Method and equipment for detecting oxidized low-density lipoprotein based on immunoturbidimetry

Abstract

The invention discloses a method and equipment for detecting oxidized low-density lipoprotein based on immunoturbidimetry, and the method comprises the following steps: preparing a macroinitiator by active esterification reaction, and preparing a block graft copolymer by atom transfer radical polymerization and radical grafting reaction; the preparation method comprises the following steps: carrying out surface modification on latex microspheres with different particle sizes by using a grafted copolymer, and then respectively coupling the microspheres with different particle sizes with different OxLDL monoclonal antibodies so as to prepare a double-particle-size latex microsphere marker; and mixing the serum sample with the double-particle-size latex microsphere marker, measuring an absorbance value, and calculating the concentration of the oxidized low-density lipoprotein in the serum sample according to a standard curve established on the basis of the standard substance solution. According to the scheme, the stability of the latex microsphere marker can be enhanced, and the binding efficiency of the latex microsphere marker and ox-LDL can be improved.

Description

Technical Field

[0001] The present application relates to the field of polymer compounds, and in particular to a method for detecting oxidized low-density lipoprotein based on immunoturbidimetry. Background Art

[0002] Oxidized low-density lipoprotein (ox-LDL) is an important marker of atherosclerosis, and its level is closely related to the occurrence and development of cardiovascular diseases. Accurate and sensitive detection of ox-LDL is of great significance for the early diagnosis and prevention of cardiovascular diseases. Existing ox-LDL detection methods mainly include enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay (CLIA) and latex-enhanced immunoturbidimetry (LEIA). Among them, the detection method based on double-size latex microsphere labeling has attracted widespread attention due to its advantages such as simple operation and fast detection speed.

[0003] However, the existing ox-LDL detection method based on double-size latex microsphere labeling still has some technical problems. First, the sensitivity and specificity of the detection need to be improved. Since the content of ox-LDL in the blood is low and its structure is similar to that of ordinary LDL, a detection method with higher sensitivity and specificity is needed. Secondly, the stability of latex microspheres is insufficient. Traditional latex microspheres are prone to aggregation or sedimentation during storage and use, affecting the accuracy and repeatability of the test results. Thirdly, the binding efficiency of latex microspheres and ox-LDL needs to be improved. Conventional labeling methods may lead to nonspecific binding and reduce the accuracy of the test. Finally, there are limitations in data analysis and result interpretation. Existing methods have errors when processing complex samples, making it difficult to accurately assess ox-LDL levels.

[0004] Therefore, there is an urgent need for a technical solution that can enhance the stability of latex microspheres and improve the binding efficiency of latex microspheres and ox-LDL. Summary of the invention

[0005] In order to solve the shortcomings of the prior art, the present application discloses a method and device for detecting oxidized low-density lipoprotein based on immunoturbidimetry. The present application solves the technical problems that the traditional latex microspheres in the prior art are prone to aggregation or sedimentation, and the conventional labeling method may cause nonspecific binding, thereby reducing the accuracy of detection.

[0006] The present application discloses a method for detecting oxidized low-density lipoprotein based on immunoturbidimetry, comprising: preparing a macromolecular initiator by active esterification reaction, and preparing a block graft copolymer by atom transfer radical polymerization and free radical grafting reaction; surface modifying latex microspheres of different particle sizes by graft copolymers, and then coupling the microspheres of different particle sizes with different OxLDL monoclonal antibodies, respectively, to prepare a double-particle-size latex microsphere marker; mixing a serum sample with the double-particle-size latex microsphere marker, and measuring the absorbance value, so as to calculate the concentration of oxidized low-density lipoprotein in the serum sample according to a standard curve established based on a standard solution.

[0007] In one possible implementation, an active esterification reaction is used to prepare a macromolecular initiator, and an atom transfer radical polymerization and a free radical grafting reaction are used to prepare a block graft copolymer, including: using an active esterification reaction to couple polyethylene glycol with a brominated alkyl compound to prepare a macromolecular initiator; using an atom transfer radical polymerization reaction to initiate polymerization of methacrylic acid monomers with the macromolecular initiator to prepare a block copolymer; and using a free radical grafting reaction to graft dimethylaminoethyl methacrylate monomers with the block copolymer as a skeleton to prepare a graft copolymer.

[0008] In one possible implementation, the surface of latex microspheres of different particle sizes is modified by using a graft copolymer, and then the microspheres of different particle sizes are coupled with different OxLDL monoclonal antibodies respectively to prepare a double-size latex microsphere marker, comprising: mixing an aqueous solution with a surfactant, introducing an inert gas to deoxygenate, and heating to a polymerization temperature; adding the graft copolymer and latex microspheres of different particle sizes to the mixture, stirring to uniformly disperse them; adding an aqueous solution of an initiator, and performing a polymerization reaction at the polymerization temperature for a preset time; cooling to room temperature, filtering to obtain different The invention discloses a method for preparing a latex microsphere dispersion of a target particle size; coupling the modified latex microspheres of large target particle size with a low-activity oxidized low-density lipoprotein monoclonal antibody under the action of a 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride coupling agent; coupling the modified latex microspheres of small target particle size with a low-activity oxidized low-density lipoprotein monoclonal antibody under the action of a 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride coupling agent; and mixing the two coupled latex microsphere markers in a preset ratio to obtain a double-size latex microsphere marker.

[0009] In a possible implementation, a serum sample is mixed with a double-size latex microsphere marker, and the absorbance value is measured to calculate the concentration of oxidized low-density lipoprotein in the serum sample according to a standard curve established based on a standard solution, including: obtaining a serum sample, separating the serum by centrifugation, and concentrating the serum sample by an ultrafiltration centrifuge device, and diluting it to a target concentration by using a buffer; preparing a tris(hydroxymethyl)aminomethane hydrochloride buffered reaction solution containing sodium chloride, disodium ethylenediaminetetraacetic acid, a surfactant, polyethylene glycol, a stabilizer and a blocking agent, wherein the pH value of the reaction solution is 4.0-6.5; preparing various concentrations of oxidized low-density lipoprotein. A protein standard solution is prepared, the standard solution and the reaction solution are incubated at 37° C. for 5 minutes, then a double-diameter latex microsphere marker is added to mix, and the mixture is incubated at 37° C. for 3 minutes; the absorbance at the target wavelength is measured by a biochemical analyzer using a two-point endpoint method, and the difference between the absorbance values ​​at the beginning and the end of the reaction is calculated, so as to establish a standard curve according to the difference between the absorbance values ​​measured by the standard solution of each concentration; a serum sample to be tested is taken, and the sample is processed according to the same operating steps as those for establishing the standard curve, the difference in the absorbance values ​​of the sample at the target wavelength is measured and calculated, and the concentration of oxidized low-density lipoprotein in the sample is calculated according to the standard curve.

[0010] In a possible implementation, polyethylene glycol and a brominated alkyl compound are coupled by an active esterification reaction to prepare a macromolecular initiator, comprising: dissolving polyethylene glycol in anhydrous tetrahydrofuran, adding triethylamine, and stirring for a predetermined time under low temperature conditions to form a reaction solution; slowly dripping a tetrahydrofuran solution of α-bromoisobutyryl bromide into the reaction solution, reacting at room temperature for a predetermined time to obtain a reaction mixture; pouring the reaction mixture into ice water to terminate the reaction, and extracting the obtained product with dichloromethane to obtain an organic phase; drying the organic phase with anhydrous magnesium sulfate, and removing the solvent by reduced pressure distillation to obtain a polyethylene glycol macromolecular initiator.

[0011] In a possible implementation, a block copolymer is prepared by initiating polymerization of methacrylic acid monomers with a macromolecular initiator through an atom transfer radical polymerization reaction, comprising: adding a monomer, a catalyst and a ligand into a reaction container in a predetermined ratio, wherein the monomer is methacrylic acid, the catalyst is cuprous bromide, and the ligand is N,N,N',N',N"-pentamethyldiethylenetriamine; dissolving the macromolecular initiator in an organic solvent, and adding the resulting solution into the reaction container, wherein the macromolecular initiator is polyethylene glycol, and the organic solvent is tetrahydrofuran; subjecting the reaction system to multiple vacuum-filling-inert gas cycle treatments, and conducting a constant temperature reaction at a preset temperature for a predetermined time, wherein the inert gas is nitrogen; pouring the reaction product into a precipitant for precipitation, collecting the precipitate by centrifugal separation, and vacuum drying the resulting precipitate to obtain a block copolymer containing the target product polyethylene glycol-b-polymethacrylic acid.

[0012] In a possible implementation, the standard curve includes: , wherein represents the absorbance value difference measured at a wavelength of 570 nm, represents the absorbance value difference offset, , , , are equation parameters, which are solved according to the test results of standard samples of various concentrations, and represent the concentration of ox-LDL.

[0013] An embodiment of the present application also discloses a device for detecting oxidized low-density lipoprotein based on immunoturbidimetry, comprising: 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, and the one or more programs include instructions, which, when executed by the processor, enable the processor to execute the method described in any one of the above embodiments.

[0014] In the method and apparatus for detecting oxidized low-density lipoprotein based on immunoturbidimetry as disclosed above, the embodiment of the present application can detect lower concentrations of ox-LDL through a multi-point binding mechanism, while reducing nonspecific interference, enhancing the stability of latex microspheres, and improving the binding efficiency of latex microspheres and ox-LDL. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0016] Figure 1 A schematic diagram of a method for detecting oxidized low-density lipoprotein based on immunoturbidimetry disclosed in an embodiment of the present application; Figure 2 A schematic diagram of a three-dimensional representation of the particle size distribution of a double-size latex microsphere disclosed in an embodiment of the present application; Figure 3 A dynamic light scattering correlation function diagram of a double-diameter latex microsphere disclosed in an embodiment of the present application; Figure 4 A simplified diagram of a hydrogen nuclear magnetic resonance spectrum of the chemical structure of a graft copolymer disclosed in an embodiment of the present application; Figure 5 A schematic diagram of a standard curve disclosed in an embodiment of the present application. DETAILED DESCRIPTION

[0017] Various exemplary embodiments of the present application will now be described in detail with reference to the accompanying drawings. It should be noted that unless otherwise specifically stated, the relative arrangement of components and steps, numerical expressions and numerical values ​​set forth in these embodiments do not limit the scope of the present application.

[0018] Those skilled in the art will appreciate that the terms "first", "second" and the like in the embodiments of the present application are only used to distinguish different steps, devices or modules, etc., and do not represent any specific technical meaning, nor do they represent the necessary logical order between them. It should also be understood that in the embodiments of the present application, "multiple" 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, in the absence of explicit limitation or contrary revelation given in the context, it can generally be understood as one or more. In addition, the term "and / or" in the present application is only a kind of association relationship describing the associated objects, indicating that there can be three kinds of relationships, for example, A and / or B, which can represent: A exists alone, A and B exist at the same time, and B exists alone. In addition, the character " / " in the present application generally indicates that the associated objects before and after are an "or" relationship. It should also be understood that the description of each embodiment in the present application emphasizes the differences between the embodiments, and the same or similar parts can refer to each other. For the sake of brevity, they will not be repeated one by one.

[0019] At the same time, it should be understood that, for ease of description, the sizes of the various parts shown in the drawings are not drawn according to the actual proportional relationship. The following description of at least one exemplary embodiment is actually only illustrative and is by no means intended to limit the present application and its application or use. The techniques, methods and devices known to ordinary technicians in the relevant fields may not be discussed in detail, but where appropriate, the techniques, methods and devices should be considered as part of the specification. It should be noted that similar numbers and letters represent similar items in the following drawings, so once an item is defined in one drawing, it does not need to be further discussed in subsequent drawings.

[0020] In order to make the purpose, technical solution and advantages of the embodiments of the present application clearer, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0021] Figure 1 The present invention discloses a method for detecting oxidized low-density lipoprotein by immunoturbidimetry in an embodiment of the present invention. The method disclosed in the embodiment of the present invention involves the synthesis of polyethylene glycol-b-polymethacrylic acid block copolymer, the preparation and surface modification of latex microspheres, and the detection of oxidized low-density lipoprotein (ox-LDL) concentration in serum using the modified latex microspheres.

[0022] like Figure 1 As shown, at step S101, a macromolecular initiator is prepared by active esterification reaction, and a block graft copolymer is prepared by atom transfer radical polymerization and free radical grafting reaction. The synthesis of polyethylene glycol-b-polymethacrylic acid block copolymer is the basis of the whole process. This synthesis usually adopts controlled / active radical polymerization technology, such as atom transfer radical polymerization (ATRP) or reversible addition-fragmentation chain transfer (RAFT) polymerization. During the synthesis process, the reaction conditions, including temperature, time and inert atmosphere, need to be strictly controlled. Specifically, the reaction system needs to be subjected to a constant temperature reaction at a preset temperature for a predetermined time, and nitrogen is usually used as an inert gas to protect the reaction system to prevent oxygen from interfering with the free radical polymerization process. After the reaction is completed, the reaction product is poured into a precipitant for precipitation, and this step can effectively separate the target product. Commonly used precipitants include but are not limited to non-polar solvents such as ether and n-hexane. The precipitate is then collected by centrifugation, and this process can use a high-speed centrifuge to optimize the separation effect by adjusting the speed and time. Finally, the obtained precipitate is subjected to vacuum drying treatment to remove the residual solvent and water, thereby obtaining a purified polyethylene glycol-b-polymethacrylic acid block copolymer.

[0023] Specifically, the method comprises: coupling polyethylene glycol with a brominated alkyl compound by an active esterification reaction to prepare a macromolecular initiator; initiating polymerization of methacrylic acid monomers by an atom transfer free radical polymerization reaction to prepare a block copolymer; and grafting dimethylaminoethyl methacrylate monomers by a free radical grafting reaction with the block copolymer as a skeleton to prepare a graft copolymer.

[0024] The method comprises the following steps: coupling polyethylene glycol with a brominated alkyl compound by an active esterification reaction to prepare a macromolecular initiator, comprising: dissolving polyethylene glycol in anhydrous tetrahydrofuran, adding triethylamine, stirring for a predetermined time under low temperature conditions to form a reaction solution; slowly dripping a tetrahydrofuran solution of α-bromoisobutyryl bromide into the reaction solution, reacting at room temperature for a predetermined time to obtain a reaction mixture; pouring the reaction mixture into ice water to terminate the reaction, and extracting the obtained product with dichloromethane to obtain an organic phase; drying the organic phase with anhydrous magnesium sulfate, and removing the solvent by reduced pressure distillation to obtain the polyethylene glycol macromolecular initiator.

[0025] The method comprises the following steps: atom transfer free radical polymerization is performed to initiate polymerization of methacrylic acid monomers with a macromolecular initiator to prepare a block copolymer, comprising: adding a monomer, a catalyst and a ligand into a reaction container in a predetermined ratio, wherein the monomer is methacrylic acid, the catalyst is cuprous bromide, and the ligand is N,N,N',N',N"-pentamethyldiethylenetriamine; dissolving the macromolecular initiator in an organic solvent, and adding the resulting solution into the reaction container, wherein the macromolecular initiator is polyethylene glycol, and the organic solvent is tetrahydrofuran; subjecting the reaction system to multiple vacuum pumping-inert gas filling cycles, and conducting a constant temperature reaction at a preset temperature for a predetermined time, wherein the inert gas is nitrogen; pouring the reaction product into a precipitant for precipitation, collecting the precipitate by centrifugal separation, and vacuum drying the resulting precipitate to obtain a block copolymer containing the target product polyethylene glycol-b-polymethacrylic acid.

[0026] In one embodiment, the raw materials are first prepared: polyethylene glycol (PEG): molecular weight 5000 Da, methacrylic acid (MAA): purity 99%, dimethylaminoethyl methacrylate (DMAEMA): purity 98%, 2-bromo-2-methylpropionyl bromide (BIBB): purity 98%, triethylamine (TEA): analytical grade, tetrahydrofuran (THF): chromatographic grade, copper (I) bromide (CuBr): purity 99.999%, N, N, N', N'', N''-pentamethyldiethylenetriamine (PMDETA): purity 99%.

[0027] Next, the synthesis of PEG-b-PMAA block copolymer was carried out: 5 g PEG was dissolved in 50 mL anhydrous THF, 1.1 equivalents of TEA were added, and the mixture was stirred at 0°C for 30 minutes. 1.1 equivalents of BIBB in THF solution were slowly added dropwise and reacted for 2 hours. The reaction mixture was poured into ice water to quench the reaction, and the product was extracted with dichloromethane. The organic phase was dried over anhydrous magnesium sulfate, and the solvent was removed by vacuum distillation to obtain the PEG macroinitiator. 10 mL MAA, 0.1 g CuBr and 0.2 mL PMDETA were added to a 50 mL round-bottom flask. The PEG macroinitiator was dissolved in 10 mL THF and added to the reaction bottle. The vacuum-nitrogen filling cycle was repeated 3 times, and the reaction was heated in an oil bath at 70°C for 4 hours. The reaction mixture was poured into methanol for precipitation, and the precipitate was collected by centrifugation and vacuum dried to obtain PEG-b-PMAA.

[0028] Then, PDMAEMA grafting was performed: 1 g PEG-b-PMAA was dissolved in 20 mL DMF, and 2 g DMAEMA and 0.05 g AIBN were added. The vacuum-nitrogen filling cycle was repeated 3 times, and the reaction was heated in an oil bath at 70°C for 6 hours. The reaction mixture was poured into ether for precipitation, and the precipitate was collected by centrifugation and vacuum dried to obtain the final product.

[0029] It should be noted that the product characterization can be carried out by using gel permeation chromatography (GPC) to determine the molecular weight and molecular weight distribution, and by using nuclear magnetic resonance hydrogen spectroscopy (1H NMR) to determine the chemical structure (such as Figure 2 The functional groups were analyzed by Fourier transform infrared spectroscopy (FT-IR).

[0030] It should be noted that all glassware must be dried in advance, water and oxygen must be strictly avoided during the reaction, BIBB is irritating, and the operation must be carried out in a fume hood and protective equipment must be worn. The polymerization reaction temperature should be controlled within the range of ±1°C to ensure the consistency of the degree of polymerization.

[0031] In step S102, the surfaces of latex microspheres of different particle sizes are modified by using graft copolymers, and then the microspheres of different particle sizes are coupled with different OxLDL monoclonal antibodies respectively to prepare double-size latex microsphere markers.

[0032] It should be understood that latex microspheres are polymer particles with uniform particle size and surface properties, which are widely used in biomedicine, materials science and other fields. In one embodiment, the particle size of the latex microspheres can be controlled by regulating the amount of surfactant and initiator. The role of the surfactant is to stabilize the monomer emulsion and prevent particle agglomeration during the polymerization process; while the initiator is used to start the polymerization reaction. Commonly used surfactants include sodium dodecyl sulfate (SDS), sodium dodecylbenzene sulfonate (SDBS), etc.; initiators can be selected from potassium persulfate (KPS), ammonium persulfate (APS), etc. By precisely controlling the dosage ratio of these two substances, latex microspheres of different particle sizes can be obtained.

[0033] In order to further improve the stability and functionality of latex microspheres, it is necessary to perform surface modification on them. The present application embodiment uses a stabilizer comprising a graft copolymer to perform surface modification on latex microspheres. This modification can change the surface properties of latex microspheres, increase their dispersion stability in aqueous solution, and provide active sites for subsequent biomolecule binding. The specific operation includes: dissolving the graft copolymer in an aqueous solution, then slowly dropping the latex microsphere dispersion, and fully combining the stabilizer with the latex microspheres under stirring conditions. In this process, the hydrophobic segment of the graft copolymer will be adsorbed on the surface of the latex microspheres, while the hydrophilic segment will extend into the aqueous phase to form a stable "brush-like" structure. Finally, a membrane separation technique (such as ultrafiltration or dialysis) is used to remove the unbound stabilizer to obtain surface-modified latex microspheres.

[0034] Specifically, the method comprises: mixing an aqueous solution with a surfactant, introducing an inert gas to deoxygenate the mixture, and heating the mixture to a polymerization temperature; adding a graft copolymer and latex microspheres of different particle sizes to the mixture, and stirring the mixture to make them uniformly dispersed; adding an aqueous solution of an initiator, and performing a polymerization reaction at the polymerization temperature for a preset time; cooling the mixture to room temperature, and filtering the mixture to obtain a dispersion of latex microspheres of different particle sizes; coupling the modified latex microspheres of large target particle size with a low-activity oxidized low-density lipoprotein monoclonal antibody under the action of a 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride coupling agent; coupling the modified latex microspheres of small target particle size with a low-activity oxidized low-density lipoprotein monoclonal antibody under the action of a 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride coupling agent; and mixing the two coupled latex microsphere markers in a preset ratio to obtain a double-particle-size latex microsphere marker.

[0035] In one embodiment, 100 nm latex microspheres are first prepared: 150 mL of deionized water and 0.1 g of sodium dodecyl sulfate (SDS) are added to a 250 mL three-necked flask. Nitrogen is introduced for 15 minutes to deoxygenate, and the temperature is raised to 70°C. 15 g of freshly distilled styrene monomer is added and stirred evenly. 0.1 g of an aqueous solution of potassium persulfate (KPS) initiator is added and reacted for 4 hours. Cool to room temperature and filter with a 0.22 μm filter membrane to obtain a latex microsphere dispersion.

[0036] Then, 500 nm latex microspheres were prepared by following the above steps, but the amount of SDS was reduced to 0.05 g and the amount of KPS was increased to 0.15 g.

[0037] Next, the surface of the latex microspheres was modified: 0.1 g of the new stabilizer was dissolved in 50 mL of deionized water and stirred evenly. 10 mL of latex microsphere dispersion (solid content 5%) was slowly added dropwise and stirred at room temperature for 2 hours. Ultrafiltration centrifuge tubes (100 kDa molecular weight cutoff) were used for centrifugation purification 3 times to remove unbound stabilizer.

[0038] In addition, the modified latex microspheres can be characterized by dynamic light scattering (DLS) to determine the particle size distribution (using a Malvern Zetasizer Nano ZS instrument), the microsphere morphology can be observed by transmission electron microscopy (TEM) (using a JEOL JEM-2100F microscope), and the amount of stabilizer grafted can be determined by thermogravimetric analysis (TGA) (using a TA Instruments Q500 instrument). It should be noted that styrene monomer needs to be purified by vacuum distillation before use. The stirring speed (400-500 rpm) needs to be strictly controlled during the emulsion polymerization process to ensure the uniformity of the particle size. Violent stirring should be avoided during surface modification to prevent aggregation of latex microspheres.

[0039] In step S103, the serum sample is mixed with the double-size latex microsphere marker, and the absorbance value is measured to calculate the concentration of oxidized low-density lipoprotein in the serum sample according to a standard curve established based on the standard solution.

[0040] After the preparation and modification of latex microspheres, the ultimate goal of the embodiments of the present application is to use these microspheres to detect the concentration of ox-LDL in serum. Ox-LDL is an important marker for atherosclerosis, and its concentration is closely related to the risk of cardiovascular disease. The detection process first requires obtaining a serum sample, which is usually separated by centrifugation. In order to improve the detection sensitivity, the serum sample can be concentrated using an ultrafiltration centrifuge and then diluted to the target concentration with a buffer.

[0041] The method comprises: obtaining a serum sample, separating the serum by centrifugation, and concentrating the serum sample by an ultrafiltration centrifugal device, so as to dilute the serum sample to a target concentration by using a buffer solution; preparing a tris(hydroxymethyl)aminomethane hydrochloride buffer reaction solution containing sodium chloride, disodium ethylenediaminetetraacetic acid, a surfactant, polyethylene glycol, a stabilizer and a blocking agent, wherein the pH value of the reaction solution is 4.0-6.5; preparing oxidized low-density lipoprotein standard solutions of various concentrations, incubating the standard solution and the reaction solution at 37°C for 5 minutes, then adding a double-diameter latex microsphere marker to mix, and continuing to incubate at 37°C for 3 minutes; measuring the absorbance at the target wavelength by using a biochemical analyzer using a two-point endpoint method, and calculating the difference in absorbance values ​​at the beginning and end of the reaction, so as to establish a standard curve according to the difference in absorbance values ​​measured by the standard solution of each concentration; taking a serum sample to be tested, processing it according to the same operating steps as those for establishing a standard curve, measuring and calculating the difference in absorbance values ​​of the sample at the target wavelength, and calculating the concentration of oxidized low-density lipoprotein in the sample according to the standard curve.

[0042] In one embodiment, human serum samples were collected and centrifuged at 3000 rpm for 10 minutes to separate serum. The serum samples were concentrated using ultrafiltration centrifuge tubes (100 kDa molecular weight cutoff). The samples were diluted to an appropriate concentration with PBST buffer (pH 7.4). A series of ox-LDL standard solutions (0-150U / L) were prepared. 3 μL of the standard solution was incubated with 180 μL of the reaction solution at 37°C for 5 minutes, and then 60 μL of the double-particle latex microsphere marker mixture was added, and the reaction was continued at 37°C for 3 minutes. The absorbance at 570 nm during the entire incubation reaction was tracked and measured using a fully automatic biochemical analyzer (such as Hitachi 7180). Then, the absorbance value A0 recorded immediately after the addition of the modified double-particle latex microspheres and the absorbance value A1 after the reaction was incubated for 3 minutes were taken, and the absorbance value difference (DA) of each concentration standard solution was calculated as DA=A1-A0. A standard curve of ox-LDL concentration and absorbance difference (DA) was drawn. Take 3 μL of the sample to be tested and follow the above steps. Calculate the concentration of ox-LDL in the sample based on the standard curve.

[0043] The standard curve (such as Figure 5 As shown) may include: , wherein represents the absorbance value difference measured at a wavelength of 570 nm, represents the absorbance value difference offset, , , , are equation parameters, which are solved according to the test results of standard samples of various concentrations, and represents the concentration of ox-LDL.

[0044] In summary, the detection method based on double-size modified latex microspheres has multiple advantages. First, the use of latex microspheres with two different particle sizes can improve the sensitivity and specificity of detection and expand the detection range. Microspheres with larger particle sizes mainly improve the detection sensitivity of low values, while microspheres with smaller particle sizes increase the upper limit of detection. Secondly, the surface-modified latex microspheres have good dispersibility and stability after antibody labeling, which can reduce nonspecific adsorption and improve the accuracy of detection. Finally, this method is easy to operate, does not require complex instruments and equipment, and is suitable for routine use in clinical laboratories.

[0045] Figure 3 This is a schematic diagram of a three-dimensional representation of the particle size distribution of a double-size latex microsphere disclosed in an embodiment of the present application. Figure 3 This is a dynamic light scattering correlation function diagram of a double-size latex microsphere disclosed in the embodiment of this application. Figure 2 As shown, it shows the dynamic particle size distribution characteristics of two latex microspheres with different particle sizes. The horizontal axis represents the particle size range of 0-1000 nm, the vertical axis is the time span of 0-10 min, and the Z axis shows the relative intensity. Two significant peak areas can be observed in three-dimensional space: the first peak area is located near 100 nm, the peak intensity is about 0.8-1.0, and the peak shape shows a normal distribution characteristic; the second peak area is located near 500 nm, and the peak intensity is relatively low, about 0.6-0.8. The half-peak widths of the two peaks in the distribution diagram are about 16 nm and 50 nm, respectively, indicating the uniformity of the particle size distribution. In the time dimension, the intensity distribution shows slight fluctuations, which is consistent with the fluctuations in particle size measurement caused by Brownian motion. The entire three-dimensional surface is marked with the viridis color scale, transitioning from blue to yellow, and the higher the intensity, the brighter the color.

[0046] Figure 3 The decay process of the autocorrelation function of latex microspheres with two particle sizes of 100 nm and 500 nm with delay time is shown. The horizontal axis is the delay time, ranging from 0-300 μs, and the vertical axis is the correlation function g2(τ). The correlation function of the 100 nm microsphere (blue curve) shows a faster exponential decay, decaying to the baseline level at about 100 μs; the 500 nm microsphere (red curve) shows a slower decay process, and has not completely decayed to the baseline at 300 μs. The decay rate of the correlation function is positively correlated with the particle size, which is consistent with the diffusion behavior predicted by the Stokes-Einstein equation.

[0047] Figure 4The following is a simplified diagram of the H NMR spectrum of the chemical structure of a graft copolymer disclosed in the examples of the present application. The examples of the present application disclose a graft copolymer stabilizer, which is formed by chemically connecting three blocks of polyethylene glycol (PEG), polymethacrylic acid (PMAA) and polydimethylaminoethyl methacrylate (PDMAEMA). The main chain is composed of a block copolymer PEG-b-PMAA formed by controlled free radical polymerization of polyethylene glycol (PEG) and methacrylic acid (MAA). On the main chain, a third monomer dimethylaminoethyl methacrylate (DMAEMA) is grafted by a free radical grafting reaction to form a PDMAEMA side chain.

[0048] The synthesis process can be divided into three main steps: Preparation of PEG macroinitiator, first, PEG is chemically modified with 2-bromo-2-methylpropionyl bromide (BIBB), and a bromoalkyl group is introduced at one end of PEG as a macroinitiator for controlled free radical polymerization. Synthesis of PEG-b-PMAA block copolymer, using copper-catalyzed controlled radical polymerization (ATRP) method, with PEG macroinitiator as initiator, MAA as monomer, controlled polymerization under copper (I) bromine / PMDETA catalytic system, to prepare PEG-b-PMAA block copolymer. Grafting of PDMAEMA side chain, using PEG-b-PMAA as macroinitiator, DMAEMA as monomer, grafting growth of PDMAEMA side chain on the main chain through free radical grafting reaction, thereby obtaining the final graft copolymer product.

[0049] In the structure of the graft copolymer, the hydrophobic PEG main chain gives the product good hydrophilicity and biocompatibility; PMAA gives the product an acidic functional group that can be used for pH response; and the PDMAEMA side chain has a quaternary ammonium salt group, which is positively charged and can interact with negative charges. Therefore, the graft copolymer can be better coupled with antibodies.

[0050] From the H NMR spectrum, it can be observed that there is an obvious strong peak at about 3.6 ppm, which can be attributed to the methylene protons (-CH2CH2O-) on the PEG main chain. The peak at about 2.8 ppm can be attributed to the methylene protons (-CH2-) in the PMAA block. The peak at about 1.2 ppm comes from the methyl protons (-N(CH3)2) in the PDMAEMA side chain.

[0051] It is noteworthy that multiple small peaks can also be observed in the range of 0.8-1.8 ppm, which correspond to the methyl protons (-CH3) on the PMAA and PDMAEMA backbones. The presence of these peaks further confirms the successful introduction of the three polymer blocks.

[0052] In addition, by integrating the relative intensities of each peak, the relative proportions of the three blocks of PEG, PMAA and PDMAEMA can be quantitatively calculated, thereby determining the precise composition of the stabilizer, which is of great significance for optimizing the performance and application of the stabilizer.

[0053] Figure 5 A schematic diagram of a standard curve disclosed in the embodiment of the present application. It should be understood that the standard curve shown in the figure is exemplary and non-restrictive. This means that the standard curve involved is not limited to a specific form, but is presented as an example. In other words, the standard curve shown in the figure can be regarded as a way of expression to clearly describe related concepts and relationships, and does not exclude other forms of standard curves. Therefore, when interpreting the standard curve in the picture, it should be understood that the model can be fitted according to different data, and various forms can be obtained, with flexibility and diversity, and its purpose is to disclose an exemplary description rather than a restrictive provision for a specific form.

[0054] like Figure 5 As shown, the standard curve includes: , wherein represents the absorbance value difference measured at a wavelength of 570 nm, represents the absorbance value difference offset, , , , are equation parameters, which are solved according to the test results of standard samples of various concentrations, and represent the concentration of ox-LDL.

[0055] The standard curve covers the ox-LDL concentration range of 0-150U / L, which is very practical for clinical applications. The curve shows a higher slope in the low concentration area (0-150U / L), indicating that the method has good sensitivity to low concentrations of ox-LDL. However, when the concentration continues to increase, the curve will gradually flatten out.

[0056] The main curve is composed of experimental data points and fitting curves, where red scattered points represent experimental data points, and each data point is marked with an error bar to reflect the systematic error and random error in the measurement process. The red solid line is the theoretical curve obtained by mathematical fitting, and the curve conforms to the equation ΔA=ΔA0+K×1 / (1+exp[-(a+b×lnC+c×C)]), where the fitting parameter values ​​are: ΔA0=85.3, K=1832.6, a=-2.31, b=0.827, c=-0.00456. The light red areas on both sides of the curve represent the 95% confidence interval range. In the range of ox-LDL concentration of 0-150U / L, the curve shows an obvious upward trend with a large slope; when the concentration approaches 150U / L, the slope of the curve gradually decreases and tends to be gentle, showing typical immunoassay reaction kinetic characteristics.

[0057] A standardized residual analysis chart is set below the main curve to evaluate the accuracy and reliability of the fitting curve. The horizontal axis maintains the same ox-LDL concentration range as the main graph (0-150 U / L), and the vertical axis is the standardized residual value. The blue scattered points in the residual graph indicate the degree of deviation between the experimental measurement value and the theoretical fitting value, and the zero line (dashed line) indicates the ideal state when fully fitted. It can be observed from the residual distribution that in the entire concentration range, the standardized residual values ​​are basically distributed within the range of ±2, and are randomly distributed on both sides of the zero line, without showing obvious systematic deviations.

[0058] Furthermore, an embodiment of the present application also discloses a device for detecting oxidized low-density lipoprotein based on immunoturbidimetry, comprising: a processor, a memory, and a system bus; the processor and the memory are connected via the system bus; the memory is used to store one or more programs, and the one or more programs include instructions, which, when executed by the processor, enable the processor to execute any of the above methods.

[0059] Furthermore, an embodiment of the present application also discloses a computer program product, which, when executed on a terminal device, enables the terminal device to execute any of the above methods.

[0060] It can be known from the description of the above implementation mode that those skilled in the art can clearly understand that all or part of the steps in the above-mentioned embodiment method 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 can be essentially or partly embodied in the form of a software product that contributes to the prior art. The computer software product can be stored in a storage medium such as ROM / RAM, a disk, an optical disk, etc., including several instructions for enabling 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 the various embodiments of the present application or certain parts of the embodiments.

[0061] It should be noted that the various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments, and the same or similar parts between the various embodiments can be referred to each other. For the device disclosed in the embodiment, since it corresponds to the method disclosed in the embodiment, the description is relatively simple, and the relevant parts can be referred to the method part description.

[0062] It should also be noted that, in the embodiments of the present application, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply that there is any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that the process, method, article or equipment including a series of elements includes not only those elements, but also includes other elements that are not clearly listed, or also includes elements inherent to such process, method, article or equipment. In the absence of more restrictions, the elements limited by the statement "comprise one..." do not exclude the presence of other identical elements in the process, method, article or equipment including the elements.

[0063] The above description of the disclosed embodiments enables professionals and technicians in the field to implement or use the present application. Various modifications to these embodiments will be apparent to professionals and technicians in the field, and 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 range 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 immunoturbidimetry, characterized in that: include: The macromolecular initiator is prepared by active esterification reaction, and the block graft copolymer is prepared by atom transfer radical polymerization and free radical grafting reaction; The surfaces of latex microspheres with different particle sizes were modified by graft copolymers, and then the microspheres with different particle sizes were coupled with different OxLDL monoclonal antibodies to prepare double-size latex microsphere markers. The serum sample was mixed with the double-size latex microsphere marker and the absorbance value was measured to calculate the concentration of oxidized low-density lipoprotein in the serum sample according to the standard curve established based on the standard solution.

2. The detection method according to claim 1, characterized in that: in, The macromolecular initiator is prepared by active esterification reaction, and the block graft copolymer is prepared by atom transfer radical polymerization and free radical grafting reaction, including: The macromolecular initiator is prepared by coupling polyethylene glycol with a brominated alkyl compound through an active esterification reaction; The block copolymer is prepared by atom transfer radical polymerization reaction, in which methacrylic acid monomer is polymerized with a macroinitiator; The graft copolymer is prepared by adopting free radical grafting reaction, taking the block copolymer as the skeleton and grafting dimethylaminoethyl methacrylate monomer.

3. The detection method according to claim 1, characterized in that: in, The surface of latex microspheres with different particle sizes is modified by using graft copolymers, and then the microspheres with different particle sizes are coupled with different OxLDL monoclonal antibodies to prepare double-size latex microsphere markers, including: After the aqueous solution is mixed with the surfactant, an inert gas is introduced to remove oxygen and the mixture is heated to the polymerization temperature; Adding the graft copolymer and latex microspheres of different particle sizes into the mixture, stirring to make them uniformly dispersed; Adding an aqueous solution of an initiator, and carrying out a polymerization reaction at the polymerization temperature for a preset time; The mixture was cooled to room temperature and filtered to obtain dispersions of latex microspheres with different particle sizes; The modified latex microspheres with large target particle size are coupled with low-activity oxidized low-density lipoprotein monoclonal antibody under the action of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride coupling agent; The modified latex microspheres with small target particle size are coupled with low-activity oxidized low-density lipoprotein monoclonal antibody under the action of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride coupling agent; The two coupled latex microsphere markers are mixed in a preset ratio to obtain a double-diameter latex microsphere marker.

4. The detection method according to claim 1, characterized in that: in, The serum sample is mixed with the dual-size latex microsphere marker and the absorbance value is measured to calculate the concentration of oxidized low-density lipoprotein in the serum sample according to a standard curve established based on the standard solution, including: Obtaining a serum sample, separating the serum by centrifugation, and concentrating the serum sample by an ultrafiltration centrifuge device, and then diluting the serum sample to a target concentration by using a buffer; A tris(hydroxymethylaminomethane) hydrochloride buffered reaction solution containing sodium chloride, disodium ethylenediaminetetraacetate, a surfactant, polyethylene glycol, a stabilizer and a blocking agent is prepared, wherein the pH value of the reaction solution is 4.0-6.5; Prepare various concentrations of oxidized low-density lipoprotein standard solutions, incubate the standard solutions with the reaction solution at 37°C for 5 minutes, then add the double-size latex microsphere marker to mix, and continue incubating at 37°C for 3 minutes; The absorbance at the target wavelength is measured by a biochemical analyzer using a two-point endpoint method, and the difference between the absorbance values ​​at the beginning and the end of the reaction is calculated, so as to establish a standard curve according to the difference in absorbance values ​​measured for standard solutions of various concentrations; Take the serum sample to be tested and process it according to the same operating steps as those for establishing the standard curve, measure and calculate the difference in the absorbance values ​​of the sample at the target wavelength, and calculate the concentration of oxidized low-density lipoprotein in the sample based on the standard curve.

5. The detection method according to claim 2, characterized in that: in, The macromolecular initiator is prepared by coupling polyethylene glycol with a brominated alkyl compound using an active esterification reaction, including: Dissolving polyethylene glycol in anhydrous tetrahydrofuran, adding triethylamine, and stirring at low temperature for a predetermined time to form a reaction solution; Slowly adding a tetrahydrofuran solution of α-bromoisobutyryl bromide to the reaction solution, reacting at room temperature for a predetermined time to obtain a reaction mixture; The reaction mixture was poured into ice water to terminate the reaction, and the obtained product was extracted with dichloromethane to obtain an organic phase; The organic phase is dried using anhydrous magnesium sulfate, and the solvent is removed by reduced pressure distillation to obtain a polyethylene glycol macromolecular initiator.

6. The detection method according to claim 2, characterized in that: in, The block copolymer is prepared by atom transfer radical polymerization reaction, using a macromolecular initiator to initiate polymerization of methacrylic acid monomers, including: Adding a monomer, a catalyst and a ligand into a reaction container in a predetermined ratio, wherein the monomer is methacrylic acid, the catalyst is cuprous bromide, and the ligand is N,N,N',N',N"-pentamethyldiethylenetriamine; Dissolving a macromolecular initiator in an organic solvent, and adding the resulting solution into the reaction container, wherein the macromolecular initiator is polyethylene glycol and the organic solvent is tetrahydrofuran; The reaction system is subjected to multiple cycles of vacuuming and filling with inert gas, and a constant temperature reaction is carried out at a preset temperature for a predetermined time, wherein the inert gas is nitrogen; The reaction product is poured into a precipitant for precipitation, the precipitate is collected by centrifugal separation, and the obtained precipitate is vacuum dried to obtain a block copolymer containing the target product polyethylene glycol-b-polymethacrylic acid.

7. The detection method according to claim 4, characterized in that: in, The standard curve comprises: ， in, It represents the difference of absorbance value measured at 570 nm wavelength. Indicates the absorbance value difference offset, , , , is the equation parameter, and is solved according to the test results of the standard products at different concentrations. Indicates the concentration of ox-LDL.

8. A device for detecting oxidized low-density lipoprotein based on immunoturbidimetry, 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 7.

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