A blood lipid dissociation agent and a blood lipid biosensor

Abstract

This invention relates to the field of biotechnology, and more particularly to a lipid dissociation agent and a lipid biosensor. The invention provides a method for dissociating lipids from lipoproteins using ester-based organic solvents as the main component of the dissociation agent. Experimental results show that ester-based organic solvents, as the main component of the dissociation agent, have good dissociation effects. A reagent layer containing the dissociation agent is loaded onto an electrode for lipid detection, resulting in a strong detection signal and high sensitivity.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more particularly to a lipid dissociation agent and a lipid biosensor. Background Technology

[0002] Blood lipids are a collective term for neutral fats (triglycerides) and lipids (phospholipids, glycolipids, sterols, and steroids) in blood plasma, widely present in the human body. They are essential substances for the basic metabolism of living cells. Generally speaking, the main components of blood lipids are triglycerides and cholesterol. Triglycerides participate in energy metabolism in the human body, while cholesterol is mainly used to synthesize cell membranes, steroid hormones, and bile acids. The levels of cholesterol and triglycerides in the blood are important indicators of the risk of coronary heart disease ("CHD"), therefore, cholesterol and triglyceride testing is of great clinical significance. Currently, methods for measuring cholesterol and triglyceride levels include fluorescence methods, chromatography and high-performance liquid chromatography, and electrochemical methods. For example, Chinese patent CN109313155A discloses a system and method for electrochemical triglyceride determination, which uses an electrochemical detection method to detect the triglyceride content in the blood. This electrochemical test strip contains a reagent mixture, mainly composed of surfactants, lipid hydrolysis reagents, glycerol kinase, glycerol-3-phosphate dehydrogenase, coenzymes, redox agents, and myocardial flavin enzymes. The mixed strip disclosed in Chinese patent CN105122017B uses an electrochemical method to detect total cholesterol, HDL cholesterol, and triglycerides. The electrochemical strip used for detection includes electrodes and enzyme reactants.

[0003] Cholesterol and triglycerides in the blood are mainly found in lipoproteins, with low levels of free cholesterol and triglycerides. Direct detection of cholesterol and triglycerides using electrochemical methods yields weak electrochemical signals, poor sensitivity, and inconsistent accuracy. Therefore, it is necessary to release cholesterol and triglycerides from lipoproteins before detection. In the aforementioned patent CN109313155A, bile acid derivatives or their salts are used as surfactants, which can release triglycerides from lipoproteins to some extent. However, the surfactants can only release a very small amount of triglycerides from lipoproteins, and the dissociation and release of triglycerides from lipoproteins remains poor, failing to significantly improve the signal intensity, sensitivity, and accuracy of triglyceride electrochemical detection.

[0004] Therefore, it is particularly necessary to explore new reagents that can be used as dissociation agents, or combinations of dissociation agents with good dissociation effects. Summary of the Invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide a lipid dissociation agent and a lipid biosensor.

[0006] A lipid dissociation agent comprising an ester organic solvent; said ester organic solvent includes one or more of the following: dimethyl glutarate, dimethyl succinate, dimethyl adipate, trimethyl phosphate, triethyl phosphate, tributyl phosphate, and / or β-propiolactone.

[0007] The lipid dissociation agent provided by this invention further includes bile acids or their derivatives or salts, and / or surfactants;

[0008] The cholic acid or its derivatives or salts include one or more of the following: cholic acid, sodium cholate, taurocholic acid, glycocholic acid, lithocholic acid, deoxycholic acid, CHAPS, CHAPSO, BIGCHAP and / or deoxyBIGCHAP.

[0009] The surfactants include one or more of the following: Triton series (TX series), sodium dodecyl sulfate (SDS), fatty alcohol polyoxyethylene lauryl ether, polyethylene glycol octylphenyl ether, aryloxy polyoxyethylene ether, fatty alcohol polyoxyethylene sulfate sodium stearate, sucrose decanoate, AEO-3, AEO-9, Tween-20, Tween-80 and / or hexadecylpyridine chloride;

[0010] The Qulaton series (TX series) includes one or more of TX-100, TX-35 and / or TX-10;

[0011] Furthermore,

[0012] The lipid dissociation agents include: sodium cholate, CHAPS, dimethyl glutarate, dimethyl succinate, dimethyl adipate, and TX-100 (dissociation agent 1); or

[0013] The lipid dissociation agents include: sodium cholate, CHAPS, tributyl phosphate, and TX-100 (dissociation agent 2).

[0014] Furthermore,

[0015] The lipid dissociation agents include: 0.01wt%~3wt% sodium cholate, 0.01wt%~3wt% CHAPS, 0.01wt%~1.5wt% dimethyl glutarate, 0.01wt%~1.5wt% dimethyl succinate, 0.01wt%~1.5wt% dimethyl adipate, and 0.01wt%~3wt% TX-100 (dissociation agent 1); or,

[0016] The lipid dissociation agents include: 0.01wt%~3wt% sodium cholate, 0.01wt%~3wt% CHAPS, 0.01wt%~1.5wt% tributyl phosphate and 0.01wt%~3wt% TX-100 (dissociation agent 2);

[0017] In a specific embodiment of the present invention,

[0018] The lipid dissociation agent comprises: 1 wt% sodium cholate, 2 wt% CHAPS, 0.5 wt% dimethyl glutarate, 0.5 wt% dimethyl succinate, 0.5 wt% dimethyl adipate, and 0.5 wt% TX-100 (lipid dissociation agent 1); or

[0019] The lipid dissociation agent comprises: 1 wt% sodium cholate, 2 wt% CHAPS, 1 wt% tributyl phosphate and 0.5 wt% TX-100 (lipid dissociation agent 2).

[0020] The present invention provides a reagent combination or reagent layer, wherein the reagent combination includes one or more of the following: lipid dissociation agent, electron mediator, buffer salt, polymer binder, magnesium-containing inorganic salt, ATP and / or stabilizer described in the present invention.

[0021] This invention provides a lipid biosensor, which includes at least one working electrode and one reference electrode, as well as a reagent layer applied to the electrode. The reagent layer includes one or more of the following: dissociating agent, enzyme, electron mediator, buffer salt, polymer binder, magnesium-containing inorganic salt, ATP precipitant, and / or stabilizer. The electrode to which the reagent layer is applied can be either a working electrode or a reference electrode, and this invention does not limit this. Furthermore, the lipid biosensor includes at least one working electrode and one reference electrode. One working electrode and one reference electrode constitute the basic structure of the lipid biosensor, and the number of working electrodes or reference electrodes can be increased based on this, and this invention does not limit this.

[0022] The enzyme includes:

[0023] A combination of lipoprotein lipase, glycerol phosphate kinase, and glycerol phosphate oxidase;

[0024] Or a combination of cholesterol esterase, cholesterol oxidase and peroxidase.

[0025] The electron mediator includes one or more of potassium ferrocyanide, potassium ferrocyanide, hexaammineruthenium trichloride, phenazine derivatives and / or phenoxazine derivatives; the phenazine derivative includes phenazine methyl sulfate; the phenoxazine derivative includes phenazine-1-carboxylic acid 1,3,4-oxadiazole thioether derivative.

[0026] The buffer salts include: Tris-HCl and / or PIPES;

[0027] The polymer binder includes: carboxymethyl cellulose and / or hydroxyethyl cellulose;

[0028] The inorganic salts containing magnesium ions include one or more of MgCl2, MgSO4 and / or MgCO3;

[0029] The precipitant includes phosphotungstic acid;

[0030] The stabilizer includes: BSA and / or trehalose;

[0031] Furthermore, in the lipid biosensor of the present invention, the reagent layer comprises:

[0032] At least one of lipid dissociation agents 1 or 2, lipoprotein lipase, glycerol phosphate kinase, glycerol phosphate oxidase, hexaammineruthenium trichloride, phenazine methyl sulfate, Tris-HCl, carboxymethyl cellulose, MgCl2, adenine nucleoside triphosphate, and BSA; or

[0033] At least one of lipid dissociation agents 1 or 2, cholesterol esterase, cholesterol oxidase, peroxidase, potassium ferrocyanide, Tris-HCl, carboxymethyl cellulose, and BSA; or

[0034] At least one of lipid dissociation agents 1 or 2, cholesterol esterase, cholesterol oxidase, peroxidase, potassium ferrocyanide, Tris-HCl, carboxymethyl cellulose, BSA, phosphotungstic acid, and MgCl2.

[0035] Furthermore, the reagent layer includes:

[0036] The following are components: at least one of lipid dissociation agents 1 or 2; 0.1wt%–4wt% lipoprotein lipase; 0.1wt%–4wt% glycerol phosphokinase; 0.1wt%–4wt% glycerol phosphooxidase; 0.1wt%–5wt% hexaammineruthenium trichloride; 0.01wt%–5wt% phenazine methyl sulfate; 0.1wt%–2wt% Tris-HCl; 0.1wt%–3wt% carboxymethyl cellulose; 0.1wt%–2wt% MgCl2; 0.1wt%–2wt% adenine nucleoside triphosphate; and 0.1wt%–5wt% BSA.

[0037] Alternatively, at least one of lipid dissociation agents 1 or 2, 0.1wt%~4wt% cholesterol esterase, 0.1wt%~4wt% cholesterol oxidase, 0.1wt%~4wt% peroxidase, 0.1wt%~5wt% potassium ferrocyanide, 0.1wt%~2wt% Tris-HCl, 0.1wt%~3wt% carboxymethyl cellulose and 0.1wt%~5wt% BSA;

[0038] Alternatively, at least one of lipid dissociation agents 1 or 2, 0.1wt%~4wt% cholesterol esterase, 0.1wt%~4wt% cholesterol oxidase, 0.1wt%~4wt% peroxidase, 0.1wt%~5wt% potassium ferrocyanide, 0.1wt%~2wt% Tris-HCl, 0.1wt%~3wt% carboxymethyl cellulose, 0.1wt%~5wt% BSA, 0.1wt%~2wt% phosphotungstic acid, and 0.1wt%~2wt% MgCl2;

[0039] In a specific embodiment of the present invention, the reagent layer includes:

[0040] 1 wt% sodium cholate, 2 wt% CHAPS, 0.5 wt% dimethyl glutarate, 0.5 wt% dimethyl succinate, 0.5 wt% dimethyl adipate, 0.5 wt% TX-100, 3 wt% lipoprotein lipase, 2 wt% glycerol phosphate kinase, 1 wt% glycerol phosphate oxidase, 2 wt% hexaammineruthenium trichloride, 0.05 wt% methyl phenazine sulfate, 1 wt% Tris-HCl, 2 wt% carboxymethyl cellulose, 0.5 wt% MgCl2, 0.6 wt% adenine triphosphate (ATP), and 0.5 wt% BSA; or

[0041] 1 wt% sodium cholate, 2 wt% CHAPS, 1 wt% tributyl phosphate, 0.5 wt% TX-100, 3% lipoprotein lipase, 2% glycerol phosphate kinase, 1% glycerol phosphate oxidase, 2 wt% hexaammineruthenium trichloride, 0.05 wt% phenazine methyl sulfate, 1 wt% Tris-HCl, 2 wt% carboxymethyl cellulose, 0.5 wt% MgCl2, 0.6 wt% adenine triphosphate (ATP), and 0.5 wt% BSA; or

[0042] 1 wt% sodium cholate, 2 wt% CHAPS, 0.5 wt% dimethyl glutarate, 0.5 wt% dimethyl succinate, 0.5 wt% dimethyl adipate, 0.5 wt% TX-100, 1 wt% cholesterol esterase, 3 wt% cholesterol oxidase, 0.5 wt% peroxidase, 3 wt% potassium ferrocyanide, 1 wt% Tris-HCl, 2 wt% carboxymethyl cellulose, and 0.5 wt% BSA; or

[0043] 1 wt% sodium cholate, 2 wt% CHAPS, 1 wt% tributyl phosphate, 0.5 wt% TX-100, 1 wt% cholesterol esterase, 3 wt% cholesterol oxidase, 0.5 wt% peroxidase, 3 wt% potassium ferrocyanide, 1 wt% Tris-HCl, 2 wt% carboxymethyl cellulose and 0.5 wt% BSA; or

[0044] 1 wt% sodium cholate, 2 wt% CHAPS, 0.5 wt% dimethyl glutarate, 0.5 wt% dimethyl succinate, 0.5 wt% dimethyl adipate, 0.5 wt% TX-100, 1 wt% cholesterol esterase, 3 wt% cholesterol oxidase, 0.5 wt% peroxidase, 3 wt% potassium ferrocyanide, 1 wt% Tris-HCl, 2 wt% carboxymethyl cellulose, 0.5 wt% BSA, 0.4 wt% phosphotungstic acid, and 0.6 wt% MgCl2; or

[0045] 1 wt% sodium cholate, 2 wt% CHAPS, 1 wt% tributyl phosphate, 0.5 wt% TX-100, 1 wt% cholesterol esterase, 3 wt% cholesterol oxidase, 0.5 wt% peroxidase, 3 wt% potassium ferrocyanide, 1% Tris-HCl, 2 wt% carboxymethyl cellulose, 0.5 wt% BSA, 0.4 wt% phosphotungstic acid and 0.6 wt% MgCl2.

[0046] In this invention, the solvent for the reagent layer is water.

[0047] This invention provides a lipid dissociation agent completely different from existing technologies. This lipid dissociation agent uses ester-based organic solvents as its main component to dissociate cholesterol and triglycerides within lipoproteins. In specific embodiments of this invention, a comparison of cholesterol and triglyceride detection using reagent layers without and with ester-based organic solvent dissociation agents revealed that adding ester-based organic solvent dissociation agents resulted in a stronger electrochemical signal and higher sensitivity for the dissociation and measurement of cholesterol and triglycerides within lipoproteins. Furthermore, by varying the types of ester-based organic solvents added, it was found that using 0.5 wt% dimethyl glutarate, 0.5 wt% dimethyl succinate, and 0.5 wt% dimethyl adipate as dissociation agents in combination with other reagents further improved the dissociation and detection performance of cholesterol and triglycerides in samples.

[0048] In this invention, the dissociation effect of blood lipids is reflected by the signal intensity and sensitivity of the electrochemical reaction. The higher the degree of dissociation of blood lipids, the better the signal intensity and sensitivity of the electrochemical reaction.

[0049] In this invention, a larger slope and a lower intercept indicate higher sensitivity;

[0050] This invention provides the application of the described dissociation agent and / or the described biosensor in blood lipid detection.

[0051] This invention provides a method for detecting blood lipids, which includes detecting a sample using the dissociation agent and / or the biosensor described in this invention.

[0052] This invention provides a method for dissociating blood lipids from lipoproteins using ester-based organic solvents as the main dissociation agent. Experimental results show that ester-based organic solvents have good dissociation effects as the main dissociation agent. A reagent layer containing the dissociation agent is loaded onto an electrode for blood lipid detection, resulting in a strong detection signal and high sensitivity. Attached Figure Description

[0053] Figure 1 Linear comparison of implementation scenarios 1, 2, and 3 for triglyceride detection;

[0054] Figure 2 Linear comparison of implementation statuses 1, 2, and 3 for total cholesterol testing;

[0055] Figure 3 Linear comparison of implementation statuses 1, 2, and 3 for high-density lipoprotein cholesterol testing. Detailed Implementation

[0056] This invention provides a lipid dissociation agent and a lipid biosensor. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0057] In this invention, the main component of the lipid dissociation agent is an ester organic solvent. This invention is the first to use an ester organic solvent for the dissociation of lipids within lipoproteins. In a specific embodiment, the lipid organic solvent may include one or more of the following: dimethyl glutarate, dimethyl succinate, dimethyl adipate, trimethyl phosphate, triethyl phosphate, tributyl phosphate, and / or β-propiolactone. For example, the present invention employs a mixture of three ester organic solvents—dimethyl glutarate, dimethyl succinate, and dimethyl adipate—and a single TNBP (tributyl phosphate) dissociation agent for the dissociation of blood lipids within lipoproteins. The present invention provides a blood lipid biosensor comprising at least one working electrode, one reference electrode, and a reagent layer mounted on the electrode. The reagent layer includes one or more of the dissociation agent, enzyme, electron mediator, buffer salt, polymer binder, magnesium-containing inorganic salt, ATP, precipitant, and / or stabilizer described in the present invention. The blood lipid sensor is used for the dissociation and determination of blood lipids within lipoproteins, exhibiting high dissociation efficiency. In reagent testing, the higher intensity of the electrochemical reaction current signal indicates the best sensitivity.

[0058] In a specific embodiment of the present invention, the lipid dissociation agent includes ester organic solvents as the main components, bile acids or their derivatives or their salts, and / or surfactants;

[0059] In specific implementation schemes, cholic acid or its derivatives or salts may be one or more of the following: cholic acid, sodium cholate, taurocholic acid, glycocholic acid, lithocholic acid, deoxycholic acid, CHAPS, CHAPSO, BIGCHAP, and deoxyBIGCHAP. The surfactant may be one or more of the following: Triton series (TX series), sodium dodecyl sulfate, fatty alcohol polyoxyethylene lauryl ether, polyethylene glycol octylphenyl ether, aryloxy polyoxyethylene ether, fatty alcohol polyoxyethylene sulfate, sodium stearate, sucrose decanoate, AEO-3, AEO-9, Tween-20, Tween-80, and / or hexadecylpyridine chloride; the Triton series (TX series) may be one or more of TX-100, TX-35, and / or TX-10.

[0060] For example, in a specific embodiment of the present invention, the cholic acid or its derivative or its salt is sodium cholate and CHAPS; the final concentration or the concentration during the reaction of the cholic acid or its derivative or its salt can be any value between 0.01wt% and 3wt%, specifically 0.01wt%, 0.02wt%, 0.03wt%, 0.04wt%, 0.05wt%, 0.06wt%, 0.07wt%, 0.08wt%, 0.09wt%, 0.10wt%, 0.11wt%, 0.12wt%, 0.13wt%, 0.14wt%, 0.15wt%, 0.16wt%, 0.17wt%, 0.18wt%, 0.19wt%, 0.20wt%, 0.21wt%, 0.2 Specific amounts include 2wt%, 0.23wt%, 0.24wt%, 0.25wt%, 0.26wt%, 0.27wt%, 0.28wt%, 0.29wt%, 0.30wt%, 0.35wt%, 0.40wt%, 0.45wt%, 0.50wt%, 0.55wt%, 0.60wt%, 0.65wt%, 0.70wt%, 0.75wt%, 0.80wt%, 0.85wt%, 0.90wt%, 0.95wt%, 1.0wt%, 1.2wt%, 1.4wt%, 1.6wt%, 1.8wt%, 1.9wt%, 2.0wt%, 2.2wt%, 2.4wt%, 2.6wt%, 2.8wt%, or 3.0wt%, etc.

[0061] For example, in a specific embodiment of the present invention, the surfactant used is TX-100; the final concentration or the concentration of the surfactant during the reaction can be any value between 0.01wt% and 3wt%, specifically 0.01wt%, 0.02wt%, 0.03wt%, 0.04wt%, 0.05wt%, 0.06wt%, 0.07wt%, 0.08wt%, 0.09wt%, 0.10wt%, 0.11wt%, 0.12wt%, 0.13wt%, 0.14wt%, 0.15wt%, 0.16wt%, 0.17wt%, 0.18wt%, 0.19wt%, 0.20wt%, 0.21wt%, 0.22wt%, 0 Specific amounts include 0.23wt%, 0.24wt%, 0.25wt%, 0.26wt%, 0.27wt%, 0.28wt%, 0.29wt%, 0.30wt%, 0.35wt%, 0.40wt%, 0.45wt%, 0.50wt%, 0.55wt%, 0.60wt%, 0.65wt%, 0.70wt%, 0.75wt%, 0.80wt%, 0.85wt%, 0.90wt%, 0.95wt%, 1.0wt%, 1.2wt%, 1.4wt%, 1.6wt%, 1.8wt%, 1.9wt%, 2.0wt%, 2.2wt%, 2.4wt%, 2.6wt%, 2.8wt%, or 3.0wt%, etc.

[0062] The dissociation agent is obtained by combining the cholic acid or its derivatives or salts, the surfactant, and the ester organic solvent. The ester organic solvent is the main component of the dissociation agent of this invention. Exemplarily, in specific embodiments of this invention, there are two specific scenarios: In the first scenario, the ester organic solvent is TNBP; in the second scenario, the ester organic solvent is dimethyl glutarate, dimethyl succinate, and dimethyl adipate. The concentration of the ester organic solvent used or during the reaction can be any value between 0.01wt% and 1.5wt%, specifically 0.01wt%, 0.02wt%, 0.03wt%, 0.04wt%, 0.05wt%, 0.06wt%, 0.07wt%, 0.08wt%, 0.09wt%, 0.10wt%, 0.11wt%, 0.12wt%, 0.13wt%, 0.14wt%, 0.15wt%, 0. Specific amounts include 20wt%, 0.25wt%, 0.30wt%, 0.35wt%, 0.40wt%, 0.45wt%, 0.50wt%, 0.55wt%, 0.60wt%, 0.65wt%, 0.70wt%, 0.75wt%, 0.80wt%, 0.85wt%, 0.90wt%, 0.95wt%, 1.0wt%, 1.1wt%, 1.2wt%, 1.3wt%, 1.4wt%, or 1.5wt%, etc.

[0063] The lipid biosensor comprises at least one working electrode and a reference electrode, and a reagent layer loaded on the electrode; the reagent layer comprises one or more of the following: dissociation agent, enzyme, electron mediator, buffer salt, polymer binder, magnesium-containing inorganic salt, ATP and / or stabilizer as described in this invention.

[0064] In specific implementation schemes, the corresponding enzyme reagent is selected according to the substance being detected. For example, when detecting triglycerides, the corresponding enzyme may be a combination of lipoprotein lipase, glycerol phosphate kinase, and glycerol phosphate oxidase; or, when detecting cholesterol, the corresponding enzyme may be a combination of cholesterol lipase, cholesterol oxidase, and peroxidase. The electron mediator may be one or more of potassium ferrocyanide, potassium ferrocyanide, hexaammineruthenium trichloride, phenazine derivatives, and phenoxazine derivatives; the buffer salt may be Tris-HCl and / or PIPES; the polymer binder may be carboxymethyl cellulose and / or hydroxyethyl cellulose; the magnesium-containing inorganic salt may be one or more of MgCl2, MgSO4, and MgCO3; the precipitant includes phosphotungstic acid; and the stabilizer may be BSA and / or trehalose.

[0065] For example, in a specific embodiment of the present invention, the enzyme is lipoprotein lipase, glycerol phosphate kinase, or glycerol phosphate oxidase; the concentration of the enzyme used or during the reaction can be any value between 0.1wt% and 4wt%, specifically 0.1wt%, 0.15wt%, 0.2wt%, 0.25wt%, 0.30wt%, 0.35wt%, 0.40wt%, 0.45wt%, 0.50wt%, 0.55wt%, 0.60wt%, 0.65wt%, 0.70wt%, 0.75wt%, 0.80wt%, 0.85wt%, 0.90wt%, 0.95wt%, 1.0wt%, 1.2wt%, 1.4wt%, 1.6wt%, 1.8wt%, 1.9wt%, 2.0wt%, 2.2wt%, 2.4wt%, 2.6wt%, 2.8wt%, 3.0wt%, 3.2wt%, 3.4wt%, 3.6wt%, 3.8wt%, or 4.0wt%, etc.

[0066] For example, in a specific embodiment of the present invention, the enzyme is cholesterol esterase, cholesterol oxidase, and peroxidase; the concentration of the enzyme used or during the reaction can be any value between 0.1wt% and 4wt%, specifically 0.1wt%, 0.15wt%, 0.2wt%, 0.25wt%, 0.30wt%, 0.35wt%, 0.40wt%, 0.45wt%, 0.50wt%, 0.55wt%, 0.60wt%, 0.65wt%, 0.70wt%, 0.75wt%, 0.80wt%, 0.85wt%, 0.90wt%, 0.95wt%, 1.0wt%, 1.2wt%, 1.4wt%, 1.6wt%, 1.8wt%, 1.9wt%, 2.0wt%, 2.2wt%, 2.4wt%, 2.6wt%, 2.8wt%, 3.0wt%, 3.2wt%, 3.4wt%, 3.6wt%, 3.8wt%, or 4.0wt%, etc.

[0067] For example, in a specific embodiment of the present invention, the electron mediator is hexaammineruthenium trichloride, methyl phenazine sulfate, and / or potassium ferrocyanide; the concentration of the electron mediator used or during the reaction can be any value between 0.1 wt% and 5 wt%, specifically 0.1 wt%, 0.15 wt%, 0.2 wt%, 0.25 wt%, 0.30 wt%, 0.35 wt%, 0.40 wt%, 0.45 wt%, 0.50 wt%, 0.55 wt%, 0.60 wt%, 0.65 wt%, 0.70 wt%, 0.7 wt%. Specific amounts include 5wt%, 0.80wt%, 0.85wt%, 0.90wt%, 0.95wt%, 1.0wt%, 1.2wt%, 1.4wt%, 1.6wt%, 1.8wt%, 1.9wt%, 2.0wt%, 2.2wt%, 2.4wt%, 2.6wt%, 2.8wt%, 3.0wt%, 3.2wt%, 3.4wt%, 3.6wt%, 3.8wt%, 4.0wt%, 4.2wt%, 4.4wt%, 4.6wt%, 4.8wt%, or 5.0wt%, etc.

[0068] For example, in a specific embodiment of the present invention, the buffer salt is Tris-HCl; the concentration of the buffer salt used or during the reaction can be any value between 0.01wt% and 2wt%, specifically 0.01wt%, 0.02wt%, 0.03wt%, 0.04wt%, 0.05wt%, 0.06wt%, 0.07wt%, 0.08wt%, 0.09wt%, 0.10wt%, 0.11wt%, 0.12wt%, 0.13wt%, 0.14wt%, 0.15wt%, 0.16wt%, 0.17wt%, 0.18wt%, 0.19wt%, 0.20wt%, 0 Specific amounts include 0.21wt%, 0.22wt%, 0.23wt%, 0.24wt%, 0.25wt%, 0.26wt%, 0.27wt%, 0.28wt%, 0.29wt%, 0.30wt%, 0.35wt%, 0.40wt%, 0.45wt%, 0.50wt%, 0.55wt%, 0.60wt%, 0.65wt%, 0.70wt%, 0.75wt%, 0.80wt%, 0.85wt%, 0.90wt%, 0.95wt%, 1.0wt%, 1.2wt%, 1.4wt%, 1.6wt%, 1.8wt%, 1.9wt%, or 2.0wt%, etc.

[0069] For example, in a specific embodiment of the present invention, the polymer binder is carboxymethyl cellulose; the final concentration or the concentration of the polymer binder during the reaction can be any value between 0.01wt% and 3wt%, specifically 0.01wt%, 0.02wt%, 0.03wt%, 0.04wt%, 0.05wt%, 0.06wt%, 0.07wt%, 0.08wt%, 0.09wt%, 0.10wt%, 0.11wt%, 0.12wt%, 0.13wt%, 0.14wt%, 0.15wt%, 0.16wt%, 0.17wt%, 0.18wt%, 0.19wt%, 0.20wt%, 0.21wt%, 0.22wt%. Specific amounts include 0.23wt%, 0.24wt%, 0.25wt%, 0.26wt%, 0.27wt%, 0.28wt%, 0.29wt%, 0.30wt%, 0.35wt%, 0.40wt%, 0.45wt%, 0.50wt%, 0.55wt%, 0.60wt%, 0.65wt%, 0.70wt%, 0.75wt%, 0.80wt%, 0.85wt%, 0.90wt%, 0.95wt%, 1.0wt%, 1.2wt%, 1.4wt%, 1.6wt%, 1.8wt%, 1.9wt%, 2.0wt%, 2.2wt%, 2.4wt%, 2.6wt%, 2.8wt%, or 3.0wt%, etc.

[0070] For example, in a specific embodiment of the present invention, the inorganic salt containing magnesium ions is MgCl2; the concentration of the inorganic salt containing magnesium ions used or during the reaction can be any value between 0.01wt% and 2wt%, specifically 0.01wt%, 0.02wt%, 0.03wt%, 0.04wt%, 0.05wt%, 0.06wt%, 0.07wt%, 0.08wt%, 0.09wt%, 0.10wt%, 0.11wt%, 0.12wt%, 0.13wt%, 0.14wt%, 0.15wt%, 0.16wt%, etc. Specific amounts may vary, including 0.17wt%, 0.18wt%, 0.19wt%, 0.20wt%, 0.25wt%, 0.30wt%, 0.35wt%, 0.40wt%, 0.45wt%, 0.50wt%, 0.55wt%, 0.60wt%, 0.65wt%, 0.70wt%, 0.75wt%, 0.80wt%, 0.85wt%, 0.90wt%, 0.95wt%, 1.0wt%, 1.2wt%, 1.4wt%, 1.6wt%, 1.8wt%, 1.9wt%, or 2.0wt%, etc.

[0071] For example, in a specific embodiment of the present invention, the concentration of adenine nucleoside triphosphate ATP used or the concentration during the reaction can be any value between 0.01wt% and 2wt%, specifically 0.01wt%, 0.02wt%, 0.03wt%, 0.04wt%, 0.05wt%, 0.06wt%, 0.07wt%, 0.08wt%, 0.09wt%, 0.10wt%, 0.11wt%, 0.12wt%, 0.13wt%, 0.14wt%, 0.15wt%, 0.16wt%, 0.17wt%. Specific amounts may vary, including 0.18wt%, 0.19wt%, 0.20wt%, 0.25wt%, 0.30wt%, 0.35wt%, 0.40wt%, 0.45wt%, 0.50wt%, 0.55wt%, 0.60wt%, 0.65wt%, 0.70wt%, 0.75wt%, 0.80wt%, 0.85wt%, 0.90wt%, 0.95wt%, 1.0wt%, 1.2wt%, 1.4wt%, 1.6wt%, 1.8wt%, 1.9wt%, or 2.0wt%, etc.

[0072] For example, in a specific embodiment of the present invention, the concentration of phosphotungstic acid used or the concentration during the reaction can be any value between 0.01wt% and 2wt%, specifically 0.1wt%, 0.15wt%, 0.2wt%, 0.25wt%, 0.30wt%, 0.35wt%, 0.40wt%, 0.45wt%, 0.50wt%, 0.55wt%, 0.60wt%, 0.65wt%, 0.70wt%, 0.75wt%, 0.80wt%, 0.85wt%, 0.90wt%, 0.95wt%, 1.0wt%, 1.2wt%, 1.4wt%, 1.6wt%, 1.8wt%, 1.9wt%, or 2.0wt%, etc.

[0073] For example, in a specific embodiment of the present invention, the stabilizer is BSA; the concentration of the stabilizer used or during the reaction can be any value between 0.1wt% and 5wt%, specifically 0.1wt%, 0.15wt%, 0.2wt%, 0.25wt%, 0.30wt%, 0.35wt%, 0.40wt%, 0.45wt%, 0.50wt%, 0.55wt%, 0.60wt%, 0.65wt%, 0.70wt%, 0.75wt%, 0.80wt%. Specific amounts include t%, 0.85wt%, 0.90wt%, 0.95wt%, 1.0wt%, 1.2wt%, 1.4wt%, 1.6wt%, 1.8wt%, 1.9wt%, 2.0wt%, 2.2wt%, 2.4wt%, 2.6wt%, 2.8wt%, 3.0wt%, 3.2wt%, 3.4wt%, 3.6wt%, 3.8wt%, 4.0wt%, 4.2wt%, 4.4wt%, 4.6wt%, 4.8wt%, or 5.0wt%, etc.

[0074] In a specific embodiment of the present invention, the reagent layer composed above is dotted onto a screen-printed carbon electrode (the carbon electrode can be at least one of a working electrode and / or a reference electrode), then dried at 50°C, and after applying double-sided tape and a hydrophilic film, it is compacted with a steel roller and then cut into the size of each electrochemical test strip with a roller cutter, for example 35mm×6mm. The test strip is inserted into the testing instrument and the instrument is automatically turned on. Blood lipid whole blood samples of different concentrations are added to the siphon channel (sample receiving port).

[0075] The term "at least one" refers to one or more, while "more" refers to two or more. "At least one" or similar expressions refer to any combination of these items, including any combination of single items or multiple items. For example, "at least one of a, b, or c," or "at least one of a, b, and c," can both mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, and c can each be a single item or multiple items.

[0076] The singular forms “a,” “the,” and “the” used in the embodiments of this application and the appended claims are also intended to include the plural forms, unless the context clearly indicates otherwise.

[0077] The terms “comprising,” “having,” or “containing,” including their grammatical synonyms, should generally be understood as open-ended and non-limiting, e.g., not excluding other unstated elements or steps unless specifically stated otherwise or understood from the context. Any and all instances or exemplary use of language such as “e.g.” or “comprising” herein are merely intended to better illustrate the invention and do not constitute a limitation on the scope of the invention unless the claims are made. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the invention.

[0078] Furthermore, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any value inevitably contains standard deviations due to individual test methods. Therefore, unless explicitly stated otherwise, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified with the word "approximately". Here, "approximately" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specific value or range.

[0079] In this invention, functionally, the reagent layer includes a lipid dissociation reagent, a lipid hydrolysis reagent, and an electroconversion reagent. The lipid dissociation reagent is responsible for dissociating lipids from lipoproteins; the lipid hydrolysis reagent and the electroconversion reagent are responsible for breaking down the lipase-catalyzed reaction into detectable intermediate products. This invention uses an electrochemical method to detect blood lipids, and the specific reaction steps can be arbitrarily shown below:

[0080] The dissociation agent rapidly dissociates blood lipids from lipoproteins, and then the enzymatic reaction in the reagent layer (lipolysis reagent and electroconversion reagent) breaks down the lipoprotein enzymatic reaction process into detectable intermediate products. In the detection of triglycerides, the enzymatic reaction includes: lipoprotein lipase (LPL, or lipoprotein lipase) hydrolyzes triglycerides into glycerol and fatty acids. Then, glycerol reacts with ATP and magnesium ions under the action of glycerol phosphokinase to generate glycerol 3-phosphate. Subsequently, glycerol 3-phosphate is oxidized to dihydroxyacetone phosphate under the action of glycerol phosphooxidase. At the same time, the generated ADP and reduced electron mediator complete the enzymatic reaction stage. In the detection of cholesterol, the enzymatic reaction includes: cholesterol esterase hydrolyzes cholesterol into free cholesterol and free fatty acids. Then, cholesterol oxidase further oxidizes free cholesterol into cholesterol-4-en-3-one, while generating hydrogen peroxide. Peroxidase is directly used for the redox reaction of hydrogen peroxide in electrochemical detection.

[0081] Following the enzymatic reaction, the resulting reduced electron mediator (a specific electron transport substance) undergoes a redox reaction on the electrode surface, generating a current signal proportional to the blood lipid concentration. By measuring the magnitude of this current signal, the blood lipid content can be quantitatively analyzed.

[0082] In some specific implementations, the method for detecting blood lipids includes providing an electrochemical test strip and placing the test strip in an analyzer, measuring the current supplied through the blood sample and the electrochemical test strip, and calculating the blood lipid level based on the current using the analyzer. Optionally, the test strip includes a working electrode, a reference electrode, and a reagent layer applied to the electrode; the reagent layer may be further optimized for pH and the concentration of each substance in the reagent layer to improve the accuracy of blood lipid determination.

[0083] In this invention, the reagent layer applied to the working electrode can be applied by printing, coating, dipping or other methods; the working electrode can also be an electrode made of various materials, including electrodes made of carbon, gold, platinum, copper or other conductive materials, which will be obvious to those skilled in the art.

[0084] The test materials used in this invention are all common commercially available products. The invention is further illustrated below with reference to embodiments:

[0085] Example 1: Triglyceride Detection

[0086] Implementation scenario 1: No organic ester solvents are added to the reagent layer.

[0087] The reagent layer comprises: 1 wt% sodium cholate, 2 wt% CHAPS, 0.5 wt% TX-100, 3 wt% lipoprotein lipase, 2 wt% glycerol phosphate kinase, 1 wt% glycerol phosphate oxidase, 2 wt% hexaammineruthenium trichloride, 0.05 wt% methyl phenazine sulfate, 1 wt% Tris-HCl, 2 wt% carboxymethyl cellulose, 0.5 wt% MgCl2, 0.6 wt% ATP, and 0.5 wt% BSA, with the balance being purified water.

[0088] Implementation Scenario 2: Adding a single organic ester solvent to the reagent layer

[0089] The reagent layer includes: an additional 1 wt% of tributyl phosphate added to “Implementation Case 1”.

[0090] Implementation Scenario 3: Multiple organic ester solvents were added to the reagent layer.

[0091] The reagent layer includes, in addition to “Implementation Case 1”, 0.5 wt% dimethyl glutarate, 0.5 wt% dimethyl succinate, and 0.5 wt% dimethyl adipate.

[0092] Prepare solutions for scenarios 1, 2, and 3 according to the above formula, apply them to the screen-printed carbon electrode, dry at 50°C, attach double-sided tape and a hydrophilic film, compact with a steel roller, and cut into 35mm×6mm electrochemical test strips. Insert the test strips into the testing instrument and turn it on automatically. Add blood samples of different concentrations of lipids to the siphon channel.

[0093] Electrochemical test strips can be tested at voltages ranging from 100 to 1000 mV. In the embodiments described in this application, the electrochemical test strips are all tested at a voltage of 200 mV.

[0094] The results of implementation status 1, 2 and 3 are as follows Figure 1 As shown, scenario 3 exhibits the largest linear slope and, at the same concentration, a relatively higher electrochemical reaction current signal intensity, indicating the best sensitivity.

[0095] Specifically, using the lipid dissociation agent in this application can release non-free lipids distributed in lipoproteins to the greatest extent, forming free lipids; thus, the concentration of lipids participating in the enzyme reaction is higher than before using the dissociation agent, the signal intensity of the enzyme electrochemical reaction is greater, and the sensitivity is higher.

[0096] Example 2 Total Cholesterol Detection

[0097] Implementation scenario 1: No organic ester solvents are added to the reagent layer.

[0098] The reagent layer comprises: 1 wt% sodium cholate, 2 wt% CHAPS, 0.5 wt% TX-100, 1 wt% cholesterol esterase, 3 wt% cholesterol oxidase, 0.5 wt% peroxidase, 3 wt% potassium ferrocyanide, 1 wt% Tris-HCl, 2 wt% carboxymethyl cellulose, 0.5 wt% BSA, and the balance being purified water.

[0099] Implementation Scenario 2: Adding a single organic ester solvent to the reagent layer

[0100] The reagent layer includes an additional 1 wt% of tributyl phosphate based on “Implementation Case 1”.

[0101] Implementation Scenario 3: Multiple organic ester solvents were added to the reagent layer.

[0102] The reagent layer includes, in addition to “Implementation Case 1”, 0.5 wt% dimethyl glutarate, 0.5 wt% dimethyl succinate, and 0.5 wt% dimethyl adipate.

[0103] Solutions for embodiments 1, 2, and 3 were prepared according to the above formula, dotted onto a screen-printed carbon electrode, dried at 50°C, and then coated with double-sided tape and a hydrophilic film. After compaction with a steel roller, the solutions were cut into 35mm × 6mm electrochemical test strips using a roller cutter. The test strips were inserted into the testing instrument, which automatically activated. Blood samples of different concentrations of lipids were added to the siphon channel. The electrochemical test strips can be tested at voltages ranging from -1000 to 0mV. In the embodiments described in this application, the electrochemical test strips all use a voltage of -250mV, specifically a positive voltage of -250mV between the working electrode and the counter electrode.

[0104] The results of implementation status 1, 2 and 3 are as follows Figure 2 As shown, scenario 3 exhibits the largest linear slope and, at the same concentration, a relatively higher electrochemical reaction current signal intensity, indicating the best sensitivity.

[0105] Example 3: Detection of High-Density Lipoprotein Cholesterol (HDL-C)

[0106] Implementation scenario 1: No organic ester solvents are added to the reagent layer.

[0107] The reagent layer comprises: 1 wt% sodium cholate, 2 wt% CHAPS, 0.5 wt% TX-100, 1 wt% cholesterol esterase, 3 wt% cholesterol oxidase, 0.5 wt% peroxidase, 3 wt% potassium ferrocyanide, 1 wt% Tris-HCl, 2 wt% carboxymethyl cellulose, 0.5 wt% BSA, 0.4 wt% phosphotungstic acid, 0.6 wt% MgCl2, and the balance being purified water.

[0108] Implementation Scenario 2: Adding a single organic ester solvent to the reagent layer

[0109] The reagent layer includes an additional 1 wt% of tributyl phosphate based on “Implementation Case 1”.

[0110] Implementation Scenario 3: Multiple organic ester solvents were added to the reagent layer.

[0111] The reagent layer includes, in addition to “Implementation Case 1”, 0.5 wt% dimethyl glutarate, 0.5 wt% dimethyl succinate, and 0.5 wt% dimethyl adipate.

[0112] Solutions for embodiments 1, 2, and 3 were prepared according to the above formula, dotted onto a screen-printed carbon electrode, dried at 50°C, and then coated with double-sided tape and a hydrophilic film. After compaction with a steel roller, the solutions were cut into 35mm × 6mm electrochemical test strips using a roller cutter. The test strips were inserted into the testing instrument, which automatically activated. Blood samples of different concentrations of lipids were added to the siphon channel. The electrochemical test strips can be tested at voltages ranging from -1000 to 0mV. In the embodiments described in this application, the electrochemical test strips all use a voltage of -250mV, specifically a positive voltage of -250mV between the working electrode and the counter electrode.

[0113] The results of implementation status 1, 2 and 3 are as follows Figure 3 As shown, scenario 3 exhibits the largest linear slope and, at the same concentration, a relatively higher electrochemical reaction current signal intensity, indicating the best sensitivity.

[0114] In summary, based on the test results of the above three different detection indicators, the lipid dissociation agent in this application can release non-free lipids distributed in lipoproteins to the greatest extent, forming free lipids. As a result, the concentration of lipids participating in the enzyme reaction is higher than before the use of the dissociation agent, the signal intensity of the enzyme electrochemical reaction is greater, and the sensitivity is higher.

[0115] In addition, the preparation steps of electrochemical test strips for detecting blood lipid levels are simple and the materials are inexpensive; the electrochemical test strips used for blood lipid detection are small in size, have short detection time, and high accuracy.

[0116] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A lipid dissociation agent, characterized in that, It contains ester organic solvents, cholic acids or their derivatives or salts thereof, and surfactants; The ester organic solvents include one or more of the following: dimethyl glutarate, dimethyl succinate, dimethyl adipate, trimethyl phosphate, triethyl phosphate, tributyl phosphate, and β-propiolactone; The cholic acid or its derivatives or salts include one or more of the following: cholic acid, sodium cholate, taurocholic acid, glycocholic acid, lithocholic acid, deoxycholic acid, CHAPS, CHAPSO, BIGCHAP, and deoxyBIGCHAP; The surfactants include one or more of the following: Triton series, sodium dodecyl sulfate, fatty alcohol polyoxyethylene lauryl ether, polyethylene glycol octylphenyl ether, aryloxy polyoxyethylene ether, fatty alcohol polyoxyethylene sulfate, sodium stearate, sucrose decanoate, AEO-3, AEO-9, Tween-20, Tween-80, and hexadecylpyridine chloride.

2. The lipid dissociation agent according to claim 1, characterized in that, The lipid-dissociating agent comprises: 0.01wt%~3wt% sodium cholate, 0.01wt%~3wt% CHAPS, 0.01wt%~1.5wt% dimethyl glutarate, 0.01wt%~1.5wt% dimethyl succinate, 0.01wt%~1.5wt% dimethyl adipate, and 0.01wt%~3wt% TX-100; or, The lipid dissociation agents include: 0.01wt%~3wt% sodium cholate, 0.01wt%~3wt% CHAPS, 0.01wt%~1.5wt% tributyl phosphate and 0.01wt%~3wt% TX-100.

3. A blood lipid biosensor, comprising at least one working electrode and one reference electrode, and a reagent layer applied to the electrodes, characterized in that, The reagent layer includes the dissociation agent as described in claim 1 or 2, and further includes one or more of the following: enzyme, electron mediator, buffer salt, polymer binder, inorganic salt containing magnesium ions, ATP, precipitant, and stabilizer.

4. The blood lipid biosensor according to claim 3, characterized in that, The enzyme includes: A combination of lipoprotein lipase, glycerol phosphate kinase, and glycerol phosphate oxidase; Or a combination of cholesterol esterase, cholesterol oxidase and peroxidase.

5. The blood lipid biosensor according to claim 4, characterized in that, The electron mediator includes one or more of the following: potassium ferrocyanide, potassium ferrocyanide, hexaammineruthenium trichloride, phenazine derivatives, and phenoxazine derivatives.

6. The blood lipid biosensor according to claim 5, characterized in that, The buffer salts include: Tris-HCl and / or PIPES; The polymer binder includes: carboxymethyl cellulose and / or hydroxyethyl cellulose; The inorganic salts containing magnesium ions include one or more of MgCl2, MgSO4, and MgCO3; The precipitant includes phosphotungstic acid; The stabilizers include BSA and / or trehalose.

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