Preparation method of dry chemical reagent for determining content of beta-hydroxybutyric acid in urine

Abstract

The invention discloses a preparation method of a dry chemical reagent for determining the content of beta-hydroxybutyric acid in urine. 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, by means of equipment, the coating reagent is coated and printed in a pore channel of a specified bottom plate base material in a volume quantification mode; 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 content of beta-hydroxybutyric acid in urine, and the dry chemical reagent is applied to a sample formed by urine.

Description

Technical Field

[0001] The invention relates to the technical field of dry chemical reagents and beta-hydroxybutyric acid determination, and in particular to a method for preparing a dry chemical reagent for determining the beta-hydroxybutyric acid content in urine. Background Art

[0002] Diabetes is a common and frequently occurring disease, and its incidence rate is increasing year by year. The main clinical manifestation is endocrine dysfunction. When the body's metabolic disorder develops into accelerated fat decomposition and a large accumulation of serum ketone bodies, it is called ketoacidosis, which is more likely to be complicated by ketoacidosis. The diagnosis and efficacy observation of ketoacidosis often require the determination of the concentration of ketone bodies in the blood. The main component of ketone bodies is β-hydroxybutyric acid, which accounts for about 78% of the total ketone bodies. Therefore, β-hydroxybutyric acid and urine ketone body content are often used as important indicators for detecting diabetes and diagnosing diabetic ketoacidosis.

[0003] Dry chemical reagents and POCT. Today, laboratory medicine has a polarized development trend. On the one hand, there are various large-scale automated and high-efficiency instruments and equipment. This type of equipment usually uses liquid reagents, which has the advantages of low price and high measurement accuracy. Disadvantages: It requires supporting large-scale equipment, and has high requirements for equipment price, floor space, and personnel training. On the other hand, it is a POCT testing model with miniaturized and handheld laboratories, simple operations, and timely test results reports. The POCT model not only meets the rescue and treatment needs of critically ill patients on the medical side, but also meets the needs of primary diagnosis and treatment institutions and outpatient clinics with small sample sizes and immediate testing. With the advancement of science, the rise of the Internet and the need for patient privacy, POCT timely detection products are gradually entering households. For single indicators or complex joint inspection indicators with obvious clinical indicators, projects that require continuous monitoring to realize value have entered a new pattern of family self-diagnosis. However, the current existing technologies still have the following technical defects:

[0004] (1) Currently, there are two detection principles for β-hydroxybutyric acid dry chemical reagents on the market: direct method and indirect method. Johnson & Johnson and Fuji's dry chemical reagents all use the indirect method, while Kyoto's β-hydroxybutyric acid dry chemical reagents use the direct method. In addition, only a few companies in the world can produce dry chemical reagents for β-hydroxybutyric acid detection. The dry chemical quantitative detection reagents for urea are imported, which makes them expensive, making it impossible for small hospitals or community clinics to afford them, and even more impossible to achieve the purpose of home self-testing and continuous monitoring.

[0005] (2) Existing dry chemical detection technologies mostly use composite structures, which are, from top to bottom, diffusion layer, reagent layer and transparent support layer. The diffusion layer is generally composed of titanium dioxide, barium sulfate, cellulose acetate and other structures, which can quickly disperse and cover the colored substances in the sample. At the same time, these reflective compounds also play a role in providing a reflective background for the color development layer. This can now be achieved through composite membrane structures such as asymmetric polyethersulfone. The reagent layer is divided into multiple functional reagent layers according to actual needs, and can be roughly divided into a reaction zone and a color development zone according to specific functions. The main function of the reaction zone is to convert the analyte into a compound that can bind to the color development substance through physical, chemical, biochemical reactions and other reactions. The main function of the color development zone is to use a specific indicator system or color developer to make the analyte react to generate a compound that can be measured by reflection. The support layer is located at the top layer of the reagent, and generally uses an optically transparent plastic substrate to allow incident light and reflected light to pass through completely, while playing a supporting role. During the test, the light source inside the instrument emits a beam of light, which passes through the transparent support layer. After the light is partially absorbed by the colored compound in the reagent layer, it is reflected on the reflective surface provided by the diffusion layer. The reflected light passes through the filter device and returns to the photometer to be read. The optical density is converted into a voltage reading to form a digital reading. During the reflection process, the colored substance is absorbed, and the analyte concentration is not proportional to the reflected light density. A direct limiting relationship cannot be generated, and the conversion must be achieved through a complex functional relationship.

[0006] (3) In the preparation process of reagent dry sheets, a process similar to film production is often used. The complex, biological enzyme, and colorimetric substrate are evenly spread on the support layer by scraping or spraying. The water in the solution is lost through a rapid drying process, and the viscous film-forming material in the liquid is tightly combined with the support layer. During the preparation process, the dispersion layer contains solids, and a screw flat coating method is required for coating, which has extremely high process requirements. It is difficult for domestic equipment to meet the process requirements, and production equipment is mostly imported, which is expensive. Large-scale production is more difficult, and the equipment cost is high, which is not suitable for small and medium-sized production enterprises.

[0007] In short, the dry chemical urea quantitative test strips have a complex structure, a difficult manufacturing process, and rely on large imported equipment, making it difficult to break through the technology. The reagent form is fixed, and it is difficult to achieve joint testing of multiple biochemical indicators. Summary of the invention

[0008] 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 the content of β-hydroxybutyric acid in urine 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.

[0009] The first aspect of the present invention is to provide a method for preparing a dry chemical reagent for determining the content of β-hydroxybutyric acid in urine, comprising:

[0010] S1, prepare buffer reagents and protective components;

[0011] S2, dissolving the biological enzyme and the chromogenic substrate to form a dissolving reagent;

[0012] S3, using a biofilm-forming material to form a coating agent;

[0013] 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;

[0014] 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;

[0015] 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.

[0016] Preferably, the S1 comprises:

[0017] S11, selecting a suitable buffer system, including: a buffer system based on Tris-HCl, MOPS, HEPES and phosphate buffer;

[0018] S12, configuring protective components.

[0019] Preferably, the S12 includes:

[0020] (1) adding a certain concentration of sucrose, trehalose, raffinose, bovine serum albumin, silk protein, casein, bovine serum and amino acids to the basic buffer system;

[0021] (2) adding a surfactant to the basic buffer system, wherein the surfactant comprises Tween-20, glycerol and PEG;

[0022] (3) Adding a certain concentration of EDTA, disodium EDTA, tetrasodium EDTA, potassium ferrocyanide, bilirubin oxidase and ascorbic acid oxidase into the basic buffer system.

[0023] Preferably, S2 includes:

[0024] S21, selecting a certain concentration of urease to be dissolved in the basic buffer system;

[0025] S22, dissolving a certain concentration of D-3-hydroxybutyrate dehydrogenase with an organic reagent and adding the resulting solution to the basic buffer system; wherein the dissolved reagent formed after dissolving the biological enzyme and the chromogenic substrate is a precipitation-free homogeneous reagent.

[0026] Preferably, S3 includes:

[0027] S31, selecting a biofilm-forming material suitable for the β-hydroxybutyrate determination methodology, and preparing the biofilm-forming material into a film-forming base liquid of a certain concentration;

[0028] 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] Preferably, the biofilm-forming material includes one or more of chitin, polysaccharides, agarose and PVA.

[0030] Preferably, S4 includes:

[0031] S41, clean and passivate the surface of the designated baseboard substrate before use;

[0032] 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.

[0033] Preferably, S5 includes:

[0034] S51, setting the wind speed, temperature and drying time of the blast drying equipment or a drying equipment with equal efficiency;

[0035] 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;

[0036] 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.

[0037] 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.

[0038] The second aspect of the present invention provides an application for determining the content of β-hydroxybutyric acid in urine prepared by the preparation method of the first aspect, which is applied to a sample formed by urine, comprising:

[0039] S1', diluting the sample with a diluent to obtain a diluted sample; the diluent is prepared by dissolving 50-500 mmol / L of a neutral buffer system with a pH value stable at 7.0-7.6 in purified water, and adding a preservative component at a concentration of 1 / 10,000 to 1 / 1000; the neutral buffer system with a stable pH value includes one or more of phosphate and Tris-HCl;

[0040] 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 3-10 minutes; the biochemical reaction can be carried out at room temperature and 37°C, and the sample color changes from colorless to blue-purple;

[0041] S3', according to the color change of the sample from colorless to blue-purple 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.

[0042] The preparation method of the present invention and the beneficial effects of its application:

[0043] With the help of biofilm-forming materials and a one-time film-forming preparation process, the original biochemical reagents or multi-layer dry chemical reagents are improved, and a one-time film-forming preparation process is used to realize a rapid preparation method for semi-quantitative and quantitative dry chemical reagents for β-hydroxybutyric acid indicators. 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 of the reagent system to remove interfering substances, 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 small-scale manual production, it can also be used in semi-automatic and fully automated production lines. The product is suitable for the single detection of creatine indicators, and is also suitable for joint detection with multiple biochemical indicators. It can be realized on traditional dry chemical technology platforms and microfluidic technology platforms. Compared with the existing technology, it provides multiple innovations in the implementation plan of the reagent preparation process, the selection and use concentration of the protection system, the components of the reagent system to remove interfering substances, the preparation plan of the diluent, and the treatment method of the substrate material. It is safe, accurate and convenient, can quickly detect the concentration of β-hydroxybutyric acid in human urine, and is simple and easy to operate. The bedside test for β-hydroxybutyrate is accurate, immediate and convenient. With its safety, accuracy and convenience, it will not be limited to the use of in-hospital patients, but will become an important tool for self-management of individual diabetic patients. Specifically, it is reflected in:

[0044] (I) Comparison of technical advantages with multi-layer dry chemical reagents:

[0045] 1. 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.

[0046] 2. The production and preparation is fast and reliable. The theoretical time from the feeding of production reagents to the output of semi-finished reagents is less than 4 hours (small batch production), which greatly shortens the production time, improves production efficiency and reduces production costs. The composition of all reagents does not contain paper products and organic film products, which greatly reduces the production and feeding costs.

[0047] 3. Liquid samples are used as reagent reaction systems, and the biofilm-forming materials dissolve after mixing with the liquid. Compared with the reactions in existing multi-layer chromatography filter papers and films, the intramolecular diffusion effect of the contents is obvious, similar to biochemical liquid reagents, which reduces the generation of color spots and color blocks in dry chemical reaction systems, improves reagent performance (especially repeatability indicators), and lays a foundation for quantitative determination.

[0048] 4. The products prepared by this technology have good stability and can be stored away from light at 2-30°C for 12 months.

[0049] (II) Comparison of advantages with freeze-dried microsphere technology:

[0050] 1. During the production and preparation process, there is no need for freeze-drying process. The freeze-drying process is a special preparation process with obvious uncontrollable factors.

[0051] 2. The freeze-drying process has special requirements for the base material. Under the condition of -30 to -40℃, it is difficult to ensure that the base material will not break or deform.

[0052] 3. The freeze-drying process usually lasts for more than 15 hours, so the time is greatly shortened.

[0053] 4. The problem of production and assembly difficulties is solved. The morphology of the freeze-dried microspheres is basically the same and they carry electric charge, which places extremely high demands on production operators. The production and assembly time of the present invention is within 4 hours (small batch production), which greatly shortens the production time, improves production efficiency, and reduces production costs. The entire reagent composition does not contain paper products and organic film products, which greatly reduces the production and feeding costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] 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.

[0055] Figure 1 A flow chart of a method for preparing a dry chemical reagent for determining the content of β-hydroxybutyric acid in urine according to an embodiment of the present invention;

[0056] Figure 2 This is a flow chart of step S1 of a method for preparing a dry chemical reagent for determining the content of β-hydroxybutyric acid in urine according to an embodiment of the present invention;

[0057] Figure 3 This is a flow chart of step S2 of a method for preparing a dry chemical reagent for determining the content of β-hydroxybutyric acid in urine according to an embodiment of the present invention;

[0058] Figure 4 This is a flow chart of step S3 of a method for preparing a dry chemical reagent for determining the content of β-hydroxybutyric acid in urine according to an embodiment of the present invention;

[0059] Figure 5 This is a flow chart of step S4 of a method for preparing a dry chemical reagent for determining the content of β-hydroxybutyric acid in urine according to an embodiment of the present invention;

[0060] Figure 6 This is a flow chart of step S5 of a method for preparing a dry chemical reagent for determining the content of β-hydroxybutyric acid in urine according to an embodiment of the present invention;

[0061] Figure 7 A flow chart of an application method of a dry chemical reagent for determining the content of β-hydroxybutyric acid in urine according to an embodiment of the present invention;

[0062] Figure 8 The figure is a schematic diagram showing actual inspection results provided according to an embodiment of the present invention. DETAILED DESCRIPTION

[0063] 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 them. 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.

[0064] 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.

[0065] 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.

[0066] The following examples use dry chemical enzyme detection technology, which can perform urine detection sensitively, specifically and quickly. The main reaction enzyme, auxiliary tool enzyme, chromogenic substrate, buffer reagent and protective component in the dry chemical reaction process are wrapped with biological film-forming materials, 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 reagents.

[0067] Embodiment 1

[0068] like Figure 1As shown, this embodiment provides a method for preparing a dry chemical reagent for determining the content of β-hydroxybutyric acid in urine, comprising:

[0069] S1, prepare buffer reagents and protective components;

[0070] like Figure 2 As shown, as a preferred embodiment, the S1 includes:

[0071] 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 D-3 hydroxybutyrate dehydrogenase;

[0072] S12, configure protection components, including:

[0073] (1) adding a certain concentration of sucrose, trehalose, raffinose, 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;

[0074] (2) adding a certain proportion of surfactants such as Tween-20, glycerol and PEG to the basic buffer system in order to make the contents in the buffer system evenly dispersed;

[0075] (3) A certain concentration of EDTA, disodium EDTA, tetrasodium EDTA, potassium ferrocyanide, bilirubin oxidase and ascorbic acid oxidase are added to the basic buffer system to remove interfering substances in the sample.

[0076] S2, dissolving the biological enzyme and the chromogenic substrate to form a dissolving reagent;

[0077] like Figure 3 As shown, as a preferred embodiment, the S2 includes:

[0078] S21, selecting a suitable concentration of urease to be dissolved in the basic buffer system;

[0079] S22, dissolving D-3-hydroxybutyrate dehydrogenase of suitable concentration with an organic reagent and adding the resulting solution to 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.

[0080] S3, using a biofilm-forming material to form a coating agent;

[0081] like Figure 4 As shown, as a preferred implementation, S3 includes:

[0082] 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;

[0083] 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.

[0084] 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;

[0085] like Figure 5 As shown, as a preferred embodiment, the S4 includes:

[0086] 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;

[0087] 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.

[0088] 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;

[0089] like Figure 6 As shown, as a preferred implementation, the S5 includes:

[0090] 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;

[0091] 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;

[0092] 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.

[0093] 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.

[0094] Embodiment 2

[0095] like Figure 7 As shown, this embodiment provides an application of a dry chemical reagent for determining the content of β-hydroxybutyric acid in urine prepared by the preparation method of Example 1, which is applied to a sample formed by urine, including:

[0096] 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 neutral buffer system with a pH stable at 7.0-7.6 such as 50-500 mmol / L phosphate, Tris-HCL, HEPES, MOPS, and the like, and adding a preservative component at a concentration of 1 part per ten thousand to 1 part per thousand.

[0097] This embodiment takes urine samples as an example: the dilution ratio is between 1:14 and 1:39.

[0098] 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 3-10 minutes; the biochemical reaction can be carried out at room temperature and 37°C, and the sample color changes from colorless to blue-purple. In this reaction system, the content of D-3 hydroxybutyrate dehydrogenase and chloronitro blue tetrazolium are both excessive. Therefore, the reaction will not terminate before the β-hydroxybutyric acid in the sample is completely consumed;

[0099] S3', according to the color of the sample after the reaction changes from colorless to blue-purple, 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 matched with the standard curve to achieve quantitative detection.

[0100] Analysis of test results of embodiment:

[0101] The actual test results are as follows Figure 8 shown.

[0102] 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.

[0103] 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 with 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 the content of β-hydroxybutyric acid in urine, 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 determining the content of β-hydroxybutyric acid in urine 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 determining the content of β-hydroxybutyric acid in urine according to claim 2, characterized in that: The S12 includes: (1) adding a certain concentration of sucrose, trehalose, raffinose, 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 comprises Tween-20, glycerol and PEG; (3) Adding a certain concentration of EDTA, disodium EDTA, tetrasodium 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 the content of β-hydroxybutyric acid in urine according to claim 3, characterized in that: The S2 includes: S21, selecting a certain concentration of urease to be dissolved in the basic buffer system; S22, dissolving a certain concentration of D-3-hydroxybutyrate dehydrogenase with an organic reagent and adding the resulting solution to the basic buffer system; wherein the dissolved 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 the content of β-hydroxybutyric acid in urine according to claim 4, characterized in that: The S3 includes: S31, selecting a biofilm-forming material suitable for the β-hydroxybutyrate 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 the content of β-hydroxybutyric acid in urine 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 the content of β-hydroxybutyric acid in urine 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. The method for preparing a dry chemical reagent for determining the content of β-hydroxybutyric acid in urine 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 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.

9. A method for preparing a dry chemical reagent for determining the content of β-hydroxybutyric acid in urine 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. An application of the method according to any one of claims 1 to 9 for determining the content of β-hydroxybutyric acid in urine, characterized in that: For use with urine samples, including: S1', diluting the sample with a diluent to obtain a diluted sample; the diluent is prepared by dissolving 50-500mmol / L of a neutral buffer system with a pH value stable at 7.0-7.6 in purified water, and adding a preservative component at a concentration of 1 / 10,000 to 1 / 1000; the neutral buffer system with a stable pH value includes one or more of phosphate and Tris-HCL; 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 3-10 minutes; the biochemical reaction can be carried out at room temperature and 37°C, and the sample color changes from colorless to blue-purple; S3', according to the color change of the sample from colorless to blue-purple 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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