A dry chemical reagent for detecting total cholesterol

Abstract

This invention discloses a dry chemical reagent for total cholesterol detection, comprising a diffusion layer, a spacer layer, and a reagent layer sequentially overlapped. The diffusion layer includes reflective particles, a binder, a surfactant, cholesterol esterase, and cholesterol oxidase. The reagent layer includes a carrier, horseradish peroxidase, an indicator composition, and a buffer solution. The dry chemical detection reagent of this invention employs a coating method, layering a premixed homogeneous slurry onto a support layer. A spacer layer blocks interlayer interference, and phenolic substances replace photosensitizers as chromogens, combined with a specific ratio of substrate and buffer system. The multi-layered, spacer-free, low-interference vertical detection system constructed by this invention significantly improves the repeatability, accuracy, and stability of the detection reagent.

Description

Technical Field

[0001] This invention relates to the field of clinical diagnostics, specifically to a dry chemical reagent for the detection of total cholesterol. Background Technology

[0002] Total cholesterol in the human body exists in two forms: free cholesterol and cholesterol esters bound to proteins. It is found in human tissues, serum, and plasma. Cholesterol plays a crucial role in assessing the development of coronary artery obstruction, atherosclerosis, myocardial infarction, and cardiovascular and cerebrovascular diseases. With social development and improved living standards, China's obesity rate is rising, and the incidence of cardiovascular and cerebrovascular diseases is showing a trend towards affecting younger people. Routine preventative cholesterol level monitoring and rapid, timely, and accurate diagnosis can effectively prevent the occurrence and deterioration of cardiovascular and other diseases. Cholesterol testing mainly falls into two categories: wet chemical methods and dry chemical methods.

[0003] Traditional wet-process testing methods require highly skilled operators and necessitate pure water cleaning systems, which are difficult and unnecessary for primary healthcare institutions. Dry chemistry methods primarily include multilayer membrane bonding (represented by Cardico) and coating methods (represented by Johnson & Johnson and Fuji). The main difference between these two methods lies in the carrier. Multilayer membrane bonding uses glass fiber or nylon as a carrier, immersing or spraying the reactants onto the membrane; while coating methods, as the name suggests, involve mixing the reactants into an inert carrier such as gelatin or agarose that does not interfere with the reaction. With multilayer membrane bonding, the varying degrees of adhesion between different membrane layers make it difficult to accurately control the gaps between them, affecting sample penetration and analyte reactions, resulting in poor repeatability and low accuracy. Furthermore, the high cost of the membranes increases the production cost of the test strips. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to provide a dry chemical reagent for total cholesterol detection, thereby improving the problems of poor repeatability and low accuracy in existing technologies.

[0005] To solve the above problems, the present invention adopts the following solution:

[0006] A dry chemical reagent for total cholesterol detection includes a diffusion layer, a spacer layer, and a reagent layer sequentially overlapped; the diffusion layer includes reflective particles, a binder, a surfactant, cholesterol esterase, and cholesterol oxidase; the reagent layer includes a carrier, horseradish peroxidase, an indicator composition, and a buffer solution.

[0007] Furthermore, the reflective particles have a particle size of 0.1-5 μm and are used in an amount of 20-500 g / m³. 2 The amount of the adhesive used is 1%-10% of the reflective particles; the amount of the surfactant used is 1-30 g / m³.2 The dosage of the cholesterol esterase is 30-100 KU / m 2 The dosage of the cholesterol oxidase is 0.1-50 KU / m 2 .

[0008] Furthermore, the reflective particles are one or more of barium sulfate, titanium dioxide, and polystyrene microspheres.

[0009] Furthermore, the adhesive is one or more combinations of cellulose acetate, polyvinylpyrrolidone, poly(N-isopropylacrylamide), and polyurethane.

[0010] Furthermore, the surfactant is one or more combinations of Triton X-100, Triton 450, Zonyl Fsn 100, Tween 20, Tween 80, and Brij 35.

[0011] Furthermore, the amount of the spacer layer used is 0.1-17.5 g / m³. 2 .

[0012] Furthermore, the spacer layer is one or more combinations of agarose, gelatin, poly(N-vinyl-2-pyrrolidone), poly(N-isopropylacrylamide), and polyacrylamide.

[0013] Furthermore, the amount of the carrier used is 1-50 g / m³. 2 The dosage of horseradish peroxidase is 5-20 KU / m³. 2 The indicator composition comprises 4-aminoantipyrine and 1,7-naphthol; the dosage of 4-aminoantipyrine is 0.1-2.5 g / m³. 2 The dosage of 1,7-naphthyldiol is 0.1-1.5 g / m³. 2 The buffer solution is a phosphate buffer or a Tris buffer.

[0014] Furthermore, the carrier is selected from hydrophilic cellulose derivatives, polysaccharides, gelatin and their derivatives.

[0015] The present invention adopts the above technical solution and has the following advantages:

[0016] The dry chemical detection reagent of the present invention adopts a coating method, in which a premixed homogeneous slurry is coated on a support layer in layers, and a spacer layer is used to block the interference between layers. Phenolic substances are used to replace photosensitizers as chromogens, and a specific ratio of substrate and buffer system is used. The multi-layered, spacerless, low-interference vertical detection system constructed by the present invention greatly improves the repeatability, accuracy and stability of the detection reagent. Attached Figure Description

[0017] Figure 1 This is a structural diagram of the dry chemical reagent tablet of the present invention;

[0018] Figure 2 The graph shows the accuracy assessment results of the reagent card in experimental group 1. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0020] The dry chemical reagents for total cholesterol detection in this invention can determine total cholesterol in body fluid samples. Body fluids can be serum, plasma, etc. Samples that have undergone appropriate pretreatment can also be used as body fluid samples.

[0021] like Figure 1 As shown, a dry chemical reagent for total cholesterol detection includes a diffusion layer 1, a spacer layer 2, and a reagent layer 3 sequentially overlapped; the diffusion layer 1 includes reflective particles, an adhesive, a surfactant, cholesterol esterase, and cholesterol oxidase; the reagent layer 3 includes a carrier, horseradish peroxidase, an indicator composition, and a buffer solution.

[0022] In the diffusion layer 1 of this invention, the reflective particles have a particle size of 0.1-5 μm and are used in an amount of 20-500 g / m². 2 Preferably 50-300g / m 2 The amount of adhesive used is 1%-10% of the reflective particles, preferably 3%-6%; the amount of surfactant used is 1-30 g / m³. 2 Preferably 5-15g / m 2 The dosage of cholesterol esterase is 30-100 KU / m 2 Preferably 30-50 KU / m 2 The dosage of cholesterol oxidase is 1-50 KU / m 2 Preferably 5-10 KU / m 2 .

[0023] The reflective particles are one or more of barium sulfate, titanium dioxide, and polystyrene microspheres; the binder is one or more of cellulose acetate, polyvinylpyrrolidone, poly(N-isopropylacrylamide), and polyurethane; the surfactant is one or more of Triton X-100, Triton 450, Zonyl Fsn 100, Tween 20, Tween 80, and Brij 35; preferably Triton X-100.

[0024] This invention controls the formation of anisotropic porous diffusion layer 1 with different porosities and pore size distributions by adjusting the size and amount of particles and the amount of binder. This layer is used for the rapid absorption, uniform distribution, metering and transport of various analytes, thereby analyzing the test substances with different molecular weight distributions and compositions in biological liquids.

[0025] The diffusion layer 1 of this invention is an isotropic porous diffusion membrane with a porosity of 25%-80%. When barium sulfate is used as reflective particles, the adhesive is concentrated on the surface of the barium sulfate microspheres and in the region adjacent to the adjacent microspheres, and the barium sulfate microspheres are bonded into a coherent three-dimensional lattice to form a high-porosity structure, so that the diffusion layer 1 has the function of efficient and uniform diffusion of liquid. Cholesterol esters in the liquid sample are saponified by esterase to release cholesterol. The released cholesterol and the original free cholesterol in the sample are oxidized to generate hydrogen peroxide in the presence of cholesterol oxidase. The small molecules of hydrogen peroxide diffuse and are transferred to the reagent layer 3 in the form of fluid.

[0026] Furthermore, the spacer layer 2 is one or more combinations of agarose, gelatin, poly(N-vinyl-2-pyrrolidone), poly(N-isopropylacrylamide), and polyacrylamide, preferably poly(N-isopropylacrylamide); the dosage is 0.1-17.5 g / m³. 2 Preferably, it is 0.5-3.5 g / m 2 .

[0027] The spacer layer 2 of this invention can block interlayer cross-interference and also plays a role in improving the adhesion between the reagent layer 3 and the diffusion layer 1.

[0028] Furthermore, in reagent layer 3, the carrier is a water-soluble polymer, used in an amount of 1-50 g / m³. 2 Preferably 10-30g / m 2 The dosage of horseradish peroxidase is 5-20 KU / m³. 2 Preferably 8-15 KU / m 2 The indicator composition comprises 4-aminoantipyrine and 1,7-naphthol, in amounts of 0.1-2.5 g / m³. 2 and 0.1-1.5g / m 2 The preferred values ​​are 1.0-1.5 g / m³. 2and 0.5-1.0 g / m 2 This is used to quantify the level of hydrogen peroxide produced during cholesterol oxidation; the buffer is phosphate buffer or Tris buffer, pH 5.0-9.0, preferably pH 6.0-7.5, and the specific pH value is determined according to the activity range of the enzyme used.

[0029] Furthermore, the carrier is selected from hydrophilic cellulose derivatives, polysaccharides, gelatin and their derivatives; preferably gelatin.

[0030] This invention also includes a support layer 4, a reagent shell upper cover 5, and a reagent shell lower cover 6. The reagent layer 3 is coated on the surface of the support layer 4, the spacer layer 2 is coated on the surface of the reagent layer 3 away from the support layer 4, and the diffusion layer 1 is coated on the surface of the spacer layer 2 away from the reagent layer 3. The diffusion layer 1, spacer layer 2, reagent layer 3, and support layer 4 are sequentially arranged between the reagent shell upper cover 5 and the reagent shell lower cover 6 from top to bottom. A sample application hole and a detection window are respectively provided in the middle of the reagent shell upper cover 5 and the reagent shell lower cover 6. The diffusion layer 1, spacer layer 2, and reagent layer 3 distributed on the support layer 4 are structural layers for the diffusion of the sample to be tested and the generation of biochemical reactions, while the support layer provides support and has light-transmitting properties. The support layer of this invention is polyethylene terephthalate (PET).

[0031] This invention also discloses a method for preparing a dry chemical reagent for total cholesterol detection, comprising the following steps:

[0032] PET material was chosen as the support layer for this invention.

[0033] The reagent coating solution is mixed with gelatin and coated onto the surface of the support layer. The wet film thickness is 50-500 μm, and the mixture is dried in the range of 15-35℃.

[0034] The spacer layer is coated on the surface of the reagent layer, with a wet film thickness of 50-200 μm, preferably 50-100 μm, and dried in the range of 15℃-35℃.

[0035] The diffusion layer slurry is coated on the spacer layer, with a wet film thickness of 200-800 μm, and dried in the range of 15-35℃.

[0036] Cut the dried slides prepared in the above steps into small pieces of 1cm×1cm, put them into the upper and lower covers of the reagent shell, and assemble them into a cholesterol detection dry chemical reagent card.

[0037] The dry chemical reagent detection method for total cholesterol detection in this invention is as follows:

[0038] 10 μL of the test liquid is dropped onto a porous diffusion layer with uniform properties and incubated at 37°C for 5 minutes. The test liquid diffuses uniformly through the diffusion layer to the reagent layer. The surfactant in the diffusion layer releases cholesterol and cholesterol esters from the lipoprotein complex. Cholesterol esters are hydrolyzed into cholesterol by cholesterol esterase. Then, all the cholesterol is catalyzed by cholesterol oxidase to generate cholesterol-4-en-3-one and hydrogen peroxide. The small molecule hydrogen peroxide diffuses and is transferred to the reagent layer in fluid form. Hydrogen peroxide reacts with dye under the action of peroxidase to produce color. The concentration of the dye produced is proportional to the total cholesterol concentration in the sample and can be determined by reflectance photometric detection.

[0039] The total cholesterol detection dry chemical reagent of the present invention has good repeatability, high accuracy, strong stability, and is easy to use and store in clinical practice.

[0040] The present invention will be further described below with reference to the embodiments.

[0041] The reagent coating solutions for experimental groups 1-6 (as shown in Table 1) were coated onto a colorless and transparent PET support layer, with a wet film thickness of 200 μm, and dried at 32 °C. Next, spacer layers for experimental groups 1-6 (as shown in Table 1) with a wet film thickness of 100 μm were coated onto the reagent layer. After drying at 32 °C, diffusion layers for experimental groups 1-6 (as shown in Table 1) were coated, with a wet film thickness of 600 μm, and dried at room temperature in a well-ventilated area. After drying, the samples were cut into 1×1 cm pieces, assembled using reagent shells, and ready for measurement. The sample volume was 10 μL, incubated at 37 °C for 5 min, and measured at a wavelength of 540 nm.

[0042] Table 1: Components of the coating solutions for reagent layers, diffusion layers, and spacer layers in experimental groups 1-6

[0043]

[0044] The reagent card from Experimental Group 1 was used to test three concentration points, with each point tested 10 times. The results are shown in Table 2. The coefficient of variation was within 3%, indicating good repeatability. Furthermore, the reagent card was used to test 10 serum samples with known concentrations. The tested values ​​showed good correlation with the nominal values, with R² = 0.9957. Figure 2 As shown.

[0045] The reagent cards in Experimental Group 1 were sealed and stored at 4℃ and 37℃ for 7 days, and then opened and stored at room temperature for 7 days before testing. Compared with day 0, the deviation of the measured values ​​was within 15%, as shown in Table 3. This indicates that the test reagent can maintain stable measured values ​​even when stored at 37℃ for 7 days and opened and stored at room temperature for 7 days.

[0046] In summary, the total cholesterol detection dry chemical reagent of the present invention has excellent performance in terms of repeatability, accuracy and stability.

[0047] Table 2 Repeatability assessment of reagent cards in Experimental Group 1

[0048] Concentration point / mM 2.31 5.25 7.54 Test 1 2.27 5.28 7.38 Test 2 2.22 5.40 7.25 Test 3 2.17 5.42 7.34 Test 4 2.17 5.33 7.29 Test 5 2.12 5.27 7.30 Test 6 2.16 5.31 7.25 Test 7 2.17 5.43 7.40 Test 8 2.15 5.40 7.40 Test 9 2.11 5.43 7.27 Test 10 2.16 5.38 7.11 coefficient of variation 2.1％ 1.1％ 1.2％

[0049] Table 3 Stability assessment of reagent cards in experimental group 1

[0050] Nominal concentration / mM 0 days / mM 4℃ for seven days / mM 37℃ for seven days / mM 7 days after opening at room temperature / mm 5.80 5.81 5.70 5.61 5.65 2.36 2.39 2.33 2.18 2.25 7.41 7.35 7.30 7.01 7.11

[0051] The coating method of this invention has better repeatability and accuracy because the carrier is inexpensive, and the different film layers in the reagent strip are coated sequentially with negligible gaps between the film layers. Furthermore, after the reagent strip is added, the carrier, such as gelatin, swells, making the entire reaction system closer to a wet liquid reaction.

[0052] The dry chemical reagent of this invention is produced using a coating method. All components of each layer are mixed and sequentially coated onto a support layer. After drying, the next layer is coated. A spacer layer is added between the reagent layer and the diffusion layer, ultimately forming a multi-layered, spacer-free, low-interference vertical detection structure. The detection reagent of this invention features good repeatability, high accuracy, and strong stability.

[0053] The above embodiments are merely illustrative of the technical concept and features of the present invention, intended to enable those skilled in the art to understand the content of the present invention and apply it accordingly, and should not be construed as limiting the scope of protection of the present invention. All equivalent substitutions or improvements made based on the essence of the present invention should be included within the scope of the claims of the present invention.

Claims

1. A dry chemical reagent for total cholesterol detection, characterized in that, It includes a diffusion layer, a spacer layer, and a reagent layer that are sequentially overlapped; the diffusion layer includes reflective particles, an adhesive, a surfactant, cholesterol esterase, and cholesterol oxidase; the reagent layer includes a carrier, horseradish peroxidase, an indicator composition, and a buffer solution.

2. The dry chemical reagent for total cholesterol detection according to claim 1, characterized in that, The reflective particles have a particle size of 0.1-5 μm and are used in amounts of 20-500 g / m². 2 The amount of the adhesive used is 1%-10% of the reflective particles; the amount of the surfactant used is 1-30 g / m³. 2 The dosage of the cholesterol esterase is 30-100 KU / m 2 The dosage of the cholesterol oxidase is 0.1-50 KU / m 2 .

3. The dry chemical reagent for total cholesterol detection according to claim 1, characterized in that, The reflective particles are one or more of barium sulfate, titanium dioxide, and polystyrene microspheres.

4. The dry chemical reagent for total cholesterol detection according to claim 1, characterized in that, The adhesive is one or more of cellulose acetate, polyvinylpyrrolidone, poly(N-isopropylacrylamide), and polyurethane.

5. The dry chemical reagent for total cholesterol detection according to claim 1, characterized in that, The surfactant is one or more of Triton X-100, Triton 450, ZonylFsn100, Tween20, Tween 80, and Brij35.

6. The dry chemical reagent for total cholesterol detection according to claim 1, characterized in that, The amount of the spacer layer used is 0.1-17.5 g / m³. 2 .

7. The dry chemical reagent for total cholesterol detection according to claim 1, characterized in that, The spacer layer is one or more of agarose, gelatin, poly(N-vinyl-2-pyrrolidone), poly(N-isopropylacrylamide), and polyacrylamide.

8. The dry chemical reagent for total cholesterol detection according to claim 1, characterized in that, The amount of the carrier used is 1-50 g / m³. 2 The dosage of horseradish peroxidase is 5-20 KU / m³. 2 The indicator composition comprises 4-aminoantipyrine and 1,7-naphthol; the amount of 4-aminoantipyrine is 0.1-2.5 g / m³. 2 The dosage of 1,7-naphthyldiol is 0.1-1.5 g / m³. 2 The buffer solution is a phosphate buffer or a Tris buffer.

9. The dry chemical reagent for total cholesterol detection according to claim 1, characterized in that, The carrier is selected from hydrophilic cellulose derivatives, polysaccharides, gelatin and their derivatives.

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