Preparation method and application of dry chemical reagent for determining creatine content

Abstract

The invention discloses a preparation method of a dry chemical reagent for creatine content determination. The preparation method comprises the following steps: S1, preparing a buffer reagent and a protective component; s2, dissolving the biological enzyme and the chromophoric substrate to form a dissolving reagent; s3, coating by using a biological film-forming material to form a coating reagent; s4, coating and printing the coating reagent in the pore channel of the specified bottom plate base material in a volume quantitative manner by virtue of equipment; the equipment comprises a single-channel type pipettor, a multi-channel type pipettor, a continuous multi-gear distributor or automatic dot printing equipment. S5, placing the specified bottom plate base material printed with the coating reagent in forced air drying equipment or drying equipment with the same efficiency; and S6, measuring the moisture content after drying and film forming, and judging whether substances containing biologically active enzymes and substrates in the reagent are completely locked to the position of the net-shaped cavernous body structure of the biological film forming material or not based on the moisture content. The invention further discloses application of the dry chemical reagent for determining the creatine content, and the dry chemical reagent is applied to a sample formed by urine, serum or tissue.

Description

Technical Field

[0001] The invention relates to the technical field of dry chemical reagents and creatine determination, and in particular to a preparation method of a dry chemical reagent for creatine content determination and application thereof. Background Art

[0002] Creatine is a nitrogen-containing organic acid with a chemical formula of C 4 H 9 N 3 O 2 , scientific name N-methylguanidinoacetic acid, naturally exists in vertebrates and can help provide energy for muscles and nerve cells. In 1832, French chemist Michel Eugène Chevreul first discovered creatine in skeletal muscle, and then named it "Creatine" based on the Greek word "Kreas" (meat). Creatine is mainly stored in muscle tissue, which can reduce muscle fatigue and tension, enhance muscle elasticity, make muscles stronger, accelerate the synthesis of human protein, reduce cholesterol, blood lipids and blood sugar, and delay the aging of the body, and play a role when energy demand is high.

[0003] Mechanism of Creatine: Creatine is synthesized from three amino acids: arginine, glycine and methionine. Creatine can be produced endogenously by the liver, kidneys and pancreas, about 1g / day; it can also be supplemented through daily diet (meat and seafood). Non-vegetarians can supplement about 1~2g of creatine from food every day. For a 70kg adult, the total amount of creatine in the body (Pcr+C) is about 120mmol / kg dry muscle mass, but generally speaking, the upper limit of creatine storage can reach 160mmol / kg. Vegetarians have lower intramuscular creatine reserves, about 90~110mmol / kg. About 1~2% of intramuscular creatine is metabolized into creatinine every day and excreted through urine. Athletes who engage in high-volume training have a higher degree of creatine metabolism, so they need to supplement additional creatine to maintain the total amount of creatine in the body. Most of the creatine in the human body (about 95%) is located in skeletal muscle, and only a small part (about 5%) is stored in the brain and testicles. About 2 / 3 of creatine in skeletal muscle is stored in the form of creatine phosphate (PCr), and the rest is stored as free creatine (C). Creatine phosphate provides energy buffering for muscle activity. Under energy metabolism requirements, creatine phosphate is hydrolyzed into creatine and phosphate, and free energy is released to phosphorylate ADP into ATP, which is then hydrolyzed to release energy, while free creatine serves as a substrate for the synthesis of creatine phosphate.

[0004] Creatine and Sarcopenia: Muscle mass decreases with age, a process known as sarcopenia. Longitudinal studies have shown that the main risk factor for sarcopenia is age. In young adult muscles, some signals stimulate protein synthesis and inhibit its breakdown, thereby maintaining muscle mass. In the elderly, however, muscles become resistant to these anabolic stimuli, resulting in impaired protein synthesis and promoted protein breakdown, which in turn promotes muscle atrophy. In 2019, a research team from the University of São Paulo in Brazil pointed out in an article that creatine supplementation as an adjunct to exercise training can enhance the adaptive response of muscles to training stimuli. Therefore, creatine may be an effective dietary strategy to combat age-related sarcopenia, and exercise is the main medium for creatine to work on the elderly. Creatine is a detection unit in the metabolism of organic acids in urine. The significance of its quantitative detection is to monitor exercise metabolism in real time and the metabolism of creatine during aerobic fitness. Reasonable and quantitative supplementation increases the intake of creatine. At the same time, for the middle-aged and elderly, long-term bedridden people and people with sarcopenia, it serves the purpose of reasonable assessment and establishing more complete metabolic monitoring.

[0005] At present, the indirect method or fluorescence spectrophotometry is mostly used to determine urine creatine in clinical practice in China. Both methods have the following technical defects: (1) Indirect method: The sensitivity is low, there are many interference factors, the heating and dehydration of creatine takes a long time and is often incomplete, and there is a big difference between the two operations. The testing laboratory prepares its own reagents, and there is a big difference between product batches.

[0006] (2) Fluorescence spectrophotometry: Fluorescence determination requires the use of an ultraviolet spectrophotometer, which is cumbersome and complicated to operate, which is not conducive to the clinical diagnosis of this project; in addition, fluorescent reagents seriously pollute the environment and are heavily dependent on ultraviolet spectrophotometers. The detection principle is: protein precipitants are often used to remove proteins and some interfering substances in the sample to be tested. Creatine in the sample combines with ninhydrin in an alkaline environment to form a fluorescent complex. Therefore, the detection laboratory prepares its own reagents, and the product batches vary greatly. Summary of the invention

[0007] The purpose of the present invention is to overcome the defects of the prior art, to address the problems in the related art, to provide a method for preparing a dry chemical reagent for determining creatine content and its application, by organically combining the use of biofilm-forming materials with traditional dry chemical determination technology. The main reaction enzyme, auxiliary tool enzyme, chromogenic substrate, buffer reagent and protective component in the dry chemical reaction process are wrapped with the biofilm-forming material, and the above mixture is printed on the bottom plate of the reaction device. The water in the mixture is completely removed by drying means. The main components are firmly locked in the pores of the reaction substrate by physical adsorption, so as to achieve the effect of drying and fixing the reagent.

[0008] The first aspect of the present invention is to provide a method for preparing a dry chemical reagent for determining creatine content, comprising: S1, prepare buffer reagents and protective components; S2, dissolving the biological enzyme and the chromogenic substrate to form a dissolving reagent; S3, using a biofilm-forming material to form a coating agent; S4, applying and printing the coating reagent in the channels of the designated bottom substrate in a volumetrically quantitative manner with the aid of a device; the device includes a single-channel pipette, a multi-channel pipette, a continuous multi-speed dispenser or an automated dot-printing device; S5, placing the designated base plate substrate printed with the coating reagent in a forced air drying device or a drying device with equivalent efficiency; S6, measuring the moisture content after drying and forming the film, and judging whether the substances containing biologically active enzymes and substrates in the reagent have been completely locked into the mesh sponge structure position of the biological film-forming material based on the moisture content.

[0009] Preferably, the S1 comprises: S11, select a suitable buffer system, including: Tris-HCL, MOPS, HEPES and phosphate buffer based buffer systems; S12, configuring protective components.

[0010] Preferably, the S12 includes: (1) adding a certain concentration of sucrose, trehalose, raffinose, inositol, bovine serum albumin, silk protein, casein, bovine serum and amino acids to the basic buffer system; (2) adding a surfactant to the basic buffer system, wherein the surfactant includes Tween-20, glycerol, PEG and Triton; (3) Adding a certain concentration of EDTA, potassium ferrocyanide, bilirubin oxidase and ascorbic acid oxidase into the basic buffer system.

[0011] Preferably, S2 includes: S21, selecting a certain concentration of creatinase to be dissolved in the basic buffer system; S22, dissolving a certain concentration of horseradish peroxidase and related coenzymes into the basic buffer system; wherein the dissolving reagent formed after dissolving the biological enzyme and the chromogenic substrate is a precipitation-free homogeneous reagent.

[0012] Preferably, S3 includes: S31, selecting a biofilm-forming material suitable for creatine determination methodology, and preparing the biofilm-forming material into a film-forming base liquid of a certain concentration; S32, after the film-forming base liquid is cooled to room temperature, the dissolving reagent is added to the film-forming base liquid at room temperature, and the mixture is thoroughly mixed.

[0013] Preferably, the biofilm-forming material includes one or more of chitin, polysaccharides, agarose and PVA.

[0014] Preferably, S4 includes: S41, clean and passivate the surface of the designated baseboard substrate before use; S42, using the equipment, the coating reagent is applied and printed into the channels of the designated bottom plate substrate in a volumetrically quantitative manner.

[0015] Preferably, S5 includes: S51, setting the wind speed, temperature and drying time of the blast drying equipment or a drying equipment with equal efficiency; S52, placing the designated base substrate after printing the coating reagent in a blast drying device or a drying device with equivalent efficiency, and drying the coating reagent printed in the pores of the designated base substrate through the air duct and the drying device; S53, determining whether the film formation after drying meets the film formation standard; the film formation standard includes: the film layer surface is smooth, without cracks, peeling and separation from the base material.

[0016] Preferably, the determining of the moisture content after drying and forming the film comprises determining the moisture content after drying and forming the film by gravimetric method or Karl Fischer method.

[0017] The second aspect of the present invention provides an application of a dry chemical reagent for determining creatine content prepared by the preparation method of the first aspect, which is applied to samples formed by urine, serum or tissue, comprising: S1', diluting the sample based on a diluent to obtain a diluted sample; the diluent is prepared by dissolving a certain concentration of a process catalyst in a neutral buffer system with a stable pH value of 50-500mmol / L, and adding a preservative component with a concentration of 1 / 10,000 to 1 / 1000; the process catalyst includes one or more of TBHB, DCHBS and 4-AA; the neutral buffer system with a stable pH value includes one or more of phosphate, Tris-HCL, HEPES and MOPS; S2', adding sample and reacting; including: adding the diluted sample to the reaction well of the corresponding reagent at a sample volume of 15-35uL per reaction system, and completing the biochemical reaction within 7-15 minutes; the biochemical reaction can be carried out at room temperature and 37°C, and the sample color changes from colorless to red; S3', according to the color change of the sample from colorless to red after the reaction, the color change is observed by naked eyes, and the color scale card established by the standard concentration sample is compared to achieve semi-quantitative detection; and / or a standard curve is established using the standard concentration sample, corresponding to the CCD, CMOS and sample color sensing equipment, and the color weight analysis is performed and then matched with the standard curve to achieve quantitative detection.

[0018] The preparation method of the present invention and the beneficial effects of its application: With the help of biofilm-forming materials and a one-time film-forming preparation process, a rapid preparation method for semi-quantitative and quantitative dry chemical reagents for creatine indicators is realized. Solid-phase reagents are convenient and easy to store. Innovations have been made in the implementation plan of the one-time film-forming preparation process, the selection and use concentration of biofilm-forming materials, the components for removing interfering substances in the reagent system, the preparation plan of the diluent, and the treatment method of the substrate material. The production and preparation process is simple and easy, and the experimental and manufacturing costs are low. While meeting the requirements of small-scale manual production, it can also be used in semi-automatic and fully automated production lines. The product is suitable for the separate detection of creatine indicators, and is also suitable for joint detection with multiple biochemical indicators. It can be achieved on both traditional dry chemical technology platforms and microfluidic technology platforms, specifically in: (1) It fills the technical gap of dry chemical enzymatic method for determining creatine content; (2) Use one-time film-forming technology to reduce the use of production equipment involved in multi-layer coating. This preparation process is suitable for small-scale manual manufacturing and also meets the design and development of fully automatic full-process preparation production lines.

[0019] (3) The liquid sample is used as the reagent reaction system, and the biofilm-forming material dissolves after mixing with the liquid. Compared with the reaction in the existing multi-layer chromatography filter paper and film, the intramolecular diffusion effect of the contents is obvious, which is similar to the biochemical liquid reagents, reducing the generation of color spots and blocks in the dry chemical reaction system, improving the reagent performance (especially the repeatability index), and laying the foundation for quantitative determination.

[0020] (4) The product produced by the technology of the present invention has good stability and can be stored in the dark at 2-8°C for 15 months. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the related technologies, the drawings required for use in the specific embodiments or the related technical descriptions will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0022] Figure 1A flow chart of a method for preparing a dry chemical reagent for creatine determination stored at room temperature according to an embodiment of the present invention; Figure 2 This is a flow chart of step S1 of a method for preparing a dry chemical reagent for creatine determination stored at room temperature according to an embodiment of the present invention; Figure 3 This is a flow chart of step S2 of a method for preparing a dry chemical reagent for creatine determination stored at room temperature according to an embodiment of the present invention; Figure 4 This is a flow chart of step S3 of a method for preparing a dry chemical reagent for creatine determination stored at room temperature according to an embodiment of the present invention; Figure 5 This is a flow chart of step S4 of a method for preparing a dry chemical reagent for creatine determination stored at room temperature according to an embodiment of the present invention; Figure 6 This is a flow chart of step S5 of a method for preparing a dry chemical reagent for creatine determination stored at room temperature according to an embodiment of the present invention; Figure 7 A flow chart of an application method of a dry chemical reagent for storing creatine assay at room temperature according to an embodiment of the present invention; Figure 8 A linear schematic diagram of product labeling provided according to an embodiment of the present invention; Fig. 9 A schematic diagram of a limiting range provided according to an embodiment of the present invention; Fig.10 This is a schematic diagram of the long-term stability of a product corresponding to 2-8°C provided according to an embodiment of the present invention. DETAILED DESCRIPTION

[0023] The technical solution of the present invention will be described clearly and completely below in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0024] In the description of the present invention, it should be noted that the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", etc., indicating the orientation or positional relationship, are based on the orientation or positional relationship shown in the drawings, and are 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 cannot be understood as limiting the present invention. In addition, the terms "first", "second", and "third" are used for descriptive purposes only, and cannot be understood as indicating or implying relative importance.

[0025] In the description of the present invention, it should be noted that, unless otherwise clearly specified and limited, the terms "installed", "connected", and "connected" 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 a direct connection, or it can be indirectly connected through an intermediate medium, or it can be the internal communication of two components. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances. Embodiment 1

[0026] like Figure 1 As shown, this embodiment provides a method for preparing a dry chemical reagent for creatine determination stored at room temperature, comprising: S1, prepare buffer reagents and protective components; like Figure 2 As shown, as a preferred embodiment, the S1 includes: S11, selecting a suitable buffer system, including: a buffer system based on Tris-HCL, MOPS, HEPES and phosphate buffers, in order to maintain the stability of the pH value of the buffer system and ensure the biological activity of the main raw materials such as creatinase; S12, configure protection components, including: (1) adding a certain concentration of sucrose, trehalose, raffinose, creatine, bovine serum albumin, silk protein, casein, bovine serum and amino acids to the basic buffer system to reduce the possibility of active enzymes being oxidized and losing their effectiveness; (2) adding a certain proportion of surfactants such as Tween-20, glycerol, PEG and Triton to the basic buffer system, in order to make the contents in the buffer system evenly dispersed; (3) A certain concentration of EDTA, potassium ferrocyanide, bilirubin oxidase and ascorbic acid oxidase are added to the basic buffer system to remove interfering substances in the sample.

[0027] S2, dissolving the biological enzyme and the chromogenic substrate to form a dissolving reagent; like Figure 3 As shown, as a preferred embodiment, the S2 includes: S21, selecting a suitable concentration of creatinase and dissolving it in the basic buffer system; S22, dissolving horseradish peroxidase and related coenzymes of suitable concentration in the basic buffer system; wherein, the dissolved reagent formed after dissolving the biological enzyme and the chromogenic substrate should be a homogeneous reagent as a whole without precipitation.

[0028] S3, using a biofilm-forming material to form a coating agent; like Figure 4As shown, as a preferred implementation, S3 includes: S31, selecting a biofilm-forming material suitable for the creatine determination methodology, preferably chitin, polysaccharide, agarose, PVA, etc.; and preparing the biofilm-forming material into a film-forming base liquid of a certain concentration. Generally, the biofilm-forming material is a high molecular weight substance, and the dissolution process is relatively difficult, requiring high temperature or other methods to assist dissolution; S32, after the film-forming base liquid is cooled to room temperature, the dissolving reagent is added to the film-forming base liquid at room temperature, and the mixture is thoroughly mixed.

[0029] S4, applying and printing the coating reagent in the channels of the designated bottom substrate in a volumetrically quantitative manner with the aid of a device; the device includes a single-channel pipette, a multi-channel pipette, a continuous multi-speed dispenser or an automated dot-printing device; like Figure 5 As shown, as a preferred embodiment, the S4 includes: S41, in order to ensure the cleanliness, flatness and surface water purity of the designated base plate substrate, the designated base plate substrate is cleaned and surface passivated before use, the purpose of which is to remove impurities and burrs of the base plate substrate and improve the hydrophilicity, which is conducive to the adhesion of the film-forming material; S42, using the equipment, the coating reagent is applied and printed into the channels of the designated bottom plate substrate in a volumetrically quantitative manner.

[0030] S5, placing the designated base plate substrate printed with the coating reagent in a forced air drying device or a drying device with equivalent efficiency; like Figure 6 As shown, as a preferred implementation, the S5 includes: S51, setting the wind speed, temperature and drying time of the blast drying equipment or drying equipment of equal efficiency to ensure that the reagent is completely dried; S52, placing the designated base substrate after printing the coating reagent in a blast drying device or a drying device with equivalent efficiency, and drying the coating reagent printed in the pores of the designated base substrate through the air duct and the drying device; S53, determining whether the film formation after drying meets the film formation standard; the film formation standard includes: the film layer surface is smooth, and there should be no cracking, peeling, separation from the base material, etc.

[0031] S6, determine the moisture content after drying and film formation by weighing method or Karl Fischer method, and judge whether the substances containing biologically active enzymes and substrates in the reagent have been completely locked into the mesh sponge structure of the biofilm-forming material based on the moisture content. The biofilm-forming material uses a three-dimensional structure similar to the sponge pores to lock the substances containing biologically active enzymes, substrates, etc. in the reagent into the mesh sponge structure of the biofilm-forming material, and can keep the reagent stable at room temperature, so as to achieve the purpose of not interfering with each other and not causing biochemical reactions. Embodiment 2

[0032] like Figure 7 As shown, this embodiment provides an application of a dry chemical reagent for determining creatine content prepared by the preparation method of Example 1, which is applied to samples formed by urine, serum or tissue, including: S1', diluting the sample based on a diluent to obtain a diluted sample; the diluent is prepared based on a suitable concentration of process catalysts such as TBHB, DCHBS, 4-AA dissolved in a pH-stable neutral buffer system such as 50-500mmol / L phosphate, Tris-HCL, HEPES, MOPS, etc., and adding a preservative component at a concentration of 1 / 10,000 to 1 / 1000.

[0033] This embodiment takes urine samples as an example: the dilution ratio is between 1:14 and 1:29.

[0034] S2', add sample and react; including: add the diluted sample to the reaction well of the corresponding reagent at a sample volume of 15-35uL per reaction system, and complete the biochemical reaction within 7-15 minutes; the biochemical reaction can be carried out at room temperature and 37°C, and the sample color changes from colorless to purple. In this reaction system, the creatinase content and coenzyme content are set to a surplus, so the reaction will not be terminated before the creatine in the sample is completely consumed; S3', according to the color change of the sample from colorless to red after the reaction, the color change is observed by naked eyes, and the color scale card established by comparing with the standard concentration sample can be used to achieve semi-quantitative detection; the standard concentration sample is used to establish a standard curve, corresponding to CCD, CMOS and sample color sensing equipment, and the color weight analysis is performed and then corresponded with the standard curve to achieve quantitative detection.

[0035] Analysis of test results of embodiment: 1. Actual data display and analysis: A. Gradient of standard curve system, standard concentrations are: 0 (purified water), 0.05mmol / L, 0.1mmol / L, 0.25mmol / L, 0.5mmol / L, 1mmol / L, 2mmol / L, 4mmol / L, 8mmol / L, 12mmol / L, the dilution ratio is 1:29.

[0036] B. Product standard linearity is shown in Table 1 and Figure 8 As shown; Table 1

[0037] C. Blank limits are shown in Table 2: Table 2

[0038] Blank limit requirement: 0.05mmol / L.

[0039] D. The limiting range is shown in Table 3 and Fig. 9 As shown: Table 3

[0040] Fig. 9 Medium requirement: R 2 >0.975.

[0041] E. Repeatability is shown in Table 4.

[0042] Table 4

[0043] Require: Repeatability: Variation dilution CV <15%.

[0044] F. The recovery rates are shown in Table 5.

[0045] Table 5

[0046] Recovery rate: 85%-115%.

[0047] G. The batch-to-batch variability of the products is shown in Table 6.

[0048] Table 6

[0049] Inter-batch difference: relative range ≤±15%.

[0050] H. Product Stability The long-term stability diagram of creatine products at 2-8°C is as follows Fig.10 As shown (the curves from top to bottom correspond to sample concentrations of 1mmol / L, 4mmol / L and 8mmol / L); this product can achieve a storage period of 15 months under 2~8℃ conditions.

[0051] Through the description of the above implementation methods, those skilled in the art can clearly understand that the above embodiments can be implemented by software, or by means of software plus necessary general hardware platforms. Based on such understanding, the technical solutions of the above embodiments can be embodied in the form of software products, 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 enabling a computer device (which can be a personal computer, a server, or a network device, etc.) to execute the methods described in the various embodiments of the present invention.

[0052] 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 aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a dry chemical reagent for determining creatine content, characterized in that: include: S1, prepare buffer reagents and protective components; S2, dissolving the biological enzyme and the chromogenic substrate to form a dissolving reagent; S3, using a biofilm-forming material to form a coating agent; S4, applying and printing the coating reagent in the channels of the designated bottom substrate in a volumetrically quantitative manner with the aid of a device; the device includes a single-channel pipette, a multi-channel pipette, a continuous multi-speed dispenser or an automated dot-printing device; S5, placing the designated base plate substrate printed with the coating reagent in a forced air drying device or a drying device with equivalent efficiency; S6, measuring the moisture content after drying and forming the film, and judging whether the substances containing biologically active enzymes and substrates in the reagent have been completely locked into the mesh sponge structure position of the biological film-forming material based on the moisture content.

2. A method for preparing a dry chemical reagent for creatine content determination according to claim 1, characterized in that: The S1 includes: S11, select a suitable buffer system, including: Tris-HCL, MOPS, HEPES and phosphate buffer based buffer systems; S12, configuring protective components.

3. A method for preparing a dry chemical reagent for creatine content determination according to claim 2, characterized in that: The S12 includes: (1) adding a certain concentration of sucrose, trehalose, raffinose, inositol, bovine serum albumin, silk protein, casein, bovine serum and amino acids to the basic buffer system; (2) adding a surfactant to the basic buffer system, wherein the surfactant includes Tween-20, glycerol, PEG and Triton; (3) Adding a certain concentration of EDTA, potassium ferrocyanide, bilirubin oxidase and ascorbic acid oxidase into the basic buffer system.

4. A method for preparing a dry chemical reagent for determining creatine content according to claim 3, characterized in that: The S2 includes: S21, selecting a certain concentration of creatinase to be dissolved in the basic buffer system; S22, dissolving a certain concentration of horseradish peroxidase and related coenzymes into the basic buffer system; wherein the dissolving reagent formed after dissolving the biological enzyme and the chromogenic substrate is a precipitation-free homogeneous reagent.

5. A method for preparing a dry chemical reagent for determining creatine content according to claim 4, characterized in that: The S3 includes: S31, selecting a biofilm-forming material suitable for creatine determination methodology, and preparing the biofilm-forming material into a film-forming base liquid of a certain concentration; S32, after the film-forming base liquid is cooled to room temperature, the dissolving reagent is added to the film-forming base liquid at room temperature, and the mixture is thoroughly mixed.

6. A method for preparing a dry chemical reagent for determining creatine content according to claim 5, characterized in that: The biofilm-forming material includes one or more of chitin, polysaccharide, agarose and PVA.

7. A method for preparing a dry chemical reagent for determining creatine content according to claim 6, characterized in that: The S4 includes: S41, clean and passivate the surface of the designated baseboard substrate before use; S42, using the equipment, the coating reagent is applied and printed into the channels of the designated bottom plate substrate in a volumetrically quantitative manner.

8. A method for preparing a dry chemical reagent for determining creatine content according to claim 7, characterized in that: The S5 includes: S51, setting the wind speed, temperature and drying time of the blast drying equipment or a drying equipment with equivalent efficiency; S52, placing the designated base substrate after printing the coating reagent in a blast drying device or a drying device with equivalent efficiency, and drying the coating reagent printed in the pores of the designated base substrate through the air duct and the drying device; S53, determining whether the film formation after drying meets the film formation standard; the film formation standard includes: the film layer surface is smooth, without cracks, peeling and separation from the base material.

9. A method for preparing a dry chemical reagent for determining creatine content according to claim 8, characterized in that: The determining of the moisture content after drying and forming the film comprises determining the moisture content after drying and forming the film by gravimetric method or Karl Fischer method.

10. Use of a dry chemical reagent for determining creatine content prepared by the preparation method according to any one of claims 1 to 9, characterized in that: Applicable to samples of urine, serum or tissue, including: S1', diluting the sample based on a diluent to obtain a diluted sample; the diluent is prepared by dissolving a certain concentration of a process catalyst in a neutral buffer system with a stable pH value of 50-500mmol / L, and adding a preservative component with a concentration of 1 / 10,000 to 1 / 1000; the process catalyst includes one or more of TBHB, DCHBS and 4-AA; the neutral buffer system with a stable pH value includes one or more of phosphate, Tris-HCL, HEPES and MOPS; S2', adding sample and reacting; including: adding the diluted sample to the reaction well of the corresponding reagent at a sample volume of 15-35uL per reaction system, and completing the biochemical reaction within 7-15 minutes; the biochemical reaction can be carried out at room temperature and 37°C, and the sample color changes from colorless to red; S3', according to the color change of the sample from colorless to red after the reaction, the color change is observed by naked eyes, and the color scale card established by the standard concentration sample is compared to achieve semi-quantitative detection; and / or a standard curve is established using the standard concentration sample, corresponding to the CCD, CMOS and sample color sensing equipment, and the color weight analysis is performed and then matched with the standard curve to achieve quantitative detection.

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