Dry chemical reagent tablet for detecting activity of isocitrate dehydrogenase and detection method

Abstract

The invention discloses a dry chemical reagent tablet for determining the activity of isocitrate dehydrogenase, which is used for determining the activity of isocitrate dehydrogenase and comprises a transparent support layer, a reagent layer, a reaction layer and a diffusion layer, the transparent supporting layer is arranged at the bottom; the reagent layer contains a reagent liquid reagent; the reaction layer contains a reaction liquid reagent formed by a hydrophilic colloid and is used for carrying out a reaction for determining the activity of isocitrate dehydrogenase; the diffusion layer is made of a hydrophilic polymer filter membrane and is used for uniformly distributing a sample and providing a reflection background at the same time; the liquid sample is uniformly distributed after being dropwise added on the diffusion layer, the bio-enzyme substance permeates into the reaction layer, and a reagent liquid reagent in the reagent layer is continuously released into the reaction layer as a dye and is subjected to a chromogenic reaction with isocitrate dehydrogenase contained in the bio-enzyme substance in the liquid sample; measuring the change of the reflectivity under the wavelength of 530-760 nm within a specific time based on a reflection spectrophotometric method; and calculating the activity of the isocitrate dehydrogenase through the standard curve. The invention further discloses a corresponding preparation method and a detection method.

Description

Technical Field

[0001] The present invention relates to the technical field of medical test determination, and particularly to a dry chemical reagent strip for determining the activity of isocitrate dehydrogenase and a detection method thereof. Background Art

[0002] Isocitrate dehydrogenase (ICD or ICDH) is an important redox enzyme with decarboxylation function in the body. There are two categories in the human body. One category uses NAD + as a coenzyme and is mainly involved in the tricarboxylic acid cycle, which has nothing to do with clinical diagnosis; the other category uses NADP + as a coenzyme. It mainly exists in the liver, heart, skeletal muscle, kidney, adrenal gland, as well as platelets and red blood cells; it has the highest content in the liver and exists in the mitochondria and cytoplasm of hepatocytes. Therefore, detecting the activity of ICD can more accurately reflect the degree of hepatocyte lesions, especially necrotic changes, and provide an accurate basis for judging hepatocyte lesions and estimating the prognosis of liver disease patients.

[0003] Currently, isocitrate dehydrogenase is generally determined using liquid reagents on large-scale fully automatic biochemical analyzers. The equipment itself is relatively expensive and not economical; at the same time, it requires professional technical personnel to operate the instrument, lacking the required universality. The detection procedures are cumbersome, the process is long, the waiting time is long, and the analysis process is relatively complex, bringing a huge time burden to patients.

[0004] Dry chemical detection technology is the main supporting technology for on-site rapid detection and has a broad application space in laboratory medicine. It has been widely used as a routine method in clinical tests and has the advantages of simplicity, rapidity, flexibility, and the need for no professional personnel to operate. As early as the 16th century, people used litmus paper to detect the acidity and alkalinity of liquids, which was the earliest application of the dry chemical method. In the early 18th century, the British chemist William Prout used dry chemical reagents to detect the pH value of urine, which was the earliest application of dry chemical reagents in the detection of biological fluids.

[0005] At present, qualitative or semi-quantitative determinations are mainly carried out in China. These dry chemical reagent tablets are all prepared by pre-fixing all reagents on sheet materials such as filter paper, cellulose sheets, and porous membranes, and the measurement linear range is not large. Dry chemical reagent cards for quantitative detection are mainly produced abroad and there are three types of products. One is the dry chemical technology of multi-coated thin films represented by Johnson & Johnson. Using the coating technology of photographic film, reagent layers such as the diffusion layer, reflection layer, and reagent layer are coated on a transparent support layer, and the change in optical density is measured from the reverse side of the support layer. The second type is the dry chemical technology of Kyoto Company in Japan. Its composition is a plastic support layer, and one end is the reaction area including the sample layer and the reagent layer. The reagent layer is a certain fabric as the carrier of the reagent. Sampling and testing are carried out on the upper part. The third type is the dry chemical product of Roche. US Pat.No.4604264A coats the reagent on multi-filament fibers or fabrics. Whole blood contacts and reacts with the upper reagent layer after being laterally filtered through glass fibers, and the change in optical density is measured by covering a transparent film on the upper part. US Pat.No.6036919A invented a dry chemical test strip for measuring whole blood. A reagent layer and a reflection layer are coated on a transparent support layer, and a synthetic fiber mesh is covered on the top as the sampling layer. For these above products, some have complex production processes and high requirements for materials and equipment, and some may have certain defects, such as the easy generation of certain bubbles and excessive liquid at the sampling end. Sampling and measurement on the same side of the sample may cause interference. Moreover, the currently applied dry biochemical analyzers are large-scale equipment, and all biochemical items are concentrated on one machine for determination. Such as the Johnson & Johnson VITROS series (VITROS-250, VITROS-950) semi-automatic and fully automatic dry biochemical analyzers and biochemical reagents; VITROS-250 and 950 are widely used in hospitals. It is not convenient to turn on the machine when the number of samples to be tested is small. For emergency items such as acute abdomen, immediate determination is required. If there are separate small machines and test strips, the urgent need can be met. This is of greater significance to some small and medium-sized hospitals lacking large-scale dry biochemical analyzers.

[0006] At present, liquid biochemical reagents are mainly used in clinical practice for the activity determination of isocitrate dehydrogenase. The detection principle can be divided into two types. One method is that isocitrate dehydrogenase catalyzes the conversion of isocitrate to α-ketoglutaric acid with the help of nicotinamide adenine dinucleotide phosphate (NADP) and Mn2 + while releasing carbon dioxide. The activity of isocitrate dehydrogenase can be determined by the amount of reduced nicotinamide adenine dinucleotide phosphate (NADPH) formed per unit time at ultraviolet range wavelengths (340 or 366 nm). The other method is to couple α-ketoglutaric acid with phenylhydrazine to obtain a color complex that can be measured by photometry.

[0007] The disadvantage of the first method is that there is no visible color change in this reactant, and it is impossible to judge the enzyme activity from the color change. Its measurement requires an optical device that can measure the change in absorbance per unit time in the ultraviolet range, and the change in NADH in biological samples is usually relatively small. Especially when the activity of isocitrate dehydrogenase in the sample is small, reverse reactions and other reactions are likely to occur, resulting in negative changes. The disadvantage of the second method is that it cannot track the reaction kinetically, and the test operation is relatively cumbersome, requiring multiple steps of reaction, which takes a lot of time and labor.

[0008] The above methods all belong to liquid reagents according to the reagent type. At present, there is no dry chemical reagent for detecting the activity of isocitrate dehydrogenase. Liquid biochemical reagents are processed in liquid form, and it is not very convenient for storage and transportation. When in use, large biochemical instruments need to be equipped for determination, which is expensive, and professional personnel are required to operate. It is generally only applicable to the centralized detection of large sample sizes in large hospitals. However, it is very inconvenient to use in emergency departments, wards, primary community hospitals, township hospitals, etc., where large equipment and professional personnel are often lacking. Summary of the Invention

[0009] The purpose of the present invention is to overcome the defects of the prior art and provide a dry chemical reagent strip and a detection method for determining the activity of isocitrate dehydrogenase in view of the problems in the related art, so as to solve the problem of inconvenient use of traditional liquid biochemical reagents for isocitrate dehydrogenase. The dry chemical reagent strip has high detection result accuracy, good precision, good specificity, good stability, long shelf life, simple operation, and wide application range.

[0010] The first aspect of the present invention lies in providing a dry chemical reagent strip for determining the activity of isocitrate dehydrogenase, which is used to determine the activity of isocitrate dehydrogenase. The dry chemical reagent strip comprises four laminated layers, which are a transparent support layer, a reagent layer, a reaction layer, and a diffusion layer in sequence; wherein:

[0011] The transparent support layer is arranged at the bottom of the dry chemical reagent strip and is used to support the reagent layer, the reaction layer, and the diffusion layer;

[0012] The reagent layer contains reagent liquid reagents and is used to provide reaction reagents for the reaction of determining the activity of isocitrate dehydrogenase;

[0013] The reaction layer contains a reaction liquid reagent formed by a hydrophilic colloid and is used to carry out the reaction of determining the activity of isocitrate dehydrogenase;

[0014] The diffusion layer is made of a hydrophilic polymer filter membrane and is used to uniformly distribute the sample and provide a reflection background at the same time;

[0015] After the liquid sample is dropped onto the diffusion layer of the dry chemical reagent strip, it is evenly distributed in the diffusion layer. The biological enzyme substances in the liquid sample penetrate into the reaction layer on the side opposite to the side of the diffusion layer that holds the liquid sample. At the same time, the reagent liquid in the reagent layer is continuously released into the reaction layer as a dye and undergoes a color reaction with the isocitrate dehydrogenase contained in the biological enzyme substances in the liquid sample. Based on reflectance spectrophotometry, the change in reflectance at a wavelength of 530 - 760 nm within a specific time is measured; based on the change in the reflectance, the isocitrate dehydrogenase activity is calculated through a standard curve.

[0016] Preferably, the material of the transparent support layer includes one of polyethylene, a polymer of polyethylene terephthalate, polyvinyl chloride, polystyrene, and polyester fiber; the thickness of the transparent support layer is 50 - 300 microns.

[0017] Preferably, the hydrophilic colloid is one or more of polyvinyl alcohol, amylopectin, cellulose ester, agarose, polyvinylpyrrolidone, polyacrylamide, hydroxypropyl methylcellulose, and methyl vinyl ether / maleic anhydride copolymer; the dosage of the hydrophilic colloid is 50 - 150 g / m 2 。

[0018] Preferably, the reagent liquid in the reagent layer includes the following components:

[0019] Dyes with oxidized and reduced states, including tetrazolium salts, cofactors, electron carriers, isocitric acid or isocitrate, divalent metal ions, buffers, additives, coenzyme stabilizers, surfactants, film formers, and preservatives.

[0020] Preferably, the type of the tetrazolium salt is sufficient to be reduced to a colored formazan, and the amount thereof is sufficient to correlate the formazan concentration with the concentration of the analyte in the liquid sample; examples of the tetrazolium salt include 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide MTT, p-iodonitrotetrazolium violet INT, nitroblue tetrazolium chloride NBT, tetranitroblue tetrazolium chloride TNBT, tetrazolium blue TB, tetrazolium violet TV, thiocarbamyl nitroblue tetrazolium chloride; triphenyltetrazolium chloride; 2-(2'-benzothiazolyl)-5-styryl-3-(4'-phthalohydrazidyl)tetrazole BSPT, 2-benzothiazolyl-(2)-3,5-diphenyltetrazole (BTDP), 2,3-bis(4-nitrophenyl)tetrazole (DNP), 2,5-diphenyl-3-(4-styrylphenyl)tetrazole (DPSP), stilbenyl nitroblue tetrazolium (DS-NBT), 2-phenyl-3-(4-carboxyphenyl)-5-methyltetrazole (PCPM), thiocarbamyl nitroblue tetrazolium (TCNBT) or a water-soluble tetrazolium salt; the water-soluble tetrazolium salt includes one or more of sodium 2-(4-iodophenyl)-3-(2,4-dinitrophenyl)-5-phenyl-3H-tetrazole-2',4'-disulfonate (IPB), 2-benzothiazolothiazolyl-3-(4-carboxy-2-methoxyphenyl)5-[4-(2-sulfoethylcarbamoyl)phenyl]-2H-tetrazole (GLT004), 2,2'-bisbenzothiazolyl-5,5'-bis[4-bis(2-sulfoethyl)aminocarbonylphenyl]-3,3'-(3,3'-dimethoxy-4,4'-biphenylyl) ditetrazole disodium salt (WST-5) and sodium 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazole monosodium salt (WST-8);

[0021] The cofactor includes β-nicotinamide adenine dinucleotide, β-nicotinamide adenine dinucleotide phosphate or any of its salts, and any of its salts includes one or more of disodium β-nicotinamide adenine dinucleotide phosphate, sodium β-nicotinamide adenine dinucleotide and lithium β-nicotinamide adenine dinucleotide;

[0022] The electron carrier includes one or more of diaphorase, phenazine methosulfate PMS, 1-methoxyphenazine methosulfate, 9-dimethylaminobenzo-a-phenoxazinium chloride and 2,6-dichlorophenol indophenol;

[0023] The divalent metal ions include one or more of magnesium ions, manganese ions, iron ions, nickel ions, zinc ions and cobalt ions;

[0024] The buffer solution includes a buffer agent capable of buffering to a pH within the range of 5 to 9, including one or more of tris(hydroxymethyl)aminomethane, tris(hydroxymethyl)methylglycine, 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid, N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid, 3-morpholinopropanesulfonic acid, 2-morpholinoethanesulfonic acid, piperazine-1,4-bis(2-ethanesulfonic acid), and bis(2-hydroxyethyl)amino(tris(hydroxymethyl)methyl)methane;

[0025] The additive includes one or more of sodium chloride, potassium chloride, sodium sulfate, and potassium sulfate, one or more of sucrose and lactose, one or more of sorbitol and mannitol, and a protein, and also includes a chelating agent, amines, basic amino acids, or heterocyclic compounds;

[0026] The coenzyme stabilizer includes one or more of sericin, carboxylated chitosan, substrate stabilizer CCD, substrate stabilizer PPCD, stabilizing protector Phos-E, complex enzyme stabilizer AES, complex enzyme stabilizer DPD, complex enzyme stabilizer AFP, complex enzyme protector HP, and protein protector TY;

[0027] The surfactant includes nonionic surfactants and anionic surfactants;

[0028] The film-forming agent includes one or at least two combinations of gelatin, dimethyl phthalate, hydroxyethyl cellulose, hydroxymethyl cellulose, polyvinylpyrrolidone K30, polyvinyl alcohol, polyacrylamide, or xanthan gum;

[0029] The preservative includes sodium azide or isothiazolinones.

[0030] Preferably, the nonionic surfactant includes one or more of polyethylene glycol monooctylphenyl ether TritonX-100, sorbitan monolaurate Span20, sorbitan monopalmitate Span40, polyoxyethylene lauryl ether Brij35, polyoxyethylene cetyl ether Brij58, polyoxyethylene sorbitan monolaurate Brij58, polyoxyethylene sorbitan monolaurate Span40, Tween 20, Tween 40, Tween 80, and Tween 60;

[0031] The anionic surfactant includes one or more of sodium dodecyl sulfate, sodium dodecylbenzenesulfonate, sodium cholate, sodium deoxycholate, and sodium lauroyl sarcosinate.

[0032] Preferably, the reagent solution in the reagent layer contains 0.01 - 20 mmol / L of tetrazolium salt; 0.5 - 200 mmol / L of β - nicotinamide adenine dinucleotide disodium salt; 1 - 1000 U / ml of diaphorase; 0.1 - 200 mmol / L of DL - trisodium isocitrate; 0.5 - 100 mmol / L of divalent magnesium ions; 0.1 - 30 wt% of additives; 0.001 to 30 wt% of coenzyme stabilizers; 0.0001 to 10 wt%, 0.001 to 1 wt% of surfactants; 0.001 to 10 wt% of film - forming agents; 0.001 to 1 wt% of preservatives; 1 - 100 mmol / L of buffer to adjust the pH to 5.0 - 10.0.

[0033] Preferably, the hydrophilic polymer filter membrane is a filter membrane prepared from one or more of cellulose acetate, polyvinylidene fluoride, polyethersulfone, and polytetrafluoroethylene; the pore size of the hydrophilic polymer filter membrane is 0.45 - 8.0 microns.

[0034] The second aspect of the present invention lies in providing a preparation method of the dry - chemical reagent tablet described in the first aspect, including the following steps:

[0035] S1, successively coat the reagent solution and the reaction solution on the reagent layer and the reaction layer, and then dry;

[0036] S2, attach the diffusion layer to the dried reaction layer;

[0037] S3, cover the dried reagent layer with the transparent support layer to obtain a dry - chemical reagent tablet successively including a transparent support layer, a reagent layer, a reaction layer, and a diffusion layer.

[0038] The third aspect of the present invention lies in providing a detection method based on the dry - chemical reagent tablet described in the first aspect, for determining the activity of isocitrate dehydrogenase in a liquid sample formed by a biological sample, including:

[0039] S1, dilute the biological sample with a diluent to prepare the liquid sample, add the liquid sample to a reaction device containing the dry - chemical reagent tablet, and heat the reaction device to a properly selected temperature, which is selected from 25°C to 37°C; wherein, the reaction device is made of ABS engineering plastic, the reaction device is provided with an isocitrate dehydrogenase detection hole, and the dry - chemical reagent tablet is assembled in the isocitrate dehydrogenase detection hole;

[0040] S2, drop the liquid sample onto the diffusion layer of the dry - chemical reagent tablet, and it is evenly distributed in the diffusion layer;

[0041] S3. The biological enzyme substances in the liquid sample penetrate into the reaction layer on the side opposite to the side of the diffusion layer that contains the liquid sample. Meanwhile, the reagent liquid in the reagent layer is continuously released into the reaction layer as a dye and reacts with isocitrate dehydrogenase contained in the biological enzyme substances in the liquid sample in the reaction layer. The reaction time is 5 - 10 min;

[0042] S4. Based on the reflectance spectrophotometry, use a reflectance spectrophotometer or a commonly used clinical automatic analyzer to measure the increasing rate of the reflectance in the detection hole at a wavelength of 530 - 760 nm within a specific time;

[0043] S5. Based on the increasing rate of the reflectance, calculate the isocitrate dehydrogenase activity through a standard curve.

[0044] Advantages of the dry chemical reagent strip, preparation method and detection method of the present invention:

[0045] (1) Liquid biochemical reagents are processed in liquid form, and are not very convenient for storage and transportation. The dry chemical reagent strip of the present invention comprises four laminated layers, namely a transparent support layer, a reagent layer, a reaction layer and a diffusion layer in sequence. The liquid sample is dropped on the diffusion layer of the reagent strip and is evenly distributed. The biological enzyme substances in the sample penetrate into the reaction layer, and the dye in the reagent layer can be continuously released into the reaction layer and reacts with isocitrate dehydrogenase in the sample. Within a specific time, the change in reflectance is measured by reflectance spectrophotometry at a wavelength of 605 nm, and the isocitrate dehydrogenase activity is calculated through a standard curve, solving the problem of inconvenient use of traditional liquid biochemical reagents for isocitrate dehydrogenase;

[0046] (2) When the sample is dropped on the diffusion layer and measured on the opposite side, the color development intensity is high, reducing the interference caused by bubbles, excessive liquid, and turbidity generated during the measurement at the sample addition end. At the same time, since the reagent layer and the reaction layer are hydrophilic colloids, they have a very high color development uniformity. Therefore, the dry chemical reagent strip of the present invention is used for quantitatively detecting the isocitrate dehydrogenase activity, can be used for rapid clinical detection, and has good economic benefits and applications;

[0047] (3) When using liquid biochemical reagents, large biochemical instruments need to be equipped for measurement, and the price is expensive. The present invention can accurately measure the isocitrate dehydrogenase activity within the visible light range without using expensive optical auxiliary tools, which can significantly broaden the application field of ICD activity measurement, especially in the medical field, and is suitable for clinical promotion and use;

[0048] (4) When using liquid biochemical reagents, professional personnel are required for operation. Generally, it is only applicable to the centralized detection of large sample sizes in large hospitals. However, in emergency departments, wards, primary community hospitals, township hospitals, etc., where large-scale equipment and professional personnel are often lacking, it is very inconvenient to use. The detection method of the present invention has a relatively simple principle for the detection instrument, low cost, and is applicable to disease screening tests in mobile and fixed medical diagnoses. It can also provide an important supplement for the dynamic continuous monitoring of many diseases and medical diagnoses in small laboratories;

[0049] (5) The testing process of the present invention is simple and fast, and patients can determine the activity of their plasma isocitrate dehydrogenase more quickly, which is essential for the diagnosis of hepatitis and other dangerous diseases;

[0050] (5) The dry chemical reagent tablets prepared by the present invention are dry chemical reagent tablets based on reflectance photometry. A polymer membrane material is used as the reagent carrier, and the dry chemical reagent tablets are prepared by adsorbing reagents. Compared with the traditional wet chemical test method, it has the advantages of simple operation, easy reagent preservation, fast detection speed, etc. The results can be detected and reported within a few minutes. Therefore, it is applicable to fields such as emergency specimen detection and has the possibility of replacing the wet chemical method for detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the related art, the following will briefly introduce the drawings required for use in the description of the specific embodiments or the related art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0052] Figure 1 It is a schematic diagram of the ability of the detection method provided by the embodiment of the present invention to measure isocitrate dehydrogenase, where the ordinate is the change amount of absorbance at 605 nm per minute, and the abscissa is the concentration of isocitrate dehydrogenase;

[0053] Figure 2 It is a schematic diagram showing the results of the clinical correlation analysis of the isocitrate dehydrogenase measurement method proposed by the present invention, with the measured values of the reference method as the ordinate and the measured values of the detection method proposed by the present invention as the abscissa. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0054] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the drawings. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art without creative efforts based on the embodiments of the present invention belong to the scope of protection of the present invention.

[0055] In the description of the present invention, it should be noted that the orientation or positional relationship indicated by the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", etc. is based on the orientation or positional relationship shown in the drawings. It is only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be construed as a limitation to the present invention. In addition, the terms "first", "second", "third" are only used for descriptive purposes and cannot be construed as indicating or implying relative importance.

[0056] In the description of the present invention, it should be noted that unless otherwise clearly specified and limited, the terms "installation", "connection", "connection" should be understood in a broad sense. For example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be directly connected or indirectly connected through an intermediate medium, and it can be the communication inside two elements. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0057] Embodiment 1

[0058] This embodiment provides a dry chemical reagent strip for measuring the activity of isocitrate dehydrogenase, which is used to measure the activity of isocitrate dehydrogenase. The dry chemical reagent strip comprises four stacked layers, which are a transparent support layer, a reagent layer, a reaction layer and a diffusion layer in sequence; wherein:

[0059] The transparent support layer is arranged at the bottom of the dry chemical reagent strip and is used to support the reagent layer, the reaction layer and the diffusion layer;

[0060] The reagent layer contains reagent liquid reagents, which are used to provide reaction reagents for the reaction of measuring the activity of isocitrate dehydrogenase;

[0061] The reaction layer contains a reaction liquid reagent formed by a hydrophilic colloid, which is used to carry out the reaction of measuring the activity of isocitrate dehydrogenase;

[0062] The material of the diffusion layer is a hydrophilic polymer filter membrane, which is used to uniformly distribute the sample and provide a reflection background at the same time;

[0063] After the liquid sample is dropped onto the diffusion layer of the dry chemical reagent strip, it is evenly distributed in the diffusion layer. The biological enzyme substances in the liquid sample penetrate into the reaction layer on the side opposite to the side of the diffusion layer that holds the liquid sample. At the same time, the reagent liquid in the reagent layer is continuously released into the reaction layer as a dye and undergoes a color reaction with the isocitrate dehydrogenase contained in the biological enzyme substances in the liquid sample. Based on reflectance spectrophotometry, the change in reflectance at a wavelength of 530 - 760 nm within a specific time is measured; based on the change in the reflectance, the isocitrate dehydrogenase activity is calculated through a standard curve.

[0064] As a preferred embodiment, the material of the transparent support layer includes one of polyethylene, polyethylene terephthalate polymer, polyvinyl chloride, polystyrene, and polyester fiber; more preferably, the material of the transparent support layer is polyethylene terephthalate polymer.

[0065] As a preferred embodiment, the thickness of the transparent support layer is 50 - 300 microns; more preferably, the thickness of the transparent support layer is 100 - 200 microns.

[0066] As a preferred embodiment, the hydrophilic colloid is one or more of polyvinyl alcohol, amylopectin, cellulose ester, agarose, polyvinylpyrrolidone, polyacrylamide, hydroxypropyl methylcellulose, and methyl vinyl ether / maleic anhydride copolymer. More preferably, the hydrophilic colloid is a mixture of cellulose ester and polyvinyl alcohol.

[0067] As a preferred embodiment, the dosage of the hydrophilic colloid is 50 - 150 g / m 2 .

[0068] As a preferred embodiment, the reagent liquid in the reagent layer includes the following components:

[0069] Dyes with oxidized and reduced states, including tetrazolium salts, cofactors, electron carriers, isocitric acid or isocitrate, divalent metal ions, buffers, additives, coenzyme stabilizers, surfactants, film formers, and preservatives; wherein,

[0070] The type of the tetrazolium salt is sufficient to be reduced to a colored formazan, and the amount thereof is sufficient to correlate the formazan concentration with the concentration of the analyte in the liquid sample; examples of the tetrazolium salt include 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT), p-iodonitrotetrazolium violet (INT), nitroblue tetrazolium chloride (NBT), tetranitroblue tetrazolium chloride (TNBT), tetrazolium blue (TB), tetrazolium violet (TV), thiocarbamyl nitroblue tetrazolium chloride; triphenyltetrazolium chloride; 2-(2'-benzothiazolyl)-5-styryl-3-(4'-phthalohydrazidyl)tetrazole (BSPT), 2-benzothiazolyl-(2)-3,5-diphenyltetrazole (BTDP), 2,3-bis(4-nitrophenyl)tetrazole (DNP), 2,5-diphenyl-3-(4-styrylphenyl)tetrazole (DPSP), distyryl nitroblue tetrazolium (DS-NBT), 2-phenyl-3-(4-carboxyphenyl)-5-methyltetrazole (PCPM), thiocarbamyl nitroblue tetrazolium (TCNBT) or a water-soluble tetrazolium salt; the water-soluble tetrazolium salt includes one or more of 2-(4-iodophenyl)-3-(2,4-dinitrophenyl)-5-phenyl-3H-tetrazole-2',4'-disulfonic acid disodium salt (IPB), 2-benzothiazolothiazolyl-3-(4-carboxy-2-methoxyphenyl)5-[4-(2-sulfoethylcarbamoyl)phenyl]-2H-tetrazole (GLT004), 2,2'-bisbenzothiazolyl-5,5'-bis[4-bis(2-sulfoethyl)aminocarbonylphenyl]-3,3'-(3,3'-dimethoxy-4,4'-biphenylyl) ditetrazole disodium salt (WST-5) and 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazole monosodium salt (WST-8);

[0071] Examples of the cofactor include β-nicotinamide adenine dinucleotide (NAD+, oxidized coenzyme I), β-nicotinamide adenine dinucleotide phosphate (NADP+, oxidized coenzyme II), or any salt thereof, such as one or more of disodium β-nicotinamide adenine dinucleotide phosphate, sodium β-nicotinamide adenine dinucleotide and lithium β-nicotinamide adenine dinucleotide;

[0072] Examples of the electron carrier include one or more of diaphorase, phenazine methosulfate (PMS), 1-methoxyphenazine methosulfate (1-methoxy-PMS), 9-dimethylaminobenzo-a-phenoxazinium chloride and 2,6-dichlorophenol indophenol (DPIP);

[0073] Examples of the divalent metal ions include metal ions such as magnesium ions, manganese ions, iron ions, nickel ions, zinc ions, and cobalt ions. The activity of isocitrate dehydrogenase requires the participation of divalent metal ions, and the ionic radius determines the reaction efficiency. Among them, manganese ions and magnesium ions are natural ligands, and magnesium ions are preferably used as the divalent metal ions in the present invention;

[0074] As a preferred embodiment, examples of the buffer solution include buffers that can be buffered to a pH in the range of 5 to 9, such as tris(hydroxymethyl)aminomethane, tris(hydroxymethyl)methylglycine, 4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid, 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid, N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid, N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid, 3-morpholinopropanesulfonic acid, 2-morpholinoethanesulfonic acid, piperazine-1,4-bis(2-ethanesulfonic acid), and bis(2-hydroxyethyl)amino(tris(hydroxymethyl)methyl)methane.

[0075] As a preferred embodiment, examples of the additives include monovalent metal salts such as chlorides and sulfates of sodium, potassium, etc., sugars such as sucrose and lactose, sugar alcohols such as sorbitol and mannitol, and proteins such as bovine serum albumin. At the same time, chelating agents, amines, basic amino acids, or heterocyclic compounds are also included.

[0076] As a preferred embodiment, examples of the coenzyme stabilizers include sericin, carboxylated chitosan, substrate stabilizer CCD, substrate stabilizer PPCD, stabilizing protector Phos-E, complex enzyme stabilizer AES, complex enzyme stabilizer DPD, complex enzyme stabilizer AFP, complex enzyme protector HP, protein protector TY, etc. These coenzyme stabilizers can be used alone or in combination of two or more.

[0077] As a preferred embodiment, examples of the surfactants include nonionic surfactants and anionic surfactants; the nonionic surfactants include one or more of polyethylene glycol monooctylphenyl ether (Triton X-100), sorbitan monolaurate (Span 20), sorbitan monopalmitate (Span 40), polyoxyethylene lauryl ether (Brij 35), polyoxyethylene cetyl ether (Brij 58), polyoxyethylene sorbitan monolaurate (Brij 58), polyoxyethylene sorbitan monolaurate (Span 40), Tween 20, Tween 40, Tween 80, and Tween 60; the anionic surfactants include one or more of sodium dodecyl sulfate, sodium dodecylbenzenesulfonate, sodium cholate, sodium deoxycholate, and sodium lauroyl sarcosinate.

[0078] As a preferred embodiment, examples of the film-forming agents include one or at least two combinations of gelatin, dimethyl phthalate, hydroxyethyl cellulose, hydroxymethyl cellulose, polyvinylpyrrolidone K30, polyvinyl alcohol, polyacrylamide, or xanthan gum.

[0079] As a preferred embodiment, examples of the preservative include sodium azide or isothiazolinones, such as ProClin 150, ProClin 200, ProClin 300 or ProClin 5000.

[0080] In a preferred embodiment of the present invention, the reagent contains 0.01 - 20 mmol / L, preferably 0.1 - 5 mmol / L of tetrazolium salt; preferably 0.5 - 200 mmol / L of β-nicotinamide adenine dinucleotide disodium salt; 1 - 1000 U / ml, preferably 10 - 200 U / ml of diaphorase; 0.1 - 200 mmol / L, preferably 1 - 100 mmol / L of trisodium DL-isocitrate; preferably 0.5 - 100 mmol / L of divalent magnesium ions; 0.1 - 30 wt% of an additive; 0.001 to 30 wt%, preferably 0.01 to 10 wt% of a coenzyme stabilizer; 0.0001 to 10 wt%, preferably 0.001 to 1 wt% of a surfactant; 0.001 to 10 wt%, preferably 0.01 to 1 wt% of a film-forming agent; preferably 0.001 to 1 wt% of a preservative; 1 - 100 mmol / L of a buffer to adjust the pH to 5.0 - 10.0. A buffer concentration of 10 - 500 mmol / L is particularly preferred, and the pH is adjusted to the range of 6.0 - 9.0.

[0081] As a preferred embodiment, the hydrophilic polymer membrane is a membrane prepared from one or more of cellulose acetate, polyvinylidene fluoride, polyethersulfone and polytetrafluoroethylene.

[0082] As a preferred embodiment, the pore size of the hydrophilic polymer membrane is 0.45 - 8.0 microns, and more preferably, the pore size of the hydrophilic polymer membrane is 0.8 - 3.0 microns.

[0083] The detection principle of the dry chemical reagent strip for measuring isocitrate dehydrogenase activity in the present invention is as follows: The dry chemical reagent strip for measuring isocitrate dehydrogenase activity comprises four laminated layers, namely a transparent support layer, a reagent layer, a reaction layer and a diffusion layer in sequence. After a liquid sample is dropped onto the diffusion layer of the dry chemical reagent strip, it is evenly distributed in the diffusion layer. The biological enzyme substances in the sample penetrate into the reaction layer, and the dye in the reagent layer is continuously released into the reaction layer, and a color reaction occurs with the isocitrate dehydrogenase in the sample. Within a specific time, the change in reflectance is measured by reflection spectrophotometry at a wavelength of 605 nm, and the isocitrate dehydrogenase activity is calculated through a standard curve. When the sample is dropped onto the diffusion layer and measured on the opposite side, the color development intensity is high, which can reduce the interference caused by bubbles, excessive liquid and turbidity generated during measurement at the sample addition end; at the same time, since the reagent layer and the reaction layer are hydrophilic colloids, they have a very high color development uniformity. Therefore, the dry chemical reagent strip of the present invention is used for quantitatively detecting isocitrate dehydrogenase activity, can be used for rapid clinical detection, and has good economic benefits and applications.

[0084] Example 2

[0085] This example provides a preparation method of the dry chemical reagent strip of Example 1, which includes the following steps:

[0086] S1, after coating the reagent liquid reagent and the reaction liquid reagent on the reagent layer and the reaction layer in sequence, drying is carried out;

[0087] S2, attaching the diffusion layer to the dried reaction layer;

[0088] S3, covering the transparent support layer on the dried reagent layer to obtain a dry chemical reagent strip including a transparent support layer, a reagent layer, a reaction layer and a diffusion layer in sequence.

[0089] Example 3

[0090] This example provides a detection method based on the dry chemical reagent strip of Example 1, which is used for measuring the isocitrate dehydrogenase activity in a liquid sample formed by a biological sample, and includes:

[0091] S1, diluting the biological sample through a diluent to prepare the liquid sample from the biological sample, adding the liquid sample to a reaction device containing the dry chemical reagent strip, and heating the reaction device to a properly selected temperature, which is selected from 25 °C to 37 °C, preferably 37 °C; wherein, the reaction device is made of ABS engineering plastic, and an isocitrate dehydrogenase detection hole is provided on the reaction device, and the dry chemical reagent strip is assembled in the isocitrate dehydrogenase detection hole;

[0092] S2, dropping the liquid sample onto the diffusion layer of the dry chemical reagent strip, and it is evenly distributed in the diffusion layer;

[0093] S3. The biological enzyme substances in the liquid sample penetrate into the reaction layer on the side opposite to the side of the diffusion layer that accommodates the liquid sample. Meanwhile, the reagent liquid in the reagent layer is continuously released into the reaction layer as a dye, and a color reaction occurs between the isocitrate dehydrogenase contained in the biological enzyme substances in the liquid sample and the reagent in the reaction layer. The reaction time is 5 - 10 minutes.

[0094] S4. Based on the reflectance spectrophotometry, use a reflectance spectrophotometer or a commonly used automatic analysis instrument in clinics to measure the increase rate of the reflectance in the detection hole at a wavelength of 530 - 760 nm (preferably 605 nm) within a specific time.

[0095] S5. Calculate the isocitrate dehydrogenase activity based on the increase rate of the reflectance through a standard curve.

[0096] Application examples and verification:

[0097] Example 1:

[0098] In this example, the transparent support layer material is made of polyethylene terephthalate, with a thickness of 0.18 mm, a length of 20 cm, and a width of 20 cm. The surface is treated by ultraviolet irradiation at a distance of 15 cm for 20 minutes.

[0099] The formulation of the isocitrate dehydrogenase reagent liquid in this example is shown in Table 1 below:

[0100] Table 1

[0101]

[0102] The formulation of the reaction liquid reagent is shown in Table 2 below:

[0103] Table 2

[0104]

[0105] The reagent liquid is sprayed on the ultraviolet - treated polyethylene terephthalate support layer and then put into a drying oven for drying. The reaction liquid is coated on the dried reagent layer and then put into a drying oven for drying.

[0106] A cellulose acetate filter membrane with a length of 20 cm, a width of 20 cm, and a pore diameter of 0.8 microns is wetted with purified water, the surface moisture is scraped off, and it is pasted on the dried reaction layer, compacted, and then put into a drying oven for drying.

[0107] It is cut into dry chemical reagent tablets of 20 mm × 20 mm size with a strip cutting machine.

[0108] Assemble the dry chemical reagent strip into the isocitrate detection hole of the reaction device and store it sealed with an aluminum foil bag.

[0109] Preparation of standards:

[0110] Prepare multiple isocitrate dehydrogenase standards with different concentration gradients for constructing a standard curve. For example, more than 5 isocitrate dehydrogenase standards with a concentration range of 1 U / L - 100 U / L can be prepared. In one example, the lyophilized isocitrate dehydrogenase standard [concentration: (100.0 ± 20.0) U / L] is dissolved in double-distilled water and then diluted to 100, 50, 25, 12.5, 6.25, 3.13, 1.57, 0.79 U / L with double-distilled water to construct a standard curve for quantification. The isocitrate dehydrogenase standard can be a commercially available quality control substance.

[0111] Determination method: Test samples of isocitrate dehydrogenase with different activities. Add 20 μL of the test sample to the detection hole of the detection device and measure it with a reflectance photometer. The test time is 10 minutes, and the absorbance change at 605 nm per minute is measured at 37°C. Calculate the isocitrate dehydrogenase activity in the sample according to the standard curve.

[0112] Each content of the above calibration samples is detected 3 times, and the correlation coefficient R value is calculated. The detection results are shown in Table 3, the results of the linearity test.

[0113] Table 3

[0114]

[0115] The results show that: R 2 The value is 0.9983, close to 1, indicating that within the range of isocitrate dehydrogenase activity of 0 - 100 U / L, the absorbance change per minute has a linear relationship with the isocitrate dehydrogenase activity.

[0116] Example 2

[0117] Precision test: Use the test method provided in Example 1 to detect samples. Use a lyophilized reconstituted solution containing 5.0 - 10.0 U / L isocitrate dehydrogenase as a low-concentration quality control product; use a lyophilized reconstituted solution containing 80.0 - 120.0 U / L isocitrate dehydrogenase as a high-concentration quality control product, and test the low-concentration quality control product with a known isocitrate dehydrogenase concentration of 6.8 U / L and the high-concentration quality control product with a known isocitrate dehydrogenase concentration of 98.5 U / L 30 times each. The isocitrate dehydrogenase quality control product can be a commercially available quality control substance.

[0118]

[0119]

[0120]

[0121] Among them, is the th measured concentration value, is the number of test times.

[0122] Calculated by the above formula, the low-concentration quality control product with an isocitrate dehydrogenase concentration of 6.8 U / L and the high-concentration quality control product with a concentration of 98.5 U / L were each tested 20 times. The average values ( ) of the measured values were 6.73 U / L and 102.38 U / L respectively, the standard deviations (SD) were 0.076 U / L and 1.82 U / L respectively, and the coefficients of variation (CV) were 3.52% and 2.87% respectively, which were less than the standard requirement of 10%. This indicates that the method for determining the activity of isocitrate dehydrogenase provided by the present invention has sufficient precision.

[0123] Example 3: Accuracy analysis of the method of the present invention

[0124] Comparative detection method: Detect with a clinically available isocitrate dehydrogenase assay kit (NAFP + redox enzyme method) on a Hitachi 7080 automatic biochemical analyzer. The automatic biochemical analyzer adds 2.5 μL of the sample and 250 μL of the reagent, reacts at 37 °C for 15 minutes, measures the absorbance value at 340 nm, and calculates the activity of isocitrate dehydrogenase in the sample according to the standard curve.

[0125] The method for determining the activity of isocitrate dehydrogenase provided in Example 1 of the present invention was used as the experimental group to detect the samples. The activity of isocitrate dehydrogenase in 30 serum samples was measured. At the same time, the activity of isocitrate dehydrogenase in these 30 identical serum samples was measured by the comparative detection method. The detection results are shown in the analysis results of Table 4.

[0126] Table 4

[0127]

[0128] The results show that the R 2 value calculated according to the detection results is 0.9971, which is greater than 0.95, indicating that there is no significant difference between the detection results of the method of the present invention and the detection results of the comparative kit, and it has high accuracy (conformity).

[0129] Through the description of the above embodiments, those skilled in the art can clearly understand that the above embodiments can be implemented by software or by means of software plus a necessary general hardware platform. Based on such an understanding, the technical solutions of the above embodiments can be embodied in the form of a software product, which can be stored in a non-volatile storage medium (which can be a CD-ROM, a USB flash drive, a mobile hard disk, etc.), including several instructions for causing a computer device (which can be a personal computer, a server, or a network device, etc.) to execute the methods described in various embodiments of the present invention.

[0130] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A dry chemical reagent sheet for isocitrate dehydrogenase activity assay, characterized in that: For determining the activity of isocitrate dehydrogenase, the dry chemical reagent sheet comprises four stacked layers, namely, a transparent support layer, a reagent layer, a reaction layer and a diffusion layer; wherein: The transparent support layer is arranged at the bottom of the dry chemical reagent sheet and is used to support the reagent layer, the reaction layer and the diffusion layer; The reagent layer contains a reagent solution reagent for providing a reaction reagent for the reaction of determining the activity of isocitrate dehydrogenase; The reaction layer contains a reaction liquid reagent formed by a hydrophilic colloid, which is used for performing a reaction to measure the activity of isocitrate dehydrogenase; The diffusion layer is made of a hydrophilic polymer filter membrane, which is used to evenly distribute the sample and provide a reflective background; After the liquid sample is dripped onto the diffusion layer of the dry chemical reagent sheet, it is evenly distributed in the diffusion layer, and the biological enzyme substances in the liquid sample penetrate into the reaction layer on the side opposite to the side of the diffusion layer accommodating the liquid sample. At the same time, the reagent solution in the reagent layer is continuously released into the reaction layer as a dye, and a color reaction occurs with the isocitrate dehydrogenase contained in the biological enzyme substances in the liquid sample. The change in reflectivity within a specific time at a wavelength of 530-760nm is measured based on the reflectance spectrophotometry; based on the change in reflectivity, the isocitrate dehydrogenase activity is calculated using a standard curve.

2. A dry chemical reagent sheet for isocitrate dehydrogenase activity assay according to claim 1, characterized in that: The material of the transparent support layer includes one of polyethylene, polyethylene terephthalate polymer, polyvinyl chloride, polystyrene and polyester fiber; the thickness of the transparent support layer is 50-300 microns.

3. A dry chemical reagent sheet for isocitrate dehydrogenase activity assay according to claim 2, characterized in that: The hydrophilic colloid is one or more of polyvinyl alcohol, pullulan, cellulose ester, agarose, polyvinyl pyrrolidone, polyacrylamide, hydroxypropyl methylcellulose and methyl vinyl ether / maleic anhydride copolymer; the amount of the hydrophilic colloid is 50-150g / m 2 .

4. The dry chemical reagent sheet for isocitrate dehydrogenase activity assay according to claim 3, characterized in that: The reagent solution in the reagent layer includes the following components: Dyes with oxidized and reduced states, including tetrazolium salts, cofactors, electron carriers, isocitrate or isocitrate salts, divalent metal ions, buffers, additives, coenzyme stabilizers, surfactants, film formers, and preservatives.

5. A dry chemical reagent sheet for isocitrate dehydrogenase activity assay according to claim 4, characterized in that: The tetrazolium salt is of a type sufficient to be reduced to a colored formazan and in an amount sufficient to correlate the concentration of the formazan with the concentration of the analyte in the liquid sample; examples of the tetrazolium salt include 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide MTT, p-iodonitrotetrazolium violet INT, nitrotetrazolyl blue chloride NBT, tetranitro blue tetrazolyl chloride TNBT, tetrazolyl blue TB, tetrazolyl violet TV, thiocarbamoyl nitro blue tetrazolyl chloride; triphenyl tetrazole; 2-(2'-benzothiazolyl)-5-phenylvinyl-3-(4'-phthaloyl) tetrazole BSPT, 2-benzothiazolyl-(2)-3,5-diphenyl tetrazole (BTDP), 2,3-di(4-nitrophenyl) tetrazole (DNP), 2,5-diphenyl-3-(4-phenylvinyl) tetrazole (DPSP), distyryl nitro blue tetrazole (DS-NBT), 2-phenyl-3-(4-carboxylic acid) tetrazole (DS-NBT), phenyl)-5-methyltetrazolium (PCPM), thiocarbamoyl nitro blue tetrazolium (TCNBT) or water-soluble tetrazolium salts; the water-soluble tetrazolium salts include 2-(4-iodophenyl)-3-(2,4-dinitrophenyl)-5-phenyl-3H-tetrazolium-2',4'-disulfonic acid sodium salt (IPB), 2-benzothiazolyl-thiazolyl-3-(4-carboxy-2-methoxyphenyl)-5-[4-(2-sulfoethylcarbamoyl)phenyl]- One or more of 2H-tetrazole (GLT004), 2,2'-dibenzothiazolyl-5,5'-bis[4-di(2-sulfoethyl)carbamoylphenyl]-3,3'-(3,3'-dimethoxy-4,4'-biphenylene)di-tetrazole disodium salt (WST-5), and 2-(2-methoxy-4-nitrobenzene)-3-(4-nitrobenzene)-5-(2,4-disulfonylphenyl)-2H-tetrazole monosodium salt (WST-8); The cofactor comprises β-nicotinamide adenine dinucleotide, β-nicotinamide adenine dinucleoside phosphate or any salt thereof, wherein any salt thereof comprises one or more of β-nicotinamide adenine dinucleotide phosphate disodium salt, β-nicotinamide adenine dinucleotide sodium salt and β-nicotinamide adenine dinucleotide lithium salt; The electron carrier comprises one or more of diaphorase, phenazine methyl sulfate PMS, 1-methoxyphenazine methyl sulfate, 9-dimethylaminobenzo-a-phenazoline chloride and 2,6-dichlorophenol indophenol; The divalent metal ions include one or more of magnesium ions, manganese ions, iron ions, nickel ions, zinc ions and cobalt ions; The buffer comprises a buffer capable of buffering to a pH in the range of 5 to 9, including one or more of tris(hydroxymethyl)aminomethane, tris(hydroxymethyl)methylglycine, 4-hydroxyethylpiperazinepropanesulfonic acid, 4-hydroxyethylpiperazineethanesulfonic acid, tris(hydroxymethyl)aminomethaneethanesulfonic acid, N,N-di(2-hydroxyethyl)-2-aminoethanesulfonic acid, 3-morpholinepropanesulfonic acid, 2-morpholineethanesulfonic acid, 1,4-piperazinediethanesulfonic acid and bis(2-hydroxyethyl)amino(trihydroxymethyl)methane; The additives include one or more of sodium chloride, potassium chloride, sodium sulfate and potassium sulfate, one or more of sucrose and lactose, one or more of sorbitol and mannitol, and protein, and also include chelating agents, amines, basic amino acids or heterocyclic compounds; The coenzyme stabilizer includes one or more of sericin, carboxylated chitosan, substrate stabilizer CCD, substrate stabilizer PPCD, stabilizer protector Phos-E, complex enzyme stabilizer AES, complex enzyme stabilizer DPD, complex enzyme stabilizer AFP, complex enzyme protector HP and protein protector TY; The surfactant includes a nonionic surfactant and an anionic surfactant; The film-forming agent includes one or a combination of at least two of gelatin, dimethyl phthalate, hydroxyethyl cellulose, hydroxymethyl cellulose, polyvinyl pyrrolidone K30, polyvinyl alcohol, polyacrylamide or xanthan gum; The preservatives include sodium azide or isothiazolinones.

6. A dry chemical reagent sheet for isocitrate dehydrogenase activity assay according to claim 5, characterized in that: The nonionic surfactant includes one or more of polyethylene glycol monooctylphenyl ether TritonX-100, sorbitol monolaurate Span20, sorbitan monopalmitate Span40, polyoxyethylene lauryl ether Brij35, polyoxyethylene cetyl ester Brij58, polyoxyethylene sorbitol monolaurate Brij58, polyoxyethylene sorbitol monolaurate Span40, Tween 20, Tween 40, Tween 80 and Tween 60; The anionic surfactant includes one or more of sodium dodecyl sulfate, sodium dodecylbenzene sulfonate, sodium cholate, sodium deoxycholate and sodium lauryl sarcosinate.

7. A dry chemical reagent sheet for isocitrate dehydrogenase activity assay according to claim 6, characterized in that: The reagent solution in the reagent layer contains 0.01-20mmol / L of tetrazolium salt; 0.5-200mmol / L of β-phosphonicotinamide adenine dinucleotide disodium salt; 1-1000U / ml diaphorase; 0.1-200mmol / L of DL-isocitrate trisodium salt; 0.5-100mmol / L of divalent magnesium ions; 0.1-30wt% additives; 0.001 to 30wt% of coenzyme stabilizers; 0.0001 to 10wt%, 0.001 to 1wt% of surfactants; 0.001 to 10wt% of film formers; 0.001 to 1wt% of preservatives; and 1-100mmol / L of buffer to adjust the pH to 5.0-10.

0.

8. The dry chemical reagent sheet for isocitrate dehydrogenase activity assay according to claim 7, characterized in that: The hydrophilic polymer filter membrane is a filter membrane made of one or more of cellulose acetate, polyvinylidene fluoride, polyether sulfone and polytetrafluoroethylene; the pore size of the hydrophilic polymer filter membrane is 0.45-8.0 microns.

9. The method for preparing the dry chemical reagent sheet according to any one of claims 1 to 8, characterized in that: The steps include: S1, coating the reagent solution reagent and the reaction solution reagent on the reagent layer and the reaction layer in sequence and then drying them; S2, laminating the diffusion layer on the dried reaction layer; S3, covering the dried reagent layer with the transparent support layer to obtain a dry chemical reagent sheet including a transparent support layer, a reagent layer, a reaction layer and a diffusion layer in sequence.

10. A detection method based on the dry chemical reagent sheet according to any one of claims 1 to 8, characterized in that: Used to determine isocitrate dehydrogenase activity in liquid samples formed from biological samples, including: S1, diluting the biological sample with a diluent to prepare the liquid sample, adding the liquid sample to a reaction device containing the dry chemical reagent sheet, and heating the reaction device to an appropriately selected temperature, the temperature being selected from 25° C. to 37° C.; wherein the reaction device is made of ABS engineering plastic, the reaction device is provided with an isocitrate dehydrogenase detection hole, and the dry chemical reagent sheet is assembled in the isocitrate dehydrogenase detection hole; S2, dropping the liquid sample into the diffusion layer of the dry chemical reagent sheet, and evenly distributing the liquid sample in the diffusion layer; S3, the biological enzyme substance in the liquid sample penetrates into the reaction layer on the side opposite to the diffusion layer containing the liquid sample, and at the same time, the reagent solution in the reagent layer is continuously released into the reaction layer as a dye, and a color reaction occurs in the reaction layer with the isocitrate dehydrogenase contained in the biological enzyme substance in the liquid sample, and the reaction time is 5-10 minutes; S4, based on the reflectance spectrophotometry method, using a reflectance spectrophotometer or an automatic analytical instrument commonly used in clinical practice to measure the rate of increase of the reflectance in the detection hole within a specific time at a wavelength of 530-760 nm; S5. Calculate the isocitrate dehydrogenase activity based on the reflectance increase rate using a standard curve.

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