Total cholesterol detection compound enzyme liquid, detection test paper and detection system

Abstract

The invention relates to the technical field of biochemistry, in particular to compound enzyme liquid, detection test paper and a detection system for total cholesterol detection. The compound enzyme liquid for total cholesterol detection comprises cholesterol oxidase, cholesterol esterase, peroxidase, an electronic mediator, a surfactant composition, a thickening agent, a protective agent, metal inorganic salt and a buffer solution. The surfactant composition comprises one or a combination of at least two of surfactants with the HLB being larger than 23 and one or a combination of at least two of surfactants with the HLB being 12-14. The compound enzyme liquid can be used for detecting total cholesterol through an electrochemical method, and is high in detection speed, high in sensitivity and high in accuracy.

Description

Technical Field

[0001] The present invention relates to the field of biochemistry, and in particular, to a total cholesterol detection composite enzyme solution, a test strip, and a detection system. Background Art

[0002] Cholesterol, also known as cholesterin, is a derivative of cyclopentane polyhydrophenanthrene. Cholesterol is the main steroid compound in mammals and plays an important role in basic cell life activities. Total cholesterol refers to the sum of cholesterol contained in all lipoproteins in the blood, including free cholesterol and cholesterol esters. Cholesterol is an important raw material for synthesizing physiologically active substances such as adrenal cortical hormones, sex hormones, bile acids, and vitamin D, and is also a major component of cell membranes. However, too high cholesterol concentration in the blood will significantly increase the risk of arterial diseases. The occurrence of most coronary heart diseases, hypertension, atherosclerosis, and lipid metabolism disorders stems from high cholesterol in the serum.

[0003] Therefore, cholesterol, as an important indicator for clinical blood lipid item detection, has important guiding significance for the diagnosis of cardiovascular diseases.

[0004] Currently, the methods for detecting total cholesterol mainly include gas-liquid chromatography-mass spectrometry, molecular luminescence method, electrochemical method, and colorimetric method.

[0005] Among them, the electrochemical test method is widely used in the field of point-of-care testing (POCT). The electrochemical test method selectively recognizes the target analyte in the test sample through biomolecules (such as enzymes) modified on the electrode surface, and converts the biorecognition signal into an electrical signal (oxidation or reduction current) that can be collected and measured, so as to realize the quantitative detection of the target analyte.

[0006] During the process of quantitatively detecting total cholesterol using the electrochemical method, total cholesterol exists in the blood in the form of lipoproteins. Lipoproteins include chylomicrons, very low density lipoproteins (VLDL), low density lipoproteins (LDL), and high density lipoproteins (HDL). The cholesterol contents of these lipoproteins are respectively called VLDL-cholesterol (VLDL-C), LDL-cholesterol (LDL-C), and HDL-cholesterol (HDL-C).

[0007] In order to allow the cholesterol in lipoproteins to fully participate in the reaction, it is necessary to first decompose lipoproteins to release cholesterol. Usually, surfactants are used to promote the dissolution of lipids. However, the rate of action of surfactants on different lipoprotein parts is different. To ensure the detection of all analytes present in the sample, it is necessary to let the surfactant react with the sample for a long enough time to release all cholesterol, which results in a longer time required to measure total cholesterol.

[0008] Therefore, it is necessary to improve the existing formula to achieve fast and accurate determination of total cholesterol. Summary of the Invention

[0009] The object of the present invention is to provide a composite enzyme solution, a test strip and a detection system for total cholesterol detection. The composite enzyme solution provided by the embodiments of the present invention can detect total cholesterol by electrochemistry, with fast detection speed, high sensitivity and high accuracy.

[0010] In a first aspect, the present invention provides a composite enzyme solution for total cholesterol detection, which includes cholesterol oxidase, cholesterol esterase, peroxidase, an electron mediator, and a surfactant composition.

[0011] Wherein the surfactant composition includes surfactant 1 and surfactant 2.

[0012] The surfactant 1 includes any one or a combination of at least two surfactants with HLB>23.

[0013] The surfactant 2 includes any one or a combination of at least two surfactants with HLB of 12-14.

[0014] The surfactant 1 includes any one or a combination of at least two of Pluronic F68, Pluronic F77, Pluronic F87, Pluronic F88, Pluronic F98, Pluronic F108, Tetronic 1107, and Tetronic 1307.

[0015] The surfactant 2 includes any one or a combination of at least two of TritonX-100, Surfynol 465, Creamophpr EL, Oleth-10, and CHEMAL LA9.

[0016] The electron mediator is a reduced electron mediator.

[0017] Wherein, the reduced electron mediator includes any one of potassium ferrocyanide, ferrocene derivatives, osmium complexes, and ruthenium complexes.

[0018] Preferably, the ferrocene derivative includes any one of potassium ferrocyanide, (ferrocenylmethyl) trimethylammonium chloride, and bis[3-trimethyl-aminopropyl] ferrocene dichloride.

[0019] In an alternative embodiment, the composite enzyme solution further includes a buffer solution, a thickener, a protective agent, and a metal inorganic salt.

[0020] In an alternative embodiment, the buffer solution is selected from any one or a combination of at least two of Tris buffer, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer, ACES buffer, sodium acetate buffer, MES buffer, sodium citrate-citric acid, PBS, Good's buffer, or glycine buffer; the pH of the buffer solution is 5.5 - 7.5.

[0021] In an alternative embodiment, the thickener includes any one or a combination of at least two of methylcellulose, hydroxypropyl methylcellulose, polyvinylpyrrolidone, hydroxyethyl cellulose, sodium starch phosphate, propylene glycol alginate, or carboxymethyl cellulose.

[0022] In an alternative embodiment, the protective agent includes any one or a combination of at least two of sucrose, mannitol, bovine serum albumin, gelatin, or trehalose.

[0023] In an alternative embodiment, the metal inorganic salt includes any one or a combination of at least two of sodium chloride, magnesium chloride, and calcium chloride.

[0024] Among them, the mass concentration of cholesterol oxidase in the complex enzyme solution is 2% - 12%, the mass concentration of cholesterol esterase is 0.5% - 4%, the mass concentration of peroxidase is 0.2 - 2%, and the mass concentration of the electron mediator is 0.5% - 5%.

[0025] The mass concentration of the surfactant is 0.3% - 1.5%. Among them, the mass concentration of surfactant 1 is 0.1% - 0.5%, and the mass concentration of surfactant 2 is 0.2% - 1%.

[0026] In an alternative embodiment, the mass concentration of the thickener is 0.1% - 3%, the mass concentration of the protective agent is 0.2% - 5%, and the mass concentration of the metal inorganic salt is 0.01 - 2.0%.

[0027] In a second aspect, the present invention provides a test strip for total cholesterol detection, which includes the complex enzyme solution for total cholesterol detection according to any one of the foregoing embodiments.

[0028] In a third aspect, the present invention provides a detection system for total cholesterol detection, which includes the test strip for total cholesterol detection according to the foregoing embodiment; a determination component that is connected to the test strip for total cholesterol detection and measures the electrochemistry reaction current value; and a calculation and display component that calculates and displays the total cholesterol content in the sample to be tested based on the total cholesterol concentration-current value standard curve; the test strip for total cholesterol detection is used to receive the sample to be tested and generate an electrical signal through an electrochemical reaction in the reaction area under the action of an excitation potential.

[0029] Compared with the prior art, the present invention has at least the following beneficial effects: (1) The present invention does not perform pre-treatment on the sample, does not set a blood filtration membrane, and directly measures whole blood, which can effectively reduce the sample volume. Only 0.8 - 2 µL is required to measure total cholesterol, having the advantage of small blood collection volume, and at the same time further shortening the detection time; (2) By adding a surfactant composition meeting the conditions to the composite enzyme solution of the present invention, it only takes 15 - 25 s to measure total cholesterol, achieving faster, more accurate, and more stable detection of total cholesterol; Description of the Drawings

[0030] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention, and thus should not be regarded as limiting the scope. For those of ordinary skill in the art, other related drawings can be obtained based on these drawings without creative efforts.

[0031] Figure 1 It is the standard curve of total cholesterol concentration - current value. Embodiment

[0032] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Those not specified in the embodiments are carried out according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments not specified by the manufacturer are all conventional products that can be obtained through commercial purchase.

[0033] The embodiments of the present invention provide a composite enzyme solution for detecting total cholesterol. The composite enzyme solution includes cholesterol oxidase, cholesterol esterase, peroxidase, electron mediator, surfactant composition, protective agent, thickening agent, metal inorganic salt, and buffer solution.

[0034] Based on the fact that the transfer rate of electrons is proportional to the total cholesterol concentration in the embodiments of the present invention, the total cholesterol concentration in the sample to be measured can be obtained, achieving the goal of detecting total cholesterol using the principle of electrochemistry, which is conducive to industrial promotion and application.

[0035] Specifically, the complex enzyme solution can cause the sample to be tested, such as a blood sample, to generate free cholesterol and fatty acids from the total cholesterol therein under the action of cholesterol esterase during detection; the free fatty acids generated during the reaction are catalyzed by cholesterol oxidase to generate cholest-4-en-3-one and hydrogen peroxide under the conditions of water and oxygen. The hydrogen peroxide and the reduced electron mediator generate an oxidized electron mediator under the action of peroxidase (POD). Under suitable voltage conditions, the oxidized electron mediator undergoes a reduction reaction on the surface of the electrode plate through the excitation potential, and the absolute value of the microcurrent generated during the process is positively correlated with the concentration of total cholesterol. Subsequently, the total cholesterol can be detected quickly and accurately through electrochemistry.

[0036] Among them, the mass concentration of the cholesterol esterase in the complex enzyme solution is 0.5% - 4.0%, for example, the mass concentration is any value between 0.5% and 4% such as 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5% and 4.0%, or a range value formed by any two values.

[0037] Among them, the mass concentration of the cholesterol oxidase in the complex enzyme solution is 2.0% - 12.0%, for example, the mass concentration is any value between 2.0% and 12.0% such as 2.0%, 3.0%, 4.0%, 5.0%, 6.0%, 7.0%, 8.0%, 9.0%, 10.0%, 11.0% and 12.0%, or a range value formed by any two values.

[0038] Among them, the mass concentration of the peroxidase in the complex enzyme solution is 0.2% - 2.0%, for example, the mass concentration is any value between 0.2% and 2.0% such as 0.20%, 0.30%, 0.40%, 0.50%, 0.60%, 0.70%, 0.80%, 0.90%, 1.00% and 2.0%, or a range value formed by any two values.

[0039] The electron mediator is a reduced electron mediator. Among them, the reduced electron mediator includes any one of potassium ferrocyanide, ferrocene derivatives, osmium complexes, and ruthenium complexes. Preferably, the electron mediator includes any one of potassium ferrocyanide, (ferrocenylmethyl) trimethylammonium chloride, and bis[3-trimethyl-aminopropyl] ferrocene dichloride. Among them, the mass concentration of the electron mediator in the complex enzyme solution is 0.5% - 5.0%, for example, the mass concentration is any value between 0.5% and 5.0% such as 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5% and 5.0%, or a range value formed by any two values.

[0040] Among them, the surfactant composition in the complex enzyme solution includes surfactant 1 and surfactant 2.

[0041] The surfactant 1 includes one or at least two combinations of surfactants with an HLB > 23.

[0042] The surfactant 2 includes one or at least two combinations of surfactants with an HLB of 12 - 14.

[0043] The surfactant 1 includes any one or at least two combinations of Pluronic F68, Pluronic F77, Pluronic F87, Pluronic F88, Pluronic F98, Pluronic F108, Tetronic 1107, and Tetronic 1307.

[0044] Preferably, the surfactant 1 includes Tetronic 1107 or / and Pluronic F88.

[0045] The surfactant 2 includes any one or at least two combinations of Triton X - 100, Surfynol 465, Creamophpr EL, Oleth - 10, and CHEMAL LA9.

[0046] Preferably, the surfactant 2 includes Triton X - 100 and / or CHEMAL LA9.

[0047] The mass concentration of the surfactant is 0.3% - 1.5%.

[0048] Among them, the mass concentration of the surfactant 1 is 0.1% - 0.5%. It should be noted that since the surfactant 1 includes one or at least two surfactants, its mass concentration is the total mass concentration of all surfactants in the surfactant 1. For example, the mass concentration is any value between 0.1% - 0.5% such as 0.10%, 0.15%, 0.20%, 0.25%, 0.30%, 0.35%, 0.40%, 0.45%, and 0.50%, or a range value formed by any at least two values.

[0049] Among them, the mass concentration of the surfactant 2 is 0.2% - 1.0%. It should be noted that since the surfactant 2 includes one or at least two surfactants, its mass concentration is the total mass concentration of all surfactants in the surfactant 2. For example, the mass concentration is any value between 0.2% and 1.0% such as 0.20%, 0.25%, 0.30%, 0.35%, 0.40%, 0.45%, 0.50%, 0.55%, 0.60%, 0.65%, 0.70%, 0.75%, 0.80%, 0.85%, 0.90%, 0.95% and 1.00%, or a range value formed by any at least two values.

[0050] Among them, the mass concentration of the protective agent in the complex enzyme solution is 0.2% - 5.0%. For example, the mass concentration is any value between 0.2% and 5.0% such as 0.2%, 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5% and 5.0%, or a range value formed by any two values.

[0051] Among them, the mass concentration of the thickener in the complex enzyme solution is 0.1% - 3.0%. For example, the mass concentration is any value between 0.1% and 3.0% such as 0.1%, 0.5%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9% and 3.0%, or a range value formed by any two values.

[0052] Among them, the mass concentration of the metal inorganic salt in the complex enzyme solution is 0.01 - 2.0%. For example, the mass concentration is any value between 0.01% and 2.0% such as 0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0% and 2.0%, or a range value formed by any two values.

[0053] Among them, the buffer solution is selected from any one or a combination of at least two of Tris buffer solution, 4 - (2 - hydroxyethyl)piperazine - 1 - ethanesulfonic acid buffer solution, ACES buffer solution, sodium acetate buffer solution, MES buffer solution, sodium citrate - citric acid, PBS, Good’s buffer solution or glycine buffer solution; and the pH of the buffer solution is 5.5 - 7.5. For example, it can be any value between 5.5 and 7.5 such as 5.5, 6, 6.5, 7 and 7.5, or a range value formed by any two values.

[0054] Since the solvent in the complex enzyme solution, except for the buffer solution, will not have a great impact on the pH of the mixed system after other materials are dissolved, the pH of the complex enzyme solution is also 5.5 - 7.5. For example, it can be any value between 5.5 and 7.5 such as 5.5, 6, 6.5, 7, and 7.5, or a range value formed by any two of these values.

[0055] Specifically, the thickener includes any one or a combination of at least two of methylcellulose, hydroxypropyl methylcellulose, hydroxyethyl cellulose, polyvinylpyrrolidone, sodium starch phosphate, propylene glycol alginate, or carboxymethyl cellulose.

[0056] The protective agent includes any one or a combination of at least two of sucrose, mannitol, bovine serum albumin, gelatin, or trehalose.

[0057] The metal inorganic salt includes any one or a combination of at least two of calcium chloride, magnesium chloride, and sodium chloride.

[0058] It should be noted that the thickener, protective agent, and surfactant used in the embodiments of the present invention are collectively referred to as non-reactive raw materials. Even though they have not participated in the reaction, they all have their different functions. Specifically as follows: The thickener can make the components in the complex enzyme solution evenly distributed. Especially during the drying process of preparing the test strip for total cholesterol electrochemical detection, the thickener can make the enzyme layer formed by the complex enzyme solution more uniform during the covering and drying processes. The protective agent can protect the activity of various enzymes in the complex enzyme solution and enhance the stability of the complex enzyme solution. Especially during the drying process of preparing the test strip for total cholesterol electrochemical detection, the protective agent can protect the enzymes to maintain their activity for a longer time under dry conditions, thereby enhancing the stability of the test strip for long-term storage. The role of the surfactant is to reduce the interfacial tension between the solid and the liquid, increase the adsorption between the solid and the liquid, and make the enzyme layer formed by the complex enzyme solution evenly dispersed on the electrode surface; at the same time, when the blood sample reacts with the enzyme solution on the test strip, the surfactant helps to dissolve lipoproteins. The purpose of adding the surfactant composition is to make different lipoproteins in the blood sample quickly dissolve within a short time, mix fully with the enzyme solution to react, and detect total cholesterol.

[0059] In the second aspect, the present invention provides a test strip for total cholesterol detection, which is prepared by using the complex enzyme solution for total cholesterol detection according to any one of the foregoing embodiments. Among them, the reaction area of the test strip is formed by adding the complex enzyme solution provided by the embodiments of the present invention and drying. According to the working principle and process of the complex enzyme solution provided by the embodiments of the present invention, the test strip for total cholesterol detection provided by the embodiments of the present invention requires a small amount of blood collection.

[0060] Specifically, the reaction that occurs when the test strip is detected is as follows:

[0061] In a third aspect, the present invention provides a detection system for total cholesterol detection, which includes the test strip for total cholesterol detection described in the foregoing embodiment; a determination component connected to the test strip for total cholesterol detection and measuring the value of the electrochemical reaction current; and a calculation and display component that calculates and displays the total cholesterol content in the sample to be tested according to the total cholesterol concentration-current value standard curve; the test strip for total cholesterol detection is used to receive the sample to be tested and generate an electrical signal through an electrochemical reaction in the reaction area under the action of an excitation potential.

[0062] Ultimately, the present invention uses the oxidation reaction of free cholesterol to generate hydrogen peroxide, and then generates oxidized electron mediators through the oxidation reaction of hydrogen peroxide and reduced electron mediators. Under appropriate voltage conditions, the oxidized electron mediators undergo a reduction reaction on the surface of the electrode plate through the excitation potential, and the absolute value of the microcurrent generated during the process is positively correlated with the concentration of total cholesterol.

[0063] The test strip provided by the embodiment of the present invention can efficiently detect total cholesterol, and has the advantages of small blood collection volume, fast detection speed, high sensitivity, high accuracy and good stability.

[0064] The embodiment of the present invention also provides a preparation method for a test strip for total cholesterol detection, including the following steps: (a) Take an insulating substrate, and then use methods such as screen mask sputtering / electroplating or laser etching to attach a conductive thin layer on the insulating substrate as the electrode layer, that is, form an electrode group, which includes a working electrode, a reference electrode, a liquid flow-in place electrode and a connection electrode.

[0065] The present invention does not make specific requirements for the positions of the respective electrodes. Those skilled in the art can make selections according to the actual situation on the basis of meeting the reaction principle of the electrochemical total cholesterol detection method.

[0066] (b) Cover a layer of enzyme solution on the electrode layer of the working electrode by the method of dropping liquid, control the loading amount to be 0.5-3.0 mg, and dry it.

[0067] (c) After the above drying operation is completed, cover the electrode layer with a hydrophilic film layer with a reagent window opened at one end by using double-sided tape or glue, so that the reagent window and the hydrophilic film layer above it together form a siphon pool capable of sucking in the sample to be tested. Finally, after pressing and cutting, the obtained electrochemical total cholesterol test strip is stored in a sealed plastic cylinder with a molecular sieve desiccant or a single-piece aluminum foil bag.

[0068] The embodiment of the present invention also provides a usage method for the above-mentioned test strip for total cholesterol detection, including the following content: Insert the exposed part of the detection test strip for the electrochemical detection of total cholesterol into the jack of the detection system. Drop 0.8 - 2.0 μL of the sample to be tested, such as blood sample, and preferably the added amount is 1.7 μL, from the inlet of the siphon pool of the detection test strip. It flows into the reaction zone through siphon action, and a voltage is applied to carry out an electrochemical reaction. During the detection process, the sample to be tested generates free cholesterol and fatty acids under the action of cholesterol esterase; the free cholesterol generated during the reaction process is oxidized by cholesterol oxidase, and hydrogen peroxide is generated under the action of water and oxygen. The oxidation reaction of hydrogen peroxide with the reduced electron mediator generates the oxidized electron mediator. Under suitable voltage conditions, the oxidized electron mediator undergoes a reduction reaction on the surface of the electrode plate through the excitation potential, and the absolute value of the microcurrent generated during the process is positively correlated with the concentration of total cholesterol. At this time, an excitation potential is applied, and the sample and the complex enzyme solution undergo an electrochemical reaction to generate a current value. By calculating through the standard curve, the total cholesterol content in the sample to be tested can be obtained.

[0069] Among them, the excitation potential is -50 to -300 mV. For example, it can be any value between -50 mV and -300 mV such as -50 mV, -100 mV, -105 mV, -110 mV, -150 mV, -180 mV, -200 mV, -250 mV, and -300 mV, or a range value formed by any two values.

[0070] To meet the conditions of surfactant 1 and surfactant 2 in the surfactant composition, surfactant 1 takes any one or a combination of at least two of Pluronic F68 with an HLB value of 29, Pluronic F77 with an HLB value of 25, Pluronic F88 with an HLB value of 28, Pluronic F108 with an HLB value of 27, Tetronic 1107 with an HLB value of 24, and Tetronic 1307 with an HLB value > 24 as examples. Surfactant 2 takes TritonX - 100 with an HLB value of 13.5, Surfynol 465 with an HLB value of 13, Creamophpr EL with an HLB value of 12 - 14, Oleth - 10 with an HLB value of 12.4, and CHEMAL LA9 with an HLB value of 13.3 as examples. At the same time, surfactants with an HLB value of around 16 - 18 are more commonly used in the prior art for the determination of total cholesterol. Therefore, Brij - 35 with an HLB value of 16.9 and Tritox - 305 with an HLB value of 17.3 are selected as controls to verify the effect of the complex enzyme solution of the present invention.

[0071] The features and properties of the present invention will be further described in detail below in conjunction with the embodiments.

[0072] Example 1: Preparation of the complex enzyme solution for total cholesterol detection First, prepare a PBS buffer solution with a concentration of 0.02 mol / L and a pH of 7.4 as the solvent; then, weigh 0.50% of hydroxypropyl cellulose, 2.0% of trehalose, 0.90% of sodium chloride, 1.0% of potassium ferrocyanide, 0.5% of Pluronic F88, and 0.5% of CHEMAL LA9 by mass percentage and mix them until completely dissolved; then, weigh 7.0% of cholesterol oxidase, 1.0% of cholesterol esterase, and 0.2% of peroxidase by mass percentage and mix them until completely dissolved. Mix the above two solutions and stir until completely dissolved to obtain an enzyme solution.

[0073] Example 2: Preparation of a composite enzyme solution for total cholesterol detection First, prepare a PBS buffer solution with a concentration of 0.02 mol / L and a pH of 7.4 as the solvent; then, weigh 0.50% of hydroxypropyl cellulose, 2.0% of trehalose, 0.90% of sodium chloride, 1.0% of potassium ferrocyanide, 0.3% of Tetronic 1107, and 0.2% of CHEMAL LA9 by mass percentage and mix them until completely dissolved; then, weigh 7.0% of cholesterol oxidase, 1.0% of cholesterol esterase, and 0.2% of peroxidase by mass percentage and mix them until completely dissolved. Mix the above two solutions and stir until completely dissolved to obtain an enzyme solution.

[0074] Example 3: Preparation of a composite enzyme solution for total cholesterol detection First, prepare a PBS buffer solution with a concentration of 0.02 mol / L and a pH of 7.4 as the solvent; then, weigh 0.50% of hydroxypropyl cellulose, 2.0% of trehalose, 0.90% of sodium chloride, 1.0% of potassium ferrocyanide, 0.3% of Tetronic 1107, 0.2% of Pluronic F88, and 0.2% of CHEMAL LA9 by mass percentage and mix them until completely dissolved; then, weigh 7.0% of cholesterol oxidase, 1% of cholesterol esterase, and 0.2% of peroxidase by mass percentage and mix them until completely dissolved. Mix the above two solutions and stir until completely dissolved to obtain an enzyme solution.

[0075] Example 4: Preparation of a composite enzyme solution for total cholesterol detection First, prepare a PBS buffer solution with a concentration of 0.02 mol / L and a pH of 7.4 as the solvent; then, weigh 0.50% of hydroxypropyl cellulose, 2.0% of trehalose, 0.90% of sodium chloride, 1.0% of potassium ferrocyanide, 0.05% of Tetronic 1107, 0.05% of Pluronic F88, and 0.5% of Surfynol 465 by mass percentage, and mix them until completely dissolved; then, weigh 7.0% of cholesterol oxidase, 1.0% of cholesterol esterase, and 0.2% of peroxidase by mass percentage, and mix them until completely dissolved. Mix the above two solutions and stir until completely dissolved to obtain the enzyme solution.

[0076] Example 5: Preparation of a Composite Enzyme Solution for Total Cholesterol Detection First, prepare a PBS buffer solution with a concentration of 0.02 mol / L and a pH of 7.4 as the solvent; then, weigh 0.50% of hydroxypropyl cellulose, 2.0% of trehalose, 0.90% of sodium chloride, 1.0% of potassium ferrocyanide, 0.3% of Tetronic 1107, 0.2% of Pluronic F88, 0.5% of CHEMAL LA9, and 0.5% of TritonX-100 by mass percentage, and mix them until completely dissolved; then, weigh 7.0% of cholesterol oxidase, 1.0% of cholesterol esterase, and 0.2% of peroxidase by mass percentage, and mix them until completely dissolved. Mix the above two solutions and stir until completely dissolved to obtain the enzyme solution.

[0077] Example 6: Preparation of a Composite Enzyme Solution for Total Cholesterol Detection First, prepare a PBS buffer solution with a concentration of 0.02 mol / L and a pH of 7.4 as the solvent; then, weigh 0.50% of hydroxypropyl cellulose, 2.0% of trehalose, 0.90% of sodium chloride, 1.0% of potassium ferrocyanide, 0.5% of Tetronic 1107, and 0.5% of TritonX-100 by mass percentage, and mix them until completely dissolved; then, weigh 7.0% of cholesterol oxidase, 1.0% of cholesterol esterase, and 0.2% of peroxidase by mass percentage, and mix them until completely dissolved. Mix the above two solutions and stir until completely dissolved to obtain the enzyme solution.

[0078] Example 7: Preparation of a Composite Enzyme Solution for Total Cholesterol Detection First, prepare a PBS buffer solution with a concentration of 0.02 mol / L and a pH of 7.4 as the solvent; then, weigh 0.50% of hydroxypropyl cellulose, 2.0% of trehalose, 0.90% of sodium chloride, 1.0% of potassium ferrocyanide, 0.3% of Tetronic 1107, 0.2% of Pluronic F88, and 0.5% of TritonX-100 by mass percentage, and mix them until completely dissolved; then, weigh 7.0% of cholesterol oxidase, 1.0% of cholesterol esterase, and 0.2% of peroxidase by mass percentage, and mix them until completely dissolved. Mix the above two solutions and stir until completely dissolved to obtain the enzyme solution.

[0079] Example 8: Preparation of a Composite Enzyme Solution for Total Cholesterol Detection First, prepare a citric acid-sodium citrate buffer solution with a concentration of 0.04 mol / L and a pH of 5.5 as the solvent; then, weigh 0.1% of methylcellulose, 0.2% of trehalose, 0.01% of calcium chloride, 0.5% of potassium ferrocyanide, 0.5% of PluronicF108, and 0.5% of Oleth-10 by mass percentage, and mix them until completely dissolved; then, weigh 2.0% of cholesterol oxidase, 0.5% of cholesterol esterase, and 0.5% of peroxidase by mass percentage, and mix them until completely dissolved. Mix the above two solutions and stir until completely dissolved to obtain the enzyme solution.

[0080] Example 9: Preparation of a Composite Enzyme Solution for Total Cholesterol Detection First, prepare a MES buffer solution with a concentration of 0.06 mol / L and a pH of 5.8 as the solvent; then, weigh 0.50% of methylcellulose, 1.0% of sucrose, 0.5% of magnesium chloride, 1.5% of (ferrocenylmethyl) trimethylammonium chloride, 0.5% of Pluronic F77, and 0.5% of Creamophpr EL by mass percentage, and mix them until completely dissolved; then, weigh 3.0% of cholesterol oxidase, 1.5% of cholesterol esterase, and 0.8% of peroxidase by mass percentage, and mix them until completely dissolved. Mix the above two solutions and stir until completely dissolved to obtain the enzyme solution.

[0081] Example 10: Preparation of a Composite Enzyme Solution for Total Cholesterol Detection First, prepare an ACES buffer solution with a concentration of 0.08 mol / L and a pH of 6.0 as the solvent; then, weigh 1.00% of polyvinylpyrrolidone, 3.0% of mannitol, 1.0% of sodium chloride, 2.0% of potassium ferrocyanide, 0.5% of Pluronic F68, and 0.5% of Surfynol 465 by mass percentage, and mix them until completely dissolved; then, weigh 5.0% of cholesterol oxidase, 2.0% of cholesterol esterase, and 1.0% of peroxidase by mass percentage, and mix them until completely dissolved. Mix the above two solutions and stir until completely dissolved to obtain the enzyme solution.

[0082] Example 11: Preparation of Composite Enzyme Solution for Total Cholesterol Detection First, prepare 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer solution with a concentration of 0.09 mol / L and a pH of 6.5 as the solvent; then, weigh 2.0% of hydroxypropyl cellulose, 4% of bovine serum albumin, 1.5% of sodium chloride, 3.0% of bis[3-(trimethylammonio)propyl]ferrocene dichloride, 0.5% of Tetronic 1307, and 0.5% of TritonX-100 according to mass percentage, and mix them until completely dissolved; then, weigh 9.0% of cholesterol oxidase, 3.0% of cholesterol esterase, and 1.5% of peroxidase according to mass percentage, and mix them until completely dissolved. Mix the above two solutions and stir until completely dissolved to obtain the enzyme solution.

[0083] Example 12: Preparation of Composite Enzyme Solution for Total Cholesterol Detection First, prepare Tris buffer solution with a concentration of 0.15 mol / L and a pH of 7.5 as the solvent; then, weigh 5.0% of carboxymethyl cellulose, 5.0% of gelatin, 2.0% of sodium chloride, 5.0% of potassium ferrocyanide, 0.5% of Pluronic F88, and 0.5% of CHEMAL LA9 according to mass percentage, and mix them until completely dissolved; then, weigh 12.0% of cholesterol oxidase, 4.0% of cholesterol esterase, and 2.0% of peroxidase according to mass percentage, and mix them until completely dissolved. Mix the above two solutions and stir until completely dissolved to obtain the enzyme solution.

[0084] Comparative Examples 1 - 11: Preparation of Composite Enzyme Solution for Total Cholesterol Detection The preparation methods provided in Comparative Examples 1 - 11 are the same as those in Example 1, except for the different material ratios, which are specifically as follows: Comparative Example 1: Except that the surfactant composition in the composite enzyme solution is adjusted from 0.5% of Pluronic F88 and 0.5% of CHEMAL LA9 to 0.5% of Tetronic 1107, the rest are the same as those in Example 1.

[0085] Comparative Example 2: Except that the surfactant composition in the composite enzyme solution is adjusted from 0.5% of Pluronic F88 and 0.5% of CHEMAL LA9 to 0.5% of Pluronic F88, the rest are the same as those in Example 1.

[0086] Comparative Example 3: Except that the surfactant composition in the composite enzyme solution is adjusted from 0.5% of Pluronic F88 and 0.5% of CHEMAL LA9 to 0.5% of CHEMAL LA9, the rest are the same as those in Example 1.

[0087] Comparative Example 4: Except that the surfactant composition in the complex enzyme solution was adjusted from 0.5% Pluronic F88 and 0.5% CHEMAL LA to 0.5% TritonX-100, the rest was the same as in Example 1.

[0088] Comparative Example 5: Except that the surfactant composition in the complex enzyme solution was adjusted from 0.5% Pluronic F88 and 0.5% CHEMAL LA to 0.3% Tetronic 1107 and 0.2% Pluronic F88, the rest was the same as in Example 1.

[0089] Comparative Example 6: Except that the surfactant composition in the complex enzyme solution was adjusted from 0.5% Pluronic F88 and 0.5% CHEMAL LA to 0.5% Brij-35, 0.3% Tetronic 1107 and 0.2% Pluronic F88, the rest was the same as in Example 1.

[0090] Comparative Example 7: Except that the surfactant composition in the complex enzyme solution was adjusted from 0.5% Pluronic F88 and 0.5% CHEMAL LA to 0.5% TritonX-305 and 0.5% CHEMAL LA9, the rest was the same as in Example 1.

[0091] Comparative Example 8: Except that the surfactant composition in the complex enzyme solution was adjusted from 0.5% Pluronic F88 and 0.5% CHEMAL LA to no surfactant, the rest was the same as in Example 1.

[0092] Comparative Example 9: Except that the surfactant composition in the complex enzyme solution was adjusted from 0.5% Pluronic F88, 0.5% CHEMAL LA to 0.3% Tetronic 1107, 0.5% Pluronic F88 and 0.2% CHEMAL LA9, the rest was the same as in Example 1.

[0093] Comparative Example 10: Except that the surfactant composition in the complex enzyme solution was adjusted from 0.5% Pluronic F88, 0.5% CHEMAL LA to 0.3% Tetronic 1107, 1.0% Pluronic F88 and 0.2% CHEMAL LA9, the rest was the same as in Example 1.

[0094] Comparative Example 11: Except that the surfactant composition in the complex enzyme solution was adjusted from 0.5% Pluronic F88 and 0.5% CHEMAL LA9 to 0.3% Tetronic 1107, 0.2% Pluronic F88, 0.5% CHEMAL LA9, and 1.0% TritonX-100, the rest was the same as in Example 1.

[0095] Application Example 1: Preparation of a test strip for total cholesterol detection Specifically as follows: (a) Take an insulating substrate, and then use the screen mask sputtering method to attach a conductive thin layer on the insulating substrate as the electrode layer.

[0096] (b) Cover a layer of the complex enzyme solution of Example 1 on the electrode layer of the working electrode by the dotting method, control the loading amount to be 2 mg, and dry it.

[0097] (c) After the above drying operation is completed, use double-sided tape to cover a hydrophilic film layer with a reagent window at one end on the electrode layer, so that the reagent window and the hydrophilic film layer above it together form a siphon pool capable of sucking in the test sample. Finally, after pressing and cutting, the obtained electrochemical total cholesterol test strip is stored in a sealed plastic cylinder with a molecular sieve desiccant or a single-piece aluminum foil bag.

[0098] Application Examples 2-12: Preparation of a test strip for total cholesterol detection In Application Examples 2-12, except that the complex enzyme solution was replaced with Examples 2-12 respectively, the rest was the same as in Application Example 1. The preparation method was referred to Application Example 1.

[0099] Application Comparative Examples 1-11: Preparation of a test strip for total cholesterol detection In Application Comparative Examples 1-11, except that the complex enzyme solution was replaced with Comparative Examples 1-11 respectively, the rest was the same as in Application Example 1. The preparation method was referred to Application Example 1.

[0100] The usage method of the above test strip for total cholesterol detection includes the following content: Insert the exposed part of the total cholesterol test strip electrode into the socket of the detection system, drop 1.7 μL of the test sample from the siphon pool inlet of the test strip, and flow into the reaction area through siphon action. Under the action of an excitation potential of -50 mV to -300 mV, after measuring the current value at the measurement site, the value of the calculation and display component that calculates and displays the total cholesterol content in the test sample according to the total cholesterol concentration-current value standard curve is the total cholesterol content in the test sample.

[0101] Now, according to the above usage method of the total cholesterol test strip, the following performance tests are carried out on the above application examples and application comparative examples: (1) Obtaining the total cholesterol concentration-current value standard curve Using the venous whole blood of normal people, different amounts of cholesterol stock solution were added to prepare 9 cholesterol whole blood samples with different concentrations. The theoretical concentrations were: 2.4 mmol / L, 3.0 mmol / L, 4.2 mmol / L, 5.4 mmol / L, 6.4 mmol / L, 7.4 mmol / L, 8.6 mmol / L, 9.7 mmol / L, 10.6 mmol / L; then, according to the aforementioned method of using the electrochemical cholesterol test strip, the excitation potential was controlled at -220 mV, and the current responses of 9 cholesterol whole blood samples with different concentrations were measured respectively. The current value at a reaction time of 25 s was taken, and each whole blood sample was measured in parallel 10 times and the average value was taken to judge the repeatability, relative sensitivity and linear range. The original data of the relevant tests in Application Example 1 are listed in Table 1, and the cholesterol concentration was used as the abscissa and the current value as the ordinate, and the Figure 1 .

[0102] Table 1 Concentration measured by biochemical analyzer / mmol / L 2.4 3.0 4.2 5.4 6.4 7.4 8.6 9.7 10.6 Current value 1 / nA -900.8 -1097.8 -1078.3 -1521.8 -1860.6 -2260.2 -2220.8 -2680.8 -2861.2 Current value 2 / nA -900.0 -998.3 -1165.0 -1489.6 -1849.4 -2195.3 -2357.3 -2721.8 -2848.8 Current value 3 / nA -932.0 -1050.1 -1102.2 -1509.8 -1881.8 -2229.8 -2264.0 -2773.4 -2622.0 Current value 4 / nA -951.0 -1092.6 -1166.4 -1525.0 -1855.2 -2298.2 -2153.0 -2690.6 -2901.3 Current value 5 / nA -910.0 -1060.3 -1144.4 -1494.2 -1832.8 -2295.0 -2165.2 -2774.6 -2849.8 Current value 6 / nA -890.3 -1003.8 -1177.4 -1503.2 -1969.0 -2190.0 -2275.3 -2669.6 -2704.0 Current value 7 / nA -857.6 -1050.2 -1176.2 -1586.6 -1855.4 -2128.8 -2334.0 -2669.8 -2798.2 Current value 8 / nA -914.0 -1012.2 -1189.4 -1542.6 -1830.8 -2173.4 -2287.2 -2704.8 -2906.8 Current value 9 / nA -865.4 -1035.8 -1154.2 -1456.0 -1873.6 -2127.6 -2286.8 -2670.8 -2779.8 Current value 10 / nA -884.4 -1056.7 -1193.2 -1536.4 -1783.0 -2227.4 -2325.2 -2726.2 -2876.5 AVG -900.6 -1045.8 -1154.7 -1516.5 -1859.2 -2212.6 -2266.9 -2708.2 -2814.8 SD 28.4 34.2 37.4 35.3 47.4 61.0 68.6 40.3 91.4 CV -3.2% -3.3% -3.2% -2.3% -2.5% -2.8% -3.0% -1.5% -3.2% A correlation analysis was performed on the detection results of this application and the detection results obtained by the method. The results are shown in Figure 1 . According to Figure 1 and Table 1, a calibration curve was established based on the average value of the total cholesterol theoretical concentration and the measured current value. The square of the correlation coefficient (R 2 ) was 0.9842, with good fitting degree, which could be used as the total cholesterol concentration-current value standard curve, and the CV of the obtained current value was below 3.3%, and the detection response time only needed 25 s.

[0103] (2) Specifically testing cholesterol samples with different concentrations Randomly select 12 blood samples with different concentrations for testing, and then evaluate them by comparing with the detection results of the biochemical analyzer. The detection results and linear correlation of Application Example 1 are shown in Table 2.

[0104] Table 2 Sample number Concentration of biochemical analyzer / mmol / L Current value in Application Example 1 / nA Total cholesterol concentration / mmol / L Relative deviation 1 2.8 -967.1 2.9 3.7% 2 3.2 -1089.7 3.4 5.4% 3 3.7 -1132.7 3.5 -3.9% 4 4.2 -1336.0 4.4 4.5% 5 4.9 -1501.7 5.0 2.1% 6 5.7 -1608.8 5.4 -3.9% 7 6.2 -1857.0 6.4 4.1% 8 6.8 -1903.2 6.6 -2.7% 9 7.7 -2100.7 7.4 -3.8% 10 8.3 -2232.1 7.9 -4.4% 11 8.9 -2580.0 9.3 4.3% 12 9.8 -2780.3 10.1 3.3% For the sake of simplicity, only the calculated results of Application Examples 2-10 and Application Comparative Examples 1-10 are given, as shown in Table 3.

[0105] Table 3 Current sensitivity (nA / mmol) <![CDATA[R 2 > Maximum CV value at each concentration Absolute value of the maximum deviation among 12 samples Application Example 1 -243.4 0.9842 3.3% 5.4% Application Example 2 -224.6 0.9656 4.6% 6.9% Application Example 3 -186.3 0.9693 5.1% 7.7% Application Example 4 -190.1 0.9672 3.9% 6.6% Application Example 5 -247.4 0.9686 4.9% 9.3% Application Example 6 -178.5 0.9631 2.3% 7.5% Application Example 7 -219.2 0.9639 3.5% 6.8% Application Example 8 -202.3 0.9839 4.7% 5.3% Application Example 9 -196.0 0.9557 3.0% 8.5% Application Example 10 -235.1 0.9582 4.1% 9.8% Application Example 11 -212.9 0.9554 3.2% 8.0% Application Example 12 -223.1 0.9601 4.6% 7.2% Application Comparative Example 1 -225.1 0.7559 8.5% 32.8% Application Comparative Example 2 -212.6 0.7583 6.8% 27.6% Application Comparative Example 3 -231.2 0.8129 4.8% 19.6% Application Comparative Example 4 -212 0.7975 7.2% 17.3% Application Comparative Example 5 -180.8 0.8863 14.8% 17.4% Application Comparative Example 6 -190.2 0.7424 6.7% 30.3% Application Comparative Example 7 -142 0.8771 6.0% 16.4% Application Comparative Example 8 -166.6 0.5772 8.8% 28.8% Application Comparative Example 9 -227 0.8489 3.8% 15.4% Application Comparative Example 10 -216 0.8262 9.7% 24.3% Application Comparative Example 11 -180.7 0.7977 8.8% 29.5% The following points can be obtained from Tables 1 to 2: (1)As can be seen from Comprehensive Application Examples 1 to 7, regardless of whether Surfactant 1 in the surfactant composition is Tetronic 1107 or Pluronic F88 or Tetronic 1107 and Pluronic F88, and Surfactant 2 is CHEMAL LA9 or TritonX-100 or Surfynol 465 or CHEMAL LA9 and TritonX-100, a good linear relationship is exhibited during the electrochemical detection process. Among them, the square of the correlation coefficient of linear fitting is greater than 0.96, and the CV of the current value repeated 10 times is within 5.1%, and the absolute value of the maximum deviation in the sample is within 9.3%. It can be seen that under the condition that Surfactant 1 conforms to HLB > 23 and Surfactant 2 conforms to HLB 12 - 14, the surfactant composition is not affected by the quantity of the surfactant; (2)As can be seen from Comprehensive Application Examples 1 to 12, the reaction enzyme solution for total cholesterol electrochemical detection provided by the present invention exhibits a good linear relationship during the electrochemical detection process. Among them, the square of the correlation coefficient of linear fitting is greater than 0.95, and the CV of the current value repeated 10 times is within 5.1%, and the absolute value of the maximum deviation in the sample is within 9.8%. Therefore, the enzyme solution within the mass concentration range described in the present invention can achieve sensitive and accurate detection of cholesterol; (3)As can be seen from Comprehensive Application Example 6 and Application Comparative Examples 1 and 4, in Application Example 6, the surfactant composition is 0.5% Tetronic 1107 and 0.5% TritonX-100, and in Application Comparative Examples 1 and 4, the surfactants are 0.5% Tetronic1107 and 0.5% TritonX-100 respectively. Compared with Application Example 6, the correlation coefficients of linear fitting in Application Comparative Examples 1 and 4 both decrease, and the deviation values also increase significantly, indicating a decrease in the accuracy of the test and a reduction in repeatability; As can be seen from Comprehensive Application Example 1 and Application Comparative Examples 2 to 3, in Application Example 1, the surfactant composition is 0.5% Pluronic F88 and 0.5% CHEMAL LA9, and in Application Comparative Examples 2 to 3, the surfactants are 0.5% Pluronic F88 and 0.5% CHEMAL LA9 respectively. Compared with Application Example 1, the correlation coefficients of linear fitting in Application Comparative Examples 2 to 3 both decrease, and the deviation values also increase significantly, indicating a decrease in the accuracy of the test and poor repeatability. The main reason is that the surfactants in Application Comparative Examples 1 to 4 are single components. Although they conform to the characteristics of Surfactant 1 or Surfactant 2, they do not satisfy the conditions of both Surfactant 1 and Surfactant 2 at the same time, resulting in a decrease in accuracy. From this, it is shown that by adding the surfactant composition that meets the conditions to the enzyme solution components, the present invention significantly improves the accuracy of the detection; From the comprehensive application example 2 and application comparative example 5, it can be seen that in application example 2, the surfactant composition is 0.3% Tetronic 1107 and 0.2% CHEMAL LA9, and in application comparative example 5, the surfactant composition is 0.3% Tetronic 1107 and 0.2% Pluronic F88. Compared with application example 2, the correlation coefficient of the linear fitting in application comparative example 5 decreases, and the deviation value also increases significantly, indicating a decrease in the accuracy of the test and poor repeatability. The main reason is that although two surfactants are added in application comparative example 5, the HLB values of both surfactants are > 23, and the conditions for surfactant 2 in the surfactant composition are not satisfied simultaneously; (5)From the comprehensive application example 7 and application comparative example 6, it can be seen that in application example 7, the surfactant composition is 0.3% Tetronic 1107, 0.2% Pluronic F88, and 0.5% TritonX-100, and in application comparative example 6, the surfactant composition is 0.3% Tetronic 1107, 0.2% Pluronic F88, and 0.5% Brij-35. Compared with application example 7, in application comparative example 6, the surfactant Brij-35 with an HLB value of 16.9 is selected as a control to replace the surfactant component TritonX-100, and other conditions remain unchanged. However, the correlation coefficient of the linear fitting in application comparative example 6 decreases significantly, and the deviation value also increases; From the comprehensive application example 1 and application comparative example 7, it can be seen that in application example 1, the surfactant composition is 0.5% Pluronic F88 and 0.5% CHEMAL LA9, and in application comparative example 7, the surfactants are 0.5% CHEMAL LA9 and 0.5% TritonX-305 respectively. Compared with application example 1, in application comparative example 7, the surfactant TritonX-305 with an HLB value of 17.3 is selected as a control to replace the surfactant component Pluronic F88, and other conditions remain unchanged. Similarly, in application comparative example 7, the correlation coefficient of the linear fitting decreases, and the deviation value increases. It can be seen that even if a surfactant composition is added, even if application comparative example 6 meets the condition that the HLB value of the surfactant is > 23 and application comparative example 7 meets the condition that the HLB value of the surfactant is between 12 - 14, the conditions that the HLB of surfactant 1 > 23 and the HLB of surfactant 2 is 12 - 14 are not satisfied simultaneously. The accuracy and repeatability of detecting total cholesterol are not as good as the results measured by the complex enzyme solution containing a surfactant composition that meets the conditions simultaneously.

[0106] (5)It can be seen from the comprehensive application example 3 and application comparative examples 9 - 10 that in application example 3, the surfactant composition is 0.3% Tetronic 1107, 0.2% Pluronic F88, and 0.2% CHEMAL LA9; in application comparative example 9, the surfactant composition is 0.3% Tetronic 1107, 0.5% Pluronic F88, and 0.2% CHEMAL LA9; in application comparative example 10, the surfactant composition is 0.3% Tetronic 1107, 1% Pluronic F88, and 0.2% CHEMAL LA9. Compared with application example 3, application comparative examples 9 and 10 adjusted the addition amount of Pluronic F88, making it exceed the range of 0.1% - 0.5% of the mass concentration of surfactant 1 in the surfactant composition; it can be seen from the comprehensive application example 5 and application comparative example 11 that in application example 5, the surfactant composition is 0.3% Tetronic 1107, 0.2% Pluronic F88, 0.5% CHEMAL LA9, and 0.5% TritonX - 100; in application comparative example 11, the surfactant composition is 0.3% Tetronic 1107, 0.2% CHEMAL LA9, and 1% TritonX - 100. Compared with application example 5, application comparative example 11 adjusted the addition amount of TritonX - 100, making it exceed the range of 0.2% - 1% of the mass concentration of surfactant 2 in the surfactant composition. According to the test results, the correlation coefficients of the linear fitting of application comparative examples 9 - 11 all decreased, and the deviation values increased, indicating that the test accuracy decreased and the repeatability was poor. It can be seen that the addition amount of the surfactant needs to satisfy that the mass concentration of surfactant 1 is 0.1% - 0.5% and the mass concentration of surfactant 2 is 0.2% - 1%. Adding too much, too little, or not adding the surfactant, such as in application comparative example 8, will cause the total cholesterol not to react fully with the complex enzyme solution and cannot achieve the best effect. Therefore, through the synergistic combination of surfactants, the present invention not only needs to satisfy the condition that the HLB of surfactant 1 > 23 and the HLB is 12 - 14, but also the addition amount needs to satisfy that the mass concentration of surfactant 1 is 0.1% - 0.5% and the mass concentration of surfactant 2 is 0.2% - 1% to finally achieve the efficient detection of total electrochemical cholesterol.

[0107] In summary, the solution of the present application has the advantages of small blood collection volume, fast detection speed, high accuracy, and good stability, and the test result can be obtained only in 15s - 25s of detection time.

[0108] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various modifications and changes. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

[0109] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0110] In addition, it should be noted that, among the various specific technical features described in the above specific embodiments, without conflict, they can be combined in any appropriate way. To avoid unnecessary repetition, the present invention will not separately describe various possible combination methods.

[0111] Furthermore, any combination can be made among various different embodiments of the present invention, as long as it does not violate the idea of the present invention, and it should also be regarded as the content disclosed by the present invention.

Claims

1. A composite enzyme solution for total cholesterol detection, characterized in that, it comprises cholesterol oxidase, cholesterol esterase, peroxidase, an electron mediator, and a surfactant composition, wherein the surfactant composition includes surfactant 1 and surfactant 2, surfactant 1 is selected from one or a combination of at least two surfactants with HLB > 23, and surfactant 2 is selected from one or a combination of at least two surfactants with HLB of 12 - 14.

2. The composite enzyme solution for total cholesterol detection according to claim 1, characterized in that, in the composite enzyme solution, the mass concentration of cholesterol oxidase is 2% - 12%, the mass concentration of cholesterol esterase is 0.5% - 4%, the mass concentration of peroxidase is 0.2% - 2%, the mass concentration of the electron mediator is 0.5% - 5%, the mass concentration of surfactant 1 is 0.1% - 0.5%, and the mass concentration of surfactant 2 is 0.2% - 1%.

3. The composite enzyme solution for total cholesterol detection according to claim 1, characterized in that, the electron mediator is a reduced electron mediator, and the reduced electron mediator includes any one of potassium ferrocyanide, ferrocene derivatives, osmium complexes, and ruthenium complexes.

4. The composite enzyme solution for total cholesterol detection according to claim 3, characterized in that, the ferrocene derivative includes any one of (ferrocenylmethyl) trimethylammonium chloride and bis[3 - trimethyl - aminopropyl] ferrocene dichloride.

5. The composite enzyme solution for total cholesterol detection according to claim 1, characterized in that, surfactant 1 includes any one or a combination of at least two of Pluronic F68, Pluronic F77, Pluronic F87, Pluronic F88, Pluronic F98, Pluronic F108, Tetronic 1107, and Tetronic 1307.

6. The composite enzyme solution for total cholesterol detection according to claim 1, characterized in that, surfactant 2 includes any one or a combination of at least two of Triton X - 100, Surfynol 465, Creamophpr EL, Oleth - 10, and CHEMAL LA9.

7. The composite enzyme solution for total cholesterol detection according to claim 1, characterized in that, the composite enzyme solution further includes a buffer solution, a thickener, a protective agent, and metal inorganic salts, the mass concentration of the thickener is 0.1% - 3%, the mass concentration of the protective agent is 0.2% - 5%, and the mass concentration of the metal inorganic salts is 0.01 - 2.0%.

8. The composite enzyme solution for total cholesterol detection according to claim 7, characterized in that, The buffer solution includes any one or a combination of at least two of Tris buffer solution, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer solution, ACES buffer solution, sodium acetate buffer solution, MES buffer solution, sodium citrate-citric acid, PBS, Good's buffer solution or glycine buffer solution; the pH of the buffer solution is 5.5 - 7.

5.

9. The complex enzyme solution for total cholesterol detection according to claim 7, characterized in that the thickener includes any one or a combination of at least two of methylcellulose, hydroxypropyl methylcellulose, polyvinylpyrrolidone, hydroxyethyl cellulose, sodium starch phosphate, propylene glycol alginate or carboxymethyl cellulose.

10. The complex enzyme solution for total cholesterol detection according to claim 7, characterized in that the protective agent includes any one or a combination of at least two of sucrose, mannitol, bovine serum albumin, gelatin or trehalose.

11. The complex enzyme solution for total cholesterol detection according to claim 7, characterized in that the metal inorganic salt includes any one or a combination of at least two of sodium chloride, magnesium chloride, calcium chloride.

12. A total cholesterol detection test strip, characterized in that the test strip includes the complex enzyme solution for total cholesterol detection according to any one of claims 1 - 11, and the complex enzyme solution is loaded on the electrode layer of the test strip.

13. A detection system for total cholesterol detection, characterized in that the detection system includes the total cholesterol detection test strip according to claim 12.

Images