Blood fat dissociation agent and blood fat biosensor

Abstract

The invention relates to the technical field of biology, in particular to a blood fat dissociation agent and a blood fat biosensor. According to the invention, the ester organic solvent is used as a main component of the dissociation agent to realize dissociation of blood fat from lipoprotein, and experimental results show that the dissociation effect of the ester organic solvent as the main component of the dissociation agent is good; the reagent layer containing the dissociation agent is loaded on an electrode and is used for detecting blood fat, the detection signal is strong, and the sensitivity is high.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a blood lipid dissociating agent and a blood lipid biosensor. Background Art

[0002] Blood lipids are a general term for neutral fats (triglycerides) and lipids (phospholipids, glycolipids, sterols, steroids) in plasma, which are 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, of which triglycerides are involved in energy metabolism in the human body, while cholesterol is mainly used to synthesize cell plasma membranes, steroid hormones and bile acids. The amount of cholesterol and triglycerides in the blood is an important indicator of the risk of coronary heart disease ("CHD"), so the detection of cholesterol and triglycerides is of great clinical significance. At present, methods for measuring the content of cholesterol and triglycerides include fluorescence, chromatography, high performance liquid chromatography, electrochemical methods, etc. For example, the system and method for electrochemical triglyceride determination disclosed in Chinese patent CN109313155A adopts an electrochemical detection method to detect the triglyceride content in the blood. The electrochemical test strip contains a reagent mixture, which is mainly composed of surfactants, blood lipid hydrolysis reagents, glycerol kinase, glycerol-3-phosphate dehydrogenase, coenzymes, redox agents, myocardial enzymes and other components. The mixed strip disclosed in Chinese patent CN105122017B uses an electrochemical method to detect total cholesterol, HDL cholesterol, and triglycerides. The electrochemical test strip used for detection includes electrodes and enzyme reactants.

[0003] Cholesterol and triglycerides in the blood are mainly present in lipoproteins, and the cholesterol and triglyceride content in the free state is low. When cholesterol and triglycerides are directly detected by electrochemical methods, the electrochemical signal obtained is weak, the detection sensitivity is poor, and the accuracy is difficult to guarantee; therefore, before detection, the cholesterol and triglycerides in the lipoprotein need to be released for electrochemical detection. In the above-mentioned patent CN109313155A, bile acid derivatives or their salts are used as surfactants, which can have a certain effect of releasing triglycerides in lipoproteins, but surfactants can only release a very small amount of triglycerides from lipoproteins, and the effect of dissociation and release of triglycerides from lipoproteins is still poor, and it cannot significantly improve the signal strength, sensitivity and accuracy of electrochemical detection of triglycerides.

[0004] Therefore, it is particularly necessary to explore new reagents that can be used as dissociation agents, or combinations of dissociation reagents with better 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 blood lipid dissociating agent and a blood lipid biosensor.

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

[0007] The blood lipid dissociating agent provided by the present invention further comprises bile acid or its derivatives or salts thereof, and / or a surfactant;

[0008] The bile acid or its derivative or salt thereof includes: one or more of bile acid, sodium cholate, taurocholic acid, glycocholic acid, lithocholic acid, deoxycholic acid, CHAPS, CHAPSO, BIGCHAP and / or deoxy BIGCHAP;

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

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

[0011] Furthermore,

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

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

[0014] Furthermore,

[0015] The blood 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 (dissociating agent 1); or,

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

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

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

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

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

[0021] The present invention provides a blood lipid biosensor, which includes at least one working electrode and a reference electrode, and a reagent layer applied to the electrode; the reagent layer includes one or more of the dissociator, enzyme, electron mediator, buffer salt, polymer binder, inorganic salt containing magnesium ions, ATP precipitant and / or stabilizer described in the present invention; the electrode to which the reagent layer is applied can be any one of a working electrode and a reference electrode, and the present invention does not limit this; further, the blood lipid biosensor includes at least one working electrode and a reference electrode, and one working electrode and a reference electrode are the basic structure of the blood lipid biosensor, and the number of working electrodes or reference electrodes can be increased on this basis, and the present invention does not limit this;

[0022] The enzymes include:

[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 derivatives include phenazine methyl sulfate; the phenoxazine derivatives include phenazine-1-carboxylic acid 1,3,4-oxadiazole sulfide derivatives;

[0026] The buffer salt includes: Tris-HCl and / or PIPES;

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

[0028] The inorganic salt containing magnesium ions includes: 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 blood lipid biosensor of the present invention, the reagent layer comprises:

[0032] At least one of lipid dissociating agents 1 or 2, lipoprotein lipase, glycerophosphokinase, glycerophosphooxidase, hexaammineruthenium trichloride, phenazine methylsulfate, Tris-HCl, carboxymethyl cellulose, MgCl2, adenosine triphosphate and BSA; or

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

[0034] At least one of lipid dissociating 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 comprises:

[0036] At least one of blood lipid dissociation agent 1 or 2, 0.1wt%~4wt% lipoprotein lipase, 0.1wt%~4wt% glycerophosphate kinase, 0.1wt%~4wt% glycerophosphate oxidase, 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% adenosine triphosphate and 0.1wt%~5wt% BSA;

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

[0038] or, at least one of lipid dissociating 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 comprises:

[0040] 1wt% sodium cholate, 2wt% CHAPS, 0.5wt% dimethyl glutarate, 0.5wt% dimethyl succinate, 0.5wt% dimethyl adipate, 0.5wt% TX-100, 3wt% lipoprotein lipase, 2wt% glycerophosphokinase, 1wt% glycerophosphooxidase, 2wt% hexaammineruthenium trichloride, 0.05wt% phenazine methylsulfate, 1wt% Tris-HCl, 2wt% carboxymethyl cellulose, 0.5wt% MgCl2, 0.6wt% adenosine triphosphate (ATP) and 0.5wt% BSA; or

[0041] 1wt% sodium cholate, 2wt% CHAPS, 1wt% tributyl phosphate, 0.5wt% TX-100, 3% lipoprotein lipase, 2% glycerophosphokinase, 1% glycerophosphooxidase, 2wt% hexaammineruthenium trichloride, 0.05wt% phenazine methylsulfate, 1wt% Tris-HCl, 2wt% carboxymethyl cellulose, 0.5wt% MgCl2, 0.6wt% adenosine triphosphate (ATP) and 0.5wt% BSA; or

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

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

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

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

[0046] In the present invention, the solvent of the reagent layer is water.

[0047] The present invention provides a completely different blood lipid dissociation agent from the prior art, wherein the blood lipid dissociation agent uses an ester organic solvent as a main component to achieve the dissociation of cholesterol and triglycerides in lipoproteins. In a specific embodiment of the present invention, by comparing the detection of cholesterol and triglycerides using a reagent layer containing an ester organic solvent dissociation agent and a reagent layer containing an ester organic solvent dissociation agent, it is found that the electrochemical signal intensity of the dissociation and determination of cholesterol and triglycerides in lipoproteins by adding an ester organic solvent dissociation agent is greater and more sensitive; further, by different types of ester organic solvents added, it is found that 0.5wt% dimethyl glutarate, 0.5wt% dimethyl succinate, and 0.5wt% dimethyl adipate as dissociation agents are used in combination with other reagents to further improve the dissociation and detection performance of cholesterol and triglycerides in samples.

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

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

[0050] The present invention provides application of the dissociating agent and / or the biosensor in blood lipid detection.

[0051] The present invention provides a method for detecting blood lipids, which comprises using the dissociation agent of the present invention and / or the biosensor of the present invention to detect a sample.

[0052] The present invention provides a method for dissociating blood lipids from lipoproteins by using ester organic solvents as the main component of the dissociation agent. Experimental results show that the ester organic solvents as the main component of the dissociation agent have a good dissociation effect. The reagent layer containing the dissociation agent is loaded on an electrode for the detection of blood lipids, and the detection signal is strong and the sensitivity is high. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 The linearity comparison of implementations 1, 2, and 3 for triglyceride detection is shown;

[0054] Figure 2 The linearity comparison of implementations 1, 2, and 3 for total cholesterol testing was performed;

[0055] Figure 3 Linearity comparison of implementations 1, 2, and 3 for HDL-C testing. DETAILED DESCRIPTION

[0056] The present invention provides a blood lipid dissociation agent and a blood lipid biosensor. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0057] In the present invention, the main component of the blood lipid dissociating agent is an ester organic solvent. The present invention is the first to use an ester organic solvent for the dissociation of blood lipids in ester proteins; in a specific embodiment, the lipid organic solvent may include: one or more of dimethyl glutarate, dimethyl succinate, dimethyl adipate, trimethyl phosphate, triethyl phosphate, tributyl phosphate and / or β-propiolactone. Exemplarily, the present invention uses a mixture of three ester organic solvents including dimethyl glutarate, dimethyl succinate and dimethyl adipate and a dissociator containing only TNBP (tributyl phosphate) for the dissociation of blood lipids in ester proteins; the present invention provides a blood lipid biosensor comprising at least one working electrode and a reference electrode, and a reagent layer loaded on the electrode; the reagent layer includes one or more of the dissociator, enzyme, electron mediator, buffer salt, polymer binder, inorganic salt containing magnesium ions, ATP, precipitant and / or stabilizer described in the present invention; the blood lipid sensor is used for the dissociation and determination of blood lipids in ester proteins, has a high dissociation efficiency, and is manifested in the reagent test as a relatively higher electrochemical reaction current signal intensity, and a relatively higher electrochemical reaction current signal intensity represents the best sensitivity.

[0058] In a specific embodiment of the present invention, the blood lipid dissociating agent includes as main components an ester organic solvent, bile acid or its derivatives or salts thereof, and / or a surfactant;

[0059] Wherein, in a specific embodiment, bile acid or its derivative or salt thereof can be: one or more of bile acid, sodium cholate, taurocholic acid, glycocholic acid, lithocholic acid, deoxycholic acid, CHAPS, CHAPSO, BIGCHAP, deoxy BIGCHAP. Surfactant can be: one or more of Triton series (TX series), sodium n-dodecyl sulfate, fatty alcohol polyoxyethylene lauryl ether, polyethylene glycol octylphenyl ether, aryloxy polyoxyethylene ether, fatty alcohol polyoxyethylene sodium sulfate, sodium stearate, sucrose succinate, AEO-3, AEO-9, Tween-20, Tween-80 and / or cetylpyridinium chloride; the Triton series (TX series) can be one or more of TX-100, TX-35 and / or TX-10;

[0060] Exemplary, in a specific embodiment of the present invention, the bile acid or its derivative or salt is sodium cholate and CHAPS; the final concentration or the concentration of the bile acid or its derivative or salt 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%, 0.2 %, 0.2wt%, 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% and the like;

[0061] Exemplarily, in a specific embodiment of the present invention, the surfactant is TX-100; the final concentration or the concentration during the reaction of the surfactant can be any value between 0.01wt% and 3wt%, and can be 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%, 0.23wt%, 0.24wt%, 0.25wt%, 0.26wt%, 0.27wt%, 0.28wt%, 0.29wt%, 0.30wt%, 0.31wt%, 0.32wt%, 0.33wt%, 0.34wt%, 0.35wt%, 0.36wt%, 0.37wt%, 0.38wt%, 0.39wt%, 0.40wt%, 0.41wt%, 0.42wt%, 0.43wt%, 0.44wt%, 0.45wt%, 0.46wt%, 0.47wt%, 0.48wt%, 0.49wt%, 0.50wt%, 0.51wt%, 0.52wt%, 0.5 %. 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% and the like;

[0062] The bile acid or its derivative or salt, the surfactant and the ester organic solvent are combined to obtain a dissociating agent; the ester organic solvent is the main component of the dissociating agent described in the present invention. Exemplarily, in the specific embodiment of the present invention, there are two specific situations; as the first specific situation, the ester organic solvent is TNBP; as another specific situation, the ester organic solvent is dimethyl glutarate, dimethyl succinate, and dimethyl adipate. The use concentration of the ester organic solvent or the concentration during the reaction can be any value between 0.01wt% and 1.5wt%, and can be 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. 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%;

[0063] The blood 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 dissociation agent, enzyme, electron mediator, buffer salt, polymer binder, inorganic salt containing magnesium ions, ATP and / or stabilizer described in the present invention;

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

[0065] Exemplary, in a specific embodiment of the present invention, the enzyme is lipoprotein lipase, glycerophosphokinase, or glycerophosphooxidase; the concentration of the enzyme used or during the reaction can be any value between 0.1wt% and 4wt%, and specifically can be 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% and the like;

[0066] Exemplary, in a specific embodiment of the present invention, the enzyme is cholesterol esterase, cholesterol oxidase and peroxidase; the enzyme is used at a concentration of 0.1wt% to 4wt%, or at a concentration during the reaction of 0.1wt% to 4wt%, and specifically may be 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% and the like;

[0067] Exemplarily, in a specific embodiment of the present invention, the electron mediator is hexaammineruthenium trichloride, phenazine methyl sulfate and / or potassium ferrocyanide; the concentration of the electron mediator used or during the reaction can be any value between 0.1wt% and 5wt%, and specifically can be 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%, 0.7 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% and the like;

[0068] Exemplarily, 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%, and specifically can be 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%, 0.23wt%, 0.24wt%, 0.25wt%, 0.26wt%, 0.27wt%, 0.28wt%, 0.29wt%, 0.30wt%, 0.31wt%, 0.32wt%, 0.33wt%, 0.34wt%, 0.35wt%, 0.36wt%, 0.37wt%, 0.38wt%, 0.39wt%, 0.40wt%, 0.41wt%, 0.42wt%, 0.43wt%, 0.44wt%, 0.45wt%, 0.46wt%, 0.47wt%, 0.48wt%, 0.49wt%, 0.50wt%, 0.51wt%, 0.52wt%, 0.53wt%, 0.54wt%, 0.55wt%, 0.56wt%, 0.57wt%, 0.58wt%, 0.59wt%, 0.60wt%, 0.61wt%, 0.62wt%, 0.63wt%, 0.64wt%, 0.65wt%, .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% and the like;

[0069] Exemplarily, in a specific embodiment of the present invention, the polymer binder is carboxymethyl cellulose; the final concentration or the concentration during the reaction of the polymer binder can be any value between 0.01wt% and 3wt%, and can be 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%, 0.23wt%, 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% and the like;

[0070] Exemplarily, 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 or the concentration during the reaction can be any value between 0.01wt% and 2wt%, and can be 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%, 0.23wt%, 0.24wt%, 0.25wt%, 0.26wt%, 0.27wt%, 0.28wt%, 0.29wt%, 0.30wt%, 0.31wt%, 0.32wt%, 0.33wt%, 0.34wt%, 0.35wt%, 0.36wt%, 0.37wt%, 0.38wt%, 0.39wt%, 0.40wt%, 0.41wt%, 0.42wt%, 0.43wt%, 0.44wt%, 0.45wt%, 0.46wt%, 0.47wt%, 0.48wt%, 0.49wt%, 0.50wt%, 0.51wt%, 0.52wt%, 0.5 %, 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%;

[0071] For example, in a specific embodiment of the present invention, the concentration of adenosine triphosphate ATP used or the concentration during the reaction can be any value between 0.01wt% and 2wt%, and can be 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%, 0.23wt%, 0.24wt%, 0.25wt%, 0.26wt%, 0.27wt%, 0.28wt%, 0.29wt%, 0.30wt%, 0.31wt%, 0.32wt%, 0.33wt%, 0.34wt%, 0.35wt%, 0.36wt%, 0.37wt%, 0.38wt%, 0.39wt%, 0.40wt%, 0.41wt%, 0.42wt%, 0.43wt%, 0.44wt%, 0.45wt%, 0.46wt%, 0.47wt%, 0.48wt%, 0.49wt%, 0.50wt%, 0.51wt%, 0.52wt%, 0.5 %, 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%;

[0072] Exemplarily, in a specific embodiment of the present invention, the concentration of the phosphotungstic acid used or the concentration during the reaction can be any value between 0.01wt% and 2wt%, and can be 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% and the like;

[0073] Exemplarily, 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%, and specifically can be 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% 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%;

[0074] In a specific embodiment of the present invention, the reagent layer composed as above is spot-coated on a screen-printed carbon electrode (the carbon electrode may be at least one of a working electrode and / or a reference electrode), and then dried at 50°C. After being affixed with double-sided tape and a hydrophilic film, it is compacted with a steel roller and cut with a rolling knife into the size of each electrochemical test strip, exemplarily 35mm×6mm. The test strip is inserted into the test instrument and automatically turned on, and whole blood samples of blood lipids of different concentrations are added to the siphon channel (sample receiving port).

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

[0076] As used in the embodiments of the present application and the appended claims, the singular forms "a," "an," "said," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0077] The use of the terms "include", "have" or "contain", including their grammatical synonyms, should generally be understood as open and non-restrictive, for example, not excluding other unrecited elements or steps, unless otherwise specifically stated or otherwise understood from the context. The use of any and all examples or exemplary language herein, such as "for example" or "including", is intended only to better illustrate the present invention and does not limit the scope of the present invention unless claims are presented. No language in this specification should be interpreted as indicating that any non-claimed element is essential to the practice of the present invention.

[0078] In addition, 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 accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, quantities, values ​​and percentages used in this disclosure are modified by "about". Here, "about" 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 the present invention, functionally, the reagent layer includes a blood lipid dissociation reagent, a blood lipid hydrolysis reagent, and an electro-conversion reagent; in the present invention, the blood lipid dissociation reagent is responsible for dissociating blood lipids from lipoproteins; the blood lipid hydrolysis reagent and the electro-conversion reagent are responsible for decomposing the blood lipid enzymatic reaction process into detectable intermediates; the present invention uses an electrochemical method to detect blood lipids, and the specific reaction process steps can be arbitrarily as follows:

[0080] The dissociating agent rapidly dissociates blood lipids from lipoproteins, and then the enzymatic reaction of the blood lipids is decomposed into detectable intermediate products through the enzymatic reaction of the reagent layer (blood lipid hydrolysis reagent and electroconversion reagent); in the detection of triglycerides, the enzymatic reaction includes: lipoprotein lipase (LPL, or also known as lipoprotein lipase) hydrolyzes triglycerides into glycerol and fatty acids, then, under the action of glycerol phosphate kinase, glycerol reacts with ATP and magnesium ions to generate 3-phosphoglycerol, then, 3-phosphoglycerol is oxidized into dihydroxyacetone phosphate under the action of glycerol phosphate oxidase, and the ADP and reduced electron mediator generated at the same time 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 cholest-4-en-3-one (Cholest-4-en-3-one), and hydrogen peroxide is generated at the same time, and peroxidase is directly used for the redox reaction of hydrogen peroxide in electrochemical detection.

[0081] After the enzymatic reaction, the generated reduced electron mediator (specific electron transfer 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 embodiments, the method for detecting blood lipids includes providing an electrochemical test strip and placing the electrochemical test strip in an analyzer, measuring the current provided through the blood sample and the electrochemical test strip; and calculating the level of blood lipids using the analyzer based on the current. Optionally, the test strip includes a working electrode, a reference electrode, and a reagent layer applied to the electrode; the reagent layer can further optimize the pH and the concentration of each substance in the reagent layer to improve the accuracy of blood lipid determination.

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

[0084] The test materials used in the present invention are all common commercial products and can be purchased on the market. The present invention is further described below in conjunction with the embodiments:

[0085] Example 1 Triglyceride Detection

[0086] Implementation 1: No organic ester solvent is added to the reagent layer

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

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

[0089] The reagent layer includes: based on "Implementation 1", 1 wt % of tributyl phosphate is additionally added.

[0090] Implementation 3: Adding multiple organic ester solvents to the reagent layer

[0091] The reagent layer includes: based on "Implementation 1", 0.5wt% of dimethyl glutarate, 0.5wt% of dimethyl succinate, and 0.5wt% of dimethyl adipate are additionally added.

[0092] Implementation cases 1, 2 and 3 were prepared into solutions according to the above formula, spot-coated on the screen-printed carbon electrode, and then dried at 50°C. After affixing double-sided tape and a hydrophilic film, they were compacted with a steel roller and cut with a rolling knife into electrochemical test strips of 35 mm × 6 mm in size each. The test strips were inserted into the test instrument and automatically turned on, and whole blood samples of different concentrations of blood lipids were added to the siphon channel.

[0093] The electrochemical test strip can be tested at a test voltage in the range of 100-1000 mV. The electrochemical test strips in the embodiments described in the present application all use a voltage of 200 mV.

[0094] The results of implementation scenarios 1, 2 and 3 are as follows Figure 1 As shown, the linear slope of implementation case 3 is the largest, and the electrochemical reaction current signal intensity is relatively higher under the same concentration, which means the sensitivity is the best.

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

[0096] Example 2 Total cholesterol detection

[0097] Implementation 1: No organic ester solvent is added to the reagent layer

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

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

[0100] The reagent layer includes: based on "Implementation 1" additionally added with 1wt% tributyl phosphate.

[0101] Implementation 3: Adding multiple organic ester solvents to the reagent layer

[0102] The reagent layer includes: based on "Implementation 1", 0.5wt% of dimethyl glutarate, 0.5wt% of dimethyl succinate, and 0.5wt% of dimethyl adipate are additionally added.

[0103] The solutions of implementation cases 1, 2 and 3 were prepared according to the above formula, and were applied on the screen-printed carbon electrode, and then dried at 50°C. After the double-sided adhesive and hydrophilic film were attached, the steel roller was compacted and cut with a roller into each electrochemical test strip with a size of 35mm×6mm. The test strip was inserted into the test instrument and automatically turned on, and whole blood samples of blood lipids of different concentrations were added to the siphon channel. The electrochemical test strip can be tested at a test voltage range of -1000~0mV. The electrochemical test strips in the embodiments described in this application all use a voltage of -250mV, and the voltage of the working electrode relative to the counter electrode is positive at -250mV.

[0104] The results of implementation scenarios 1, 2 and 3 are as follows Figure 2 As shown, the linear slope of implementation case 3 is the largest, and the electrochemical reaction current signal intensity is relatively higher under the same concentration, which means the sensitivity is the best.

[0105] Example 3 High-density lipoprotein cholesterol (HDL-C) detection

[0106] Implementation 1: No organic ester solvent is added to the reagent layer

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

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

[0109] The reagent layer includes: based on "Implementation 1" additionally added with 1wt% tributyl phosphate.

[0110] Implementation 3: Adding multiple organic ester solvents to the reagent layer

[0111] The reagent layer includes: based on "Implementation 1", 0.5wt% of dimethyl glutarate, 0.5wt% of dimethyl succinate, and 0.5wt% of dimethyl adipate are additionally added.

[0112] The solutions of implementation cases 1, 2 and 3 were prepared according to the above formula, and were applied on the screen-printed carbon electrode, and then dried at 50°C. After the double-sided adhesive and hydrophilic film were attached, the steel roller was compacted and cut with a roller into each electrochemical test strip with a size of 35mm×6mm. The test strip was inserted into the test instrument and automatically turned on, and whole blood samples of blood lipids of different concentrations were added to the siphon channel. The electrochemical test strip can be tested at a test voltage range of -1000~0mV. The electrochemical test strips in the embodiments described in this application all use a voltage of -250mV, and the voltage of the working electrode relative to the counter electrode is positive at -250mV.

[0113] The results of implementation scenarios 1, 2 and 3 are as follows Figure 3 As shown, the linear slope of implementation case 3 is the largest, and the electrochemical reaction current signal intensity is relatively higher under the same concentration, which means the sensitivity is the best.

[0114] In summary, according to the tests of the above three different detection indicators, the use of the lipid dissociator in the present application can release the non-free lipids distributed in the lipoproteins to the greatest extent, forming free lipids; in this way, the lipid concentration participating in the enzyme reaction is higher than before the use of the dissociator, and the signal intensity of the enzyme electrochemical reaction is greater and the sensitivity is higher.

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

[0116] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A blood lipid dissociating agent, characterized in that: Contains an ester organic solvent; the ester organic solvent includes: one or more of dimethyl glutarate, dimethyl succinate, dimethyl adipate, trimethyl phosphate, triethyl phosphate, tributyl phosphate and β-propiolactone.

2. The blood lipid dissociating agent according to claim 1, characterized in that It also includes bile acid or its derivatives or salts thereof, and / or surfactants.

3. The blood lipid dissociating agent according to claim 2, characterized in that The bile acid or its derivative or salt thereof includes one or more of bile acid, sodium cholate, taurocholic acid, glycocholic acid, lithocholic acid, deoxycholic acid, CHAPS, CHAPSO, BIGCHAP and deoxy BIGCHAP.

4. The blood lipid dissociating agent according to claim 2, characterized in that The surfactant includes: one or more of Triton series, sodium n-dodecyl sulfate, fatty alcohol polyoxyethylene lauryl ether, polyethylene glycol octylphenyl ether, aryloxy polyoxyethylene ether, fatty alcohol polyoxyethylene sodium sulfate, sodium stearate, sucrose decanoate, AEO-3, AEO-9, Tween-20, Tween-80 and cetylpyridinium chloride.

5. The blood lipid dissociating agent according to any one of claims 2 to 4, characterized in that The blood 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 blood lipid dissociating agent comprises: 0.01wt%~3wt% sodium cholate, 0.01wt%~3wt% CHAPS, 0.01wt%~1.5wt% tributyl phosphate and 0.01wt%~3wt% TX-100.

6. A blood lipid biosensor comprising at least one working electrode and a reference electrode, and a reagent layer applied to the electrodes, characterized in that: The reagent layer comprises the dissociation agent according to any one of claims 2 to 5, and further comprises one or more of an enzyme, an electron mediator, a buffer salt, a polymer binder, an inorganic salt containing magnesium ions, ATP, a precipitant and a stabilizer.

7. The blood lipid biosensor according to claim 6, characterized in that: The enzymes include: A combination of lipoprotein lipase, glycerophosphate kinase, and glycerophosphate oxidase; or a combination of cholesterol esterase, cholesterol oxidase and peroxidase.

8. The blood lipid biosensor according to claim 6, characterized in that: The electron mediator includes one or more of potassium ferrocyanide, potassium ferrocyanide, hexaammineruthenium trichloride, phenazine derivatives and phenoxazine derivatives.

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

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