Composite enzyme solution for total cholesterol detection, test paper and detection system

Abstract

The present invention relates to the technical field of biochemistry, in particular to a composite enzyme solution for total cholesterol detection, a test paper and a detection system. The composite enzyme solution for total cholesterol detection comprises cholesterol oxidase, cholesterol esterase, peroxidase, an electron mediator, a surfactant composition, a thickening agent, a protective agent, a metal inorganic salt and a buffer solution, wherein the surfactant composition comprises one of or a combination of at least two of surfactants with an HLB>23 and one of or a combination of at least two of surfactants with an HLB of 12-14. The composite enzyme solution can be used for detecting total cholesterol by means of an electrochemical method, exhibiting fast detection speed, high sensitivity, and high accuracy.

Description

Total cholesterol detection complex enzyme solution, test strips and detection system Technical Field

[0001] The present application relates to the field of biochemical technology, and in particular to a total cholesterol detection complex enzyme solution, a detection test strip, and a detection system. Background Art

[0002] Cholesterol, also known as cholesterol, is a derivative of cyclopentaphenanthrene. Cholesterol is the primary steroid compound in mammals and plays an important role in basic cellular life. 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 the synthesis of physiologically active substances such as adrenocortical hormones, sex hormones, bile acids, and vitamin D. It is also a major component of cell membranes. However, high cholesterol concentrations in the blood significantly increase the risk of arterial disease. Most cases of coronary heart disease, hypertension, atherosclerosis, and lipid metabolism dysfunction are caused by high serum cholesterol.

[0003] Therefore, cholesterol, as an important indicator in clinical blood lipid testing, 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 colorimetry.

[0005] Among them, electrochemical testing is widely used in the field of point-of-care testing (POCT). Electrochemical testing selectively recognizes the target analyte in the sample to be tested by modifying the biological molecules (such as enzymes) on the electrode surface, and converts the biological recognition signal into an electrical signal (oxidation or reduction current) that can be collected and measured, thereby achieving quantitative detection of the target analyte.

[0006] In the process of quantitative detection of total cholesterol using electrochemical methods, total cholesterol exists in the blood in the form of lipoproteins, including chylomicrons, very low-density lipoproteins (VLDL), low-density lipoproteins (LDL) and high-density lipoproteins (HDL). The cholesterol contents of these lipoproteins are called VLDL-cholesterol (VLDL-C), LDL-cholesterol (LDL-C) and HDL-cholesterol (HDL-C), respectively.

[0007] In order for the cholesterol in lipoproteins to fully participate in the reaction, the lipoproteins must first be broken down to release the cholesterol. Surfactants are usually used to promote the solubility of lipids, but surfactants act on different lipoprotein parts at different rates. To ensure that all analytes present in the sample are detected, the surfactant needs to react with the sample long enough to release all the cholesterol, which results in a longer time for the determination of total cholesterol.

[0008] Therefore, there is a need to improve existing formulations to achieve rapid and accurate determination of total cholesterol.

[0009] Summary of the Invention

[0010] The following is a summary of the subject matter described in detail herein. This summary is not intended to limit the scope of the claims.

[0011] The present application provides a complex enzyme solution, a test strip, and a detection system for total cholesterol detection. The complex enzyme solution provided in the embodiments of the present application can detect total cholesterol using electrochemistry, with fast detection speed, high sensitivity, and high accuracy.

[0012] In a first aspect, the present application provides a complex enzyme solution for total cholesterol detection, wherein the complex enzyme solution comprises cholesterol oxidase, cholesterol esterase, peroxidase and an electron mediator surfactant composition.

[0013] The surfactant composition comprises surfactant 1 and surfactant 2.

[0014] The surfactant 1 includes any one or a combination of at least two surfactants having a hydrophilic-lipophilic balance (HLB) value greater than 23.

[0015] The surfactant 2 includes any one or a combination of at least two surfactants having an HLB of 12-14.

[0016] The surfactant 1 includes any one of Pluronic F68, Pluronic F77, Pluronic F87, Pluronic F88, Pluronic F98, Pluronic F108, Tetronic 1107 or Tetronic 1307, or a combination of at least two of them.

[0017] The surfactant 2 includes any one of TritonX-100, Surfynol 465, Creamophr EL, Oleth-10 or CHEMAL LA9, or a combination of at least two thereof.

[0018] The electron mediator is a reduced electron mediator.

[0019] Wherein, the reduced electron mediator includes any one of potassium ferrocyanide, ferrocene derivatives, osmium complexes or ruthenium complexes.

[0020] Preferably, the ferrocene derivative includes any one of (ferrocenylmethyl)trimethylammonium chloride or bis[3-trimethyl-aminopropyl]ferrocene dichloride.

[0021] In an optional embodiment, the complex enzyme solution further includes a buffer solution, a thickener, a protective agent and a metal inorganic salt.

[0022] In an optional embodiment, the buffer solution is selected from any one or a combination of at least two of Tris buffer, 4-hydroxyethylpiperazineethanesulfonic acid buffer, ACES buffer, sodium acetate buffer, MES buffer, sodium citrate-citric acid buffer, PBS buffer, Good's buffer or glycine buffer; the pH of the buffer solution is 5.5-7.5.

[0023] In an optional embodiment, the thickener includes any one of methyl cellulose, hydroxypropyl methyl cellulose, polyvinyl pyrrolidone, hydroxyethyl cellulose, sodium starch phosphate, propylene glycol alginate or carboxymethyl cellulose, or a combination of at least two thereof.

[0024] In an optional embodiment, the protective agent includes any one of sucrose, mannitol, bovine serum albumin, gelatin or trehalose, or a combination of at least two of them.

[0025] In an optional embodiment, the metal inorganic salt includes any one of sodium chloride, magnesium chloride or calcium chloride, or a combination of at least two of them.

[0026] Wherein, the mass concentration of the cholesterol oxidase in the complex enzyme solution is 2%-12%, the mass concentration of the cholesterol esterase is 0.5%-4%, the mass concentration of the peroxidase is 0.2%-2%, and the mass concentration of the electron mediator is 0.5%-5%.

[0027] The mass concentration of the surfactant is 0.3%-1.5%; wherein the mass concentration of the surfactant 1 is 0.1%-0.5%, and the mass concentration of the surfactant 2 is 0.2%-1%.

[0028] In an optional 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%.

[0029] In a second aspect, the present application provides a test strip for total cholesterol detection, which includes the complex enzyme solution for total cholesterol detection described in any one of the aforementioned embodiments.

[0030] In a third aspect, the present application provides a detection system for total cholesterol detection, which includes the test paper for total cholesterol detection as described in the aforementioned embodiment; a measuring component connected to the test paper for total cholesterol detection and measuring the current value of the electrochemical reaction; 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 paper for total cholesterol detection is used to receive the sample to be tested and to generate an electrochemical reaction in the reaction area under the action of the excitation potential to form an electrical signal.

[0031] Compared with the related art, this application has at least the following beneficial effects:

[0032] (1) This application does not pre-treat the sample, does not set up a blood filter membrane, and directly measures whole blood, which can effectively reduce the sample volume. Only 0.8-2 μL is needed to measure total cholesterol, which has the advantage of a small blood volume and further shortens the detection time; and

[0033] (2) By adding a qualified surfactant composition to the complex enzyme solution of the present application, total cholesterol can be measured in only 15-25 seconds, achieving faster, more accurate and more stable total cholesterol detection.

[0034] Still other aspects will become apparent upon reading and understanding the accompanying drawings and detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following is a brief introduction to the drawings required for use in the embodiments. It should be understood that the following drawings only show certain embodiments of the present application and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without creative work.

[0036] Figure 1 is a standard curve of total cholesterol concentration-current value. DETAILED DESCRIPTION

[0037] To make the purpose, technical solutions and advantages of the examples of the present application clearer, the technical solutions in the examples of the present application will be described clearly and completely below. Where specific conditions are not specified in the examples, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0038] In a first aspect, an embodiment of the present application provides a complex enzyme solution for total cholesterol detection, wherein the complex enzyme solution includes cholesterol oxidase, cholesterol esterase, peroxidase, an electron mediator, a surfactant composition, a protective agent, a thickener, a metal inorganic salt and a buffer solution.

[0039] The embodiment of the present application is based on the fact that the electron transfer rate is proportional to the total cholesterol concentration, and the total cholesterol concentration in the sample to be tested can be obtained, thereby achieving the goal of detecting total cholesterol using the principle of electrochemistry, which is conducive to industrial promotion and application.

[0040] Specifically, the complex enzyme solution can convert the total cholesterol in a sample to be tested, such as a blood sample, into free cholesterol and fatty acids under the action of cholesterol esterase. The free fatty acids produced during the reaction are catalyzed by cholesterol oxidase to produce cholester-4-en-3-one and hydrogen peroxide in the presence of water and oxygen. Hydrogen peroxide reacts with a reduced electron mediator under the action of peroxidase (POD) to produce an oxidized electron mediator. Under appropriate voltage conditions, the oxidized electron mediator undergoes a reduction reaction on the electrode plate surface through the excitation potential. The absolute value of the microcurrent generated during the process is positively correlated with the total cholesterol concentration, enabling rapid and accurate electrochemical detection of total cholesterol.

[0041] Wherein, 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%-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.

[0042] Wherein, the mass concentration of the cholesterol oxidase in the complex enzyme solution is 2.0%-12.0%, for example, the mass concentration is 2.0%, 3.0%, 4.0%, 5.0%, 6.0%, 7.0%, 8.0%, 9.0%, 10.0%, 11.0% and 12.0%, or any value between 2.0% and 12.0%, or a range value formed by any two values.

[0043] 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 formed by any two values.

[0044] The electron mediator is a reduced electron mediator; wherein the reduced electron mediator includes any one of potassium ferrocyanide, ferrocene derivatives, osmium complexes or ruthenium complexes; preferably, the electron mediator includes any one of potassium ferrocyanide, (ferrocenylmethyl)trimethylammonium chloride or bis[3-trimethyl-aminopropyl]ferrocene dichloride; wherein 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%-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.

[0045] Wherein, the surfactant composition in the complex enzyme solution includes surfactant 1 and surfactant 2.

[0046] The surfactant 1 includes one or a combination of at least two surfactants with HLB>23.

[0047] The surfactant 2 includes one or a combination of at least two surfactants having an HLB of 12-14.

[0048] The surfactant 1 includes any one of Pluronic F68, Pluronic F77, Pluronic F87, Pluronic F88, Pluronic F98, Pluronic F108, Tetronic 1107 or Tetronic 1307, or a combination of at least two of them.

[0049] Preferably, the surfactant 1 includes Tetronic 1107 and / or Pluronic F88.

[0050] The surfactant 2 includes any one of TritonX-100, Surfynol 465, Creamophr EL, Oleth-10 or CHEMAL LA9, or a combination of at least two thereof.

[0051] Preferably, the surfactant 2 includes TritonX-100 and / or CHEMAL LA9.

[0052] The mass concentration of the surfactant is 0.3%-1.5%.

[0053] 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% and 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.

[0054] Wherein, 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 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%, etc., any value between 0.2% and 1.0%, or a range value formed by any at least two values.

[0055] 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 formed by any two values.

[0056] Wherein, the mass concentration of the thickener in the complex enzyme solution is 0.1%-3.0%, for example, the mass concentration is 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 any value between 0.1% and 3.0%, or a range value formed by any two values.

[0057] Wherein, the mass concentration of the metal inorganic salt in the complex enzyme solution is 0.01%-2.0%, for example, the mass concentration is 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 any value between 0.01% and 2.0%, or a range formed by any two values.

[0058] The buffer solution is selected from any one or a combination of at least two of Tris buffer, 4-hydroxyethylpiperazineethanesulfonic acid buffer, ACES buffer, sodium acetate buffer, MES buffer, sodium citrate-citric acid buffer, PBS buffer, Good's buffer or glycine buffer; and the pH of the buffer solution is 5.5-7.5, for example, it can be any value between 5.5-7.5, such as 5.5, 6, 6.5, 7 and 7.5, or a range value formed by any two values.

[0059] Since the solvent in the complex enzyme solution, except for the buffer solution, other materials will not have much impact on the pH of the mixed system after dissolution, the pH of the complex enzyme solution is also 5.5-7.5, for example, it can be any value between 5.5-7.5, such as 5.5, 6, 6.5, 7 and 7.5, or a range value formed by any two values.

[0060] Specifically, the thickener includes any one of methyl cellulose, hydroxypropyl methyl cellulose, hydroxyethyl cellulose, polyvinyl pyrrolidone, sodium starch phosphate, propylene glycol alginate or carboxymethyl cellulose, or a combination of at least two thereof.

[0061] The protective agent includes any one of sucrose, mannitol, bovine serum albumin, gelatin or trehalose, or a combination of at least two of them.

[0062] The metal inorganic salt includes any one of calcium chloride, magnesium chloride or sodium chloride, or a combination of at least two of them.

[0063] It should be noted that the thickener, protective agent and surfactant used in the embodiments of the present application are collectively referred to as non-reactive raw materials. Even if they have not participated in the reaction, they all have different effects, as follows: the thickener can make the distribution of the components in the composite enzyme solution uniform and consistent, especially in the drying process of the test paper for electrochemical detection of total cholesterol, the thickener can make the enzyme layer formed by the composite enzyme solution more uniform during the covering and drying process; the protective agent can protect the activity of various enzymes in the composite enzyme solution and enhance the stability of the composite enzyme solution, especially in the drying process of the test paper for electrochemical detection of total cholesterol, the protective agent can protect the enzyme from maintaining activity for a longer time under dry conditions, thereby enhancing the stability of the test paper for long-term storage; and the role of the surfactant is to reduce the interfacial tension between the solid and the liquid, increase adsorption between the solid and the liquid, so that the enzyme layer formed by the composite enzyme solution is evenly dispersed on the electrode surface, and when the blood sample reacts with the enzyme solution on the test paper, the surfactant helps to dissolve lipoproteins. The purpose of adding the surfactant composition is to make different lipoproteins in the blood sample quickly dissolve in a relatively short time, fully react with the enzyme solution and detect total cholesterol.

[0064] In a second aspect, embodiments of the present application provide a test strip for total cholesterol detection, which is prepared using the complex enzyme solution for total cholesterol detection described in any of the aforementioned embodiments. The reaction zone of the test strip is formed by adding the complex enzyme solution provided in embodiments of the present application and drying it. Based on the working principle and process of the complex enzyme solution provided in embodiments of the present application, the test strip for total cholesterol detection provided in embodiments of the present application requires a small amount of blood.

[0065] Specifically, the reaction of the test strip during testing is as follows:

[0066] In a third aspect, an embodiment of the present application provides a detection system for total cholesterol detection, which includes the detection test paper for total cholesterol detection described in the aforementioned embodiment; a measuring component connected to the total cholesterol detection test paper and measuring the current value of the electrochemical reaction; 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 total cholesterol detection test paper is used to receive the sample to be tested and to generate an electrochemical reaction in the reaction area under the action of the excitation potential to form an electrical signal.

[0067] This application ultimately uses the oxidation reaction of free cholesterol to generate hydrogen peroxide, and then generates an oxidized electron mediator through the oxidation reaction of hydrogen peroxide and a reduced electron mediator. Under appropriate voltage conditions, the oxidized electron mediator undergoes a reduction reaction on the surface of the electrode plate through the excitation potential. The absolute value of the microcurrent generated in the process is positively correlated with the concentration of total cholesterol.

[0068] The test strips provided in the embodiments of the present application can efficiently detect total cholesterol and have the advantages of small blood collection volume, fast detection speed, high sensitivity, high accuracy and good stability.

[0069] The present invention also provides a method for preparing a total cholesterol test strip, comprising the following steps:

[0070] (a) An insulating substrate is prepared and then a conductive thin layer is attached to the insulating substrate as an electrode layer by sputtering / electroplating with a screen mask or laser etching, thereby forming an electrode assembly comprising a working electrode, a reference electrode, a flow-in electrode, and a contact electrode;

[0071] This application does not make specific requirements for the position of each electrode. Those skilled in the art can select according to actual conditions based on the reaction principle of the electrochemical total cholesterol detection method.

[0072] (b) coating the electrode layer of the working electrode with a layer of enzyme solution by dotting, controlling the loading amount to 0.5-3.0 mg, and drying; and

[0073] (c) After the drying operation is completed, the hydrophilic film layer with a reagent window at one end is covered on the electrode layer using double-sided tape or glue, so that the hydrophilic film layer at the reagent window and the hydrophilic film layer above the electrode layer together form a siphon pool capable of absorbing the sample to be tested. Finally, after lamination and cutting, the obtained electrochemical total cholesterol test paper is placed in a sealed plastic tube with a molecular sieve desiccant or a single piece of aluminum foil bag.

[0074] The present application also provides a method for using the above-mentioned test paper for total cholesterol detection, including the following contents:

[0075] Insert the exposed portion of the electrode of the test strip for electrochemical detection of total cholesterol into the socket of the detection system, and drip the sample to be tested, such as 0.8-2.0 μL of blood sample, preferably 1.7 μL, from the inlet of the siphon pool of the test strip. The sample flows into the reaction zone by siphon action, and a voltage is applied to perform 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 is oxidized by cholesterol oxidase and generates hydrogen peroxide under the action of water and oxygen. The oxidation reaction of hydrogen peroxide with the reduced electron mediator generates an oxidized electron mediator. Under appropriate voltage conditions, the oxidized electron mediator undergoes a reduction reaction on the surface of the electrode plate through the excitation potential. 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 undergoes an electrochemical reaction with the complex enzyme solution to generate a current value. The total cholesterol content in the sample to be tested can be obtained by calculation through the standard curve.

[0076] Among them, the excitation potential is -50 to -300mV, for example, it can be any value between -50 and -300mV, such as -50mV, -100mV, -105mV, -110mV, -150mV, -180mV, -200mV, -250mV and -300mV, or a range value formed by any two values.

[0077] In order to meet the conditions of surfactant 1 and surfactant 2 in the surfactant composition, surfactant 1 is exemplified by 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, or Tetronic 1307 with an HLB value greater than 24; surfactant 2 is exemplified by Triton X-100 with an HLB value of 13.5, Surfynol 465 with an HLB value of 13, Creamophr EL with an HLB value of 12-14, Oleth-10 with an HLB value of 12.4, or CHEMAL with an HLB value of 13.3. Taking any one or a combination of at least two of LA9 as an example, and in the related art, surfactants with an HLB value of about 16-18 are mostly used to determine total cholesterol, so 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 application.

[0078] The features and performance of the present application are further described in detail below with reference to the embodiments.

[0079] Example 1: Preparation of complex enzyme solution for total cholesterol detection

[0080] First, a PBS buffer solution with a concentration of 0.02 mol / L and a pH of 7.4 was prepared as a solvent; then, 0.50% hydroxypropyl cellulose, 2.0% trehalose, 0.90% sodium chloride, 1.0% potassium ferrocyanide, 0.5% Pluronic F88, and 0.5% CHEMAL LA9 were weighed according to mass percentage and mixed until completely dissolved; then, 7.0% cholesterol oxidase, 1.0% cholesterol esterase, and 0.2% peroxidase were weighed according to mass percentage and mixed until completely dissolved; and the above two were mixed and stirred until completely dissolved to obtain an enzyme solution.

[0081] Example 2: Preparation of complex enzyme solution for total cholesterol detection

[0082] First, a PBS buffer solution with a concentration of 0.02 mol / L and a pH of 7.4 was prepared as a solvent; then, 0.50% hydroxypropyl cellulose, 2.0% trehalose, 0.90% sodium chloride, 1.0% potassium ferrocyanide, 0.3% Tetronic 1107, and 0.2% CHEMAL LA9 were weighed according to mass percentage and mixed until completely dissolved; then, 7.0% cholesterol oxidase, 1.0% cholesterol esterase, and 0.2% peroxidase were weighed according to mass percentage and mixed until completely dissolved; and the above two were mixed and stirred until completely dissolved to obtain an enzyme solution.

[0083] Example 3: Preparation of complex enzyme solution for total cholesterol detection

[0084] First, a PBS buffer solution with a concentration of 0.02 mol / L and a pH of 7.4 was prepared as a solvent; then, 0.50% hydroxypropyl cellulose, 2.0% trehalose, 0.90% sodium chloride, 1.0% potassium ferrocyanide, 0.3% Tetronic 1107, 0.2% Pluronic F88, and 0.2% CHEMAL LA9 were weighed according to mass percentage and mixed until completely dissolved; then, 7.0% cholesterol oxidase, 1% cholesterol esterase, and 0.2% peroxidase were weighed according to mass percentage and mixed until completely dissolved; and the above two were mixed and stirred until completely dissolved to obtain an enzyme solution.

[0085] Example 4: Preparation of complex enzyme solution for total cholesterol detection

[0086] First, a PBS buffer solution with a concentration of 0.02 mol / L and a pH of 7.4 was prepared as a solvent; then, 0.50% hydroxypropyl cellulose, 2.0% trehalose, 0.90% sodium chloride, 1.0% potassium ferrocyanide, 0.05% Tetronic 1107, 0.05% Pluronic F88, and 0.5% Surfynol 465 were weighed according to mass percentage and mixed until completely dissolved; then, 7.0% cholesterol oxidase, 1.0% cholesterol esterase, and 0.2% peroxidase were weighed according to mass percentage and mixed until completely dissolved; and the above two were mixed and stirred until completely dissolved to obtain an enzyme solution.

[0087] Example 5: Preparation of complex enzyme solution for total cholesterol detection

[0088] First, a PBS buffer solution with a concentration of 0.02 mol / L and a pH of 7.4 was prepared as a solvent; then, 0.50% hydroxypropyl cellulose, 2.0% trehalose, 0.90% sodium chloride, 1.0% potassium ferrocyanide, 0.3% Tetronic 1107, 0.2% Pluronic F88, 0.5% CHEMAL LA9, and 0.5% Triton X-100 were weighed according to mass percentage and mixed until completely dissolved; then, 7.0% cholesterol oxidase, 1.0% cholesterol esterase, and 0.2% peroxidase were weighed according to mass percentage and mixed until completely dissolved; and the above two were mixed and stirred until completely dissolved to obtain an enzyme solution.

[0089] Example 6: Preparation of complex enzyme solution for total cholesterol detection

[0090] First, a PBS buffer solution with a concentration of 0.02 mol / L and a pH of 7.4 was prepared as a solvent; then, 0.50% hydroxypropyl cellulose, 2.0% trehalose, 0.90% sodium chloride, 1.0% potassium ferrocyanide, 0.5% Tetronic 1107, and 0.5% Triton X-100 were weighed according to mass percentage and mixed until completely dissolved; then, 7.0% cholesterol oxidase, 1.0% cholesterol esterase, and 0.2% peroxidase were weighed according to mass percentage and mixed until completely dissolved; and the above two were mixed and stirred until completely dissolved to obtain an enzyme solution.

[0091] Example 7: Preparation of complex enzyme solution for total cholesterol detection

[0092] First, a PBS buffer solution with a concentration of 0.02 mol / L and a pH of 7.4 was prepared as a solvent; then, 0.50% hydroxypropyl cellulose, 2.0% trehalose, 0.90% sodium chloride, 1.0% potassium ferrocyanide, 0.3% Tetronic 1107, 0.2% Pluronic F88, and 0.5% Triton X-100 were weighed according to mass percentage and mixed until completely dissolved; then, 7.0% cholesterol oxidase, 1.0% cholesterol esterase, and 0.2% peroxidase were weighed according to mass percentage and mixed until completely dissolved; and the above two were mixed and stirred until completely dissolved to obtain an enzyme solution.

[0093] Example 8: Preparation of complex enzyme solution for total cholesterol detection

[0094] First, a citric acid-sodium citrate buffer solution with a concentration of 0.04 mol / L and a pH of 5.5 is prepared as a solvent; then, 0.1% methylcellulose, 0.2% trehalose, 0.01% calcium chloride, 0.5% potassium ferrocyanide, 0.5% Pluronic F108, and 0.5% Oleth-10 are weighed according to mass percentage and mixed until completely dissolved; 2.0% cholesterol oxidase, 0.5% cholesterol esterase, and 0.5% peroxidase are weighed according to mass percentage and mixed until completely dissolved; the above two are mixed and stirred until completely dissolved to obtain an enzyme solution.

[0095] Example 9: Preparation of complex enzyme solution for total cholesterol detection

[0096] First, prepare MES buffer with a concentration of 0.06 mol / L and a pH of 5.8 as a solvent; then weigh 0.50% methylcellulose, 1.0% sucrose, 0.50% magnesium chloride, 1.5% (ferrocenylmethyl)trimethylammonium chloride, 0.5% Pluronic F77, and 0.5% Creamophr EL according to mass percentage and mix until completely dissolved; then weigh 3.0% cholesterol oxidase, 1.5% cholesterol esterase, and 0.8% peroxidase according to mass percentage and mix until completely dissolved; and then stir the above two ingredients until completely dissolved to obtain an enzyme solution.

[0097] Example 10: Preparation of complex enzyme solution for total cholesterol detection

[0098] First, an ACES buffer solution with a concentration of 0.08 mol / L and a pH of 6.0 was prepared as a solvent; then, 1.00% polyvinyl pyrrolidone, 3.0% mannitol, 1.0% sodium chloride, 2.0% potassium ferrocyanide, 0.5% Pluronic F68, and 0.5% Surfynol 465 were weighed according to mass percentage and mixed until completely dissolved; 5.0% cholesterol oxidase, 2.0% cholesterol esterase, and 1.0% peroxidase were weighed according to mass percentage and mixed until completely dissolved; and the above two were mixed and stirred until completely dissolved to obtain an enzyme solution.

[0099] Example 11: Preparation of complex enzyme solution for total cholesterol detection

[0100] First, a 4-hydroxyethylpiperazineethanesulfonic acid buffer solution with a concentration of 0.09 mol / L and a pH of 6.5 is prepared as a solvent; then, 2.0% hydroxypropyl cellulose, 4% bovine serum albumin, 1.5% sodium chloride, 3.0% bis[3-trimethylaminopropyl]ferrocene dichloride, 0.5% Tetronic 1307, and 0.5% Triton X-100 are weighed according to mass percentage and mixed until completely dissolved; then, 9.0% cholesterol oxidase, 3.0% cholesterol esterase, and 1.5% peroxidase are weighed according to mass percentage and mixed until completely dissolved; and the above two are mixed and stirred until completely dissolved to obtain an enzyme solution.

[0101] Example 12: Preparation of complex enzyme solution for total cholesterol detection

[0102] First, a Tris buffer solution with a concentration of 0.15 mol / L and a pH of 7.5 was prepared as a solvent; then, 5.0% carboxymethyl cellulose, 5.0% gelatin, 2.0% sodium chloride, 5.0% potassium ferrocyanide, 0.5% Pluronic F88, and 0.5% CHEMAL LA9 were weighed according to mass percentage and mixed until completely dissolved; then, 12.0% cholesterol oxidase, 4.0% cholesterol esterase, and 2.0% peroxidase were weighed according to mass percentage and mixed until completely dissolved; and the above two were mixed and stirred until completely dissolved to obtain an enzyme solution.

[0103] Comparative Example 1-11: Preparation of complex enzyme solution for total cholesterol detection

[0104] The preparation methods provided in Comparative Examples 1-11 are the same as those in Example 1, except that the material ratios are different, as follows:

[0105] Comparative Example 1: The process was the same as that of Example 1 except that the surfactant composition in the complex enzyme solution was adjusted from 0.5% of Pluronic F88 and 0.5% of CHEMAL LA9 to 0.5% of Tetronic 1107.

[0106] Comparative Example 2: The process was the same as that of Example 1 except that the surfactant composition in the complex enzyme solution was adjusted from Pluronic F88 0.5% and CHEMAL LA9 0.5% to Pluronic F88 0.5%.

[0107] Comparative Example 3: The process was the same as that of Example 1 except that the surfactant composition in the complex enzyme solution was adjusted from Pluronic F88 0.5% and CHEMAL LA9 0.5% to CHEMAL LA9 0.5%.

[0108] Comparative Example 4: Except that the surfactant composition in the complex enzyme solution was adjusted from Pluronic F88 0.5% and CHEMAL LA9 0.5% to Triton X-100 0.5%, the rest was the same as Example 1.

[0109] Comparative Example 5: The same as Example 1 except that the surfactant composition in the complex enzyme solution was adjusted from Pluronic F88 0.5% and CHEMAL LA9 0.5% to Tetronic 1107 0.3% and Pluronic F88 0.2%.

[0110] Comparative Example 6: Except that the surfactant composition in the complex enzyme solution was adjusted from Pluronic F88 0.5% and CHEMAL LA9 0.5% to Brij-35 0.5%, Tetronic 1107 0.3% and Pluronic F88 0.2%, the rest was the same as Example 1.

[0111] Comparative Example 7: Except that the surfactant composition in the complex enzyme solution was adjusted from Pluronic F88 0.5% and CHEMAL LA9 0.5% to Triton X-305 0.5% and CHEMAL LA9 0.5%, the rest was the same as Example 1.

[0112] Comparative Example 8: Except that the surfactant composition in the complex enzyme solution was adjusted from Pluronic F88 0.5% and CHEMAL LA9 0.5% to no surfactant, the rest was the same as Example 1.

[0113] Comparative Example 9: Except that the surfactant composition in the complex enzyme solution was adjusted from Pluronic F88 0.5% and CHEMAL LA9 0.5% to Tetronic 1107 0.3%, Pluronic F88 0.5% and CHEMAL LA9 0.2%, the rest was the same as Example 1.

[0114] Comparative Example 10: Except that the surfactant composition in the complex enzyme solution was adjusted from Pluronic F88 0.5% and CHEMAL LA9 0.5% to Tetronic 1107 0.3%, Pluronic F88 1.0% and CHEMAL LA9 0.2%, the rest was the same as Example 1.

[0115] Comparative Example 11: Except that the surfactant composition in the complex enzyme solution was adjusted from Pluronic F88 0.5% and CHEMAL LA9 0.5% to Tetronic 1107 0.3%, Pluronic F88 0.2%, CHEMAL LA9 0.5% and TritonX-100 1.0%, the rest was the same as Example 1.

[0116] Application Example 1: Preparation of Test Strips for Total Cholesterol Detection

[0117] The details are as follows:

[0118] (a) An insulating substrate is prepared and then a conductive thin layer is deposited on the insulating substrate as an electrode layer using a screen mask sputtering method;

[0119] (b) coating the electrode layer of the working electrode with a layer of the complex enzyme solution of Example 1 by dot-coating, controlling the loading amount to 2 mg, and drying; and

[0120] (c) After the drying operation is completed, the hydrophilic film layer with a reagent window at one end is covered on the electrode layer using double-sided tape, so that the hydrophilic film layer at the reagent window and the hydrophilic film layer above the electrode layer together form a siphon pool capable of absorbing the sample to be tested. Finally, after lamination and cutting, the obtained electrochemical total cholesterol test paper is placed in a sealed plastic tube with a molecular sieve desiccant or a single piece of aluminum foil bag.

[0121] Application Example 2-12: Preparation of Test Strips for Total Cholesterol Detection

[0122] Application Examples 2-12 are the same as Application Example 1 except that the complex enzyme solution is replaced by that of Examples 2-12. The preparation method is the same as that of Application Example 1.

[0123] Comparative Application Examples 1-11: Preparation of Test Paper for Total Cholesterol Detection

[0124] Comparative Application Examples 1-11 are the same as Application Example 1 except that the complex enzyme solution is replaced by that of Comparative Application Examples 1-11. The preparation method is the same as that of Application Example 1.

[0125] The method of using the above-mentioned test strips for total cholesterol detection includes the following:

[0126] Insert the exposed electrode of the total cholesterol test strip into the connector of the detection system. 1.7μL of the sample to be tested is dripped into the siphon pool entrance of the test strip and flows into the reaction area by siphon action. Under the action of the excitation potential of -50mV to -300mV, the current value is detected by the measuring part. The total cholesterol content in the sample to be tested is calculated and displayed according to the total cholesterol concentration-current value standard curve. The value of the calculation and display component is the total cholesterol content in the sample to be tested.

[0127] Now, according to the above-mentioned total cholesterol test strip usage method, the following performance tests are performed on the above-mentioned application examples and application comparison examples:

[0128] (1) Obtaining a total cholesterol concentration-current value standard curve

[0129] Using normal human venous whole blood, different amounts of cholesterol mother solution were added to prepare 9 cholesterol whole blood samples with different concentrations, with theoretical concentrations of 2.4mmol / L, 3.0mmol / L, 4.2mmol / L, 5.4mmol / L, 6.4mmol / L, 7.4mmol / L, 8.6mmol / L, 9.7mmol / L, and 10.6mmol / L, respectively. Then, according to the above-mentioned electrochemical cholesterol test strip method, the excitation potential was controlled at -220mV, and the current response of each of the 9 cholesterol whole blood samples with different concentrations was tested. The current value at a reaction time of 25s was taken. Each whole blood sample was tested in parallel 10 times and the average value was taken to determine the repeatability, relative sensitivity, and linear range. The raw data of the relevant tests of Application Example 1 are listed in Table 1, with cholesterol concentration as the horizontal axis and current value as the vertical axis. Figure 1 was obtained by fitting the trend line.

[0130] Table 1

[0131] The correlation analysis of the test results of the present application and the test results obtained by the method is shown in Figure 1. According to Figure 1 and Table 1, the calibration curve is established by the average value of the total cholesterol theoretical concentration and the current value obtained by the test. The square of the correlation coefficient of the linear fit (R 2 ) is 0.9842, which has a good fit and can be used as a standard curve of total cholesterol concentration-current value. The CV of the obtained current value is below 3.3%, and the detection response time is only 25s.

[0132] (2) Specific testing of cholesterol samples with different concentrations

[0133] Twelve blood samples of different concentrations were randomly selected for testing and then compared with the test results of the biochemical analyzer for evaluation. The test results and linear correlation of Application Example 1 are shown in Table 2.

[0134] Table 2

[0135] For the sake of simplicity, only the calculated results are given for Application Examples 2-10 and Comparative Application Examples 1-10, as shown in Table 3.

[0136] Table 3

[0137] From Table 1 and Table 2, we can draw the following conclusions:

[0138] (1) Comprehensive application examples 1-7 show that, 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 electrochemical detection, wherein the square correlation coefficient of the linear fit 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 the surfactant composition is not affected by the amount of surfactant under the conditions that surfactant 1 meets HLB>23 and surfactant 2 meets HLB 12-14;

[0139] (2) Comprehensive application examples 1-12 show that the enzyme solution for electrochemical detection of total cholesterol provided by the present application has a good linear relationship in the electrochemical detection process, wherein the square correlation coefficient of the linear fit 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 application can achieve sensitive and accurate detection of cholesterol;

[0140] (3) From the perspective of Application Example 6 and Comparative Examples 1 and 4, it can be seen that the surfactant composition in Application Example 6 is Tetronic 1107 0.5% and TritonX-100 0.5%, while the surfactants in Comparative Examples 1 and 4 are Tetronic 1107 0.5% and TritonX-100 0.5%, respectively. Compared with Application Example 6, the correlation coefficients of the linear fitting of Comparative Examples 1 and 4 are both reduced, and the deviation values ​​are also greatly increased, indicating that the accuracy of the test is reduced and the repeatability is reduced. From the perspective of Application Example 1 and Comparative Examples 2-3, it can be seen that the surfactant composition in Application Example 1 is Pluronic F88 0.5% and CHEMAL LA9 0.5%, while the surfactants in Comparative Examples 2-3 are Pluronic F88 0.5% and CHEMAL LA9 0.5%. Compared with Application Example 1, the correlation coefficients of the linear fitting of Application Comparative Examples 2-3 all decreased, and the deviation values ​​also increased greatly, indicating that the accuracy of the test decreased and the repeatability was poor. The main reason is that the surfactant in Application Comparative Examples 1-4 is a single component. Although it meets the characteristics of Surfactant 1 or Surfactant 2, it fails to meet the conditions of Surfactant 1 and Surfactant 2 at the same time, resulting in reduced accuracy. This shows that the present application significantly improves the accuracy of the test by adding a surfactant composition that meets the conditions to the enzyme solution component;

[0141] (4) From the comprehensive analysis of Application Example 2 and Application Comparative Example 5, it can be seen that the surfactant composition in Application Example 2 is Tetronic 1107 0.3% and CHEMAL LA9 0.2%, while the surfactant composition in Application Comparative Example 5 is Tetronic 1107 0.3% and Pluronic F88 0.2%. Compared with Application Example 2, the correlation coefficient of the linear fitting of Application Comparative Example 5 decreases and the deviation value also increases greatly, indicating that the accuracy of the test decreases and the repeatability is poor. The main reason is that although two surfactants are also added in Application Comparative Example 5, the HLB values ​​of the two surfactants are both greater than 23, which fails to simultaneously meet the conditions of surfactant 2 in the surfactant composition;

[0142] (5) From the combined application example 7 and the application comparative example 6, it can be seen that the surfactant composition in the application example 7 is Tetronic 1107 0.3%, Pluronic F88 0.2% and TritonX-100 0.5%, and the surfactant composition in the application comparative example 6 is Tetronic 1107 0.3%, Pluronic F88 0.2% and Brij-35 0.5%. Compared with the application example 7, the application comparative example 6 selects the surfactant Brij-35 with an HLB value of 16.9 as the control, and replaces the surfactant component TritonX-100. Other conditions remain unchanged, but the correlation coefficient of the linear fitting of the application comparative example 6 decreases significantly, and the deviation value also increases. From the combined application example 1 and the application comparative example 7, it can be seen that the surfactant composition in the application example 1 is Pluronic F88 0.5% and CHEMAL LA9 0.5%, and the surfactant composition in the application comparative example 7 is CHEMAL LA9 0.5% and TritonX-305 0.5%. Compared with Application Example 1, Comparative Example 7 selected Triton X-305, a surfactant with an HLB value of 17.3, as a control, and replaced the surfactant component Pluronic F88. Other conditions remained unchanged. Comparative Example 7 also showed a decrease in the correlation coefficient of the linear fit and an increase in the deviation value. It can be seen that even if the surfactant composition is added, even if Comparative Example 6 meets the requirement that the HLB value of the surfactant is greater than 23 and Comparative Example 7 meets the requirement that the HLB value of the surfactant is between 12 and 14, the conditions of surfactant 1 having an HLB greater than 23 and surfactant 2 having an HLB of 12 to 14 are not met at the same time. The accuracy and repeatability of the total cholesterol detection are not as good as the results of the composite enzyme solution containing the surfactant composition that meets the conditions at the same time.

[0143] (6) From the comprehensive application example 3 and the application comparative examples 9-10, it can be seen that the surfactant composition in the application example 3 is Tetronic 1107 0.3%, Pluronic F88 0.2% and CHEMAL LA9 0.2%, the surfactant composition in the application comparative example 9 is Tetronic 1107 0.3%, Pluronic F88 0.5% and CHEMAL LA9 0.2%, and the surfactant composition in the application comparative example 10 is Tetronic 1107 0.3%, Pluronic F88 1% and CHEMAL LA9 0.2%. Compared with the application example 3, the addition amount of Pluronic F88 in the application comparative examples 9 and 10 is adjusted to exceed the mass concentration range of 0.1%-0.5% of the surfactant 1 in the surfactant composition; from the comprehensive application example 5 and the application comparative example 11, it can be seen that the surfactant composition in the application example 5 is Tetronic 1107 0.3%, Pluronic F88 0.2%, CHEMAL LA9 0.5% and TritonX-100 0.5%, the surfactant composition in Comparative Example 11 is Tetronic 1107 0.3%, CHEMAL LA9 0.2% and TritonX-100 1%. Compared with Application Example 5, the amount of TritonX-100 added in Comparative Example 11 is adjusted to exceed the mass concentration range of 0.2%-1% of surfactant 2 in the surfactant composition. According to the test results, the correlation coefficients of the linear fits of Comparative Examples 9-11 are all decreased, and the deviation values ​​are increased, indicating that the accuracy of the test is decreased and the repeatability is poor. It can be seen that the amount of surfactant added must meet the mass concentration of surfactant 1 of 0.1%-0.5% and the mass concentration of surfactant 2 of 0.2%-1%. If too much, too little or no surfactant is added, such as in Comparative Example 8, the total cholesterol will not be able to fully react with the complex enzyme solution, and the best effect cannot be achieved. Therefore, the synergistic combination of surfactants in this application not only needs to meet the conditions that HLB of surfactant 1 is greater than 23 and HLB is 12-14, but the added amount also needs to meet the mass concentration of surfactant 1 of 0.1%-0.5% and the mass concentration of surfactant 2 of 0.2%-1%, in order to finally achieve efficient electrochemical detection of total cholesterol.

[0144] 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 results can be obtained in only 15-25 seconds.

[0145] The above description is merely a preferred embodiment of the present application and is not intended to limit the present application. Various modifications and variations are possible for those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present application shall be included within the scope of protection of the present application.

[0146] The preferred embodiments of the present application are described in detail above. However, the present application is not limited to the specific details of the above embodiments. Within the technical concept of the present application, various simple modifications can be made to the technical solution of the present application, and these simple modifications all fall within the scope of protection of the present application.

[0147] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any appropriate manner unless there is any contradiction. In order to avoid unnecessary repetition, this application will not further describe various possible combinations.

[0148] In addition, the various implementation methods of the present application may be arbitrarily combined, and as long as they do not violate the concept of the present application, they should also be regarded as the contents disclosed in the present application.

Claims

1. A total cholesterol detection complex enzyme solution, comprising cholesterol oxidase, cholesterol esterase, peroxidase, an electron mediator and a surfactant composition, wherein the surfactant composition comprises a surfactant 1 and a surfactant 2; the surfactant 1 is selected from one or a combination of at least two surfactants with an HLB of 12-14; the surfactant 2 is selected from one or a combination of at least two surfactants with an HLB of 12-14.

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

3. The total cholesterol detection complex enzyme solution according to claim 1, wherein The electron mediator is a reduced electron mediator, and the reduced electron mediator includes any one of potassium ferrocyanide, ferrocene derivatives, osmium complexes or ruthenium complexes.

4. The total cholesterol detection complex enzyme solution according to claim 3, wherein: The ferrocene derivative includes any one of (ferrocenylmethyl)trimethylammonium chloride or bis[3-trimethyl-aminopropyl]ferrocene dichloride.

5. The total cholesterol detection complex enzyme solution according to claim 1, wherein The surfactant 1 includes any one of Pluronic F68, Pluronic F77, Pluronic F87, Pluronic F88, Pluronic F98, Pluronic F108, Tetronic 1107 or Tetronic 1307, or a combination of at least two thereof.

6. The total cholesterol detection complex enzyme solution according to claim 1, wherein The surfactant 2 includes any one of TritonX-100, Surfynol 465, Creamophr EL, Oleth-10 or CHEMAL LA9, or a combination of at least two thereof.

7. The total cholesterol detection complex enzyme solution according to claim 1, wherein: The complex enzyme solution also includes a buffer solution, a thickener, a protective agent and a metal inorganic salt; the mass concentration of the thickener is 0.1%-3%; the mass concentration of the protective agent is 0.2%-5%; the mass concentration of the metal inorganic salt is 0.01-2.0%.

8. The total cholesterol detection complex enzyme solution according to claim 7, wherein: The buffer solution includes any one of Tris buffer, 4-hydroxyethylpiperazineethanesulfonic acid buffer, ACES buffer, sodium acetate buffer, MES buffer, sodium citrate-citric acid buffer, PBS buffer, Good's buffer or glycine buffer, or a combination of at least two thereof; the pH of the buffer solution is 5.5-7.

5.

9. The total cholesterol detection complex enzyme solution according to claim 7, wherein: The thickener includes any one of methyl cellulose, hydroxypropyl methyl cellulose, polyvinyl pyrrolidone, hydroxyethyl cellulose, sodium starch phosphate, propylene glycol alginate or carboxymethyl cellulose, or a combination of at least two thereof.

10. The composite enzyme solution for detecting total cholesterol according to claim 7, wherein: The protective agent includes any one of sucrose, mannitol, bovine serum albumin, gelatin or trehalose, or a combination of at least two of them.

11. The composite enzyme solution for detecting total cholesterol according to claim 7, wherein: The metal inorganic salt includes any one of sodium chloride, magnesium chloride or calcium chloride, or a combination of at least two of them.

12. A total cholesterol detection test strip, comprising the total cholesterol detection composite enzyme solution according to any one of claims 1 to 11, wherein the composite enzyme solution is loaded on an electrode layer of the detection test strip.

13. A detection system for total cholesterol detection, comprising the total cholesterol detection test strip according to claim 12.

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