyme domain in FIG. 6B contains at least one enzyme that catalyzes one or more catalytic reactions, ultimately producing hydrogen peroxide in an amount capable of electrochemically converting a signal corresponding to the concentration of an analyte(s). In one example, the generated hydrogen peroxide diffuses through layer EZL2 156 and through inner layer EZL1 155 to reach the WE surface and undergo oxidation-reduction at a potential P2, where P2 ≠ P1. In this manner, electron transfer and electrolysis (redox) can be selectively controlled by controlling the potentials P1 and P2 applied to the same WE surface. Any applied potential duration, e.g., equal / periodic duration, staggered duration, random duration, as well as various potential difference sequences, cyclic voltammetry, etc., can be used for P1 and P2. In some examples, impedimetric sensing can be used. In one example, a phase shift (e.g., a time lag) can result from detecting two signals from two different working electrodes, each signal generated by a different EZL (EZL1, EZL2, 155, 156) associated with each electrode. The two (or more) signals can be decomposed into components to detect the individual signals and signal artifacts generated by each of EZL1 155 and EZL2 156 in response to the detection of the two analytes. In some examples, each EZL detects a different analyte. In other examples, both EZLs detect the same analyte.

[0297] In another alternative exemplary configuration, the multienzyme domain configuration described above is provided for a continuous multi-analyte sensor device using a single WE with two or more active surfaces, as shown in Figures 6C and 6D. In one example, the multienzyme domain configuration discussed herein is formed on a planar substrate. In another example, the single WE is coaxial, e.g., configured as a wire, with two or more active surfaces positioned around the longitudinal axis of the wire. The additional wire can be used, for example, as a reference electrode and / or counter electrode. In another example, the single WE is a conductive trace on a substrate, with two or more active surfaces positioned around the longitudinal axis of the trace. At least a portion of the two or more active surfaces are discontinuous, providing at least two physically separated WE surfaces on the same WE wire or trace (e.g., WE1, WE2). In one example, the first analyte detected by WE1 is glucose, and the second analyte detected by WE2 is lactate. In another example, the first analyte detected by WE1 is glucose and the second analyte detected by WE2 is a ketone.

[0298] 6C and 6D show an exemplary configuration of a continuous multi-analyte sensor construct in which EZL1 155, EZL2 156, and RL 152 (resistance domains) as described above are disposed, e.g., by sequential dip-coating techniques, on a single coaxial wire containing spatially separated electrode surfaces WE1 and WE2. One or more parameters of the enzyme domain, resistance domain, etc., can be independently controlled along the longitudinal axis of the WE, e.g., thickness, length along the axis from the distal end of the wire, etc. In one example, at least a portion of the spatially separated electrode surfaces are of the same composition. In another example, at least a portion of the spatially separated electrode surfaces are of different compositions. In FIGS. 6C and 6D, WE1 represents a first working electrode surface, e.g., configured to operate at P1, electrically isolated from a second working electrode surface, WE2, configured to operate at P2, and RE represents a reference electrode RE electrically isolated from both WE1 and WE2. In the configuration of FIG. 6C, one resistive domain is provided covering the reference electrode and WE1 and WE2. In the configuration of FIG. 6D, an additional resistive domain is provided that extends essentially only over WE2. Additional electrodes, such as a counter electrode, can be used. Such configurations (whether single-wire or dual-wire) can also be used to measure the same analyte using two different techniques. Data collected from two different measurement modes using different signal generation sequences and different RLs provides increased fidelity, improved performance, and device life. A non-limiting example is a glucose oxidase (H2O2-producing) and glucose dehydrogenase (electrically coupled) configuration. Measuring glucose from two different electrodes at two potentials provides more data points and precision. Such an approach may not be required for glucose sensing, but can be applied across the biomarker sensing spectrum for other analytes, alone or in combination with glucose sensing, such as ketone sensing, ketone / lactate sensing, and ketone / glucose sensing.

[0299] In an alternative configuration to that shown in FIGS. 6C and 6D , two or more wire electrodes are presented, which may be collinear, wrapped, or otherwise juxtaposed, with WE1 separated from WE2, e.g., another elongated electrode. An insulating layer electrically insulates WE1 from WE2. In this configuration, independent electrode potentials may be applied to corresponding electrode surfaces, and independent electrode potentials may be provided to WE1 and WE2 simultaneously, sequentially, or randomly. In one embodiment, the electrode potentials provided to corresponding electrode surfaces WES1 and WES2 are different. One or more additional electrodes, such as a reference electrode and / or counter electrode, may be present. In one embodiment, WES2 is positioned longitudinally distal from WES1 in an elongated configuration. For example, using a dip-coating method, WES1 and WES2 are coated with the enzyme domain EZL1, while WES2 is coated with a different enzyme domain EZL2. Multilayer enzyme domains, each layer independently containing a different loading and / or composition of enzyme and / or cofactor, mediator, or mediator, can be employed based on immersion parameters or different thicknesses of the enzyme domain. Similarly, one or more resistance domains (RLs) can be applied, each of different thicknesses along the longitudinal axis of the electrode, across different electrodes and enzyme domains, for example, by controlling immersion length and other parameters. Referring to Figure 6D, such an arrangement of RLs is depicted, with an additional RL 152' adjacent to WES2 but substantially absent from WES1.

[0300] In one example for measuring two different analytes, the configuration includes an enzyme domain EZL1 155 containing one or more enzymes and one or more mediators for at least one enzyme in EZL1 to provide direct electron transfer to WES1, thereby determining the concentration of at least a first analyte. Additionally, an enzyme domain EZL2 156 can include at least one enzyme that provides peroxide (e.g., hydrogen peroxide) or consumes oxygen during catalytic reaction with its substrate. The peroxide or oxygen produced in EZL2 156 migrates to WES2, providing a detectable signal directly or indirectly corresponding to the second analyte. For example, WES2 can be carbon wired to glucose dehydrogenase to measure glucose, while WES1 can be platinum to measure peroxide produced from lactate oxidase / lactate in EZL2 156. The combinations of electrode materials and enzymes disclosed herein are exemplary and non-limiting.

[0301] In one example, the P1 and P2 potentials can be separated by an amount of potential such that both signals (from direct electron transfer from EZL1 155 and from hydrogen peroxide oxidation-reduction at the WE) can be activated and measured separately. In one example, the sensor electronics module can continuously switch between the two sensing potentials in a continuous or semi-continuous cyclic manner, e.g., for a period (t1) at potential P1 and a period (t2) at potential P2, optionally with a rest period during which no potential is applied. The extracted signals can then be analyzed to measure the concentrations of two different analytes. In another example, the sensor electronics module can undergo cyclic voltammetry, and the change in current when swiping across the P1 and P2 potentials can be correlated to the transduced signal coming from either direct electron transfer or hydrogen peroxide electrolysis, respectively. In one example, the sensing modality is non-limiting and can include different amperometric techniques, e.g., cyclic voltammetry. In one embodiment, an alternative configuration is provided in which hydrogen peroxide production in EZL2 is replaced by another suitable electrolytic compound that maintains the P2≠P1 relationship, such as oxygen, and at least one enzyme-substrate combination that provides the other electrolytic compound.

[0302] For example, a continuous multianalyte sensor configuration for choline and glucose was prepared in which enzyme domains EZL1 155 and EZ2 156 were associated with different WEs, e.g., platinum WE2 and gold WE1. In this exemplary case, EZL1 155 contained glucose oxidase and a mediator bound to WE1 to facilitate direct electron transfer during glucose catalysis, and EZL2 156 contained choline oxidase, which catalyzed choline to generate hydrogen peroxide for electrolysis at WE2. The EZLs were coated with resistive domains, and upon curing and preparation, they underwent cyclic voltammetry in the presence of glucose and choline. The glucose oxidase enzyme wired to the gold electrode was capable of transducing a signal at 0.2 volts; therefore, by analyzing the current change at 0.2 volts, glucose concentration could be determined. The data also demonstrate that choline concentration can also be inferentially detected at the WE2 platinum electrode when the CV trace is analyzed at voltage P2.

[0303] In one example, either electrode WE1 or WE2 can be a composite material, such as a gold electrode with platinum ink deposited on top, a carbon / platinum mixture, and / or a trace of carbon on top of platinum, or a porous carbon coating on a platinum surface. In one example, an electrode surface can contain two different materials, such as a wired enzyme and carbon used for electron transfer, while platinum is used for hydrogen peroxide oxidation-reduction and detection. As shown in FIG. 6E, an example of such a composite electrode surface is shown, in which an extended platinum-coated wire 157 is half-coated with carbon 158 to facilitate multiple sensing on two different surfaces of the same electrode. In one example, WE2 can be grown on or extend from a portion of the surface or distal end of WE1, for example, by evaporation, sputtering, or electrolytic deposition.

[0304] Further examples include composite electrode materials that can be used to form one or both of WE1 and WE2. In one example, a platinum-carbon electrode WE1 containing EZL1 with glucose dehydrogenase is wired to the carbon surface, and an outer electrode EZL2 contains lactate oxidase, which produces hydrogen peroxide detectable by the platinum surface of the same WE1 electrode. Another example of this configuration can include ketone sensing (electrolytically coupled beta-hydroxybutyrate dehydrogenase enzyme in EZL1 155) and glucose sensing (glucose oxidase in EZL2 156). Other membranes can be used in the aforementioned configurations, such as electrodes, resistors, biointerfaces, and drug-releasing membranes. In other examples, one or both of the working electrodes (WE1, WE2) can be gold-carbon (Au-C), palladium-carbon (Pd-C), iridium-carbon (Ir-C), rhodium-carbon (Rh-C), or ruthenium-carbon (Ru-C). In some examples, the carbon in the working electrodes discussed herein may alternatively or additionally comprise other materials suitable for forming working electrodes, such as graphene, graphene oxide, or commercially available carbon inks.

[0305] Figure 6F graphically depicts the sweep traces obtained for the configuration shown in Figure 6B, plotting the current at 0.7 V for each swipe of the CV. Glucose was spiked from 50 to 200 mg / dL, followed by choline from 0.5 to 14.5 mg / dL. At 0.7 V, the data show that the platinum WE2 electrode (n = 4) is sensitive to both glucose and choline, while the trace from the gold WE1 electrode (n = 4) shows only a change in current at 0.7 V upon choline spiking. This change may be due to the formation of hydrogen peroxide in the electrically uncoupled outer EZL2 layer 156 and partial detection of hydrogen peroxide at the gold electrode. Nevertheless, in combination with the detection of glucose at P1 (0.2 V) on the gold WE1 electrode, both glucose and choline can be detected using a continuous multi-analyte sensor.

[0306] In one example, first layer EZL 1155 includes an oxidase enzyme that does not produce hydrogen peroxide. Such enzymes include, but are not limited to, lactate dehydrogenase, glucose dehydrogenase, beta-hydroxybutyrate dehydrogenase, diaphorase, and the like. In one example, these dehydrogenase enzymes are wired to at least a portion of the WE1 electrode to reduce or eliminate crosstalk, reduce potential, and minimize or eliminate interfering signals. In one example, EZL 1155 can include any enzyme capable of providing electron transfer while wired or covalently attached to the electrode surface or in the presence of any type of redox mediator, and EZL 2156 can include any oxidoreductase enzyme that produces hydrogen peroxide or other suitable compounds that undergo oxidation-reduction or electrolysis at the electrode surface at an applied potential.

[0307] In one example, the continuous creatinine sensor configuration described above may be combined with any one of the continuous alcohol, uric acid, cholesterol, bilirubin / ascorbic acid, ketone, ketone and glucose, or ketone and lactate, and other sensor configurations described above to provide a continuous multi-analyte sensor device as described further below, which may further include continuous glucose monitoring capability.

[0308] Glycerol sensor configuration As shown in FIG. 7A, an exemplary continuous glycerol sensor configuration is depicted in which a first enzyme domain EZL1 160, comprising galactose oxidase, is located proximal to at least a portion of the WE surface. A second enzyme domain EZL2 161, comprising glucose oxidase and catalase, is located more distal to the WE. As shown in FIG. 7A, one or more resistance domains (RLs) 152 are located between EZL1 160 and EZL2 161. Additional RLs can be employed, for example, adjacent to EZL2 161. Modifications of one or more RL membranes to attenuate the flux of either analyte and increase the glycerol-to-galactose sensitivity ratio are envisioned. The above glycerol sensing configuration provides a glycerol sensor that can be combined with one or more additional sensor configurations as disclosed herein.

[0309] Glycerol can be catalyzed by the enzyme galactose oxidase (GalOx), which has an activity ratio of 1% to 5% relative to glycerol. In one embodiment, the activity of GalOx on this secondary analyte, glycerol, can be utilized. The relative concentrations of glycerol in vivo are much higher than those of galactose (approximately 2 μmol / L for galactose and approximately 100 μmol / L for glycerol), which complements the previous configuration.

[0310] Unless otherwise functionally limited, GalOx present in the EZL1 160 membrane catalyzes most, if not all, of the glycerol passing through one or more RLs. The signal contribution from the glycerol present is higher compared to the signal contribution from galactose. In one example, one or more RLs are chemically configured to provide a higher flux of glycerol or a lower flux of galactose.

[0311] In another example, a glycol sensor configuration is provided using multiple working electrodes (WEs) that utilize signals converted from both WEs. Utilizing signals converted from both WEs can provide increased selectivity. In one example, EZL1 160 and EZL2 161 contain the same oxidase enzyme (e.g., galactose oxidase) with different enzyme loading ratios, and / or different immobilization polymers, and / or different numbers and layers of RLs on the WEs. Such a configuration provides measurement of the same target analyte with different sensitivities, resulting in dual measurements. Using a mathematical algorithm to correct for noise and interference from a first signal and inputting a first signal from one sensing electrode with a first analyte sensitivity ratio into the mathematical algorithm allows decoupling of a second signal corresponding to the desired analyte contribution. Modification of the sensitivity ratio of one or more EZLs to distinguish between signals from interfering species and the analyte of interest can be provided by adjusting one or more of the enzyme source, enzyme loading in the EZL, the chemistry / diffusion properties of the EZL, the chemistry / diffusion properties of at least one RL, and combinations thereof.

[0312] As discussed herein, a secondary enzyme domain can be utilized to catalyze non-target analytes, reducing their concentration and limiting their diffusion toward the sensing electrode through the adjacent membrane containing the primary enzyme and necessary additives. In this example, the most distal enzyme domain, EZL2, 161, is configured to catalyze non-target analytes that would otherwise react with EZL1, thus providing a potentially less accurate reading of the target analyte (glycerol) concentration. This secondary enzyme domain can act as a "selective diffusion-exclusion membrane" by itself, or in some other configurations, can be positioned above or below the resistive layer (RL) 152. In this example, the target analyte is glycerol, and GalOX is used to catalyze the glycerol to form a measurable species (e.g., hydrogen peroxide).

[0313] Figure 7B shows the sensitivity change of a glycerol sensor in which galactose oxidase is used as the primary enzyme to convert glycerol, and in some cases, a secondary "selective diffusion-exclusion membrane" is used, with or without an additional RL layer. As shown in Figure 7B, the application of a selective secondary enzyme domain can improve the selectivity of the sensing platform for the target analyte. For example, as shown, the sensitivity ratio (sensitivity to target analyte / sensitivity to non-target analytes) can be improved by utilizing a RL-selective secondary enzyme domain. Figure 7B shows four immersion configurations: a configuration without an enzyme or RL (labeled "No RL"), in which neither a secondary enzyme-selective layer nor a RL is present; a configuration without a RL (labeled "No RL + GOX-CAT"), which includes a second enzyme domain for catalyzing non-target analytes; a configuration without an enzyme (labeled "RL"), which includes only a resistance domain; and a final configuration, which includes a RL layer positioned below the selective secondary enzyme domain (which can be described as being positioned between two EZLs (labeled "RL + GOX-CAT"). "No RL" and "RL" in Figure 7B represent examples without and with a resistance domain, respectively. "No RL + GOXCAT" and "RL + GOXCAT" in Figure 7B represent exemplary sensors configured with an outer layer containing GOX and catalase, without and with a RL, respectively. Compared to the "RL + no GOXCAT" configuration without a RL, the RL + GOXCAT configuration demonstrates an increased glycerol / galactose sensitivity ratio with a smaller variance relative to the RL-immersed configuration.

[0314] In one example, a continuous glycerol sensor configuration is provided using at least glycerol oxidase, which provides hydrogen peroxide upon reaction and catalysis of glycerol. Thus, in one example, an enzyme domain containing glycerol oxidase can be positioned adjacent to at least a portion of a WE surface, and hydrogen peroxide is detected amperometrically. In another example, an enzyme domain containing glycerol oxidase can be used to sense oxygen level changes, for example, in a Clark-type electrode setup. Alternatively, at least a portion of the WE surface can be coated with one or more layers of electrically coupled polymers, such as the mediator system discussed below, to provide a coated WE capable of electron transfer from the enzyme at a lower potential. The coated WE can then be operated at a different, lower voltage to measure its correlation to oxygen and glycerol concentrations.

[0315] In another example, a glycerol sensor configuration is provided using glycerol-3-phosphate oxidase in the enzyme domain. In one example, ATP is used as a cofactor. Thus, as shown in Figures 7C and 7D, in one example (Figure 7C), an exemplary sensor configuration is depicted in which one or more cofactors (e.g., ATP) 162 are proximal to at least a portion of the WE surface. One or more enzyme domains 163 including glycerol-3-phosphate oxidase (G3PD), lipase, and / or glycerol kinase (GK), and one or more regenerating enzymes capable of continuously regenerating the cofactors, are contained in the enzyme domains adjacent to the cofactors or more distal from the WE surface than the cofactor layer 162. Examples of regenerating enzymes that can be used to provide ATP regeneration include, but are not limited to, ATP synthase, pyruvate kinase, acetate kinase, and creatine kinase. The one or more regenerating enzymes can be contained in one or more enzyme domains or in separate layers.

[0316] An alternative configuration is shown in Figure 7D, in which one or more enzyme domains 163 containing G3PD, at least one cofactor, and at least one regenerating enzyme are positioned proximal to at least a portion of the WE surface, one or more cofactor reservoirs 162 are adjacent to the G3PD-containing enzyme domains and more distal to the WE surface, and one or more RL's 152 are positioned adjacent to the cofactor reservoirs. In either of these configurations, an additional enzyme domain containing a lipase can be included to indirectly measure triglycerides, as the lipase produces glycerol for detection by the glycerol sensor configuration described above.

[0317] FIG. 7E graphically illustrates the measured current response to stepwise addition of glycerol levels using the glycerol kinase, glycerol-3-phosphate oxidase, and ATP sensor configuration described in FIG. 7D, using current measurements at 0.6 volts and a low range of physiological concentrations. The "low" range in this example may be based on expected glycerol values ​​of about 5 μM to about 100 μM, and thus the low value may be a value less than about 50 μM. In other examples, the low glycerol value may be a value less than about 25 μM. In yet other examples, the low glycerol value may be less than about 10 μM. In yet another example, the low glycerol value may be less than about 5 μM.

[0318] In another example, a glycerol sensor configuration is provided using a dehydrogenase enzyme with a cofactor and a regenerating enzyme. In one example, the cofactors that can be incorporated into one or more enzyme domains include one or more of NAD(P)H, NADP+, and ATP. In one example, for example, for the use of NAD(P)H, the regenerating enzyme can be NADH oxidase or diaphorase to convert NADH, the product of dehydrogenase catalysis, back to NAD(P)H. Similar methodologies can be used to create other glycerol sensors; for example, glycerol dehydrogenase combined with NADH oxidase or diaphorase can be configured to measure glycerol or oxygen.

[0319] In one example, mathematical modeling can be used to identify and remove interfering signals and measure very low analyte concentrations, signal error, and noise reduction to improve and increase the end-of-life of a multi-analyte sensor. For example, using a two WE electrode configuration where WE1 is coated with a first EZL and WE2 is coated with two or more different EZLs, optionally using one or more resistive domains (RLs), mathematical correction for such interference can be corrected, providing for increased accuracy of the measurement.

[0320] Variations in enzyme loading, immobilization polymer, and resistance domain properties across each analyte sensing region can result in different sensitivity ratios between two or more target analytes and interfering species. When signals are collected and analyzed using mathematical modeling, more accurate concentrations of target analytes can be calculated.

[0321] One example where the use of mathematical modeling can be useful is glycerol sensing, where galactose oxidase is sensitive to both galactose and glycerol. The sensitivity ratio of galactose oxidase to glycerol is approximately 1% to 5% of its sensitivity to galactose. In such cases, modifying the sensitivity ratio to the two analytes is possible by adjusting one or more parameters, such as the enzyme source, enzyme loading, enzyme domain (EZL) diffusion properties, RL diffusion properties, and combinations thereof. When two WEs are operating in a sensor system, signal correction and analysis from both WEs using mathematical modeling provides a high degree of fidelity and target analyte concentration measurement.

[0322] In the above configuration, the proximity of one or more of these enzyme immobilization layers discussed herein to the WE may be different or reversed; for example, this configuration may be used where the one closest to the WE enzyme domain provides hydrogen peroxide.

[0323] In some embodiments, a target analyte can be measured using one or more enzymes working in concert. In one embodiment, ATP can be immobilized in one or more EZL membranes, or added alone or in combination with a secondary cofactor to an adjacent layer, or can be regenerated / recycled for use in the same EZL or an adjacent third EZL. This configuration can further include a cofactor-regenerating enzyme, such as alcohol dehydrogenase or NADH oxidase, to regenerate NAD(P)H. Other examples of cofactor-regenerating enzymes that can be used to regenerate ATP include ATP synthase, pyruvate kinase, acetate kinase, creatine kinase, etc.

[0324] In one example, the continuous glycerol sensor configuration described above can be combined with any one of the continuous alcohol, uric acid, cholesterol, bilirubin / ascorbic acid, ketone, and choline sensor configurations described above to provide a continuous multi-analyte sensor device as described further below. This continuous multi-analyte sensor device can further include continuous glucose monitoring capabilities. Other configurations can be used in the continuous glycerol sensor configuration described above, such as electrodes, resistors, biointerfaces, and drug-releasing membranes.

[0325] Creatinine sensor configuration In one embodiment, a continuous creatinine sensor configuration is provided, which contains one or more enzymes and / or cofactors. The creatinine sensor configuration is an example of a continuous analyte sensing system that generates intermediates, interference products, and these intermediates / interfering substances are also present in the sampled biological fluid. The present disclosure provides a solution to address these technical issues and, alone or in combination with other continuous multi-analyte sensor configurations, provides accurate and stable continuous creatinine monitoring.

[0326] During use, creatinine sensors are subject to the alteration of numerous physiologically occurring intermediates / interfering products, such as sarcosine and creatine, which may affect the correlation between the transduced signal and creatinine concentration. For example, the physiological concentration range of sarcosine is an order of magnitude lower than that of creatinine or creatine, so signal contributions from circulating sarcosine are typically minimal. However, changes in local physiological creatine concentrations can affect the creatinine sensor signal. In one embodiment, a method is provided for eliminating or reducing such signal contributions.

[0327] Thus, in one embodiment, eliminating or reducing the creatine signal contribution of a creatinine sensor involves using at least one enzyme that consumes a non-target interfering analyte, in this case, creatine. For example, two enzyme domains located adjacent to each other are used. At least a portion of the first enzyme domain is located proximal to at least a portion of the WE surface, and the first enzyme domain includes one or more enzymes selected from creatinine amidohydrolase (CNH), creatine amidohydrolase (CRH), and sarcosine oxidase (SOX). A second enzyme domain adjacent to the first enzyme domain and more distal from the WE surface includes one or more enzymes that use creatine as their substrate so as to eliminate or reduce creatine diffusion toward the WE. In one example, the enzyme combination includes CRH, SOX, creatine kinase, and catalase, with the enzyme ratio adjusted to provide a sufficient number of units so that circulating creatine is at least partially consumed by CRH to provide sarcosine and urea, while the produced sarcosine is at least partially consumed by SOX to provide an oxidized form of glycine (e.g., glycinaldehyde), which is at least partially consumed by catalase. In the above alternative configuration, the urea produced by CRH catalysis can be at least partially consumed by urease to provide ammonia, and the aqueous form (NH4+) is detected via an ion-selective electrode (e.g., a non-actin ionophore). Such an alternative potentiometric sensing configuration may provide an alternative to amperometric peroxide detection (e.g., improved sensitivity, detection limits, and lack of reference electrode depletion, alternative pathways / mechanisms). An example of this dual-analyte sensing may include a creatinine-potassium sensor with potentiometric sensing at two different working electrodes. In this example, interfering signals can be identified and corrected for.In an alternative embodiment, the above configuration can include a multimodal sensing architecture that uses a combination of amperometry and potentiometry to detect concentrations of peroxide and ammonium ions, which are measured using amperometry and potentiometry, respectively, and correlated to measure the concentration of creatinine. In one embodiment, the above configuration can further include one or more structures (e.g., no enzyme) separating the two enzyme domains to provide complementary or supplemental diffusion separations and barriers.

[0328] In yet another example, a method for separating the signal and essentially measuring only creatinine is to use a second WE that measures an interfering species (e.g., creatine) and then corrects the signal using mathematical modeling. Thus, for example, the signal from a WE that interacts with creatine is used as a reference signal. A signal from another WE that interacts with creatinine is then corrected for the signal from the WE that interacts with creatinine to selectively determine the creatinine concentration.

[0329] In yet another embodiment, creatinine sensing is provided by electrochemically measuring oxygen level changes, for example, in a Clark-type electrode setup, or by using one or more electrodes coated with layers of different polymers, such as NAFION™, to correlate changes in potential based on oxygen changes, which indirectly correlate with the concentration of creatinine.

[0330] In yet another embodiment, creatinine sensing is provided by using sarcosine oxidase wired to at least one WE using one or more electrically coupled mediators, in which the concentration of creatinine is indirectly correlated with the electron transfer-generated signal collected from the WE.

[0331] In the aforementioned creatinine sensor configurations based on hydrogen peroxide and / or oxygen measurement, the one or more enzymes can be in a single enzyme domain, or the one or more enzymes can be independently in one or more enzyme domains, or any other combination thereof with at least one enzyme in each layer. In the aforementioned creatinine sensor configurations based on the use of an electrically coupled sarcosine oxidase-containing layer, the layer is located adjacent to the electrode and is electrically coupled to at least a portion of the electrode surface using a mediator.

[0332] In another example, the creatinine sensor configuration described above can be sensed potentiometrically by using a urease enzyme (UR) to create ammonium from urea, which is created from creatine by CRH, and creatine is formed from the interaction of creatinine with CNH. Thus, ammonium can be measured by the configuration and correlated with creatinine concentration. Alternatively, creatine amidohydrolase (CI) or creatinine deiminase can be used to create ammonia gas, which provides ammonium ions for signal transduction under physiological conditions in a transcutaneous sensor.

[0333] In yet another embodiment, creatinine sensing is provided by using one or more enzymes and one or more cofactors. Some non-limiting examples of such configurations include creatinine deaminase (CD), which provides ammonium from creatinine, and glutamate dehydrogenase (GLDH), which provides peroxide from ammonium, with the hydrogen peroxide correlating with the level of creatinine present. The above configurations can further include a third enzyme, glutamate oxidase (GLOD), to further decompose glutamate formed from GDLH and create additional hydrogen peroxide. Such combinations of enzymes can be independently present in one or more enzyme domains, or any other combination thereof, with at least one enzyme present in each domain or layer.

[0334] In yet another embodiment, creatinine sensing is provided by a combination of creatinine amidohydrolase (CNH), creatine kinase (CK), and pyruvate kinase (PK), and the pyruvate produced by PK can be detected by one or more of either lactate dehydrogenase (LDH) or pyruvate oxidase (POX) enzymes configured independently, where one or more of the aforementioned enzymes are present in one layer, or multiple layers each contain at least one enzyme, or any other combination thereof.

[0335] In such sensor configurations where one or more cofactors and / or regenerating enzymes for the cofactors are used, providing an excess of one or more of NADH, NAD(P)H, and ATP in any one of one or more configurations may be used, and one or more diffusion resistance domains may be introduced to limit or prevent the flux of the cofactors from their respective membranes. Other configurations may be used in the aforementioned configurations, such as electrodes, resistors, biointerfaces, and drug-releasing membranes.

[0336] In yet another example, creatinine detection is provided by using creatinine deiminase in one or more enzyme domains and providing ammonium to the enzyme domains through the catalytic action of creatinine. Ammonium ions can then be detected potentiometrically or by using a composite electrode that undergoes oxidation-reduction when exposed to ammonium ions, such as a NAFION™ / polyaniline composite electrode, where polyaniline undergoes oxidation-reduction in the presence of ammonium at the electrode under potential. The ammonium concentration can then be correlated with the creatinine concentration.

[0337] FIG. 8A shows an exemplary continuous sensor configuration for creatinine. In the example of FIG. 8A, the sensor includes a first enzyme domain 164 containing CNH, CRH, and SOX adjacent to a working electrode (WE, e.g., platinum). A second enzyme domain 165 is located adjacent to the first enzyme domain and more distal from the WE. One or more resistor domains (RLs) 152 can be located adjacent to the second enzyme domain or between the first and second layers. Creatinine can diffuse through the RL and second enzyme domain to the first enzyme domain, where it is converted to peroxide and transduces a signal corresponding to its concentration. Creatine can diffuse through the RL and be converted to sarcosine and urea in the second enzyme domain. Sarcosine is consumed by sarcosine oxidase, and the generated peroxide is consumed by catalase, thus preventing transmission of the creatine signal.

[0338] Figure 8B shows experimental results of an exemplary creatinine sensor with the (CNH, CRH, SOX) configuration discussed above, using a platinum electrode surface. The data demonstrate reproducible sensitivity in the physiological range of creatinine, with drift stability in the physiological range for up to 7 days (Figure 8C). The CNH, CRH, and SOX enzyme combinations can be independently present in one or more enzyme domains, or any other combination thereof with at least one enzyme present in each domain or layer. Furthermore, such sensor configurations can include one or more electrode domains, interference membranes, and resistive domains to modify and tailor the sensor's sensitivity, selectivity, and stability.

[0339] For example, variations on the above configurations are possible for continuously monitoring creatinine alone or in combination with one or more other analytes. Thus, one alternative approach to sensing creatinine could be electrochemical sensing of changes in oxygen levels, for example, in a Clark-type electrode configuration. In one example, the WE could be coated with layers of different polymers, such as NAFION™, and the concentration of creatinine could be correlated based on changes in potential oxygen changes. In yet another example, one or more enzymes closest to the WE, i.e., sarcosine oxidase, could be "wired" to the electrode using one or more mediators. Each of the different enzymes in the above configurations could be distributed within a polymer matrix or domain to provide an enzyme domain. In another example, one or more of the different enzymes discussed herein could be formed as enzyme domains, with each layer having at least one enzyme present. In an example of a "wired" enzyme configuration with a multilayer membrane, the wired enzyme domain is closest to the electrode. One or more interferent layers can be deposited between the multi-layer enzyme configurations to block non-target analytes from reaching the electrodes.

[0340] In one example, the continuous creatinine sensor configuration described above may be combined with any one of the continuous alcohol, uric acid, cholesterol, bilirubin / ascorbic acid, ketone, choline, and glycerol sensor configurations described above to provide a continuous multi-analyte sensor device as described further below, which may further include continuous glucose monitoring capability.

[0341] Lactose sensor configuration In one embodiment, a continuous lactose sensor configuration is provided, alone or in combination with another analyte sensing configuration comprising one or more enzymes and / or cofactors. In a general sense, a lactose sensing configuration using at least one enzyme domain comprising a lactase enzyme is used to produce glucose and galactose from lactose. The produced glucose or galactose is then enzymatically converted to peroxide for signal transduction at an electrode. Thus, in one embodiment, at least one enzyme domain EZL1 comprising lactase is located proximate to at least a portion of a WE surface capable of electrolyzing hydrogen peroxide. In one embodiment, a glucose oxidase enzyme (GOX) is included in EZL1 along with one or more cofactors or electrically coupled mediators. In another embodiment, a galactose oxidase enzyme (GalOx) is included in EZL1, optionally along with one or more cofactors or mediators. In one embodiment, both the glucose oxidase enzyme and the galactose oxidase enzyme are included in EZL1. In one example, the glucose oxidase enzyme and the galactose oxidase enzyme are both included in EZL1, optionally along with one or more cofactors or electrically coupled mediators.

[0342] One or more additional EZLs (e.g., EZL2) can be located adjacent to EZL1, with at least a portion of EZL2 being more distal from at least a portion of the WE than EZL1. In one embodiment, one or more layers can be located between EZL1 and EZL2, and such layers can include enzymes, cofactors, or mediators, or can essentially lack one or more of the enzymes, cofactors, or mediators. In one embodiment, one or more layers located between EZL1 and EZL2 essentially lack enzymes, e.g., do not include intentionally added enzymes. In one embodiment, one or more layers can be located adjacent to EZL2, more distal from at least a portion of EZL1 than EZL2, and include one or more enzymes present in either EZL1 or EZL2.

[0343] In one example of the aforementioned lactose sensor configuration, a peroxide-generating enzyme can be electrically coupled to an electrode using a binding mediator, and the transduced peroxide signal from the aforementioned lactose sensor configuration can be correlated with the level of lactose present.

[0344] Figures 9A, 9B, 9C, and 9D show an alternative continuous lactose sensor configuration. Thus, the enzyme domain EZL1 164, which is most proximal to the WE (G1), containing GalOx and lactase, provides a lactose sensor that is sensitive to galactose and lactose concentration changes and essentially does not transduce glucose concentration. As shown in Figures 9B, 9C, and 9D, additional layers are used, including a non-enzyme-containing layer 159, a lactase enzyme-containing layer 165, and, optionally, electrodes, resistors, biointerfaces, and drug-releasing membranes (not shown). Because changes in physiological galactose concentration are minimal, the transduced signal is essentially from physiological lactose fluctuations. Figure 9E shows a linear response to lactose from the above configuration shown in Figure 9C.

[0345] In one example, the continuous lactose sensor configuration described above can be combined with any one of the continuous alcohol, uric acid, cholesterol, bilirubin / ascorbic acid, ketone, choline, glycerol, and creatinine sensor configurations described above to provide a continuous multi-analyte sensor device as described further below. This continuous multi-analyte sensor device can further include continuous glucose monitoring capabilities. Other membranes can be used in the sensor configurations described above, such as electrodes, resistors, biointerfaces, and drug-releasing membranes.

[0346] Urea Sensor Configuration A similar approach to that described above can also be used to create a continuous urea sensor. For example, urease (UR), which can decompose urea to provide ammonium, can be used in an enzyme domain configuration. Ammonium can be detected potentiometrically or by using a composite electrode, such as an electrode that undergoes redox when exposed to ammonium. Exemplary electrodes for ammonium signaling include, but are not limited to, NAFION™ / polyaniline composite electrodes, where polyaniline undergoes redox in the presence of ammonium at an applied potential, providing an essentially direct correlation between the signal and the level of ammonium present in the environment. This method can also be used to measure other analytes, such as glutamate, using the enzyme glutaminase (GLUS).

[0347] In one embodiment, the above-described continuous uric acid sensor configuration can be combined with any one of the above-described continuous alcohol sensor configuration, continuous uric acid sensor configuration, continuous cholesterol sensor configuration, continuous bilirubin / ascorbic acid sensor configuration, continuous ketone sensor configuration, continuous choline sensor configuration, continuous glycerol sensor configuration, continuous creatinine sensor configuration, and / or continuous lactose sensor configuration to provide a continuous multi-analyte sensor device as described further below. This continuous multi-analyte sensor device can further include continuous glucose monitoring capability. Other membranes can be used in the above-described uric acid sensor configuration, such as electrodes, resistors, biointerfaces, and drug-releasing membranes.

[0348] mediator One or more mediators may be employed to facilitate the electrolysis of one or more analytes or of a second compound that correlates with or interferes with the signaling of one or more analytes. Non-polymeric and polymeric redox mediators may be used in the continuous multi-analyte sensor devices of the present disclosure.

[0349] In one example, zwitterionic compounds / polymers, Prussian blue, Medora blue, methylene blue, methylene green, methyl viologen, ferrocyanide, ferrocene, cobalt ions, and cobalt phthalocyanine may be used as a coating on one or more WEs to facilitate or otherwise assist in the transmission and transduction of detection signals corresponding to one or more analytes. In one example, a transition metal complex is attached to one or more polymer backbones as a redox mediator. In one example, the transition metal complex includes at least one substituted or unsubstituted biimidazole ligand. In another example, the transition metal complex includes at least one substituted or unsubstituted biimidazole ligand and a substituted or unsubstituted bipyridine or pyridylimidazole ligand.

[0350] In one example, the mediator is a metal compound or complex of one or more of ruthenium, osmium, iron (e.g., polyvinylferrocene or hexacyanoferrate), or cobalt, including, for example, metallocene compounds thereof. In one example, the mediator is bound or otherwise attached to the conductive material of one of the reference electrode, working electrode, or counter electrode. In one example, a non-polymeric or polymeric mediator can be adsorbed or covalently attached to the conductive material of an electrode, such as a carbon surface or the surface of gold, platinum, palladium, rhodium, and their alloys. In one example, the mediator is quaternized.

[0351] Polymeric or non-polymeric mediators may be immobilized on the electrode surface using a variety of methods, such as adsorptive immobilization with or without crosslinking, vapor deposition, functionalization of at least a portion of the electrode surface, and then chemically bonding (ionic or covalent) the mediator polymer to the functional groups on the electrode surface. In one example, poly(4-vinylpyridine) or polyvinylpyridine-co-styrene or polyvinylimidazole is at least partially bonded to the electrode surface, where bpy is 2,2'-bipyridine [Os(bpy)2Cl]. + / 2+ In one example, at least a portion of the pyridine rings of poly(4-vinylpyridine) or polyvinylpyridine-co-styrene are reacted with 2-bromoethylamine and then crosslinked using a diepoxide such as polyethylene glycol diglycidyl ether. Other polymeric and / or non-polymeric intermediaries, such as PVI and PVP-ruthenium(phenanthrolidinedione), can be used.

[0352] Carbon surfaces can be modified for attachment of one or more polymeric and / or non-polymeric mediators, for example, by electroreduction of a diazonium salt, followed by activation with a carbodiimide, e.g., 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride, and then coupled with an amine-functionalized mediator, e.g., the osmium-containing polymers described above or 2-aminoethylferrocene, to form a mediator pair.

[0353] Similarly, gold can be functionalized with thiols or amines such as cysteamine, and the mediator [Os(bpy)2(pyridine-4-carboxylate)Cl]0 / + can be activated with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride to form a reactive O-acylisourea that reacts with the gold-bound amine to form an amide.

[0354] Modified enzymes In other examples, genetic variants of any one of the aforementioned enzymes are used, e.g., variants that improve thermostability, e.g., storage or preservation stability and / or operational stability. Examples of genetic mutations to improve enzyme thermostability include, but are not limited to, the addition of stabilizers such as substrates and similar ligands, sugars, polymers, specific and non-specific ionic species, and small uncharged organic molecules, immobilization, protein engineering (e.g., site-directed mutagenesis), and / or chemical modification. In one example, enzymes isolated from anaerobic hyperthermophiles, such as NADH oxidases isolated from Clostridium thermohydrosulfuricum, Thermus thermophilus, Thermoanaerobium brockii, Streptococcus mutans, Pyrococcus horikoshii, and Bacillus licheniformis, are used to confer at least some thermal operational stability to the sensor, e.g., up to about 80°C.

[0355] Additional single and multi-electrode configurations The multi-analyte sensor configurations described above can be adapted into a continuous multi-analyte sensor electrode configuration. FIGS. 10A-10C illustrate one embodiment (e.g., the in vivo portion) of a continuous multi-analyte sensor 100 including an elongated conductive body 102. The elongated conductive body 102 includes a core 110 (see FIG. 10B ) and a first conductive layer 112 at least partially surrounding the core. The first layer includes a working electrode (e.g., located within a window 106) and a membrane 108 located over the working electrode, the membrane 108 being configured and arranged for multi-axis bending. In some embodiments, the core and the first layer can be a single material (e.g., platinum). In some embodiments, the elongated conductive body is a composite of at least two materials, such as a composite of two conductive materials or a composite of at least one conductive material and at least one non-conductive material. In some embodiments, the elongated conductive body includes multiple layers. In certain embodiments, there are at least two concentric (e.g., annular) layers, such as a core formed from a first material and a first layer formed from a second material. However, in some embodiments, additional layers can be included. In some embodiments, the layers are coaxial.

[0356] The elongated conductive body may be long and thin, yet flexible and strong. For example, in some embodiments, the smallest dimension of the elongated conductive body is less than about 0.1 inches, 0.75 inches, 0.05 inches, 0.25 inches, 0.01 inches, 0.004 inches, or 0.002 inches. While the elongated conductive body is illustrated in FIGS. 10A-10C as having a circular cross-section, in other embodiments, the cross-section of the elongated conductive body may be oval, rectangular, triangular, polyhedral, star-shaped, C-shaped, T-shaped, X-shaped, Y-shaped, irregular, etc. In one embodiment, a conductive wire electrode is used as the core. Two additional conductive layers may be added to such a coated electrode (e.g., with an intervening insulating layer to provide electrical isolation). The conductive layers may be made of any suitable material. In certain embodiments, it may be desirable to use a conductive layer comprising conductive particles (i.e., particles of a conductive material) in a polymer or other binder.

[0357] In one embodiment, the material used to form the elongated conductive body (e.g., stainless steel, titanium, tantalum, platinum, platinum-iridium, iridium, certain polymers, and / or the like) can be strong and stiff, and therefore resistant to breakage. For example, in some embodiments, the ultimate tensile strength of the elongated conductive body can be from about 80 kPsi to about 500 kPsi. In other embodiments, the Young's modulus of the elongated conductive body can be from about 160 GPa to about 220 GPa. In still other embodiments, the yield strength of the elongated conductive body can be from about 60 kPsi to about 2200 MPa. Ultimate tensile strength, Young's modulus, and yield strength are discussed in more detail elsewhere herein. In some embodiments, the small diameter of the sensor provides (e.g., allows for) flexibility for these materials, and therefore for the entire sensor.

[0358] FIG. 10B is a perspective schematic diagram of the in vivo portion of an analyte sensor in one embodiment. In some embodiments, the sensor further includes a third layer 114 comprising a conductive material. In further embodiments, the third layer 114 may comprise a reference electrode, which may be formed from a silver-containing material applied over the second layer 104 (e.g., an insulator). The silver-containing material may include any of a variety of materials and may be in various forms, such as commercially available Ag / AgCl-polymer paste, paint, polymer-based conductive mixture, and / or ink. The third layer 114 may be processed using a paste / dip / coating step, for example, using a die-metered dip coating process. In one illustrative embodiment, the Ag / AgCl polymer paste is applied to the elongated body by dip-coating the elongated body (e.g., using a meniscus coating technique) and then drawing the body through a die to adjust the coating to a precise thickness. In some embodiments, multiple coating steps are used to build the coating to a predetermined thickness. In one example, the third (or additional layer(s)) layer 114 includes one or more of a resistive membrane, a biointerface membrane, and a drug-releasing membrane.

[0359] 10C is a schematic side view showing the in vivo portion of a continuous multi-analyte sensor. As described above with reference to FIG. 10A and as shown in FIG. 10C, an insulator 104 is disposed on (e.g., disposed over, covering) at least a portion of an elongated conductive body 102. In some embodiments, the sensor is configured and arranged such that the elongated body includes a core 110 and a first conductive layer 112, with a portion of the first conductive layer 112 exposed through a window 106 in the insulator 104. In other embodiments, the sensor is configured and arranged such that the elongated conductive body 102 includes a core embedded in an insulator, with a portion of the core exposed through a window in the insulator. For example, in some embodiments, an insulating material is applied (e.g., by screen printing, dip printing, inkjet printing, and / or block printing) to the elongated conductor 102 in a configuration designed to leave a portion of the surface of the first conductive layer 112 (or the surface of the core 110) exposed. For example, the insulating material used for insulator 104, or other insulating layers discussed herein, may be formed in a pattern that does not cover a portion of the elongate body. In another embodiment, a portion of the elongate body is masked before application of the insulating material. Removing the mask after application of the insulating material exposes a portion of the elongate body. In another embodiment, a series of conductive and insulating layers may be plated, dipped, or otherwise formed.

[0360] In some embodiments, the insulator 104 is applied as a single layer of material. In other embodiments, the insulator 104 is applied as two or more layers of either the same or different materials. In some embodiments, the insulating material includes at least one of polyurethane, polyimide, and parylene. In one embodiment, the insulating material includes parylene, which can be a polymer coating advantageous for its strength, lubricity, and electrical insulating properties. Generally, parylene is produced by vapor deposition and polymerization of paraxylylene (or its substituted derivatives). However, any suitable insulating material may be used, such as, but not limited to, a dielectric ink, paste, or paint, such as a fluorinated polymer, polyethylene terephthalate (PET), polyurethane, polyimide, or other non-conductive polymer.

[0361] In some embodiments, the insulator 104 comprises a polymer, such as a non-conductive (e.g., dielectric) polymer. Dip coating, spray coating, vapor deposition, printing, and / or other thin and / or thick film coating or deposition techniques may be used to deposit the insulating material onto the elongate body and / or core.

[0362] 10C and 10D show the results of this removal / cutting process in side / cross-sectional views. The removal process can be accomplished by various methods. In one embodiment, the removal step is performed, for example, by laser skiving, and can be performed on a continuous strand in a reel-to-reel process. The removed area can be stepped, for example, by removing different layers at different lengths (FIG. 10D). In such manufacturing methods involving a continuous strand, the sensor can be singulated after the removal step. In some embodiments, if the core is metallic, an end cap of insulating or other isolating material may be employed on the tip, for example, by dipping, spraying, shrink tubing, crimp wrapping, etc. If the core is polymeric (e.g., a hydrophobic material), an end cap may not be necessary. For example, in the sensor shown in FIG. 10D, an end cap (e.g., of a polymeric or insulating material) or other structure may be provided over the core (e.g., if the core 110 is not insulating).

[0363] FIG. 10F can be thought of as building on the general structure shown in FIG. 10B in that two or more additional layers are added to create one or more additional electrodes. Also, methods of selectively removing two or more windows to form two or more electrodes can be used. For example, by adding another conductive layer 122′ and insulating layer 224 below the reference electrode domain 115, two electrodes (first working electrode 114′ and second working electrode 114″) can be formed, resulting in a dual-electrode sensor. The same concept can be applied to create, for example, a counter electrode, an electrode for measuring an additional analyte (e.g., oxygen), etc.

[0364] 10G illustrates another embodiment in which selective removal of various layers is staged to expose working electrodes along the length of the elongate body (shown as exposed portions 112, 122′ and insulators 104, 224). In one example, a dual-electrode sensor can include a first working electrode (e.g., formed from first conductive layer 112) configured to detect glucose and a second working electrode (e.g., formed from second conductive layer 122′) configured to detect lactate. In one example, a dual-electrode sensor can include a first working electrode (e.g., formed from first conductive layer 112) configured to detect glucose and a second working electrode (e.g., formed from second conductive layer 122′) configured to detect ketone. In another example, both working electrodes are configured to detect the same analyte.

[0365] Figure 11 is a schematic cross-sectional view of an exemplary membrane configuration of the multi-analyte sensor of Figure 10A taken along line 11-11. In one example, the membrane system includes multiple domains, such as an electrode domain 602, an interference domain 304, a transducer domain 606 (e.g., an enzyme, an aptamer, an ionophore, etc.), and / or a resistance domain 608, as shown in Figure 11, and may include a high oxygen solubility domain and / or a bioprotective domain and / or a drug-release domain, as described in more detail in U.S. Patent Application Publication No. 2005-0245799 (A1). The membrane system(s) may be independently deposited onto the exposed electroactive surface using known thin-film techniques (e.g., vapor deposition, spraying, electrodeposition, dipping, etc.).

[0366] FIG. 12A is a perspective view of the in vivo portion of a single-electrode, dual-working electrode surface multi-analyte sensor (e.g., configured and arranged for multi-axis bending). In this example, an insulated elongate body includes three conductive cores 110A, 110B, 110C positioned (e.g., embedded, coated) within an insulator 104. In this example, multiple windows are formed in and / or through the insulator, each window exposing a portion of a core. As a non-limiting example, window 106A is formed in the insulator such that a portion of core 110A is exposed. Similarly, window 106B is formed in the insulator such that a portion of core 110B is exposed. The windows can be staggered and / or non-staggered along the longitudinal length of the sensor. In a further example, each conductive core includes an inner core and an outer core as described elsewhere herein.

[0367] 12B is a perspective view of an in vivo portion of an analyte sensor including an elongated body (e.g., configured and arranged for multi-axis bending) formed from an insulator 104, first and second conductive cores 110A, 110B embedded in the insulator, and a membrane 108. The first conductive core is formed from platinum, platinum-iridium, gold, palladium, iridium, graphite, carbon, a conductive polymer, and / or an alloy, and the first window 106A is configured and arranged to expose the electroactive portion of the first conductive core. The second conductive core is formed from a silver-containing material (e.g., a silver or silver / silver chloride wire, or a body of silver-containing material in the form of a silver-containing wire), and the second window 106B is configured and arranged to expose the electroactive portion of the second conductive core. In some embodiments, instead of a bulk metal wire, the first conductive core comprises an inner core and an outer core. For example, to reduce material costs, the inner core may be formed from a material relatively less expensive than platinum, such as stainless steel, titanium, tantalum, and / or a polymer, and the outer core may be formed from a material that provides a suitable electroactive surface, such as, but not limited to, platinum, platinum-iridium, gold, palladium, iridium, graphite, carbon, a conductive polymer, and / or an alloy. In some embodiments, a membrane covers the exposed electroactive portion of the first conductive core. In further embodiments, the membrane covers the in vivo portion of the sensor. In some embodiments, a third conductive core is embedded in an insulator. In some embodiments, the third conductive core is configured and arranged as a second working electrode, which may be configured as a redundant working electrode, a non-analyte signal measuring working electrode (e.g., without a transduction element as described below), a counter working electrode for detecting a second analyte, etc.

[0368] 13A is a perspective view of an in vivo portion of another embodiment of a multi-electrode sensor system 800 including two working electrodes and at least one reference / counter electrode. The sensor system 800 includes first and second elongate bodies E1 and E2, each formed from a conductive core or a core onto which a conductive layer is deposited. In this particular embodiment, an insulating layer 810, a conductive layer 820, and one of the aforementioned films (not shown) are deposited on the elongate bodies E1 and E2. The insulating layer 810 separates the conductive layer 820 from the elongate bodies. The material selected to form the insulating layer 810 may include any of the insulating materials described elsewhere herein, including polyurethane and polyimide. The material selected to form the conductive layer 820 may include any of the conductive materials described elsewhere herein, including silver / silver chloride, platinum, gold, etc. The working electrodes 802', 802'' are formed by removing portions of the conductive layer 820 and the insulating layer 810, thereby exposing the electroactive surfaces of the elongate bodies E1, E2, respectively. Figure 13B provides a close-up perspective view of the distal portions of the elongate bodies E1, E2.

[0369] In one example, the two elongate bodies shown in FIG. 13A are fabricated to have substantially the same shape and dimensions. In some examples, the working electrodes are fabricated to have the same characteristics, thereby providing signal measurement redundancy or providing a sensor system capable of providing unique signals representative of two or more different analytes. In other examples, each of the working electrodes associated with elongate bodies E1, E2 may have one or more characteristics that distinguish it from the other working electrode. For example, in one example, each of elongate bodies E1, E2 may have a different conductive surface, such that each working electrode has different electrochemical properties from the other working electrode. Additionally, in one example, each of elongate bodies E1, E2 may be covered with different membrane(s) such that each working electrode has different membrane properties from the other working electrode. For example, one of the working electrodes may have a membrane including a first transducer element, and the other working electrode may have a membrane including a layer with an inactivated form of the transducer element or without the transducer element, aptamer(s), or cofactor(s). Additional sensor system configurations possible using multiple working electrodes (e.g., sensor elements) are described in U.S. Provisional Application No. 61 / 222,716, filed July 2, 2009, and U.S. Patent Application No. 12 / 829,264, filed July 1, 2010, entitled "ANALYTE SENSOR," each of which is incorporated herein by reference in its entirety.

[0370] Although not shown in FIGS. 13A-13B, in certain embodiments, the distal ends 830′, 830″ of the core portions of elongate bodies E1, E2 may be covered with an insulating material (e.g., polyurethane or polyimide). In alternative embodiments, the exposed core portions 830′, 830″ may be coated with any of the membrane systems described above and / or may serve as additional working electrode surface area.

[0371] Regarding the fabrication of the sensor system shown in FIGS. 13A-13B, in one embodiment, elongated bodies E1, E2 may be formed as elongated conductive cores, or alternatively, as cores (conductive or non-conductive) onto which at least one conductive material is deposited. Next, an insulating layer 810 is deposited on elongated bodies E1, E2. Thereafter, a conductive layer 820 is deposited on the insulating layer 810. The conductive layer 820 may function as a reference electrode / counter electrode and may be formed from silver / silver chloride or any other material that can be used for a reference electrode. In alternative embodiments, the conductive layer 820 may be formed from a different conductive material and may be used as a separate working electrode. After these steps, a layer removal process is performed to remove portions of the deposited layers (i.e., the conductive layer 820 and / or the insulating layer 810). Any of the techniques described elsewhere herein (e.g., laser ablation, chemical etching, grit blasting) may be used. 13A and 13B, layers of the conductive layer 820 and the insulating layer 810 are removed to form the working electrodes 802′, 802″. In the illustrated embodiment, layer removal occurs around the entire cross-sectional perimeter (e.g., circumference) of the deposited layer, although it is contemplated that in other embodiments, layer removal may occur around a preselected portion of the cross-sectional perimeter instead of the entire cross-sectional perimeter.

[0372] Contacts 804′, 804″ used to provide electrical connection between the working electrode and other components of the sensor system may be similarly formed. As shown, contacts 804′ and 804″ are isolated from one another to prevent electrical connection therebetween. Because the layer removal process is performed on each individual elongate body E1, E2 rather than a single, geometrically complex elongate body, this particular sensor design (i.e., two elongate bodies arranged side by side) may offer ease of manufacturing compared to the manufacturing processes associated with other multi-electrode systems having other geometries.

[0373] After the conductive and insulating layers are deposited on the elongate body and after selected portions of the deposited layers are removed, a membrane is applied over at least a portion of the elongate body. In some embodiments, any of the aforementioned membrane systems is applied only to the working electrode, while in other embodiments, any of the aforementioned membrane systems is applied to the entire elongate body. In one embodiment, any of the aforementioned membrane systems is simultaneously deposited on two working electrodes while they are placed together (e.g., by bundling), while in another embodiment, any of the aforementioned membrane systems is first deposited on each individual working electrode, and then the two working electrodes are placed together. Thus, in one embodiment, any of the aforementioned membrane systems is designed for a sensitivity of about 1 pA / mg / dL to about 100 pA / mg / dL. In other embodiments, the sensitivity is about 5 pA / mg / dL to about 25 pA / mg / dL. In further embodiments, the sensitivity is about 4 to about 7 pA / mg / dL. Without wishing to be bound by any theory, it is believed that membrane systems designed with sensitivity in the above ranges enable measurement of one or more analyte signals in low analyte and / or low reactant / co-reactant situations, respectively. Decreased measurement accuracy in the low analyte range can be problematic due to low availability of analyte to the sensor, and / or increased signal noise has been shown in the high analyte range due to insufficient oxygen required to react with the amount of analyte being measured. Therefore, without wishing to be bound by theory, it is believed that the membrane systems of the present disclosure, combined with the electronic circuit design and exposed electrochemically reactive surface area design, support measurement of analytes in the picoampere range and below, thereby enabling improved levels of resolution and accuracy in both the low and high analyte ranges.

[0374] Biointerface membrane / layer In one embodiment, the sensor includes a porous material disposed over some portion thereof, which modifies the host tissue response to the sensor. In some embodiments, the porous material surrounding the sensor advantageously enhances and extends the performance and lifespan of the sensor in the short term by slowing or reducing cellular migration into the sensor and associated degradation that would be caused by cellular invasion if the sensor were directly exposed to an in vivo environment. Alternatively, the porous material may provide stabilization of the sensor via tissue ingrowth into the porous material in the long term.Suitable porous materials include silicone, polytetrafluoroethylene, expanded polytetrafluoroethylene, ethylene tetrafluoroethylene copolymers, polyolefins, polyesters, polycarbonates, biostable polytetrafluoroethylene, homopolymers, copolymers, and terpolymers of polyurethane, polypropylene (PP), polyvinyl chloride (PVC), polyvinylidene fluoride (PVDF), polyvinyl alcohol (PVA), polybutylene terephthalate (PBT), polymethyl methacrylate (PMMA), polyether ether ketone (PEEK), polyamides, polyurethanes, cellulosic polymers, poly(ethylene oxide), poly(propylene oxide) and copolymers and blends thereof, polysulfone and its block copolymers (such as diblock, triblock, alternating, random, and graft copolymers), as well as metals, ceramics, cellulose, hydrogel polymers, poly(2-hydroxyethyl methacrylate, pHEMA), hydroxyethyl methacrylate, (hydroxyethyl methacrylate, HEMA), polyacrylonitrile-polyvinyl chloride (polyvinyl chloride), and the like. chloride, PAN-PVC), high density polyethylene, acrylic copolymers, nylon, polyvinyl fluoride, polyanhydrides, poly(L-lysine), poly(L-lactic acid), hydroxyethyl methacrylate, hydroxyapeptite, alumina, zirconia, carbon fiber, aluminum, calcium phosphate, titanium, titanium alloys, nintinol, stainless steel, and CoCr alloys, as described, for example, in U.S. Pat. No. 7,875,293 to Shults et al. and U.S. Pat. No. 7,192,450 to Brauker et al.

[0375] In some embodiments, porous materials surrounding a sensor offer inherent advantages that can be used to enhance and extend the performance and lifespan of the sensor. However, such materials may also provide advantages over a period of time (e.g., for sensor durability over periods of 14, 15, or 21 days or more). In particular, the in vivo portion of the sensor (the portion of the sensor that is implanted in the host's tissue) is encased (partially or completely) in a porous material. The porous material can be wrapped around the sensor (e.g., by wrapping the porous material around the sensor or by inserting the sensor into a section of the porous material sized to receive the sensor). Alternatively, the porous material can be deposited on the sensor (e.g., by electrospinning a polymer directly onto it). In yet other alternative embodiments, the sensor is inserted into a selected section of porous biomaterial. Other methods for surrounding the in vivo portion of the sensor with porous material can also be used, as will be understood by those skilled in the art.

[0376] The porous material surrounding the sensor advantageously slows or reduces cell migration toward the sensor and the associated degradation that would be caused by cell invasion if the sensor were directly exposed to the in vivo environment. That is, the porous material provides a barrier that makes cell migration toward the sensor more tortuous and therefore slower (providing a service life advantage). This is believed to reduce or delay the loss of sensitivity typically observed in sensors over time.

[0377] In embodiments where the porous material is a high-oxygen solubility material, such as porous silicone, the high-oxygen solubility porous material partially or entirely surrounds the in vivo portion of the sensor. In some embodiments, a lower ratio of oxygen to glucose can be sufficient to provide excess oxygen by using a high-oxygen solubility domain (e.g., a silicone- or fluorocarbon-based material) to enhance oxygen delivery / transport to the enzyme domain and / or electroactive surface. In some embodiments, it is believed that some signal noise typically seen with sensors may be due to oxygen deficiency. Silicone has high oxygen permeability, thereby facilitating oxygen delivery to the enzyme domain. By enhancing oxygen delivery through the use of a silicone composition, for example, glucose concentration may no longer be a limiting factor. In other words, if more oxygen is delivered to the enzyme and / or electroactive surface, more glucose can also be delivered to the enzyme without creating an oxygen-limiting excess. Without being bound by any particular theory, it is believed that silicone materials offer enhanced biostability compared to other polymeric materials, such as polyurethane.

[0378] In another embodiment, the porous material further comprises a bioactive agent that is released upon insertion. In one embodiment, the porous structure provides access for glucose permeation while allowing for drug release / elution. In one embodiment, as the bioactive agent is released / eluted from the porous structure, glucose transport may increase to offset any attenuation of glucose transport from, for example, the aforementioned immune response factors.

[0379] In these examples, the porous material is a biointerface membrane comprising a first domain having an architecture including cavity size, configuration, and / or overall thickness that modifies the host tissue response, for example, by forming fluid pockets, promoting vascularized tissue ingrowth, disrupting underlying tissue contracture, resisting fibrous tissue growth adjacent to the device, and / or preventing barrier cell formation. The biointerface membrane, in one example, can be any shape or size, including covering at least the sensing mechanism of the sensor and covering or surrounding the sensing mechanism or sensor uniformly, asymmetrically, or axisymmetrically.

[0380] A second domain of the biointerface membrane is optionally provided that is impermeable to cells and / or cell processes. A bioactive agent incorporated into at least one of the first domain, the second domain, the sensing membrane, or other portions of the implantable device is optionally provided, the bioactive agent being configured to modify a host tissue response. In one example, the biointerface membrane includes a bioactive agent incorporated into at least one of the first and second domains of the biointerface membrane or into the device and adapted to diffuse through the first and / or second domains to modify a host tissue response to the membrane.

[0381] Due to the small dimensions of the sensors (sensing mechanisms) of the present disclosure, some conventional methods of porous membrane formation and / or porous membrane adhesion are inappropriate for forming a biointerface membrane on a sensor as described herein. Therefore, the following examples illustrate systems and methods for forming and / or adhering a biointerface membrane on a miniature structured sensor as defined herein. For example, the biointerface membrane or release membrane of the present disclosure can be formed on a sensor using techniques such as electrospinning, molding, weaving, direct writing, freeze-drying, wrapping, and the like.

[0382] In embodiments where the biointerface membrane is written directly onto the sensor, the dispenser dispenses the polymer solution using a nozzle having a valve or the like, as described, for example, in U.S. Patent No. 7,857,756. Generally, a variety of nozzles and / or dispensers can be used to dispense the polymer material to form the woven or nonwoven fibers of the biointerface membrane.

[0383] Drug-releasing domains - Inflammatory response control Drug release domains, which may include at least one membrane having one or more layers, may be included in the membrane systems discussed herein. Generally, the inflammatory response to a biomaterial implant can be divided into two phases. The first phase consists of mast cell recruitment, followed by infiltration of primarily polymorphonuclear (PMN) cells. This phase is referred to as the acute inflammatory phase. Over days to weeks, chronic cell types, comprising the second phase of inflammation, replace PMNs. Macrophages and lymphocytes predominate during this phase. While not wishing to be bound by any particular theory, it is believed that limiting vasodilation and / or blocking pro-inflammatory signaling, stimulating angiogenesis, or inhibiting scar formation or barrier cell layer formation may provide protection from scar tissue formation and / or reduce acute inflammation, thereby providing a stable platform for, for example, sustained maintenance of an altered foreign body response.

[0384] Thus, bioactive intervention may modify the foreign body response in the early weeks of foreign body capsule formation and alter the long-term behavior of the foreign body capsule. Additionally, in some situations, the biointerface membranes and / or drug-releasing membranes of the present disclosure may benefit from bioactive intervention to overcome the sensitivity of the biointerface membranes and / or drug-releasing membranes to implant procedures, implant movement, or other factors known to otherwise cause inflammation, scar formation, or interfere with device function in vivo.

[0385] In general, bioactive agents that are believed to modify tissue responses include anti-inflammatory agents, anti-infective agents, antiproliferative agents, antihistamines, anesthetics, inflammatory agents, growth factors, angiogenic (growth) factors, adjuvants, immunosuppressants, antiplatelet agents, anticoagulants, ACE inhibitors, cytotoxic agents, anti-barrier cell compounds, angiogenic compounds, antisense molecules, etc. In some examples, bioactive agents include S1P (sphingosine-1-phosphate), monobuty...

Claims

1. A continuous analyte sensor device, An analyte sensor operably coupled to a signal converter, wherein the analyte sensor comprises at least one membrane system adjacent to the signal converter, and the at least one membrane system is independently, A first domain including at least one first conversion element, A second domain adjacent to the first domain, which is the same as or different from the first domain, A continuous analyte sensor device comprising at least one regenerative cofactor.

2. The continuous analyte sensor device according to claim 1, wherein the analyte sensor is a multi-analyte sensor.

3. The continuous analyte sensor device according to claim 2, wherein the multi-analyte sensor is a glucose and ketone analyte sensor, or a glucose and creatinine analyte sensor, or a ketone and potassium ion analyte sensor.

4. The continuous analyte sensor device according to claim 1, wherein the signal converter comprises at least one electrode.

5. The continuous analyte sensor device according to claim 1, wherein the first domain includes at least one first conversion element, and the second domain includes at least one second conversion element, the second conversion element being different from the first conversion element.

6. The continuous analyte sensor device according to claim 1, wherein the at least one regenerative cofactor is one or more of NAD, NADH, NAD(P)H, NAD(P)+, ATP, flavin adenine dinucleotide (FAD), magnesium (Mg++), pyrroloquinoline quinone (PQQ), pyrroloquinoline quinone (PQQ), and functionalized derivatives thereof.

7. The continuous analyte sensor device according to any one of claims 1, further comprising at least one mediating substance present in the first domain, the second domain, or both the first and second domains.

8. The continuous analyte sensor device according to claim 7, wherein the mediating substance is one or more of the following: 2,2'-bipridin, poly-1,10-phenanthroline-5,6-dione, polyvinylferrocene, hexacyanoferrate, phthalocyanine or organometallic compounds thereof, organometallic compounds of osmium or ruthenium, and functionalized derivatives thereof, and complexes of one or more transition metals with one or more polymers or ligands and salts thereof.

9. The continuous analyte sensor device according to claim 7, wherein the first domain comprises the at least one mediating material and the at least one first conversion element, and the second domain comprises the at least one regenerating cofactor, or the first domain comprises the at least one regenerating cofactor and the at least one first conversion element, and the second domain comprises the at least one mediating material.

10. The continuous analyte sensor device according to claim 5, wherein the at least one first conversion element and the at least one second conversion element are independently a dehydrogenase enzyme, a reductase enzyme, a kinase enzyme, a peroxidase enzyme, an esterase enzyme, an (amide) hydrolase enzyme, an oxidase enzyme, or a combination thereof.

11. The continuous analyte sensor device according to claim 5, wherein the at least one first conversion element and the at least one second conversion element are independently beta-hydroxybutyrate dehydrogenase, alcohol dehydrogenase, lipase, amide hydrolase, glycerol kinase, creatinine kinase, creatine amide hydrolase, alcohol oxidase, cholesterol oxidase, galactose oxidase, choline oxidase, glutamate oxidase, glycerol-3-phosphate oxidase, bilirubin oxidase, urease, pyruvate oxidase, xanthine oxidase, glucose oxidase, lactate oxidase, sarcosine oxidase, malate dehydrogenase, formaldehyde dehydrogenase, glutathione reductase, glutathione peroxidase, 3-hydroxysteroid dehydrogenase, NADH oxidase, or a combination thereof.

12. The continuous analyte sensor device according to claim 5, wherein the at least one first conversion element is a beta-hydroxybutyrate dehydrogenase, and the at least one second conversion element is an NADH oxidase.

13. The at least one electrode comprises platinum or palladium, An interference domain is deposited on the at least one electrode, The first domain is adjacent to the interference domain, and the first domain contains beta-hydroxybutyrate dehydrogenase, NADH oxidase, and the cofactor. The second domain is adjacent to the first domain, and the second domain contains a polyvinylpyridine polymer or copolymer. The continuous analyte sensor device according to claim 4, wherein the continuous analyte sensor device is configured to provide a continuous analyte signal without a transition metal-containing mediating substance.

14. The continuous analyte sensor device according to claim 13, wherein the at least one regenerative cofactor is NAD.

15. The continuous analyte sensor device according to claim 13, wherein the interference domain is configured to block the diffusion of at least one of the following from the electrode: acetaminophen, ascorbic acid, bilirubin, cholesterol, creatinine, dopamine, ephedrine, ibuprofen, L-dopa, methyldopa, salicylic acid, tetracycline, trazamide, tolbutamide, triglycerides, and uric acid.

16. The continuous analyte sensor device according to claim 13, wherein the interference domain comprises polyurethane, polyurethane-zwitterionic polymer, polymer having pendant ionic groups, NAFION®, chitosan, cellulose, alternating layers of polyallylamine and polyacrylate, or a combination or blend thereof.

17. The continuous analyte sensor device according to claim 1, wherein the first domain or the second domain comprises an amphiphilic polymer or copolymer.

18. The continuous analyte sensor device according to claim 1, wherein the first domain or the second domain comprises a heterocyclic polymer or copolymer, or at least partially quaternized heterocyclic polymer or copolymer.

19. The continuous analyte sensor device according to claim 1, further comprising a transmitter configured to wirelessly transmit data to a paired display device or drug delivery device.

20. The continuous analyte sensor device according to claim 1, wherein the continuous analyte sensor device is sterilized.

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