Glycated protein sensor, measurement method, program, and sensor manufacture method

Abstract

To provide a glycated protein sensor, a glycated protein measurement method, and a method for producing a glycated protein sensor, which enable high-accuracy measurement and rapid measurement.SOLUTION: Provided is a glycated protein sensor comprising immobilized protease, immobilized ketoamine oxidase, and a hydrogen peroxide detection section.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure relates to a glycated protein sensor, a measurement method, a program, and a method for manufacturing the sensor. [Background technology]

[0002] Glycated proteins are measured as indicators for diagnosing diabetes and managing blood glucose control. For example, glycated hemoglobin and glycated albumin are frequently measured in clinical settings. Known methods for measuring glycated proteins include electrophoresis, ion exchange chromatography, affinity chromatography, immunoassays, and enzymatic methods. However, due to the demand for accurate, simple, and rapid measurements, enzymatic methods have become the mainstream in recent years.

[0003] A typical enzymatic method for measuring glycated proteins involves first, in the first step, breaking down proteins into amino acids using protease, then, in the second step, treating only the glycated amino acids with ketoamine oxidase to generate hydrogen peroxide, and in the third step, converting the hydrogen peroxide into a color-developing reaction and measuring the absorbance. Summary of the Invention

[0004] In conventional glycated protein measurement methods, the coexistence of protease and ketoamine oxidase causes a decomposition reaction between the enzymes, making it difficult to measure with high accuracy. Furthermore, conventional glycated protein measurement methods require multiple steps for measurement, making it difficult to perform rapid measurements and prone to measurement errors depending on the technique of the tester.

[0005] According to one embodiment of the present disclosure, a glycated protein or fructosamine sensor comprises an immobilized protease, an immobilized ketoamine oxidase, and a hydrogen peroxide detector.

[0006] The test sample may be a solution. The solution may be a body fluid, a solution derived from a body fluid, or a dilution of a body fluid. The solution may be a non-body fluid (non-body fluid-derived) solution, or a mixture of a body fluid or a body fluid-derived solution and a non-body fluid-derived solution. The solution may be a solution used for sample measurement, or a solution used for calibration measurement. For example, the solution may be a standard solution or a calibration solution.

[0007] "Body fluid" may be blood, serum, plasma, lymph, tissue fluid such as interstitial fluid, intercellular fluid, or interstitial fluid, or body cavity fluid, serous cavity fluid, pleural fluid, peritoneal fluid, pericardial fluid, cerebrospinal fluid (spinal fluid), joint fluid (synovial fluid), or aqueous humor (aqueous humor). Body fluid may be digestive fluid such as saliva, gastric juice, bile, pancreatic juice, or intestinal fluid, or may be sweat, tears, nasal mucus, urine, semen, vaginal fluid, amniotic fluid, or milk. Body fluid may be animal body fluid or human body fluid. "Body fluid" may be liquid in foods containing animal-derived protein (e.g., milk, dairy products, etc.). Body fluid may be plant body fluid, plant biofluid, or plant-derived liquid. For example, body fluid may be plant juice, nectar, or sap. "Body fluid" may also be a solution.

[0008] In some embodiments, the solution may include a physiological buffer solution. The solution may include a measurement target. The buffer solution may be a so-called Good's buffer. The buffer solution may include phosphate buffered saline (PBS) or N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid buffer (TES). The buffer solutions were 2-morpholinoethanesulfonic acid (MES), 3-morpholinopropanesulfonic acid (MOPS), 2-hydroxy-3-morpholinopropanesulfonic acid (MOPSO), piperazine-1,4-bis(2-hydroxy-3-propanesulfonic acid) dihydrate (POPSO), N-(2-acetamido)iminodiacetic acid (ADA), piperazine-1,4-bis(2-ethanesulfonic acid) monosodium salt (PIPES), N-(2-acetamido)-2-aminoethanesulfonic acid (ACES), cholamine hydrochloride, N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 2-[4- Sodium (2-hydroxyethyl)-1-piperazinyl]ethanesulfonate (HEPES-Na), Acetamidoglycine, Tricine, Glycinamide, Bicine, Bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane (Bis-Tris), N-Cyclohexyl-3-aminopropanesulfonic acid (CAPS), N-Cyclohexyl-2-hydroxy-3-aminopropanesulfonic acid (CAPSO), N-Cyclohexyl-2-aminoethanesulfonic acid (CHES), 3-[N,N-Bis(2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid (DIPSO), 3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulfonic acid (HEPPS), 2-Hydroxy-3-[4-(2-hydroxyethyl)-1-piperazinyl] It may contain any one or a mixture of propanesulfonic acid monohydrate (HEPPSO), N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS), and 2-hydroxy-N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPSO).

[0009] The solution may contain a substance to be measured. For example, the solution may be tears, and the substance to be measured may be glycoalbumin contained in tears. Alternatively, the substance to be measured may be albumin, glycoalbumin, hemoglobin, or glycohemoglobin in blood or serum, albumin or glycoalbumin in interstitial fluid, albumin or glycoalbumin in tears, albumin or glycoalbumin in urine, or albumin or glycoalbumin in saliva.

[0010] In some embodiments, the sensing target may be fructosamine. In some embodiments, the sensor may be a fructosamine sensor. Fructosamine may be a glycated protein, a glycated peptide, or a glycated amino acid. The glycated protein may be glycated albumin or glycated hemoglobin. The glycated protein may be AGE (Advanced Glycation End Products). In some embodiments, the sensing target may be a glycated lipid.

[0011] "Immobilization" means immobilizing an enzyme (protease, ketoamine oxidase) or the like to a substrate or base material. These enzymes may be immobilized on a base material that is immobilized on the substrate. In some embodiments, the enzyme may be immobilized directly or indirectly to the device body, the inner wall of a flow channel, or the wall surface of a chamber or storage section. The enzyme may be immobilized to an immobilization target via a member for the purpose of immobilization. The enzyme may be immobilized to an immobilization target via one or more members that are not primarily intended for immobilization. In some embodiments, the immobilization target to which the enzyme is immobilized may be substantially movable relative to the device body or the like. For example, the enzyme may be immobilized on beads, and the beads may be movable relative to the flow channel along with the movement of the solution.

[0012] Immobilization methods include, for example, covalent bonding, physical adsorption, ionic bonding, cross-linking, entrapment, and biochemical specific binding. Depending on the enzyme used, an immobilization method that does not inactivate the enzyme may be selected, or multiple immobilization methods may be used in combination. In some embodiments, a protein may be used as the substrate, and the enzyme may be mixed with the protein and then solidified with a cross-linking agent such as glutaraldehyde. This allows for cost reduction even when using relatively expensive enzymes. In some embodiments, immobilization may be performed using fluororesins, hydraulic resins, photocurable resins, solid polymer electrolytes, or polyion complexes. Entrapment of enzymes may be performed using water-insoluble semipermeable membranes such as nylon, ethyl cellulose, acetyl cellulose, or polystyrene, or in liposomes or reverse micelles using phospholipids.

[0013] An adhesive such as a silane coupling agent may be used to fix the substrate to the base material. The adhesive may be formed as a layer (silane coupling layer) between the substrate and the base material. The hydrogen peroxide detection unit and the layer thereon may be bonded via, for example, a bonding agent. Various bonding agents may be used as long as they do not substantially interfere with the measurement principle of the present disclosure. The bonding agent may, for example, contain a material that bonds inorganic materials and organic materials. The bonding agent may, for example, be a silane coupling agent. Examples of silane coupling agents include the following: Vinyl-based: vinyltrimethoxysilane, vinyltriethoxysilane, 7-octenyltrimethoxysilane, vinyldimethylethoxysilane, vinylmethyldimethoxysilane, vinylmethyldiethoxysilane, vinyltris(2-methoxyethoxy)silane, vinyltris(trimethylsiloxy)silane, 4-vinylphenyltrimethoxysilane, allyltrimethoxysilane, allyltriethoxysilane, 5-(triethoxysilyl)-2-norbornene; Styryl series: p-styryltrimethoxysilane; Methacrylic: 3-methacryloxypropyltrimethoxysilane, 3-methacryloxypropylmethyldimethoxysilane, 3-methacryloxypropyltriethoxysilane, 3-methacryloxypropylmethyldiethoxysilane, 3-methacryloxypropyltris(trimethylsiloxy)silane, 3-methacryloxypropyltriallylsilane, 8-methacryloxyoctyltrimethoxysilane; Acrylic: 3-acryloxypropyltrimethoxysilane, 3-acryloxypropyldimethylmethoxysilane, 3-acryloxypropyltriallylsilane; Epoxy-based: 3-glycidoxypropyltrimethoxysilane, 3-glycidoxypropylmethyldimethoxysilane, 3-glycidoxypropylmethyldiethoxysilane, 8-glycidoxyoctyltrimethoxysilane, 2-(3,4-epoxycyclohexyl)ethyltrimethoxysilane; Amino-based: N-2-(aminoethyl)-3-aminopropyltrimethoxysilane, N-2-(aminoethyl)-3-aminopropylmethyldimethoxysilane, N-2-(aminoethyl)-3-aminopropyltriethoxysilane, N-2-(aminoethyl)-8-aminooctyltrimethoxysilane, N-6-(aminohexyl)-3-aminopropyltrimethoxysilane, 3-aminopropyltrimethoxysilane, 3-aminopropylmethyldimethoxysilane, 3-aminopropyltriethoxysilane Triethoxysilane (APTES), 3-aminopropylmethyldiethoxysilane, 3-triethoxysilyl-N-(1,3-dimethyl-butylidene)propylamine, N-phenyl-3-aminopropyltrimethoxysilane, N-methyl-3-aminopropyltrimethoxysilane, N,N-dimethyl-3-aminopropyltrimethoxysilane, bis[3-(trimethoxysilyl)-propyl]amine, N-(vinylbenzyl)-2-aminoethyl-3-aminopropyltrimethoxysilane hydrochloride; Ureido-based: 3-ureidopropyltrimethoxysilane, 3-ureidopropyltriethoxysilane; Azide-based: 11-azidoundecyltrimethoxysilane; Isocyanate-based: 3-isocyanatepropyltrimethoxysilane, 3-isocyanatepropyltriethoxysilane; Isocyanurate series: tris-(trimethoxysilylpropyl) isocyanurate; Mercapto-based: 3-mercaptopropyltrimethoxysilane, 3-mercaptopropylmethyldimethoxysilane, 3-mercaptopropyltriethoxysilane; Or, acid anhydride: 3-trimethoxysilylpropylsuccinic anhydride.

[0014] The substrate may contain SiO2 as a main component, or may be a glass substrate. The substrate may contain a polymer or a resin. The substrate may contain a transparent polymer material. The substrate may be composed of a resin material such as polyethylene terephthalate (PET), polyethylene naphthalate (PEN), or cycloolefin polymer (COP). The substrate may be a transparent substrate or an opaque substrate. The substrate may be a flexible substrate. The substrate may be a sapphire substrate.

[0015] The main component of the substrate may be a polymer. The main component of the substrate may be a biopolymer, an organic polymer, or an inorganic polymer. For example, the substrate may be or contain porous inorganic materials such as silica gel, glass, alumina, molecular sieves, celite, and charcoal; kaolinite, ceramics, and ceramic materials such as hydroxyapatite; clays such as bentonite; acid clay; polyacrylamide gel; polyvinyl alcohol resin; urethane polymer; silicone resin; styrene resin; perfluorosulfonic acid resin; lacquer; cellulose; agarose (agar), alginic acid; polysaccharides such as carrageenan; collagen (glue or gelatin); chitin; chitosan; polypeptide; or polylysine.

[0016] The main component of the substrate may be a protein. The main component of the substrate may be bovine serum albumin (BSA). When the substrate is composed of a protein, a crosslinker is generally used to crosslink and immobilize the enzyme and the protein of the substrate. The protein that is the main component of the substrate and the crosslinker may collectively be referred to as the "substrate." The crosslinker may be a photocurable resin, a water-curable resin, or a thermosetting resin. The crosslinker may be a bifunctional reagent such as glutaraldehyde. The crosslinker may be formaldehyde or a bifunctional reagent selected from glyoxal, malondialdehyde, succinaldehyde, and glutaraldehyde.

[0017] The main component of the substrate may be an ionic matrix or a polyion complex (hereinafter simply referred to as an ionic complex). By mixing a polyanion and a polycation in an aqueous solution, a polyion complex is formed due to strong electrostatic interactions. The ionic matrix may be electrostatically immobilized on the substrate. The ionic matrix can electrostatically confine an enzyme within the matrix. For example, by mixing polymers with opposite charges (cations and anions), such as polyamino acids, a complex is formed through electrostatic interactions, and an enzyme or the like can be confined within it. Because the ionic matrix does not directly bind to the enzyme, it can reduce or avoid a decrease in enzyme activity. The ionic matrix may also be used for enzymes that are inactivated by crosslinking.

[0018] The substrate may be beads. The beads may be carbon particles (carbon beads) or silica (SiO2) particles (silica beads). The beads may be polymeric beads. The material of the beads may be a polymeric polysaccharide such as chitin, chitosan, or alginic acid. The beads may contain metal particles or a magnetizable substance, or may be magnetic beads. The average particle size of the beads may be 10 nm or more and 200 nm or less. The enzyme may be crosslinked to the beads. The beads may be directly fixed to a substrate or other substrate, or may be arranged so as to be contained within a predetermined volume without being fixed to a substrate or substrate. The beads may be movable with the flow of a solution without being fixed to the device body. The beads may be arranged so as not to substantially leave the container that contains them, even if not directly fixed to the container. This allows the beads to be maintained in place while unnecessary substances are removed, for example, by washing or rinsing with a diluent or cleaning solution. This allows, for example, repeated use.

[0019] The substrate may include a porous material. The porous material may be a ceramic or a carbon material. The substrate may be a zeolite. The substrate may be a metal-organic framework.

[0020] "Protease" is generally a general term for peptide bond hydrolases that hydrolyze and catabolize proteins and polypeptides. Proteases may also be enzymes that break down proteins into peptide fragments. When a protein contains glycated amino acid residues, the peptide fragments generated by the action of a protease may include peptide fragments containing glycated amino acid residues and peptide fragments that are not glycated at all.

[0021] The "protease" may be an animal-derived protease, a plant-derived protease, or a microbial-derived protease. The protease may be an exopeptidase or an endopeptidase. The protease may be an aspartic protease, a metalloprotease, a serine protease, or a thiol protease.

[0022] The term "protease" may include multiple types or kinds of proteases, or may include a single type or kind of protease. For example, the protease may include either a proteinase or a peptidase, or both. Mixing multiple proteases may increase the decomposition efficiency. The protease may include a modified protease or a modified protease. The protease may be used together with an additive. The additive may be, for example, a surfactant or urea. The additive may, for example, be capable of destabilizing or denaturing the protein. The use of a modified protease or an additive may, for example, be capable of improving the decomposition efficiency of the protein or the substrate selectivity, for example, but not limited to these.

[0023] The animal-derived protease may be trypsin, chymotrypsin, pepsin, elastase, bovine pancreatic protease, cathepsin, calpain, protease type-I, protease type-XX, aminopeptidase N, carboxypeptidase, pancreatin (a mixture of multiple enzymes such as proteases and amylases), or the like.

[0024] The plant-derived protease may be papain, bromelain, gingipain, kallikrein, ficin, chymopapain, actinidin, carboxypeptidase W, or the like.

[0025] The protease derived from a microorganism may be a protease derived from the genus Bacillus (or derived from), the genus Geobacillus, the genus Paenibacillus, the genus Aspergillus, the genus Penicillium, the genus Streptomyces, the genus Lysobacter, a protease derived from yeast, the genus Tritirachium, the genus Thermus, the genus Pseudomonas, the genus Achromobacter, the genus Rhizopus, or the genus Staphylococcus.

[0026] The protease may be selected based on its efficiency in digesting or degrading proteins, for example, by measuring absorbance. In some embodiments, a protease may be used that produces a difference in absorbance of 100 mAbs or more before and after degrading albumin (HSA). The difference in absorbance of 90 mAbs or more before and after degrading albumin (HSA) may also be used. The difference in absorbance of 110 mAbs or more before and after degrading albumin (HSA) may also be used.

[0027] The protease may be selected from the group consisting of protease type XXIV, orientase 22BF, orientase 90N, Toyozyme NEP-160, and alkalophilic proteases. The protease may be selected from the group consisting of protease type XXIV, orientase 22BF, orientase 90N, Toyozyme NEP-160, and alkalophilic proteases. The protease may be selected from the group consisting of pronase and protease type XIV.

[0028] The protease may be a neutral protease, an acidic protease, or an alkaline protease. For example, when measuring tears, a neutral or weakly alkaline protease may be used. The protease may be a heat-resistant protease (e.g., thermolysin) that maintains its activity even at high temperatures (e.g., 60°C or higher). The protease may also be a cold-active protease that maintains its activity even at low temperatures (e.g., 30°C or lower).

[0029] In some embodiments, the protease may be disposed in a dry state within the sensor. The protease may be formed by, but is not limited to, air drying, freeze drying, or spray drying. In some embodiments, the protease may be disposed in a dissolved state in a solution. The protease dissolved in the solution may be stored refrigerated until use. The protease may be frozen with the solution and then thawed at the time of use. The protease may not be dried, but may be maintained in a moist environment. For example, the protease may be disposed in a gel.

[0030] "Ketoamine oxidase" is an oxidase that recognizes the ketoamine structure of a glycated amino acid or a peptide or peptide fragment containing a glycated amino acid residue and oxidizes the glycated amino acid to produce an amino acid, glucosone (α-ketoaldehyde), and hydrogen peroxide. Thus, ketoamine oxidase produces an amount of hydrogen peroxide that is proportional to or related to the amount of the glycated amino acid or peptide or peptide fragment containing the glycated amino acid residue that it recognizes.

[0031] The ketoamine oxidase may be a dehydrogenase, a kinase, or an oxidase, and may be fructosyl amino acid oxidase (FAOD), fructosyl peptide oxidase, fructosyl valylhistidine oxidase, fructosyl amine oxidase, amadoriase, fructosyl amine deglycase, or modified forms thereof.

[0032] In some embodiments, the ketoamine oxidase may be an oxidase that acts on an amino acid or peptide whose ε-amino group is glycated. The amino acid may be lysine. A glycoalbumin sensor can be constructed using an oxidase that selectively acts on an amino acid or peptide whose ε-amino group is glycated.

[0033] In some embodiments, the ketoamine oxidase may be an oxidase that acts on an amino acid or peptide whose α-amino group is glycated. The amino acid may be valine. A glycated hemoglobin sensor or a glycated hemoglobin A1c (HbA1c) sensor can be constructed using an oxidase that acts on an amino acid or peptide whose α-amino group is glycated.

[0034] In some embodiments, the sensor may include a detection unit. The detection unit may be a hydrogen peroxide detection unit. The "hydrogen peroxide detection unit" (hydrogen peroxide sensor) may be an electrochemical electrode or a hydrogen peroxide electrode. The hydrogen peroxide electrode may have a counter electrode, a reference electrode, and a working electrode. In some embodiments, the detection unit may detect oxygen. For example, it may detect the amount or concentration of oxygen reduced by an enzyme reaction. Oxygen detection is considered to be relatively insensitive to molecules and ions that are noise sources and resistant to interference. Oxygen consumption may be measured by oxygen detection. Since tears are saturated with air when collected, the detection unit may be used to sense enzymes in tears. The detection unit may be configured to perform multiple detection methods selectively or in combination.

[0035] Hydrogen peroxide may be detected optically. Optical detection may include measuring absorbance or luminescence. For example, by adding peroxidase, 4-aminoantipyrine, and a color developer, the color change of a quinone dye caused by oxidative condensation may be measured, for example, from the back side of a transparent substrate. In some embodiments, the detection unit may include a luminescent reagent and a photodetector. For example, luminol may be used as the luminescent reagent. Luminol may be provided in powder form. Hydrogen peroxide may be reacted with luminol, and the intensity of luminescence (wavelength 460 nm) resulting from the luminol reaction may be measured. The reagent may further include potassium hexacyanoferrate, sodium hydroxide, or the like. The luminol reaction may be measured by electrochemiluminescence using an AC-driven electrode, such as a gold electrode, a platinum electrode, or a transparent indium tin oxide electrode (ITO electrode). To detect a fluorescent reaction, a combination of an oxalate ester and a fluorescent substance may be used, or lucigenin (acridinium, bis(N-methylacridinium)) may be used. The detection unit may be a hydrogen peroxide sensor of another type.

[0036] "On the detection unit" may refer to a portion of the surface of the detection unit, or may be arranged so as to cover the entire detection unit. It may also refer to a detection unit formed on a substrate. It may cover the entire surface of the substrate, or it may cover a portion of the substrate while covering the detection unit.

[0037] The sensor may have a liquid storage portion. The liquid storage portion may include one, two, or all of an immobilized protease, an immobilized ketoamine oxidase, and a detection portion. The liquid storage portion may extend in a longitudinal direction. The volume of the liquid storage portion may be less than or equal to 1 mL, 500 μL, 300 μL, 200 μL, 100 μL, 50 μL, 30 μL, 20 μL, 10 μL, 5 μL, 4 μL, 3 μL, 2 μL, 1 μL, 0.9 μL, 0.8 μL, 0.7 μL, 0.6 μL, 0.5 μL, 0.4 μL, 0.3 μL, 0.2 μL, or 0.1 μL. The liquid storage portion may have a liquid inlet. The liquid storage portion may have a liquid outlet. The liquid storage portion may have an air hole. The air hole may have the function of discharging gas that was in the liquid storage portion to the outside of the sensor when liquid is introduced into the liquid storage portion.

[0038] Exemplary or potential effects of the glycated protein sensor and the like of the present disclosure will be described. Proteases can degrade other proteases. Proteases can also degrade (ketoamine) oxidase. Unimmobilized proteases can move through a liquid by diffusion or other means, encountering and degrading other proteases or ketoamine oxidases. Protease solutions are not suitable for storage, and solid proteases must be weighed each time. Pre-immobilizing proteases in a device can streamline the measurement procedure. Immobilized proteases can be reused. Immobilizing the ketoamine oxidase and proteases of the present invention enables repeated measurements, significantly reducing the running cost per measurement. The device may be cleaned between measurements. The glycated protein sensor of the present disclosure can be miniaturized. Furthermore, the proximity of the hydrogen peroxide detection unit and the ketoamine oxidase immobilization layer shortens the diffusion distance of hydrogen peroxide, making it less susceptible to interferences in body fluids that react with hydrogen peroxide. When the immobilized layers of ketoamine oxidase and protease are formed as thin films of, for example, 1 micrometer (μm) or less on the hydrogen peroxide display area, they are in close proximity to each other, enabling highly sensitive measurements. Furthermore, the required sensitivity can be achieved even with a small amount of the expensive enzyme as a reagent, which also reduces manufacturing costs.

[0039] It should be noted that embodiments in which one or more of the features of the present disclosure described above are combined in any manner are also included within the scope of the present disclosure. [Brief explanation of the drawings]

[0040] [Figure 1] Schematic cross-sectional view of a sensor according to an embodiment of the present disclosure. [Figure 2] Schematic cross-sectional view of a sensor according to an embodiment of the present disclosure. [Figure 3] Schematic cross-sectional view of a sensor according to an embodiment of the present disclosure. [Figure 4] Schematic cross-sectional view of a sensor according to an embodiment of the present disclosure. [Figure 5] Schematic cross-sectional view of a sensor according to an embodiment of the present disclosure. [Figure 6]Schematic cross-sectional view of a sensor according to an embodiment of the present disclosure. [Figure 7] Schematic cross-sectional view of a sensor according to an embodiment of the present disclosure. [Figure 8] Schematic cross-sectional view of a sensor according to an embodiment of the present disclosure. [Figure 9] Schematic cross-sectional view of a sensor according to an embodiment of the present disclosure. [Figure 10] Schematic cross-sectional view of a sensor according to an embodiment of the present disclosure. [Figure 11] Schematic cross-sectional view of a sensor according to an embodiment of the present disclosure. [Figure 12] Schematic cross-sectional view of a sensor according to an embodiment of the present disclosure. [Figure 13] Schematic diagram showing the configuration of a sensor according to an embodiment of the present disclosure. [Figure 14] Schematic diagram showing the configuration of a sensor according to an embodiment of the present disclosure. [Figure 15] Schematic diagram showing the configuration of a sensor according to an embodiment of the present disclosure. [Figure 16] 1 is a schematic plan view of a sensor according to an embodiment of the present disclosure; [Figure 17] Schematic diagram showing the configuration of a sensor according to an embodiment of the present disclosure. [Figure 18] 1 is a schematic plan view of a sensor chip according to an embodiment of the present disclosure; [Figure 19] 1 is a schematic plan view of a sensor chip according to an embodiment of the present disclosure; [Figure 20] 1 is a schematic plan view of a sensor chip according to an embodiment of the present disclosure; [Figure 21] 1 is a schematic plan view of a sensor chip according to an embodiment of the present disclosure; [Figure 22] 1 is a schematic plan view of a sensor chip according to an embodiment of the present disclosure; [Figure 23A] 1A to 1C are cross-sectional views illustrating a manufacturing process of a sensor according to an embodiment of the present disclosure. [Figure 23B] 1A to 1C are cross-sectional views illustrating a manufacturing process of a sensor according to an embodiment of the present disclosure. [Figure 23C] 1A to 1C are cross-sectional views illustrating a manufacturing process of a sensor according to an embodiment of the present disclosure. [Figure 23D]1A to 1C are cross-sectional views illustrating a manufacturing process of a sensor according to an embodiment of the present disclosure. [Figure 23E] 1A to 1C are cross-sectional views illustrating a manufacturing process of a sensor according to an embodiment of the present disclosure. [Figure 23F] 1A to 1C are cross-sectional views illustrating a manufacturing process of a sensor according to an embodiment of the present disclosure. [Figure 23G] 1A to 1C are cross-sectional views illustrating a manufacturing process of a sensor according to an embodiment of the present disclosure. [Figure 23H] 1A to 1C are cross-sectional views illustrating a manufacturing process of a sensor according to an embodiment of the present disclosure. [Figure 23I] 1A to 1C are cross-sectional views illustrating a manufacturing process of a sensor according to an embodiment of the present disclosure. [Figure 24] 1 is a schematic diagram illustrating a configuration of a portion of a sensor according to an embodiment of the present disclosure; [Figure 25] 1 is a schematic diagram illustrating a configuration of a portion of a sensor according to an embodiment of the present disclosure; [Figure 26] Graph showing the digestibility of each protease [Figure 27] Graph showing sensor output for each protease [Figure 28] Graph showing the temperature dependence of the activity of proteases and ketoamine oxidases [Figure 29] Graph showing the dependence of sensor output on glycated albumin concentration DETAILED DESCRIPTION OF THE INVENTION

[0041] 1. Sensor structure The configuration of a glycated protein sensor according to one embodiment of the present disclosure will be described with reference to FIG. 1 . The sensor 100 shown in FIG. 1 includes a protease 101, a ketoamine oxidase 102, and a hydrogen peroxide detection unit 103. The protease 101 and the ketoamine oxidase 102 are cross-linked to a base material, bovine serum albumin 105, using a cross-linking agent, glutaraldehyde 106. The bovine serum albumin 105 is also cross-linked to itself. These form an immobilization layer 104. The immobilization layer 104 is connected to the hydrogen peroxide detection unit 103 by a silane coupling agent 107. As a result, the protease 101 and the ketoamine oxidase 102 are immobilized to the hydrogen peroxide detection unit 103 or the entire sensor 100. The immobilization layer 104, together with the protease 101 and the ketoamine oxidase 102 immobilized thereto, forms an enzyme layer 111.

[0042] A glycated protein 151 is introduced into the sensor 100. The glycated protein 151 has a structure in which a sugar 153 is bound to a protein 152. The glycated protein 151 is decomposed by a protease 101, generating peptide fragments.

[0043] The peptide fragments include glycated peptide fragments 154 and non-glycated peptide fragments 155. These peptide fragments are thought to diffuse through the immobilization membrane 104. When the glycated peptide fragments 154 react with ketoamine oxidase 102, glucosone (not shown) and hydrogen peroxide 156 are produced.

[0044] It is believed that this hydrogen peroxide 156 also diffuses within the immobilization film 104. The hydrogen peroxide detection unit 103 detects this hydrogen peroxide 156 and outputs a signal related to its concentration.

[0045] When the hydrogen peroxide detection unit 103 is a hydrogen peroxide electrode, hydrogen peroxide 156 is decomposed at the hydrogen peroxide electrode, and the released electrons are detected as a current. This reaction can be described as follows: H2O2 → 2H + + O2+ 2e-

[0046] Hydrogen peroxide 156 is consumed by the hydrogen peroxide electrode 103. Therefore, the concentration of hydrogen peroxide 156 in the immobilization film 104 is lowest near the hydrogen peroxide detection unit 103 and increases with increasing distance from the hydrogen peroxide detection unit 103. In other words, a concentration gradient of hydrogen peroxide 156 exists within the immobilization film 104. This concentration gradient changes immediately after the start of measurement and becomes approximately stable after a predetermined time. When this concentration gradient stabilizes, the concentration of hydrogen peroxide 156 detected by the hydrogen peroxide electrode 103 is related to the concentration of glycated protein 151 introduced into the sensor 100. The relationship between the concentration of glycated protein 151 and the current value generated at the hydrogen peroxide electrode 103 is determined in advance. When an actual measurement is performed, the concentration of glycated protein 151 in the sample solution can be calculated from the current value generated at the hydrogen peroxide electrode 103 based on this calibration.

[0047] In some embodiments, the sensor may have the protease and the ketoamine oxidase immobilized on the same substrate (integrated).

[0048] 2 shows the structure of an integrated sensor according to some embodiments (second embodiment). Sensor 200 has protease 201, ketoamine oxidase 202, substrate 204, and hydrogen peroxide detection unit 203. Protease 201 and ketoamine oxidase 202 are immobilized on substrate 204, which is in turn immobilized on hydrogen peroxide detection unit 203. In sensor 200 shown in FIG. 2, the same substrate 204 is formed as a layer or film on hydrogen peroxide detection unit 203, and an enzyme layer 211 in which protease 201 and ketoamine oxidase 202 are immobilized on substrate 204 is formed on hydrogen peroxide detection unit 203. Protease 201 and ketoamine oxidase 202 are immobilized in this same substrate 204.

[0049] FIG. 3 shows the structure of a sensor according to some embodiments (third embodiment). The sensor 300 includes a substrate 304 on which a protease 301 and a ketoamine oxidase 302 are immobilized, and a hydrogen peroxide detection unit 303 on which the substrate 304 is immobilized. In the sensor 300 shown in FIG. 3, the same substrate 304 is formed as a layer or film on the hydrogen peroxide detection unit 303, and an enzyme layer 311 in which the protease 301 and the ketoamine oxidase 302 are immobilized on the substrate 304 is formed on the hydrogen peroxide detection unit 303. The sensor 300 shown in FIG. 3 further includes an adhesive or bonding agent 305 between the substrate 304 and the hydrogen peroxide detection unit 303 to bond them together. The bonding agent 305 may be a silane coupling agent. When the hydrogen peroxide detection unit 303 has a metal (not shown) such as an electrode on its surface, the silane coupling agent can relatively firmly bond the metal to the organic substrate 304.

[0050] 4 shows the structure of a sensor according to some embodiments (fourth embodiment). Sensor 400 includes protease 401, ketoamine oxidase 402, substrates 404 and 414 on which they are immobilized, and hydrogen peroxide detection unit 403 on which substrate 414 is immobilized. More specifically, a layer of substrate 414 containing ketoamine oxidase 402 (ketoamine oxidase layer 412) is formed on hydrogen peroxide detection unit 403. A layer of substrate 404 containing protease 401 (protease layer 411) is formed on ketoamine oxidase layer 412, i.e., on the surface opposite to hydrogen peroxide detection unit 403. That is, ketoamine oxidase layer 412 and protease layer 411 are layered in this order on hydrogen peroxide detection unit 403.

[0051] In the sensors of the present disclosure, the protease and ketoamine oxidase may or may not be layered on the sensing portion, and in some embodiments, the protease and ketoamine oxidase may be disposed adjacent to the sensing portion or near the sensing portion.

[0052] In some embodiments, the thickness of the protease, ketoamine oxidase, or enzyme layer may be less than or equal to 100 μm, 50 μm, 20 μm, 10 μm, 1 μm, 500 nm, 300 nm, 250 nm, 200 nm, 100 nm, 50 nm, etc. In some embodiments, the thickness of the protease, ketoamine oxidase, or enzyme layer may be greater than or equal to 10 nm, 20 nm, 25 nm, 30 nm, 50 nm, 100 nm, etc.

[0053] In some embodiments, the sensor may be arranged so that the ketoamine oxidase portion surrounds the protease portion (surrounding type).

[0054] 5 shows the structure of a sensor (fifth embodiment) according to some embodiments. Sensor 500 has protease 501, ketoamine oxidase 502, substrates 504 and 514 on which these are immobilized, a hydrogen peroxide detection unit 503 on which substrate 514 is immobilized, and substrate 506. More specifically, hydrogen peroxide detection unit 503 is provided on substrate 506. Substrate 514 on which ketoamine oxidase 502 is immobilized (immobilized ketoamine oxidase unit 512) is formed on hydrogen peroxide detection unit 503. Substrate 504 on which protease 501 is immobilized (immobilized protease unit 511) is formed so as to surround immobilized ketoamine oxidase unit 512. In other words, immobilized ketoamine oxidase section 512 is disposed on substrate 506 in a portion substantially corresponding to hydrogen peroxide detection section 503, and immobilized protease section 511 is disposed so as to cover immobilized ketoamine oxidase section 512 and the surface of substrate 506 where hydrogen peroxide detection section 503 is not present. This structure allows the amount of protease immobilized to be relatively large compared with the amount of ketoamine oxidase immobilized. This makes it possible to improve the response speed and detection sensitivity of the sensor, for example, in cases where the decomposition or digestion of large protein molecules by proteases is rate-limiting.

[0055] The enzyme may be immobilized within the substrate or on the outer surface of the substrate (direct conjugation).

[0056] The sensor 600 shown in Fig. 6 is of a direct bonding type and includes a protease 601, a ketoamine oxidase 602, and a hydrogen peroxide detection unit 603 to which these are immobilized. More specifically, a substrate 614 (ketoamine oxidase layer 612) to which ketoamine oxidase 602 is immobilized is formed on the hydrogen peroxide detection unit 603. The protease 601 is immobilized on the upper surface of this ketoamine oxidase layer 612 by a cross-linking agent 604. The protease 601 in Fig. 6 is immobilized on the substrate 614. The protease 601 may be cross-linked to the ketoamine oxidase 602.

[0057] Fig. 7 shows an example of a sensor structure having a hydrogen peroxide electrode. In Fig. 7, a layered structure as shown in Fig. 4 is shown as an example of immobilizing a protease and a ketoamine oxidase. However, the mode of immobilizing a protease and a ketoamine oxidase is not limited to this, and as exemplarily explained above, a layered structure, a structure other than that shown in Fig. 7, or a non-layered structure may also be used.

[0058] 7 includes a hydrogen peroxide electrode 730 formed on an insulating substrate 706, ketoamine oxidase 702 arranged on the hydrogen peroxide electrode 730 and a substrate 714 (ketoamine oxidase layer 712) on which it is immobilized, and protease 701 arranged on the ketoamine oxidase layer 712 and a substrate 704 (protease layer 711) on which it is immobilized. A silane coupling agent 705 is applied between the hydrogen peroxide electrode 730 and the ketoamine oxidase layer 712 to bond the surface of the electrode 730 to the substrate 714.

[0059] The hydrogen peroxide electrode 730 shown in Figure 7 is an electrode for use in a three-electrode method, and is configured to include a counter electrode 731, a reference electrode 732, and a working electrode 733. The sensor 700 shown in Figure 7 further includes an electric circuit 770 connected to the hydrogen peroxide electrode 730. This electric circuit 770 includes an operational amplifier 771, a voltage generating circuit 772, and a current measuring circuit 773. The output (OUT) of the operational amplifier is connected to the counter electrode 731, the inverting input (-IN) is connected to the reference electrode 732, and the non-inverting input (+IN) is connected to the voltage generating circuit 772.

[0060] The three-electrode method involves placing a counter electrode, a reference electrode, and a working electrode in contact with a solution or body fluid, creating a predetermined potential difference between the counter electrode and the working electrode, and measuring the current flowing from the counter electrode to the working electrode. Generally, when a substance such as a metal or metal oxide enters an electrolyte solution, a potential difference called the interfacial potential occurs between the substance and the electrolyte. Applying a voltage between the counter electrode and the working electrode, taking this potential difference into account, causes a current to flow from the counter electrode, which can change the potential difference between the counter electrode and the solution. This change in potential difference can prevent the desired voltage from being accurately applied to the solution. To avoid this, the three-electrode method uses a reference electrode to measure the potential applied to the counter electrode, and the voltage applied to the counter electrode can be controlled so that the potential is set to the desired value. A feedback circuit is also included to feed back the potential measured by the reference electrode to the circuit controlling the counter electrode.

[0061] 2. Reduction of noise caused by impurities A solution may contain impurities. For example, when measuring proteins in a body fluid, measurement noise occurs due to various impurities such as proteins other than the protein being measured, peptide fragments, nucleic acids, and ions. The noise caused by these impurities can cause measurement errors and measurement inaccuracies. Therefore, sensors according to some embodiments of the present disclosure may have a configuration that reduces the effect of impurities on the measurement signal.

[0062] <Noise reduction using ion exchange resin> Some embodiments of the sensor may have an ion exchange resin on the detector.

[0063] 8 includes a hydrogen peroxide electrode 803, ketoamine oxidase 802 arranged on the hydrogen peroxide electrode 803 and a substrate 814 (ketoamine oxidase layer 812) on which it is immobilized, and protease 801 arranged on the ketoamine oxidase layer 814 and a substrate 804 (protease layer 811) on which it is immobilized. Sensor 800 shown in FIG. 8 further includes an ion exchange resin 807 between ketoamine oxidase layer 812 and the hydrogen peroxide electrode 803.

[0064] For example, cation exchange resins such as Nafion (registered trademark) can be used to suppress or prevent ascorbic acid and uric acid, particularly negative ions, present in body fluids from permeating and reaching the detection unit, while anion exchange resins such as polypyrrole can be used to suppress or prevent dopamine, particularly positive ions, from permeating and reaching the detection unit.

[0065] The ion exchange resin may comprise one, multiple, or at least one type of ion exchange resin. The ion exchange resin may be configured with one, multiple, or at least one type of layer.

[0066] In Figure 8, as an example of immobilization of a protease and a ketoamine oxidase, a layered structure as shown in Figure 4 is shown. However, the mode of immobilization of a protease and a ketoamine oxidase is not limited to this, and as exemplarily explained above, a layered structure, a structure other than that shown in Figure 8, or a non-layered structure may also be used.

[0067] <Noise reduction using differential sensors> In some embodiments, the sensor may be a pair or set of differential or differential sensors. The sensor may include multiple pairs or sets of differential or differential sensors. The set of differential sensors in this differential sensor may be configured with a main sensor and a sub-sensor. The main sensor includes an immobilized protease, an immobilized ketoamine oxidase, and a detection unit. The sub-sensor has low sensitivity to the main analyte (test substance) detected by the main sensor, but has substantially the same or similar sensitivity to noise-causing molecules as the main sensor.

[0068] As an example, consider a sensor for measuring glycated proteins in body fluids. Body fluids contain proteins other than the test substance, such as proteins, peptides, vitamin C, and ions. For example, peptide fragments are recognized by ketoamine oxidase, which causes the generation of hydrogen peroxide. Furthermore, ions in body fluids can be detected by a hydrogen peroxide detector.

[0069] Therefore, the sub-sensor may be a sensor that detects noise sources, including these, to the same extent as the main sensor or in a manner that has some correlation therewith. The sub-sensor may, for example, not contain protease (protease-free) compared to the main sensor, but may otherwise have the same structure. For example, the surface area and height dimensions of the substrate and enzyme-containing portion of the sub-sensor, the type of substrate, and the manufacturing method may be similar to those of the main sensor. The materials, structure, manufacturing method, etc., other than the protease, of the main sensor and the sub-sensor do not need to be the same. In this case, the correlation between them can be determined by calibration or the like.

[0070] In some embodiments, the hydrogen peroxide detector in the main sensor and / or subsensor of the differential sensor may be covered with ion exchange resin, or a layer or membrane of ion exchange resin may be placed on the surface of the hydrogen peroxide detector.

[0071] In some embodiments, the main sensor configuration may be an enzyme layer integrated type, for example as shown in FIG.

[0072] The sensor 1000 shown in FIG. 9 includes a main sensor 1000a and a sub-sensor 1000b that are integrated with an enzyme layer.

[0073] The main sensor 1000a shown in Fig. 9 is an enzyme layer-integrated type like that shown in Fig. 2 or 3. Specifically, the main sensor 1000a has a substrate 1004 to which protease 1001 and ketoamine oxidase 1002 are immobilized, and a hydrogen peroxide detection unit 1003a to which the substrate 1004 is immobilized. In the main sensor 1000a shown in Fig. 9, the same substrate 1004 is formed as a layer or film on the hydrogen peroxide detection unit 1003a, and an enzyme layer 1011a in which the protease 1001 and ketoamine oxidase 1002 are immobilized on the substrate 1004 is formed on the hydrogen peroxide detection unit 1003a. The enzyme molecules of the protease 1001 and ketoamine oxidase 1002 are immobilized in this same substrate 1004.

[0074] 9 has a configuration similar to that of the main sensor 1000a, but does not contain (non-containing) the protease 1001. Specifically, the subsensor 1000b has the same ketoamine oxidase 1002, substrate 1014, and hydrogen peroxide detection unit 1003b as the main sensor 1000a. The enzyme layer 1011b of the subsensor 1000b, which has the ketoamine oxidase 1002 and substrate 1014, may have substantially the same dimensions as the enzyme layer 1011a of the main sensor.

[0075] The hydrogen peroxide detectors 1003a, 1003b of the main sensor 1000a and the sub-sensor 1000b are connected to measuring electrical circuits 1070a, 1070b, respectively. These electrical circuits 1070a, 1070b receive output signals, such as current, from the hydrogen peroxide detectors 1003a, 1003b, convert the signals into digital values, and send them to a computing unit 1030, such as a CPU. In some embodiments, transmission from the electrical circuits 1070a, 1070b to the computing unit 1030 may be via electrical or optical wires, or wirelessly.

[0076] The calculation unit 1030 may perform calculations such as a difference between the signals from the main sensor 1000a and the sub-sensor 1000b. By removing noise, such as impurities, from the output signal of the main sensor 1000a based on the signal from the sub-sensor 1000b, the signal of the substance to be measured can be detected with higher accuracy. The calculation unit 1030 may further include a transmitter (not shown) or may be connected to an external transmitter. The transmitter can transmit signals optically, electrically, or electromagnetically, via wire or wirelessly. The transmitted signals may be signals after a difference calculation has been performed by the calculation unit 1030. In another embodiment, the transmitter may transmit the signals from the main sensor 1000a and the sub-sensor 1000b separately. A calculation such as a difference may be performed at the destination. The configuration or function of these calculation units is not limited to this embodiment and may be applied to other embodiments. The calculation unit may include an internal storage medium or may be connected to or configured to be connected to a storage medium located outside the calculation unit.

[0077] In the differential sensor, the main sensor may have a stacked structure as shown in FIG.

[0078] The sensor 1100 shown in FIG. 10 has a stacked main sensor 1100a and a sub-sensor 1100b.

[0079] Main sensor 1100a shown in Fig. 10 is of the laminated type as shown in Fig. 4. Main sensor 1100a shown in Fig. 10 has protease 1101, ketoamine oxidase 1102, substrates 1104 and 1114 to which these are immobilized, and hydrogen peroxide detection unit 1103a to which substrate 1114 is immobilized. More specifically, a layer of substrate 1114 containing ketoamine oxidase 1102 (ketoamine oxidase layer 1112a) is formed on hydrogen peroxide detection unit 1103a. A layer of substrate 1104 containing protease 1101 (protease layer 1111a) is formed on ketoamine oxidase layer 1112a, that is, on the surface opposite to hydrogen peroxide detection unit 1103a. That is, a ketoamine oxidase layer 1112a and a protease layer 1111a are laminated in this order on a hydrogen peroxide detection section 1103a.

[0080] In some embodiments, the substrates for each layer may be different materials, for example, the ketoamine oxidase layer may use a protein-based substrate and the protease layer may use a photocrosslinkable resin-based substrate.

[0081] 10 has a configuration similar to that of the main sensor 1100a, but does not contain or does not include the protease 1101 or its substrate 1104. Specifically, the subsensor 1100b has the same ketoamine oxidase 1102, substrate 1124, and hydrogen peroxide detection unit 1103b as the sensor 1100a. The enzyme layer 1112b of the subsensor 1100b, which has the ketoamine oxidase 1102 and substrate 1124, may have approximately the same dimensions as the entire enzyme layers 1111a and 1112b of the main sensor.

[0082] The thickness of the enzyme layer may be different between the main sensor and the subsensor. In some embodiments, the thickness of the ketoamine oxidase layer of the subsensor may be the same as the thickness of the ketoamine oxidase layer of the main sensor. The film thickness of each sensor may be adjusted based on correlations such as signal strength obtained using the same or different calibration solutions. The concentration or total amount of the enzyme may be the same or different between the main sensor and the subsensor, and may be adjusted depending on the sensor. The material of the substrate may also be the same or different between the main sensor and the subsensor.

[0083] Hydrogen peroxide detectors 1103a and 1103b of main sensor 1100a and sub-sensor 1100b are connected to measuring electrical circuits 1170a and 1170b, respectively. These electrical circuits 1170a and 1170b receive output signals such as current from each of the hydrogen peroxide detectors 1103a and 1103b and send the digital values ​​to a calculation unit 1130 such as a CPU.

[0084] In some embodiments, the main sensor in a differential sensor may have an enclosed type configuration, for example, as shown in FIG.

[0085] In some embodiments, the main sensor in the differential sensor may be of a direct junction type, for example, as shown in FIG.

[0086] The sensor 1200 shown in FIG. 11 includes a direct-bonded main sensor 1200a and a sub-sensor 1200b.

[0087] The main sensor 1200a shown in Fig. 11 is a direct-bonded sensor like that shown in Fig. 4, and includes a protease 1201, a ketoamine oxidase 1202, and a hydrogen peroxide detection unit 1203a to which these are immobilized. More specifically, a substrate 1214 (ketoamine oxidase layer 1212a) to which the ketoamine oxidase 1202 is immobilized is formed on the hydrogen peroxide detection unit 1203a. The protease 1201 is immobilized on the upper surface of this ketoamine oxidase layer 1212a by a cross-linking agent 1204. The protease 1201 in Fig. 11 is immobilized on the substrate 1214. The protease 1201 may be cross-linked to the ketoamine oxidase 1202.

[0088] 11 does not contain or does not contain protease 1201. Specifically, subsensor 1200b has ketoamine oxidase 1202, substrate 1224, and hydrogen peroxide detection unit 1203b, similar to main sensor 1200a. In other words, enzyme layer 1212b containing ketoamine oxidase 1202 and substrate 1224 is formed on hydrogen peroxide detection unit 1203b.

[0089] In main sensor 1200a, protease 1201 is bonded directly onto ketoamine oxidase layer 1212a. In contrast, the outermost surface of subsensor 1200b is essentially composed of substrate 1224. As a result, main sensor 1200a and subsensor 1200b may not necessarily be identical or may differ in properties, such as the permeability of molecules and ions at the outermost surface into the substrate. Therefore, the configuration of enzyme layer 1212b of subsensor 1200b, such as thickness, type of substrate 1224, concentration of ketoamine oxidase 1202, manufacturing method, etc., may be relatively adjusted so that the properties of main sensor 1200a and subsensor 1200b are substantially the same or related to each other.

[0090] Hydrogen peroxide detectors 1203a and 1203b of main sensor 1200a and sub-sensor 1200b are connected to measuring electrical circuits 1270a and 1270b, respectively. These electrical circuits 1270a and 1270b receive output signals such as current from hydrogen peroxide detectors 1203a and 1203b, and send the digital values ​​to a calculation unit 1230 such as a CPU.

[0091] The main sensor and sub-sensor of the differential sensor may each include a hydrogen peroxide electrode in the hydrogen peroxide detection section. For example, hydrogen peroxide electrode 730 and electrical circuit 770 shown in Figure 7 may be arranged in the main sensor, and a hydrogen peroxide electrode and electrical circuit of a similar or relatively different configuration may be arranged in the sub-sensor.

[0092] The main sensor and sub-sensor of the differential sensor may share some of the configuration of the hydrogen peroxide detection unit. For example, if a hydrogen peroxide electrode is disposed in the hydrogen peroxide detection unit, the working electrode may be disposed in each sensor, and the counter electrode and reference electrode may be disposed in one of the sensors and shared.

[0093] In the differential sensor 1300 shown in FIG. 12, a main sensor 1300 a and a sub-sensor 1300 b are arranged on a substrate 1306 .

[0094] In main sensor 1300a shown in Fig. 12, enzyme layer 1311a is disposed on hydrogen peroxide electrode 1330a with silane coupling agent 1305a interposed therebetween. Enzyme layer 1311a contains protease 1301 and ketoamine oxidase 1302 in substrate 1304. In subsensor 1300b shown in Fig. 12, enzyme layer 1311b is disposed on hydrogen peroxide electrode 1330b with silane coupling agent 1305b interposed therebetween. Enzyme layer 1311b does not contain protease 1301, but contains ketoamine oxidase 1302 in substrate 1324.

[0095] The hydrogen peroxide electrode 1330a of the main sensor 1300a includes a main sensor working electrode 1333a and a counter electrode 1331. On the other hand, the hydrogen peroxide electrode 1330b of the sub-sensor 1300b includes a sub-sensor working electrode 1333b and a reference electrode 1332. These electrodes are connected to an electric circuit 1370.

[0096] 12 are electrodes for the three-electrode method, and the working electrodes 1333a and 1333b are disposed in the main sensor 1300a and the sub-sensor 1300b, respectively. On the other hand, the counter electrode 1331 and the reference electrode 1332 are disposed in only one of the main sensor 1300a and the sub-sensor 1300b.

[0097] 12 further includes an electric circuit 1370 connected to the hydrogen peroxide electrodes 1330a and 1330b. The electric circuit 1370 includes an operational amplifier 1371, a voltage generating circuit 1372, and current measuring circuits 1373a and 1373b connected to the main sensor 1300a and the sub-sensor 1300b, respectively. The output (OUT) of the operational amplifier is connected to the counter electrode 1331, the inverting input (-IN) is connected to the reference electrode 1332, and the non-inverting input (+IN) is connected to the voltage generating circuit 1372.

[0098] During measurement, a solution (not shown) containing a test substance (substance to be measured) comes into contact with both the main sensor 1300a and the sub-sensor 1300b. Therefore, the counter electrode 1331 generates a constant potential difference relative to the potential of the reference electrode 1332, and can apply a desired voltage to both the main sensor working electrode 1333a and the sub-sensor working electrode 1333b. Current measurement circuits 1373a and 1373b detect output signals from the main sensor 1300a and the sub-sensor 1300b, respectively, and provide the output signals to an external device (not shown). This configuration reduces the area of ​​the electrodes, enabling the sensor and device to be miniaturized.

[0099] The enzyme layer of the main sensor 1300a in Fig. 12 has an integrated structure as shown in Fig. 2, but is not limited to this. The sub-sensor 1300b in Fig. 12 is also not limited to this structure. The main sensor 1300a and the sub-sensor 1300b may be configured in any other manner.

[0100] 12, current measurement circuits 1373a and 1373b are provided for main sensor 1300a and sub-sensor 1300b, respectively, but this configuration is not limited to this. One current measurement circuit and switching circuit may be provided, and signals from main sensor working electrode 1333a and sub-sensor working electrode 1333b may be supplied to the current measurement circuit alternately or at a predetermined timing using the switching circuit for detection or measurement.

[0101] The protease, ketoamine oxidase, and detection unit may be stacked, or may not be located in the same location or in close proximity. In some embodiments, the ketoamine oxidase and detection unit may be stacked or located in close proximity, with the protease located at a distance therefrom. In another embodiment, the protease, ketoamine oxidase, and detection unit may be located at a distance from one another. By immobilizing and locating the protease at a distance from other components, various conditions of the protease-mediated degradation reaction, including the time and temperature, can be made more efficient or optimized. After the protease-mediated degradation reaction has been carried out sufficiently, the peptide fragments that are the reaction products can be sent to the ketoamine oxidase.

[0102] In some embodiments, the protease, ketoamine oxidase, and detection unit may be arranged in this order from upstream to downstream of the solution, in other words, according to the direction of solution introduction or the order of reactions, thereby enabling efficient transport of the reaction product from each reaction to the next reaction.

[0103] In some embodiments, the protease, ketoamine oxidase, and detection unit may be contained in separate chambers. Each reaction occurs in a corresponding chamber, and some reactions can be separated from other reactions. This reduces the limitations and influences of other reaction conditions or preparation conditions, allowing the desired reaction to occur efficiently. For example, the protease can be allowed sufficient time to break down glycated proteins into peptide fragments, and after a desired time, the peptide fragments can be sent to the ketoamine oxidase.

[0104] 13, the protease, ketoamine oxidase, and detection unit are arranged spaced apart from one another. Furthermore, the protease, ketoamine oxidase, and detection unit are arranged in this order from upstream to downstream according to the direction of solution introduction or the direction of reaction order. Furthermore, the protease, ketoamine oxidase, and detection unit are contained in respective defined storage units (chambers) and are fluidically connected to one another.

[0105] FIG. 13 will be explained in more detail. Liquid entering through liquid inlet 1452 enters protease containing portion 1455. The liquid may enter through a channel up to liquid inlet 1452. The liquid may be transported to solution inlet 1452 by capillary action. Protease containing portion 1455 contains protease 1401 immobilized on beads 1404. In some embodiments, beads 1404 may be immobilized on the inner wall of containing portion 1455. In other embodiments, beads 1404 may not be directly immobilized in containing portion 1455. For example, by narrowing the channel for sufficiently large beads 1404, beads 1404 are substantially immobilized within containing portion 1455.

[0106] A heater 1460 is disposed in the protease storage section 1455 shown in FIG. 13. The heater 1460 heats or controls the temperature of the solution or protease 1401 in the protease storage section 1455, thereby increasing the rate of the decomposition reaction by the protease and thereby making the reaction more efficient or optimized. While FIG. 13 illustrates a heater that uses electric current, this is not limiting. A heating method other than electricity may also be used for the heater. In another embodiment, a temperature regulator may be disposed.

[0107] In some embodiments, the temperature of the protease may be controlled. In some embodiments, the temperatures of the protease and ketoamine oxidase may be controlled together or individually. In some embodiments, the temperature of a portion or the entire sensor or sensor chip may be controlled. In some embodiments, the temperature of the protease-containing portion may be controlled. The heating time and temperature profile of the protease, etc. may be controlled.

[0108] In some embodiments, the temperature of the protease may be greater than or equal to any of 20° C., 25° C., 30° C., 35° C., 40° C., 45° C., 50° C., 55° C., and 60° C. In some embodiments, the temperature of the protease may be less than or equal to any of 80° C., 75° C., 70° C., 65° C., 60° C., 55° C., 50° C., 45° C., 40° C., and 35° C. In some embodiments, the temperature of the protease may be within the optimum temperature range for the protease. In some embodiments, the temperature of the protease may be lower than the inactivation temperature.

[0109] The peptide fragments produced in protease reservoir 1455 are transported to ketoamine oxidase reservoir 1456 where ketoamine oxidase 1402 is immobilized on substrate 1414 .

[0110] In some embodiments, the transport of the protease in solution state from the protease storage portion 1455 to the ketoamine oxidase storage portion 1456 may be achieved by capillary action. For example, the residence time of the protease in solution state in the protease storage portion 1455 and the timing of delivery to the ketoamine oxidase storage portion 1456 can be adjusted by selecting or adjusting the shape of the flow channels between each storage portion, such as the cross-sectional area and distance, and the material of the inner wall, such as hydrophilicity or hydrophobicity.

[0111] In some embodiments, protease-containing portion 1455 may be made of a flexible or elastic material such as silicone so that its volume is variable, and may be deformed by applying external pressure at a predetermined timing to discharge liquid from protease-containing portion 1455. At this time, liquid inlet 1452 may be configured to be closed so that liquid does not flow back into liquid inlet 1452.

[0112] In the ketoamine oxidase containing section 1456, ketoamine oxidase 1402 is immobilized on a substrate 1414. In Fig. 13, the substrate 1414 and ketoamine oxidase 1402 are formed as an immobilization layer in the ketoamine oxidase containing section 1456. The peptide fragment transported into the ketoamine oxidase containing section 1456 reacts with the ketoamine oxidase 1402, resulting in the generation of hydrogen peroxide.

[0113] In some embodiments, the temperature of the ketoamine oxidase may be controlled. In some embodiments, the temperature of the ketoamine oxidase container may be controlled. In some embodiments, the temperatures of the protease and the ketoamine oxidase may be controlled so that they are different from each other. In some embodiments, the temperature of the protease may be controlled so that it is higher than the temperature of the ketoamine oxidase, or the temperature of the ketoamine oxidase may be controlled so that it is lower than the temperature of the protease. In some embodiments, the temperature control of the protease and the ketoamine oxidase may be performed simultaneously, at different times, or so that they partially overlap. In some embodiments, the heating time or temperature profile of the ketoamine oxidase, etc. may be controlled.

[0114] In some embodiments, the controlled temperature of the ketoamine oxidase may be greater than or equal to any of 10° C., 12° C., 15° C., 20° C., 25° C., 30° C., 35° C., and 37° C. In some embodiments, the controlled temperature of the ketoamine oxidase may be less than or equal to any of 70° C., 60° C., 55° C., 50° C., 45° C., 40° C., 37° C., and 35° C. The temperature of the ketoamine oxidase may be controlled by heating, cooling, or a combination of heating and cooling.

[0115] In some embodiments, the ketoamine oxidase may be maintained at room temperature during measurement. In some embodiments, the temperature of the ketoamine oxidase during measurement may be measured, and temperature correction may be applied to the results obtained by the detector. For example, measurement may be performed while the ketoamine oxidase is maintained at room temperature, and temperature correction may be applied to the measurement results. In some embodiments, the temperature of the protease during measurement or peptide fragmentation may be measured, and temperature correction may be applied to the results obtained by the detector. In some embodiments, temperature correction may be applied to the measurement results based on the temperature of the protease during measurement or peptide fragmentation and the temperature of the ketoamine oxidase during measurement. Illustratively, temperature correction can simplify temperature control of at least one of the ketoamine oxidase and the protease. This allows, for example, the power source to be a battery, thereby miniaturizing the sensor configuration.

[0116] The hydrogen peroxide is transported to a detection reactor 1457 and detected by a detector 1431. In Figure 13, a reagent 1431 containing luminol is placed inside the detection reactor 1457, and a photodetector (photodiode) 1432 is placed outside to detect luminescence due to the luminol reaction.

[0117] The ketoamine oxidase may not be immobilized on a substrate in an immobilization layer, but may be immobilized on beads.

[0118] The sensor 1500 shown in FIG. 14 has a configuration similar to that of the sensor 1400 shown in FIG. 13, but the ketoamine oxidase 1502 is immobilized on beads 1504 and contained in a ketoamine oxidase containing portion 1556 .

[0119] In the sensor 1400 shown in Fig. 13 and the sensor 1500 shown in Fig. 14, the detector is configured to include a reagent for the luminol reaction and a photodetector that detects luminescence. However, other detectors may be used in a similar configuration. Other photodetectors may also be used as the detector, and a hydrogen peroxide electrode may also be used.

[0120] In some embodiments, the ketoamine oxidase and the hydrogen peroxide detector may be in contact with or adjacent to each other, and the protease may be spaced apart from them. Placing the ketoamine oxidase and the hydrogen peroxide detector in close proximity is one way to reduce the influence of noise factors on the detection by the hydrogen peroxide detector or to improve the detection sensitivity of hydrogen peroxide.

[0121] In the sensor 1600 shown in FIG. 15, a ketoamine oxidase 1602 is immobilized adjacent to a hydrogen peroxide detector 1603, and a protease 1601 is positioned at a distance therefrom.

[0122] The liquid that has entered through the liquid inlet 1652 enters the protease containing section 1655. The protease containing section 1655 contains protease 1601 in a state where it is immobilized on beads 1604.

[0123] A heater 1660 is disposed in the protease storage section 1655 shown in Figure 15. The heater 1660 heats or controls the temperature of the solution or protease 1601 in the protease storage section 1655, thereby making the decomposition reaction by the protease more efficient or optimal. While Figure 15 shows a heater that uses electric current, this is not limiting. A heating method other than electricity may also be used for the heater. In another embodiment, a temperature regulator may be disposed.

[0124] The peptide fragments produced in the protease reservoir 1655 are transported to the ketoamine oxidase reservoir 1656 which contains immobilized ketoamine oxidase 1602 and hydrogen peroxide detector 1603 .

[0125] In the ketoamine oxidase containing section 1656, ketoamine oxidase 1602 is immobilized on a substrate 1614 to form an enzyme layer 1611. In Figure 15, the substrate 1614 and ketoamine oxidase 1602 are formed as an immobilization layer in the ketoamine oxidase containing section 1656. The peptide fragments transported into the ketoamine oxidase containing section 1656 react with the ketoamine oxidase 1602, resulting in the generation of hydrogen peroxide. The generated hydrogen peroxide is detected by a hydrogen peroxide detector 1603 located nearby.

[0126] Even in a configuration in which the protease and the ketoamine oxidase are spaced apart, noise sources such as substances in the solution may exist. The sensor according to the present disclosure may further include a mechanism for reducing such noise, or may be configured to be connected to the mechanism.

[0127] In some embodiments, the hydrogen peroxide detector may be coated with an ion exchange resin, or a layer or membrane of ion exchange resin may be placed on the surface of the hydrogen peroxide detector.

[0128] In some embodiments, the sensor may be a pair or set of differential or differential sensors having a configuration that spaces the protease and the ketoamine oxidase apart.

[0129] In some embodiments of the differential sensor, the hydrogen peroxide detector may be coated with an ion exchange resin, or a layer or membrane of ion exchange resin may be placed on the surface of the hydrogen peroxide detector.

[0130] In the sensor 1700 shown in Figure 16, the protease is separated from the ketoamine oxidase and placed in a separate reservoir.

[0131] The solution introduced from inlet 1752 is divided into main sensor 1700a and sub-sensor 1700b. In main sensor 1700a, the solution is introduced into protease containing section 1755a. Protease containing section 1755a is provided with enzyme layer 1711a on which protease is immobilized. Peptide fragments produced in protease containing section 1755a are transported through a flow path to ketoamine oxidase containing container 1756a. In ketoamine oxidase containing container 1756a shown in FIG. 16, ketoamine oxidase layer 1712a on which ketoamine oxidase is immobilized and hydrogen peroxide detector 1703a are provided. Due to the reaction in ketoamine oxidase containing container 1756a, glycated peptide fragments are ultimately detected as hydrogen peroxide.

[0132] On the other hand, the flow path and storage section of sub-sensor 1700b are configured similarly to main sensor 1700a, but no protease is disposed therein. That is, chamber 1755b, into which the solution is first introduced, has a structure similar to protease storage section 1755a of main sensor 1700a, but does not store protease. A ketoamine oxidase storage container 1756b is disposed as the chamber (storage section) next to chamber 1755b without protease. Inside ketoamine oxidase storage container 1756b of sub-sensor 1700b, a ketoamine oxidase layer 1712b on which ketoamine oxidase is immobilized and a hydrogen peroxide detector 1703b are disposed, similar to ketoamine oxidase storage container 1756a of main sensor 1700a.

[0133] The protease containing section 1755a of the main sensor 1700a and the corresponding chamber 1755b of the sub-sensor 1700b are provided with a heater 1760. This increases the speed of the decomposition reaction caused by the protease in the protease containing section 1755a, making the reaction more efficient or optimized. Furthermore, the configuration and conditions of the flow path of the sub-sensor 1700b can be made as similar as possible to those of the main sensor 1700a, except for the absence of protease.

[0134] A differential signal can be calculated between the output signal from hydrogen peroxide detector 1703a of main sensor 1700a and the output signal from hydrogen peroxide detector 1703b of sub-sensor 1700b. From this differential calculation, the concentration of hydrogen peroxide, which is the target in hydrogen peroxide detector 1712a, and the associated concentration of glycated protein, which is the test substance in the original solution, can be determined.

[0135] In some embodiments, the temperatures of the protease and the ketoamine oxidase may be controlled separately. In some embodiments, a temperature control device may be provided for each of the protease and the ketoamine oxidase. The temperature control device may be a heating device, a cooling device, or both, or may be a device that can be controlled to be hot or cold.

[0136] In some embodiments, the sensor may have a temperature-controlling container between the protease container and the ketoamine oxidase container. In some embodiments, when the operating temperatures of the protease and the ketoamine oxidase differ, the temperature-controlling container may change or control the temperature of the solution delivered from the protease container to bring it closer to the operating temperature of the ketoamine oxidase. In some embodiments, a heating device may be disposed relative to the protease, and a cooling device may be disposed in the temperature-controlling container. In some embodiments, a heating device may be disposed relative to the ketoamine oxidase.

[0137] 17 includes a protease containing section 1855, a cooling solution containing section (cooling section) 1858, and a ketoamine oxidase containing section 1856, which are fluidly connected in series. The protease containing section 1855 includes a liquid inlet 1852 through which a solution can be introduced into the protease containing section 1855. The protease containing section 1855 contains protease 1801 immobilized on beads 1804.

[0138] 17 is provided with a heater 1860. The heater 1860 heats or controls the temperature of the solution or protease 1801 in the protease-containing portion 1855, thereby making it possible to increase the rate of the protease reaction and thereby improve or optimize the efficiency of the reaction.

[0139] The peptide fragments produced in the protease reservoir 1855 are transferred to the ketoamine oxidase reservoir 1856, which is fluidly connected to the protease reservoir 1855 via the cooling solution reservoir 1858. The ketoamine oxidase reservoir 1856 contains immobilized ketoamine oxidase 1802 and a hydrogen peroxide detector 1803. The hydrogen peroxide detector 1803 is connected to a measuring device (not shown) and can transmit an electrical signal thereto.

[0140] In the ketoamine oxidase containing section 1856, ketoamine oxidase 1802 is immobilized on a substrate 1814 to form an enzyme layer 1811. In Figure 17, the substrate 1814 and ketoamine oxidase 1802 are formed as an immobilization layer in the ketoamine oxidase containing section 1856. The peptide fragment transported into the ketoamine oxidase containing section 1856 reacts with the ketoamine oxidase 1802, resulting in the generation of hydrogen peroxide. The generated hydrogen peroxide is detected by a hydrogen peroxide detector 1803 located nearby.

[0141] A heater 1861 is disposed in the ketoamine oxidase containing section 1856. The heater 1861 heats or controls the temperature of the solution or ketoamine oxidase 1802 in the ketoamine oxidase containing section 1856, thereby making it possible to increase the rate of the enzymatic reaction and thereby to make the reaction more efficient or optimized.

[0142] In Figure 17, individual heaters 1860, 1861 are provided for the protease-containing portion 1855 and the ketoamine oxidase-containing portion 1856, respectively. This allows for individual temperature control or heating of the protease 1801 and the ketoamine oxidase 1802. This allows for independent temperature control, for example, over time, temperature, or both. In some embodiments, the temperature of the protease-containing portion 1855 or the protease 1801 can be controlled to be higher than the temperature of the ketoamine oxidase-containing portion 1856 or the ketoamine oxidase 1802. In some embodiments, the temperature of the protease-containing portion 1855 or the protease 1801 can be controlled to be lower than the temperature of the ketoamine oxidase-containing portion 1856 or the ketoamine oxidase 1802.

[0143] If the solution heated in the protease containing section 1855 is sent to the ketoamine oxidase containing section 1856, it may take a long time for the solution to reach the appropriate temperature for the ketoamine oxidase 1802, which may result in inefficiency or inaccurate measurement. For this reason, the solution may be cooled before being introduced into the ketoamine oxidase containing section 1856. The sensor 1800 shown in FIG. 17 can retain the solution heated in the heated protease containing section 1855 in the cooling solution containing section 1858. The solution is cooled using a cooling device (circuit / element) 1862 (e.g., a Peltier element) to bring it closer to the optimal temperature or operating temperature of the ketoamine oxidase 1802. The solution can then be sent to the ketoamine oxidase containing section 1856. This allows, for example, for efficient or accurate measurement.

[0144] The sensor 1800 shown in FIG. 17 may be used as follows, for example. That is, first, a solution containing a substance to be measured is introduced into the protease containing portion 1855. Heating of the protease containing portion 1855 may begin before or after the introduction of the solution. After peptide fragmentation is performed in the protease containing portion 1855, the solution is sent to the cooling solution containing portion 1858. Cooling of the cooling solution containing portion 1858 may begin before or after the introduction of the solution. After cooling to a sufficient or appropriate temperature in the cooling solution containing portion 1858, the solution may be sent to the ketoamine oxidase containing portion 1856. Heating of the ketoamine oxidase containing portion 1856 may begin after the introduction of the solution.

[0145] In some embodiments, the solution may be delivered by applying pressure from solution inlet 1852. In some embodiments, the solution may be delivered by changing the volume of each of storage sections 1855, 1856, and 1858 or by applying pressure to each storage section. Protease storage section 1855 may have an air vent (not shown). In some embodiments, the pressure during solution delivery may be positive pressure. In some embodiments, the pressure during solution delivery may be negative pressure. In some embodiments, a valve may be disposed near each or both of the inlet and outlet of each storage section. The valve may function to remove bubbles from the flow path that may be generated due to differences in temperature between the storage sections, etc.

[0146] In some embodiments, the protease and beads may exit the protease reservoir when the solution is removed from the protease reservoir. For example, the protease and beads may enter the cooling reservoir or the ketoamine oxidase reservoir. In some embodiments, the sensor may be configured such that the protease and beads substantially remain in the protease reservoir during delivery.

[0147] 17 illustrates heaters 1860, 1861 and cooling device 1862 that use electric current, but this is not limiting. Heaters 1860, 1861 may employ a heating method other than electricity. Cooling device 1862 may employ a cooling method other than a Peltier element. In other embodiments, other temperature regulators may be provided.

[0148] In some embodiments, the solution inlet 1852, the protease containing portion 1855, the cooling solution containing portion 1858, and the ketoamine oxidase containing portion 1856 may be a single component, such as a cassette or a disposable fluidic device. In some embodiments, the heaters 1860, 1861, and the cooling device 1862 may be fixed to a main body. The sensor 1800 may be configured so that the fluidic devices are inserted into or fixed to the main body. A thermal insulator may be disposed between the heaters 1860, 1861 and the cooling device 1862. This may, for example, improve the efficiency of temperature control.

[0149] 3. Hydrogen Peroxide Electrode Placement The layout or arrangement of the hydrogen peroxide electrodes can be variously configured, and the configuration of the hydrogen peroxide electrodes will be described below as an example.

[0150] <Example 1 of electrode arrangement> The hydrogen peroxide electrode disclosed herein is not limited to applications for measuring glycated proteins or fructosamine, but can also be used for other applications, including electrochemical measurement of solutions. That is, a sensor or sensor chip according to an embodiment of the present disclosure includes a hydrogen peroxide electrode. The reference electrode of the hydrogen peroxide electrode may be sandwiched between a counter electrode and a working electrode. The sensor chip may have a liquid storage section. The liquid storage section may extend in the longitudinal direction. The volume of the liquid storage section may be less than or equal to 10 μL, 5 μL, 4 μL, 3 μL, 2 μL, 1 μL, 0.9 μL, 0.8 μL, 0.7 μL, 0.6 μL, 0.5 μL, 0.4 μL, 0.3 μL, 0.2 μL, or 0.1 μL. The liquid storage section may have a liquid inlet. The liquid storage section may have a liquid outlet. The liquid storage section may have an air hole. The air hole may have a function of discharging gas that is present in the liquid storage portion to the outside of the sensor chip when liquid is introduced into the liquid storage portion. The sensor chip may have an electric circuit or may be configured to be connected to the electric circuit. The sensor chip may have an output terminal for connection to the electric circuit.

[0151] Fig. 18 is a top view schematically showing the electrode configuration of a sensor chip according to one embodiment. In sensor chip 2000 shown in Fig. 18, counter electrode 2031, reference electrode 2032, and working electrode 2033 are arranged on substrate 2006 so as to be in contact with the introduced solution, and enzyme layer 2011 is arranged to cover these electrodes.

[0152] The sensor chip 2000 may further include a member that defines the flow path or the liquid storage portion, such as a chip cover, or may be formed as a sensor chip without such a chip cover. In Fig. 18, a sensor chip cover (not shown) that defines the flow path and the liquid storage portion is placed on the top surface of the sensor chip. A portion 2051 on the sensor chip that corresponds to the liquid storage portion is defined, and liquid is introduced into the liquid storage portion 2051 from an inlet 2052. The chip cover has an air hole 2053 on the opposite side of the liquid inlet 2052 from the liquid storage portion 2051.

[0153] The hydrogen peroxide electrodes 2031, 2032, and 2033 are arranged parallel to one another and extend longitudinally along the liquid storage portion 2051. On the liquid inlet 2052 side, the end of the working electrode 2033 is configured to be shorter than the ends of the other electrodes, namely the counter electrode 2031 and the reference electrode 2032. The liquid introduced from the inlet 2052 advances longitudinally within the liquid storage portion 2051. If the liquid comes into contact with the working electrode 2033 first or simultaneously with the other electrodes, a large current will flow, potentially damaging the electrode. This configuration makes it possible to avoid electrode damage.

[0154] The sensor chip 2000 shown in FIG. 18 further includes a liquid detection electrode 2034 in the liquid storage portion. In FIG. 18, the electrode 2034 is disposed at a position that the liquid last comes into contact with when the liquid enters the liquid storage portion 2051. By detecting the presence or absence of liquid with the liquid detection electrode 2034, it is possible to confirm that the liquid storage portion 2051 or the hydrogen peroxide electrodes 2031, 2032, and 2033 are sufficiently filled with liquid. If the liquid detection electrode 2034 detects liquid, the user may be notified that liquid collection has ended. Alternatively, if the liquid detection electrode 2034 does not detect liquid even after waiting for a predetermined time or longer, the measurement may not be started because the liquid was not collected sufficiently, or the user may be notified to try collecting liquid again or to use another chip.

[0155] The sensor chip 2000 shown in Fig. 18 has output terminals 2041, 2042, 2043, and 2044 that correspond to and are electrically connected to a counter electrode 2031, a reference electrode 2032, a working electrode 2033, and a liquid detection electrode 2034. The sensor chip may be configured to be pluggable or detachably connectable to an electric circuit or another device having an electric circuit (none of which are shown). In the sensor chip 2000 shown in Fig. 18, the output terminals are arranged in these detachable sections.

[0156] FIG. 18 shows only one detection electrode. However, in some embodiments, multiple detection electrodes may be arranged. Multiple electrodes may be installed in the flow path in the direction of liquid flow. For example, detection electrodes may be arranged at the point where the detection unit first comes into contact with the liquid, at the point where the detection unit has completely come into contact with the liquid, or somewhere in between. For example, the first detection electrode can detect when liquid has begun to enter the detection unit, the flow path, or the liquid storage unit. The intermediate electrode can detect whether liquid has entered. If the liquid has not entered the intermediate electrode, this may be used to notify that measurement is impossible because only a portion of the detection unit is being used, or that the measurement may have a large error.

[0157] Other arrangements and variations will be described below as examples. Some arrangements and descriptions may be omitted. Other configurations may be applied to each embodiment and example.

[0158] <Multiple electrode placement> The liquid storage section of the sensor chip may be provided with multiple or different types of sensors or hydrogen peroxide electrodes. The multiple sensors may be of the same type, or may be a combination of a main sensor and a sub-sensor for differential measurement as described above.

[0159] <Example of electrode arrangement 2> 19, a sensor chip 2100 has multiple sensors 2100a and 2100b arranged parallel to one another in the longitudinal direction within a liquid storage portion 2151. The reference electrodes and counter electrodes of the multiple sensors 2100a and 2100b are connected together in a common circuit, with the reference electrodes and working electrodes connected together. Liquid is introduced through a fluid inlet 2152 and, due to capillary action and the action of an immobilizing membrane that also functions as a hydrophilic, water-containing polymer, progresses through the liquid storage portion 2151, filling it while pushing out the air inside through an air hole 2153, and finally reaches the detection electrode 2134.

[0160] <Example 3 of electrode arrangement> In the sensor chip 2200 shown in Fig. 20, a portion of the hydrogen peroxide electrode as shown in Fig. 12 is shared by multiple sensors. That is, the sensor chip 2200 shown in Fig. 20 has a main sensor 2220a and a sub-sensor 2220b. In the main sensor 2220a, a working electrode 2233a and a counter electrode 2131 are disposed on a substrate 2206, and an enzyme layer 2221a for the main sensor is disposed on these electrodes. In the sub-sensor 2220b, a working electrode 2233b and a reference electrode 2232 are disposed on a substrate 2206, and an enzyme layer 2221b for the sub-sensor is disposed on these electrodes. The reference electrode 2232 is disposed or sandwiched between the counter electrode 2231 and the working electrode 2233b on the substrate 2206.

[0161] <Multiple sensor assembly type> When multiple sensors are arranged on the same sensor chip, it is not necessary to form all the sensors directly on the main substrate of the sensor chip. For example, each sensor may be bonded to the main substrate after it has been fabricated.

[0162] In the sensor chip 2300 shown in FIG. 21, two already fabricated sensors 2300a and 2300b are bonded onto a main substrate 2360.

[0163] The enzyme layers 2311a, 2311b or hydrogen peroxide electrodes 2330a, 2330b of the sensors 2300a, 2300b are formed so as to extend in the longitudinal direction of the liquid storage portion 2351, extending from the liquid inlet 2352 toward the air hole 2353.

[0164] 21, bonding pads 2340a, 2340b, 2341a, and 2341b are formed on the main substrate 2360 and on each sensor, respectively. These bonding pads are electrically connected after or during bonding of the sensors 2300a and 2300b to the main substrate 2360. For example, the bonding pads may be connected by wire bonding.

[0165] 21, output terminals 2340 connectable to an electric circuit (not shown) or the like are arranged on a main substrate 2360. These output terminals 2340 are connected to corresponding bonding pads 2341a, 2341b by wiring. The counter electrode and reference electrode may be short-circuited via the sensor chip 2300 or an electric circuit. With this configuration, for example, when it is difficult to simultaneously manufacture two sensors in one chip due to the durability of the enzyme layer or the configuration of other layers, or when the yields of the two sensors are significantly different, it is possible to reduce the total manufacturing cost by manufacturing them separately and combining non-defective products.

[0166] <Other electrode directions> The hydrogen peroxide electrode may be formed along a direction other than the longitudinal direction of the elongated liquid containing portion.

[0167] For example, as in the sensor chip 2400 shown in FIG. 22, electrodes extending perpendicular to the longitudinal direction of the liquid storage portion 2451 may be arranged on the substrate 2406. More specifically, the hydrogen peroxide electrode of FIG. 22 has a counter electrode 2431, a reference electrode 2432, and a working electrode 2433 arranged in the liquid storage portion 2451 in a direction perpendicular to the longitudinal direction from the liquid inlet 2452 toward the air hole 2453. The reference electrode 2432 is sandwiched between the counter electrode 2431 and the working electrode 2433. The working electrode 2433 is arranged at the last position of the three electrodes of the hydrogen peroxide electrode in the direction of liquid advancement during liquid introduction. A liquid detection electrode 2434 is arranged behind these hydrogen peroxide electrodes. An enzyme layer 2411 is formed on the hydrogen peroxide electrode. In this electrode configuration, the reference electrode 2432 is sandwiched between the counter electrode 2431 and the working electrode 2433, which, for example, reduces external noise, and by having the liquid come into contact with the working electrode 2433 last when it enters the liquid storage section 2451, excessive current flow is avoided, enabling safe measurement.

[0168] 4. Sensor manufacturing method A method for producing a dual glycated protein sensor according to one embodiment of the present disclosure will be described below with reference to the cross-sectional schematic views of FIGS. 23A to 23I.

[0169] <Formation of hydrogen peroxide electrode> As shown in Figure 23A, two pairs of electrodes, namely, first hydrogen peroxide electrode 3030a and second hydrogen peroxide electrode 3030b, are formed on insulating substrate 3006. Insulating substrate 3006 may be a substrate whose main component is glass, quartz, or ceramic. Insulating substrate 3006 may also be made of polyethylene terephthalate (PET), polyethylene naphthalate (PEN), cycloolefin polymer (COP) resin, or the like. These materials are known for their excellent water resistance, heat resistance, and chemical resistance, as well as their excellent adhesion to the electrodes.

[0170] Each pair of hydrogen peroxide electrodes 3030a, 3030b comprises a working electrode made of a Ti / Pt laminate, a reference electrode made of a Ti / Pt / Ag / AgCl laminate, and a counter electrode made of a Ti / Pt laminate. These laminated structures are not shown in the figure. These electrodes can be formed using, for example, sputtering, ion plating, vacuum deposition, chemical vapor deposition (CVD), electrolysis, screen printing, and other methods. Sputtering can form a thin platinum film with high precision in a relatively short time. For example, first, titanium (Ti) and platinum (Pt) films are deposited and patterned in this order. Next, the working electrode and counter electrode are covered, and a silver (Ag) film is deposited on the reference electrode. Finally, the silver surface is chlorinated. This results in the laminated electrode structure described above. Note that Ti is often deposited to enhance adhesion between the substrate and the Pt. Therefore, depending on the substrate and film-forming conditions, the Ti layer of the hydrogen peroxide electrode may be omitted. For example, when an insulating substrate made of a flexible sheet of PET resin or COP resin is used, the Ti layer of the hydrogen peroxide electrode may be omitted.

[0171] <Formation of enzyme layer for sub-sensor> First, as shown in FIG. 23B, a positive photoresist 3009 used in photolithography processes such as semiconductor manufacturing is spin-coated onto insulating substrate 3006 to cover two pairs of hydrogen peroxide electrodes 3030a and 3030b.

[0172] Subsequently, as shown in FIG. 23C, resist 3009 is patterned by exposure, development, and etching to form an opening above second hydrogen peroxide electrode 3030b.

[0173] As shown in Figure 23D, a solution of 3-aminopropyltriethoxysilane (APTES), which is a silane coupling agent 3005b, is spin-coated at 3,000 rpm for 30 seconds and then dried. A solution containing BSA and ketoamine oxidase 3002, which will form the substrate 3014, is then mixed with glutaraldehyde. This mixture is then spin-coated onto the substrate. The ketoamine oxidase 3002 is solidified by a cross-linking reaction of glutaraldehyde.

[0174] Resist 3009 is lifted off by ultrasonic treatment in acetone. An enzyme membrane 3011b containing BSA and ketoamine oxidase 3002, which serves as substrate 3014, remains only on second hydrogen peroxide electrode 3030b, via silane coupling agent 3005b. This forms sub-sensor 3000b on insulating substrate 3006, exposing first hydrogen peroxide electrode 3030a for the main sensor (FIG. 23E).

[0175] 23B, resist 3091 is again spin-coated over the entire insulating substrate 3006. After coating, resist 3091 is hardened to prevent the enzyme from being inactivated (FIG. 23F).

[0176] <Formation of enzyme layer for main sensor> First, resist 3091 is patterned by exposure, development, and etching to form an opening above the first hydrogen peroxide electrode (FIG. 23G).

[0177] With the opening on the first hydrogen peroxide electrode 3030a, a silane coupling agent 3005a (3-aminopropyltriethoxysilane (APTES) solution) is spin-coated at 3,000 rpm for 30 seconds and dried, as in FIG. 23D. A solution containing BSA, ketoamine oxidase 3002, and protease 3001, which will form the base material 3004, is then mixed with glutaraldehyde. This mixture is then spin-coated onto the substrate. The ketoamine oxidase 3002 and protease 3001 are solidified by a cross-linking reaction with glutaraldehyde (FIG. 23H).

[0178] Resist 3091 is lifted off by ultrasonic treatment in acetone. Thus, a sensor was fabricated: enzyme layer 3011a containing protease 3001 and ketoamine oxidase 3002 immobilized on BSA 3004, respectively, and enzyme layer 3011b containing only ketoamine oxidase 3002 immobilized on BSA 3014, via silane coupling agents 3005a and 3005b, respectively, on first and second hydrogen peroxide electrodes 3330a and 3330b on insulating substrate 3006. That is, main sensor 3000a and sub-sensor 3000b were formed on insulating substrate 3006. In this way, sensor 3000 having a differential dual glycated albumin sensor enzyme layer, which differs only in the presence or absence of protease 3001, can be fabricated (FIG. 23I).

[0179] Although this example shows a manufacturing method using resist patterning, it is also possible to use a metal mask having an opening above the first hydrogen peroxide electrode and a metal mask having an opening above the second hydrogen peroxide electrode.

[0180] In some embodiments, the glycated protein sensor (detection unit, detector) may include an albumin sensor (detection unit, detector). The glycated protein sensor and the albumin sensor may be collectively referred to as a sensor, and a system configured to measure both glycated proteins and albumin may be referred to as a glycated protein sensor. The sensor may be configured so that a body fluid or liquid obtained from the same collection is applied to both the glycated protein sensor and the albumin sensor. In another embodiment, the sensor may be configured so that body fluids or liquids collected at different times are supplied to the glycated protein sensor and the albumin sensor. In some embodiments, the glycated protein sensor may be configured to be connected to the albumin sensor. By cooperating with the albumin sensor, glycated albumin (GA) levels can be measured more simply and efficiently.

[0181] The albumin sensor can be used for measurement by, for example, a dye binding method or an electrochemical method that utilizes the change in absorption wavelength or absorption spectrum when albumin binds to a dye such as bromocresol green (BCG) or bromocresol purple (BCP). Alternatively, it may be a device or apparatus based on an immunoassay using an antibody.

[0182] In some embodiments, the enzyme (ketoamine oxidase and / or protease) may be covered with a protective agent, which may be a protective layer, a protective film, or a protective coating.

[0183] As shown in Figure 24, a protective agent may be disposed so as to cover the enzyme layer. In Figure 24, only the ketoamine oxidase portion is shown, and the protease and other elements and components are omitted. Hydrogen peroxide detector 4003 is disposed on substrate 4006, and ketoamine oxidase 4002 is encapsulated in base material 4014 thereon to form ketoamine oxidase layer 4012. Protective film 4008 is disposed so as to cover ketoamine oxidase 4002 or ketoamine oxidase layer 4012. Protective film 4008 may also be disposed so as to cover hydrogen peroxide detector 4003.

[0184] As shown in Figure 25, the protective agent may be disposed to cover each enzyme molecule, or may be disposed to cover portions of the enzyme or clusters of enzyme molecules. In Figure 25, only the ketoamine oxidase portion is shown, and the protease and other elements and components are omitted. A hydrogen peroxide detector 4103 is disposed on a substrate 4106, and ketoamine oxidase 4102 is encapsulated in a base material 4114 thereon to form a ketoamine oxidase layer 4112. The protective coating may cover ketoamine oxidase 4102 individually or on a molecular basis. It may also be formed to cover multiple ketoamine oxidases 4102.

[0185] In some embodiments, the enzyme protectant may be selected from the material of the substrate. In some embodiments, the enzyme protectant may be made of the same material as the enzyme substrate. If the enzyme substrate and the protectant are made of the same material, peeling or breakage due to thermal stress caused by differences in thermal expansion coefficients, for example, due to heating during measurement or changes in room temperature, can be avoided. The protectant can, for example, but is not limited to, avoid enzyme deterioration or extend its lifespan. For example, protease that is not immobilized on the device body, or a portion of the immobilized protease, may come into contact with ketoamine oxidase and decompose or denature it. The protectant can, for example, avoid or suppress such decomposition of ketoamine oxidase. In some embodiments, the ketoamine oxidase protectant may be made of a material that is resistant to protease degradation.

[0186] The protective agent may be formed by applying a FOAD film (e.g., spin coating or bar coating), crosslinking (leaving, crosslinking time), drying, and then forming a protective film. The protective film may be formed to have a uniform thickness. Depending on the properties of the solution used, the protective film may be applied by spin coating, bar coating, blade coating, spray coating, or dipping. The applied protective film may be held at a predetermined temperature for a predetermined time. The required film thickness may be achieved by post-application treatment.

[0187] The protective agent may be, but is not limited to, a fluororesin, a photocurable resin, a water-curable resin, cellulose (e.g., ethyl cellulose, acetyl cellulose), nylon, polystyrene, a non-protein resin, a polyion complex, a complex, an inorganic polymer, a solid polymer electrolyte, a porous polymer metal complex (e.g., ZIF-8), a metal-organic framework, or a phospholipid. In some embodiments, the protective agent for ketoamine oxidase may contain albumin or may consist essentially of albumin. In some embodiments, the albumin used as the protective agent may be BSA. In a system in which the lifespan of ketoamine oxidase was one week without a protective agent, it was confirmed that covering the ketoamine oxidase with a BSA protective agent extended the lifespan of the ketoamine oxidase to about one month and doubled the time to half its output.

[0188] A protective agent can, as a non-limiting example, reduce the impact on the lifespan of thermal vibrations caused by the liquid. The enzyme may be immobilized by the substrate, partially immobilized, or coated by the substrate. As one interpretation, the protective agent may protect the portion of the enzyme molecule exposed on the surface of the substrate. As one interpretation, protecting the enzyme with a protective agent can be considered to extend the lifespan. As a non-limiting example, the impact of contaminants on the enzyme activity, enzyme reaction efficiency, etc. can be reduced. As a non-limiting example, a protective film can prevent or reduce the enzyme from falling off or detaching from the substrate. As a non-limiting example, a protective film can prevent denaturation (protein denaturation) caused by sudden changes in parameters such as external pressure, pH, and salt concentration that occur during liquid transfer, etc.

[0189] Hereinafter, proteases and ketoamine oxidases will be described using several examples.

[0190] Example 1 The performance characteristics of various proteases were compared. The proteases used were Orientase 22BF (manufactured by HIV Corporation) 16 mg, Nucleisin (manufactured by HIV Corporation) 133 mg, Orientase AY (manufactured by HIV Corporation) 12.8 mg, Orientase OP (manufactured by HIV Corporation) 8.0 mg, Sumiteam MP (manufactured by Shin Nippon Chemical Industry Co., Ltd.) 16 mg, and Protease XIV (Sigma-Aldrich Japan LLC) 4 mg. Each was dissolved in 400 μL of TES buffer and dialyzed overnight to prepare the protease solution. 4 mg of thermolysin (Fujifilm Wako Pure Chemical Industries, Ltd.), 4 mg of trypsin (Fujifilm Wako Pure Chemical Industries, Ltd.), 4 mg of α-chymotrypsin (Tokyo Chemical Industry Co., Ltd.), 4 mg of papain (Nacalai Tesque Inc.), and 4 mg of bromelain (Fujifilm Wako Pure Chemical Industries, Ltd.) were also dissolved in 400 μL of TES buffer and used as is.

[0191] 12 μL of GA-L calibrator (GA concentration: 14.9 mg / mL, albumin concentration: 47.9 mg / mL) from the Glycoalbumin Kit Lucica GA-L (Asahi Kasei Pharma Corporation) was mixed with 93 μL of TES buffer, and 15 μL of protease solution was added. The mixture was heated at 37°C or 60°C for 10 minutes in an aluminum block incubator. The mixture was removed from the aluminum block and immediately placed in a refrigerator to prevent residual heat or digestion from progressing at room temperature, and then stored at a low temperature. This is referred to as the GA digestion sample. Meanwhile, the protease solution was mixed with TES buffer. This mixture was similarly heat-treated. This is referred to as the protease autolysis sample.

[0192] Absorbance measurements were performed using a microplate reader and the commercially available glycoalbumin kit Lusica ALB R-2 solution. The albumin digestion rate of each protease was determined from the change in absorbance before and after digestion of the GA-digested sample.

[0193] Figure 26 shows the digestibility at heating temperatures of 37°C and 60°C. At 37°C, Orientase 22BF and Protease XIV showed relatively high digestibility. At 60°C, Orientase 22BF, Protease XIV, Sumizyme MP, and Thermolysin showed relatively high digestibility. Orientase 22BF, Protease XIV, Sumizyme MP, and Thermolysin are all bacterial proteases. One interpretation is that bacterial proteases have higher digestibility than animal- and plant-derived proteases, or alternatively, that animal- and plant-derived proteases have lower digestibility than bacterial proteases. This interpretation represents one idea, and other theories or experimental results may be supported. Based on these experimental results, in some embodiments, a protease selected from the group consisting of Orientase 22BF, Protease XIV, Sumizyme MP, and Thermolysin may be used to digest albumin. Alternatively, a combination of two or more of these proteases may be used.

[0194] The current value of the GA digested sample was measured using an FAOD electrode, as shown in Figure 27. The net output from the digested GA is shown as the difference between the output from the digested GA sample and the output from the protease autolysis sample. As shown in Figure 27, Orientase 22BF, Protease XIV, and Smithyme MP gave relatively high outputs. One interpretation is that Orientase 22BF, Protease XIV, and Smithyme MP are preferred proteases for digesting albumin to glycated peptide fragments of a size that can be reacted with FAOD. This interpretation represents one idea, and other theories or experimental results may be supported.

[0195] <Example 2> The operating temperature of the protease was investigated. 16 mg of Orientase 22BF (manufactured by HIVI Corporation) was dissolved in 400 μL of TES buffer and dialyzed overnight to prepare a protease solution.

[0196] 12 μL of GA-L calibrator (GA concentration: 14.8 mg / mL, albumin concentration: 47.5 mg / mL) from the Glycoalbumin Kit Lucica GA-L (Asahi Kasei Pharma Corporation) was mixed with 93 μL of TES buffer, and 15 μL of protease solution was added. At the same time, sample solutions containing only protease and only calibrator were also prepared. These were kept in an aluminum block incubator at temperatures of 20°C, 30°C, 40°C, 50°C, 60°C, and 70°C for 2.5 minutes or 10 minutes. Afterwards, each solution was removed from the aluminum block and immediately placed in a refrigerator to prevent residual heat or digestion from progressing at room temperature. They were then stored at low temperatures.

[0197] The output current of the FAOD electrode was measured when the GA digestion sample was introduced. The difference between the output current of the protease-only sample solution (protease autolysis sample) and the output current of the GA-only sample solution (GA digestion sample) was calculated from the output current of the GA digestion sample. This difference was calculated as follows: [output current of the GA digestion sample] - [output current of the protease-only sample solution] - [output current of the GA-only sample solution]. Figure 28 plots the output current of the net digested GA as a function of the digestion temperature (digestion temperature) (a).

[0198] As shown in FIG. 28, the output current value from the FAOD electrode was highest near 60°C or between 60°C and 70°C. In some embodiments, the digestion temperature of the protease may be set in a range where the output current value from the FAOD electrode is high. For example, the digestion temperature of the protease may be set near 60°C or between 60°C and 70°C, where the output current value from the FAOD electrode is high in FIG. 28. The digestion temperature of the protease may be higher than 50°C, 55°C, or 60°C. The digestion temperature of the protease may be lower than or equal to the inactivation temperature of the protease. In some embodiments, the digestion temperature of the protease may be different from the optimal temperature range of the FAOD.

[0199] Example 3 The working temperature of ketoamine oxidase (FAOD) was investigated. N-ε-(1-deoxyfructosyl)-L-lysine (FK) trifluoroacetate (Peptide Institute, Inc.) was dissolved in TES buffer to prepare a 12.8 μM FK solution. The output current from the FAOD electrode upon introduction of this FK solution was measured at temperatures of 10°C, 20°C, 30°C, 40°C, 50°C, and 60°C (Figure 28(b)).

[0200] As shown in Figure 28, the output current value from the FAOD electrode was highest near 40°C. One interpretation is that the optimum temperature range for the FAOD electrode is near 40°C. The temperature of the FAOD electrode or its vicinity may be set to near 40°C. In some embodiments, the temperature of the FAOD electrode or its vicinity may be different from the optimum temperature or temperature range for the protease.

[0201] Example 4 The dependence of the output current of the FAOD electrode on the GA concentration was investigated. Albumin powders with different GA values ​​were dissolved in TES buffer to prepare GA sample solutions. 4 mg of Protease XIV (Sigma-Aldrich Japan LLC) and 16 mg of Orientase 22BF (manufactured by HIVI Corporation) were each dissolved in 400 μL of TES buffer, and the solution was dialyzed overnight to prepare a protease solution.

[0202] 40 μL of latex bead (IMMUTEX P0113, Cosmo Bio Co., Ltd.) dispersion was mixed with 400 μL of TES buffer, and 50 mg of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM, Tokyo Chemical Industry Co., Ltd.) was added and dissolved. The solution was stirred at room temperature for 2 hours. The mixture was centrifuged (12,000 rpm, 20 minutes) and the supernatant was removed. 400 μL of TES buffer was added, and the solution was stirred. This procedure was repeated three times to wash the beads and remove excess DMT-MM. After the third removal of the supernatant, 400 μL of dialyzed protease solution was added, and the solution was stirred in a refrigerator for 4 hours. 400 μL of TES buffer was added, centrifuged (12,000 rpm, 20 minutes), and the supernatant was removed. 800 μL of TES buffer was added and the solution was stirred. The same procedure was repeated three times to wash the beads and remove excess protease. After removing the supernatant for the third time, 200 μL of TES buffer was added and the solution was stored in the refrigerator.

[0203] 60 μL of 1.67 mg / mL GA sample solution and 20 μL of protease-immobilized bead solution were mixed thoroughly. This mixture was heated at 37°C for 30 minutes in an aluminum block incubator. After 30 minutes, 40 μL of TES buffer was added, and the mixture was centrifuged (12,000 rpm, 30 minutes). The supernatant was then collected. This solution was used as the digested GA sample.

[0204] A digested GA sample solution was introduced and the output current from the FAOD electrode was measured. Figure 29 shows this output current as a function of GA concentration. As shown in Figure 29, although the intercept value differed depending on the protease, there was a linear relationship between the GA concentration and the output current from the FAOD electrode using the digested GA sample solution. Furthermore, when a GA solution was digested using protease immobilized on beads, the output current from the FAOD electrode using the digested solution was found to vary depending on the GA concentration in the system. This demonstrates that GA concentration can be measured using immobilized protease and an FAOD electrode.

[0205] 6.Measurement method Hereinafter, several embodiments of the measurement method according to the present disclosure will be described.

[0206] A test solution containing glycated proteins is introduced into the sensor. The immobilized protease comes into contact with the test solution containing glycated proteins and fragments the glycated proteins to generate peptide fragments. After generation, the peptide fragments diffuse to the immobilized ketoamine oxidase. The immobilized ketoamine oxidase generates hydrogen peroxide from the peptide fragments containing glycated amino acid residues. The hydrogen peroxide detector detects the hydrogen peroxide generated by the ketoamine oxidase.

[0207] In some embodiments, the glycated protein measured may be glycated albumin.

[0208] In some embodiments, a glycated protein sensor including an immobilized protease, an immobilized ketoamine oxidase, and a hydrogen peroxide detector is first prepared. A measurement solution containing glycated proteins is then prepared. The measurement solution is then introduced into the glycated protein sensor. The glycated protein in the measurement solution is detected using the glycated protein sensor.

[0209] In some embodiments, the concentration of the glycated protein may be determined from the output signal of the hydrogen peroxide detector. Detecting the glycated protein in the measurement solution may include determining or calculating the concentration of the glycated protein in the measurement solution.

[0210] The concentration of glycated protein associated with the output signal from the hydrogen peroxide detector may be determined. The association, correlation, function, etc. between the concentration of glycated protein in the calibration solution and the output signal from the hydrogen peroxide detector may be determined in advance or before the intended measurement. The concentration of glycated protein in the test solution may be determined from the output signal from the hydrogen peroxide detector obtained from the test solution using or with reference to the reference data, reference table, association, correlation, function, etc. obtained in this manner. For example, if the detector measures current, a calculation may be performed to determine the concentration of glycated protein from the current value measured by the electrical circuit based on a table (calibration line) correlating the current value with the concentration of glycated protein. The correlation may be a calculation to determine the difference between a concentration conversion value based on the current value from the main (first) sensor and a concentration conversion value based on the current value from the sub (second) sensor.

[0211] In some embodiments, glycated proteins may be detected, the presence of glycated proteins may be detected when the glycated proteins are at or above a certain threshold, the concentration of glycated proteins may be determined, the glycated proteins may be quantified, or the degree of glycosylation of proteins may be determined.

[0212] In some embodiments, the total amount of the glycated protein (glycated protein) to be measured in the measurement solution and its corresponding non-glycated protein, or the concentration of the total protein in the solution, may be determined. The glycated protein may be glycated albumin.

[0213] In some embodiments, the ratio or proportion of the concentration of glycated protein (glycated portion of target protein) to the concentration of the total amount of target protein may be determined. In some embodiments, this ratio may be further converted to determine the average blood glucose level for the period from the previous measurement to the current measurement. In some embodiments, the ratio determined from two consecutive measurements may be converted to determine the average blood glucose level for the corresponding period. In some embodiments, the ratio obtained from multiple measurements may be used to determine the average blood glucose level for the corresponding period. In some embodiments, the average blood glucose level may be determined by dividing the sum of the ratios obtained from multiple measurements by the number of measurements. In some embodiments, the average blood glucose level may be determined as a weighted function of the ratio obtained from each measurement, and as an example, the weight may be set greater for the ratio obtained in a more recent measurement.

[0214] In some embodiments, the user may be notified, for example by a warning, that the glycated protein concentration has been measured.

[0215] A user may be a person who actually uses a glycated protein sensor, or may refer to a person who is measuring glycated proteins of themselves or others, a person who plans to measure them, or a person who is required or recommended to measure them. A user may be an individual, one or more people, or at least one person. A user may also be one, multiple, or at least one organization such as a corporation or company.

[0216] Notification to the user may be made at a predetermined or appropriate timing. For measurements after the first one, notification may be made at regular or irregular intervals. The interval between any two measurements may be 3 days, 5 days, 6 days, 7 days, 10 days, 15 days, 20 days, 30 days, 40 days, 50 days, 60 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or other intervals. For measurements of glycated albumin, notification may be made at intervals of 7 days, 10 days, 15 days, 20 days, 1 week, 2 weeks, 3 weeks, etc.

[0217] In some embodiments, washing may be performed between measurements, and washing may involve flushing all or part of the immobilized protease, immobilized ketoamine oxidase, and detection unit with a buffer solution such as pure water, saline, or TES.

[0218] A program or computer program according to some embodiments of the present disclosure may cause a computer to execute each step included in the measurement method described herein, or may be read and executed by a computer. A computer-readable storage medium according to some embodiments of the present disclosure may store a program or computer program for causing a computer to execute each step included in the measurement method described herein.

[0219] A01 an immobilized protease; an immobilized ketoamine oxidase; a hydrogen peroxide detection unit; A glycated protein sensor comprising: A01a the immobilized ketoamine oxidase is positioned adjacent to the hydrogen peroxide detection unit; A glycated protein sensor according to embodiment A01. A01b the immobilized protease is positioned adjacent to the immobilized ketoamine oxidase; A glycated protein sensor according to embodiment A01a. A01c Further provided with a liquid storage section that stores a liquid to be measured, A glycated protein sensor according to any one of embodiments A01 to A01b. A01d The volume of the liquid storage portion is 100 μL or less. A glycated protein sensor according to embodiment A01c. A02 a hydrogen peroxide detection unit; an enzyme layer disposed on the hydrogen peroxide detection unit and including an immobilized protease and an immobilized ketoamine oxidase; A glycated protein sensor comprising: A03 the enzyme layer has a protease layer containing an immobilized protease and a ketoamine oxidase layer containing an immobilized ketoamine oxidase stacked together; A glycated protein sensor according to embodiment A02. A04 the ketoamine oxidase layer and the protease layer are laminated in this order from the side closer to the hydrogen peroxide detection unit; A glycated protein sensor according to embodiment A03. A05 the ketoamine oxidase layer comprises a first ketoamine oxidase layer and a second ketoamine oxidase layer; the first ketoamine oxidase layer, the protease layer, and the second ketoamine oxidase layer are laminated in this order; A glycated protein sensor according to embodiment A03. A06 the enzyme layer comprises a ketoamine oxidase layer containing immobilized ketoamine oxidase, and a protease immobilized on a surface of the ketoamine oxidase layer opposite to the hydrogen peroxide detection section; A glycated protein sensor according to embodiment A02. A07 The hydrogen peroxide detector further includes an ion exchange resin disposed between the hydrogen peroxide detector and the enzyme layer. A glycated protein sensor according to any one of embodiments A02 to A06. A08 the immobilized protease is spaced apart from the immobilized ketoamine oxidase; A glycated protein sensor according to embodiment A01. A09 a protease storage section that stores the immobilized protease; a ketoamine oxidase reservoir containing the immobilized ketoamine oxidase and in fluid communication with the protease reservoir; a liquid delivery mechanism for delivering a liquid from the protease storage section to the ketoamine oxidase storage section; Equipped with A glycated protein sensor described in embodiment A08. A10 further comprising a heater for heating the protease and / or the ketoamine oxidase; A glycated protein sensor according to embodiment A08 or A09. A11 Further provided is a protease heater for heating the protease storage section. A glycated protein sensor according to embodiment A08 or A09. A12 The protease heater is configured to heat the protease storage section to 40°C or higher or 50°C or higher. A glycated protein sensor according to embodiment A11. A13 Further provided is a protease temperature sensor for measuring the temperature of the protease storage section or the protease. A glycated protein sensor according to embodiment A11 or A12. A14 further comprising a temperature control mechanism that controls the heat generation amount of the protease heater based on information from the protease temperature sensor; A glycated protein sensor according to any one of embodiments A11 to A13. A15 the enzyme-containing apparatus further includes a ketoamine oxidase heater for heating the ketoamine oxidase-containing section; A glycated protein sensor according to any one of embodiments 11 to A14. A16 the ketoamine oxidase heater is configured to heat the ketoamine oxidase containing section to a temperature of not less than room temperature and not more than 50°C; A glycated protein sensor according to embodiment A15. A17 a temperature sensor for ketoamine oxidase for measuring the temperature of the ketoamine oxidase-containing section or the ketoamine oxidase; A glycated protein sensor according to embodiment A15 or A16. A18 a temperature control mechanism for controlling the amount of heat generated by the ketoamine oxidase heater based on information from the ketoamine oxidase temperature sensor; A glycated protein sensor according to any one of embodiments A15 to A17. A19 a cooling unit disposed between the protease-accommodating unit and the ketoamine oxidase-accommodating unit and fluidly connected to both the protease-accommodating unit and the ketoamine oxidase-accommodating unit, the cooling unit having a cooling mechanism; A glycated protein sensor according to any one of embodiments A11 to A18. A21 From the upstream direction of the solution containing the test substance, the immobilized protease, the immobilized ketoamine oxidase; the hydrogen peroxide detection unit; Arranged in the order of A glycated protein sensor according to embodiment 1. A22 The protease is immobilized on a substrate. A glycated protein sensor according to any one of embodiments A01 to A21. A23 The protease is immobilized on beads. A glycated protein sensor according to embodiment A22. A24 The hydrogen peroxide detection unit includes a hydrogen peroxide electrode. A glycated protein sensor according to any one of embodiments A01 to A23. A25 The hydrogen peroxide detector includes a photodetector. A glycated protein sensor according to any one of embodiments A01 to A23. A26 The hydrogen peroxide detector includes a luminescent reagent that reacts with hydrogen peroxide and a photodetector. A glycated protein sensor according to any one of embodiments A01 to A23. A27 a first immobilized protease; and a first immobilized ketoamine oxidase; a first hydrogen peroxide detection unit; a first sensor comprising: a second immobilized ketoamine oxidase; and a second hydrogen peroxide detection unit; a second sensor comprising: A glycated protein sensor comprising: A28 a first hydrogen peroxide detection unit; a first enzyme layer disposed on the first hydrogen peroxide detection portion and including an immobilized protease and an immobilized ketoamine oxidase; a second hydrogen peroxide detection unit; a second enzyme layer disposed on the second hydrogen peroxide detection element and containing an enzyme consisting essentially of the immobilized ketoamine oxidase; A glycated protein sensor comprising: A29 The first hydrogen peroxide detection unit and the second hydrogen peroxide detection unit include a hydrogen peroxide electrode. A glycated protein sensor according to embodiment A28. A30 the first hydrogen peroxide detection unit includes a first working electrode and one of a counter electrode and a reference electrode; the second hydrogen peroxide detection unit includes a second working electrode and the other of the counter electrode and the reference electrode; A glycated protein sensor according to embodiment A29 A31 The device further includes a current measuring circuit that applies a potential to the counter electrode and measures a current flowing through the first working electrode and a current flowing through the second working electrode. A glycated protein sensor according to embodiment A29 or A30. A32 The glycated protein includes glycated albumin. A glycated protein sensor according to any one of embodiments A01 to A31. A33 Further comprising an albumin sensor. A glycated protein sensor according to embodiment A32. B01 Providing a measurement solution containing glycated proteins; directing the test solution to the immobilized protease; fragmenting the glycated protein using the immobilized protease to generate peptide fragments; generating hydrogen peroxide from a peptide fragment containing a glycated amino acid residue among the peptide fragments using an immobilized ketoamine oxidase; detecting the hydrogen peroxide generated by the ketoamine oxidase using a hydrogen peroxide detector; A method for measuring glycated proteins, comprising: B02 an immobilized protease; an immobilized ketoamine oxidase; a hydrogen peroxide detection unit; providing a glycated protein sensor comprising: Providing a measurement solution containing glycated proteins; introducing the measurement solution into the glycated protein sensor; detecting the glycated protein in the measurement solution using the glycated protein sensor; A method for measuring glycated proteins, comprising: B03 and determining a concentration of glycated protein associated with the output signal from the hydrogen peroxide detection unit. A method for measuring glycated proteins according to embodiment B02. B04 the glycated protein is glycated albumin or glycated hemoglobin, The method comprises: determining the concentration of albumin or the concentration of hemoglobin; determining a glycated albumin value or a glycated hemoglobin value, which is a ratio of the glycated albumin concentration or the glycated hemoglobin concentration to the albumin concentration or the hemoglobin concentration; Further comprising: The method for measuring glycated proteins according to embodiment B02 or B03. B05 notifying the user to measure the glycated protein concentration every week, every two weeks, every three weeks, every four weeks, or every month; Further comprising: A method for measuring glycated proteins according to any one of embodiments B02 to B04. B06 The glycated protein includes glycated albumin. A method for measuring glycated proteins according to any one of embodiments B02 to B05. B07 The method further comprises calculating an average blood glucose level from a ratio of the albumin concentration or the hemoglobin concentration measured multiple times. A method for measuring glycated proteins according to embodiment B06. B08 A program for causing a computer to execute each step included in the measurement method according to any one of embodiments B02 to B07. B09 A computer-readable storage medium storing the program described in embodiment B07. B10 a first hydrogen peroxide detection unit; a first enzyme layer disposed on the first hydrogen peroxide detection portion and including an immobilized protease and an immobilized ketoamine oxidase; a second enzyme layer disposed on the second hydrogen peroxide detection element and containing an enzyme consisting essentially of the immobilized protease; providing a glycated protein sensor comprising: introducing a measurement solution containing a glycated protein into the glycated protein sensor; determining, from the output signal from the first hydrogen peroxide detection unit and the output signal from the second hydrogen peroxide detection unit, the concentration of a glycated protein associated with these output signals; A method for measuring glycated proteins, comprising: C01 Preparing the substrate; disposing a hydrogen peroxide detection unit on the substrate; immobilizing ketoamine oxidase on the substrate; Immobilizing a protease on the substrate; A method for manufacturing a glycated protein sensor, comprising: C02 immobilizing ketoamine oxidase on the substrate includes immobilizing ketoamine oxidase on the hydrogen peroxide detection unit; Immobilizing a protease on the substrate includes immobilizing a protease on the hydrogen peroxide detection unit. A method for producing a glycated protein sensor according to embodiment C01. C03 Immobilizing ketoamine oxidase on the hydrogen peroxide detection unit includes forming a ketoamine oxidase layer on a first substrate to which ketoamine oxidase is immobilized; immobilizing a protease on the hydrogen peroxide detection unit includes forming a protease layer on the ketoamine oxidase layer, the protease being immobilized on a second substrate; The manufacturing method described in embodiment C02. C04 and immobilizing ketoamine oxidase on the hydrogen peroxide detection unit further comprises forming a second ketoamine oxidase layer on the protease layer, the second ketoamine oxidase layer having ketoamine oxidase immobilized on a third substrate. The manufacturing method described in embodiment C03. C05 immobilizing ketoamine oxidase on the hydrogen peroxide detection section and immobilizing protease on the hydrogen peroxide section includes mixing the ketoamine oxidase and the protease with a common base material to form a uniform enzyme layer on the hydrogen peroxide section; The manufacturing method described in embodiment C02. C06 Preparing the substrate; forming a first hydrogen peroxide detection unit and a second hydrogen peroxide detection unit on the substrate; immobilizing a first ketoamine oxidase and a protease on the first hydrogen peroxide detection unit; immobilizing a second ketoamine oxidase on the second hydrogen peroxide detection unit; Equipped with A method for manufacturing a glycated protein sensor. C07 providing the substrate comprises providing a first substrate and a second substrate; forming a first hydrogen peroxide detector and a second hydrogen peroxide detector on the substrate comprises forming a first hydrogen peroxide detector on the first substrate and a second hydrogen peroxide detector on the second substrate; Prepare the main board, Bonding the first substrate and the second substrate onto the main substrate; Further comprising: The manufacturing method described in embodiment C06.

[0220] Several embodiments and examples of the present disclosure have been described above, but these embodiments and examples exemplify the present disclosure. For example, the above embodiments have been described in detail to clearly explain the present disclosure, and additional changes in dimensions, configurations, materials, and circuits may be made as necessary. The claims encompass numerous modifications to the embodiments without departing from the technical spirit of the present disclosure. Therefore, the embodiments and examples disclosed herein are presented for illustrative purposes only and should not be considered to limit the scope of the present disclosure. [Explanation of symbols]

[0221] 100 sensors 101 Protease 102 Ketoamine oxidase 103 Hydrogen peroxide detector 104 Fixed layer 105 Base material 106 Crosslinking Agent 107 Silane coupling agents 111 Enzyme Layer 151 Glycated Proteins 152 Proteins 153 Sugar 154 Glycated Peptide Fragments 155 Non-glycosylated peptide fragments 156 Hydrogen Peroxide 200 sensors 201 Protease 202 Ketoamine oxidase 203 Hydrogen peroxide detector 204 Base material 211 Enzyme Layer 300 sensors 301 Protease 302 Ketoamine oxidase 303 Hydrogen peroxide detector 304 Base material 305 Bonding agent 311 Enzyme Layer 400 sensors 401 Protease 402 Ketoamine oxidase 403 Hydrogen peroxide detector 404,414 Base material 411 Protease layer 412 Ketoamine oxidase layer 500 sensors 501 Protease 502 Ketoamine oxidase 503 Hydrogen peroxide detector 506 board 504,514 Base material 511 Immobilized protease part 512 Immobilized ketoamine oxidase part 600 sensors 601 Protease 602 Ketoamine oxidase 603 Hydrogen peroxide detector 604 Crosslinking Agent 612 Ketoamine oxidase layer 614 Base material 700 Sensors 701 Protease 702 Ketoamine oxidase 704,714 Base material 705 Silane coupling agents 706 Insulating substrate 711 Protease layer 712 Ketoamine oxidase layer 730 Hydrogen Peroxide Electrode 731 Opposite 732 Reference pole 733 Working electrode 770 Electrical Circuits 771 Operational Amplifier 772 Voltage Generator Circuit 773 Current measurement circuit 800 sensors 801 Protease 802 Ketoamine oxidase 804,814 Base material 807 Ion exchange resin 811 Protease layer 812 Ketoamine oxidase layer 1000 sensors 1000a main sensor 1000b sub-sensor 1001 Protease 1002 Ketoamine oxidase 1003a, 1003b Hydrogen peroxide detection unit 1004,1014 Base material 1011a,1011b Enzyme layer 1030 Arithmetic unit 1070a, 1070b Electrical circuit 1100 Sensor 1100a main sensor 1100b sub sensor 1101 Protease 1102 Ketoamine oxidase 1103a, 1103b Hydrogen peroxide detection unit 1104,1114,1124 Base material 1111a,1111b Protease layer 1112a Ketoamine oxidase layer 1011a,1011b Enzyme layer 1130 Arithmetic unit 1170a, 1170b Electrical circuit 1200 Sensors 1200a main sensor 1200b sub sensor 1201 Protease 1202 Ketoamine oxidase 1203a, 1203b Hydrogen peroxide detection unit 1204 Crosslinking agent 1214,1224 Base material 1212a Ketoamine oxidase layer 1212b Enzyme layer 1230 Arithmetic unit 1270a, 1270b Electrical Circuit 1300 Sensors 1300a Main Sensor 1300b sub sensor 1301 Protease 1302 Ketoamine oxidase 1304,1324 Base material 1305a, 1305b Silane coupling agent 1306 PCB 1311a,1311b enzyme layer 1330a, 1330b Hydrogen peroxide electrode 1331 Opposite 1332 Reference pole 1333a,1333b Working electrode 1370 Electrical Circuits 1371 operational amplifier 1372 Voltage Generator Circuit 1373a,1373b Current measurement circuit 1400 Sensors 1401 Protease 1402 Ketoamine oxidase 1404 beads 1414 Base material 1431 Reagents 1432 Photodetector 1452 Liquid inlet 1455 Protease storage section 1456 Ketoamine oxidase housing 1457 Detection Reactor 1460 Heater 1500 sensors 1502 Ketoamine oxidase 1504 beads 1600 Sensors 1601 Protease 1602 Ketoamine oxidase 1603 Hydrogen Peroxide Detector 1604 beads 1611 Enzyme layer 1614 Base material 1652 Liquid inlet 1655 Protease storage section 1656 Ketoamine oxidase housing 1660 Heater 1700 Sensors 1700a main sensor 1700b sub sensor 1703a, 1703b Hydrogen peroxide detector 1711a Enzyme layer 1712a, 1712b Ketoamine oxidase layer 1752 Liquid inlet 1755a Protease storage section 1755b Protease-free chamber 1756a, 1756b Ketoamine oxidase container 1760 heater 1800 sensors 1801 Protease 1802 Ketoamine oxidase 1803 Hydrogen Peroxide Detector 1804 beads 1811 Enzyme layer 1814 Base material 1852 Liquid inlet 1855 Protease storage section 1856 Ketoamine oxidase housing 1858 Cooling unit / cooling solution storage unit 1860,1861 heater 1862 Cooling circuit / element 2000 sensor chips 2006 PCB 2011 Enzyme Layer 2031 Opposite 2032 Reference pole 2033 Working electrode 2034 Liquid detection electrode 2041, 2042, 2043, 2044 output terminals 2051 Liquid storage section 2052 Fluid inlet 2053 Air vent 2100 sensor chip 2100a, 2100b Sensor 2134 Liquid detection electrode 2151 Liquid storage section 2152 Fluid inlet 2153 Air vent 2200 sensor chip 2200a Main Sensor 2200b Sub-sensor 2206 board 2221a,2221b Enzyme layer 2231 Opposite 2232 Reference pole 2233a,2233b Working electrode 2300 sensor chip 2300a, 2300b sensors 2311a,2311b Enzyme layer 2330a, 2330b Hydrogen peroxide electrode 2340 output terminal 2340a, 2340b, 2341a, 2341b Bonding Pads 2351 Liquid storage section 2352 Fluid inlet 2353 Air vent 2360 board 2400 sensor chip 2406 board 2411 Enzyme layer 2431 Opposite 2432 Reference pole 2433 Working electrode 2434 Liquid detection electrode 2451 Liquid storage unit 2452 Fluid inlet 2453 Air vent 3000 sensors 3000a main sensor 3000b sub-sensor 3001 Protease 3002 Ketoamine oxidase 3004,3014 Base material (BSA) 3005a, 3005b Silane coupling agent 3006 Insulating substrate 3009,3091 Resist 3011a,3011b Enzyme layer 3030a, 3030b Hydrogen peroxide electrode 4002,4102 Ketoamine oxidase 4003,4103 Hydrogen peroxide detector 4006,4106 board 4008,4108 protection 4012,4112 Ketoamine oxidase layer 4014,4114 Base material

Claims

1. an immobilized protease; an immobilized ketoamine oxidase; a hydrogen peroxide detection unit; A glycated protein sensor comprising:

2. the immobilized ketoamine oxidase is positioned adjacent to the hydrogen peroxide detection unit; The glycated protein sensor according to claim 1 .

3. the immobilized protease is positioned adjacent to the immobilized ketoamine oxidase; The glycated protein sensor according to claim 2 .

4. Further provided with a liquid storage section that stores a liquid to be measured, The glycated protein sensor according to claim 1 .

5. The volume of the liquid storage portion is 100 μL or less. The glycated protein sensor according to claim 4 .

6. a hydrogen peroxide detection unit; an enzyme layer disposed on the hydrogen peroxide detection unit and including an immobilized protease and an immobilized ketoamine oxidase; A glycated protein sensor comprising:

7. the enzyme layer has a protease layer containing an immobilized protease and a ketoamine oxidase layer containing an immobilized ketoamine oxidase stacked together; The glycated protein sensor according to claim 6 .

8. the ketoamine oxidase layer and the protease layer are laminated in this order from the side closer to the hydrogen peroxide detection unit; The glycated protein sensor according to claim 7 .

9. the ketoamine oxidase layer comprises a first ketoamine oxidase layer and a second ketoamine oxidase layer; the first ketoamine oxidase layer, the protease layer, and the second ketoamine oxidase layer are laminated in this order; The glycated protein sensor according to claim 7 .

10. the enzyme layer comprises a ketoamine oxidase layer containing immobilized ketoamine oxidase, and a protease immobilized on a surface of the ketoamine oxidase layer opposite to the hydrogen peroxide detection section; The glycated protein sensor according to claim 6 .

11. The hydrogen peroxide detector further includes an ion exchange resin disposed between the hydrogen peroxide detector and the enzyme layer. The glycated protein sensor according to any one of claims 6 to 10.

12. the immobilized protease is spaced apart from the immobilized ketoamine oxidase; The glycated protein sensor according to claim 1 .

13. a protease storage section that stores the immobilized protease; a ketoamine oxidase reservoir containing the immobilized ketoamine oxidase and in fluid communication with the protease reservoir; a liquid delivery mechanism for delivering a liquid from the protease storage section to the ketoamine oxidase storage section; Equipped with The glycated protein sensor according to claim 12.

14. further comprising a heater for heating the protease section and / or the ketoamine oxidase section; The glycated protein sensor according to claim 12 or 13.

15. Further provided is a protease heater for heating the protease storage section. The glycated protein sensor according to claim 12 or 13.

16. The protease heater is configured to heat the protease storage section to 40°C or higher. The glycated protein sensor according to claim 15.

17. Further provided is a protease temperature sensor for measuring the temperature of the protease storage section or the protease. The glycated protein sensor according to claim 15 or 16.

18. further comprising a temperature control mechanism that controls the heat generation amount of the protease heater based on information from the protease temperature sensor; The glycated protein sensor according to any one of claims 15 to 17.

19. the enzyme-containing apparatus further includes a ketoamine oxidase heater for heating the ketoamine oxidase-containing section; The glycated protein sensor according to any one of claims 15 to 18.

20. the ketoamine oxidase heater is configured to heat the ketoamine oxidase containing section to a temperature of 40°C or less; The glycated protein sensor according to claim 19.

21. a temperature sensor for ketoamine oxidase for measuring the temperature of the ketoamine oxidase-containing section or the ketoamine oxidase; The glycated protein sensor according to claim 19 or 20.

22. a temperature control mechanism for controlling the amount of heat generated by the ketoamine oxidase heater based on information from the ketoamine oxidase temperature sensor; The glycated protein sensor according to any one of claims 19 to 21.

23. a cooling unit disposed between the protease-accommodating unit and the ketoamine oxidase-accommodating unit and fluidly connected to both the protease-accommodating unit and the ketoamine oxidase-accommodating unit, the cooling unit having a cooling mechanism; The glycated protein sensor according to any one of claims 15 to 22.

24. From the upstream direction of the solution containing the test substance, the immobilized protease, the immobilized ketoamine oxidase; the hydrogen peroxide detection unit; Arranged in the order of The glycated protein sensor according to claim 1 .

25. The protease is immobilized on a substrate. The glycated protein sensor according to claim 24.

26. The protease is immobilized on beads. The glycated protein sensor according to claim 25.

27. The hydrogen peroxide detection unit includes a hydrogen peroxide electrode. A glycated protein sensor according to any one of claims 1 to 26.

28. The hydrogen peroxide detector includes a photodetector. A glycated protein sensor according to any one of claims 1 to 26.

29. The hydrogen peroxide detector includes a luminescent reagent that reacts with hydrogen peroxide and a photodetector. A glycated protein sensor according to any one of claims 1 to 26.

30. a first immobilized protease; and a first immobilized ketoamine oxidase; a first hydrogen peroxide detection unit; a first sensor comprising: a second immobilized ketoamine oxidase; and a second hydrogen peroxide detection unit; a second sensor comprising: A glycated protein sensor comprising:

31. a first hydrogen peroxide detection unit; a first enzyme layer disposed on the first hydrogen peroxide detection portion and including an immobilized protease and an immobilized ketoamine oxidase; a second hydrogen peroxide detection unit; a second enzyme layer disposed on the second hydrogen peroxide detection element and containing an enzyme consisting essentially of the immobilized ketoamine oxidase; A glycated protein sensor comprising:

32. The first hydrogen peroxide detection unit and the second hydrogen peroxide detection unit include a hydrogen peroxide electrode. The glycated protein sensor according to claim 31.

33. the first hydrogen peroxide detection unit includes a first working electrode and one of a counter electrode and a reference electrode; the second hydrogen peroxide detection unit includes a second working electrode and the other of the counter electrode and the reference electrode; The glycated protein sensor according to claim 32.

34. The device further includes a current measuring circuit that applies a potential to the counter electrode and measures a current flowing through the first working electrode and a current flowing through the second working electrode. The glycated protein sensor according to claim 32 or 33.

35. The glycated protein includes glycated albumin. A glycated protein sensor according to any one of claims 1 to 34.

36. Further comprising an albumin sensor. The glycated protein sensor according to claim 35.

37. preparing a measurement solution containing glycated proteins; introducing the test solution into an immobilized protease; fragmenting the glycated protein using the immobilized protease to generate peptide fragments; generating hydrogen peroxide from a peptide fragment containing a glycated amino acid residue among the peptide fragments using an immobilized ketoamine oxidase; detecting the hydrogen peroxide generated by the ketoamine oxidase using a hydrogen peroxide detector; A method for measuring glycated proteins, comprising:

38. an immobilized protease; an immobilized ketoamine oxidase; a hydrogen peroxide detection unit; providing a glycated protein sensor comprising: preparing a measurement solution containing glycated proteins; introducing the measurement solution into the glycated protein sensor; detecting the glycated protein in the measurement solution using the glycated protein sensor; A method for measuring glycated proteins, comprising:

39. and determining a concentration of glycated protein associated with the output signal from the hydrogen peroxide detection unit. The method for measuring glycated proteins according to claim 38.

40. the glycated protein is glycated albumin or glycated hemoglobin, The method comprises: determining the concentration of albumin or the concentration of hemoglobin; determining a glycated albumin value or a glycated hemoglobin value, which is a ratio of the glycated albumin concentration or the glycated hemoglobin concentration to the albumin concentration or the hemoglobin concentration; Further comprising: A method for measuring a glycated protein according to claim 38 or 39.

41. notifying the user to measure the glycated protein concentration every week, every two weeks, every three weeks, every four weeks, or every month; Further comprising: A method for measuring a glycated protein according to any one of claims 38 to 40.

42. determining the albumin concentration or hemoglobin concentration; determining a glycated albumin value or a glycated hemoglobin value, which is a ratio of the glycated albumin concentration or the glycated hemoglobin concentration to the albumin concentration or the hemoglobin concentration; The method further comprises repeatedly performing A method for measuring a glycated protein according to any one of claims 38 to 41.

43. The method further comprises calculating an average blood glucose level from a ratio of the albumin concentration or the hemoglobin concentration measured multiple times. The method for measuring glycated proteins according to claim 42.

44. A program for causing a computer to execute each step included in the measurement method according to any one of claims 38 to 41.

45. The glycated protein includes glycated albumin. A method for measuring a glycated protein according to any one of claims 41 to 44.

46. a first hydrogen peroxide detection unit; a first enzyme layer disposed on the first hydrogen peroxide detection portion and including an immobilized protease and an immobilized ketoamine oxidase; a second enzyme layer disposed on the second hydrogen peroxide detection element and containing an enzyme consisting essentially of the immobilized ketoamine oxidase; providing a glycated protein sensor comprising: introducing a measurement solution containing a glycated protein into the glycated protein sensor; determining, from the output signal from the first hydrogen peroxide detection unit and the output signal from the second hydrogen peroxide detection unit, the concentration of a glycated protein associated with these output signals; A method for measuring glycated proteins, comprising:

47. Preparing the substrate; disposing a hydrogen peroxide detection unit on the substrate; immobilizing ketoamine oxidase on the substrate; Immobilizing a protease on the substrate; A method for manufacturing a glycated protein sensor, comprising:

48. immobilizing ketoamine oxidase on the substrate includes immobilizing ketoamine oxidase on the hydrogen peroxide detection unit; Immobilizing a protease on the substrate includes immobilizing a protease on the hydrogen peroxide detection unit. The method for producing a glycated protein sensor according to claim 47.

49. Immobilizing ketoamine oxidase on the hydrogen peroxide detection unit includes forming a ketoamine oxidase layer on a first substrate to which ketoamine oxidase is immobilized; immobilizing a protease on the hydrogen peroxide detection unit includes forming a protease layer on the ketoamine oxidase layer, the protease being immobilized on a second substrate; The method of claim 48.

50. and immobilizing ketoamine oxidase on the hydrogen peroxide detection unit further comprises forming a second ketoamine oxidase layer on the protease layer, the second ketoamine oxidase layer having ketoamine oxidase immobilized on a third substrate.

50. The method of claim 49.

51. immobilizing ketoamine oxidase on the hydrogen peroxide detection unit and immobilizing protease on the hydrogen peroxide detection unit include forming a single enzyme layer on the hydrogen peroxide detection unit, in which the ketoamine oxidase and the protease are immobilized on a common substrate; The method of claim 48.

52. Preparing the substrate; forming a first hydrogen peroxide detection unit and a second hydrogen peroxide detection unit on the substrate; immobilizing a first ketoamine oxidase and a protease on the first hydrogen peroxide detection unit; immobilizing a second ketoamine oxidase on the second hydrogen peroxide detection unit; Equipped with A method for manufacturing a glycated protein sensor.

53. providing the substrate comprises providing a first substrate and a second substrate; forming a first hydrogen peroxide detector and a second hydrogen peroxide detector on the substrate comprises forming a first hydrogen peroxide detector on the first substrate and a second hydrogen peroxide detector on the second substrate; Prepare the main board, Bonding the first substrate and the second substrate onto the main substrate; Further comprising:

53. The method of claim 52.

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