Glycated protein sensor

Abstract

Provided is a glycated protein sensor provided with an immobilized protease, an immobilized ketoamine oxidase, and a hydrogen peroxide detection unit.

Description

[0001] This application is a divisional application of a Chinese patent application with an application number of 201980041212.5 (international application number PCT / JP2019 / 019640), an entry date of December 18, 2020 (international filing date May 17, 2019), and an invention title of "Glycated Protein Sensor, Measurement Method, Program, and Method for Manufacturing the Sensor". Technical Field

[0002] The present invention relates to a glycated protein sensor, a measurement method, a program, and a method for manufacturing the sensor. Background Art

[0003] As management indicators for the diagnosis of diabetes and blood glucose control, the measurement of glycated proteins is performed. As an example, glycated hemoglobin and glycated albumin are frequently measured in the clinical field. As methods for measuring glycated proteins, electrophoresis, ion exchange chromatography, affinity chromatography, immunoassay, and enzymatic methods are known. However, accurate, simple, and rapid measurement is desired, and therefore, in recent years, the enzymatic method has become the mainstream.

[0004] In a general enzymatic method for measuring glycated proteins, first, in the first step, a protease is used to decompose proteins into amino acids, and in the second step, a ketamine oxidase acts only on the glycated amino acids among these amino acids to generate hydrogen peroxide. In the third step, the hydrogen peroxide is converted into a color reaction and the absorbance is measured. Summary of the Invention

[0005] In conventional glycated protein measurement methods, decomposition reactions between enzymes occur due to the coexistence of a protease and a ketamine oxidase, making it difficult to perform high-precision measurement. In addition, in conventional glycated protein measurement methods, the number of steps for measurement is large, and thus there are problems that rapid measurement is difficult and measurement errors are likely to occur depending on the technique of the examiner.

[0006] According to one embodiment of the present invention, a glycated protein or fructosamine sensor includes an immobilized protease, an immobilized ketamine oxidase, and a hydrogen peroxide detection unit.

[0007] The test sample can be a solution. The solution can be a body fluid, a solution derived from a body fluid, or a dilution of a body fluid. The solution can be a solution that is not a body fluid (non-body fluid source), or a mixture of a body fluid or a solution derived from a body fluid and a solution from a non-body fluid source. The solution can be a solution used in sample measurement or a solution used in measurement for calibration. For example, the solution can be a standard solution or a calibration solution.

[0008] "Body fluid" can be blood, serum, plasma, lymph fluid, can be interstitial fluid, intercellular fluid, interstitial fluid and other tissue fluids, can be body cavity fluid, serous cavity fluid, pleural effusion, ascites, pericardial fluid, cerebrospinal fluid (medullary fluid), synovial fluid (synovial fluid), aqueous humor (aqueous humor). Body fluid can be digestive juices such as saliva, gastric juice, bile, pancreatic juice, intestinal juice, can be sweat, tears, nasal mucus, urine, semen, vaginal secretion, amniotic fluid, milk. Body fluid can be the body fluid of animals, can be the body fluid of humans. "Body fluid" can be the liquid in foods containing proteins of animal origin (such as milk, dairy products, etc.). Body fluid can be the body fluid of plants, the living body fluid of plants or the liquid of plant origin. For example, body fluid can be the fruit juice, honey juice, sap of plants. "Body fluid" can be a solution.

[0009] In some embodiments, the solution may contain a physiological buffer. The solution may contain the analyte. The buffer can be a so-called buffer known as Good's buffer. The buffer can contain phosphate buffered saline (PBS), N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid buffer (TES). The buffer can contain 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 (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), sodium 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonate (HEPES-Na), acetamide glycine, tris(hydroxymethyl)methylglycine (Tricine), glycine amide, N,N-dihydroxyethylglycine (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]propanesulfonic acid monohydrate (HEPPSO), N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS) and 2-hydroxy-N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPSO), either alone or as a mixture.

[0010] The solution may contain the analyte. For example, the solution may be tear fluid and the analyte may be glycated albumin contained in the tear fluid. Alternatively, the analyte may be albumin, glycated albumin, hemoglobin, glycated hemoglobin in blood or serum, albumin, glycated albumin in interstitial fluid, albumin, glycated albumin in tear fluid, albumin, glycated albumin in urine, or albumin, glycated albumin in saliva.

[0011] In some embodiments, the analyte to be sensed 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 analyte to be sensed may be a glycated lipid.

[0012] "Immobilization" means immobilizing an enzyme (such as a protease or a ketoamine oxidase) on a substrate or a base material. These enzymes can be immobilized on a base material fixed on the substrate. In some embodiments, the enzyme can be directly or indirectly immobilized on the device body, the inner wall of the flow path, the wall surface of the chamber or the housing part. For the purpose of immobilization, the enzyme can be immobilized on the object to be fixed through a component. The enzyme can be immobilized on the object to be fixed through one or more components that are not originally intended for immobilization. In some embodiments, the object to be fixed with the immobilized enzyme can be capable of moving substantially relative to the device body or the like. For example, the enzyme can be immobilized on a microbead, and the microbead can be capable of moving relative to the flow path as the solution moves.

[0013] As immobilization methods, exemplary ones include covalent bonding method, physical adsorption method, ionic bonding method, crosslinking method, encapsulation method, biochemical specific binding method, etc. The immobilization method that does not inactivate the enzyme can be selected according to the enzyme used, or multiple immobilization methods can be used in combination. In some embodiments, a protein can be used as the base material, and after mixing the enzyme therein, a crosslinking agent such as glutaraldehyde is used for curing. Thus, even if an expensive enzyme is used, the cost can be reduced. In some embodiments, a fluorine-based resin, a hydraulic resin, a photocurable resin, a solid polymer electrolyte, or a polyelectrolyte complex can be used for immobilization, a water-insoluble semipermeable membrane such as nylon, ethyl cellulose, cellulose acetate, or polystyrene can be used to enclose the enzyme, and a phospholipid can be used to enclose it in a liposome or a reverse micelle.

[0014] To fix the substrate to the base plate, adhesives such as silane coupling agents can be used. The adhesive can be formed as a layer (silane coupling layer) between the base plate and the substrate. The hydrogen peroxide detection unit and the layer thereon can be joined, for example, via an adhesive. Regarding the adhesive, various adhesives can be used as long as they do not substantially interfere with the measurement principle of the present invention. The adhesive can contain, for example, materials that bond inorganic materials and organic materials. The adhesive can be, for example, a silane coupling agent. The following substances can be illustratively listed as silane coupling agents.

[0015] Vinyl-based: vinyltrimethoxysilane, vinyltriethoxysilane, 7-octenyltrimethoxysilane, vinyldimethylethoxysilane, vinylmethyldimethoxysilane, vinylmethyldiethoxysilane, vinyltris(2-methoxyethoxy)silane, vinyltris(trimethylsiloxy)silane, 4-vinylphenyltrimethoxysilane, allyltrimethoxysilane, allyltriethoxysilane, 5-(triethoxysilyl)-2-norbornene;

[0016] Styryl-based: p-styryltrimethoxysilane;

[0017] Methacrylic acid-based: 3-methacryloxypropyltrimethoxysilane, 3-methacryloxypropylmethyldimethoxysilane, 3-methacryloxypropyltriethoxysilane, 3-methacryloxypropylmethyldiethoxysilane, 3-methacryloxypropyltris(trimethylsiloxy)silane, 3-methacryloxypropyltriallylsilane, 8-methacryloxyoctyltrimethoxysilane;

[0018] Acrylic acid-based: 3-acryloxypropyltrimethoxysilane, 3-acryloxypropyldimethylmethoxysilane, 3-acryloxypropyltriallylsilane;

[0019] Epoxy-based: 3-glycidoxypropyltrimethoxysilane, 3-glycidoxypropylmethyldimethoxysilane, 3-glycidoxypropylmethyldiethoxysilane, 8-glycidoxyoctyltrimethoxysilane, 2-(3,4-epoxycyclohexyl)ethyltrimethoxysilane;

[0020] Amino group series: 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 (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;

[0021] Urea group series: 3-ureidopropyltrimethoxysilane, 3-ureidopropyltriethoxysilane;

[0022] Azide group series: 11-azidoundecyltrimethoxysilane;

[0023] Isocyanate group series: 3-isocyanatopropyltrimethoxysilane, 3-isocyanatopropyltriethoxysilane;

[0024] Isocyanurate group series: tris-(trimethoxysilylpropyl)isocyanurate;

[0025] Mercapto group series: 3-mercaptopropyltrimethoxysilane, 3-mercaptopropylmethyldimethoxysilane, 3-mercaptopropyltriethoxysilane; or

[0026] Anhydride: 3-trimethoxysilylpropyl succinic anhydride.

[0027] The substrate may contain SiO 2 as the main component, or it may be a glass substrate. The substrate may contain polymers or resins. The substrate may contain transparent polymer materials. For example, the substrate may be composed of resin materials such as polyethylene terephthalate (PET), polyethylene naphthalate (PEN), and 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.

[0028] The main component of the substrate can be a polymer. The main component of the substrate can be a biopolymer, an organic polymer, or an inorganic polymer. For example, the substrate can be a porous inorganic material such as silica gel, glass, alumina, molecular sieve, diatomaceous earth, charcoal, a ceramagnetic substance such as kaolinite, ceramic, hydroxyapatite, a clay 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, carrageenan and other polysaccharides, collagen (animal glue, gelatin), chitin, chitosan, polypeptide, polylysine, or may contain the above substances.

[0029] The main component of the substrate can be a protein. The main component of the substrate can be bovine serum albumin (BSA). In the case where the substrate is composed of a protein, a crosslinking agent is usually used to crosslink the enzyme and the protein of the substrate for immobilization. The protein that is the main component of the substrate and the crosslinking agent may also be collectively referred to as the "substrate". The crosslinking agent can be a photo-curable resin, a water-curable resin, or a heat-curable resin. The crosslinking agent can be a bifunctional reagent such as glutaraldehyde. The crosslinking agent can be formaldehyde, or a bifunctional reagent selected from glyoxal, malonaldehyde, succinaldehyde, and glutaraldehyde.

[0030] The main component of the substrate can be an ionic matrix, or a polyion complex (hereinafter simply referred to as an ion complex). By mixing a polyanion and a polycation in an aqueous solution, a polyion complex is formed by strong electrostatic interaction. The ionic matrix can be electrostatically fixed to the substrate. The ionic matrix can electrostatically enclose the enzyme within the matrix. For example, a complex can be formed by mixing polymers with opposite charges (cation and anion) such as polyamino acids through electrostatic interaction, and the enzyme and the like can be enclosed therein. Since the ionic matrix does not directly bind to the enzyme, a decrease or avoidance of the enzyme activity can be achieved. The ionic matrix can be used for enzymes that are inactivated in the crosslinking method.

[0031] The substrate can be microbeads. The microbeads can be carbon microparticles (carbon microbeads), or silica (SiO 2)Particles (silica microspheres). The microspheres can be polymer microspheres. The material of the microspheres can be high molecular polysaccharides such as chitin, chitosan, and alginic acid. The microspheres can be metal particles, microspheres containing magnetizable substances, and can be magnetic microspheres. Regarding the size of the microspheres, the average particle diameter can be 10 nm or more and can be 200 nm or less. The enzyme can be cross-linked with the microspheres. The microspheres can be directly fixed to a substrate or other base materials, or can be configured in a manner that is not fixed to a substrate or base material, etc., but is accommodated in a specified volume. The microspheres can be mobile with the flow of the solution without being fixed to the device body. The microspheres can be configured in a manner that they will not substantially depart from the accommodating portion even if not directly fixed to the accommodating portion that houses them. Thus, for example, unwanted substances can be removed by flushing or rinsing with a diluent, washing solution, etc., and the microspheres can be maintained in their positions. Thus, for example, reuse can be achieved.

[0032] The base material can include a porous material. The porous material can be a ceramic and can be a carbon material. The base material can be a zeolite. The base material can be a metal-organic structure.

[0033] "Protease" generally refers to a general term for peptide hydrolases that hydrolyze and dissimilate proteins and polypeptides. The protease can be an enzyme that decomposes proteins into peptide fragments. When the protein contains glycated amino acid residues, peptide fragments containing glycated amino acid residues and completely unglycated peptide fragments may be present in the peptide fragments generated by the action of the protease.

[0034] "Protease" can be a protease of animal origin, a protease of plant origin, or a protease of microbial origin. The protease can be an exopeptidase or an endopeptidase. The protease can be an aspartic protease, a metalloprotease, a serine protease, or a thiol protease.

[0035] "Protease" can include two or more types or species of proteases, or can include one type or species of protease. For example, the protease can include one of a proteolytic enzyme and a peptidase, or can include both. Sometimes, the decomposition efficiency is improved by mixing two or more proteases. The protease can include a modified protease or a protease that has been modified. The protease can also be used together with an additive. The additive can be, for example, a surfactant or urea. The additive can, for example, make the protein unstable or denatured, but is not limited thereto. By using a modified protease or an additive, for example, the decomposition efficiency of the protein and the selectivity of the substrate can be improved, but is not limited thereto.

[0036] Animal-derived proteases can be trypsin, chymotrypsin, pepsin, elastase, bovine trypsin, cathepsin, calpain, protease type-I, protease type-XX, aminopeptidase N, carboxypeptidase, pancreatin (a mixture of various enzymes such as protease and amylase), etc.

[0037] Plant-derived proteases can be papain, bromelain, zingibain, kallikrein, ficin, chymopapain, actinidin, carboxypeptidase W, etc.

[0038] Microorganism-derived proteases can be from Bacillus (or source, the same below), Geobacillus, Paenibacillus, Aspergillus, Penicillium, Streptomyces, Lysobacter, yeast source, Tritirachium, Thermus, Pseudomonas, Achromobacter, Rhizopus, Staphylococcus, etc.

[0039] For example, proteases can be selected based on the digestion or decomposition efficiency of proteins obtained by measuring absorbance. In several embodiments, proteases with an absorbance difference of 100 mAbs or more before and after the decomposition of albumin (HSA) are used. The absorbance difference before and after the decomposition of albumin (HSA) can be 90 mAbs or more. The absorbance difference before and after the decomposition of albumin (HSA) can be 110 mAbs or more.

[0040] The protease can be selected from the group consisting of protease-XXIV, Orientase 22BF, Orientase 90N, Toyozyme NEP-160, and alkaline protease. The protease can be selected from the group consisting of protease-XXIV, Orientase 22BF, Orientase 90N, Toyozyme NEP-160, and alkaline protease. The protease can be selected from the group consisting of pronase and protease-XIV.

[0041] The protease can be any one of a neutral protease, an acidic protease, and a basic protease. For example, in the case of measuring tears, a neutral or weakly basic protease can be used. The protease can be a heat-resistant protease (such as thermolysin) that remains active even in a high-temperature range (e.g., 60 °C or higher). The protease can be a low-temperature active protease that remains active even in a low-temperature range (e.g., 30 °C or lower).

[0042] In some embodiments, the protease can be disposed in the sensor in a dry state. For example, it can be formed by natural drying, freeze-drying method, or spray-drying method, but is not limited thereto. In some embodiments, it can be disposed in a state dissolved in a solution. The protease dissolved in the solution can be stored cooled until use. The protease can be frozen together with the solution and dissolved before use. The protease can be kept in a moisture-containing environment without being dried. For example, the protease can be disposed in a gel.

[0043] "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, glucuronone (α-ketoaldehyde), and hydrogen peroxide. Therefore, ketoamine oxidase generates an amount of hydrogen peroxide proportional to or related to the amount of the recognized glycated amino acid or the peptide or peptide fragment containing a glycated amino acid residue.

[0044] The ketoamine oxidase can be a dehydrogenase, can be a kinase, and can be an oxidase. The ketoamine oxidase can be fructosyl amino acid oxidase (FAOD), fructosyl peptide oxidase, fructosyl valyl histidine oxidase, fructosyl amine oxidase, Amadoriase, fructosyl amine deglycase, and modified forms thereof.

[0045] In some embodiments, the ketoamine oxidase can be an oxidase that acts on an amino acid or peptide in which the ε-amino group is glycated. The amino acid can be lysine. By using an oxidase that selectively acts on an amino acid or peptide in which the ε-amino group is glycated, a glycated albumin sensor can be constructed.

[0046] In some embodiments, the ketoamine oxidase can be an oxidase that acts on an amino acid or peptide in which the α-amino group is glycated. The amino acid can be valine. By using an oxidase that acts on an amino acid or peptide in which the α-amino group is glycated, a glycated hemoglobin sensor or a glycated hemoglobin A1c (HbA1c) sensor can be constructed.

[0047] 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, specifically 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 that decreases due to an enzymatic reaction. Generally, the sensitivity of oxygen detection is relatively low and the interference is strong with respect to molecules and ions that are noise sources. By detecting oxygen, the consumption of oxygen can be measured. Since tears are already saturated with air during collection, the detection unit can be used for sensing enzymes in tears. The detection unit may be configured in such a way that multiple detection methods can be selectively or combinatorially performed.

[0048] The detection of hydrogen peroxide may be an optical detection. Optical detection may include the measurement of absorbance or luminescence. For example, the color change of a quinone dye generated by oxidative condensation by adding peroxidase, 4-aminoantipyrine, and a chromogenic agent can be measured from the back of a transparent substrate, for example. 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 configured in powder form. The reaction of hydrogen peroxide with luminol can be carried out, and the intensity of the luminescence (wavelength 460 nm) caused by the luminol reaction can be measured. The reagent may also include potassium hexacyanoferrate, sodium hydroxide, etc. In the luminol reaction, a gold electrode, a platinum electrode, or an indium tin oxide transparent electrode (ITO electrode) can be used, and the measurement can be carried out by an alternating current-driven electrochemiluminescence method. In addition, in the case of detecting a fluorescence reaction, a combination of oxalate and a fluorescent substance can be used, and lucigenin (acridine 、bis(N-methylacridine )) can be used. The detection unit may be a hydrogen peroxide sensor other than the above.

[0049] "On the detection unit" may be the upper part of a part of the surface of the detection unit and may be configured to cover the entire detection unit. Additionally, it may be on the detection unit formed on a substrate. It may cover the entire surface of the substrate, cover the detection unit, and cover a part of the substrate.

[0050] The sensor may have a liquid containment portion. The liquid containment portion may contain one, two, or all of immobilized protease, immobilized ketamine oxidase, and a detection portion. The liquid containment portion may extend along the longitudinal direction. The volume of the liquid containment portion may be 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, 0.1 μL or less than the above values. The liquid containment portion may have a liquid inlet. The liquid containment portion may have a liquid outlet. The liquid containment portion may have a gas pore. The gas pore may have the following function: when introducing liquid into the liquid containment portion, discharging the gas present in the liquid containment portion to the outside of the sensor.

[0051] Illustrate the exemplary or potential effects brought by the glycated protein sensor of the present invention and the like.

[0052] Protease can decompose other proteases. In addition, protease can also decompose (ketamine) oxidase. Unimmobilized protease moves in the liquid by diffusion or the like and meets other proteases and ketamine oxidase, and may decompose these. A solution of protease is not suitable for storage, and it is necessary to weigh solid protease each time. By immobilizing protease in the device in advance, the measurement steps can be made efficient. The immobilized protease can be reused. The ketamine oxidase and protease of the present invention can achieve repeated measurement by immobilization, and the running cost per measurement can be significantly reduced. The inside of the device can be washed between measurements. The glycated protein sensor of the present invention can be miniaturized. In addition, when the hydrogen peroxide detection portion is close to the ketamine oxidase immobilization layer, the diffusion distance of hydrogen peroxide becomes shorter, and it becomes less susceptible to interfering substances in the body fluid that react with hydrogen peroxide. When the immobilization layers of ketamine oxidase and protease are each formed as a thin film of, for example, 1 micrometer (μm) or less and formed on the hydrogen peroxide detection portion, since they are close to each other, highly sensitive measurement can be achieved. Moreover, even if the amount of the enzyme, which is relatively expensive as a reagent, is small, a specified sensitivity can be obtained, and the manufacturing cost can be reduced.

[0053] It should be noted that embodiments obtained by arbitrarily combining one or more of the above-listed features of the present invention are also included within the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 A cross-sectional schematic view of a sensor according to an embodiment of the present invention.

[0055] Figure 2 A cross-sectional schematic view of a sensor according to an embodiment of the present invention.

[0056] Figure 3 Cross-sectional schematic diagram of a sensor according to an embodiment of the present invention.

[0057] Figure 4 Cross-sectional schematic diagram of a sensor according to an embodiment of the present invention.

[0058] Figure 5 Cross-sectional schematic diagram of a sensor according to an embodiment of the present invention.

[0059] Figure 6 Cross-sectional schematic diagram of a sensor according to an embodiment of the present invention.

[0060] Figure 7 Cross-sectional schematic diagram of a sensor according to an embodiment of the present invention.

[0061] Figure 8 Cross-sectional schematic diagram of a sensor according to an embodiment of the present invention.

[0062] Figure 9 Cross-sectional schematic diagram of a sensor according to an embodiment of the present invention.

[0063] Figure 10 Cross-sectional schematic diagram of a sensor according to an embodiment of the present invention.

[0064] Figure 11 Cross-sectional schematic diagram of a sensor according to an embodiment of the present invention.

[0065] Figure 12 Cross-sectional schematic diagram of a sensor according to an embodiment of the present invention.

[0066] Figure 13 Schematic diagram showing the structure of a sensor according to an embodiment of the present invention.

[0067] Figure 14 Schematic diagram showing the structure of a sensor according to an embodiment of the present invention.

[0068] Figure 15 Schematic diagram showing the structure of a sensor according to an embodiment of the present invention.

[0069] Figure 16 Planar schematic diagram of a sensor according to an embodiment of the present invention.

[0070] Figure 17 Schematic diagram showing the structure of a sensor according to an embodiment of the present invention.

[0071] Figure 18 Planar schematic diagram of a sensor chip according to an embodiment of the present invention.

[0072] Figure 19 A plan view of a sensor chip according to an embodiment of the present invention.

[0073] Figure 20 A plan view of a sensor chip according to an embodiment of the present invention.

[0074] Figure 21 A plan view of a sensor chip according to an embodiment of the present invention.

[0075] Figure 22 A plan view of a sensor chip according to an embodiment of the present invention.

[0076] Figure 23A A cross-sectional view showing the manufacturing process of a sensor according to an embodiment of the present invention.

[0077] Figure 23B A cross-sectional view showing the manufacturing process of a sensor according to an embodiment of the present invention.

[0078] Figure 23C A cross-sectional view showing the manufacturing process of a sensor according to an embodiment of the present invention.

[0079] Figure 23D A cross-sectional view showing the manufacturing process of a sensor according to an embodiment of the present invention.

[0080] Figure 23E A cross-sectional view showing the manufacturing process of a sensor according to an embodiment of the present invention.

[0081] Figure 23F A cross-sectional view showing the manufacturing process of a sensor according to an embodiment of the present invention.

[0082] Figure 23G A cross-sectional view showing the manufacturing process of a sensor according to an embodiment of the present invention.

[0083] Figure 23H A cross-sectional view showing the manufacturing process of a sensor according to an embodiment of the present invention.

[0084] Figure 23I A cross-sectional view showing the manufacturing process of a sensor according to an embodiment of the present invention.

[0085] Figure 24 A schematic diagram showing a partial structure of a sensor according to an embodiment of the present invention.

[0086] Figure 25 A schematic diagram showing a partial structure of a sensor according to an embodiment of the present invention.

[0087] Figure 26 A graph showing the digestibility of each protease.

[0088] Figure 27 A graph showing the sensor output for each protease.

[0089] Figure 28 A graph showing the dependence of protease and ketoamine oxidase on the working temperature.

[0090] Figure 29 A graph showing the dependence of the sensor output on the glycated albumin concentration. Detailed Description of the Invention

[0091] 1. Structure of the Sensor

[0092] Use Figure 1 to illustrate the constitution of the glycoprotein sensor according to an embodiment of the present invention. Figure 1 The sensor 100 shown has a protease 101, a ketoamine oxidase 102, and a hydrogen peroxide detection unit 103. The protease 101 and the ketoamine oxidase 102 are crosslinked with bovine serum albumin 105 as a substrate through glutaraldehyde 106 as a crosslinking agent. The bovine serum albumin 105 is also crosslinked with each other. These form an immobilized layer 104. The immobilized layer 104 is connected to the hydrogen peroxide detection unit 103 through a silane coupling agent 107. Thus, the protease 101 and the ketoamine oxidase 102 are immobilized relative to the hydrogen peroxide detection unit 103 or the entire sensor 100. The immobilized layer 104 and the protease 101 and the ketoamine oxidase 102 immobilized on this layer together form an enzyme layer 111.

[0093] 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 the protease 101 to generate peptide fragments.

[0094] Among the peptide fragments, there are glycated peptide fragments 154 and non-glycated peptide fragments 155. It is considered that these peptide fragments will diffuse within the immobilized membrane 104. If the glycated peptide fragment 154 reacts with the ketoamine oxidase 102, glucuronone (not shown) and hydrogen peroxide 156 are generated.

[0095] It is considered that the hydrogen peroxide 156 will also diffuse within the immobilized membrane 104. The hydrogen peroxide detection unit 103 detects the hydrogen peroxide 156 and outputs a signal related to its concentration.

[0096] When the hydrogen peroxide detection unit 103 is a hydrogen peroxide electrode, the hydrogen peroxide 156 is decomposed at the hydrogen peroxide electrode, and the released electrons are detected in the form of an electric current. This reaction can be described as follows.

[0097] H2 O 2 → 2H + + O 2 + 2e -

[0098] Hydrogen peroxide 156 is consumed at the hydrogen peroxide electrode 103. Therefore, the concentration of hydrogen peroxide 156 in the immobilized film 104 is the lowest near the hydrogen peroxide detection unit 103 and increases as it moves away from the hydrogen peroxide detection unit 103. That is, there is a concentration gradient of hydrogen peroxide 156 within the immobilized film 104. This concentration gradient changes immediately after the start of measurement and becomes basically stable after a specified time. At this stable moment, 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 correlation between the concentration of glycated protein 151 and the current value generated in the hydrogen peroxide electrode 103 is obtained in advance. When performing actual measurement, based on this calibration, the concentration of glycated protein 151 in the test solution can be calculated from the current value generated in the hydrogen peroxide electrode 103.

[0099] In the sensor according to some embodiments, the protease and the ketoamine oxidase can be immobilized on the same substrate (integrated type).

[0100] Figure 2 The structure of the integrated type sensor according to some embodiments (second embodiment) is shown. The sensor 200 has a protease 201, a ketoamine oxidase 202, a substrate 204, and a hydrogen peroxide detection unit 203. The protease 201 and the ketoamine oxidase 202 are immobilized on the substrate 204, and the substrate 204 is immobilized on the hydrogen peroxide detection unit 203. Figure 2 In the shown sensor 200, the same substrate 204 is formed as a layer or a film on the hydrogen peroxide detection unit 203, and an enzyme layer 211 in which the protease 201 and the ketoamine oxidase 202 are immobilized on the substrate 204 is formed on the hydrogen peroxide detection unit 203. The protease 201 and the ketoamine oxidase 202 are immobilized in the same substrate 204.

[0101] Figure 3 The structure of the sensor according to some embodiments (third embodiment) is shown. The sensor 300 has a protease 301, a ketoamine oxidase 302, a substrate 304 on which these are immobilized, and a hydrogen peroxide detection unit 303 on which the substrate 304 is immobilized. Figure 3 In the shown sensor 300, the same substrate 304 is formed as a layer or a 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. Figure 3The illustrated sensor 300 further has an adhesive or bonding agent 305 between the substrate 304 and the hydrogen peroxide detection unit 303 that bonds the two. This bonding agent 305 can be a silane coupling agent. In the case where the hydrogen peroxide detection unit 303 has a metal such as an electrode on its surface (not shown), the silane coupling agent can bond the metal and the substrate 304 formed of an organic material relatively firmly.

[0102] Figure 4 The structure of a sensor (fourth embodiment) showing several embodiments is shown. The sensor 400 has a protease 401, a ketamine oxidase 402, substrates 404 and 414 on which these are immobilized, and a hydrogen peroxide detection unit 403 on which the substrate 414 is immobilized. More specifically, a layer of the substrate 414 containing the ketamine oxidase 402 (ketamine oxidase layer 412) is formed on the hydrogen peroxide detection unit 403. A layer of the substrate 404 containing the protease 401 (protease layer 411) is formed on the ketamine oxidase layer 412, that is, on the surface on the side opposite to the hydrogen peroxide detection unit 403. That is, the ketamine oxidase layer 412 and the protease layer 411 are stacked in sequence on the hydrogen peroxide detection unit 403.

[0103] In the sensor of the present invention, the protease and the ketamine oxidase may or may not be stacked on the detection unit. In several embodiments, the protease and the ketamine oxidase may be arranged close to the detection unit or may be arranged near the detection unit.

[0104] In several embodiments, the thickness of the protease layer, the ketamine oxidase layer, or the enzyme layer may be a value of 100 μm, 50 μm, 20 μm, 10 μm, 1 μm, 500 nm, 300 nm, 250 nm, 200 nm, 100 nm, 50 nm, etc. or less than the above value. In several embodiments, the thickness of the protease layer, the ketamine oxidase layer, or the enzyme layer may be a value of 10 nm, 20 nm, 25 nm, 30 nm, 50 nm, 100 nm, etc. or more than the above value.

[0105] According to the sensor of several embodiments, the ketamine oxidase portion may be arranged so as to surround the protease portion (surrounding type).

[0106] Figure 5Structure of a sensor (fifth embodiment) showing several embodiments. The sensor 500 has a protease 501, a ketamine oxidase 502, substrates 504, 514 to which these are immobilized, a hydrogen peroxide detection unit 503 to which the substrate 514 is immobilized, and a substrate 506. More specifically, the hydrogen peroxide detection unit 503 is provided on the substrate 506. A substrate 514 (immobilized ketamine oxidase unit 512) to which the ketamine oxidase 502 is immobilized is formed on the hydrogen peroxide detection unit 503. A substrate 504 (immobilized protease unit 511) to which the protease 501 is immobilized is formed so as to surround the immobilized ketamine oxidase unit 512. In other words, on the substrate 506, the immobilized ketamine oxidase unit 512 is arranged in a portion where the hydrogen peroxide detection unit 503 is substantially arranged, and the immobilized protease unit 511 is arranged so as to cover the surface of the immobilized ketamine oxidase unit 512 and the substrate 506 without the hydrogen peroxide detection unit 503. According to such a structure, the amount of immobilized protease can be relatively increased compared to the amount of immobilized ketamine oxidase. Thereby, for example, when the protease-based decomposition or digestion of a macromolecular protein becomes rate-limiting, etc., the response speed, detection sensitivity, etc. of the sensor can be improved.

[0107] Enzymes can be immobilized within a substrate or on the outer surface of the substrate (direct bonding type).

[0108] Figure 6 The shown sensor 600 is of the direct bonding type and has a protease 601, a ketamine oxidase 602, and a hydrogen peroxide detection unit 603 to which these are immobilized. More specifically, a substrate 614 (ketamine oxidase layer 612) to which the ketamine oxidase 602 is fixed is formed on the hydrogen peroxide detection unit 603. The protease 601 is fixed to the upper surface of the ketamine oxidase layer 612 through a crosslinking agent 604. Figure 6 The protease 601 is fixed to the substrate 614. The protease 601 can be crosslinked with the ketamine oxidase 602.

[0109] Figure 7 An example of a sensor structure having a hydrogen peroxide electrode is shown. Figure 7 In it, as an example of the immobilization of protease and ketamine oxidase, Figure 4 such a laminated structure is shown. However, the method of immobilizing protease and ketamine oxidase is not limited to this. As illustratively described above, even in the case of a laminated structure, it can be Figure 7 a structure other than this and can be a non-laminated structure.

[0110] Figure 7The sensor 700 shown has: a hydrogen peroxide electrode 730 formed on an insulating substrate 706, a ketamine oxidase 702 disposed on the hydrogen peroxide electrode 730 and a substrate 714 (ketamine oxidase layer 712) for fixing the same, and a protease 701 disposed on the ketamine oxidase layer 712 and a substrate 704 (protease layer 711) for fixing the same. A silane coupling agent 705 that bonds the surface of the electrode 730 and the substrate 714 is coated between the hydrogen peroxide electrode 730 and the ketamine oxidase layer 712.

[0111] Figure 7 The hydrogen peroxide electrode 730 shown is an electrode for a three - electrode method and includes a counter electrode 731, a reference electrode 732, and a working electrode 733. Figure 7 The sensor 700 shown also has a circuit 770 connected to the hydrogen peroxide electrode 730. The circuit 770 includes an operational amplifier 771, a voltage generation circuit 772, and a current measurement 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 generation circuit 772.

[0112] The three - electrode method is as follows: The counter electrode, the reference electrode, and the working electrode are arranged in contact with a solution or body fluid, a predetermined potential difference is provided between the counter electrode and the working electrode, and the current flowing from the counter electrode into the working electrode is measured. Generally, when substances such as metals and metal oxides enter the electrolyte solution, a potential difference called an interfacial potential is generated between the substance and the electrolyte solution. If a voltage is applied between the counter electrode and the working electrode while also considering this potential difference, current flows out from the counter electrode, and the potential difference between the counter electrode and the solution may change. Sometimes, due to this change in potential difference, it may not be possible to accurately apply the desired voltage to the solution. In the three - electrode method, to avoid this situation, the reference electrode can measure the potential applied to the counter electrode and control the voltage applied to the counter electrode in such a way that the potential is fixed at the desired value. Additionally, there is a feedback circuit that returns (feeds back) the potential measured by the reference electrode to the circuit that controls the counter electrode.

[0113] 2. Reduction of noise caused by inclusions

[0114] The solution may contain inclusions. For example, when measuring proteins in body fluids, measurement noise may be generated due to various inclusions such as proteins other than the protein being measured, peptide fragments, nucleic acids, and ions. The noise caused by these inclusions can be the cause of measurement errors and measurement inaccuracies. Therefore, the sensors of some embodiments of the present invention may have a configuration that reduces the influence of inclusions on the measurement signal.

[0115] <Noise reduction using an ion - exchange resin>

[0116] Sensors according to some embodiments may have an ion exchange resin on the detector.

[0117] Figure 8 The illustrated sensor 800 has a hydrogen peroxide electrode 803, a ketamine oxidase 802 disposed on the hydrogen peroxide electrode 803, and a substrate 814 (ketamine oxidase layer 812) for immobilizing it, and a protease 801 and a substrate 804 (protease layer 811) for immobilizing it disposed on the ketamine oxidase layer 814. Figure 8 The illustrated sensor 800 also has an ion exchange resin 807 between the ketamine oxidase layer 812 and the hydrogen peroxide electrode 803.

[0118] For example, by using a cation exchange resin represented by Nafion (registered trademark), it is possible to suppress or prevent ascorbic acid, uric acid, especially negative ions, etc. present in body fluid from passing through and reaching the detection unit. For example, by using an anion exchange resin such as polypyrrole, it is possible to suppress or prevent dopamine, etc., especially positive ions, etc. from passing through and reaching the detection unit.

[0119] The ion exchange resin may include one, multiple, or at least one ion exchange resin. The ion exchange resin may be composed of one, multiple, or at least one layer.

[0120] Figure 8 Shown Figure 4 such a stacked structure as an example of the immobilization of protease and ketamine oxidase. However, the method of immobilizing protease and ketamine oxidase is not limited to this. As exemplarily described above, even in the case of a stacked structure, it may be Figure 8 other structures and may be a non-stacked structure.

[0121] <Reduction of noise using a differential sensor>

[0122] Sensors according to some embodiments may be a pair or a group of differential or difference sensors. The sensor may include multiple pairs or groups of differential or difference sensors. A group of differential sensors included in the differential type sensor may be composed of a main sensor and a sub-sensor. The main sensor has an immobilized protease, an immobilized ketamine oxidase, and a detection unit. The sub-sensor has a lower sensitivity to the main measurement object (substance to be measured) detected by the main sensor, but has substantially the same or similar sensitivity to the molecules that cause noise as the main sensor.

[0123] As an example, as a sensor, a sensor for measuring glycated proteins in body fluid is considered. Body fluid contains proteins, peptides, vitamin C, ions, etc. other than the protein as the substance to be measured. For example, peptide fragments are recognized by ketamine oxidase and cause the generation of hydrogen peroxide. In addition, ions in body fluid may be detected by the hydrogen peroxide detection unit.

[0124] Therefore, the sub-sensor can be a sensor that detects the cause of noise including the above in a manner that is substantially the same as or has a certain correlation with the main sensor. For example, the sub-sensor can be free of protease (protease-free) relative to the main sensor, and other structures are substantially the same. For example, for the sub-sensor, the surface area and height dimensions of the part containing the substrate and the enzyme, the type of the substrate, the manufacturing method, etc. can be substantially the same as the corresponding items of the main sensor. The materials, structures, manufacturing methods, etc. other than protease of the main sensor and the sub-sensor can also be different. In this case, the correlation between them can be obtained through calibration or the like.

[0125] In some embodiments, the hydrogen peroxide detector of both or one of the main sensor and the sub-sensor of the differential sensor can be covered with an ion exchange resin, and a layer or film of the ion exchange resin can be disposed on the surface of the hydrogen peroxide detector.

[0126] In some embodiments, the configuration of the main sensor can be, for example, Figure 3 of the enzyme layer integrated type.

[0127] Figure 9 The sensor 1000 has a main sensor 1000a and a sub-sensor 1000b of the enzyme layer integrated type.

[0128] Figure 9 The main sensor 1000a shown is Figure 2 or Figure 3 of the enzyme layer integrated type such as that. Specifically, the main sensor 1000a has a protease 1001, a ketamine oxidase 1002, a substrate 1004 to which these are immobilized, and a hydrogen peroxide detection unit 1003a to which the substrate 1004 is immobilized. Figure 9 In the main sensor 1000a shown, the same substrate 1004 is formed in a layer or film on the hydrogen peroxide detection unit 1003a, and an enzyme layer 1011a in which the protease 1001 and the ketamine 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 the ketamine oxidase 1002 are immobilized in the same substrate 1004.

[0129] On the other hand, Figure 9 The sub-sensor 1000b shown can have a configuration substantially the same as that of the main sensor 1000a and be free of (free of) the protease 1001. Specifically, the sub-sensor 1000b has the same ketamine oxidase 1002, substrate 1014, and hydrogen peroxide detection unit 1003b as the main sensor 1000a. The enzyme layer 1011b of the sub-sensor 1000b having the ketamine oxidase 1002 and the substrate 1014 can have substantially the same size as the enzyme layer 1011a of the main sensor.

[0130] The hydrogen peroxide detectors 1003a and 1003b of the main sensor 1000a and the sub-sensor 1000b are respectively connected to the measurement circuits 1070a and 1070b. These circuits 1070a and 1070b receive output signals such as current from the respective hydrogen peroxide detectors 1003a and 1003b, convert their values into digital form, and send them to an arithmetic unit 1030 such as a CPU. In some embodiments, the transmission from the circuits 1070a and 1070b to the arithmetic unit 1030 can be a wired method using electricity or light, or a wireless method.

[0131] The arithmetic unit 1030 can perform operations such as differential operations on the signals from the main sensor 1000a and the sub-sensor 1000b. By removing the output equivalent to noise such as inclusions from the output signal of the main sensor 1000a based on the signal from the sub-sensor 1000b, the signal regarding the measurement object can be detected with higher accuracy. The arithmetic unit 1030 may further include a transmission unit (not shown), and may also be connected to an external transmission unit. The transmission unit can send the signal in a wired or wireless manner by light, electricity, or electromagnetic means. The sent signal can be the signal after differential operation in the arithmetic unit 1030. In another embodiment, the transmission unit can send the signals from the main sensor 1000a and the sub-sensor 1000b separately. Differential operations etc. can also be performed at the delivery location. The configuration or function of these arithmetic units is not limited to this embodiment and can also be used in other embodiments. The arithmetic unit can have a storage medium inside, or can be configured in a manner of being connected or connected to a storage medium arranged outside the arithmetic unit.

[0132] In a differential type sensor, the configuration of the main sensor can be, for example, Figure 4 such a stacked type.

[0133] Figure 10 The shown sensor 1100 has a stacked main sensor 1100a and a sub-sensor 1100b.

[0134] Figure 10 The shown main sensor 1100a is Figure 4 such a stacked type. Figure 10The main sensor 1100a shown has a protease 1101, a ketoamine oxidase 1102, substrates 1104 and 1114 to which these are immobilized, and a hydrogen peroxide detection unit 1103a to which the substrate 1114 is immobilized. More specifically, a layer of the substrate 1114 containing the ketoamine oxidase 1102 (ketoamine oxidase layer 1112a) is formed on the hydrogen peroxide detection unit 1103a. A layer of the substrate 1104 containing the protease 1101 (protease layer 1111a) is formed on the ketoamine oxidase layer 1112a, that is, on the surface on the side opposite to the hydrogen peroxide detection unit 1103a. That is, the ketoamine oxidase layer 1112a and the protease layer 1111a are stacked in this order on the hydrogen peroxide detection unit 1103a.

[0135] In some embodiments, substrates in each layer may be made of different materials. For example, a substrate mainly composed of protein may be used in the ketoamine oxidase layer, and a substrate mainly composed of a photocrosslinkable resin may be used in the protease layer.

[0136] On the other hand, Figure 10 The sub-sensor 1100b shown has a configuration substantially the same as that of the main sensor 1100a and does not include or contains no protease 1101 and its substrate 1104. Specifically, the sub-sensor 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 sub-sensor 1100b having the ketoamine oxidase 1102 and the substrate 1124 may have substantially the same size as the entire enzyme layers 1111a and 1112b of the main sensor.

[0137] Between the main sensor and the sub-sensor, the thickness of the enzyme layer may be different. In some embodiments, the thickness of the ketoamine oxidase layer of the sub-sensor 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 the correlation of signal intensities and the like obtained using the same or different calibration solutions. The concentration and total amount of the enzyme may be the same or different between the main sensor and the sub-sensor and may be adjusted according to the sensor. The material of the substrate may also be the same or different between the main sensor and the sub-sensor.

[0138] The hydrogen peroxide detectors 1103a and 1103b of the main sensor 1100a and the sub-sensor 1100b are respectively connected to measurement circuits 1170a and 1170b. These circuits 1170a and 1170b receive output signals such as current from each hydrogen peroxide detector 1103a and 1103b and send the values thereof in digital form to an arithmetic unit 1130 such as a CPU.

[0139] In some embodiments, in a differential-type sensor, the configuration of the main sensor may be, for example, Figure 5 such an enclosed type.

[0140] In some embodiments, in the differential sensor, the main sensor may be configured as, for example, Figure 6 a direct bonding type as such.

[0141] Figure 11 The sensor 1200 shown has a direct bonding type main sensor 1200a and a sub-sensor 1200b.

[0142] Figure 11 The main sensor 1200a shown is Figure 4 a direct bonding type sensor as such, having a protease 1201, a ketamine oxidase 1202, and a hydrogen peroxide detection unit 1203a to which these are immobilized. More specifically, on the hydrogen peroxide detection unit 1203a, a substrate 1214 (ketamine oxidase layer 1212a) to which the ketamine oxidase 1202 is fixed is formed. The protease 1201 is fixed to the upper surface of the ketamine oxidase layer 1212a through a crosslinking agent 1204. Figure 11 The protease 1201 is fixed to the substrate 1214. The protease 1201 is crosslinked with the ketamine oxidase 1202.

[0143] On the other hand, Figure 11 the sub-sensor 1200b shown does not contain or is free of the protease 1201. Specifically, the sub-sensor 1200b has the same ketamine oxidase 1202, substrate 1224, and hydrogen peroxide detection unit 1203b as the main sensor 1200a. In other words, an enzyme layer 1212b containing the ketamine oxidase 1202 and the substrate 1224 is formed on the hydrogen peroxide detection unit 1203b.

[0144] In the main sensor 1200a, the protease 1201 is directly bonded to the ketamine oxidase layer 1212a. In contrast, the outermost surface of the sub-sensor 1200b may be substantially composed of the substrate 1224. Thus, it is considered that the properties such as the permeability of molecules and ions on the outermost surface into the substrate inside the main sensor 1200a and the sub-sensor 1200b are not necessarily the same or may be different. Therefore, the composition of the enzyme layer 1212b of the sub-sensor 1200b, such as thickness, type of the substrate 1224, concentration of the ketamine oxidase 1202, manufacturing method, etc., can be relatively adjusted so that the properties of the main sensor 1200a and the sub-sensor 1200b are substantially the same or have a correlation.

[0145] The hydrogen peroxide detectors 1203a and 1203b of the main sensor 1200a and the sub-sensor 1200b are respectively connected to the measurement circuits 1270a and 1270b. These circuits 1270a and 1270b receive output signals such as current from the respective hydrogen peroxide detectors 1203a and 1203b, and send their values in digital form to an arithmetic unit 1230 such as a CPU.

[0146] The main sensor and the sub-sensor of the differential sensor can each include a hydrogen peroxide electrode in the hydrogen peroxide detection section. For example, Figure 7 the hydrogen peroxide electrode 730 and the circuit 770 shown can be arranged in the main sensor, and a hydrogen peroxide electrode and a circuit that are the same or relatively different can be arranged in the sub-sensor.

[0147] In the main sensor and the sub-sensor of the differential sensor, a partial configuration of the hydrogen peroxide detection section can be shared. For example, when a hydrogen peroxide electrode is arranged in the hydrogen peroxide detection section, the working electrode can be arranged in each sensor, and the counter electrode and the reference electrode can be arranged in any one of the sensors and shared.

[0148] Figure 12 In the differential sensor 1300 shown, the main sensor 1300a and the sub-sensor 1300b are arranged on the substrate 1306.

[0149] Figure 12 In the main sensor 1300a shown, an enzyme layer 1311a is arranged on the hydrogen peroxide electrode 1330a via a silane coupling agent 1305a. The enzyme layer 1311a contains a protease 1301 and a ketamine oxidase 1302 in the substrate 1304. Figure 12 In the sub-sensor 1300b shown, an enzyme layer 1311b is arranged on the hydrogen peroxide electrode 1330b via a silane coupling agent 1305b. The enzyme layer 1311b does not contain the protease 1301 and contains the ketamine oxidase 1302 in the substrate 1324.

[0150] The hydrogen peroxide electrode 1330a of the main sensor 1300a includes a working electrode 1333a for the main sensor and a counter electrode 1331. On the other hand, the hydrogen peroxide electrode 1330b of the sub-sensor 1300b includes a working electrode 1333b for the sub-sensor and a reference electrode 1332. These electrodes are connected to the circuit 1370.

[0151] In other words, Figure 12 the hydrogen peroxide electrodes 1330a and 1330b shown are electrodes for the three-electrode method, and the working electrodes 1333a and 1333b are respectively arranged in the main sensor 1300a and the sub-sensor 1300b. On the other hand, the counter electrode 1331 and the reference electrode 1332 are arranged in only one of the main sensor 1300a and the sub-sensor 1300b.

[0152] Figure 12 The sensor 1300 shown also has a circuit 1370 connected to the hydrogen peroxide electrodes 1330a and 1330b. The circuit 1370 includes current measurement circuits 1373a and 1373b respectively connected to an operational amplifier 1371, a voltage generation circuit 1372, a main sensor 1300a, and a sub-sensor 1300b. 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 generation circuit 1372.

[0153] During measurement, a solution (not shown) containing the substance to be measured (measurement object, measured substance) comes into contact with both the main sensor 1300a and the sub-sensor 1300b. Therefore, the counter electrode 1331 can generate a constant potential difference with respect to the potential of the reference electrode 1332 and apply a desired voltage to both the working electrode 1333a for the main sensor and the working electrode 1333b for the sub-sensor. The current measurement circuits 1373a and 1373b respectively detect the output signals from the main sensor 1300a and the sub-sensor 1300b and provide the output signals to the outside (not shown). With such a configuration, the area of the electrodes can be reduced and the sensor and device can be miniaturized.

[0154] Figure 12 The enzyme layer of the main sensor 1300a has Figure 2 an integrated configuration like this, but is not limited thereto. Figure 12 The same applies to the sub-sensor 1300b and is not limited thereto. The main sensor 1300a and the sub-sensor 1300b can be configured in any other way.

[0155] Figure 12 In, current measurement circuits 1373a and 1373b are respectively arranged in the main sensor 1300a and the sub-sensor 1300b, but are not limited to this configuration. One current measurement circuit and a switching circuit can be arranged, and the signals from the working electrode 1333a for the main sensor and the working electrode 1333b for the sub-sensor are provided to the current measurement circuit alternately or at a specified timing by the switching circuit for detection or measurement.

[0156] The protease, the ketoamine oxidase, and the detection unit can be configured in a stacked manner or disposed close to each other at the same location. In some embodiments, the ketoamine oxidase and the detection unit can be stacked or disposed close to each other, and the protease can be disposed separately from these. In another embodiment, the protease, the ketoamine oxidase, and the detection unit can be disposed separately from each other. By separating and fixedly disposing the protease from other components, various conditions including time and temperature for the decomposition reaction based on the protease can be made efficient or optimized. After allowing the decomposition reaction based on the protease to proceed sufficiently, the peptide fragments as reaction products can be sent to the ketoamine oxidase.

[0157] In some embodiments, the protease, the ketoamine oxidase, and the detection unit can be arranged in the order of the protease, the ketoamine oxidase, and the detection unit from the upstream of the solution, or from the upstream side to the downstream side, that is, along the direction of solution introduction or the direction of reaction sequence. Thereby, the reaction products in each reaction can be efficiently transported to the next reaction.

[0158] In some embodiments, the protease, the ketoamine oxidase, and the detection unit can be respectively housed in defined housing portions (chambers). Each reaction can occur in the corresponding housing portion, and in addition, a certain reaction can be carried out separately from other reactions. Thereby, the limitations and influences caused by the conditions of other reactions and preparation conditions can be reduced, and the desired reaction can be carried out efficiently. For example, sufficient time for decomposing glycated proteins into peptide fragments using the protease can be obtained, and the peptide fragments can be sent to the ketoamine oxidase after the desired time.

[0159] Figure 13 In the illustrated sensor 1400, the protease, the ketoamine oxidase, and the detection unit are disposed separately from each other. In addition, the protease, the ketoamine oxidase, and the detection unit are arranged in the order of the protease, the ketoamine oxidase, and the detection unit from the upstream side to the downstream side along the direction of solution introduction or the direction of reaction sequence. Moreover, the protease, the ketoamine oxidase, and the detection unit are respectively housed in defined housing portions (chambers) and are fluidly connected to each other.

[0160] For Figure 13 A more detailed description will be given. The liquid that enters from the liquid inlet 1452 enters the protease housing portion 1455. The liquid can enter the liquid inlet 1452 via a flow path. The liquid can also be sent to the solution inlet 1452 using capillary action. In the protease housing portion 1455, the protease 1401 is housed in a state fixed to the beads 1404. In some embodiments, the beads 1404 can be fixed to the inner wall of the housing portion 1455. In another embodiment, the beads 1404 may not be directly fixed to the housing portion 1455. For example, by narrowing the flow path with respect to the beads 1404 that are large enough, a state in which the beads 1404 are substantially fixed within the housing portion 1455 is formed.

[0161] Figure 13 A heater 1460 is disposed in the protease housing portion 1455 shown. The heater 1460 can heat the solution or protease 1401 in the protease housing portion 1455, control its temperature, increase the rate of the decomposition reaction based on the protease, etc., so as to make the reaction efficient or optimized. Figure 13 A heater using an electric current is described, but it is not limited thereto. The heater can adopt a heating method other than electricity. In another embodiment, a temperature regulator can be disposed.

[0162] In some embodiments, the temperature of the protease can be controlled. In some embodiments, the temperatures of the protease and the ketamine oxidase can be controlled uniformly or separately. In some embodiments, the temperature of a part or the whole of the sensor or the sensor chip can be controlled. In some embodiments, the temperature of the protease housing portion can be controlled. The heating time and temperature distribution of the protease and the like can be controlled.

[0163] In some embodiments, the temperature of the protease can be any temperature above 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, and 60°C or higher than any of the above temperatures. In some embodiments, the temperature of the protease can be any temperature below 80°C, 75°C, 70°C, 65°C, 60°C, 55°C, 50°C, 45°C, 40°C, and 35°C or lower than any of the above temperatures. In some embodiments, the temperature of the protease can be within the optimum temperature range of the protease. In some embodiments, the temperature of the protease can be lower than the temperature at which inactivation occurs.

[0164] In the protease housing portion 1455, the generated peptide fragments are transported to the ketamine oxidase housing portion 1456 where the ketamine oxidase 1402 is immobilized on the substrate 1414.

[0165] In some embodiments, the capillary phenomenon can be utilized to transport the protease in a solution state from the protease housing portion 1455 to the ketamine oxidase housing portion 1456. For example, the cross-sectional area and distance of each housing portion and the flow path therebetween, the shape such as hydrophilicity or hydrophobicity of the inner wall, and other materials can be selected or adjusted to adjust the residence time of the protease in a solution state in the protease housing portion 1455, the timing of liquid feeding to the ketamine oxidase housing portion 1456, and the like.

[0166] In some embodiments, the protease housing portion 1455 can be formed of a flexible or elastic material such as silicone in a volume-variable manner, and pressure is applied from the outside at a specified timing to deform it, so that the liquid is discharged from the protease housing portion 1455. It can be configured such that the liquid inlet 1452 is closed at this time to prevent the liquid from flowing back to the liquid inlet 1452.

[0167] In the ketamine oxidase housing unit 1456, the ketamine oxidase 1402 is immobilized on the substrate 1414. Figure 13 In the housing unit 1456, the substrate 1414 and the ketamine oxidase 1402 are formed in the form of an immobilized layer. The peptide fragments transported into the ketamine oxidase housing unit 1456 react with the ketamine oxidase 1402, and as a result, hydrogen peroxide is produced.

[0168] In some embodiments, the temperature of the ketamine oxidase can be controlled. In some embodiments, the temperature of the ketamine oxidase housing unit can be controlled. In some embodiments, the temperatures of the protease and the ketamine oxidase can be controlled in such a way that they are different. In some embodiments, the temperatures of the protease and the ketamine oxidase can be controlled in such a way that the temperature of the protease is higher than that of the ketamine oxidase, or the temperature of the ketamine oxidase can be controlled in such a way that it is lower than that of the protease. In some embodiments, the temperature control of the protease and the ketamine oxidase can be carried out simultaneously, in different time ranges, or in a partially overlapping time range. In some embodiments, the heating time and temperature distribution of the ketamine oxidase and the like can be controlled.

[0169] In some embodiments, the controlled temperature of the ketamine oxidase can be any temperature above 10°C, 12°C, 15°C, 20°C, 25°C, 30°C, 35°C, and 37°C or higher than any of the above temperatures. In some embodiments, the controlled temperature of the ketamine oxidase can be any temperature below 70°C, 60°C, 55°C, 50°C, 45°C, 40°C, 37°C, and 35°C or lower than any of the above temperatures. The temperature control of the ketamine oxidase can be carried out by heating, cooling, or a combination of heating and cooling.

[0170] In some embodiments, the ketamine oxidase can be maintained at room temperature during measurement. In some embodiments, the temperature of the ketamine oxidase during measurement can be measured, and the results obtained by the detector can be temperature-corrected. For example, measurement can be carried out while maintaining the ketamine oxidase at room temperature, and the measurement results can be temperature-corrected. In some embodiments, the temperature of the protease during measurement or peptide fragmentation can be measured, and the results obtained by the detector can be temperature-corrected. In some embodiments, the measurement results can be temperature-corrected based on the temperature of the protease during measurement or peptide fragmentation and the temperature of the ketamine oxidase during measurement. As an example, by performing temperature correction, the temperature control of at least one of the ketamine oxidase and the protease can be simplified. Thus, for example, the power supply can be a battery, and the sensor can be made smaller.

[0171] The hydrogen peroxide is transported to the detection reactor 1457 and detected using the detector 1431. Figure 13In [the device], a reagent 1431 containing luminol is disposed inside the detection reactor 1457, and a light detector (photodiode) 1432 is disposed outside, thereby detecting the luminescence based on the luminol reaction.

[0172] The ketamine oxidase may not be immobilized on the substrate within the immobilized layer. The ketamine oxidase may be immobilized on microbeads.

[0173] Figure 14 The sensor 1500 shown is Figure 13 partially similar in configuration to the sensor 1400 shown. However, the ketamine oxidase 1502 is immobilized on the microbeads 1504 and is housed within the ketamine oxidase housing portion 1556.

[0174] Figure 13 In the sensor 1400 shown, Figure 14 In the sensor 1500 shown, the detector is composed of a reagent for the luminol reaction and a light detector for detecting luminescence. However, in the same configuration, the detector can be set to other detectors. As the detector, other light detectors can be used, or a hydrogen peroxide electrode can be used.

[0175] In some embodiments, the ketamine oxidase can be brought into contact with or close to the hydrogen peroxide detector, and the protease can be disposed separately from these. The method of disposing the ketamine oxidase close to the hydrogen peroxide detector is a method for reducing the influence of noise factors in the detection by the hydrogen peroxide detector or for increasing the detection sensitivity of hydrogen peroxide.

[0176] Figure 15 In the sensor 1600 shown, the ketamine oxidase 1602 is fixed close to the hydrogen peroxide detector 1603, and the protease 1601 is disposed separately from these.

[0177] The liquid introduced from the liquid inlet 1652 enters the protease housing portion 1655. In the protease housing portion 1655, the protease 1601 is housed in a state of being immobilized on the microbeads 1604.

[0178] Figure 15 A heater 1660 is disposed in the protease housing portion 1655 shown. The heater 1660 can heat the solution or the protease 1601 within the protease housing portion 1655 or control its temperature to make the decomposition reaction based on the protease efficient or optimized. Figure 15 A heater using an electric current is described in [the reference], but it is not limited thereto. The heater can also adopt a heating method other than electricity. In another embodiment, a temperature regulator can be disposed.

[0179] In the protease housing section 1655, the generated peptide fragments are transported to the ketoamine oxidase housing section 1656 that houses the immobilized ketoamine oxidase 1602 and the hydrogen peroxide detector 1603.

[0180] In the ketoamine oxidase housing section 1656, the ketoamine oxidase 1602 is immobilized on the substrate 1614 to form the enzyme layer 1611. Figure 15 In the ketoamine oxidase housing section 1656, the substrate 1614 and the ketoamine oxidase 1602 are formed in the form of an immobilized layer. The peptide fragments transported into the ketoamine oxidase housing section 1656 react with the ketoamine oxidase 1602, and as a result, hydrogen peroxide is generated. The generated hydrogen peroxide is detected by the nearby hydrogen peroxide detector 1603.

[0181] In a configuration where the protease and the ketoamine oxidase are separately arranged, there may be noise sources in substances in solution, etc. The sensor of the present invention may further have a mechanism for reducing such noise and may be configured in a manner connected to the mechanism.

[0182] In some embodiments, the hydrogen peroxide detector may be covered with an ion exchange resin, and a layer or film of the ion exchange resin may be disposed on the surface of the hydrogen peroxide detector.

[0183] The sensor of some embodiments may be a pair or a group of differential or difference sensors having a configuration in which the protease and the ketoamine oxidase are separately arranged.

[0184] In the differential sensor of some embodiments, the hydrogen peroxide detector may be covered with an ion exchange resin, and a layer or film of the ion exchange resin may be disposed on the surface of the hydrogen peroxide detector.

[0185] Figure 16 In the shown sensor 1700, the protease and the ketoamine oxidase are separated and arranged in separate housing sections.

[0186] The solution introduced from the inlet 1752 is split into the main sensor 1700a and the sub-sensor 1700b. The solution is introduced into the protease housing section 1755a in the main sensor 1700a. An enzyme layer 1711a immobilized with the protease is disposed in the protease housing section 1755a. The peptide fragments generated in the protease housing section 1755a are transported through the flow path to the ketoamine oxidase housing container 1756a. In Figure 16 In the shown ketoamine oxidase housing container 1756a, a ketoamine oxidase layer 1712a immobilized with the ketoamine oxidase and the hydrogen peroxide detector 1703a are disposed. Through the reaction in the ketoamine oxidase housing container 1756a, the glycated peptide fragments finally form hydrogen peroxide and are detected.

[0187] On the other hand, the flow path and the housing part of the sub-sensor 1700b are configured in the same manner as those of the main sensor 1700a, but no protease is disposed. That is, the chamber 1755b into which the solution is first introduced has the same structure as the protease housing part 1755a of the main sensor 1700a, but does not house protease. As the chamber (housing part) after the protease-free chamber 1755b, a ketamine oxidase housing container 1756b is disposed. In the ketamine oxidase housing container 1756b of the sub-sensor 1700b, a ketamine oxidase layer 1712b in which ketamine oxidase is immobilized and a hydrogen peroxide detector 1703b are disposed in the same manner as in the ketamine oxidase housing container 1756a of the main sensor 1700a.

[0188] In the protease housing part 1755a of the main sensor 1700a and the corresponding chamber 1755b of the sub-sensor 1700b, a heater 1760 is provided. Thereby, the rate of the decomposition reaction based on protease in the protease housing part 1755a can be increased, and the reaction can be made efficient or optimized. Moreover, the configuration and conditions of the flow path of the sub-sensor 1700b can be made as identical or the same as possible to those of the flow path of the main sensor 1700a except for the absence of protease.

[0189] A differential signal of the output signal from the hydrogen peroxide detector 1703a of the main sensor 1700a and the output signal from the hydrogen peroxide detector 1703b of the sub-sensor 1700b can be calculated. By this differential operation, the concentration of the target hydrogen peroxide in the hydrogen peroxide detector 1712a can be obtained, and then the concentration of the glycated protein as the analyte in the original solution related thereto can be obtained.

[0190] In some embodiments, temperature control can be performed on protease and ketamine oxidase respectively. In some embodiments, temperature control devices for protease and ketamine oxidase can be disposed respectively. The temperature control device can be a heating device, can be a cooling device, can have both of them, and can be a device capable of performing heating and cooling control.

[0191] In some embodiments, the sensor can have a temperature control housing part between the protease housing part and the ketamine oxidase housing part. In some embodiments, when the operating temperatures of protease and ketamine oxidase are different, the temperature control housing part can change or control the temperature of the solution sent from the protease housing part so as to make it close to the operating temperature of ketamine oxidase. In some embodiments, a heating device can be disposed for protease and a cooling device can be disposed in the temperature control housing part. In some embodiments, a heating device can be disposed for ketamine oxidase.

[0192] Figure 17The illustrated sensor 1800 has a protease containment section 1855, a cooling solution containment section (cooling section) 1858, and a ketamine oxidase containment section 1856 that are fluid-connected in series. The protease containment section 1855 has a liquid inlet 1852 through which a solution can be introduced into the protease containment section 1855. In the protease containment section 1855, a protease 1801 is contained in a state fixed to microbeads 1804.

[0193] Figure 17 A heater 1860 is disposed in the illustrated protease containment section 1855. The heater 1860 heats the solution or the protease 1801 in the protease containment section 1855 or controls its temperature, which can increase the speed of the protease reaction, etc., and make the reaction efficient or optimized.

[0194] The peptide fragments generated in the protease containment section 1855 are sent to the fluid-connected ketamine oxidase containment section 1856 via the cooling solution containment section 1858. In the ketamine oxidase containment section 1856, an immobilized ketamine oxidase 1802 and a hydrogen peroxide detector 1803 are contained. The hydrogen peroxide detector 1803 is connected to a measuring device (not shown) and can send an electrical signal to it.

[0195] In the ketamine oxidase containment section 1856, the ketamine oxidase 1802 is fixed to a substrate 1814 to form an enzyme layer 1811. Figure 17 In it, a substrate 1814 and a ketamine oxidase 1802 are formed in the form of an immobilized layer in the ketamine oxidase containment section 1856. The peptide fragments transported into the ketamine oxidase containment section 1856 react with the ketamine oxidase 1802, and as a result, hydrogen peroxide is produced. The produced hydrogen peroxide is detected by the proximately arranged hydrogen peroxide detector 1803.

[0196] A heater 1861 is disposed in the ketamine oxidase containment section 1856. The heater 1861 heats the solution or the ketamine oxidase 1802 in the ketamine oxidase containment section 1856 or controls its temperature, which can increase the speed of the enzyme reaction, etc., and make the reaction efficient or optimized.

[0197] Figure 17In this case, heaters 1860 and 1861 are respectively arranged in the protease accommodating section 1855 and the ketoamine oxidase accommodating section 1856. Thus, the protease 1801 and the ketoamine oxidase 1802 can be respectively temperature-controlled or heated. Thus, for example, independent temperature control can be performed in terms of time or temperature, or both. In some embodiments, the temperature of the protease accommodating section 1855 or the protease 1801 can be controlled to be higher than the temperature of the ketoamine oxidase accommodating section 1856 or the ketoamine oxidase 1802. In some embodiments, the temperature of the protease accommodating section 1855 or the protease 1801 can be controlled to be lower than the temperature of the ketoamine oxidase accommodating section 1856 or the ketoamine oxidase 1802.

[0198] If the solution heated in the protease accommodating section 1855 is sent to the ketoamine oxidase accommodating section 1856, it takes time for the solution to reach a suitable temperature for the ketoamine oxidase 1802, which is inefficient or sometimes cannot be accurately measured. Therefore, the solution can be temporarily cooled before being introduced into the ketoamine oxidase accommodating section 1856. Figure 17 The sensor 1800 as shown can keep the solution heated in the heated protease accommodating section 1855 in the cooling solution accommodating section 1858. The solution is cooled by a cooling device (circuit / element) 1862 (such as a Peltier element) to make it close to the optimum temperature or the operating temperature of the ketoamine oxidase 1802. Thereafter, the solution can be sent to the ketoamine oxidase accommodating section 1856. Thus, for example, the measurement can be performed efficiently or accurately.

[0199] As an example, Figure 17 The sensor 1800 as shown can be used as follows. That is, first, a solution containing the object to be measured is introduced into the protease accommodating section 1855. The protease accommodating section 1855 can start heating before the solution is introduced or after the solution is introduced. After peptide fragmentation is performed in the protease accommodating section 1855, the solution is sent to the cooling solution accommodating section 1858. The cooling solution accommodating section 1858 can start cooling before the solution is introduced or after the solution is introduced. After sufficient cooling or cooling to a suitable temperature in the cooling solution accommodating section 1858, the solution can be sent to the ketoamine oxidase accommodating section 1856. The ketoamine oxidase accommodating section 1856 can start heating after the solution is introduced.

[0200] In some embodiments, the liquid delivery can be performed by applying pressure from the solution inlet 1852. In some embodiments, the liquid delivery can be performed by changing the volume of each storage unit 1855, 1856, 1858 or applying pressure to each storage unit. The protease storage unit 1855 may have air holes (not shown). In some embodiments, the pressure during liquid delivery can be a positive pressure. In some embodiments, the pressure during liquid delivery can be a negative pressure. In some embodiments, valves can be respectively arranged near the inlet and outlet of each storage unit, or valves can be arranged near both of them. The valve can perform the following function: removing bubbles that may be generated due to temperature differences in each storage unit from the flow path.

[0201] In some embodiments, when the solution leaves the protease storage unit, the protease and the beads can also leave the protease storage unit. For example, the protease and the beads can enter the cooling storage unit and can enter the ketamine oxidase storage unit. In some embodiments, the sensor can be configured such that the protease and the beads substantially remain in the protease storage unit during liquid delivery.

[0202] Figure 17 The heaters 1860, 1861 and the cooling device 1862 using electric current are described, but are not limited thereto. The heaters 1860, 1861 can adopt heating methods other than electricity. The cooling device 1862 can adopt cooling methods other than Peltier elements. In another embodiment, other temperature regulators can be arranged.

[0203] In some embodiments, the solution inlet 1852, the protease storage unit 1855, the cooling solution storage unit 1858 and the ketamine oxidase storage unit 1856 can be a single component, such as a cartridge or a disposable fluid device. In some embodiments, the heaters 1860, 1861 and the cooling device 1862 can be fixed to the main body. The sensor 1800 can be configured such that the above-mentioned fluid device is inserted or fixed to the main body. An adiabatic material can be arranged between the heaters 1860, 1861 and the cooling device 1862. Thus, for example, the efficiency of temperature control can be improved.

[0204] 3. Configuration of Hydrogen Peroxide Electrode

[0205] The layout or configuration of the hydrogen peroxide electrode can have various structures. The configuration of the hydrogen peroxide electrode is illustratively described below.

[0206] <Electrode Configuration Example 1>

[0207] The hydrogen peroxide electrode shown in the present invention is not limited to the use for the determination of glycated proteins or fructosamines, and can also be used for other purposes including the electrochemical determination of solutions. That is, the sensor or sensor chip according to an embodiment of the present invention includes a hydrogen peroxide electrode. The reference electrode of the hydrogen peroxide electrode may be sandwiched between the counter electrode and the working electrode. The sensor chip may have a liquid storage portion. The liquid storage portion may extend along the length direction. The volume of the liquid storage portion may be 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, 0.1 μL or less than this value. The liquid storage portion may have a liquid inlet. The liquid storage portion may have a liquid outlet. The liquid storage portion may have a gas hole. The gas hole may have the following function: when the liquid is introduced into the liquid storage portion, the gas existing in the liquid storage portion is discharged to the outside of the sensor chip. The sensor chip may have a circuit and may be configured in a manner connected to the circuit. The sensor chip may have an output terminal for connecting to the circuit.

[0208] Figure 18 FIG. is a plan view schematically showing the electrode configuration of a sensor chip as an embodiment. Figure 18 In the sensor chip 2000, the counter electrode 2031, the reference electrode 2032, and the working electrode 2033 are arranged on the substrate 2006 in contact with the introduced solution, and the enzyme layer 2011 is arranged to cover these electrodes.

[0209] The sensor chip 2000 may further have a member for partitioning 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. Figure 18 In, a sensor chip cover (not shown) that defines the flow path and the liquid storage portion covers the upper surface of the sensor chip. A portion 2051 on the sensor chip corresponding to the liquid storage portion is defined, and the liquid is introduced into the liquid storage portion 2051 from the inlet 2052. For the liquid storage portion 2051, the chip cover has a gas hole 2053 on the side opposite to the liquid inlet 2052.

[0210] The hydrogen peroxide electrodes 2031, 2032, and 2033 are arranged parallel to each other and extend along the length direction of the liquid storage portion 2051. On the side of the liquid inlet 2052, the end of the working electrode 2033 is formed shorter than the ends of the other electrodes, that is, the counter electrode 2031 and the reference electrode 2032. The liquid introduced from the inlet 2052 advances along the length direction in the liquid storage portion 2051. If the liquid contacts the working electrode 2033 first or simultaneously with the other electrodes, a large current may flow and damage the counter electrode. By adopting such a configuration, damage to the electrodes can be avoided.

[0211] Figure 18 The sensor chip 2000 shown further has a liquid detection electrode 2034 in the liquid storage portion. Figure 18 Among them, the electrode 2034 is arranged at the position where the liquid finally contacts when the liquid enters the liquid storage portion 2051. By detecting whether there is liquid at the liquid detection electrode 2034, it can be confirmed that the liquid storage portion 2051 or the hydrogen peroxide electrodes 2031, 2032, 2033 have been sufficiently filled with liquid. When liquid is detected at the liquid detection electrode 2034, it is possible to notify the user that the collection of the liquid has been completed. Alternatively, when liquid is not detected at the liquid detection electrode 2034 even after waiting for a prescribed time or more, it is possible to notify that the liquid has not been sufficiently collected and the measurement cannot be started, or to notify the user to collect the liquid again, use another chip, etc.

[0212] Figure 18 The sensor chip 2000 shown has output terminals 2041, 2042, 2043, 2044 corresponding to and electrically connected to the counter electrode 2031, the reference electrode 2032, the working electrode 2033, and the liquid detection electrode 2034. The sensor chip can be connected to a circuit or other devices having a circuit (not shown in both cases) in a plug-in or detachable manner to form a structure. Figure 18 In the sensor chip 2000 shown, the above output terminals are arranged in its detachable portion.

[0213] Figure 18 Only one detection electrode is described therein. However, in some embodiments, two or more detection electrodes can be arranged. Two or more electrodes can be arranged along the advancing direction of the liquid in the flow path. For example, the detection electrodes can be arranged at the position where the liquid first contacts the detection portion, the position where the liquid is in full contact with the detection portion, and the middle of the two. For example, the first detection electrode can detect the point where the liquid starts to enter the detection portion, the flow path, or the liquid storage portion. The middle electrode can detect whether the liquid has entered this point. When the liquid has not entered the middle electrode, only a part of the detection portion is used, so it can be used to notify that the measurement cannot be performed or to notify that the measurement error is large.

[0214] Hereinafter, other configurations and modification examples will be illustratively described. Sometimes, some configurations and descriptions may be omitted. In each embodiment and illustration, other configurations can be applied.

[0215] <Two or more electrode arrangements>

[0216] In the liquid storage portion of the sensor chip, two or more or two or more types of sensors or hydrogen peroxide electrodes can be arranged. Two or more sensors can be of the same type, or can be a combination of a main sensor and a sub-sensor for differential measurement as described above.

[0217] <Example of Electrode Arrangement 2>

[0218] Figure 19 In the sensor chip 2100 shown, two or more sensors 2100a and 2100b are arranged parallel to each other along the length direction within the liquid containment portion 2151. It should be noted that for the reference electrodes and counter electrodes of the two or more sensors 2100a and 2100b, the reference electrodes are connected to each other and the working electrodes are connected to each other and are in communication in the circuit. The liquid is introduced from the fluid inlet 2152 and advances in the liquid containment portion 2151 by means of capillary action and the action of the immobilized film that also acts as a hydrophilic water-containing polymer, etc., squeezing out the internal air from the air holes 2153 and filling the liquid containment portion 2151, and finally reaching the detection electrode 2134.

[0219] <Example of Electrode Arrangement 3>

[0220] Figure 20 In the sensor chip 2200 shown, a part of the hydrogen peroxide electrode as shown is shared among two or more sensors. That is, Figure 12 as shown, Figure 20 the sensor chip 2200 shown 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 arranged on the substrate 2206, and an enzyme layer 2221a for the main sensor is arranged on these electrodes. In the sub-sensor 2220b, a working electrode 2233b and a reference electrode 2232 are arranged on the substrate 2206, and an enzyme layer 2221b for the sub-sensor is arranged on these electrodes. The reference electrode 2232 is arranged on the substrate 2206 or sandwiched between the counter electrode 2231 and the working electrode 2233b.

[0221] <Assembly Type of Two or More Sensors>

[0222] When two or more sensors are arranged in the same sensor chip, it is not necessary to directly form all the sensors on the main substrate of the sensor chip. For example, each manufactured sensor can be joined to the main substrate.

[0223] Figure 21 In the sensor chip 2300 shown, two manufactured sensors 2300a and 2300b are joined to the main substrate 2360.

[0224] The enzyme layers 2311a and 2311b or the hydrogen peroxide electrodes 2330a and 2330b of the sensors 2300a and 2300b are formed in a manner that extends along the length direction of the liquid containment portion 2351 from the liquid inlet 2352 to the air hole 2353.

[0225] Figure 21 In the sensor chip 2300, corresponding bonding pads 2340a, 2340b, 2341a, and 2341b are respectively formed on the main body substrate 2360 and on each sensor. After the sensors 2300a and 2300b are bonded to the main body substrate 2360 or during the bonding process, these bonding pads are electrically bonded. For example, the bonding pads can be connected by wire bonding.

[0226] Figure 21 In the sensor chip 2300, output terminals 2340 capable of being connected to a circuit (not shown) etc. are arranged on the main body substrate 2360. These output terminals 2340 are connected to the corresponding bonding pads 2341a and 2341b through wirings. The counter electrode and the reference electrode can be short-circuited via the sensor chip 2300 or the circuit. According to such a configuration, for example, in cases where it is difficult to fabricate two sensors in one chip simultaneously due to the durability of the enzyme layer and the constitution of other layers, or where the yield rates of the two sensors are significantly different, the total manufacturing cost can be suppressed by fabricating them separately and combining the qualified products.

[0227] <Other electrode directions>

[0228] The hydrogen peroxide electrode can be formed in a direction other than the length direction of the elongated liquid storage portion.

[0229] For example, as Figure 22 shown in the sensor chip 2400, each electrode extending in a direction perpendicular to the length direction of the liquid storage portion 2451 can be arranged on the substrate 2406. More specifically, Figure 22 in the hydrogen peroxide electrode, the counter electrode 2431, the reference electrode 2432, and the working electrode 2433 are arranged in the liquid storage portion 2451 in a direction perpendicular to the length direction from the liquid inlet 2452 to 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 among the three electrodes of the hydrogen peroxide electrode in the liquid advancing direction during liquid introduction. A liquid detection electrode 2434 is arranged after these hydrogen peroxide electrodes. An enzyme layer 2411 is formed on the hydrogen peroxide electrode. By sandwiching the reference electrode 2432 between the counter electrode 2431 and the working electrode 2433 as in this electrode configuration, for example, external noise can be reduced, and when the liquid enters the liquid storage portion 2451, it finally contacts the working electrode 2433, thereby avoiding an excessive current from flowing and enabling safe measurement.

[0230] 4. Manufacturing method of the sensor

[0231] The following uses Figures 23A to 23IThe cross-sectional schematic diagram illustrates the manufacturing method of a dual glycated protein sensor as an embodiment of the present invention.

[0232] <Formation of Hydrogen Peroxide Electrodes>

[0233] As Figure 23A shown, two sets of electrodes, namely a first hydrogen peroxide electrode 3030a and a second hydrogen peroxide electrode 3030b, are formed on an insulating substrate 3006. The insulating substrate 3006 can be a substrate mainly composed of glass, quartz, or ceramic. The insulating substrate 3006 can be polyethylene terephthalate (PET), polyethylene naphthalate (PEN), cycloolefin polymer (COP) resin, etc. These are known as materials with excellent water resistance, heat resistance, chemical resistance, and adhesion to the above electrodes.

[0234] For each set of hydrogen peroxide electrodes 3030a and 3030b, a working electrode formed by laminating Ti / Pt, a reference electrode formed by laminating Ti / Pt / Ag / AgCl, and a counter electrode formed by laminating Ti / Pt are formed. These laminated structures are not shown in the figure. As an example, these electrodes can be formed using sputtering, ion plating, vacuum evaporation, chemical vapor deposition (CVD), electrolysis, screen printing, etc. The sputtering method can form a thin platinum film with high precision in a short time. For example, first, films are formed and patterned in the order of titanium (Ti) and platinum (Pt). Then, the working electrode and the counter electrode are covered, and a silver (Ag) film is formed on the reference electrode. Finally, the surface of silver is chlorinated. Thus, the above-mentioned laminated electrode structure can be formed. It should be noted that in most cases, Ti is deposited to improve the adhesion between the substrate and Pt. Therefore, depending on the substrate and film formation conditions, the Ti layer of the hydrogen peroxide electrode can be omitted. For example, in the case of using a flexible sheet-like insulating substrate such as PET resin or COP resin, the Ti layer of the hydrogen peroxide electrode can be omitted.

[0235] <Formation of Enzyme Layer for Sub-Sensor>

[0236] First, as Figure 23B shown, a positive photoresist 3009 used in a lithography process such as semiconductor manufacturing is spin-coated on the insulating substrate 3006 to cover the two sets of hydrogen peroxide electrodes 3030a and 3030b.

[0237] Next, as Figure 23C shown, the photoresist 3009 is patterned by exposure, development, and etching to form an opening on the second hydrogen peroxide electrode 3030b.

[0238] As Figure 23DAs shown, a 3-aminopropyltriethoxysilane (APTES) solution 3005b of 3-aminopropyltriethoxysilane as a silane coupling agent was spin-coated at 3000 rpm for 30 seconds and dried. Then, a solution containing BSA as the substrate 3014 and ketamine oxidase 3002 was mixed with glutaraldehyde. Then, this mixture was spin-coated onto the substrate. The ketamine oxidase 3002 was immobilized by the cross-linking reaction of glutaraldehyde.

[0239] The resist 3009 was ultrasonically treated in acetone for lift-off. Through the silane coupling agent 3005b, only the enzyme film 3011b containing BSA as the substrate 3014 and ketamine oxidase 3002 remained on the second hydrogen peroxide electrode 3030b. Thus, the sub-sensor 3000b was formed on the insulating substrate 3006, and the first hydrogen peroxide electrode 3030a for the main sensor was exposed ( Figure 23E ).

[0240] And Figure 23B In the same manner as the process described in, the resist 3091 was again spin-coated over the entire insulating substrate 3006. After coating, the resist 3091 was cured under conditions that did not inactivate the enzyme ( Figure 23F ).

[0241] <Formation of Enzyme Layer for Main Sensor>

[0242] First, the resist 3091 was patterned by exposure, development, and etching to form an opening on the first hydrogen peroxide electrode ( Figure 23G ).

[0243] With the opening present on the first hydrogen peroxide electrode 3030a, in the same manner as Figure 23D , a 3-aminopropyltriethoxysilane (APTES) solution 3005a of 3-aminopropyltriethoxysilane as a silane coupling agent was spin-coated at 3000 rpm for 30 seconds and dried. Thereafter, a solution containing BSA as the substrate 3004, ketamine oxidase 3002, and protease 3001 was mixed with glutaraldehyde. Then, this mixture was spin-coated onto the substrate. The ketamine oxidase 3002 and protease 3001 were immobilized by the cross-linking reaction of glutaraldehyde ( Figure 23H ).

[0244] The resist 3091 is subjected to ultrasonic treatment in acetone to be peeled off. Thus, a sensor is prepared in which an enzyme layer 3011a and an enzyme layer 3011b remain on the first and second hydrogen peroxide electrodes 3330a and 3330b on the insulating substrate 3006 respectively through the silane coupling agents 3005a and 3005b, respectively. The enzyme layer 3011a includes a protease 3001 and a ketoamine oxidase 3002 fixed to BSA3004 as a substrate, and the enzyme layer 3011b includes only ketoamine oxidase 3002 as an enzyme and is fixed to BSA3014 as a substrate. That is, a main sensor 3000a and a sub-sensor 3000b are formed on the insulating substrate 3006. By the above operation, a sensor 3000 (having a differential type dual glycated albumin sensor enzyme layer that differs only in the presence or absence of the protease 3001) can be prepared. Figure 23I ).

[0245] It should be noted that, although the manufacturing method using patterning with a resist is described in this embodiment, a metal mask having an opening on the first hydrogen peroxide electrode and a metal mask having an opening on the second hydrogen peroxide electrode may be used.

[0246] In some embodiments, a glycated protein sensor (detection unit, detector) may include an albumin sensor (detection unit, detector). Glycated protein sensors and albumin sensors may be collectively referred to as sensors, and a system configured to measure both glycated protein and albumin may be referred to as a glycated protein sensor. The sensor may be configured in such a way that body fluids or liquids obtained from the same sampling are used for both the glycated protein sensor and the albumin sensor. In another embodiment, the sensor may be configured in such a way 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 in such a way that it is connected to an albumin sensor. By working in conjunction with an albumin sensor, the glycated albumin (GA) value may be determined more simply and efficiently.

[0247] As an albumin sensor, for example, the measurement can be performed by a dye binding method utilizing a change in the absorption wavelength or absorption spectrum when albumin binds to a dye such as bromocresol chloride (BCG) or bromocresol purple (BCP), or an electrochemical method. In addition, a device or apparatus based on an immunological method using an antibody or the like can be used.

[0248] In some embodiments, the enzyme (ketoamine oxidase and / or protease) may be covered by a protective agent. The protective agent may be a protective layer, a protective film or a protective coating.

[0249] like Figure 24 As shown, the protective agent can be configured in such a way as to cover the enzyme layer. Figure 24The ketamine oxidase section is shown, and the protease and other elements and configurations are omitted. A hydrogen peroxide detector 4003 is disposed on a substrate 4006, and on it, a ketamine oxidase layer 4012 is formed by including a ketamine oxidase 4002 in a substrate 4014. A protective film 4008 is disposed so as to cover the ketamine oxidase 4002 or the ketamine oxidase layer 4012. The protective film 4008 can be disposed so as to also cover the hydrogen peroxide detector 4003.

[0250] As Figure 25 shown, the protective agent can be disposed so as to cover each enzyme molecule, or can be disposed so as to cover a part of the enzyme or an aggregate of enzyme molecules. Figure 25 The ketamine oxidase section is shown, and the protease and other elements and configurations are omitted. A hydrogen peroxide detector 4103 is disposed on a substrate 4106, and on it, a ketamine oxidase layer 4112 is formed by including a ketamine oxidase 4102 in a substrate 4114. The protective coating can cover the ketamine oxidase 4102 in a molecule-by-molecule or one-by-one manner. It can be formed so as to cover two or more ketamine oxidases 4102.

[0251] In some embodiments, the protective agent for the enzyme can be selected from the material of the substrate. In some embodiments, the protective agent for the enzyme can be the same material as the substrate of the enzyme. When the substrate of the enzyme and the protective agent are formed of the same material, for example, peeling and breakage caused by thermal stress due to differences in thermal expansion coefficients caused by heating during measurement or temperature changes at room temperature can be avoided. The protective agent can, for example, prevent enzyme deterioration or extend the lifespan, but is not limited thereto. For example, when a protease not immobilized on the device main body or a part of the immobilized protease detaches and flows out and comes into contact with the ketamine oxidase, it may decompose or denature it. The protective agent can, for example, prevent or inhibit such decomposition of the ketamine oxidase. In some embodiments, the protective agent for the ketamine oxidase can be formed of a material that is not easily decomposed by protease.

[0252] As a method for forming the protective agent, after coating (e.g., spin coating, bar coating) a FOAD film and crosslinking it (standing, crosslinking time), and drying, a protective film can be formed. The protective film can be formed so as to have a uniform film thickness. Depending on the nature of the solution used, the protective film can be coated by a spin coating method, a bar coating method, a doctor blade method, a spraying method, or a dipping method. The coated protective film can be held for a specified time and at a specified temperature. Through the post-treatment after coating, the desired film thickness can be obtained.

[0253] The protective agent can be, for example, a fluorine-based resin, a photocurable resin, a water-curable resin, cellulose (such as ethyl cellulose, cellulose acetate, etc.), nylon, polystyrene, a non-protein-based resin, a polyelectrolyte complex, a complex, an inorganic polymer, a solid polymer electrolyte, a porous polymer metal complex (such as ZIF-8), a metal-organic structure, or a phospholipid, but not limited thereto. In some embodiments, the protective agent of ketamine oxidase can include albumin and can be substantially formed of albumin. In some embodiments, the albumin as the protective agent can be BSA. It has been confirmed that for a system where the lifespan of ketamine oxidase without a protective agent is 1 week, by covering it with a protective agent formed of BSA, the lifespan of ketamine oxidase is extended to 1 month, and the time when the output becomes half is doubled.

[0254] As a non-limiting example, the protective agent can reduce the influence of heat vibration and the like caused by the liquid on the lifespan. The enzyme can be wrapped and fixed by the substrate, can be partially fixed, or can be coated by the substrate. As an explanation, the protective agent can protect the part of the enzyme molecules exposed on the surface of the substrate. As an explanation, it can also be considered that the lifespan of the enzyme is extended when it is protected by the protective agent. For example, it can reduce the influence of inclusions on the enzyme activity, enzyme reaction efficiency, etc., but not limited thereto. For example, a protective film or the like can prevent or reduce the detachment or separation of the enzyme from the substrate, but not limited thereto. For example, the protective film can prevent denaturation (protein denaturation) caused by drastic changes in parameters such as external pressure, pH, and salt concentration during liquid feeding, etc., but not limited thereto.

[0255] The protease and ketamine oxidase will be described below using several examples.

[0256] <Example 1>

[0257] The working characteristics of various proteases were compared.

[0258] Dissolve 16 mg of Orientase 22BF (manufactured by HBI Co., Ltd.) as a protease, 133 mg of Nucleicin (manufactured by HBI Co., Ltd.), 12.8 mg of Orientase AY (manufactured by HBI Co., Ltd.), 8.0 mg of Orientase OP (manufactured by HBI Co., Ltd.), 16 mg of Sumizyme MP (manufactured by Shin Nippon Chemical Industry Co., Ltd.), and 4 mg of protease XIV (Sigma-Aldrich Japan K.K.) separately in 400 μL of TES buffer, and dialyze overnight. Use the resulting solution as the protease solution. Dissolve 4 mg of thermolysin (manufactured by Fujifilm Wako Pure Chemical Corporation), 4 mg of trypsin (manufactured by Fujifilm Wako Pure Chemical Corporation), 4 mg of α-chymotrypsin (manufactured by Tokyo Chemical Industry Co., Ltd.), 4 mg of papain (manufactured by Nacalai Tesque, Inc.), and 4 mg of bromelain (manufactured by Fujifilm Wako Pure Chemical Corporation) separately in 400 μL of TES buffer and use directly.

[0259] Mix 12 μL of the GA-L calibrator (GA concentration: 14.9 mg / mL, albumin concentration: 47.9 mg / mL) of the commercially available glycated albumin kit Lucica GA-L (manufactured by Asahi Kasei Pharma Corporation) and 93 μL of TES buffer, and add 15 μL of the protease solution thereto. Heat the resulting mixed solution in an aluminum block incubator at 37 °C or 60 °C for 10 minutes. Remove the mixed solution from the aluminum block and immediately place it in the refrigerator to avoid digestion at residual heat and room temperature, and then store it at low temperature thereafter. This is called the GA digestion sample. On the other hand, mix the protease solution and TES buffer. Perform the same heat treatment on this mixed solution. This is called the protease autodigestion sample.

[0260] Use the commercially available ALB R-2 solution of the Lucica glycated albumin kit and a microplate reader to measure the absorbance. Calculate the digestion rate of albumin of each protease from the change in absorbance before and after digestion of the GA digestion sample.

[0261] Figure 26The digestibility at heating temperatures of 37°C and 60°C is shown. 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 proteases of bacterial origin. As an explanation, it can be said that the digestibility of proteases of bacterial origin is higher than that of proteases of animal origin or plant origin, or the digestibility of proteases of animal origin or plant origin is lower than that of proteases of bacterial origin. This explanation shows an idea and can also be other theories or experimental results. Based on the results of this experiment, in several embodiments, as the protease for digesting albumin, a protease selected from the group consisting of Orientase 22BF, protease XIV, Sumizyme MP, and thermolysin can be used. Alternatively, two or more of these proteases can be used in combination.

[0262] Figure 27 In this case, the current value of the GA digestion sample was measured using a FAOD electrode. The net output of the digested GA was represented by the difference between the output of the digested GA sample and the output of the protease autodigestion sample.

[0263] As Figure 27 shown, Orientase 22BF, protease XIV, and Sumizyme MP provided relatively high outputs. As an explanation, it can also be said that Orientase 22BF, protease XIV, and Sumizyme MP are ideal proteases for digesting albumin into glycopeptide fragments of a size that can react with FAOD. This explanation shows an idea and can also be other theories or experimental results.

[0264] <Example 2>

[0265] The working temperature of the protease was studied.

[0266] 16 mg of Orientase 22BF (manufactured by HBI Co., Ltd.) was dissolved in 400 μL of TES buffer and dialyzed overnight, and the resulting solution was used as the protease solution.

[0267] Mix 12 μL of the GA-L calibrator (GA concentration: 14.8 mg / mL, albumin concentration: 47.5 mg / mL) of the glycated albumin kit Lucica GA-L (Asahi Kasei Pharma Corporation) with 93 μL of TES buffer, and add 15 μL of protease solution thereto. At the same time, also prepare a sample solution containing only protease and a sample solution containing only the calibrator. Keep these in an aluminum block incubator at each temperature of 20°C, 30°C, 40°C, 50°C, 60°C, and 70°C for 2.5 minutes or 10 minutes. Then remove each solution from the aluminum block, and immediately place it in a refrigerator to avoid digestion at the residual heat and room temperature, and then store it at a low temperature thereafter.

[0268] Measure the output current value of the FAOD electrode when introducing the GA-digested sample. Based on the output current value of this GA-digested sample, take the difference between the output current value of the sample solution containing only protease (protease autodigested sample) and the output current value of the sample solution containing only GA (GA-digested sample), that is, [output current value of GA-digested sample] - [output current value of sample solution containing only protease] - [output current value of sample solution containing only GA] as the output current value of the remaining digested GA. Figure 28 In it, the output current value (a) of the remaining digested GA is plotted in the form of a function of the digestion temperature.

[0269] As Figure 28 shown, the output current value from the FAOD electrode is the highest around 60°C or between 60°C and 70°C. In some embodiments, the digestion temperature of the protease can be set within the range where the output current value from the FAOD electrode is high. For example, the digestion temperature of the protease can be set around 60°C or between 60°C and 70°C where the output current value from the FAOD electrode is high. The digestion temperature of the protease can be 50°C, 55°C, or higher than 60, or even higher. The digestion temperature of the protease can be below or lower than the inactivation temperature of the protease. In some embodiments, the digestion temperature of the protease can be different from the optimal temperature range of the FAOD. Figure 28 In it, around 60°C or between 60°C and 70°C where the output current value from the FAOD electrode is high. The digestion temperature of the protease can be 50°C, 55°C, or higher than 60, or even higher. The digestion temperature of the protease can be below or lower than the inactivation temperature of the protease. In some embodiments, the digestion temperature of the protease can be different from the optimal temperature range of the FAOD.

[0270] <Example 3>

[0271] Studied the working temperature of flavin adenine dinucleotide-dependent amine oxidase (FAOD).

[0272] Dissolve the trifluoroacetate salt of N-ε-(1-deoxyfructosyl)-L-lysine (FK) (Peptide Institute, Inc.) in TES buffer to prepare a 12.8 μM FK solution. At each temperature of 10°C, 20°C, 30°C, 40°C, 50°C, and 60°C, measure the output current value from the FAOD electrode when introducing this FK solution ( Figure 28 of (b)).

[0273] As Figure 28 shown, the output current value from the FAOD electrode is highest around 40°C. As an explanation, it can be said that the optimal temperature range of the FAOD electrode is around 40°C. The temperature of the FAOD electrode or its vicinity can be set to around 40°C. In some embodiments, the temperature of the FAOD electrode or its vicinity can be different from the optimal temperature or temperature range of the protease.

[0274] <Example 4>

[0275] The dependence of the output current of the FAOD electrode on the GA concentration was investigated.

[0276] Solutions prepared by dissolving albumin powder with different GA values in TES buffer were used as GA sample solutions.

[0277] 4 mg of protease XIV (Sigma-Aldrich Japan Co., Ltd.) and 16 mg of Orientase 22BF (manufactured by HBI Co., Ltd.) were separately dissolved in 400 μL of TES buffer and dialyzed overnight, and the resulting solutions were prepared as protease solutions.

[0278] 40 μL of a latex bead (IMMUTEX P0113, Cosmo Bio Co., Ltd.) dispersion solution was mixed with 400 μL of TES buffer, and 50 mg of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine hydrochloride (DMT-MM, Tokyo Chemical Industry Co., Ltd.) was added thereto and dissolved, and the solution was stirred at room temperature for 2 hours. Centrifugation (12,000 rpm, 20 minutes) was performed to remove the supernatant. 400 μL of TES buffer was added, and the solution was stirred. This operation was repeated 3 times to wash the beads and remove excess DMT-MM. After removing the supernatant for the third time, 400 μL of the dialyzed protease solution was added, and the solution was stirred in a refrigerator for 4 hours. 400 μL of TES buffer was added, and after centrifugation (12,000 rpm, 20 minutes), the supernatant was removed. 800 μL of TES buffer was added, and the solution was stirred. This operation was repeated 3 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 a refrigerator.

[0279] 60 μL of a 1.67 mg / mL GA sample solution and 20 μL of the protease-immobilized bead solution were thoroughly mixed. The mixed solution was heated at 37°C for 30 minutes using an aluminum block incubator. After 30 minutes, 40 μL of TES buffer was added, and after centrifugation (12,000 rpm, 30 minutes), the supernatant was taken out. This solution was used as the digested GA sample.

[0280] Introduce the digested GA sample solution and measure the output current value from the FAOD electrode. Figure 29 In, this output current value is shown as a function of the GA concentration. As Figure 29 shown, although the value of the intercept varies depending on the protease, there is a linear relationship between the GA concentration and the output current value from the FAOD electrode of the digested GA sample solution. Moreover, it is known that when digestion is performed using a protease in which a GA solution is immobilized on microbeads, the output current value from the FAOD electrode of this digestion solution changes depending on the GA concentration in the system. This indicates that it is possible to measure the GA concentration using the immobilized protease and the FAOD electrode.

[0281] 6. Measurement method

[0282] Several embodiments of the measurement method of the present invention will be described below.

[0283] Introduce the measurement solution containing glycated protein into the sensor. The immobilized protease contacts the measurement solution containing glycated protein, fragmenting the glycated protein to generate peptide fragments. After generation, the peptide fragments diffuse and reach the immobilized ketamine oxidase. The immobilized ketamine oxidase generates hydrogen peroxide from the peptide fragments containing amino acid residues that have been glycated. The hydrogen peroxide detector detects the hydrogen peroxide generated by the above-mentioned ketamine oxidase.

[0284] In several embodiments, the glycated protein to be measured can be glycated albumin.

[0285] In several embodiments, first prepare a glycated protein sensor equipped with an immobilized protease, an immobilized ketamine oxidase, and a hydrogen peroxide detection unit. In addition, prepare a measurement solution containing glycated protein. Then, introduce the measurement solution into the glycated protein sensor. Use the glycated protein sensor to detect the glycated protein in the measurement solution.

[0286] In several embodiments, the concentration of the glycated protein can be determined from the output signal of the hydrogen peroxide detector. The step of detecting the glycated protein in the measurement solution may include the step of determining or calculating the concentration of the glycated protein in the measurement solution.

[0287] The concentration of glycated protein associated with the output signal from the hydrogen peroxide detection unit can be determined. The correlation, correlation relationship, function, etc. between the concentration of glycated protein in the calibration solution and the output signal of the hydrogen peroxide detector can be determined in advance or before the target measurement. The reference data, reference table, correlation, correlation relationship, function, etc. obtained therefrom can be used or referred to, and the concentration of glycated protein in the measurement solution can be determined from the output signal of the hydrogen peroxide detector obtained from the measurement solution. For example, when the detector measures current, based on a table (standard curve) that associates the current value with the concentration of glycated protein, referring to this table, an operation to determine the concentration of glycated protein can be performed from the current value measured in the circuit. The correlation, etc. can be an operation to determine the difference between the concentration conversion value based on the current value from the main (first) sensor and the concentration conversion value based on the current value from the sub (second) sensor.

[0288] In some embodiments, glycated protein can be detected, glycated protein when it is above or higher than a certain threshold can be detected, the concentration of glycated protein can be determined, glycated protein can be quantified, and the degree of glycation of the protein can be determined.

[0289] In some embodiments, the amount or concentration in solution of the total protein obtained by adding the glycated protein (the glycated protein to be measured) in the measurement solution and its corresponding non - glycated protein can be determined. The glycated protein can be glycated albumin.

[0290] In some embodiments, the ratio or proportion of the concentration of glycated protein (the protein obtained by glycating the target protein) to the total amount of the target protein can be determined. In some embodiments, the average blood glucose value during the period from the previous measurement to the current measurement can be further calculated by converting from this ratio. In some embodiments, the average blood glucose value for the corresponding period can be calculated by converting from the ratio obtained from two consecutive measurements. In some embodiments, the average blood glucose value for the corresponding period can be calculated by the ratio obtained from multiple measurements. In some embodiments, the average blood glucose value can be determined by dividing the sum of the ratios obtained from multiple measurements by the number of times. In some embodiments, the average blood glucose value can be determined in the form of a function obtained by weighting each ratio obtained, and as an example of this function, it can be determined in a function form where the weight is set to be larger for the ratio obtained in more recent measurements.

[0291] In some embodiments, notifications such as reminders for measuring the concentration of glycated protein can be given to the user.

[0292] The user can be a person who actually uses the glycated protein sensor, which refers to a person who measures the glycated protein of himself or others, a person who is scheduled to measure, a person who is required to measure or is recommended to measure, etc. The user can be an individual, can be one person or multiple people, and can be at least one person. The user can be one, multiple or at least one legal person, company or other organization.

[0293] The notification to the user can be made at a prescribed or appropriate timing. For measurements after the first time, the notification can be made at fixed or non-fixed intervals. The interval between any two measurements can 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, and can be other periods. For the measurement of glycated albumin, the notification can be made at intervals such as 7 days, 10 days, 15 days, 20 days, 1 week, 2 weeks, 3 weeks, etc.

[0294] In several embodiments, washing can be performed between multiple measurements. The washing can include: flushing the whole or part of the immobilized protease, the immobilized ketamine oxidase and the detection unit with pure water, physiological saline, buffer solutions such as TES, etc.

[0295] The program or computer program of several embodiments of the present invention can cause a computer to execute each step included in the measurement method described in this application, and can cause the computer to read and execute. The computer-readable storage medium of several embodiments of the present invention can store a program or computer program, and the program or computer program is used to cause a computer to execute each step included in the measurement method described in this application.

[0296] A01

[0297] A glycated protein sensor, which comprises an immobilized protease, an immobilized ketamine oxidase and a hydrogen peroxide detection unit.

[0298] A01a

[0299] According to the glycated protein sensor described in Embodiment A01, wherein the above-mentioned immobilized ketamine oxidase is arranged close to the above-mentioned hydrogen peroxide detection unit.

[0300] A01b

[0301] According to the glycated protein sensor described in Embodiment A01a, wherein the above-mentioned immobilized protease is arranged close to the above-mentioned immobilized ketamine oxidase.

[0302] A01c

[0303] The glycated protein sensor according to any one of Embodiments A01 to A01b, further comprising a liquid containing portion for containing a liquid to be measured.

[0304] A01d

[0305] The glycated protein sensor according to Embodiment A01c, wherein the volume of the liquid containing portion is 100 μL or less.

[0306] A02

[0307] A glycated protein sensor, comprising:

[0308] A hydrogen peroxide detection portion; and

[0309] An enzyme layer disposed on the hydrogen peroxide detection portion and containing an immobilized protease and an immobilized ketamine oxidase.

[0310] A03

[0311] The glycated protein sensor according to Embodiment A02, wherein the enzyme layer has a protease layer containing an immobilized protease and a ketamine oxidase layer containing an immobilized ketamine oxidase in a stacked manner.

[0312] A04

[0313] The glycated protein sensor according to Embodiment A03, wherein the ketamine oxidase layer and the protease layer are stacked in the order of the ketamine oxidase layer and the protease layer from the side closer to the hydrogen peroxide detection portion.

[0314] A05

[0315] The glycated protein sensor according to Embodiment A03, wherein

[0316] the ketamine oxidase layer contains a first ketamine oxidase layer and a second ketamine oxidase layer,

[0317] the first ketamine oxidase layer, the protease layer, and the second ketamine oxidase layer are stacked in this order.

[0318] A06

[0319] The glycated protein sensor according to Embodiment A02, wherein the enzyme layer has a ketamine oxidase layer containing an immobilized ketamine oxidase and a protease immobilized on the surface of the ketamine oxidase layer opposite to the hydrogen peroxide detection portion.

[0320] A07

[0321] The glycated protein sensor according to any one of Embodiments A02 to A06, further comprising an ion exchange resin disposed between the hydrogen peroxide detection unit and the enzyme layer.

[0322] A08

[0323] The glycated protein sensor according to Embodiment A01, wherein the immobilized protease and the immobilized ketoamine oxidase are disposed separately.

[0324] A09

[0325] The glycated protein sensor according to Embodiment A08, comprising:

[0326] a protease housing unit that houses the immobilized protease;

[0327] a ketoamine oxidase housing unit that houses the immobilized ketoamine oxidase and is fluidly connected to the protease housing unit; and

[0328] a liquid feeding mechanism that feeds a liquid from the protease housing unit to the ketoamine oxidase housing unit.

[0329] A10

[0330] The glycated protein sensor according to Embodiment A08 or A09, further comprising a heater that heats the protease and / or the ketoamine oxidase.

[0331] A11

[0332] The glycated protein sensor according to Embodiment A08 or A09, further comprising a protease heater that heats the protease housing unit.

[0333] A12

[0334] The glycated protein sensor according to Embodiment A11, wherein the protease heater is configured to heat the protease housing unit to 40 °C or higher or 50 °C or higher.

[0335] A13

[0336] The glycated protein sensor according to any one of Embodiments A11 to A13, further comprising a protease temperature sensor that measures the temperature of the protease housing unit or the protease.

[0337] A14

[0338] The glycated protein sensor according to any one of Embodiments A11 to A13, further comprising a temperature control mechanism that controls the calorific value of the protease heater based on information from the protease temperature sensor.

[0339] A15

[0340] The glycated protein sensor according to any one of Embodiments 11 to A14, further comprising a heater for ketoamine oxidase that heats the ketoamine oxidase housing portion.

[0341] A16

[0342] The glycated protein sensor according to Embodiment A15, wherein the heater for ketoamine oxidase is configured to heat the ketoamine oxidase housing portion at a temperature equal to or higher than room temperature and equal to or lower than 50°C.

[0343] A17

[0344] The glycated protein sensor according to Embodiment A15 or A16, further comprising a temperature sensor for ketoamine oxidase that measures the temperature of the ketoamine oxidase housing portion or ketoamine oxidase.

[0345] A18

[0346] The glycated protein sensor according to any one of Embodiments A15 to A17, further comprising a temperature control mechanism that controls the calorific value of the heater for ketoamine oxidase based on information from the temperature sensor for ketoamine oxidase.

[0347] A19

[0348] The glycated protein sensor according to any one of Embodiments A11 to A18, further comprising a cooling unit that is disposed between the protease housing portion and the ketoamine oxidase housing portion in a fluidly connected manner and has a cooling mechanism.

[0349] A21

[0350] The glycated protein sensor according to Embodiment 1, wherein the immobilized protease, the immobilized ketoamine oxidase, and the hydrogen peroxide detection unit are sequentially arranged in the upstream direction of the solution containing the analyte.

[0351] A22

[0352] The glycated protein sensor according to any one of Embodiments A01 to A21, wherein the protease is immobilized on a substrate.

[0353] A23

[0354] The glycated protein sensor according to Embodiment A22, wherein the protease is immobilized on microbeads.

[0355] A24

[0356] The glycated protein sensor according to any one of Embodiments A01 to A23, wherein the hydrogen peroxide detection unit includes a hydrogen peroxide electrode.

[0357] A25

[0358] The glycated protein sensor according to any one of Embodiments A01 to A23, wherein the hydrogen peroxide detector includes a photodetector.

[0359] A26

[0360] The glycated protein sensor according to any one of Embodiments A01 to A23, wherein the hydrogen peroxide detector includes a luminescent reagent that reacts with hydrogen peroxide and a photodetector.

[0361] A27

[0362] A glycated protein sensor, comprising:

[0363] A first sensor having a first immobilized protease, a first immobilized ketoamine oxidase, and a first hydrogen peroxide detection unit; and

[0364] A second sensor having a second immobilized ketoamine oxidase and a second hydrogen peroxide detection unit.

[0365] A28

[0366] A glycated protein sensor, comprising:

[0367] A first hydrogen peroxide detection unit;

[0368] A first enzyme layer disposed on the first hydrogen peroxide detection unit and including an immobilized protease and an immobilized ketoamine oxidase;

[0369] A second hydrogen peroxide detection unit; and

[0370] A second enzyme layer disposed on the second hydrogen peroxide detection unit and including an enzyme substantially composed of the immobilized ketoamine oxidase.

[0371] A29

[0372] The glycated protein sensor according to Embodiment A28, wherein the first hydrogen peroxide detection unit and the second hydrogen peroxide detection unit include hydrogen peroxide electrodes.

[0373] A30

[0374] The glycated protein sensor according to Embodiment A29, wherein

[0375] the first hydrogen peroxide detection unit includes one of a counter electrode and a reference electrode and a first working electrode,

[0376] The second hydrogen peroxide detection unit described above includes the counter electrode and the reference electrode, and the other one of them, as well as the second working electrode.

[0377] A31

[0378] The glycated protein sensor according to Embodiment A29 or A30, further comprising a current measurement circuit that applies a potential to the counter electrode and measures the current flowing through the first working electrode and the current flowing through the second working electrode.

[0379] A32

[0380] The glycated protein sensor according to any one of Embodiments A01 to A31, wherein the glycated protein includes glycated albumin.

[0381] A33

[0382] The glycated protein sensor according to Embodiment A32, further comprising an albumin sensor.

[0383] B01

[0384] A method for measuring glycated protein, comprising:

[0385] A step of preparing a measurement solution containing glycated protein;

[0386] A step of introducing the measurement solution into an immobilized protease;

[0387] A step of fragmenting the glycated protein with the immobilized protease to generate peptide fragments;

[0388] A step of generating hydrogen peroxide from the peptide fragments containing glycated amino acid residues in the peptide fragments using an immobilized ketoamine oxidase; and

[0389] A step of detecting the hydrogen peroxide generated by the ketoamine oxidase using a hydrogen peroxide detector.

[0390] B02

[0391] A method for measuring glycated protein, comprising:

[0392] A step of preparing a glycated protein sensor, the glycated protein sensor including an immobilized protease, an immobilized ketoamine oxidase, and a hydrogen peroxide detection unit;

[0393] A step of preparing a measurement solution containing glycated protein;

[0394] The step of introducing the above-mentioned measurement solution into the above-mentioned glycated protein sensor; and

[0395] The step of detecting the glycated protein in the above-mentioned measurement solution using the above-mentioned glycated protein sensor.

[0396] B03

[0397] The method for measuring glycated protein according to Embodiment B02, further comprising: the step of obtaining the concentration of glycated protein associated with the output signal from the above-mentioned hydrogen peroxide detection unit.

[0398] B04

[0399] The method for measuring glycated protein according to Embodiment B02 or B03, wherein

[0400] The above-mentioned glycated protein is glycated albumin or glycated hemoglobin,

[0401] The above-mentioned method further comprises:

[0402] The step of obtaining the concentration of albumin or hemoglobin; and

[0403] The step of obtaining the glycated albumin value or glycated hemoglobin value, which is the ratio of the concentration of the above-mentioned glycated albumin or the above-mentioned glycated hemoglobin to the concentration of the above-mentioned albumin or the above-mentioned hemoglobin.

[0404] B05

[0405] The method for measuring glycated protein according to any one of Embodiments B02 to B04, further comprising: the step of notifying the user to measure the glycated protein concentration every 1 week, every 2 weeks, every 3 weeks, every 4 weeks or every 1 month.

[0406] B06

[0407] The method for measuring glycated protein according to any one of Embodiments B02 to B05, wherein the above-mentioned glycated protein includes glycated albumin.

[0408] B07

[0409] The method for measuring glycated protein according to Embodiment B06, further comprising: the step of calculating the average blood glucose value by converting the ratio of the concentration of the above-mentioned albumin or the above-mentioned hemoglobin multiple times.

[0410] B08

[0411] A program for causing a computer to execute each step included in the measurement method according to any one of Embodiments B02 to B07.

[0412] B09

[0413] A computer-readable storage medium stores a program according to Embodiment B07.

[0414] B10

[0415] A method for measuring glycated protein, comprising:

[0416] A step of preparing a glycated protein sensor, the glycated protein sensor comprising a first hydrogen peroxide detection unit, a first enzyme layer disposed on the first hydrogen peroxide detection unit and containing immobilized protease and immobilized ketamine oxidase, and a second enzyme layer disposed on the second hydrogen peroxide detection unit and containing an enzyme substantially composed of the immobilized protease;

[0417] A step of introducing a measurement solution containing glycated protein into the glycated protein sensor; and

[0418] Calculating the concentration of glycated protein associated with these output signals from the output signal from the first hydrogen peroxide detection unit and the output signal from the second hydrogen peroxide detection unit.

[0419] C01

[0420] A method for manufacturing a glycated protein sensor, comprising:

[0421] A step of preparing a substrate;

[0422] A step of disposing a hydrogen peroxide detection unit on the substrate;

[0423] A step of immobilizing ketamine oxidase on the substrate; and

[0424] A step of immobilizing protease on the substrate.

[0425] C02

[0426] According to the method for manufacturing a glycated protein sensor described in Embodiment C01, wherein

[0427] The step of immobilizing ketamine oxidase on the substrate includes a step of immobilizing ketamine oxidase on the hydrogen peroxide detection unit,

[0428] The step of immobilizing protease on the substrate includes a step of immobilizing protease on the hydrogen peroxide detection unit.

[0429] C03

[0430] The manufacturing method according to Embodiment C02, wherein the step of immobilizing ketamine oxidase on the hydrogen peroxide detection unit includes: forming a ketamine oxidase layer in which ketamine oxidase is immobilized on a first substrate,

[0431] The step of immobilizing protease on the hydrogen peroxide detection unit includes: forming a protease layer in which protease is immobilized on a second substrate on the ketamine oxidase layer.

[0432] C04

[0433] The manufacturing method according to Embodiment C03, wherein the step of immobilizing ketamine oxidase on the hydrogen peroxide detection unit further includes: forming a second ketamine oxidase layer in which ketamine oxidase is immobilized on a third substrate on the protease layer.

[0434] C05

[0435] The manufacturing method according to Embodiment C02, wherein the step of immobilizing ketamine oxidase on the hydrogen peroxide detection unit and the step of immobilizing protease on the hydrogen peroxide unit include: mixing the ketamine oxidase and the protease with a common substrate to form the same enzyme layer on the hydrogen peroxide unit.

[0436] C06

[0437] A manufacturing method of a glycated protein sensor, comprising:

[0438] A step of preparing a substrate;

[0439] A step of forming a first hydrogen peroxide detection unit and a second hydrogen peroxide detection unit on the substrate;

[0440] A step of immobilizing a first ketamine oxidase and protease on the first hydrogen peroxide detection unit; and

[0441] A step of immobilizing a second ketamine oxidase on the second hydrogen peroxide detection unit.

[0442] C07

[0443] According to the manufacturing method described in Embodiment C06, wherein,

[0444] The step of preparing the substrate includes: a step of preparing a first substrate and a second substrate,

[0445] The step of forming a first hydrogen peroxide detection unit and a second hydrogen peroxide detection unit on the substrate includes: a step of forming a first hydrogen peroxide detection unit on the first substrate and forming a second hydrogen peroxide detection unit on the second substrate,

[0446] The manufacturing method further includes:

[0447] Steps of preparing a main body substrate; and

[0448] Steps of bonding the first substrate and the second substrate on the above-mentioned main body substrate.

[0449] Several embodiments and examples of the present invention have been described above, but these embodiments and examples are illustrative of the present invention. For example, each of the above embodiments has been described in detail for the purpose of easily understanding the present invention, but the dimensions, configurations, materials, and circuits can also be added and changed as needed. The claims include a number of deformation modes for the embodiments without departing from the technical concept of the present invention. Therefore, the embodiments and examples disclosed in this specification are shown for illustration purposes and should not be considered as limiting the scope of the present invention.

[0450] Symbol description

[0451] 100 Sensor

[0452] 101 Protease

[0453] 102 Ketamine oxidase

[0454] 103 Hydrogen peroxide detection unit

[0455] 104 Immobilized layer

[0456] 105 Substrate

[0457] 106 Crosslinking agent

[0458] 107 Silane coupling agent

[0459] 111 Enzyme layer

[0460] 151 Glycated protein

[0461] 152 Protein

[0462] 153 Sugar

[0463] 154 Glycated peptide fragment

[0464] 155 Unglycated peptide fragment

[0465] 156 Hydrogen peroxide

[0466] 200 Sensor

[0467] 201 Protease

[0468] 202 Ketamine oxidase

[0469] 203 Hydrogen peroxide detection unit

[0470] 204 Substrate

[0471] 211 Enzyme layer

[0472] 300 Sensor

[0473] 301 Protease

[0474] 302 Ketamine oxidase

[0475] 303 Hydrogen peroxide detection unit

[0476] 304 Substrate

[0477] 305 Adhesive

[0478] 311 Enzyme layer

[0479] 400 Sensor

[0480] 401 Protease

[0481] 402 Ketamine oxidase

[0482] 403 Hydrogen peroxide detection unit

[0483] 404, 414 Substrate

[0484] 411 Protease layer

[0485] 412 Ketamine oxidase layer

[0486] 500 Sensor

[0487] 501 Protease

[0488] 502 Ketamine oxidase

[0489] 503 Hydrogen peroxide detection unit

[0490] 506 Substrate

[0491] 504, 514 Substrate

[0492] 511 Immobilized protease section

[0493] 512 Immobilized ketamine oxidase section

[0494] 600 Sensor

[0495] 601 Protease

[0496] 602 Ketamine oxidase

[0497] 603 Hydrogen peroxide detection unit

[0498] 604 Crosslinking agent

[0499] 612 Ketamine oxidase layer

[0500] 614 Substrate

[0501] 700 Sensor

[0502] 701 Protease

[0503] 702 Ketamine oxidase

[0504] 704, 714 Substrate

[0505] 705 Silane coupling agent

[0506] 706 Insulating substrate

[0507] 711 Protease layer

[0508] 712 Ketamine oxidase layer

[0509] 730 Hydrogen peroxide electrode

[0510] 731 Counter electrode

[0511] 732 Reference electrode

[0512] 733 Working electrode

[0513] 770 Circuit

[0514] 771 Operational amplifier

[0515] 772 Voltage generation circuit

[0516] 773 Current measurement circuit

[0517] 800 Sensor

[0518] 801 Protease

[0519] 802 Ketamine oxidase

[0520] 804, 814 Substrate

[0521] 807 Ion exchange resin

[0522] 811 Protease layer

[0523] 812 Ketamine oxidase layer

[0524] 1000 Sensor

[0525] 1000a Main sensor

[0526] 1000b Sub - sensor

[0527] 1001 Protease

[0528] 1002 Ketamine oxidase

[0529] 1003a, 1003b Hydrogen peroxide detection unit

[0530] 1004, 1014 Substrate

[0531] 1011a, 1011b Enzyme layer

[0532] 1030 Operation unit

[0533] 1070a, 1070b Circuit

[0534] 1100 Sensor

[0535] 1100a Main sensor

[0536] 1100b Sub - sensor

[0537] 1101 Protease

[0538] 1102 Ketamine oxidase

[0539] 1103a, 1103b Hydrogen peroxide detection unit

[0540] 1104, 1114, 1124 Substrate

[0541] 1111a, 1111b Protease layer

[0542] 1112a Ketamine oxidase layer

[0543] 1011a, 1011b Enzyme layer

[0544] 1130 Operation unit

[0545] 1170a, 1170b Circuit

[0546] 1200 Sensor

[0547] 1200a Main sensor

[0548] 1200b Sub - sensor

[0549] 1201 Protease

[0550] 1202 Ketamine oxidase

[0551] 1203a, 1203b Hydrogen peroxide detection unit

[0552] 1204 Cross - linker

[0553] 1214, 1224 Substrate

[0554] 1212a Ketamine Oxidase Layer

[0555] 1212b Enzyme Layer

[0556] 1230 Operation Unit

[0557] 1270a, 1270b Circuits

[0558] 1300 Sensor

[0559] 1300a Main Sensor

[0560] 1300b Sub - Sensor

[0561] 1301 Protease

[0562] 1302 Ketamine Oxidase

[0563] 1304, 1324 Substrates

[0564] 1305a, 1305b Silane Coupling Agents

[0565] 1306 Substrate

[0566] 1311a, 1311b Enzyme Layers

[0567] 1330a, 1330b Hydrogen Peroxide Electrodes

[0568] 1331 Counter Electrode

[0569] 1332 Reference Electrode

[0570] 1333a, 1333b Working Electrodes

[0571] 1370 Circuit

[0572] 1371 Operational Amplifier

[0573] 1372 Voltage Generation Circuit

[0574] 1373a, 1373b Current Measurement Circuits

[0575] 1400 Sensor

[0576] 1401 Protease

[0577] 1402 Ketamine Oxidase

[0578] 1404 Microbeads

[0579] 1414 Substrate

[0580] 1431 Reagent

[0581] 1432 Photodetector

[0582] 1452 Liquid inlet

[0583] 1455 Protease containment section

[0584] 1456 Ketamine oxidase containment section

[0585] 1457 Detection reactor

[0586] 1460 Heater

[0587] 1500 Sensor

[0588] 1502 Ketamine oxidase

[0589] 1504 Microbeads

[0590] 1600 Sensor

[0591] 1601 Protease

[0592] 1602 Ketamine oxidase

[0593] 1603 Hydrogen peroxide detector

[0594] 1604 Microbeads

[0595] 1611 Enzyme layer

[0596] 1614 Substrate

[0597] 1652 Liquid inlet

[0598] 1655 Protease containment section

[0599] 1656 Ketamine oxidase containment section

[0600] 1660 Heater

[0601] 1700 Sensor

[0602] 1700a Main sensor

[0603] 1700b Sub - sensor

[0604] 1703a, 1703b Hydrogen peroxide detector

[0605] 1711a Enzyme layer

[0606] 1712a, 1712b Ketamine oxidase layer

[0607] 1752 Liquid inlet

[0608] 1755a Protease containment section

[0609] 1755b Protease-Free Chamber

[0610] 1756a, 1756b Ketamine Oxidase Containment Vessel

[0611] 1760 Heater

[0612] 1800 Sensor

[0613] 1801 Protease

[0614] 1802 Ketamine Oxidase

[0615] 1803 Hydrogen Peroxide Detector

[0616] 1804 Microbeads

[0617] 1811 Enzyme Layer

[0618] 1814 Substrate

[0619] 1852 Liquid Inlet

[0620] 1855 Protease Containment Section

[0621] 1856 Ketamine Oxidase Containment Section

[0622] 1858 Cooling Section / Cooling Solution Containment Section

[0623] 1860, 1861 Heater

[0624] 1862 Cooling Circuit / Component

[0625] 2000 Sensor Chip

[0626] 2006 Substrate

[0627] 2011 Enzyme Layer

[0628] 2031 Counter Electrode

[0629] 2032 Reference Electrode

[0630] 2033 Working Electrode

[0631] 2034 Liquid Detection Electrode

[0632] 2041, 2042, 2043, 2044 Output Terminals

[0633] 2051 Liquid Containment Section

[0634] 2052 Fluid Inlet

[0635] 2053 Air Hole

[0636] 2100 Sensor Chip

[0637] Sensors 2100a and 2100b

[0638] Liquid detection electrode 2134

[0639] Liquid containment section 2151

[0640] Fluid inlet 2152

[0641] Air hole 2153

[0642] Sensor chip 2200

[0643] Main sensor 2200a

[0644] Sub - sensor 2200b

[0645] Substrate 2206

[0646] Enzyme layers 2221a and 2221b

[0647] Counter electrode 2231

[0648] Reference electrode 2232

[0649] Working electrodes 2233a and 2233b

[0650] Sensor chip 2300

[0651] Sensors 2300a and 2300b

[0652] Enzyme layers 2311a and 2311b

[0653] Hydrogen peroxide electrodes 2330a and 2330b

[0654] Output terminal 2340

[0655] Bonding pads 2340a, 2340b, 2341a, and 2341b

[0656] Liquid containment section 2351

[0657] Fluid inlet 2352

[0658] Air hole 2353

[0659] Substrate 2360

[0660] Sensor chip 2400

[0661] Substrate 2406

[0662] Enzyme layer 2411

[0663] Counter electrode 2431

[0664] 2432 Reference electrode

[0665] 2433 Working electrode

[0666] 2434 Liquid detection electrode

[0667] 2451 Liquid containment section

[0668] 2452 Fluid inlet

[0669] 2453 Air hole

[0670] 3000 Sensor

[0671] 3000a Main sensor

[0672] 3000b Sub - sensor

[0673] 3001 Protease

[0674] 3002 Ketamine oxidase

[0675] 3004, 3014 Substrate (BSA)

[0676] 3005a, 3005b Silane coupling agent

[0677] 3006 Insulating substrate

[0678] 3009, 3091 Resist

[0679] 3011a, 3011b Enzyme layer

[0680] 3030a, 3030b Hydrogen peroxide electrode

[0681] 4002, 4102 Ketamine oxidase

[0682] 4003, 4103 Hydrogen peroxide detector

[0683] 4006, 4106 Substrate

[0684] 4008, 4108 Protection

[0685] 4012, 4112 Ketamine oxidase layer

[0686] 4014, 4114 Substrate

Claims

1. A glycated protein sensor, comprising: A hydrogen peroxide detection unit; Ketamine oxidase that is fixed and close to the hydrogen peroxide detection unit; An ion exchange resin disposed between the hydrogen peroxide detection unit and the fixed ketamine oxidase; and Protease that is fixed separately from the fixed ketamine oxidase, The glycated protein sensor comprises: A protease housing unit that houses the fixed protease; A ketamine oxidase housing unit that houses the fixed ketamine oxidase and is fluidly connected to the protease housing unit; and A liquid feeding mechanism that conveys liquid from the protease housing unit to the ketamine oxidase housing unit.

2. The glycated protein sensor according to claim 1, wherein, It further comprises a temperature control mechanism configured to control the temperature of the protease and the temperature of the ketamine oxidase respectively.

3. The glycated protein sensor according to claim 2, wherein, The temperature control mechanism is configured to heat the protease housing unit to 40 °C or higher.

4. The glycated protein sensor according to claim 2, wherein, It further comprises a temperature sensor for protease that measures the temperature of the protease.

5. The glycated protein sensor according to claim 2, wherein, The temperature control mechanism is configured to heat the ketamine oxidase housing unit at a temperature below 40 °C.

6. The glycated protein sensor according to claim 2, wherein, It further comprises a temperature sensor for ketamine oxidase that measures the temperature of the ketamine oxidase.

7. The glycated protein sensor according to claim 1, wherein, It further comprises a cooling unit that is disposed between the protease housing unit and the ketamine oxidase housing unit in a fluidly connected manner to both and has a cooling mechanism.

8. The glycated protein sensor according to claim 1, wherein, The protease is fixed to a substrate.

9. The glycated protein sensor according to claim 1, wherein, The protease is fixed to microbeads.

10. The glycated protein sensor according to claim 1, wherein, The hydrogen peroxide detection unit includes a hydrogen peroxide electrode.

11. The glycated protein sensor according to claim 1, wherein, The glycated protein includes glycated albumin.

12. The glycated protein sensor according to claim 11, wherein, It further comprises an albumin sensor.

Images