Copolymer-coated biosensors and uses thereof

Abstract

The present invention relates to copolymer-coated biosensors and their uses. Methods for preparing biosensors are also provided. [Selected figure] Figure 1

Description

[Technical Field]

[0001] The present invention relates to copolymer-coated biosensors and their uses. Methods for preparing biosensors are also provided. [Background technology]

[0002] Biometric systems and electrochemical biosensors that utilize electrochemical leakage offer the potential for rapid, real-time analysis, making them particularly suitable for rapid measurements in the point-of-care industry. The outer membrane of a biosensor is of great importance because it represents the interface between the sensor and the analyte medium. The purpose of this interfacial membrane is to allow diffusion of the analyte to the detection layer while excluding potential interfering species that may be present in the analyte medium. Therefore, there is a need for biosensors with improved interfacial membranes. Summary of the Invention [Problem to be solved by the invention]

[0003] An object of the present invention is to provide a biosensor. [Means for solving the problem]

[0004] In one aspect, the invention provides a biosensor comprising a substrate, a working electrode on one side of the substrate, a sensing layer on one side of the working electrode, the sensing layer comprising metal nanoparticles, polydopamine, and a peptide probe, and a biocompatible membrane on one side of the sensing layer, the biocompatible membrane comprising a reblock polymer AbBbC, where A is a hydrophilic soft segment, B is a hydrophobic hard segment, C is a flexible polymer segment, and b is a chain extender. In some embodiments, the working electrode according to the above aspects comprises carbon, graphene, gold, or platinum. In some embodiments, the metal nanoparticles according to any of the above aspects are platinum nanoparticles, gold nanoparticles, or iridium nanoparticles. In some embodiments, the metal nanoparticles according to any of the above aspects have a particle size of about 1 to about 100 nm. In some embodiments, the peptide probe of any of the above aspects comprises an enzyme, an antibody, or a polymer comprising a peptide. In some embodiments, the peptide probe of any of the above aspects comprises an oxidoreductase.

[0005] In some embodiments, the peptide probe of any of the above aspects comprises glucose oxidase, glucose dehydrogenase, or horseradish peroxidase.

[0006] In some embodiments, the metal nanoparticles of any of the above aspects are coated with polydopamine and peptide probes. In some embodiments, the metal nanoparticles of any of the above aspects are mixed with polydopamine and peptide probes.

[0007] In some embodiments, the hydrophilic soft segment of any of the above aspects comprises a polymer selected from the group consisting of polyethylene glycol (PEG), polypropylene glycol (PPG), and polyetheramine (PEA).

[0008] In some embodiments, the hydrophobic hard segment according to any of the above aspects comprises a polymer selected from the group consisting of polycarbonate (PC) and poly(methyl methacrylate) (PMMA).

[0009] In some embodiments, the flexible polymer segment according to any of the above aspects comprises a polymer selected from the group consisting of polydimethylsiloxane (PDMS) and poly(2-hydroxyethyl methacrylate) (PHEMA). In some embodiments, the chain extender of the biocompatible membrane of any of the above aspects is derived from an isocyanate-containing compound.

[0010] In some embodiments, each chain extender according to any of the above aspects is independently derived from methylene diphenyl diisocyanate (MDI), hexamethylene diisocyanate (HDI), or bis(4-isocyanatocyclohexyl)methane.

[0011] In some embodiments, the number average molecular weight of A according to any of the above aspects is about 200 to about 10,000, the number average molecular weight of B is about 1,000 to about 20,000, and the number average molecular weight of C is about 1,000 to about 20,000.

[0012] In some embodiments of preparing a biosensor according to any of the above embodiments, the biocompatible membrane comprises about 1 to about 10 parts by weight of A, about 1 to about 5 parts by weight of B, about 1 to about 5 parts by weight of C, and about 1 to about 3 parts by weight of b.

[0013] In some embodiments, the linkages of Ab, Bb, and Cb themselves according to any of the above aspects are independently urea or carbamate linkages.

[0014] In some embodiments, the biosensor of any of the above aspects further comprises an adhesive layer between the sensing layer and the biocompatible membrane, the adhesive layer comprising a polymer comprising a first monomeric unit comprising at least two amine moieties crosslinked with a second monomeric unit comprising at least two formyl moieties.

[0015] In some embodiments, the first monomer unit according to any of the above aspects is 1,6-diaminohexane and the second monomer unit is glutaraldehyde.

[0016] In some embodiments, the biosensor of any of the above aspects further includes a blank electrode, a counter electrode, and a reference electrode that are substantially identical to the working electrode, and the blank electrode is directly covered by the biocompatible membrane. In some embodiments, the blank electrode is directly covered by an adhesive layer that is covered by the biocompatible membrane. In some embodiments, the minimum distance between the working electrode and the blank electrode according to any of the above aspects is about 5 mm or less.

[0017] In another aspect, the present invention provides a method for preparing a biosensor according to any of the previous aspects, comprising: (1) forming a working electrode on a substrate; (2) forming a sensing layer on one side of the working electrode, the sensing layer comprising metal nanoparticles, polydopamine, and a peptide probe; and (3) forming a reblock polymer AbBbC on one side of the sensing layer, where A is a hydrophilic soft segment, B is a hydrophobic hard segment, C is a flexible polymer segment, and b is a chain extender. In some embodiments of preparing a biosensor according to any of the above embodiments, the working electrode comprises carbon, graphene, gold, or platinum.

[0018] In some embodiments of preparing a biosensor according to any of the above aspects, step (1) comprises forming a working electrode on one of the substrates by etching or screen printing.

[0019] In some embodiments of preparing a biosensor according to any of the above aspects, the metal nanoparticles are platinum nanoparticles, gold nanoparticles, or iridium nanoparticles. In some embodiments of preparing a biosensor according to any of the above embodiments, the metal nanoparticles have a particle size of about 1 to about 100 nm.

[0020] In some embodiments of preparing a biosensor according to any of the above embodiments, the peptide probe comprises an enzyme, an antibody, or a polymer comprising a peptide. In some embodiments of preparing a biosensor according to any of the above embodiments, the peptide probe comprises an oxidoreductase.

[0021] In some embodiments of preparing a biosensor according to any of the above embodiments, the peptide probe comprises glucose oxidase, glucose dehydrogenase, or horseradish peroxidase.

[0022] In some embodiments of preparing a biosensor according to any of the above embodiments, the hydrophilic soft segment comprises a polymer selected from the group consisting of polyethylene glycol (PEG), polypropylene glycol (PPG), and polyetheramine (PEA).

[0023] In some embodiments of preparing a biosensor according to any of the above embodiments, the hydrophobic hard segment comprises a polymer selected from the group consisting of polycarbonate (PC) and poly(methyl methacrylate) (PMMA).

[0024] In some embodiments of preparing a biosensor according to any of the above embodiments, the flexible polymer segment comprises a polymer selected from the group consisting of polydimethylsiloxane (PDMS) and poly(2-hydroxyethyl methacrylate) (PHEMA).

[0025] In some embodiments of preparing a biosensor according to any of the above embodiments, the chain extender in the biocompatible membrane is derived from an isocyanate-containing compound.

[0026] In some embodiments of preparing a biosensor according to any of the above embodiments, each chain extender is independently derived from methylene diphenyl diisocyanate (MDI), hexamethylene diisocyanate (HDI), or bis(4-isocyanatocyclohexyl)methane.

[0027] In some embodiments for preparing a biosensor according to any of the above embodiments, the number average molecular weight of A is about 200 to about 10,000, the number average molecular weight of B is about 1,000 to about 20,000, and the number average molecular weight of C is about 1,000 to about 20,000.

[0028] In some embodiments of preparing a biosensor according to any of the above embodiments, the biocompatible membrane comprises about 1 to about 10 parts by weight of A, about 1 to about 5 parts by weight of B, about 1 to about 5 parts by weight of C, and about 1 to about 3 parts by weight of b.

[0029] In some embodiments of preparing a biosensor according to any of the above embodiments, the bond of each of Ab, Bb, and Cb is independently a urea bond or a carbamate bond.

[0030] In some embodiments of preparing a biosensor according to any of the above embodiments, step (2) comprises (a) mixing a peptide probe, dopamine or a derivative thereof, and a metalate in water, thereby forming a solution comprising metal nanoparticles having a coating comprising polydopamine and the peptide probe, the metalate being an oxidizing agent, and (b) depositing the metal nanoparticles with a coating comprising polydopamine and the peptide probe on one of the working electrodes by an electrochemical oxidation reaction.

[0031] In some embodiments of preparing a biosensor according to any of the above embodiments, (i) the concentration of the peptide probe in the solution is about 0.1 to about 10 mg / mL; (ii) the concentration of dopamine or a derivative thereof in the solution is about 1 to about 10 g / L; (iii) the metalate comprises chloroplatinic acid, chloroauric acid, or hexachloroiridic acid, and the concentration of the metalate is about 0.1 to about 1 mg / L; (iv) the pH of the solution is about 7 to about 9; (v) the dissolved oxygen concentration in the solution is less than about 1% saturation; (vi) the temperature is about 20°C to about 40°C; and / or (vii) the potential applied to the working electrode relative to a silver / silver chloride reference electrode is about 0 to about 0.8 V.

[0032] In some embodiments of preparing a biosensor according to any of the above embodiments, step (2) comprises (a) mixing a peptide probe, dopamine or a derivative thereof, and a metalate in water, thereby forming a solution comprising metal nanoparticles having a coating comprising polydopamine and the peptide probe, the metalate being an oxidizing agent, and (b) depositing the metal nanoparticles with a coating comprising polydopamine and the peptide probe on one of the working electrodes by an electrochemical oxidation reaction.

[0033] In some embodiments of preparing a biosensor according to any of the above embodiments, (i) the metal nanoparticles have a particle size of about 1 to about 100 nm; (ii) the concentration of the metal nanoparticles is about 1000 ppm to about 5000 ppm; (iii) the concentration of the peptide probe in the solution is 0.1 to about 10 mg / mL; (iv) the concentration of dopamine or its derivative in the solution is about 1 to about 10 g / L; (v) the pH of the solution is about 7 to about 9; (vi) the dissolved oxygen concentration in the solution is less than about 1% saturation; (vii) the temperature is about 20°C to about 40°C; and / or (viii) the potential applied to the working electrode relative to a silver / silver chloride reference electrode is about -0.5 to about 0.8 V. In some embodiments of preparing a biosensor according to any of the above embodiments, dopamine is used in step (2).

[0034] In some embodiments of preparing a biosensor according to any of the above embodiments, a derivative of dopamine is used in step (2), wherein the derivative of dopamine is formed by oxidizing dopamine or by reducing dopamine. In some embodiments of preparing a biosensor according to any of the above embodiments, the derivative of dopamine is levodopa or dihydroxyindole.

[0035] In some embodiments of preparing a biosensor according to any of the above embodiments, step (3) includes: (a) mixing A, B, and C in an organic solvent at a temperature of about 30 to about 45°C; (b) adding a catalyst and adding an isocyanate-containing compound dropwise to the solution formed in step (a); raising the temperature of the solution to about 55 to 70°C; and reacting the solution at that temperature for about 12 to about 20 hours; and (c) adding deionized water to the solution formed in step (b); and reacting the resulting mixture for about 12 to about 18 hours.

[0036] In some embodiments of preparing a biosensor according to any of the above embodiments, (i) the organic solvent is tetrahydrofuran (THF), cyclohexanone, isobutanol, or a mixture thereof, and (ii) the ratio of organic solvent to the total mass of A, B, and C is about 2 to about 10 mL:1 g.

[0037] In some embodiments of preparing a biosensor according to any of the above embodiments, the catalyst comprises triethylenediamine or dibutyltin bis(2-ethylhexanoate).

[0038] In some embodiments of preparing a biosensor according to any of the above embodiments, the ratio of the volume of deionized water added in step (c) to the total mass of A, B, and C is about 1 to about 10 mL:1 g.

[0039] In some embodiments of preparing a biosensor according to any of the above embodiments, step (3) comprises forming an adhesive layer over the detection layer and forming a triblock polymer over the adhesive layer, the adhesive layer comprising a polymer comprising a crosslink of a first monomer unit comprising at least two amine moieties and a second monomer unit comprising at least two formyl moieties.

[0040] In some embodiments of preparing a biosensor according to any of the above embodiments, the first monomer unit is 1,6-diaminohexane and the second monomer unit is glutaraldehyde.

[0041] In some embodiments of preparing a biosensor according to any of the above embodiments, the method of crosslinking the first monomer unit and the second monomer unit includes (i) applying the first monomer unit to the sensing layer in ethanol, and (2) applying the second monomer unit to the sensing layer in the vapor phase at a temperature of about 40 to about 55°C.

[0042] In other aspects, methods of using the biosensors described herein are provided. In some embodiments, the biosensors described herein are suitable for use in systems for evaluating analytes in a sample stream. In some embodiments, the systems may provide methods for evaluating a sample fluid for a target analyte. Evaluation may range from detecting the presence of the analyte to determining the concentration of the analyte. The analyte and sample fluid may be any for which the test system is suitable. In some embodiments, the analyte is glucose and the sample fluid is blood or interstitial fluid. [Brief explanation of the drawings]

[0043] [Figure 1] 1 illustrates an exemplary method for forming a sensing layer on top of a working electrode. [Figure 2] Another exemplary method for forming a sensing layer on top of a working electrode is shown. [Figure 3] 1 shows the current output over time for different biosensors at different glucose concentrations. DETAILED DESCRIPTION OF THE INVENTION

[0044] definition

[0045] Unless otherwise defined, all technical and scientific terms used herein shall have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. All patents, published patent applications, patent applications, and non-patent publications mentioned herein are incorporated herein by reference in their entirety. To the extent that a definition set forth in this section contradicts or is otherwise inconsistent with a definition set forth in an application, published application, or other published document incorporated herein by reference, the definition set forth in this section shall take precedence over the definition incorporated herein by reference.

[0046] As used herein, and unless otherwise specified, the terms "about" and "approximately," when used in reference to a dose, amount, or weight percent of a component of a composition or dosage form, mean a dose, amount, or weight percent that would be recognized by one of ordinary skill in the art as providing an equivalent pharmacological effect to that resulting from the specified dose, amount, or weight percent. Specifically, the terms "about" and "approximately," when used in this context, contemplate a dose, amount, or weight percent that is within 15%, 10%, 5%, 4%, 3%, 2%, 1%, or 0.5% of the specified dose. As used herein, "a" or "an" means "at least one" or "one or more."

[0047] As used herein, the terms "including," "containing," and "comprising" are used in an open, non-limiting sense. For clarity of disclosure, and not by way of limitation, the detailed description of the invention is divided into the subsections that follow. Biosensors

[0048] In one aspect, the invention provides a biosensor, the biosensor comprising a substrate, a working electrode on top of the substrate, a sensing layer on top of the working electrode, the sensing layer comprising metal nanoparticles, polydopamine, and a peptide probe, and a biocompatible membrane on top of the sensing layer, the biocompatible membrane comprising a reblock polymer AbBbC, where A is a hydrophilic soft segment, B is a hydrophobic hard segment, C is a flexible polymer segment, and b is a chain extender. i. Substrate

[0049] Examples of these substrate materials include, but are not limited to, inorganic materials such as glass and silicon wafers, and organic materials such as polyimides and polydimethylsiloxanes. In some embodiments, the substrate comprises glass. In some embodiments, the substrate comprises a silicon wafer. In some embodiments, the substrate comprises a polyimide. In some embodiments, the substrate comprises a polydimethylsiloxane. ii. Working electrode

[0050] In some embodiments, the working electrode may be prepared using any suitable conductive material. In some embodiments, the working electrode comprises carbon, graphene, gold, or platinum. In some embodiments, the working electrode comprises carbon. In some embodiments, the working electrode comprises graphene. In some embodiments, the working electrode comprises gold. In some embodiments, the working electrode comprises platinum. iii. Detection layer

[0051] In some embodiments, the detection layer comprises metal nanoparticles, polydopamine, and peptide probes. In some embodiments, the term "nanoparticle" refers to nanoscale particles having a size measured in nanometers. In some embodiments, the metal nanoparticles are platinum nanoparticles, gold nanoparticles, or iridium nanoparticles. In some embodiments, the metal nanoparticles are platinum nanoparticles. In some embodiments, the metal nanoparticles are gold nanoparticles. In some embodiments, the metal nanoparticles are iridium nanoparticles.

[0052] In some embodiments, the particle size of the metal nanoparticles is about 1 to about 900, about 1 to about 800, about 1 to about 700, about 1 to about 600, about 1 to about 500, about 1 to about 400, about 1 to about 300, about 1 to about 200, about 1 to about 100, about 1 to about 50, about 50 to about 900, about 50 to about 800, about 50 to about 700, about 50 ~ about 600, about 50 to about 500, about 50 to about 400, about 50 to about 300, about 50 to about 200, about 50 to about 100, about 100 to about 900, about 200 to about 800, about 200 to about 700, about 200 to about 600, about 200 to about 500, about 200 to about 400, about 200 to about 300, 300 to about 900, about 300 to about 800, about 300 to about 700, about 300 to about 600, about 300 to about 500, about 300 to about 400, 400 to about 900, about 400 to about 800, about 400 to about 700, about 400 to about 600, about 400 to about 500, about 500 to about 900, about 500 to about 800, about 500 to about 700, about 50 In some embodiments, the particle size of the metal nanoparticles is less than about 900, about 800, about 600, about 700, about 400, about 300, about 200, about 100, about 900, about 600, about 800, about 700, about 900, about 1 to about 90, about 1 to about 80, about 1 to about 70, about 1 to about 60, about 1 to about 50, about 1 to about 40, about 1 to about 30, about 1 to about 20, or about 1 to about 10 nm. In some embodiments, the metal nanoparticles have a particle size of at least about 900, about 800, about 700, about 600, about 500, about 400, about 300, about 200, about 100, about 90, about 80, about 70, about 60, about 50, about 40, about 30, about 20, about 10, or about 1 nm. In some embodiments, the metal nanoparticles have a particle size of about 900, about 800, about 700, about 600, about 500, about 400, about 300, about 200, about 100, about 90, about 80, about 70, about 60, about 50, about 40, about 30, about 20, about 10, or about 1 nm. In some embodiments, the metal nanoparticles have a particle size of about 1 to about 100 nm.

[0053] In some embodiments, the peptide probe comprises an enzyme, an antibody, or a peptide-containing polymer. In some embodiments, the peptide probe comprises an enzyme. In some embodiments, the peptide probe comprises an oxidoreductase. In some embodiments, the peptide probe comprises an oxidase, such as glucose oxidase, glutamate oxidase, alcohol oxidase, lactate oxidase, ascorbate oxidase, cholesterol oxidase, or choline oxidase. In some embodiments, the peptide probe comprises a dehydrogenase, such as alcohol dehydrogenase, glutamate dehydrogenase, glucose dehydrogenase, or lactate dehydrogenase. In some embodiments, the peptide probe comprises a peroxidase, such as horseradish peroxidase. In some embodiments, the peptide probe comprises glucose oxidase, glutamate oxidase, alcohol oxidase, lactate oxidase, ascorbic acid oxidase, cholesterol oxidase, choline oxidase, alcohol dehydrogenase, glutamate dehydrogenase, glucose dehydrogenase, lactate dehydrogenase, or horseradish peroxidase. In some embodiments, the peptide probe comprises glucose oxidase, glucose dehydrogenase, or horseradish peroxidase. In some embodiments, the peptide probe comprises an antibody, such as a hepatitis B antibody. In some embodiments, the peptide probe comprises a polymer comprising a peptide.

[0054] In some embodiments, the metal nanoparticles are coated with polydopamine and peptide probes. In some embodiments, the metal nanoparticles are mixed with polydopamine and peptide probes. iv. Biocompatible membrane

[0055] In some embodiments, the biocompatible membrane comprises a triblock polymer AbBbC, where A is a hydrophilic soft segment. In some embodiments, the hydrophilic soft segment comprises a polymer selected from the group consisting of polyethylene glycol (PEG), polypropylene glycol (PPG), and polyetheramine (PEA). In some embodiments, the hydrophilic soft segment comprises PEG. In some embodiments, the hydrophilic soft segment comprises PPG. In some embodiments, the hydrophilic soft segment comprises PEA. In some embodiments, the hydrophilic soft segment comprises at least two polymers selected from the group consisting of PEG, PPG, and PEA. In some embodiments, the hydrophilic soft segment comprises PEG, PPG, and PEA.

[0056] In some embodiments, the biocompatible membrane comprises a triblock polymer AbBbC, where B is a hydrophobic hard segment. In some embodiments, the hydrophobic hard segment comprises a polymer selected from the group consisting of polycarbonate (PC) and poly(methyl methacrylate) (PMMA). In some embodiments, the hydrophobic hard segment comprises PC. In some embodiments, the hydrophobic hard segment comprises PMMA. In some embodiments, the hydrophobic hard segment comprises PC and PMMA.

[0057] In some embodiments, the biocompatible membrane comprises a triblock polymer AbBbC, where C is a flexible polymer segment. In some embodiments, the flexible polymer segment comprises a polymer selected from the group consisting of polydimethylsiloxane (PDMS) and poly(2-hydroxyethyl methacrylate) (PHEMA). In some embodiments, the flexible polymer segment comprises PDMS. In some embodiments, the flexible polymer segment comprises PHEMA. In some embodiments, the flexible polymer segment comprises PDMS and PHEMA.

[0058] In some embodiments, the biocompatible membrane comprises a triblock polymer AbBbC, where b is a chain extender. In some embodiments, the chain extender of the biocompatible membrane is derived from a compound containing an isocyanate (i.e., an —NCO group). In some embodiments, each chain extender is independently derived from methylene diphenyl diisocyanate (MDI), hexamethylene diisocyanate (HDI), or bis(4-isocyanatocyclohexyl)methane. In some embodiments, the chain extender is MDI. In some embodiments, the chain extender is HDI. In some embodiments, the chain extender is bis(4-isocyanatocyclohexyl)methane.

[0059] In some embodiments, the molecular weight of each of A, B, and C is determined by measuring the molecular weight of n polymer molecules, summing the weights, and dividing the total mass by n (i.e., the number average molecular weight). In some embodiments, the number average molecular weight of A is about 100 to about 10,000, about 200 to about 10,000, about 500 to about 10,000, about 1,000 to about 10,000, about 2,000 to about 10,000, or about 5,000 to about 10,000. In some embodiments, the number average molecular weight of A is at least about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1000, about 2000, about 3000, about 4000, about 5000, about 6000, about 7000, about 8000, about 9000, about 10000, about 15000, or about 20000. In some embodiments, the number average molecular weight of A is less than about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1000, about 2000, about 3000, about 4000, about 5000, about 6000, about 7000, about 8000, about 9000, about 10000, about 15000, or about 20000. In some embodiments, the number average molecular weight of A is about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1000, about 2000, about 3000, about 4000, about 5000, about 6000, about 7000, about 8000, about 9000, about 10000, about 15000, or about 20000. In some embodiments, the number average molecular weight of A is about 200 to about 10000.

[0060] In some embodiments, the number average molecular weight of B is about 100 to about 20,000, about 200 to about 20,000, about 500 to about 20,000, about 1,000 to about 20,000, about 2,000 to about 20,000, or about 5,000 to about 20,000. In some embodiments, the number average molecular weight of B is at least about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1,000, about 2,000, about 3,000, about 4,000, about 5,000, about 6,000, about 7,000, about 8,000, about 9,000, about 10,000, about 15,000, or about 20,000. In some embodiments, the number average molecular weight of B is less than about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1000, about 2000, about 3000, about 4000, about 5000, about 6000, about 7000, about 8000, about 9000, about 10000, about 15000, or less than about 20000. In some embodiments, the number average molecular weight of B is about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1000, about 2000, about 3000, about 4000, about 5000, about 6000, about 7000, about 8000, about 9000, about 10000, about 11000, about 12000, about 13000, about 14000, about 15000, about 16000, about 17000, about 18000, about 19000, or about 20000. In some embodiments, the number average molecular weight of B is about 1000 to about 20000.

[0061] In some embodiments, the number average molecular weight of C is about 100 to about 20,000, about 200 to about 20,000, about 500 to about 20,000, about 1,000 to about 20,000, about 2,000 to about 20,000, or about 5,000 to about 20,000. In some embodiments, the number average molecular weight of C is at least about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1,000, about 2,000, about 3,000, about 4,000, about 5,000, about 6,000, about 7,000, about 8,000, about 9,000, about 10,000, about 15,000, or about 20,000. In some embodiments, the number average molecular weight of C is less than about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1000, about 2000, about 3000, about 4000, about 5000, about 6000, about 7000, about 8000, about 9000, about 10000, about 15000, or less than about 20000. In some embodiments, the number average molecular weight of C is about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1000, about 2000, about 3000, about 4000, about 5000, about 6000, about 7000, about 8000, about 9000, about 10000, about 11000, about 12000, about 13000, about 14000, about 15000, about 16000, about 17000, about 18000, about 19000, or about 20000. In some embodiments, the number average molecular weight of C is about 1000 to about 20000.

[0062] In some embodiments of preparing a biosensor according to any of the above embodiments, the biocompatible membrane comprises about 1 to about 10 parts by weight of A, about 1 to about 5 parts by weight of B, about 1 to about 5 parts by weight of C, and about 1 to about 3 parts by weight of b.

[0063] In some embodiments, the bonds between Ab, Bb, and Cb themselves according to any of the above aspects are independently urea or carbamate bonds. In some embodiments, the bond between Ab is a urea bond. In some embodiments, the bond between Ab is a carbamate bond. In some embodiments, the bond between Bb is a urea bond. In some embodiments, the bond between Bb is a carbamate bond. In some embodiments, the bond between Cb is a urea bond. In some embodiments, the bond between Cb is a carbamate bond. v. Adhesive layer

[0064] In some embodiments, the biosensor of any of the above aspects further comprises an adhesive layer positioned between the sensing layer and the biocompatible membrane, the adhesive layer comprising a polymer including a first monomeric unit comprising at least two amine moieties crosslinked with a second monomeric unit comprising at least two formyl moieties.

[0065] In some embodiments, the first monomer unit comprises at least 2, 3, 4, or 5 amine moieties. In some embodiments, the first monomer unit comprises two amine moieties. In some embodiments, the first monomer unit has the structure HN-alkylene-NH. "Alkylene" refers to a divalent aliphatic hydrocarbyl group, either linear or branched, preferably having 1 to 8 carbon atoms. Examples of alkylene include, but are not limited to, methylene (-CH-), ethylene (-CHCH-), n-propylene (-CHCHCH-), iso-propylene (-CHCH(CH)-), -C(CH)CHCH-, -C(CH)CH-, and the like. In some embodiments, the first monomer unit is 1,6-diaminohexane.

[0066] In some embodiments, the second monomer unit comprises at least 2, 3, 4, or 5 formyl moieties. In some embodiments, the second monomer unit comprises 2 formyl moieties. In some embodiments, the second monomer unit is glyoxal, malondialdehyde, succindialdehyde, glutaraldehyde, or phthalaldehyde. In some embodiments, the second monomer unit is glutaraldehyde.

[0067] In some embodiments, the biosensor of any of the above aspects further includes a blank electrode, a counter electrode, and a reference electrode that are substantially identical to the working electrode, and the blank electrode is directly covered by a biocompatible membrane or directly covered by an adhesive layer that is covered by the biocompatible membrane. In some embodiments, the working electrode and the blank electrode are made of substantially the same material, i.e., the same or nearly the same material is used, or both the working electrode and the blank electrode are substantially sized so that both electrodes have the same or nearly the same electron mobility. In some embodiments, the difference in electron mobility between the two electrodes is less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0.5%. In some embodiments, the working electrode and the blank electrode are made of the same material, and there is no difference in their electron mobility. In some embodiments, the working electrode and the counter electrode are made of substantially the same material, i.e., the same or nearly the same material is used for both the working electrode and the counter electrode so that both electrodes have the same or nearly the same electron mobility. In some embodiments, the difference in electron mobility between the two electrodes is less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0.5%. In some embodiments, the working electrode and counter electrode are made of the same material and have no difference in electron mobility between them.

[0068] In some embodiments, the minimum distance between the working electrode and the blank electrode is about 1 mm or less, about 2 mm or less, about 3 mm or less, about 4 mm or less, about 5 mm or less, about 6 mm or less, about 7 mm or less, about 8 mm or less, about 9 mm or less, or about 10 mm or less. In some embodiments, the minimum distance between the working electrode and the blank electrode is less than about 1 mm, about 2 mm, about 3 mm, about 4 mm, about 5 mm, about 6 mm, about 7 mm, about 8 mm, about 9 mm, or about 10 mm. In some embodiments, the minimum distance between the working electrode and the blank electrode is about 1 mm, about 2 mm, about 3 mm, about 4 mm, about 5 mm, about 6 mm, about 7 mm, about 8 mm, about 9 mm, or about 10 mm. Preparation method

[0069] In another aspect, the present invention provides a method for preparing a biosensor according to any of the above aspects, comprising: (1) forming a working electrode on a substrate; (2) forming a sensing layer on top of the working electrode, the sensing layer comprising metal nanoparticles, polydopamine, and a peptide probe; and (3) forming a reblock polymer AbBbC on top of the sensing layer, where A is a hydrophilic soft segment, B is a hydrophobic hard segment, C is a flexible polymer segment, and b is a chain extender, and the working electrode, sensing layer, and reblock polymer AbBbC are described in detail herein. In some embodiments, step (1) includes forming a working electrode on top of a substrate by etching or screen printing.

[0070] In some embodiments, step (2) comprises (a) mixing a peptide probe, dopamine or a derivative thereof, and a metalate in water, thereby forming a solution comprising metal nanoparticles having a coating comprising polydopamine and the peptide probe, the metalate being an oxidizing agent, and (b) depositing the metal nanoparticles with a coating comprising polydopamine and the peptide probe on one of the working electrodes by an electrochemical oxidation reaction.

[0071] In some embodiments of preparing a biosensor according to any of the above embodiments, (i) the metal nanoparticles have a particle size of about 1 to about 100 nm; (ii) the concentration of the metal nanoparticles is about 1000 ppm to about 5000 ppm; (iii) the concentration of the peptide probe in the solution is 0.1 to about 10 mg / mL; (iv) the concentration of dopamine or its derivative in the solution is about 1 to about 10 g / L; (v) the pH of the solution is about 7 to about 9; (vi) the dissolved oxygen concentration in the solution is less than about 1% saturation; (vii) the temperature is about 20°C to about 40°C; and / or (viii) the potential applied to the working electrode relative to a silver / silver chloride reference electrode is about -0.5 to about 0.8 V.

[0072] In some embodiments of preparing a biosensor according to any of the above embodiments, the concentration of the peptide probe in the solution in step (2) is from about 0.1 to about 50, from about 0.1 to about 40, from about 0.1 to about 30, from about 0.1 to about 20, from about 0.1 to about 10, from about 0.1 to about 9, from about 0.1 to about 8, from about 0.1 to about 7, from about 0.1 to about 6, from about 0.1 to about 5, from about 0.1 to about 4, from about 0.1 to about 3, from about 0.1 to about 2, from about 0.1 to about 1, from about 0.1 to about 0.5, from about 0.5 to about 50, from about 0.5 to about 40, from about 0.5 to about about 1 to about 30, about 0.5 to about 20, about 0.5 to about 10, about 0.5 to about 9, about 0.5 to about 8, about 0.5 to about 7, about 0.5 to about 6, about 0.5 to about 5, about 0.5 to about 4, about 0.5 to about 3, about 0.5 to about 2, about 0.5 to about 1, about 1 to about 50, about 1 to about 40, about 1 to about 30, about 1 to about 20, about 1 to about 10, about 1 to about 9, about 1 to about 8, about 1 to about 7, about 1 to about 6, about 1 to about 5, about 1 to about 4, about 1 to about 3, about 1 to about 2, about 5 to about 50, about 5 to about 40, about 5 to about 30, about 5 to about 20, or about 5 to about 10 mg / mL. In some embodiments, the concentration of the peptide probe in the solution of step (2) is at least about 0.01, about 0.05, about 0.1, about 0.5, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 mg / mL. In some embodiments, the concentration of the peptide probe in the solution of step (2) is less than about 0.01, about 0.05, about 0.1, about 0.5, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 mg / mL. In some embodiments, the concentration of the peptide probe in the solution of step (2) is about 0.01, about 0.05, about 0.1, about 0.5, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 mg / mL. In some embodiments, the concentration of the peptide probe in the solution in step (2) is about 0.1 to about 10 mg / mL.

[0073] In some embodiments of preparing a biosensor according to any of the above embodiments, the concentration of dopamine or a derivative thereof in the solution in step (2) is about 0.5 to about 50, about 0.5 to about 40, about 0.5 to about 30, about 0.5 to about 20, about 0.5 to about 10, about 0.5 to about 9, about 0.5 to about 8, about 0.5 to about 7, about 0.5 to about 6, about 0.5 to about 5, In some embodiments, the concentration of dopamine or a derivative thereof in the solution of step (2) is at least about 0.01, about 0.05, about 0.1, about 0.5, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, or about 21. In some embodiments, the concentration of dopamine or a derivative thereof in the solution of step (2) is at least about 0.01, about 0.05, about 0.1, about 0.5, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 g / L. In some embodiments, the concentration of dopamine or a derivative thereof in the solution of step (2) is less than about 0.01, about 0.05, about 0.1, about 0.5, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 g / L. In some embodiments, the concentration of dopamine or a derivative thereof in the solution of step (2) is about 0.01, about 0.05, about 0.1, about 0.5, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 g / L. In some embodiments, the concentration of dopamine or a derivative thereof in the solution of step (2) is about 1 to about 10 g / L.

[0074] In some embodiments of preparing a biosensor according to any of the above embodiments, the metalate comprises chloroplatinic acid, chloroauric acid, or chlorophenylphosphoric acid. In some embodiments, the metalate comprises chloroplatinic acid. In some embodiments, the metalate comprises chloroauric acid. In some embodiments, the metalate comprises chloroiridic acid.

[0075] In some embodiments of preparing a biosensor according to any of the above embodiments, the concentration of metalate in the solution in step (2) is from about 0.01 to about 10, from about 0.01 to about 5, from about 0.01 to about 1, from about 0.01 to about 0.5, from about 0.01 to about 0.1, from about 0.05 to about 10, from about 0.05 to about 5, from about 0.05 to about 1, from about 0.05 to about 0.5, from about 0.05 to about 0.1, from about 0.1 to about 10 , about 0.1 to about 9, about 0.1 to about 8, about 0.1 to about 7, about 0.1 to about 6, about 0.1 to about 5, about 0.1 to about 4, about 0.1 to about 3, about 0.1 to about 2, about 0.1 to about 1, about 0.1 to about 0.5, about 0.5 to about 10, about 0.5 to about 9, about 0.5 to about 8, about 0.5 to about 7, about 0.5 to about 6, about 0.5 to about 5, about 0.5 to about 4, about 0.5 to about 3, about 0.5 to about 2, or about 0.5 to about 1 mg / mL. In some embodiments, the concentration of the metalate in the solution of step (2) is at least about 0.01, about 0.05, about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 mg / mL. In some embodiments, the concentration of the metalate in the solution of step (2) is less than about 0.01, about 0.05, about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 mg / mL. In some embodiments, the concentration of the metalate in the solution of step (2) is about 0.01, about 0.05, about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 mg / mL. In some embodiments, the concentration of the metalate is about 0.1 to about 1 mg / L.

[0076] In some embodiments of preparing a biosensor according to any of the above embodiments, the pH of the solution in step (2) is about 6 to about 10, about 6 to about 9.5, about 6 to about 9, about 6 to about 8.5, about 6 to about 8, about 6 to about 7.5, about 6 to about 7, about 6 to about 6.5, about 6.5 to about 10, about 6.5 to about 9.5, about 6.5 to about 9, about 6.5 to about 8.5, about 6.5 to about 8, about 6.5 to about 7 0.5, about 6.5 to about 7, about 7 to about 10, about 7 to about 9.5, about 7 to about 9, about 7 to about 8.5, about 7 to about 8, about 7 to about 7.5, about 7.5 to about 10, about 7.5 to about 9.5, about 7.5 to about 9, about 7.5 to about 8.5, about 7.5 to about 8, about 8 to about 10, about 8 to about 9.5, about 8 to about 9, about 8 to about 8.5, about 8.5 to about 10, about 8.5 to about 9.5, or about 8.5 to about 9. In some embodiments, the pH of the solution in step (2) is at least about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, or about 10. In some embodiments, the pH of the solution in step (2) is less than about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, or about 10. In some embodiments, the pH of the solution in step (2) is about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, or about 10. In some embodiments, the pH of the solution in step (2) is about 7 to about 9.

[0077] In some embodiments of preparing a biosensor according to any of the above embodiments, the dissolved oxygen concentration saturation in the solution of step (2) is less than about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, about 0.5%, about 0.1%, about 0.05%, or about 0.01%. In some embodiments, the dissolved oxygen concentration saturation in the solution of step (2) is less than about 1%.

[0078] In some embodiments of preparing a biosensor according to any of the above embodiments, the temperature at which step (2) is carried out is about 10 to about 50, about 15 to about 50, about 20 to about 50, about 25 to about 50, about 30 to about 50, about 35 to about 50, about 40 to about 50, about 45 to about 50, about 10 to about 45, about 15 to about 45, about 20 to about 45, about 25 to about 45, about 30 to about 45, about 35 to about 45, about 40 to about 45, about 10 to about 40, about 15 to about 40, about 20 to about 40, about 25 to about 40, about 30 to about 40, about 35 to about 40, about 10 to about 35, about 15 to about 35, about 20 to about 35, about 25 to about 35, about 30 to about 35, about 10 to about 30, about 15 to about 30, about 20 to about 30, about 25 to about 30, about 10 to about 25, about 15 to about 25, about 20 to about 25, about 10 to about 20, or about 15 to about 20° C. In some embodiments, the temperature is at least about 10, about 15, about 20, about 25, about 30, about 35, about 40, or about 45° C. In some embodiments, the temperature is less than about 15, about 20, about 25, about 30, about 35, about 40, about 45, or about 50° C. In some embodiments, the temperature is about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, or about 50° C. In some embodiments, the temperature is between about 20° C. and about 40° C.

[0079] In some embodiments of preparing a biosensor according to any of the above embodiments, the potential applied to the working electrode relative to the silver / silver chloride reference solution electrode in step (2) is about −0.5 to about 1.2, about −0.5 to about 1, about −0.5 to about 0.8, about −0.5 to about 0.6, about −0.5 to about 0.4, about −0.5 to about 0.2, about −0.5 to about 0, about 0 to about 1.2, about 0 to about 1, about 0 to about 0.8, about 0 to about 0.6, about 0 to about 0.4, about 0 to about 0.2, about 0.2 to about 1.2, about 0.2 to about 1, about 0.2 to about 0.8, about 0.2 to about 0.6, about 0.2 to about 0.4, about 0.4 to about 1, about 0.4 to about 0.8, about 0.4 to about 0.6, about 0.6 to about 1, about 0.6 to about 0.8, or about 0.8 to about 1 V. In some embodiments, the potential applied to the working electrode relative to a silver / silver chloride reference electrode is at least about -0.5, about 0, about 0.2, about 0.4, about 0.6, about 0.8, or about 1 V. In some embodiments, the potential applied to the working electrode relative to a silver / silver chloride reference solution electrode is less than about 0, about 0.2, about 0.4, about 0.6, about 0.8, about 1, or about 1.2 V. In some embodiments, the potential applied to the working electrode relative to a silver / silver chloride reference solution electrode is about −0.5, about −0.4, about −0.2, about 0, about 0.2, about 0.4, about 0.6, about 0.8, about 1, or about 1.2 V. In some embodiments, the potential applied to the working electrode relative to a silver / silver chloride reference solution electrode is from about 0 to about 0.8 V.

[0080] In some embodiments of preparing a biosensor according to any of the above embodiments, the dimensions of the metal nanoparticles in the solution in step (2) are as detailed herein. In some embodiments, the particle size of the metal nanoparticles is about 1 to about 100 nm.

[0081] In some embodiments of preparing a biosensor according to any of the above embodiments, the concentration of metal nanoparticles in the solution in step (2) is from about 500 ppm to about 8000 ppm, from about 1000 ppm to about 8000 ppm, from about 2000 ppm to about 8000 ppm, from about 3000 ppm to about 8000 ppm, from about 4000 ppm to about 8000 ppm, from about 5000 ppm to about 8000 ppm, from about 6000 ppm to about 8000 ppm, from about 7000 ppm to about 8000 ppm, from about 500 ppm to about 7000 ppm, from about 1000 ppm to about 7000 ppm, from about 2000 ppm to about 7000 ppm, from about 3000 ppm to about 7000 ppm, from about 4000 ppm to about 7000 ppm, from about 5000 ppm to about 7000 ppm, from about 6000 ppm to about 7000 ppm, from about 500 ppm to about 6000 ppm ppm, about 1000 ppm to about 6000 ppm, about 2000 ppm to about 6000 ppm, about 3000 ppm to about 6000 ppm, about 4000 ppm to about 6000 ppm, about 5000 ppm to about 6000 ppm, about 500 ppm to about 5000 ppm, about 1000 ppm to about 5000 ppm, about 2000 ppm to about 5000 ppm, about 3000 ppm to about 5000 ppm, about 4000 ppm to about 5000 ppm, about 500 ppm to about 4000 ppm, about 1000 ppm to about 4000 ppm, about 2000 ppm to about 4000 ppm, about 3000 ppm to about 4000 ppm, about 500 ppm to about 3000 ppm, about 1000 ppm to about 3000 ppm, about 2000 ppm to about 3000 ppm, about 500 ppm to about 2000 ppm, about 1000 ppm to about 2000 ppm, or about 500 ppm to about 1000 ppm. In some embodiments, the concentration of metal nanoparticles in the solution of step (2) is at least about 500, about 1000, about 2000, about 3000, about 4000, about 5000, about 6000, or about 7000 ppm. In some embodiments, the concentration of metal nanoparticles in the solution of step (2) is less than about 1000, about 2000, about 3000, about 4000, about 5000, about 6000, about 7000, or about 8000 ppm.In some embodiments, the concentration of the metal nanoparticles in the solution of step (2) is about 500, about 1000, about 2000, about 3000, about 4000, about 5000, about 6000, about 7000, or about 8000 ppm. In some embodiments, the concentration of the metal nanoparticles in the solution of step (2) is about 1000 ppm to about 5000 ppm.

[0082] In some embodiments of preparing a biosensor according to any of the above embodiments, dopamine is used in step (2). In some embodiments, a derivative of dopamine is used in step (2). In some embodiments, the derivative of dopamine is formed by oxidizing dopamine or reducing dopamine. In some embodiments, the derivative of dopamine is formed by oxidizing dopamine. In some embodiments, the derivative of dopamine is formed by reducing dopamine. In some embodiments, the derivative of dopamine is levodopa or dihydroxyindole. In some embodiments, the derivative of dopamine is levodopa. In some embodiments, the derivative of dopamine is dihydroxyindole.

[0083] In some embodiments of preparing a biosensor according to any of the above embodiments, step (3) includes: (a) mixing A, B, and C in an organic solvent at a temperature of about 30 to about 45°C; (b) adding a catalyst and adding an isocyanate-containing compound dropwise to the solution formed in step (a); raising the temperature of the solution to about 55 to 70°C; and reacting the solution at that temperature for about 12 to about 20 hours; and (c) adding deionized water to the solution formed in step (b); and reacting the resulting mixture for about 12 to about 18 hours. Examples of organic solvents include, but are not limited to, hexane, pentane, cyclopentane, cyclohexane, benzene, toluene, 1,4-dioxane, dichloromethane (DCM), chloroform, ethyl acetate, tetrahydrofuran (THF), cyclohexanone, dichloromethane, acetone, acetonitrile (MeCN), dimethylformamide (DMF), dimethyl sulfoxide (DMSO), 1,3-dimethyl-2-imidazolidinone (DMI), acetic acid, isobutanol, n-butanol, isopropanol, n-propanol, ethanol, and methanol. In some embodiments, the organic solvent is tetrahydrofuran (THF), cyclohexanone, isobutanol, or a mixture thereof. In some embodiments, the organic solvent is THF. In some embodiments, the organic solvent is cyclohexanone. In some embodiments, the organic solvent is isobutanol. In some embodiments, the organic solvent is a mixture of two or three of THF, cyclohexanone, isobutanol, and isobutanol.

[0084] In some embodiments, the ratio of the volume of organic solvent to the total weight of A, B, and C is from about 0.1 to about 20, from about 0.1 to about 15, from about 0.1 to about 10, from about 0.1 to about 9, from about 0.1 to about 8, from about 0.1 to about 7, from about 0.1 to about 6, from about 0.1 to about 5, from about 0.1 to about 4, from about 0.1 to about 3, from about 0.1 to about 2, from about 0.1 to about 1, from about 0.1 to about 0.5, from about 1 to about 20, from about 1 to about 15, from about 1 to about 10, from about 1 to about 9, from about 1 to about 8, from about 1 to about 7, from about 1 to about 6, from about 1 to about 5, from about 1 to about 4, from about 1 to about 3, about 1 to about 2, about 2 to about 20, about 2 to about 15, about 1 to about 10, about 2 to about 9, about 2 to about 8, about 2 to about 7, about 2 to about 6, about 2 to about 5, about 2 to about 4, about 2 to about 3, about 4 to about 20, about 4 to about 15, about 4 to about 10, about 4 to about 9, about 4 to about 8, about 4 to about 7, about 4 to about 6, about 4 to about 5, about 6 to about 20, about 6 to about 15, about 6 to about 10, about 6 to about 9, about 6 to about 8, about 6 to about 7, about 8 to about 20, about 8 to about 15, about 8 to about 10, about 8 to about 9, about 10 to about 20, or about 10 to about 15 mL: 1 g. In some embodiments, the ratio of the total mass of A, B, and C to the volume of organic solvent is at least about 0.1, about 0.5, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, or about 15 mL:1 g. In some embodiments, the ratio of the volume of organic solvent to the total weight of A, B, and C is less than about 0.1, about 0.5, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, or about 15 mL:1 g. In some embodiments, the ratio of the volume of organic solvent to the total weight of A, B, and C is about 0.1, about 0.5, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, or about 15 mL:1 g. In some embodiments, the ratio of the volume of organic solvent to the total weight of A, B, and C is about 2 to about 10 mL:1 g.

[0085] In some embodiments of preparing a biosensor according to any of the above embodiments, the catalyst used in step (b) comprises triethylenediamine or dibutyltin bis(2-ethylhexanoate). In some embodiments, the catalyst comprises triethylenediamine. In some embodiments, the catalyst comprises dibutyltin bis(2-ethylhexanoate). In some embodiments, the catalyst comprises a mixture of triethylenediamine and dibutyltin bis(2-ethylhexanoate).

[0086] In some embodiments of preparing a biosensor according to any of the above embodiments, the ratio of the volume of deionized water added in step (c) to the total weight of A, B, and C is from about 0.1 to about 20, from about 0.1 to about 15, from about 0.1 to about 10, from about 0.1 to about 9, from about 0.1 to about 8, from about 0.1 to about 7, from about 0.1 to about 6, from about 0.1 to about 5, from about 0.1 to about 4, from about 0.1 to about 3, from about 0.1 to about 2, from about 0.1 to about 1, from about 0.1 to about 0.5, from about 1 to about 20, from about 1 to about 15, from about 1 to about 10, from about 1 to about 9, from about 1 to about 8, from about 1 to about 7, from about 1 to about 6, about 1 to about 5, about 1 to about 4, about 1 to about 3, about 1 to about 2, about 2 to about 20, about 2 to about 15, about 1 to about 10, about 2 to about 9, about 2 to about 8, about 2 to about 7, about 2 to about 6, about 2 to about 5, about 2 to about 4, about 2 to about 3, about 4 to about 20, about 4 to about 15, about 4 to about 10, about 4 to about 9, about 4 to about 8, about 4 to about 7, about 4 to about 6, about 4 to about 5, about 6 to about 20, about 6 to about 15, about 6 to about 10, about 6 to about 9, about 6 to about 8, about 6 to about 7, about 8 to about 20, about 8 to about 15, about 8 to about 10, about 8 to about 9, about 10 to about 20, or about 10 to about 15 mL:1g. In some embodiments, the ratio of the volume of deionized water to the total weight of A, B, and C is at least about 0.1, about 0.5, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, or about 15 mL:1 g. In some embodiments, the ratio of the volume of deionized water to the total mass of A, B, and C is about 0.1, about 0.5, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, or about 15 mL:1 g. In some embodiments, the ratio of the volume of deionized water to the total weight of A, B, and C is about 0.1, about 0.5, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, or about 15 mL:1 g. In some embodiments, the ratio of the volume of deionized water to the total mass of A, B, and C is about 1 to about 10 mL:1 g.

[0087] In some embodiments of preparing a biosensor according to any of the above embodiments, step (3) includes forming an adhesive layer on the detection layer and forming a triblock polymer on the adhesive layer, wherein the adhesive layer is described in detail herein. In some embodiments, the first monomer unit is 1,6-diaminohexane and the second monomer unit is glutaraldehyde. In some embodiments of preparing a biosensor according to any of the above embodiments, the method of crosslinking the first monomer unit and the second monomer unit includes (i) applying the first monomer unit to the detection layer in ethanol, and (2) applying the second monomer unit to the detection layer in the vapor phase at a temperature of about 40 to about 55°C. In some embodiments, the temperature is from about 20 to about 60, from about 25 to about 60, from about 30 to about 60, from about 35 to about 60, from about 40 to about 60, from about 45 to about 60, from about 50 to about 60, from about 55 to about 60, from about 20 to about 55, from about 25 to about 55, from about 30 to about 55, from about 35 to about 55, from about 40 to about 55, from about 45 to about 55, from about 50 to about 55, from about 20 to about 50, from about 25 to about 50, from about 30 to about 55 The temperature is about 50° C., about 35 to about 50° C., about 40 to about 50° C., about 45 to about 50° C., about 20 to about 45° C., about 25 to about 45° C., about 30 to about 45° C., about 35 to about 45° C., about 40 to about 45° C., about 20 to about 40° C., about 25 to about 40° C., about 30 to about 40° C., about 35 to about 40° C., about 20 to about 35° C., about 25 to about 35° C., about 30 to about 35° C., about 20 to about 30° C., about 25 to about 30° C., or about 20 to about 25° C. In some embodiments, the temperature is at least about 20, about 25, about 30, about 35, about 40, about 45, about 50, or about 55° C. In some embodiments, the temperature is less than about 25, about 30, about 35, about 40, about 45, about 50, about 55, or about 60° C. In some embodiments, the temperature is about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, or about 60° C. In some embodiments, the temperature is about 40° C. to about 55° C. (Example) The following examples are provided to illustrate the present invention, but not to limit the biosensors and preparation methods disclosed herein. Example 1. Formation of a detection layer on an electrode An exemplary method for forming a sensing layer on an electrode is shown in FIG. 1 and described in more detail further below. Step 1: Platinum electrodes were formed on the glass substrate by etching.

[0088] Step 2: Peptide probe molecules (glucose oxidase), dopamine, and chloroplatinic acid were added to water at 30°C. The concentrations of glucose oxidase, dopamine, and chloroplatinic acid were 5 mg / mL, 5 g / L, and 5 mg / L, respectively. The pH of the solution was adjusted to 8, and the dissolved oxygen concentration in the solution was adjusted to less than 1% saturation. This resulted in the formation of metal nanoparticles with a coating containing polydopamine and the peptide probe in the solution.

[0089] Step 3 - The platinum electrode prepared in step 1 was placed in the solution of step 2, and the metal nanoparticles formed in step 2 were deposited on top of the electrode by electrochemical oxidation. The potential applied to the electrode was 0.4 V relative to the silver / silver chloride reference solution electrode. Example 2. Formation of a detection layer on an electrode Another exemplary method for forming a sensing layer on an electrode is shown in FIG. 2 and described in more detail further below. Step 1—Gold electrodes were formed on polydimethylsiloxane substrates by screen printing.

[0090] Step 2: Gold nanoparticles, peptide probe molecules (hepatitis B antibodies), and dopamine were added to water at 35°C. The size of the gold nanoparticles was approximately 50 nm. The concentrations of the gold nanoparticles, peptide probe molecules, and dopamine were 25,000 ppm, 4 mg / mL, and 6 g / L, respectively. The pH of the solution was adjusted to 7, and the dissolved oxygen concentration in the solution was adjusted to less than 1% saturation. The gold electrode prepared in step 1 was immersed in the solution. A detection layer containing polydopamine, gold nanoparticles, and peptide probes was formed on the electrode by electrochemical oxidation. The potential applied to the electrode was 0.6 V relative to a silver / silver chloride reference electrode. Example 3. Formation of a biocompatible membrane iv. Example 3.1

[0091] Step 1 - Polyetheramine (number average molecular weight: 1000, 25 g), polycarbonate diol (number average molecular weight: 5000, 10 g), diamino-terminated polydimethylsiloxane (number average molecular weight: 5000, 15 g) were added to 100 mL of tetrahydrofuran at 40°C and mixed thoroughly.

[0092] Step 2 - Triethylenediamine was added to the solution from Step 1. Then, 12 g of methylenediphenyldiisocyanate was added dropwise. The mixture was reacted at 65°C for 12 hours. Step 3 - To the solution from step 2, 50 mL of deionized water was added and the mixture was allowed to react for 12 hours. The resulting triblock polymer was applied to the detection layer formed in Example 1 or 2 using an appropriate method. ii. Example 3.2

[0093] Step 1 - Amino-terminated polyethylene glycol (number average molecular weight: 2000, 20 g), polycarbonate diol (number average molecular weight: 2000, 15 g), poly(methyl methacrylate) (number average molecular weight: 2000, 15 g), and diamino-terminated polydimethylsiloxane (number average molecular weight: 8000, 15 g) were added to 500 mL of tetrahydrofuran at 30°C and mixed thoroughly.

[0094] Step 2 - Triethylenediamine was added to the solution from Step 1. Then, a mixture of methylenediphenyldiisocyanate and bis(4-isocyanatocyclohexyl)methane was added dropwise. The mixture was reacted at 55°C for 14 hours. Step 3 - To the solution from step 2, 500 mL of deionized water was added and the mixture was allowed to react for 18 hours. The resulting triblock polymer was applied to the detection layer formed in Example 1 or 2 using an appropriate method. iii. Example 3.3

[0095] Step 1—Amino-terminated polypropylene glycol (molecular weight: 500, 15 g), polyetheramine (molecular weight: 600, 10 g), poly(bisphenol A polycarbonate) (molecular weight: 5000, 25 g), diamino-terminated polydimethylsiloxane (molecular weight: 20000, 10 g), and poly(2-hydroxyethyl methacrylate) (molecular weight: 5000, 5 g) were added to 150 mL of isobutanol at 35° C. and mixed thoroughly.

[0096] Step 2 - Dibutyltin bis(2-ethylhexanoate) was added to the solution from Step 1. Then, 15 g of hexamethylene diisocyanate was added dropwise. The mixture was reacted at 60°C for 16 hours. Step 3 - 150 mL of deionized water was added to the solution from step 2 and the mixture was allowed to react for 14 hours. The resulting triblock polymer was applied to the detection layer formed in Example 1 or 2 using an appropriate method. iv. Example 3.4

[0097] Step 1—Amino-terminated polyethylene glycol (number average molecular weight: 10,000, 30 g), polycarbonate diol (number average molecular weight: 2,000, 5 g), poly(methyl methacrylate) (number average molecular weight: 2,000, 5 g), and poly(2-hydroxyethyl methacrylate) (molecular weight: 20,000, 15 g) were added to 600 mL of isobutanol at 35° C. and mixed thoroughly.

[0098] Step 2: Dibutyltin bis(2-ethylhexanoate) was added to the solution from Step 1. Then, 20 g of bis(4-isocyanatocyclohexyl)methane was added dropwise. The mixture was reacted at 70 °C for 16 hours. The resulting triblock polymer was applied to the detection layer formed in Example 1 or 2 using an appropriate method. Example 4. Formation of adhesive layer Step 1 - 10 g of 1,6-diaminohexane was dissolved in 100 mL of ethanol.

[0099] Step 2 - The substrate with the detection layer formed in Example 1 or 2 was immersed in the solution of Step 1 for 10 minutes, rinsed three times with ethanol, immersed in ethanol for 10 minutes, and dried. Step 3 - The substrate prepared in step 2 was contacted with glutaraldehyde in the vapor phase at 40°C for 10 minutes. Step 4 - The solution formed in any one of Examples 3.1-3.4 was applied to the substrate prepared in Step 3 to form a biocompatible film by spin coating. Example 5.

[0100] Biosensors with only the sensing layer described herein, biocompatible membranes deposited by conventional methods described herein, and sensing layers alone, as well as biosensors with the sensing layer, biocompatible membrane, and adhesive layer described herein, were contacted with glucose solutions. A constant potential was applied to the working electrode for each biosensor, and the current output at the working electrode was measured at six glucose concentrations: 0 mmol / L, 5 mmol / L, 10 mmol / L, 15 mmol / L, 20 mmol / L, and 25 mmol / L. Figure 3 shows the current output over time for each biosensor at different glucose concentrations. As shown in Figure 3, the biosensors with the sensing layer, biocompatible membrane, and adhesive layer described herein exhibited more stable current output over time and better linearity as the glucose concentration increased.

[0101] The foregoing specification of the invention will enable one of ordinary skill in the art to make and use the biosensors and methods described herein, and one of ordinary skill in the art will understand and recognize that there are variations, combinations, and equivalents of the specific embodiments, methods, and examples herein. Accordingly, the biosensors and methods provided herein are not limited by the above-described embodiments, methods, and examples, but by all embodiments and methods within the scope and spirit of the compounds, uses, and methods provided herein.

Claims

1. 1. A biosensor comprising: A substrate; a working electrode on top of the substrate; a sensing layer comprising metal nanoparticles, polydopamine, and a peptide probe, the sensing layer being on the working electrode; a biocompatible membrane on top of the detection layer, the biocompatible membrane comprising a triblock polymer A-b-B-b-C (A, B, C are in any order), where: A is a hydrophilic soft segment, B is a hydrophobic hard segment; C is a flexible polymer segment, b is a chain extender, The biosensor further comprises an adhesive layer between the sensing layer and the biocompatible membrane, the adhesive layer having a first monomeric unit comprising at least two amine moieties and a second monomeric unit comprising at least two or at least two aldehyde groups; A biosensor, wherein an aldehyde group from the second monomer unit reacts with an amine moiety in the first monomer unit and with an amine moiety in a peptide probe in the detection layer to form a crosslinked polymer.

2. The biosensor of claim 1 , wherein the working electrode comprises carbon, graphene, gold, or platinum.

3. The biosensor of claim 1 or 2, wherein the metal nanoparticles are platinum nanoparticles, gold nanoparticles, or iridium nanoparticles.

4. The biosensor according to any one of claims 1 to 3, wherein the metal nanoparticles have a particle size of 1 to 100 nm.

5. The biosensor of any one of claims 1 to 4, wherein the peptide probe comprises an enzyme, an antibody, or a polymer containing a peptide.

6. The biosensor of claim 1 , wherein the peptide probe comprises an oxidoreductase.

7. The biosensor of any one of claims 1 to 6, wherein the peptide probe comprises glucose oxidase, glucose dehydrogenase, or horseradish peroxidase.

8. The biosensor of any one of claims 1 to 7, wherein the metal nanoparticles are coated with polydopamine and a peptide probe.

9. The biosensor of any one of claims 1 to 7, wherein the metal nanoparticles are mixed with polydopamine and the peptide probe.

10. The biosensor of any one of claims 1 to 9, wherein the hydrophilic soft segment comprises a polymer selected from the group consisting of polyethylene glycol (PEG), polypropylene glycol (PPG), and polyetheramine (PEA).

11. The biosensor of any one of claims 1 to 10, wherein the hydrophobic hard segment comprises a polymer selected from the group consisting of polycarbonate (PC) and poly(methyl methacrylate) (PMMA).

12. The biosensor of any one of claims 1 to 11, wherein the flexible polymer segment comprises a polymer selected from the group consisting of polydimethylsiloxane (PDMS) and poly(2-hydroxyethyl methacrylate) (PHEMA).

13. The biosensor of any one of claims 1 to 12, wherein the chain extender of the biocompatible membrane is derived from an isocyanate-containing compound.

14. 14. The biosensor of any one of claims 1 to 13, wherein each chain extender is independently derived from methylene diphenyl diisocyanate (MDI), hexamethylene diisocyanate (HDI), or bis(4-isocyanatocyclohexyl)methane.

15. The biosensor according to any one of claims 1 to 14, The number average molecular weight of A is 200 to 10,000; The number average molecular weight of B is 1,000 to 20,000; and A biosensor in which C has a number average molecular weight of 1,000 to 20,000.

16. The biosensor according to any one of claims 1 to 15, The biocompatible membrane 1 to 10 parts by weight of A; 1 to 5 parts by weight of B; 1 to 5 parts by weight of C, and A biosensor comprising 1 to 3 parts by weight of b.

17. The biosensor of any one of claims 1 to 16, wherein each of the bonds Ab, Bb, and Cb is independently a urea bond or a carbamate bond.

18. 2. The biosensor of claim 1, wherein the first monomer unit is 1,6-diaminohexane and the second monomer unit is glutaraldehyde.

19. The biosensor of any one of claims 1 to 18, further comprising a blank electrode substantially identical to the working electrode, a counter electrode, and a reference electrode, wherein the blank electrode is directly covered by the biocompatible membrane.

20. 10. The biosensor of claim 1, further comprising a blank electrode, a counter electrode, and a reference electrode substantially identical to the working electrode, the blank electrode being covered by the adhesive layer, and the adhesive layer being covered by the biocompatible membrane.

21. The biosensor of claim 19 or 20, wherein the minimum distance between the working electrode and the blank electrode is 5 mm or less.

22. 1. A method for preparing a biosensor, comprising: (1) forming a working electrode on the substrate; (2) The detection layer comprises metal nanoparticles, polydopamine, and a peptide probe. forming a detection layer on the working electrode; (3) forming a triblock polymer A-b-B-b-C (A, B, and C are in any order) on the detection layer, wherein: A is a hydrophilic soft segment, B is a hydrophobic hard segment; C is a flexible polymer segment, b is a chain extender, The step (3) includes forming an adhesive layer on the detection layer and forming a triblock polymer on the adhesive layer, the adhesive layer having a first monomer unit including at least two amine moieties and a second monomer unit including at least two kinds or at least two aldehyde groups; A method of preparing a biosensor, wherein an aldehyde group from the second monomer unit reacts with an amine moiety in the first monomer unit and with an amine moiety in a peptide probe in a detection layer to form a crosslinked polymer.

23. 23. The method of claim 22, wherein the working electrode comprises carbon, graphene, gold, or platinum elemental.

24. 24. The method of claim 22 or 23, wherein the step (1) comprises forming a working electrode on top of a substrate by etching or screen printing.

25. The method of any one of claims 22 to 24, wherein the metal nanoparticles are platinum nanoparticles, gold nanoparticles, or iridium nanoparticles.

26. The method according to any one of claims 22 to 25, wherein the metal nanoparticles have a particle size of 1 to 100 nm.

27. The method of any one of claims 23 to 27, wherein the peptide probe comprises an enzyme, an antibody, or a peptide-containing polymer.

28. The method of any one of claims 22 to 27, wherein the peptide probe comprises an oxidoreductase.

29. The method of any one of claims 22 to 28, wherein the peptide probe comprises glucose oxidase, glucose dehydrogenase, or horseradish peroxidase.

30. 30. The method of any one of claims 22 to 29, wherein the hydrophilic soft segment comprises a polymer selected from the group consisting of polyethylene glycol (PEG), polypropylene glycol (PPG), and polyetheramine (PEA).

31. The method of any one of claims 22 to 30, wherein the hydrophobic hard segment comprises a polymer selected from the group consisting of polycarbonate (PC) and poly(methyl methacrylate) (PMMA).

32. The method of any one of claims 22 to 31, wherein the flexible polymer segment comprises a polymer selected from the group consisting of polydimethylsiloxane (PDMS) and poly(2-hydroxyethyl methacrylate) (PHEMA).

33. The method of any one of claims 22 to 32, wherein the chain extender of the biocompatible membrane is derived from an isocyanate-containing compound.

34. Each of the chain extenders is independently selected from methylene diphenyl diisocyanate (MDI), hexamethylene diisocyanate (HDI), or bis(4-isocyanatocyclohexyl 34. The method of any one of claims 22 to 33, wherein the olefin is derived from methane.

35. The method according to any one of claims 22 to 34, comprising the steps of: The number average molecular weight of A is 200 to 10,000; The number average molecular weight of B is 1,000 to 20,000; and The number average molecular weight of C is 1,000 to 20,000.

36. The method according to any one of claims 22 to 35, wherein the biocompatible membrane comprises 1 to 10 parts by weight of A; 1 to 5 parts by weight of B; 1 to 5 parts by weight of C, and The process comprising 1 to 3 parts by weight of b.

37. 37. The method of any one of claims 22 to 36, wherein each of the bonds Ab, Bb, and Cb is independently a urea bond or a carbamate bond.

38. The method according to any one of claims 22 to 37, wherein step (2) (a) mixing the peptide probe, dopamine or a derivative thereof, and a metalate in water to form metal nanoparticles in solution having a coating comprising polydopamine and the peptide probe; (b) depositing metal nanoparticles on the working electrode by an electrochemical oxidation reaction; forming two detection layers of

39. 39. The method of claim 38, (i) the concentration of the peptide probe in the solution is 0.1 to 10 mg / mL; (ii) the concentration of dopamine or a derivative thereof in the solution is 1 g / L to 10 g / L, and the concentration of the metalate is 0.1 mg / L to 1 mg / L; (iii) the metalate comprises chloroplatinic acid, chloroauric acid, or hexachloroiridic (IV) acid hydrate, and the metalate has a concentration of 0.1 mg / L to 1 mg / L after forming a solution containing metal nanoparticles; (iv) the pH of the solution is from 7 to 9; (v) the dissolved oxygen concentration saturation in the solution is less than 1%; (vi) the temperature of the solution is between 20° C. and 40° C.; and / or (vii) A method in which the potential applied to the working electrode relative to a silver / silver chloride reference solution electrode is 0 to 0.8 V.

40. The method according to any one of claims 22 to 37, wherein step (2) (a) mixing the metal nanoparticles, a peptide probe, and dopamine or a derivative thereof in water; (b) contacting the working electrode with the solution formed in step (a); and (c) forming a sensing layer on the working electrode by an electrochemical oxidation reaction.

41. 41. The method of claim 40, (i) the metal nanoparticles have a particle size of 1 to 100 nm; (ii) the concentration of the metal nanoparticles in the solution is 1000 ppm to 5000 ppm; (iii) the concentration of the peptide probe in the solution is 0.1 to 10 mg / mL; (iv) the concentration of dopamine or a derivative thereof in the solution is 1 g / L to 10 g / L; (v) the pH of the solution is from 7 to 9; (vi) the dissolved oxygen concentration saturation in the solution is less than 1%; (vii) the temperature of the solution is between 20° C. and 40° C.; and / or (viii) The method, wherein the potential applied to the working electrode relative to a silver / silver chloride reference solution electrode is between −0.5 V and 0.8 V.

42. The method according to any one of claims 38 to 41, wherein dopamine is used in step (2).

43. 42. The method of any one of claims 38 to 41, wherein a derivative of dopamine is used in step (2), and the derivative of dopamine is formed by oxidizing dopamine or reducing dopamine.

44. 44. The method of claim 43, wherein the derivative of dopamine is levodopa or dihydroxyindole.

45. The method according to any one of claims 22 to 44, wherein step (3) (a) mixing A, B, and C in an organic solvent at a temperature of 30-45° C.; (b) adding a catalyst to the solution formed in step (a), adding an isocyanate-containing compound dropwise, raising the temperature of the solution to 55-70° C., and reacting the solution for 12-20 hours; (c) adding deionized water to the solution formed in step (b) and allowing the resulting mixture to react for 12 to 18 hours.

46. 46. ​​The method of claim 45, (i) the organic solvent is tetrahydrofuran (THF), cyclohexanone, isobutanol, or a mixture thereof; and (ii) The method, wherein the ratio of the volume of the organic solvent to the total mass of A, B and C is 2-10 mL:1 g.

47. 47. The method of claim 45 or 46, wherein the catalyst comprises triethylenediamine or dibutyltin bis(2-ethylhexanoate).

48. 48. The method of any one of claims 45 to 47, wherein the ratio of the volume of deionized water added in step (c) to the total mass of A, B, and C is 1-10 mL:1 g.

49. 23. The method of claim 22, wherein the first monomer unit is 1,6-diaminohexane and the second monomer unit is glutaraldehyde.

50. 23. The method of claim 22, (i) applying the first monomer unit to the substrate in ethanol; (ii) applying said second monomer unit to said substrate in the gas phase at a temperature of from 40 to 55°C.

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