A kit and method for measuring glycated hemoglobin concentration

Abstract

The present invention provides a kit for measuring the concentration of glycated hemoglobin and a method thereof, wherein the kit comprises: a detection reagent, comprising: polystyrene microspheres coated with aminophenylboronic acid derivatives; a surfactant; a protective agent; a hemoglobin precipitant; and a washing liquid. The present invention solves the technical problem that the current glycated hemoglobin kit is inconvenient to operate, and achieves the technical effects of good stability of the kit, convenient operation, and fast detection speed.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to a kit and a method for measuring the concentration of glycosylated hemoglobin. Background Art

[0002] Glycated hemoglobin (HbA1c) is the product of the combination of hemoglobin in red blood cells and blood sugar. Since the reaction is irreversible, proportional to blood sugar concentration, and can be maintained for about 120 days, HbA1c can reflect the average blood sugar level in the past 2-4 months, which is of great reference significance for the treatment of diabetes and the monitoring of the condition of diabetic patients. The Asia-Pacific Diabetes Prevention and Treatment Guidelines launched by the International Diabetes Federation clearly stipulate that glycated hemoglobin is the internationally recognized "gold standard" for diabetes monitoring.

[0003] There are many methods for detecting glycated hemoglobin, such as HPLC, latex immunoturbidimetry, enzyme method, etc. HPLC is widely used as the gold standard for detecting glycated hemoglobin, but the cost of this method is relatively high; latex immunoturbidimetry is accurate and the price is lower than HPLC, but its operation is more complicated and the test time is long, which is suitable for high-throughput testing; the precision and accuracy of enzyme detection is poorer than that of HPLC and latex immunoturbidimetry.

[0004] Phenylboronic acid has the property of reversibly generating cyclic esters with cis-dihydroxy molecules, and cis-dihydroxy molecules exist in glycoproteins, so it can be used for the determination of glycosylated proteins based on this property. Some people have proposed a method for preparing phenylboronic acid derivatives for detecting glycosylated proteins, and some people have mentioned a kit for determining glycated hemoglobin using phenylboronic acid derivatives. Glycated hemoglobin is analyzed in the presence of phenylboronic acid derivatives and hemoglobin precipitants. Since phenylboronic acid derivatives and hemoglobin precipitants have poor stability when they coexist, the product has a short shelf life and the reliability of the determination is reduced.

[0005] In order to solve the above problems, a kit for measuring glycated hemoglobin has been proposed, in which phenylboronic acid derivatives and zinc ions are present as different reagents in the kit, which can improve the stability of the kit and increase the reliability of the measurement. However, it increases the components of the kit, which makes it inconvenient for users to use.

[0006] Therefore, it is particularly important to provide a simple and easy-to-operate kit for measuring the concentration of glycated hemoglobin on the basis of ensuring the stability of the kit. Summary of the invention

[0007] The present invention solves the technical problem that the current glycosylated hemoglobin test kit is inconvenient to operate, and achieves the technical effects that the test kit has good stability, is easy to operate, and has a fast detection speed.

[0008] To solve the above problems, the present invention provides a kit for measuring the concentration of glycosylated hemoglobin, comprising: a detection reagent, including: polystyrene microspheres coated with an aminophenylboronic acid derivative; a surfactant; a protective agent; a hemoglobin precipitant; and a washing solution.

[0009] Compared with the prior art, the technical effects achieved by the technical solution are as follows: surfactant is used to lyse the red blood cell membrane and release hemoglobin; protective agent is used to stabilize aminophenylboronic acid derivatives; hemoglobin precipitant precipitates hemoglobin; washing liquid is used to remove free aminophenylboronic acid derivatives on polystyrene microspheres. By coating the aminophenylboronic acid derivatives on blue polystyrene microsphere particles, the two are covalently linked, so when the aminophenylboronic acid derivatives and the hemoglobin precipitant coexist, the aminophenylboronic acid derivatives are not easily damaged, which solves the problem of poor stability when the aminophenylboronic acid derivatives and the hemoglobin precipitant coexist, and improves the stability of the aminophenylboronic acid derivatives. In addition, the kit provided by the present invention is a single detection reagent, and the phenylboronic acid derivatives and the hemoglobin precipitant coexist, which reduces the types of detection reagents in the kit, making the user operation more convenient. The user only needs to mix the test sample with one detection reagent, and there is no need to mix multiple detection reagents, which reduces the detection steps and is more convenient to operate.

[0010] In one embodiment of the present invention, the molecular formula of the aminophenylboronic acid derivative is

[0011]

[0012] Wherein, R is an amino group, and n is 0 to 5.

[0013] Compared with the prior art, the technical effect achieved by adopting this technical solution is as follows: there are cis-dihydroxy molecules in glycoproteins, and the above-mentioned aminophenylboronic acid can reversibly generate cyclic esters with cis-dihydroxy molecules, so it can be used for the determination of glycosylated proteins based on this characteristic, and the detection is convenient.

[0014] In one example of the present invention, the polystyrene microspheres are carboxyl polystyrene microspheres encapsulating blue dyes, and the particle size of the polystyrene microspheres is 100-200 nm.

[0015] Compared with the prior art, the technical effect achieved by adopting this technical solution is: the blue dye is encapsulated inside the polystyrene microspheres, and aminophenylboronic acid and glycoprotein reversibly generate cyclic esters, and the cyclic esters are marked in blue, that is, the glycated hemoglobin bound to the aminophenylboronic acid derivative is marked in blue, which can be distinguished from the red total hemoglobin, making it easier to detect and observe in the follow-up. The selection of polystyrene microspheres with a particle size range of 100 to 200 nm is conducive to improving the sensitivity of the reagent and increasing the linear range in the preparation process of polystyrene microspheres coated with aminophenylboronic acid derivatives.

[0016] In one embodiment of the present invention, the surfactant includes Triton X-100 and Igepal CA-630.

[0017] Compared with the existing technology, the technical effect achieved by adopting this technical solution is as follows: Triton X-100 and Igepal CA-630 can dissolve lipids, play a role in dissociating membrane proteins from the cell membrane of red blood cells, have a good effect in lysing the red blood cell membrane, and then release hemoglobin for subsequent detection of hemoglobin.

[0018] In one embodiment of the present invention, the protecting agent includes a p-toluenesulfonic acid derivative.

[0019] Compared with the existing technology, the technical effect achieved by adopting this technical solution is that the benzenesulfonic acid derivatives can keep the aminophenylboronic acid derivatives stable, thereby extending the shelf life of the test kit and avoiding affecting the credibility of the test.

[0020] In one embodiment of the present invention, the protective agent is one or more of p-aminobenzenesulfonic acid, 3-aminobenzenesulfonic acid, p-carboxybenzenesulfonic acid, and 3-carboxybenzenesulfonic acid.

[0021] Compared with the prior art, the technical effect achieved by adopting this technical scheme is as follows: selecting one or more of p-aminobenzenesulfonic acid, 3-aminobenzenesulfonic acid, p-carboxybenzenesulfonic acid, and 3-carboxybenzenesulfonic acid can keep the aminophenylboronic acid derivatives stable, thereby extending the shelf life of the test kit and avoiding affecting the credibility of the test.

[0022] In one embodiment of the present invention, the hemoglobin precipitant contains zinc ions, and the hemoglobin precipitant includes any one of zinc acetate, zinc sulfate, and zinc chloride.

[0023] Compared with the existing technology, the technical effect achieved by adopting this technical solution is as follows: zinc ions can denature hemoglobin and then precipitate it, which is convenient for subsequent detection, and zinc acetate, zinc sulfate and zinc chloride are easily available and can quickly precipitate hemoglobin, thereby increasing the detection speed.

[0024] In one embodiment of the present invention, the cleaning solution includes glycerol and a surfactant.

[0025] Compared with the existing technology, the technical effect achieved by adopting this technical solution is as follows: during the washing process, the surfactant can fully release hemoglobin, allowing it to combine more fully with the glycated protein, while propylene glycol can remove the free aminophenylboronic acid derivatives on the polystyrene microspheres, avoiding their impact on subsequent detection, making the test results more accurate.

[0026] In one embodiment of the present invention, the pH value of the detection reagent is 7.5-8.0, and the pH value of the washing solution is 8.5-9.5.

[0027] Compared with the prior art, the technical effects achieved by adopting this technical solution are as follows: the detection reagent is weakly alkaline, which is more conducive to the reaction between aminophenylboronic acid and glycoprotein; the washing solution is alkaline, which can more fully remove the free aminophenylboronic acid derivatives on the polystyrene microspheres, thereby more accurately determining the concentration of glycated protein.

[0028] The present invention also provides a method for determining the concentration of glycated hemoglobin, using any one of the kits in the above embodiments, the kit also includes a test card, and the method includes the following steps: S10: obtaining a test sample; S20: mixing the test sample and the test reagent to obtain a mixed solution; S30: dropping the mixed solution into the test card; S40: dropping a detergent into the test card; S50: measuring the light reflectance R1 of the test card at a wavelength of 620nm and the light reflectance R2 at a wavelength of 470nm, calculating the ratio of R1 to R2, and obtaining the concentration of glycated hemoglobin in the test sample.

[0029] Compared with the prior art, the technical effect achieved by adopting the technical scheme is as follows: detecting the reflectivity R1 of the blue glycosylated hemoglobin combined with the aminophenylboronic acid derivative under light with a wavelength of 620nm, and the reflectivity R2 of the red total hemoglobin under light with a wavelength of 470nm, and calculating the ratio of R1 to R2, which is the concentration of HbA1C in the test sample. The detection method is simple, easy to operate, and has a fast detection speed. DETAILED DESCRIPTION

[0030] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, a detailed description is given below in conjunction with specific embodiments of the present invention.

[0031] [Example 1]

[0032] The present invention provides a kit for measuring the concentration of glycosylated hemoglobin, comprising:

[0033] Detection reagents, including:

[0034] Polystyrene microspheres coated with aminophenylboronic acid derivatives;

[0035] Surfactants;

[0036] Protective agent;

[0037] Hemoglobin precipitants;

[0038] Detergent.

[0039] Specifically, the test kit also includes a test card, which includes a porous membrane capable of retaining hemoglobin precipitates, absorbent paper, and a plastic card slot with a sample addition hole.

[0040] Specifically, phenylboronic acid and its derivatives can selectively promote the transport of glucose through lipid bilayers and are increasingly used as molecular recognition units, in particular, to design and synthesize boron lectins (glycoproteins) and carbohydrate sensors, thereby being used to detect glycated proteins.

[0041] Specifically, the surfactant is used to lyse the red blood cell membrane and release hemoglobin; the protective agent is used to stabilize the aminophenylboronic acid derivative; the hemoglobin precipitant precipitates the hemoglobin; and the washing liquid is used to remove the free aminophenylboronic acid derivative on the polystyrene microspheres. The detection reagent is mixed with the blood sample, and the mixture is loaded onto the porous membrane. The membrane is washed with a detergent, and the precipitated glycated and non-glycated hemoglobin is retained on the membrane, while the free aminophenylboronic acid derivative on the polystyrene microspheres is washed off the membrane by the washing liquid. After washing the membrane, the light reflection of the membrane is measured, that is, the hemoglobin captured on the membrane, and the concentration of glycated hemoglobin can be obtained. The operation is simple and fast, and the detection speed is fast.

[0042] The aminophenylboronic acid derivative is coated on blue polystyrene microsphere particles and the two are covalently linked. Therefore, when the aminophenylboronic acid derivative and the hemoglobin precipitant coexist, the aminophenylboronic acid derivative is not easily destroyed, thereby solving the problem of poor stability when the aminophenylboronic acid derivative and the hemoglobin precipitant coexist, and improving the stability of the aminophenylboronic acid derivative. In addition, the kit provided by the present invention is a single detection reagent, and the phenylboronic acid derivative and the hemoglobin precipitant coexist, thereby reducing the types of detection reagents in the kit, making it more convenient for users to operate. Users only need to mix the test sample with one detection reagent without mixing multiple detection reagents, thereby reducing the detection steps and making the operation more convenient.

[0043] Further, the molecular formula of the aminophenylboronic acid derivative is

[0044]

[0045] Wherein, R is an amino group, and n is 0 to 5.

[0046] Specifically, there are cis-dihydroxy molecules in glycoproteins, and the aminophenylboronic acid can reversibly generate cyclic esters with cis-dihydroxy molecules. Therefore, based on this property, it can be used for the determination of glycosylated proteins, and the detection is convenient.

[0047] Furthermore, the polystyrene microspheres are carboxyl polystyrene microspheres with blue dye wrapped inside, and the particle size of the polystyrene microspheres is 100-200 nm.

[0048] Specifically, the blue dye is wrapped inside the polystyrene microspheres, and aminophenylboronic acid reversibly reacts with glycoprotein to form cyclic esters, which are marked in blue. That is, the glycated hemoglobin bound to the aminophenylboronic acid derivative is marked in blue, which can be distinguished from the red total hemoglobin, making subsequent detection and observation more convenient.

[0049] Furthermore, polystyrene microspheres coated with aminophenylboronic acid derivatives are prepared by the following method: the surface carboxyl groups of blue polystyrene microspheres are activated with carbodiimide hydrochloride to form an activated ester structure, and then the aminophenylboronic acid derivatives are added, and the amino groups of the boronic acid derivatives are covalently linked to the carboxyl groups on the blue polystyrene microspheres through the activated ester, thereby obtaining polystyrene microspheres coated with aminophenylboronic acid derivatives.

[0050] Specifically, the selection of polystyrene microspheres with a particle size range of 100 to 200 nm is beneficial to improving the sensitivity of the reagent and increasing the linear range during the preparation process.

[0051] Further, the surfactant includes Triton X-100 and Igepal CA-630.

[0052] Specifically, Triton X-100 and Igepal CA-630 can dissolve lipids and play a role in dissociating membrane proteins from the cell membrane of red blood cells. They have a good effect in lysing the red blood cell membrane and then releasing hemoglobin for subsequent detection of hemoglobin.

[0053] Furthermore, the protecting agent includes a p-toluenesulfonic acid derivative.

[0054] Specifically, the benzenesulfonic acid derivatives can stabilize the aminophenylboronic acid derivatives, thereby extending the shelf life of the test kit and avoiding affecting the reliability of the test.

[0055] Furthermore, the protective agent is one or more of p-aminobenzenesulfonic acid, 3-aminobenzenesulfonic acid, p-carboxybenzenesulfonic acid, and 3-carboxybenzenesulfonic acid.

[0056] Specifically, the selection of one or more of p-aminobenzenesulfonic acid, 3-aminobenzenesulfonic acid, p-carboxybenzenesulfonic acid, and 3-carboxybenzenesulfonic acid can stabilize the aminophenylboronic acid derivative, thereby extending the shelf life of the kit and avoiding affecting the credibility of the test.

[0057] Furthermore, the hemoglobin precipitant contains zinc ions, and the hemoglobin precipitant includes any one of zinc acetate, zinc sulfate, and zinc chloride.

[0058] Specifically, zinc ions can denature hemoglobin and cause it to precipitate, which is convenient for subsequent detection. Zinc acetate, zinc sulfate, and zinc chloride are easily available and can quickly precipitate hemoglobin, thereby increasing the detection speed.

[0059] Furthermore, the washing liquid includes glycerol and a surfactant.

[0060] Specifically, during the washing process, the surfactant can fully release hemoglobin, allowing it to combine more fully with the glycated protein, while propylene glycol can remove free aminophenylboronic acid derivatives on the polystyrene microspheres to prevent them from affecting subsequent tests, making the test results more accurate.

[0061] Furthermore, the pH value of the detection reagent is 7.5-8.0, and the pH value of the washing solution is 8.5-9.5.

[0062] Specifically, the detection reagent is weakly alkaline, which is more conducive to the reaction between aminophenylboronic acid and glycoprotein, and the washing solution is alkaline, which can more fully remove the free aminophenylboronic acid derivatives on the polystyrene microspheres, thereby more accurately determining the concentration of glycated protein.

[0063] The present invention also provides a method for determining the concentration of glycated hemoglobin, using any one of the kits in the above embodiments, the kit also includes a test card, and the method includes the following steps: S10: obtaining a test sample; S20: mixing the test sample and the test reagent to obtain a mixed solution; S30: dropping the mixed solution into the test card; S40: dropping a detergent into the test card; S50: measuring the light reflectance R1 of the test card at a wavelength of 620nm and the light reflectance R2 at a wavelength of 470nm, calculating the ratio of R1 to R2, and obtaining the concentration of glycated hemoglobin in the test sample.

[0064] Specifically, a glycated hemoglobin detector is used to detect the reflectivity R1 of blue glycated hemoglobin combined with aminophenylboronic acid derivatives under light with a wavelength of 620nm, and the reflectivity R2 of red total hemoglobin under light with a wavelength of 470nm. The ratio of R1 to R2 is calculated to be the concentration of HbA1C in the test sample. The detection method is simple, easy to operate, and has a fast detection speed.

[0065] [Example 2]

[0066] Based on Example 1, this example provides a method for labeling blue polystyrene microspheres with aminophenylboronic acid derivatives, and the labeling method is as follows:

[0067] 30 μL of blue polystyrene microspheres were measured and diluted with 0.5 mL of buffer, the buffer was 10 mmol / L, pH 6.5 4-morpholineethanesulfonic acid, EDC was added with 7.5 μL of carbodiimide (10 mg / mL aqueous solution), and incubated at 25°C for 30 minutes;

[0068] Weigh 50 mg of aminophenylboronic acid and dilute it with 0.5 mL of buffer solution (10 mmol / L, pH 7.8, hydroxyethylpiperazine ethanesulfonic acid), then add it to the above blue polystyrene microsphere reaction solution and incubate at 25°C for 16 hours;

[0069] Measure 20 μl of bovine serum albumin solution (50 mg / mL aqueous solution) and add it to the above reaction solution. React at 25°C for 1 hour. Centrifuge at 15000 rpm for 30 minutes, discard the supernatant, add 1 mL of preservation solution to resuspend the precipitate. The preservation solution is 10 mmol / L, pH 7.8 hydroxyethylpiperazine ethanesulfonic acid, and disperse by ultrasound to obtain polystyrene microspheres coated with aminophenylboronic acid derivatives, which are stored at 4°C for future use.

[0070] [Example 3]

[0071] Detection reagent 1 includes: 0.1g blue polystyrene microspheres coated with aminophenylboronic acid derivatives, 0.02g p-aminobenzenesulfonic acid, 0.2g Triton X-100, 0.3g zinc sulfate, 0.9g sodium chloride, 0.05g sodium azide, 1.26g hydroxyethylpiperazinepropanesulfonic acid, add water to dissolve and make up to 100ml, adjust pH to 7.8 with sodium hydroxide and set aside.

[0072] Washing solution 1 includes: 0.1g propylene glycol, 0.5g Triton X-100, 1.0g 2-(cyclohexylamino)-1-ethanesulfonic acid, 1.2g sodium chloride, 0.05g sodium azide, which are dissolved in water and fixed to 100ml, and the pH is adjusted to 9.0 with sodium hydroxide.

[0073] In a specific embodiment, the method for determining the concentration of glycated hemoglobin comprises the following steps:

[0074] S10: Obtain a blood sample, mix 10 μl of the blood sample with detection reagent 1 for 2 minutes to obtain a mixed solution;

[0075] S20: Pipette 25 μl of the mixed solution and drop it into the sample well of the test card;

[0076] S30: Pipette 40 μl of washing solution and add one drop to the sample well;

[0077] S40: Determine the light reflectance R1 of the porous membrane in the sample adding hole of the test card at a wavelength of 620nm and the light reflectance R2 at a wavelength of 470nm, calculate the ratio of R1 to R2, and obtain the ratio of glycated hemoglobin to total hemoglobin in the measured blood sample.

[0078] [Example 4]

[0079] Detection reagent 2 includes: 0.15g blue polystyrene microspheres coated with aminophenylboronic acid derivatives, 0.025g p-aminobenzenesulfonic acid, 0.2g Igepal CA-630, 0.3g zinc acetate, 0.9g sodium chloride, 0.05g sodium azide, 1.26g hydroxyethylpiperazinepropanesulfonic acid, add water to dissolve and make up to 100ml, adjust pH to 7.5 with sodium hydroxide and set aside.

[0080] Washing solution 2 includes: 0.1g propylene glycol, 0.5g Igepal CA-630, 1.0g 2-(cyclohexylamino)-1-ethanesulfonic acid, 1.2g sodium chloride, 0.05g sodium azide, add water to dissolve and make up to 100ml, adjust pH to 9.5 with sodium hydroxide and set aside.

[0081] In a specific embodiment, the method for determining the concentration of glycated hemoglobin comprises the following steps:

[0082] S10: Obtain a blood sample, mix 10 μl of the blood sample with detection reagent 2 for 2 minutes to obtain a mixed solution;

[0083] S20: Pipette 25 μl of the mixed solution and drop it into the sample well of the test card;

[0084] S30: Pipette 40 μl of washing solution and add two drops into the sample well;

[0085] S40: Determine the light reflectance R1 of the porous membrane in the sample adding hole of the test card at a wavelength of 620nm and the light reflectance R2 at a wavelength of 470nm, calculate the ratio of R1 to R2, and obtain the ratio of glycated hemoglobin to total hemoglobin in the measured blood sample.

[0086] [Example 5]

[0087] Detection reagent three: 0.12g blue polystyrene microspheres coated with aminophenylboronic acid derivatives, 0.02g p-aminobenzenesulfonic acid, 0.2g Triton X-100, 0.3g zinc chloride, 1.2g sodium chloride, 2.0g magnesium chloride, 0.05g sodium azide, 1.26g hydroxyethylpiperazinepropanesulfonic acid, dissolve in water and make up to 100ml, adjust pH to 7.5 with sodium hydroxide and set aside.

[0088] Washing solution three: 0.2g propylene glycol, 0.5g Triton X-100, 0.5g 2-(cyclohexylamino)-1-ethanesulfonic acid, 0.9g sodium chloride, 0.05g sodium azide, add water to dissolve and make up to 100ml, adjust pH to 9.0 with sodium hydroxide and set aside.

[0089] In a specific embodiment, the method for determining the concentration of glycated hemoglobin comprises the following steps:

[0090] S10: Obtain a blood sample, and mix 10 μl of the blood sample with detection reagent 3 for 2 minutes to obtain a mixed solution;

[0091] S20: Pipette 25 μl of the mixed solution and drop it into the sample well of the test card;

[0092] S30: Pipette 40 μl of washing solution and add three drops into the sample well;

[0093] S40: Determine the light reflectance R1 of the porous membrane in the sample adding hole of the test card at a wavelength of 620nm and the light reflectance R2 at a wavelength of 470nm, calculate the ratio of R1 to R2, and obtain the ratio of glycated hemoglobin to total hemoglobin in the measured blood sample.

[0094] [Example 6]

[0095] This example provides the stability verification results of a kit for determining the glycosylation ratio of hemoglobin.

[0096] In Example 3, Example 4 and Example 5, 6 test samples were tested respectively, and the test kits were packaged and divided into two groups, which were stored at 2-8°C and 37°C for 7 days respectively. After 7 days, the 6 whole blood samples of Examples 3 to 5 were tested respectively. The results are shown in Table 1.

[0097] Table 1

[0098]

[0099]

[0100] Table 1 shows the accelerated stability verification results of the kit. It can be seen from Table 1 that after the sample detection kits of Examples 3, 4, and 5 were stored at 4°C and 37°C for 7 days, the detection results were not much different, which shows that the kits are stable in storage.

[0101] Although the present invention is disclosed as above, the present invention is not limited thereto. Any person skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the scope defined by the claims.

Claims

1. A kit for measuring the concentration of glycosylated hemoglobin, characterized in that: include: Detection reagents and washing solutions; The detection reagent is a solution, and the solution includes: Polystyrene microspheres coated with aminophenylboronic acid derivatives, Surfactant, the surfactant includes TritonX-100 or IgepalCA-630, A protective agent, wherein the protective agent includes a p-toluenesulfonic acid derivative, and the protective agent is one or more of p-aminobenzenesulfonic acid, 3-aminobenzenesulfonic acid, p-carboxybenzenesulfonic acid, and 3-carboxybenzenesulfonic acid. A hemoglobin precipitant, wherein the hemoglobin precipitant contains zinc ions and is any one of zinc acetate, zinc sulfate, and zinc chloride; The washing liquid comprises glycerol and the surfactant; The pH value of the detection reagent is 7.5-8.0, and the pH value of the washing solution is 8.5-9.

5.

2. The kit according to claim 1, characterized in that The molecular formula of the aminophenylboronic acid derivative is or ; Wherein, R is an amino group, and n is 0 to 5.

3. The kit according to claim 1, characterized in that The polystyrene microspheres are carboxyl polystyrene microspheres with blue dye wrapped inside, and the particle size of the polystyrene microspheres is 100-200nm.

4. A method for measuring the concentration of glycosylated hemoglobin, the method not being intended for disease diagnosis, characterized in that: Using the kit according to any one of claims 1 to 3, the kit further comprises a test card, and the method comprises the following steps: S10: Obtain test samples; S20: mixing the test sample and the test reagent to obtain a mixed solution; S30: adding the mixed solution dropwise to the test card; S40: dropping the washing solution into the test card; S50: measuring the light reflectance R1 of the test card at a wavelength of 620 nm and the light reflectance R2 at a wavelength of 470 nm, calculating the ratio of R1 to R2, and obtaining the concentration of glycated hemoglobin in the test sample.

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