Oxidized Low-Density Lipoprotein Detection Method Based on Stabilizers and Blocking Agents

Abstract

The present application discloses a method for detecting oxidized low-density lipoprotein based on a stabilizer and a blocker, which relates to the field of macromolecular compounds. The method includes: copolymerizing a hydrophilic monomer, a hydrophobic monomer and a monomer containing an antioxidant functional group to form a multifunctional polymer stabilizer, characterizing its structure and properties by nuclear magnetic resonance spectroscopy, gel permeation chromatography and dynamic light scattering method, and optimizing the monomer composition through molecular dynamics simulation; based on the stabilizer, preparing a sample reaction solution; collecting a serum sample, and preparing a sample for detecting oxidized low-density lipoprotein through the stabilization treatment of the sample reaction solution; using a monoclonal antibody against oxidized low-density lipoprotein labeled with latex microspheres as an antigen capturer, incubating and reacting with the sample, calculating the difference in absorbance values before and after the reaction, and calculating the concentration of oxidized low-density lipoprotein according to the standard curve. The present application can improve the stability of ox-LDL samples and can also be used as an efficient blocker to reduce non-specific binding.

Description

Technical Field

[0001] This application relates to the field of high molecular compounds, and particularly to a method for detecting oxidized low-density lipoprotein based on a stabilizer and a blocker. Background Art

[0002] Oxidized low-density lipoprotein (ox-LDL), as an important marker of atherosclerosis, its detection is of great significance in the prevention, diagnosis, and treatment of cardiovascular diseases. In the prior art, the methods for detecting ox-LDL mainly include enzyme-linked immunosorbent assay (ELISA), chemiluminescence immunoassay (CLIA), radioimmunoassay (RIA), etc. Although these methods have achieved certain results in clinical applications, they still face problems such as poor sample stability, insufficient detection specificity, and limited sensitivity.

[0003] Specifically, the molecular structure of ox-LDL is complex and it is prone to further oxidation or degradation in the in vitro environment, resulting in inaccurate detection results. At the same time, due to the similar molecular structures of ox-LDL and other lipoprotein molecules, cross-reactions often occur in the existing detection methods, affecting the detection specificity. In addition, traditional blockers (such as bovine serum albumin BSA) have limited effects in reducing non-specific binding and are difficult to meet the requirements of high-sensitivity detection.

[0004] Therefore, there is an urgent need for a technical solution that can improve the stability of ox-LDL samples and also act as an efficient blocker to reduce non-specific binding. Summary of the Invention

[0005] To solve the deficiencies of the prior art, the embodiments of this application provide a method for detecting oxidized low-density lipoprotein based on a stabilizer and a blocker. This application solves the technical problems such as poor sample stability and insufficient detection specificity in the prior art.

[0006] The embodiments of this application provide a method for detecting oxidized low-density lipoprotein based on a stabilizer and a blocker, including: copolymerizing a hydrophilic monomer, a hydrophobic monomer, and a monomer containing an antioxidant functional group to form a multifunctional polymer stabilizer, characterizing its structure and properties by nuclear magnetic resonance spectroscopy, gel permeation chromatography, and dynamic light scattering, and optimizing the monomer composition through molecular dynamics simulation; based on the stabilizer, adding a blocker, a surfactant, a flocculant, a metal ion chelator, and a preservative to prepare a sample reaction solution; collecting a serum sample, and preparing a sample for detecting oxidized low-density lipoprotein through the stabilization treatment of the sample reaction solution; using a monoclonal antibody against oxidized low-density lipoprotein labeled with latex microspheres as an antigen capturer, incubating with the sample treated with the reaction solution containing the stabilizer and the blocker, calculating the difference in absorbance values before and after the reaction, and calculating the concentration of oxidized low-density lipoprotein according to the standard curve.

[0007] In one possible implementation, based on a stabilizer, a blocker, a surfactant, a flocculant promoter, a metal ion chelator, and a preservative are added to prepare a sample reaction solution, including:

[0008] The sample reaction solution contains 20 mmol / L Tris buffer, 0.5 g / L stabilizer, 10 g / L sodium chloride, 20 mmol / L disodium ethylenediaminetetraacetate, 0.01%-0.05% surfactant, 4 g / L-10 g / L flocculant promoter, 0.05-0.2 g / L blocker, 0.05% proclin 300, and the pH value is 4.0-6.5; the blocker is an active blocker, specifically HBR-X or HBR-ULTRA; the surfactant is Triton X-100 or Tween-20; the flocculant promoter is PEG6000 or PEG8000.

[0009] In one possible implementation, a serum sample is collected, and a sample for oxidized low-density lipoprotein detection is prepared through stabilization treatment with the sample reaction solution, including: collecting a blood sample, separating the serum, dispensing the separated serum into a container and storing it at low temperature; mixing and diluting the serum sample with the sample reaction solution to a target concentration range so that it is within the linear range of detection; incubating at 37°C for 5 min to allow the stabilizer to coat the oxidized low-density lipoprotein particles.

[0010] In one possible implementation, a monoclonal antibody against oxidized low-density lipoprotein labeled with latex microspheres is used as an antigen capturer, which is incubated with a sample treated with a reaction solution containing a stabilizer and a blocker, and the difference in absorbance values before and after the reaction is calculated. The concentration of oxidized low-density lipoprotein is calculated according to a standard curve, including: using a monoclonal antibody against oxidized low-density lipoprotein labeled with double-sized latex microspheres as an antigen capturer, adding it to a sample treated with a sample reaction solution containing a stabilizer, and performing an incubation reaction for 3 min; latex microspheres of different sizes are cross-linked together after the formation of the antigen-antibody complex, recording the absorbance value A0 at the start of the reaction and the absorbance value A1 after 3 min of the incubation reaction, and calculating the difference in absorbance values DA before and after the reaction; drawing a standard curve based on the standard product concentration and the difference in absorbance values, and calculating the concentration of oxidized low-density lipoprotein in the sample.

[0011] In one possible implementation, a predetermined amount of hydrophilic monomers, hydrophobic monomers, and monomers containing antioxidant functional groups are mixed, an organic solvent is added, inert gas is introduced to remove oxygen, a radical initiator is added, and radical copolymerization reaction is carried out at a set temperature for a predetermined time, including: adding a predetermined amount of polyethylene glycol methacrylate monomers, dodecyl methacrylate monomers, and vitamin E methacrylate monomers into a reaction vessel; adding a predetermined amount of tetrahydrofuran solvent into the reaction vessel and introducing inert gas for deoxygenation treatment; adding a predetermined amount of azobisisobutyronitrile initiator into the deoxygenated reaction mixture; placing the reaction vessel in a constant temperature heating device for polymerization reaction, and maintaining inert gas protection throughout the reaction process.

[0012] In one possible implementation, the reaction solution is poured into an excessive precipitant to precipitate the target product, the precipitate is collected by centrifugation, washed repeatedly with the precipitant, and dried to a constant weight under reduced pressure to obtain a multifunctional polymer stabilizer, including: pouring the reaction solution into an excessive aprotic organic solvent to precipitate the polymer product; collecting the obtained precipitate by centrifugation and washing it three times with the aprotic organic solvent; placing the washed product in a vacuum drying device and drying it at a lower temperature for a preset time to obtain the target multifunctional polymer stabilizer.

[0013] In one possible implementation, the method for calculating the coating efficiency of the stabilizer includes: , where represents the coating efficiency of the stabilizer, represents the time-dependent reaction rate constant of the stabilizer concentration, and its value range is 0.11–0.72 min -1 ·(mL / mg), represents the stabilizer concentration, represents the coating time, represents the reaction rate constant related to the molecular weight and hydrophilicity of the stabilizer, and its value range is 10 -5 - 10 -3 mol / g, represents the molecular weight of the stabilizer, represents the hydrophilicity influence factor, represents the hydrophilicity index of the stabilizer.

[0014] The embodiments of the present application also provide an oxidized low-density lipoprotein detection device, including: a processor, a memory, and a system bus; wherein, the processor and the memory are connected through the system bus; the memory is used to store one or more programs, and the one or more programs include instructions, and when the instructions are executed by the processor, the processor executes the method according to any one of the above embodiments.

[0015] In the method and device for detecting oxidized low-density lipoprotein based on a stabilizer and a blocker provided above, in the embodiments of the present application, by synthesizing an amphiphilic copolymer with multiple functions, the stability of the ox-LDL sample can be improved, and it can also be used as an efficient blocker to reduce non-specific binding. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] To more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present application. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0017] Figure 1 It is a schematic flow chart of a method for detecting oxidized low-density lipoprotein based on a stabilizer and a blocker provided by an embodiment of the present application;

[0018] Figure 2 It is a schematic diagram of a simulation trajectory of molecular dynamics provided by an embodiment of the present application;

[0019] Figure 3 It is a schematic diagram of the coating efficiency of a stabilizer provided by an embodiment of the present application. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0020] Now, various exemplary embodiments of the present application will be described in detail with reference to the drawings. It should be noted that: Unless otherwise specifically stated, the relative arrangements, numerical expressions, and numerical values of the components and steps described in these embodiments do not limit the scope of the present application.

[0021] Those skilled in the art can understand that terms such as "first" and "second" in the embodiments of the present application are only used to distinguish different steps, devices or modules, etc., and neither represent any specific technical meaning nor indicate an inevitable logical order between them. It should also be understood that in the embodiments of the present application, "a plurality" may refer to two or more, and "at least one" may refer to one, two or more. It should also be understood that for any component, data or structure mentioned in the embodiments of the present application, without clear limitation or contrary indication in the context, it can generally be understood as one or more. In addition, the term "and / or" in the present application is merely a description of the association relationship of associated objects, indicating that three relationships can exist. For example, A and / or B can represent: A exists alone, A and B exist simultaneously, and B exists alone. In addition, the character " / " in the present application generally represents an "or" relationship between the associated objects before and after. It should also be understood that the description of each embodiment in the present application emphasizes the differences between the embodiments, and their similarities can be referred to each other. For the sake of brevity, they will not be elaborated one by one.

[0022] Meanwhile, it should be understood that for the sake of description, the sizes of the various parts shown in the drawings are not drawn in actual proportional relationships. The following description of at least one exemplary embodiment is actually merely illustrative and in no way restricts the present application or its application or use. Technologies, methods and devices known to those of ordinary skill in the relevant art may not be discussed in detail, but where appropriate, the said technologies, methods and devices should be regarded as part of the description. It should be noted that: like reference numerals and letters denote like items in the following drawings, and thus, once an item is defined in one drawing, it need not be further discussed in subsequent drawings.

[0023] To make the objectives, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are some but not all of the embodiments of the present application. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present application without creative efforts fall within the scope of protection of the present application.

[0024] Figure 1Schematic flow chart of a method for detecting oxidized low-density lipoprotein based on a stabilizer and a blocker provided by an embodiment of the present application. The purpose of this method is to solve the technical problems such as poor stability and insufficient specificity existing in the existing detection methods for oxidized low-density lipoprotein (ox-LDL). The main innovation of the embodiment of the present application lies in the development of a multifunctional polymer stabilizer, which can not only improve the stability of ox-LDL samples, but also act as a blocker to reduce non-specific binding, thereby improving the accuracy and sensitivity of detection.

[0025] The oxidized low-density lipoprotein refers to low-density lipoprotein that has undergone oxidative modification and plays an important role in the occurrence and development of cardiovascular diseases such as atherosclerosis. The detection of ox-LDL is of great significance for evaluating the risk of cardiovascular diseases. However, due to the instability of ox-LDL and the limitations of detection methods, there are some problems in the existing ox-LDL detection methods, such as poor sample stability and insufficient detection specificity, which affect the accuracy and reliability of detection results.

[0026] The detection method provided by the embodiment of the present application includes but is not limited to the following key steps: First, design and synthesize an amphiphilic copolymer stabilizer. The copolymer is composed of a hydrophilic monomer and a hydrophobic monomer copolymerized to form a polymer chain with an amphiphilic structure. Among them, the hydrophilic monomer can be but is not limited to polyethylene glycol methacrylate, and the hydrophobic monomer can be but is not limited to dodecyl methacrylate. This amphiphilic structure enables the stabilizer to interact with both the lipid and protein parts on the surface of ox-LDL particles simultaneously, forming a stable coating layer, thereby improving the stability of ox-LDL samples.

[0027] Second, introduce antioxidant functional groups, such as but not limited to vitamin E derivatives, on the copolymer molecular chain. These antioxidant functional groups can further improve the stability of ox-LDL samples and prevent them from undergoing further oxidation during storage and detection. The introduction of antioxidant functional groups can be achieved by but is not limited to the following methods: copolymerizing a monomer containing an antioxidant group with a hydrophilic monomer and a hydrophobic monomer; or grafting an antioxidant group onto the copolymer molecular chain by chemical modification methods after copolymer synthesis.

[0028] Third, introduce an epitope-mimicking structure on the copolymer molecular chain that can competitively bind with the detection antibody. This enables the stabilizer to also have the function of a blocker, effectively reducing non-specific binding and improving the specificity of detection. The epitope-mimicking structure can be but is not limited to a mimetic peptide segment of a specific antigenic determinant on the surface of ox-LDL or other structures that can specifically bind to the detection antibody. These structures can be directly introduced into the copolymer molecular chain by chemical synthesis methods or connected to the copolymer through bioconjugation techniques.

[0029] To achieve the best stability and blocking effect, the embodiments of the present application also utilize computational chemistry and molecular dynamics simulation methods to optimize the composition and structure of the copolymer. This includes but is not limited to: simulating the effects of different monomer ratios on the properties of the copolymer; predicting the interaction between the copolymer and ox-LDL; evaluating the optimal distribution of antioxidant functional groups and epitope-mimicking structures in the copolymer, etc. Through these computational simulations, the properties of the copolymer can be predicted and optimized before actual synthesis, thereby improving the R & D efficiency.

[0030] Specifically for the implementation steps, as Figure 1 shown, at step S101, a hydrophilic monomer, a hydrophobic monomer, and a monomer containing an antioxidant functional group are copolymerized to form a multifunctional polymer stabilizer. Nuclear magnetic resonance spectroscopy, gel permeation chromatography, and dynamic light scattering are used to characterize its structure and properties, and the monomer composition is optimized through molecular dynamics simulation. This step can be carried out by methods including but not limited to free radical polymerization, controlled / living free radical polymerization, ionic polymerization, etc. The polymerization reaction can be carried out in a suitable solvent, such as water, an organic solvent, or a mixture thereof. The conditions of the polymerization reaction, such as temperature, time, initiator type, etc., need to be optimized according to the specific monomer combination. The synthesized multifunctional polymer stabilizer needs to be characterized to confirm its structure and properties. The embodiments of the present application use but are not limited to the following methods for characterization: Nuclear magnetic resonance spectroscopy (NMR) is used to determine the chemical structure and composition of the copolymer; Gel permeation chromatography (GPC) is used to measure the molecular weight and its distribution of the copolymer; Dynamic light scattering (DLS) is used to measure the particle size and dispersibility of the copolymer in solution. In addition, other characterization methods, such as Fourier transform infrared spectroscopy (FTIR), ultraviolet-visible spectroscopy (UV-Vis), etc., can also be used to obtain more comprehensive structural information.

[0031] Specifically, it includes: mixing a predetermined amount of a hydrophilic monomer, a hydrophobic monomer, and a monomer containing an antioxidant functional group, adding an organic solvent, purging with an inert gas to remove oxygen, adding a free radical initiator, and carrying out a free radical copolymerization reaction at a set temperature for a predetermined time; pouring the reaction solution into an excess of a precipitating agent to precipitate the target product, collecting the precipitate by centrifugation, washing it repeatedly with the precipitating agent, and drying it to a constant weight under reduced pressure to obtain a multifunctional polymer stabilizer; using nuclear magnetic resonance spectroscopy to determine the composition of the copolymer, using gel permeation chromatography to measure the molecular weight and its distribution, and characterizing the particle size distribution of the copolymer in an aqueous solution through dynamic light scattering; using molecular dynamics simulation methods to simulate the interaction between the copolymer and oxidized low-density lipoprotein under physiological conditions, and optimizing the molar ratio of the monomers by analyzing the simulation trajectory (as Figure 2 shown), to determine the optimal copolymer composition.

[0032] Among them, a predetermined amount of hydrophilic monomer, hydrophobic monomer, and monomer containing an antioxidant functional group are mixed, an organic solvent is added, inert gas is introduced to remove oxygen, a radical initiator is added, and a radical copolymerization reaction is carried out at a set temperature for a predetermined time, including: adding a predetermined amount of polyethylene glycol methacrylate monomer, dodecyl methacrylate monomer, and vitamin E methacrylate monomer into a reaction vessel; adding a predetermined amount of tetrahydrofuran solvent into the reaction vessel, and introducing inert gas for deoxygenation treatment; adding a predetermined amount of azobisisobutyronitrile initiator into the deoxygenated reaction mixture; placing the reaction vessel in a constant temperature heating device for polymerization reaction, and maintaining inert gas protection throughout the reaction process.

[0033] Among them, the reaction solution is poured into an excessive amount of precipitant to precipitate the target product, the precipitate is collected by centrifugation, washed repeatedly with the precipitant, and dried to constant weight under reduced pressure to obtain a multifunctional polymer stabilizer, including: pouring the reaction solution into an excessive amount of aprotic organic solvent to precipitate the polymer product; collecting the obtained precipitate by centrifugation, and washing it three times with the aprotic organic solvent; placing the washed product in a vacuum drying device and drying it at a lower temperature for a preset time to obtain the target multifunctional polymer stabilizer.

[0034] In one embodiment, first, raw material preparation is carried out. Hydrophilic monomer: polyethylene glycol methacrylate (PEGMA, Mn = 500 g / mol); hydrophobic monomer: dodecyl methacrylate (DDMA); antioxidant functional group: α-tocopherol methacrylate (VE-MA); initiator: azobisisobutyronitrile (AIBN); solvent: tetrahydrofuran (THF).

[0035] Synthesis steps: Add PEGMA (5 g, 10 mmol), DDMA (2.54 g, 10 mmol), and VE-MA (0.5 g, 1 mmol) into a 100 mL round-bottom flask. Add 50 mL of THF as the solvent, and introduce nitrogen to remove oxygen for 15 minutes. Add AIBN (0.033 g, 0.2 mmol) as the initiator. React in an oil bath at 65 °C for 24 hours, while maintaining nitrogen protection during the reaction. After the reaction is completed, pour the reaction solution into an excessive amount of ice-cold diethyl ether to precipitate the polymer. Centrifuge to collect the precipitate, and wash it 3 times with diethyl ether. Dry the product in a vacuum drying oven at 40 °C for 24 hours to obtain the final multifunctional polymer stabilizer.

[0036] For characterization, nuclear magnetic resonance hydrogen spectroscopy (1H NMR) can be used: The composition of the copolymer was determined using a Bruker AVANCE III 400 MHz nuclear magnetic resonance spectrometer. Gel permeation chromatography (GPC): The molecular weight and molecular weight distribution were determined using a Waters Alliance e2695 system. Dynamic light scattering (DLS): The particle size distribution of the copolymer in aqueous solution was determined using a Malvern Zetasizer Nano ZS.

[0037] Next, the composition and structure of the copolymer were optimized using computational chemistry and molecular dynamics simulation methods. Molecular dynamics simulations were performed using the GROMACS software package, and the simulation conditions were as follows: Force field: OPLS-AA; Temperature: 310 K (physiological temperature); Pressure: 1 atm; Simulation time: 100 ns; Time step: 2 fs. By analyzing the simulation trajectory, the interaction energy between the copolymer and the surface of the simulated ox-LDL was calculated to optimize the composition ratio of the copolymer. For example, according to the optimization results, the optimal monomer molar ratio was found to be PEGMA: DDMA: VE-MA = 10:10:1.

[0038] In step S102, based on the stabilizer, a blocker, a surfactant, a flocculant promoter, a metal ion chelator, and a preservative were added to prepare a sample reaction solution. The following steps can be adopted but are not limited to:

[0039] The sample reaction solution contains 20 mmol / L Tris buffer, 0.5 g / L stabilizer, 10 g / L sodium chloride, 20 mmol / L disodium ethylenediaminetetraacetate, 0.01%-0.05% surfactant, 4 g / L - 10 g / L flocculant promoter, 0.05 - 0.2 g / L blocker, 0.05% proclin300, and the pH value is 4.0 - 6.5;

[0040] The blocker is an active blocker, and specifically, HBR-X or HBR-ULTRA can be selected; in this embodiment, 0.05 g / L HBR-ULTRA is preferably used;

[0041] The surfactant can be selected from TritonX-100 or Tween-20; in this embodiment, 0.02% TritonX - 100 is preferably used;

[0042] The flocculant promoter can be selected from PEG6000 or PEG8000; in this embodiment, 10 g / L PEG6000 is preferably used;

[0043] Among them, the stabilizer can quickly adsorb on the antigen surface, reduce the further oxidation of the OxLDL antigen, expose the antibody recognition epitope, and facilitate the rapid reaction between the latex microsphere-labeled antibody and the OxLDL antigen.

[0044] In step S103, a serum sample is collected, and through the stabilization treatment of the sample reaction solution, a sample for the detection of oxidized low-density lipoprotein is prepared.

[0045] In this step S103, it specifically includes: collecting a blood sample, separating the serum, and sub-packaging the separated serum into a container for low-temperature storage; mixing and diluting the serum sample with the sample reaction solution to a target concentration range so that it is within the linear range of detection; incubating at 37 °C for 5 min to coat the oxidized low-density lipoprotein particles with the stabilizer.

[0046] The following steps can be adopted but are not limited to: First, collect a whole blood sample from the vein of the subject and obtain serum by centrifugation. Then, dilute the serum sample with a buffer according to a predetermined ratio, and the dilution ratio can be adjusted according to the expected ox-LDL concentration range. Next, add an appropriate amount of multifunctional polymer stabilizer to the diluted serum sample and mix well to allow the stabilizer to fully interact with the ox-LDL in the sample to form a stable complex. Specifically, it includes: collecting a blood sample, separating the serum, and sub-packaging the separated serum into a container for low-temperature storage; mixing and diluting the serum sample with the buffer to a target concentration range so that it is within the linear range of detection; mixing the diluted serum sample with the multifunctional polymer stabilizer solution according to the target volume ratio, and incubating at room temperature to coat the oxidized low-density lipoprotein particles with the stabilizer.

[0047] Among them, the calculation method of the coating efficiency of the stabilizer (as Figure 3 shown) includes: , where represents the coating efficiency of the stabilizer, represents the time-dependent reaction rate constant of the stabilizer concentration, and its value range is 0.11 – 0.72 min -1 ·(mL / mg), represents the stabilizer concentration, represents the coating time, represents the reaction rate constant related to the molecular weight and hydrophilicity of the stabilizer, and its value range is 10 -5 - 10 -3 mol / g, represents the molecular weight of the stabilizer, represents the hydrophilicity influence factor, represents the hydrophilicity index of the stabilizer.

[0048] In an implementation scenario, for the sample pretreatment, first, serum sample collection is required: Use a vacuum blood collection tube to collect 5 mL of fasting venous blood from the subject. After standing at room temperature for 30 minutes, centrifuge at 3000 rpm for 15 minutes to separate the serum. Aliquot the separated serum into 1.5 mL centrifuge tubes and store at -80 °C for later use.

[0049] In step S104, a monoclonal antibody against oxidized low-density lipoprotein labeled with latex microspheres is used as an antigen capture agent, and it is incubated with a sample treated with a reaction solution containing a stabilizer and a blocker. The difference in absorbance values before and after the reaction is calculated, and the concentration of oxidized low-density lipoprotein is calculated according to the standard curve.

[0050] This step S104 specifically includes: using a monoclonal antibody against oxidized low-density lipoprotein labeled with dual-sized latex microspheres as an antigen capture agent, adding it to a sample treated with a sample reaction solution containing a stabilizer, and performing an incubation reaction for 3 min.

[0051] Latex microspheres of different particle sizes are cross-linked together after the formation of the antigen-antibody complex. The absorbance value A0 at the start of the reaction and the absorbance value A1 after 3 min of incubation reaction are recorded, and the difference in absorbance values ΔA before and after the reaction is calculated. A standard curve is plotted based on the standard product concentration and the difference in absorbance values, and the concentration of oxidized low-density lipoprotein in the sample is calculated.

[0052] Example 1: As an example of the sample reaction solution described in the present invention, the components of the sample reaction solution in this example are as follows:

[0053] 20 mmol / L Tris buffer (pH 6.0), 10 g / L sodium chloride, 20 mmol / L disodium ethylenediaminetetraacetate, 0.02% Triton X-100, 10 g / L polyethylene glycol 6000, 0.05% proclin 300.

[0054] Example 2: As an example of the sample reaction solution described in the present invention, the components of the sample reaction solution in this example are as follows:

[0055] 20 mmol / L Tris buffer (pH 6.0), 0.5 g / L stabilizer, 10 g / L sodium chloride, 20 mmol / L disodium ethylenediaminetetraacetate, 0.02% Triton X-100, 10 g / L polyethylene glycol 6000, 0.05% proclin 300.

[0056] Example 3: As an example of the sample reaction solution described in the present invention, the components of the sample reaction solution in this example are as follows:

[0057] 20 mmol / L Tris buffer (pH 6.0), 0.5 g / L stabilizer, 10 g / L sodium chloride, 20 mmol / L disodium ethylenediaminetetraacetate, 0.02% Triton X-100, 10 g / L polyethylene glycol 6000, 0.05 g / L HBR-ULTRA, 0.05% proclin 300.

[0058] Among them, HBR-ULTRA was purchased from Scantibodies Laboratory, Inc., product number: 3KC102.

[0059] Example 4: As an example of the sample reaction solution described in the present invention, the components of the sample reaction solution in this example are as follows:

[0060] 20 mmol / L Tris buffer (pH 6.0), 0.5 g / L stabilizer, 10 g / L sodium chloride, 20 mmol / L disodium ethylenediaminetetraacetate, 0.02% TritonX-100, 10 g / L polyethylene glycol 6000, 0.05 g / L HBR-X, 0.05% proclin300.

[0061] Among them, HBR-X was purchased from Scantibodies Laboratory, Inc., product number: 3KC029.

[0062] Example 5: As an example of the sample reaction solution described in the present invention, the components of the sample reaction solution in this example are as follows:

[0063] 20 mmol / L Tris buffer (pH 6.0), 0.5 g / L stabilizer, 10 g / L sodium chloride, 20 mmol / L disodium ethylenediaminetetraacetate, 0.02% Tween-20, 10 g / L polyethylene glycol 6000, 0.05 g / L HBR-X, 0.05% proclin300.

[0064] Example 6: As an example of the sample reaction solution described in the present invention, the components of the sample reaction solution in this example are as follows:

[0065] 20 mmol / L Tris buffer (pH 6.0), 0.5 g / L stabilizer, 10 g / L sodium chloride, 20 mmol / L disodium ethylenediaminetetraacetate, 0.02% TritonX-100, 10 g / L polyethylene glycol 8000, 0.05 g / L HBR-X, 0.05% proclin300.

[0066] Example 7: As an example of the sample reaction solution described in the present invention, the components of the sample reaction solution in this example are as follows:

[0067] 20 mmol / L Tris buffer (pH 4.2), 0.5 g / L stabilizer, 10 g / L sodium chloride, 20 mmol / L disodium ethylenediaminetetraacetate, 0.02% TritonX-100, 10 g / L polyethylene glycol 8000, 0.05 g / L HBR-X, 0.05% proclin300.

[0068] Example 8: Commonly used surfactants are selected. The surfactant can increase the dispersibility of OxLDL. In this example, two surfactants, Tween-20 and TritonX-100, are used to prepare R1 reaction solutions with their contents of 0.01%, 0.05%, 0.1%, and 0.2% respectively. Then, calibration tests are carried out using calibration products at different concentrations (blank, 5 U / L, 10 U / L, 20 U / L, 40 U / L, 80 U / L, 120 U / L). The test results of each group are shown in Table 1 and Table 2:

[0069] Table 1 Test results of calibration products with different concentrations of Tween-20

[0070]

[0071] Table 2 Test results of calibration products with different concentrations of TritonX-100

[0072]

[0073] It can be seen from the test results of surfactant optimization that: with the increase of the addition amount of Tween-20 and TritonX-100, the reactivity value shows a significant downward trend, indicating that with the increase of the surfactant concentration, its dispersing effect on the antigen weakens, thereby affecting the occurrence of antigen-antibody reaction and resulting in a decreasing trend of the reactivity; from the discrimination degree of each point, with the increase of the surfactant concentration, the discrimination degree of each point increases significantly, but after increasing to more than 0.1%, the discrimination degree of the low value becomes poor, affecting the sensitivity of the product. From the comparison results of the two surfactants, the influence of TritonX-100 on the signal value is slightly less than that of Tween-20, and it can be preferentially selected. Therefore, both Tween-20 and TritonX-100 can be applied to the R1 reaction solution, and their use concentrations are relatively beneficial within the range of 0.01%-0.05% (i.e., 0.1 g / L - 0.5 g / L).

[0074] Example 9: According to the influence of two flocculants, PEG6000 and PEG8000, on the detection results, the dosages of the two flocculants are optimized and tested. The dosages of the flocculants are set at 0.2%, 0.4%, 0.8%, 1%, and 1.5%, and the R1 reaction solution is prepared according to the set dosages. After preparation, calibration tests are carried out with calibration products at each concentration (blank, 5 U / L, 10 U / L, 20 U / L, 40 U / L, 80 U / L, 120 U / L), and the reactivity of each concentration is recorded and the discrimination degree of each concentration point is calculated. The test results are recorded in Table 3 and Table 4 as follows:

[0075] Table 3 Test results of PEG6000

[0076]

[0077] Table 4 Test Results of PEG8000

[0078]

[0079] From the test results of different dosages of EPG6000, it can be seen that when the dosage is below 1% PEG6000, the reactivity decreases to a certain extent, and the discrimination at each point can meet the requirements; when the dosage is 1.5%, the reactivity increases significantly and the discrimination decreases. When the dosage of PEG6000 is 0.1%, the reactivity is the lowest and the discrimination decreases to a certain extent, indicating that the effect of the flocculation promoter is not good. When the content of the flocculation promoter PEG6000 is in the range of 0.4% - 1%, its reactivity and discrimination at each point can meet the test requirements. Therefore, the dosage of PEG6000 should be in the range of 0.4% - 1.0%, that is, 4 g / L - 10 g / L.

[0080] From the test results of different dosages of PEG8000, it can be seen that the reactivity of the calibrator test increases with the increase of the dosage. When the dosage is 1.5%, the discrimination in the high-value region decreases; when the dosage is 0.1%, the high-value discrimination also decreases; when the dosage is in the range of 0.4 - 1.0%, the reactivity and discrimination can meet the test requirements. Therefore, the appropriate dosage of PEG8000 is in the range of 0.4 - 1.0%, that is, 4 g / L - 10 g / L.

[0081] Example 10: The initial screening of the blockers is HBR-X and HBR-ULTRA, and the optimization test of their usage amounts is carried out in this experiment. The optimized dosages of the two blockers are set as 0.02 mg / ml, 0.05 mg / ml, 0.1 mg / ml, and 0.2 mg / ml. The R1 reaction solutions are prepared with the blockers at the above different dosages respectively. After preparation, first, a calibrator with a fixed value result is used for calibration testing, and the calibration reactivity and the discrimination at each point are recorded. Then, 10 clinical samples (assigned values using the Mercodia oxidized LDLELISA Kit) are selected for testing, and the relative deviation analysis is carried out between the test results and the ELISA results. The data are recorded as shown in Table 5 and Table 6 below, and the test results of the two blockers for clinical samples and the deviation analysis are shown in Table 7 and Table 8 below.

[0082] Table 5 Calibration Data of Each Group of HBX-X

[0083]

[0084] Table 6 Calibration Data of Each Group of HBR-ULTRA

[0085]

[0086] Table 7 Test Results of Two Blockers for Clinical Samples

[0087]

[0088] Table 8 Deviation Analysis of Clinical Samples Tested with Two Blockers

[0089]

[0090] The calibration test results of the two blockers show that different dosages of the blockers have relatively little impact on the reactivity of calibration products at various concentrations, and the resolution changes at each point are not obvious. From the test results of clinical samples, it can be seen that when using a blocker at a concentration of 0.02 mg / ml, the test results of some clinical samples are on the high side. When using blockers at concentrations of 0.05 - 0.2 mg / ml, the results of clinical samples are close to the assigned results of the ELISA control group, and the relative deviation meets the clinical use requirements. Therefore, the dosage of blockers HBR-X and HBR-ULTRA should be preferably 0.05 - 0.2 mg / ml. Considering cost factors, a dosage of 0.05 mg / ml is preferably selected for the experiment.

[0091] Example 11: Optimize the pH value of the R1 reaction solution for testing. In the experiment, hydrochloric acid was used to adjust the pH values to 8.4, 7.4, 6.5, 5.0, and 4.0 to obtain R1 reaction solutions with different pH values. After preparation, calibration products at various concentrations (blank, 5 U / L, 10 U / L, 20 U / L, 40 U / L, 80 U / L, 120 U / L) were used for testing. The test results of each group are shown in Table 9 below:

[0092] Table 9 Test Results of Each Group under Different pH Conditions

[0093]

[0094] From the test results at different pH values, it can be seen that within the range of pH 4.0 - 8.4, the reactivity value decreases to a certain extent with the increase of pH value, and the decrease range is relatively small; from the resolution of each point, after the pH value rises above 6.5, the resolution of high-value points gradually decreases, and the resolution becomes worse. Therefore, the more suitable range for the pH value reaction is 4.0 - 6.5.

[0095] Example 12: This example is a detection method for oxidized low-density lipoprotein, including the following content:

[0096] Collect human serum samples, centrifuge at 3000 rpm for 10 minutes to separate the serum. Use an ultrafiltration centrifuge tube (with a molecular weight cut-off of 100 kDa) to concentrate the serum samples. Dilute the samples to an appropriate concentration with PBST buffer (pH 7.4). Prepare a series of ox-LDL standard solutions with concentrations ranging from 0 - 150 U / L.

[0097] Take 3 μL of the standard solution or sample, add it to 180 μL of the sample reaction solution, mix well, incubate at 37 °C for 5 min to obtain a stabilized sample. Then immediately add 60 μL of the double-sized latex microsphere labeling mixture and continue to incubate at 37 °C for 3 min. Use an automatic biochemical analyzer (such as Hitachi 7180) to track and measure the absorbance at 570 nm throughout the incubation reaction process. Then take the absorbance value A0 recorded immediately after adding the modified double-sized latex microspheres and the absorbance value A1 after the 3-min incubation reaction ends, and calculate the absorbance difference (DA) for each concentration standard solution as DA = A1 - A0.

[0098] Data processing can be used to plot a standard curve: with the concentration of the ox-LDL standard as the abscissa and the absorbance difference (DA) as the ordinate, plot a five-parameter Logistic curve. Calculate the concentration of ox-LDL in the sample according to the standard curve equation.

[0099] For each test, quality control products at high, medium, and low concentrations are set. The within-batch coefficient of variation (CV) should be less than 10%, and the between-batch CV should be less than 15%. Detection sensitivity and linear range: Detection sensitivity: 5 U / L. Linear range: 5 - 150 U / L. In addition, all reagents should be fully mixed and restored to room temperature before use. Strictly control the temperature and time of each step to ensure the accuracy and repeatability of the detection.

[0100] Example 9: Select 20 clinical samples and test them according to the test method of Example 8 using the sample reaction solution prepared in Examples 1 - 7 and latex microsphere-labeled oxidized low-density lipoprotein. At the same time, test using the control reagent Oxidized LDL ELISA KIT (product number 10 - 1143 - 01) from Swedish Mercodia AB. Calculate the relative deviation of each example from the control reagent, and the results are shown in the following table: unit U / L

[0101]

[0102] The relative deviation results of each example from the control group are shown in the following table:

[0103]

[0104] For samples numbered 1 - 15, the samples are normal samples. Compared with Example 1, after adding stabilizers in Examples 2 - 7, the test result deviation has been significantly improved; for samples numbered 16 - 20 which are interference samples, compared with Example 2, after adding blockers in Examples 3 - 7, the test result deviation has been significantly improved. Examples 3, 5 - 7 show that surfactants, flocculants, and pH have little effect on the test results.

[0105] In summary, the multifunctional polymer stabilizer described in the embodiments of the present application can not only improve the stability of ox-LDL samples, but also act as a blocker to reduce non-specific binding. This is because the epitope-mimicking structure introduced on the molecular chain of the stabilizer can compete with the detection antibody for binding, thereby reducing the binding of the detection antibody to non-specific sites. This dual-functional design significantly improves the accuracy and sensitivity of the detection.

[0106] Figure 2 This is a schematic diagram of the simulation trajectory of molecular dynamics provided by the embodiments of the present application. The embodiments of the present application relate to a method for detecting oxidized low-density lipoprotein (ox-LDL) based on a stabilizer and a blocker, and one of the key steps is the synthesis and optimization of a multifunctional polymer stabilizer. The figure shows the molecular dynamics simulation trajectory of the interaction between the polymer stabilizer and the surface of ox-LDL, and this simulation is of great significance for understanding and optimizing the performance of the stabilizer.

[0107] Overall, the graph shows how the particles start to move at different initial heights and gradually aggregate towards the surface. It can be seen from the figure that the simulation system contains three different types of monomer units, represented by blue, red, and green respectively. These three colors correspond to the hydrophilic monomer polyethylene glycol methacrylate (PEGMA), the hydrophobic monomer dodecyl methacrylate (DDMA), and the antioxidant-functionalized α-tocopherol methacrylate (VE-MA). The selection of these three monomers reflects the design concept of the multifunctional polymer stabilizer in the embodiments of the present application, that is, to achieve efficient stabilization and protection of ox-LDL by reasonably combining monomers with different functions.

[0108] The simulation trajectory shows the movement of these monomer units in three-dimensional space, and each colored line represents the movement trajectory of a monomer unit during the simulation. From the distribution of the trajectories, it can be observed that different types of monomer units exhibit different movement characteristics and spatial distributions. This difference reflects the different roles of each monomer unit in the polymer chain: PEGMA provides good water solubility and biocompatibility, DDMA imparts a certain hydrophobicity to the polymer to enhance the interaction with ox-LDL, and VE-MA plays an antioxidant protection role in the whole structure.

[0109] The gray plane at the bottom of the figure represents the simulated surface of ox-LDL. It can be observed that the trajectories of many polymer chains tend to approach this surface, indicating that the polymer stabilizer can indeed effectively interact with the surface of ox-LDL. This interaction is the key to achieving the stabilization of ox-LDL, because it can prevent the further oxidation and aggregation of ox-LDL, thereby maintaining its structural integrity and function.

[0110] The simulation system adopted periodic boundary conditions, which can be seen from the continuity of the trajectories in the x and y directions. This treatment method can effectively simulate the behavior of large systems while reducing the consumption of computing resources. The time scale and space scale of the simulation have also been optimized to balance computational efficiency and simulation accuracy.

[0111] From the density and distribution of the trajectories, it can be inferred that the polymer chains show a certain degree of flexibility and dynamics in the solution. This property is crucial for the function of the stabilizer because it allows the polymer chains to adapt to different configurations on the surface of ox-LDL, thus achieving more effective coverage and protection. At the same time, the diversity of the trajectories also reflects the thermodynamic equilibrium state of the system, indicating that the simulation has fully sampled the configuration space.

[0112] By analyzing the spatial distribution and motion characteristics of different types of monomer units, it is possible to evaluate whether the current monomer ratio is optimal. For example, if it is observed that the PEGMA units are overly aggregated or the VE-MA units are unevenly distributed, it may be necessary to adjust the molar ratio of the monomers. This optimization process based on the simulation results is an important part of the design of the polymer stabilizer in the embodiments of this application.

[0113] The simulation results can also be used to calculate the interaction energy between the polymer chain and the surface of ox-LDL. By analyzing the particle density and trajectory characteristics in different regions, the main binding sites between the polymer chain and the surface can be identified. This information can guide further structural optimization, such as adjusting the chain length of the hydrophobic monomers or introducing new functional groups.

[0114] It should be noted that this simulation adopted a simplified Lennard-Jones potential function to describe the interaction between particles. Although this treatment method makes a trade-off between computational efficiency and physical accuracy, it can still capture the key features of the system. In practical applications, more complex force field models, such as the OPLS-AA force field, can be introduced according to needs to obtain more accurate simulation results.

[0115] Generally speaking, this molecular dynamics simulation provides an important theoretical basis for the design and optimization of the multifunctional polymer stabilizer in the embodiments of this application. By visualizing the behavior of the polymer chain and its interaction with the surface of ox-LDL, the working mechanism of the stabilizer can be deeply understood and improved targeted. This computationally assisted design method can not only accelerate the development process but also reduce experimental costs and improve the performance of the final product.

[0116] Figure 3Schematic diagram of the coating efficiency of the stabilizer provided by the embodiments of the present application. The embodiments of the present application provide a sample pretreatment method for the detection of oxidized low-density lipoprotein (ox-LDL), and the key steps include stabilizing the serum sample with a multifunctional polymer stabilizer. Figure 3 It shows the change curve of the coating efficiency of the stabilizer over time, which is one of the core technical points of the embodiments of the present application. From Figure 3 it can be seen that the coating efficiency (SE) of the stabilizer shows a non-linear growth trend over time, growing relatively fast in the initial stage and then gradually slowing down and tending to be stable.

[0117] Figure 3 Three curves are shown in . It can be observed that as increases, the overall curve moves upward, indicating that a higher stabilizer concentration can achieve a higher coating efficiency. This phenomenon is consistent with the theoretical model of the embodiments of the present application, that is, the stabilizer concentration is one of the key factors affecting the coating efficiency. However, it should be noted that too high a stabilizer concentration may lead to an increase in non-specific binding or interference with subsequent detection steps, so the stabilization effect and detection sensitivity need to be weighed in practical applications. As shown in the figure, when = 0.1 mg / mL, a relatively high coating efficiency can be achieved within a reasonable time, which is consistent with the recommended stabilizer concentration in the embodiments of the present application.

[0118] On the time axis, Figure 3 the position of 3 minutes is specially marked. The selection of this time point is of great significance because at this time, all three curves have approached their respective stable levels. This indicates that an incubation time of 3 minutes is sufficient for the stabilizer to fully play its role and achieve effective coating of ox-LDL particles, and the coating rate reaches a basically saturated state within 5 minutes. Although extending the incubation time can slightly improve the coating efficiency, the gain effect is not significant, and it may instead increase the complexity and time cost of sample processing. Therefore, the embodiments of the present application select 3 minutes as the optimal incubation time, which not only ensures sufficient stabilization effect but also takes into account the convenience and efficiency of operation.

[0119] By optimizing these molecular parameters, the embodiments of the present application have designed a multifunctional polymer stabilizer that can not only effectively stabilize the sample but also has the function of blocking non-specific binding, thus simplifying the sample pretreatment process.

[0120] It should be noted that the curves in the figure all show a relatively high final coating efficiency, which can reach more than 80% at most. This feature highlights the high efficiency of the stabilizer used in the embodiments of the present application, and a good stabilization effect can be achieved even at a relatively low concentration. This is not only conducive to reducing the reagent cost, but also minimizes the potential interference of the stabilizer on subsequent detection steps. At the same time, the high-efficiency coating process also means that the ox-LDL particles in the sample can be quickly and comprehensively protected, effectively preventing degradation or denaturation during storage and detection, thereby improving the accuracy and reliability of the detection results.

[0121] The method for calculating the coating efficiency of the stabilizer in the embodiments of the present application not only considers the time and concentration factors, but also introduces the influence of molecular weight and hydrophilicity. This multi-parameter model can more comprehensively describe the kinetic characteristics of the coating process, providing theoretical guidance for the design and optimization of the stabilizer. By adjusting these parameters, the most suitable stabilization strategy can be customized for different types of biomarkers. For example, for easily degradable protein biomarkers, the molecular weight and hydrophilicity of the stabilizer can be increased to provide stronger protection; while for small molecule biomarkers, these parameters can be appropriately reduced to minimize the impact on the properties of the analyte itself.

[0122] Furthermore, the embodiments of the present application also provide an oxidized low-density lipoprotein detection device, including: a processor, a memory, and a system bus; the processor and the memory are connected through the system bus; the memory is used to store one or more programs, and the one or more programs include instructions, and when the instructions are executed by the processor, the processor executes any one of the above methods.

[0123] Furthermore, the embodiments of the present application also provide a computer program product, and when the computer program product runs on a terminal device, the terminal device executes any one of the above methods.

[0124] From the description of the above embodiments, those skilled in the art can clearly understand that all or part of the steps in the above embodiment methods can be implemented by means of software plus a necessary general hardware platform. Based on such an understanding, the technical solution of the present application, in essence, or the part that contributes to the prior art, can be embodied in the form of a software product. The computer software product can be stored in a storage medium, such as ROM / RAM, magnetic disk, optical disc, etc., including several instructions for causing a computer device (which can be a personal computer, a server, or a network communication device such as a media gateway, etc.) to execute the methods described in various embodiments or some parts of the embodiments of the present application.

[0125] It should be noted that the embodiments in this specification are described in a progressive manner. Each embodiment focuses on the differences from other embodiments. For the same or similar parts among the embodiments, reference can be made to each other. For the devices disclosed in the embodiments, since they correspond to the methods disclosed in the embodiments, the description is relatively simple. For related parts, reference can be made to the description in the method part.

[0126] It should also be noted that in the embodiments of the present application, relational terms such as "first" and "second" are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the term "comprising", "including" or any other variant thereof is intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements not only includes those elements, but also includes other elements not explicitly listed, or further includes elements inherent to such process, method, article or device. Without further limitation, an element defined by the statement "including one..." does not exclude the existence of additional identical elements in the process, method, article or device including the said element.

[0127] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present application. Various modifications to these embodiments will be obvious to those skilled in the art. The general principles defined in the embodiments of the present application can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to these embodiments shown in the embodiments of the present application, but will conform to the widest scope consistent with the principles and novel features disclosed in the embodiments of the present application.

Claims

1. A method for detecting oxidized low-density lipoprotein based on stabilizers and blockers, wherein the method is for non-diagnostic purposes and is characterized in that: include: Adding predetermined amounts of polyethylene glycol methacrylate monomer, dodecyl methacrylate monomer, and vitamin E methacrylate monomer into a reaction vessel; Adding a predetermined amount of tetrahydrofuran solvent into the reaction container, and introducing an inert gas to perform deoxygenation treatment; adding a predetermined amount of azobisisobutyronitrile initiator to the deoxygenated reaction mixture; Placing the reaction container in a constant temperature heating device to carry out a polymerization reaction, and maintaining inert gas protection throughout the reaction; The reaction solution is poured into an excess of aprotic organic solvent to precipitate the polymer product; The obtained precipitate is collected by centrifugation and washed three times with an aprotic organic solvent; The washed product is placed in a vacuum drying device and dried at 40° C. for a preset time to obtain the target multifunctional polymer stabilizer; The structure and properties of the target multifunctional polymer stabilizer were characterized by nuclear magnetic resonance spectroscopy, gel permeation chromatography, and dynamic light scattering, and the monomer composition was optimized by molecular dynamics simulation; Based on the stabilizer, a blocking agent, a surfactant, a flocculant, a metal ion chelating agent, and a preservative are added to prepare a sample reaction solution, wherein the sample reaction solution comprises 20 mmol / L Tris buffer, 0.5 g / L stabilizer, 10 g / L sodium chloride, 20 mmol / L disodium ethylenediaminetetraacetic acid, 0.01%-0.05% surfactant, 4 g / L-10 g / L flocculant, 0.05-0.2 g / L blocking agent, 0.05% proclin300, and the pH value is 4.0-6.5; The blocker is an active blocker, and the active blocker is HBR-X or HBR-ULTRA; The surfactant is TritonX-100 or Tween-20; The flocculant is PEG6000 or PEG8000; collecting serum samples, and preparing samples for oxidized low-density lipoprotein detection by stabilizing the sample reaction solution; The monoclonal antibody of oxidized low-density lipoprotein labeled with latex microspheres is used as an antigen capture agent, and is incubated with the sample treated with the sample reaction solution containing the stabilizer and the blocker to react, and the difference of the absorbance value before and after the reaction is calculated, and the concentration of oxidized low-density lipoprotein is calculated according to the standard curve.

2. The method for detecting oxidized low-density lipoprotein according to claim 1, characterized in that: in, The method uses nuclear magnetic resonance spectroscopy, gel permeation chromatography and dynamic light scattering to characterize the structure and performance of the target multifunctional polymer stabilizer, and optimizes the monomer composition through molecular dynamics simulation, including: The composition of the target multifunctional polymer stabilizer is determined by nuclear magnetic resonance spectroscopy, the molecular weight and its distribution are determined by gel permeation chromatography, and the particle size distribution of the target multifunctional polymer stabilizer in aqueous solution is characterized by dynamic light scattering; Molecular dynamics simulation method was used to simulate the interaction between the target multifunctional polymer stabilizer and oxidized low-density lipoprotein under physiological conditions. The molar ratio of the monomers was optimized by analyzing the simulation trajectory to determine the optimal composition of the target multifunctional polymer stabilizer.

3. The method for detecting oxidized low-density lipoprotein according to claim 1, characterized in that: in, Collect serum samples, and prepare samples for oxidized low-density lipoprotein detection by stabilizing the sample reaction solution, including: Collect blood samples, separate serum, and store the separated serum in containers at low temperatures; The serum sample and the sample reaction solution are mixed and diluted to the target concentration range so that it is within the linear range of the test; The stabilizer was coated on the oxidized low-density lipoprotein particles by incubating at 37°C for 5 minutes.

4. The method for detecting oxidized low-density lipoprotein according to claim 1, characterized in that: in, Using latex microsphere-labeled oxidized low-density lipoprotein monoclonal antibody as an antigen capture agent, incubating with the sample treated with the sample reaction solution containing the stabilizer and the blocking agent, calculating the difference in absorbance before and after the reaction, and calculating the oxidized low-density lipoprotein concentration according to the standard curve, including: Using a monoclonal antibody against oxidized low-density lipoprotein labeled with double-diameter latex microspheres as an antigen capture agent, adding the sample treated with the sample reaction solution containing the stabilizer and the blocker, and incubating the reaction for 3 minutes; Latex microspheres of different particle sizes are cross-linked together after the formation of antigen-antibody complexes. The absorbance value A0 at the beginning of the reaction and the absorbance value A1 after 3 minutes of incubation are recorded, and the absorbance difference DA before and after the reaction is calculated. A standard curve is drawn based on the standard concentration and the absorbance difference, and the concentration of oxidized low-density lipoprotein in the sample is calculated.

5. An oxidized low-density lipoprotein detection device, characterized in that: include: A processor, a memory, and a system bus; wherein the processor and the memory are connected via the system bus; The memory is used to store one or more programs, wherein the one or more programs include instructions, and when the instructions are executed by the processor, the processor executes the method according to any one of claims 1 to 4.

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