Dry chemical test paper and application thereof

Abstract

The invention discloses dry chemical test paper and application thereof. The dry chemical test paper comprises a supporting layer, a blood filtering layer, a filtering layer and a reaction layer, the filtering layer is treated by an R1 reagent, and the reaction layer is treated by an R2 reagent; the R1 reagent comprises a 3-morpholine propanesulfonate buffer solution, polyanions, an Mg < 2 + > reagent and a thickening agent; the R2 reagent comprises a 3-morpholine propanesulfonate buffer solution, a nonionic surfactant, an auxiliary surfactant, a saccharide stabilizer, a protein stabilizer, an auxiliary stabilizer, a preservative, a color developing agent, a chromogen substrate, peroxidase, cholesterol oxidase and cholesterol esterase. The dry chemical test paper can directly detect the low-density lipoprotein cholesterol on the dry chemical film, is not influenced by the concentration of triglyceride in a sample, and is accurate in detection result.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biology and new medicine technology, and relates to a dry chemical test paper and application thereof. BACKGROUND

[0002] Low-density lipoprotein cholesterol (LDL-c) detection techniques are divided into reference methods, direct methods and indirect methods. The reference methods include ultracentrifugation method and electrophoresis method. The ultracentrifugation method separates LDL particles based on density difference, and the beta quantitative method determines cholesterol, which has high accuracy, but the equipment is expensive and time-consuming. The electrophoresis method separates lipoproteins by charge difference, and quantifies by optical density scanning after staining, which can distinguish LDL, Lp(a) and VLDL subtypes, but the operation is complicated and is only limited to scientific research. The direct method includes immunosorbent method, chemical removal method and homogeneous method. The immunosorbent method uses anti-ApoB-100 or anti-LDL antibody to specifically bind LDL, and determines the cholesterol content after physical separation, which has high specificity and sensitivity, but the cost is high and the operation is complex. The chemical removal method selectively dissolves non-LDL lipoprotein by surfactant, and retains LDL for cholesterol determination, which is simple and fast in operation, but cannot distinguish LDL from lipoprotein(a) (Lp(a)). The homogeneous method selectively inhibits non-LDL cholesterol through chemical modification or enzymatic reaction, and directly determines LDL-c by color development, without separation step, which is suitable for emergency detection, but is easily interfered by high concentration of bilirubin and hemolysis, and needs to be detected by professional operators with large instruments. The indirect method is mainly based on rapid dry detection, using Friedewald or Martin-Hopkins modified formula, calculating LDL-c based on total cholesterol (TC), high-density lipoprotein cholesterol (HDL-c) and triglyceride (TG). This detection method has more outstanding advantages, low cost, convenient testing, and can be popularized to home scene, however, the method of calculating LDL has large error when TG level is high, and the result cannot be referred. Therefore, it is urgent to develop a method that can directly detect LDL concentration by detecting markers in the POCT rapid detection field, to make up for the defects of existing dry detection and provide more extensive clinical application space. SUMMARY

[0003] In view of the deficiencies of the prior art and actual needs, the present application provides a dry chemical test paper and application thereof, which can directly detect low-density lipoprotein cholesterol on a dry chemical film, and is not affected by the concentration of triglyceride in the sample, and the detection result is accurate.

[0004] To achieve the purpose of the present application, the following technical solutions are adopted:

[0005] In a first aspect, the present application provides a dry chemical test paper, which comprises a support layer, a blood filtration layer, a filter layer treated with R1 reagent, and a reaction layer treated with R2 reagent; the R1 reagent comprises a buffer, a polyanion reagent, Mg2 + a thickening agent; and the R2 reagent comprises a buffer, a non-ionic surfactant, an auxiliary surfactant, a saccharide stabilizer, a protein stabilizer, an auxiliary stabilizer, a preservative, a color developing agent, a chromogenic substrate, a peroxidase, a cholesterol oxidase, and a cholesterol esterase.

[0006] The dry chemical test paper of the present application can be stored and transported at room temperature, and can rapidly and quantitatively detect the concentration of low-density lipoprotein cholesterol (LDL-c) within 3 minutes without being interfered by the concentration of triglyceride. The sample is added from the support layer, and first passes through the blood filtration layer to remove blood cells under the action of gravity, and then passes through the filter layer treated with the reagent R1, and the chylomicron (CM) and very low-density lipoprotein (VLDL) in the sample are combined with the polyanion reagent in the filter layer to become a precipitate. Then, the high-density lipoprotein (HDL) and the low-density lipoprotein (LDL) all reach the reaction layer treated with the reagent R2, and under the action of the surfactant, the enzyme solution (cholesterol esterase, cholesterol oxidase), and the inhibitor in the reaction layer, the HDL is shielded, the LDL normally performs the coupling end-point colorimetric reaction and develops color, and the content of LDL-c in the sample can be quantitatively detected according to the color development intensity. The main principle is to use the polyanion precipitation method, to specifically shield the reaction activity of CM, VLDL, and cholesterol esterase, cholesterol oxidase by the polyanion reagent in the R1 reagent, and to specifically shield the combination of HDL and cholesterol esterase by the non-ionic inhibitor in the R2 reagent, and then under the action of the surfactant, the low-density lipoprotein particles are specifically dissolved to release LDL-c, and finally the LDL-c performs a specific color development reaction with the cholesterol esterase and the color developing agent on the reaction film.

[0007] The polyanion reagent in the R1 reagent and Mg2 + After binding, a complex with strong negative charge is formed, which preferentially adsorbs the apolipoprotein (such as ApoB-48, ApoE) on the surface of CM / VLDL through electrostatic interaction. The sulfonic acid group is combined with the polar head of the phospholipid bilayer on the surface of the lipoprotein to form a stable spatial barrier, which prevents the cholesterol oxidase (CHOD) and the cholesterol esterase (CHER) from approaching the cholesterol of CM / VLDL.

[0008] The non-ionic surfactant in the R2 reagent of the present application can be inserted into the HDL phospholipid bilayer to destroy the structural stability of the HDL, so that the HDL cholesterol ester (CE) cannot be enzymatically hydrolyzed. The hydrophilic segment forms a micelle to wrap the HDL fragments, preventing them from contacting the chromogenic reagent. The LDL particles (containing ApoB-100) have a low surface charge density and weak affinity with the polyanion reagent, and are not completely covered by the complex, so that the reactivity of cholesterol is retained. The LDL particles not covered by the polyanion reagent are completely dissolved under the action of the surfactant, releasing free cholesterol (FC) and cholesterol ester (CE). The CHER hydrolyzes CE to FC, and then CHOD catalyzes FC to generate Δ4-cholestenone and hydrogen peroxide (H2O2). H2O2 reacts with the chromogenic reagent under the catalysis of peroxidase, and the absorbance is positively correlated with the concentration of LDL-C. The concentration of LDL-C can be quantified by a standard curve.

[0009] Preferably, the buffer includes any one of or a combination of citrate buffer, phosphate buffer, acetate buffer, carbonate buffer, Tris-HCl buffer, or 3-morpholinopropanesulfonic acid buffer.

[0010] Preferably, the final concentration of the buffer in the R1 reagent is 10-100 mmol / L (for example, 10 mmol / L, 20 mmol / L, 30 mmol / L, 50 mmol / L, or 100 mmol / L), and the pH is 6.7-6.8 (for example, 6.7, 6.75, or 6.8).

[0011] Preferably, the final concentration of the buffer in the R2 reagent is 10-100 mmol / L (for example, 10 mmol / L, 20 mmol / L, 30 mmol / L, 50 mmol / L, or 100 mmol / L), and the pH is 6.7-6.8 (for example, 6.7, 6.75, or 6.8).

[0012] Preferably, the polyanion reagent in the R1 reagent includes any one of or a combination of polysaccharide polyanion, nucleic acid polyanion, polyamino acid polyanion, or synthetic polyanion.

[0013] Preferably, the polysaccharide polyanion reagent includes any one of or a combination of sodium alginate, hyaluronic acid, cyclodextrin salt, or carrageenan.

[0014] Preferably, the nucleic acid polyanion reagent includes any one of or a combination of deoxyribonucleic acid sodium salt, polyinosinic acid-cytidylic acid, or phosphorothioate oligonucleotide.

[0015] Preferably, the polyamino acid-based polyanionic agent comprises any one or a combination of at least two of polyglutamic acid, polyaspartic acid, or polylysine-graft-polyethylene glycol.

[0016] Preferably, the synthetic-based polyanionic agent comprises any one or a combination of at least two of polyacrylic acid, sodium polystyrene sulfonate, or perfluorosulfonic acid polymer.

[0017] Preferably, the Mg2+ agent in the R1 agent has a final concentration of 2-20 mmol / L (e.g., 2 mmol / L, 3 mmol / L, 5 mmol / L, 10 mmol / L, 12 mmol / L, 15 mmol / L, or 20 mmol / L). + Preferably, the Mg2+ agent in the R1 agent has a final concentration of 2-20 mmol / L (e.g., 2 mmol / L, 3 mmol / L, 5 mmol / L, 10 mmol / L, 12 mmol / L, 15 mmol / L, or 20 mmol / L).

[0018] Preferably, the thickening agent in the R1 agent has a final concentration of 2-6 g / L (e.g., 2 g / L, 3 g / L, 4 g / L, 5 g / L, or 6 g / L).

[0019] Preferably, the polyanionic agent in the R1 agent has a final concentration of 0.5-16 mmol / L (e.g., 0.5 mmol / L, 1 mmol / L, 2 mmol / L, 3 mmol / L, 5 mmol / L, 10 mmol / L, 12 mmol / L, 15 mmol / L, or 16 mmol / L).

[0020] Preferably, the Mg2+ agent in the R1 agent has a final concentration of 2-20 mmol / L (e.g., 2 mmol / L, 3 mmol / L, 5 mmol / L, 10 mmol / L, 12 mmol / L, 15 mmol / L, or 20 mmol / L). + Preferably, the Mg2+ agent in the R1 agent has a final concentration of 2-20 mmol / L (e.g., 2 mmol / L, 3 mmol / L, 5 mmol / L, 10 mmol / L, 12 mmol / L, 15 mmol / L, or 20 mmol / L).

[0021] Preferably, the thickening agent in the R1 agent has a final concentration of 2-6 g / L (e.g., 2 g / L, 3 g / L, 4 g / L, 5 g / L, or 6 g / L).

[0022] Preferably, the non-ionic surfactant in the R2 agent comprises any one or a combination of at least two of the Tetronic series, the Pluronic series, EO-PO block copolymer, or the Plantaren series.

[0023] Preferably, the co-surfactant in the R2 agent comprises any one or a combination of at least two of sodium cholate, sodium dodecyl sulfate, Triton X-100, Tween-20, 3-[(3-cholamido)propyl]dimethyl-(mono-hydroxyethyl)ammonium, or SB-10.

[0024] Preferably, the saccharide stabilizer in the R2 agent comprises any one or a combination of at least two of sucrose, glucose, lactose, or trehalose.

[0025] Preferably, the proteinaceous stabilizer in the R2 reagent comprises BSA.

[0026] Preferably, the auxiliary stabilizer in the R2 reagent comprises any one or a combination of at least two of glycerol, sorbitol, or polyethylene glycol.

[0027] Preferably, the preservative in the R2 reagent comprises any one or a combination of at least two of sodium benzoate, potassium sorbate, sodium propionate, or sodium azide.

[0028] Preferably, the chromogenic agent in the R2 reagent comprises any one of ADOS, TOOS, TOPS, MADB, MAOS, DAOS, HDAOS, ADPS, or ALPS.

[0029] Preferably, the chromogenic substrate in the R2 reagent comprises any one of 4-AAP or MBTH.

[0030] Preferably, the peroxidase in the R2 reagent comprises horseradish peroxidase.

[0031] Preferably, the source of cholesteryl esterase in the R2 reagent comprises Pseudomonas fluorescens.

[0032] Preferably, the source of cholesteryl oxidase in the R2 reagent comprises the genus Rhodococcus.

[0033] Preferably, the final concentration of non-ionic surfactant in the R2 reagent is 4-20 g / L (e.g., 4 g / L, 5 g / L, 6 g / L, 10 g / L, 15 g / L, or 20 g / L).

[0034] Preferably, the final concentration of auxiliary surfactant in the R2 reagent is 4-20 g / L (e.g., 4 g / L, 5 g / L, 6 g / L, 10 g / L, 15 g / L, or 20 g / L).

[0035] Preferably, the final concentration of saccharide stabilizer in the R2 reagent is 1-5 g / L (e.g., 1 g / L, 2 g / L, 3 g / L, 4 g / L, or 5 g / L).

[0036] Preferably, the final concentration of proteinaceous stabilizer in the R2 reagent is 3-5 g / L (e.g., 3 g / L, 4 g / L, or 5 g / L).

[0037] Preferably, the final concentration of auxiliary stabilizer in the R2 reagent is 1-4 g / L (e.g., 1 g / L, 2 g / L, 3 g / L, or 4 g / L).

[0038] Preferably, the final concentration of preservative in the R2 reagent is 1-5 g / L (e.g., 1 g / L, 2 g / L, 3 g / L, 4 g / L, or 5 g / L).

[0039] Preferably, the final concentration of chromogenic agent in the R2 reagent is 1.2-1.5 mmol / L (e.g. 1.2 mmol / L, 1.3 mmol / L, 1.4 mmol / L or 1.5 mmol / L).

[0040] Preferably, the final concentration of chromogenic substrate in the R2 reagent is 2-3 g / L (e.g. 2 g / L, 2.5 g / L or 3 g / L).

[0041] Preferably, the final concentration of peroxidase in the R2 reagent is 25-35 KU / L (e.g. 25 KU / L, 30 KU / L or 35 KU / L).

[0042] Preferably, the final concentration of cholesterol esterase in the R2 reagent is 0.5-2 KU / L (e.g. 0.5 KU / L, 1 KU / L or 2 KU / L).

[0043] Preferably, the final concentration of cholesterol oxidase in the R2 reagent is 2-5 KU / L (e.g. 2 KU / L, 3 KU / L, 4 KU / L or 5 KU / L).

[0044] Preferably, the blood flow improving agent is any one or a combination of at least two of trehalose, sorbitol, glycerol or polyethylene glycol.

[0045] Preferably, the final concentration of the blood flow improving agent is 5-25 mmol / L (e.g. 5 mmol / L, 10 mmol / L, 15 mmol / L, 20 mmol / L or 25 mmol / L).

[0046] Preferably, the treatment of the filtration layer comprises soaking or spraying the membrane with the R1 reagent after preparation of the R1 reagent and drying after soaking or spraying, the R1 reagent comprising 3-morpholinopropanesulfonate buffer, polyanion reagent, Mg2 + Reagents and thickening agents.

[0047] Preferably, the treatment of the reaction layer comprises soaking or spraying the membrane with the R2 reagent after preparation of the R2 reagent and drying after soaking or spraying, the R2 reagent comprising 3-morpholinopropanesulfonate buffer, non-ionic surfactant, auxiliary surfactant, saccharide stabilizer, protein stabilizer, auxiliary stabilizer, preservative, chromogenic agent, chromogenic substrate, peroxidase, cholesterol oxidase and cholesterol esterase.

[0048] In a second aspect, the present application provides a reagent card for detecting low density lipoprotein cholesterol, the reagent card for detecting low density lipoprotein cholesterol comprising the dry chemistry test paper of the first aspect.

[0049] In a third aspect, the application provides the use of the dry chemistry test paper of the first aspect or the reagent card for detecting low density lipoprotein cholesterol in the preparation of a blood lipid detection product.

[0050] Compared with the prior art, the application has the following beneficial effects:

[0051] (1) The dry chemistry test paper and the reagent card of the application can directly detect low density lipoprotein cholesterol on a dry chemistry film, without the need to calculate the content value through other three indicators (total cholesterol, triglyceride and high density lipoprotein cholesterol), and the detection result is accurate and is not affected by the concentration of triglyceride in the sample;

[0052] (2) The dry chemistry test paper of the application can effectively prevent cross interference of reagents by layering and fixing reagent R1 and reagent R2;

[0053] (3) The dry chemistry test paper and the reagent card of the application can end the reaction within 3 minutes, and the correlation of the measured value and the value measured by a biochemical instrument is 0.985, so that the low density lipoprotein cholesterol can be quickly detected and the result is accurate. DETAILED DESCRIPTION

[0054] In order to further illustrate the technical means adopted by the application and its effects, the application is further described by using examples. It can be understood that the specific embodiments described herein are only used to explain the application, and not to limit the application.

[0055] If a specific technology or condition is not specified in the examples, the technology or condition described in the literature in the art or according to the product instruction is used. If the manufacturer of the reagent or instrument is not specified, it is a conventional product that can be commercially available through a regular channel.

[0056] In order to make the application more easily understood, some terms are first defined. As used in the application, unless otherwise defined, all technical and scientific terms used in the application have the same meaning as commonly understood by one of ordinary skill in the art to which the application belongs. All patents and publications referred to in the application are incorporated by reference in their entirety.

[0057] The term "Tetronic series" refers to a branched block polyether.

[0058] The term "Pluronic series" refers to a linear block polyether.

[0059] The term "EO-PO block copolymer" refers to an organic copolymer composed of alternating arrangement of ethylene oxide (EO) and propylene oxide (PO) blocks.

[0060] The term "Plantaren series" refers to an alkyl polyglycoside surfactant.

[0061] The term "SB-10" refers to a zwitterionic surfactant.

[0062] The term "DOS, TOOS, TOPS, MADB, MAOS, DAOS, HDAOS, ADPS, ALPS" refers to different kinds of chromogenic substrate name abbreviations, wherein DOS refers to N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline sodium salt; TOOS refers to N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline sodium salt; TOPS refers to N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline sodium salt; MADB refers to 3,5 dimethoxy-N-ethyl-N-(2-hydroxy-3-sulfopropyl) aniline sodium salt; MAOS refers to N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methoxyaniline sodium salt; DAOS refers to N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline sodium salt; HDAOS refers to N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline sodium salt; ADPS refers to N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-amino-4-methoxyaniline sodium salt; ALPS refers to N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline sodium salt.

[0063] The term "4-AAP and MBTH" refers to 4-aminoantipyrine and 3-methyl-2-benzothiazolone hydrazone hydrochloride.

[0064] Example 1

[0065] The present example provides a dry-chemistry test paper for detecting low-density lipoprotein cholesterol, as follows:

[0066] The dry-chemistry test paper of the present example includes a support layer, a blood filtration layer, a filter layer treated with an R1 reagent, and a reaction layer treated with an R2 reagent, and the blood filtration layer is treated with a blood flow improvement agent.

[0067] R1 reagent: buffer (3-morpholinopropanesulfonate buffer 50 mmol / L), polyanion reagent (cyclodextrin salt 2 mmol / L), Mg2 + reagent (magnesium sulfate 5 mmol / L), thickening agent (polyvinylpyrrolidone 6 g / L).

[0068] R2 reagent: buffer (3-morpholinopropanesulfonic acid buffer 100 mmol / L), nonionic surfactant (Pluronic series F-68 4 g / L), auxiliary surfactant (sodium dodecyl sulfate 4 g / L), saccharide stabilizer (sucrose 1 g / L), protein stabilizer (BSA 3 g / L), auxiliary stabilizer (sorbitol 1 g / L), preservative (sodium benzoate 1 g / L), color developer (MAOS 1.2 mmol / L), chromogenic substrate (4-AAP 2 g / L), peroxidase 25 KU / L, cholesterol esterase 0.5 KU / L, and cholesterol oxidase 2 KU / L.

[0069] Blood flow improver: sorbitol 5 mmol / L.

[0070] Example 2

[0071] This example provides a dry chemical test paper for detecting low-density lipoprotein cholesterol, which is as follows:

[0072] The dry chemical test paper of this example includes a support layer, a blood filtration layer, a filter layer treated with Rl reagent, and a reaction layer treated with R2 reagent, and the blood filtration layer is treated with a blood flow improver.

[0073] Rl reagent: buffer (3-morpholinopropanesulfonic acid buffer 10 mmol / L), polyanion reagent (cyclodextrin salt 0.5 mmol / L), Mg2 + reagent (magnesium sulfate 2 mmol / L), thickening agent (polyvinylpyrrolidone 2 g / L).

[0074] R2 reagent: buffer (3-morpholinopropanesulfonic acid buffer 100 mmol / L), nonionic surfactant (Pluronic series F-68 4 g / L), auxiliary surfactant (sodium dodecyl sulfate 4 g / L), saccharide stabilizer (sucrose 1 g / L), protein stabilizer (BSA 3 g / L), auxiliary stabilizer (sorbitol 1 g / L), preservative (sodium benzoate 1 g / L), color developer (MAOS 1.2 mmol / L), chromogenic substrate (4-AAP 2 g / L), peroxidase 25 KU / L, cholesterol esterase 0.5 KU / L, and cholesterol oxidase 2 KU / L.

[0075] Blood flow improver: sorbitol 5 mmol / L.

[0076] Example 3

[0077] This example provides a dry chemical test paper for detecting low-density lipoprotein cholesterol, which is as follows:

[0078] The dry chemical test paper of the present embodiment includes a support layer, a blood filtration layer, a filter layer treated with an Rl reagent, and a reaction layer treated with an R2 reagent, the blood filtration layer being treated with a blood flow improvement agent.

[0079] Rl reagent: buffer (3-morpholinopropanesulfonate buffer 100 mmol / L), polyanion reagent (cyclodextrin salt 16 mmol / L), Mg2 + reagent (magnesium sulfate 20 mmol / L), thickening agent (polyvinylpyrrolidone 6 g / L).

[0080] R2 reagent: buffer (3-morpholinopropanesulfonate buffer 100 mmol / L), non-ionic surfactant (Pluronic series F-68 20 g / L), auxiliary surfactant (sodium dodecyl sulfate 20 g / L), sugar stabilizer (sucrose 5 g / L), protein stabilizer (BSA 5 g / L), auxiliary stabilizer (sorbitol 4 g / L), preservative (sodium benzoate 5 g / L), color developing agent (MAOS 1.5 mmol / L), chromogenic substrate (4-AAP 3 g / L), peroxidase 35 KU / L, cholesterol esterase 2 KU / L, and cholesterol oxidase 5 KU / L.

[0081] Blood flow improvement agent: sorbitol 25 mmol / L.

[0082] Example 4

[0083] The present embodiment provides a dry chemical test paper for detecting low-density lipoprotein cholesterol, which is different from Example 1 only in that the polyanion reagent (cyclodextrin salt) is not contained in the Rl reagent, the mass percentage of which is proportionally distributed to the Mg2 + reagent (magnesium sulfate) and the thickening agent (polyvinylpyrrolidone).

[0084] Example 5

[0085] The present embodiment provides a dry chemical test paper for detecting low-density lipoprotein cholesterol, which is different from Example 1 only in that the Mg2 + reagent (magnesium sulfate) is not contained in the Rl reagent, the mass percentage of which is proportionally distributed to the polyanion (cyclodextrin salt) and the thickening agent (polyvinylpyrrolidone).

[0086] Example 6

[0087] The present embodiment provides a dry chemical test paper for detecting low-density lipoprotein cholesterol, which is different from Example 1 only in that the thickening agent (polyvinylpyrrolidone) is not contained in the Rl reagent, the mass percentage of which is proportionally distributed to the polyanion reagent (cyclodextrin salt) and the Mg2 + reagent (magnesium sulfate).

[0088] Example 7

[0089] The present example provides a dry chemical test paper for detecting low density lipoprotein cholesterol, which is different from Example 1 only in that the 3-morpholine propyl sulfonate buffer in the Rl reagent and the R2 reagent is replaced by a phosphate buffer in the same mass percentage.

[0090] Example 8

[0091] The present example provides a dry chemical test paper for detecting low density lipoprotein cholesterol, which is different from Example 1 only in that the non-ionic surfactant (Pluronic series F-68) in the R2 reagent is proportionally distributed to the auxiliary surfactant (sodium dodecyl sulfate) in terms of mass percentage.

[0092] Example 9

[0093] The present example provides a dry chemical test paper for detecting low density lipoprotein cholesterol, which is different from Example 1 only in that the sugar stabilizer (sucrose) in the R2 reagent is proportionally distributed to the protein stabilizer (BSA) and the auxiliary stabilizer (sorbitol) in terms of mass percentage.

[0094] Example 10

[0095] The present example provides a dry chemical test paper for detecting low density lipoprotein cholesterol, which is different from Example 1 only in that the protein stabilizer (BSA) in the R2 reagent is proportionally distributed to the sugar stabilizer (sucrose) and the auxiliary stabilizer (sorbitol) in terms of mass percentage.

[0096] Example 11

[0097] The present example provides a dry chemical test paper for detecting low density lipoprotein cholesterol, which is different from Example 1 only in that the preservative (sodium benzoate) in the R2 reagent is proportionally distributed to the sugar stabilizer (sucrose), the protein stabilizer (BSA) and the auxiliary stabilizer (sorbitol) in terms of mass percentage.

[0098] Test Example 1

[0099] The actual values of the low density lipoprotein cholesterol concentrations of the clinical plasma samples with the concentrations of 0.70 mmol / L, 1.50 mmol / L, 2.30 mmol / L, 3.30 mmol / L, 4.40 mmol / L and 5.30 mmol / L were dropped on the support layers of the dry chemical test papers of the embodiments of the present application, and the detection values of the low density lipoprotein cholesterol concentrations were obtained after the reaction. The low density lipoprotein cholesterol values were detected by using the Mindray BS280 biochemical instrument, the deviation rates from the biochemical instrument were calculated, and the times required for the reaction results on the dry chemical test papers were recorded. The correlation coefficient R of the biochemical instrument detection values and the measurement values of the dry chemical test papers of the present application was calculated. 2The results are shown in Table 1.

[0100] Table 1

[0101]

[0102] From Table 1, it can be seen that the dry chemical test paper for detecting low density lipoprotein cholesterol in Examples 1-3 can directly detect the concentration of low density lipoprotein cholesterol on the dry chemical film, the detection result is accurate, the deviation rate is less than 15%, and the detection time is short, which can be completed in 3 minutes. Examples 4-5 show that the poly-anion and magnesium ions in the poly-anion reagent need to coexist to synergistically play a shielding effect, wherein the poly-anion and Mg2 + After combination, a complex with strong negative electricity is formed, which is preferentially adsorbed by electrostatic interaction

[0103] The apolipoprotein (such as ApoB-48, ApoE) on the surface of CM / VLDL, so that the CM / VLDL components are precipitated, and the cholesterol therein is prevented from reacting with cholesterol oxidase (CHOD) and cholesterol esterase (CHER). Example 6 shows that the thickening agent PVP (polyvinylpyrrolidone) can increase the upper limit of the detection of low density lipoprotein cholesterol, because the presence of the thickening agent prolongs the reaction time of the sample and the film reagent, which can make the non- LDL components in the sample be fully precipitated, and if there is no PVP, the test value is high. Example 7 shows that compared with other buffer solutions, 3-morpholine propyl sulfonate buffer helps to improve the detection accuracy of the reagent strip and reduce the average absolute coefficient of variation. Example 8 shows that the non-ionic surfactant can combine with the polar head of the phospholipid bilayer on the surface of the lipoprotein to form a stable spatial barrier, preventing cholesterol oxidase (CHOD) and cholesterol esterase (CHER) from approaching the cholesterol containing non- LDL components. Examples 9-10 show that the three of sugar stabilizer, protein stabilizer BSA, and preservative synergistically play a role to keep the enzyme activity in the reagent strip stable during the test.

[0104] Test Example 2

[0105] The dry chemical test paper for detecting low density lipoprotein cholesterol in Example 1 was tested to test 10 groups of clinical plasma samples containing high value of triglyceride (TG>4.5mmol / L). The values of total cholesterol (TC), triglyceride (TG), high density lipoprotein cholesterol (HDL-c), and low density lipoprotein cholesterol (LDL-c) of the 10 groups of clinical plasma samples were tested by Mindray BS280 biochemical instrument, and the concentration deviation rate of LDL-c was calculated by the formula method (LDL-c=TC-HDL-c-TG / 2.2) using three indicators, and compared with the test value deviation rate of the test paper test method in Example 1 and the biochemical instrument. The test results are shown in Table 2.

[0106] Table 2

[0107]

[0108] From Table 2, it can be seen that the dry chemical test paper for detecting low density lipoprotein cholesterol in Example 1 can detect samples with high triglyceride values (TG>4.5mmol / L), and the deviation rate is not more than 15%, while the formula method is more likely to have calculation deviation when calculating samples with high TG values.

[0109] In summary, the dry chemical test paper of the present application can directly detect low density lipoprotein cholesterol on a dry chemical film, is not affected by the concentration of triglyceride in the sample, and the detection result is accurate.

[0110] The applicant declares that the detailed method of the present application is illustrated by the above examples, but the present application is not limited to the above detailed method, that is, it does not mean that the present application must rely on the above detailed method to be implemented. It should be understood by those skilled in the art that any improvement of the present application, equivalent replacement of each raw material of the product of the present application, addition of auxiliary ingredients, selection of specific methods, etc. fall within the protection scope and disclosure scope of the present application.

Claims

1. A dry chemical test paper, characterized in that, The dry chemical test strip includes a support layer, a blood filtration layer, a filter layer, and a reaction layer. The filter layer is treated with reagent R1, and the reaction layer is treated with reagent R2. The R1 reagent includes buffer solution, polyanionic reagent, and Mg. 2+ Reagents and thickeners; The R2 reagent includes buffer solution, nonionic surfactant, auxiliary surfactant, carbohydrate stabilizer, protein stabilizer, auxiliary stabilizer, preservative, chromogenic agent, chromogenic substrate, peroxidase, cholesterol oxidase and cholesterol esterase.

2. The dry chemical test paper according to claim 1, characterized in that, The buffer solution includes any one or a combination of at least two of the following: citrate buffer, phosphate buffer, acetate buffer, carbonate buffer, tris(hydroxymethyl)aminomethane-hydrochloric acid buffer, or 3-morpholine propanesulfonate buffer. Preferably, the final concentration of the buffer solution in reagent R1 is 10-100 mmol / L, and the pH is 6.7-6.8; Preferably, the final concentration of the buffer solution in the R2 reagent is 10-100 mmol / L, and the pH is 6.7-6.8; Preferably, the polyanionic reagent in the R1 reagent includes any one or a combination of at least two of the following: polysaccharide polyanionic reagents, nucleic acid polyanionic reagents, polyamino acid polyanionic reagents, or synthetic polyanionic reagents. Preferably, the polysaccharide polyanionic reagent includes any one or a combination of at least two of sodium alginate, hyaluronic acid, cyclodextrin salt, or carrageenan; Preferably, the nucleic acid polyanionic reagent includes any one or a combination of at least two of sodium deoxyribonucleic acid, polyinosinic acid-cytidine, or phosphate thioester oligonucleotide; Preferably, the polyamino acid-based polyanionic reagent includes any one or a combination of at least two of polyglutamic acid, polyaspartic acid, or polylysine-grafted-polyethylene glycol; Preferably, the synthetic polyanionic reagent comprises any one or a combination of at least two of polyacrylic acid, sodium polystyrene sulfonate, or perfluorosulfonic acid polymers; Preferably, the Mg in the R1 reagent 2+ The reagents include any one or a combination of at least two of magnesium chloride, magnesium sulfate, or magnesium phosphate. Preferably, the thickener in the R1 reagent includes any one or a combination of at least two of the following: hydroxymethylcellulose, hydroxypropylcellulose, maleic anhydride, polyvinylpyrrolidone, polyethylene glycol, or methylcellulose.

3. The dry chemical test paper according to claim 1 or 2, characterized in that, The final concentration of the polyanionic reagent in reagent R1 is 0.5-16 mmol / L; Preferably, Mg2+ in reagent R1 + The final concentration of the reagent is 2-20 mmol / L; Preferably, the final concentration of the thickener in the R1 reagent is 2-6 g / L.

4. The dry chemical test paper according to any one of claims 1-3, characterized in that, The nonionic surfactant in the R2 reagent includes any one or a combination of at least two of the Tetronic series, Pluronic series, EO-PO block copolymers, or Plantaren series. Preferably, the auxiliary surfactant in the R2 reagent includes any one or a combination of at least two of sodium cholate, sodium dodecyl sulfate, Triton X-100, Tween-20, 3-[(3-cholamido)propyl]dimethylmono(hydroxyethyl)ammonium or SB-10; Preferably, the sugar stabilizer in the R2 reagent includes any one or a combination of at least two of sucrose, glucose, lactose or trehalose; Preferably, the protein stabilizer in the R2 reagent includes BSA; Preferably, the auxiliary stabilizer in the R2 reagent includes any one or a combination of at least two of glycerol, sorbitol, or polyethylene glycol; Preferably, the preservative in the R2 reagent includes any one or a combination of at least two of sodium benzoate, potassium sorbate, sodium propionate, or sodium azide; Preferably, the colorimetric reagent in the R2 reagent includes any one of ADOS, TOOS, TOPS, MADB, MAOS, DAOS, HDAOS, ADPS, or ALPS; Preferably, the chromogenic substrate in the R2 reagent includes either 4-AAP or MBTH; Preferably, the peroxidase in the R2 reagent includes horseradish peroxidase; Preferably, the source of cholesterol esterase in the R2 reagent includes *Pseudomonas fluorescens*; Preferably, the cholesterol oxidase in the R2 reagent is derived from Rhodococcus spp.

5. The dry chemical test paper according to claims 1-4, characterized in that, The final concentration of the nonionic surfactant in reagent R2 is 4-20 g / L; Preferably, the final concentration of the auxiliary surfactant in reagent R2 is 4-20 g / L; Preferably, the final concentration of the carbohydrate stabilizer in reagent R2 is 1-5 g / L; Preferably, the final concentration of the protein stabilizer in reagent R2 is 3-5 g / L; Preferably, the final concentration of the auxiliary stabilizer in reagent R2 is 1-4 g / L; Preferably, the final concentration of the preservative in reagent R2 is 1-5 g / L; Preferably, the final concentration of the colorimetric reagent in reagent R2 is 1.2-1.5 mmol / L; Preferably, the final concentration of the chromogen substrate in the R2 reagent is 2-3 g / L; Preferably, the final concentration of peroxidase in reagent R2 is 25-35 KU / L; Preferably, the final concentration of cholesterol esterase in reagent R2 is 0.5-2 KU / L; Preferably, the final concentration of cholesterol oxidase in the R2 reagent is 2-5 KU / L.

6. The dry chemical test paper according to claims 1-5, characterized in that, The filtration layer is treated with a blood flow improver, which includes any one or a combination of at least two of trehalose, sorbitol, glycerin, or polyethylene glycol. Preferably, the final concentration of the blood flow improver is 5-25 mmol / L.

7. The dry chemical test paper according to any one of claims 1-6, characterized in that, The filter layer is treated by: preparing R1 reagent and then soaking or spraying with R1 reagent followed by drying. R1 reagent includes 3-morpholine propanesulfonate buffer, polyanionic reagent, and Mg2+. + Reagents and thickeners.

8. The dry chemical test paper according to any one of claims 1-7, characterized in that, The treatment method of the reaction layer includes: preparing R2 reagent, soaking or spraying with R2 reagent, and then drying. The R2 reagent includes 3-morpholine propanesulfonate buffer, nonionic surfactant, auxiliary surfactant, sugar stabilizer, protein stabilizer, auxiliary stabilizer, preservative, chromogenic agent, chromogenic substrate, peroxidase, cholesterol oxidase and cholesterol esterase.

9. A reagent card for detecting low-density lipoprotein cholesterol, characterized in that, The reagent card for detecting low-density lipoprotein cholesterol includes the dry chemical test paper according to any one of claims 1-8.

10. The use of the dry chemical test paper according to any one of claims 1-8 or the reagent card for detecting low-density lipoprotein cholesterol according to claim 9 in the preparation of blood lipid detection products.

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