Method and apparatus for detecting oxidized low density lipoprotein based on immunoturbidimetry

Abstract

The application discloses a method and equipment for detecting oxidized low-density lipoprotein based on an immune turbidimetry method, and the method comprises the following steps: preparing a macromolecular initiator by adopting an active esterification reaction, and preparing a block graft copolymer by means of an atom transfer radical polymerization and a radical grafting reaction; the surface of different particle size latex microspheres is modified by using the graft copolymer, then the microspheres with different particle sizes are coupled with different OxLDL monoclonal antibodies respectively, so as to prepare double-particle-size latex microsphere markers; a serum sample is mixed with the double-particle-size latex microsphere markers, and an absorbance value is measured, and the concentration of oxidized low-density lipoprotein in the serum sample is calculated according to a standard curve established based on a standard sample solution. Through the scheme, the stability of the latex microsphere marker can be enhanced, and the binding efficiency of the latex microsphere marker and ox-LDL is improved.

Description

TECHNICAL FIELD

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

[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 early diagnosis and prevention of cardiovascular diseases. Existing ox-LDL detection methods mainly include enzyme-linked immunosorbent assay (ELISA), chemiluminescence immunoassay (CLIA), and latex-enhanced immunoturbidimetry (LEIA), etc. Among them, the detection method based on double-particle-size latex microspheres labeling has attracted widespread attention due to its simple operation and fast detection speed.

[0003] However, the existing ox-LDL detection method based on double-particle-size latex microspheres 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 blood is low, and its structure is similar to that of ordinary LDL, a detection method with higher sensitivity and specificity is needed. Second, the stability of the latex microspheres is insufficient. Traditional latex microspheres are prone to aggregation or sedimentation during storage and use, affecting the accuracy and repeatability of the detection results. Third, the binding efficiency of the latex microspheres and ox-LDL needs to be improved. Conventional labeling methods may cause non-specific binding, reducing the accuracy of the detection. Finally, there are limitations in data analysis and result interpretation. The existing method has errors when dealing with complex samples, making it difficult to accurately assess the level of ox-LDL.

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

[0005] To solve the problems of the prior art, the embodiments of the present application disclose a method and device for detecting oxidized low-density lipoprotein based on immunoturbidimetry. The present application solves the technical problems of the prior art, such as the aggregation or sedimentation of traditional latex microspheres, non-specific binding caused by conventional labeling methods, and reduced accuracy of detection.

[0006] The embodiment of the application discloses a method for detecting oxidized low density lipoprotein based on immune nephelometry, comprising the following steps: preparing a macromolecular initiator by adopting active esterification reaction, and preparing a block graft copolymer by adopting atom transfer radical polymerization and radical grafting reaction; modifying the surface of different particle size latex microspheres by using the graft copolymer, 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 an implementation manner, the macromolecular initiator is prepared by adopting active esterification reaction, and the block graft copolymer is prepared by adopting atom transfer radical polymerization and radical grafting reaction, comprising the following steps: coupling polyethylene glycol and a bromoalkyl compound by adopting active esterification reaction to prepare a macromolecular initiator; preparing a block copolymer by initiating polymerization of a methyl methacrylic acid monomer with the macromolecular initiator through atom transfer radical polymerization reaction; and preparing a graft copolymer by grafting dimethylaminoethyl methacrylate monomers on the block copolymer as a skeleton through radical grafting reaction.

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

[0009] In one possible implementation, wherein the serum sample is mixed with the double-size latex microsphere marker, and the absorbance value is measured to calculate the oxidized low-density lipoprotein concentration in the serum sample according to a standard curve established based on a standard solution, the method comprises the following steps: obtaining a serum sample, separating the serum by centrifugation, and concentrating the serum sample by using an ultrafiltration centrifuge device, and diluting to a target concentration by using a buffer; configuring a tris-hydroxymethyl aminomethane hydrochloride buffer reaction solution containing sodium chloride, disodium ethylenediaminetetraacetate, a surfactant, polyethylene glycol, a stabilizer, and a blocking agent, and the pH value of the reaction solution is 4.0-6.5; preparing a plurality of concentrations of oxidized low-density lipoprotein standard solution, incubating the standard solution with the reaction solution at 37°C for 5 min, then adding the double-size latex microsphere marker for mixing, and continuing to incubate at 37°C for 3 min; measuring the absorbance at the target wavelength by using a biochemical analyzer by using a two-point endpoint method, and calculating the difference between the absorbance values at the beginning and the end of the reaction, to establish a standard curve according to the measured absorbance value difference of each concentration standard solution; taking the serum sample to be measured, processing according to the same operation steps as establishing the standard curve, measuring and calculating the difference between the 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.

[0010] In one possible implementation, wherein the polyethylene glycol is coupled with a bromoalkyl compound by using an active ester reaction to prepare a macromolecular initiator, the method comprises the following steps: dissolving the polyethylene glycol in anhydrous tetrahydrofuran, adding triethylamine, stirring at a low temperature for a predetermined time to form a reaction solution; slowly adding an alpha-bromo-isobutyryl bromide tetrahydrofuran solution to 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 by using dichloromethane to obtain an organic phase; drying the organic phase by using anhydrous magnesium sulfate, and removing the solvent by using a reduced-pressure distillation method to obtain a polyethylene glycol macromolecular initiator.

[0011] In one possible implementation, the block copolymer is prepared by atom transfer radical polymerization reaction, in which a macroinitiator is used to initiate polymerization of a methacrylic monomer, comprising: adding monomer, catalyst and ligand into a reaction vessel in a predetermined ratio, wherein the monomer is methacrylic acid, the catalyst is cuprous bromide, and the ligand is N,N,N',N',N"-pentamethyl diethylene triamine; dissolving a macroinitiator in an organic solvent and adding the obtained solution into the reaction vessel, wherein the macroinitiator is polyethylene glycol and the organic solvent is tetrahydrofuran; performing multiple vacuum-inert gas circulation treatment on the reaction system, and performing 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 performing vacuum drying treatment on the obtained precipitate to obtain a block copolymer containing the target product polyethylene glycol-b-poly(methacrylic acid).

[0012] In one possible implementation, the standard curve comprises: , wherein, represents a difference value of absorbance measured at a wavelength of 570 nm, represents a difference value of absorbance, , , , is an equation parameter, which is solved according to test results of each concentration standard, represents the concentration of ox-LDL.

[0013] The application further discloses a device for detecting oxidized low-density lipoprotein based on an immunoturbidimetry method, comprising: a processor, a memory, and a system bus; wherein the processor and the memory are connected through the system bus; the memory is used for storing one or more programs, the one or more programs comprising instructions, which, when executed by the processor, cause the processor to perform the method in any one of the above embodiments.

[0014] In the method and device for detecting oxidized low-density lipoprotein based on an immunoturbidimetry method as disclosed above, the application can detect lower concentration of ox-LDL through a multi-point binding mechanism, reduce non-specific interference, enhance the stability of latex microspheres, and improve the binding efficiency of latex microspheres and ox-LDL. BRIEF DESCRIPTION OF DRAWINGS

[0015] In order 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 needed to be used in the embodiments or prior art description. Obviously, the drawings in the following description are some embodiments of the present application, and for those skilled in the art, other drawings can also be obtained without creative labor.

[0016] Figure 1 A flowchart of a method for detecting oxidized low-density lipoprotein based on immunoturbidimetry according to an embodiment of the present application;

[0017] Figure 2 A three-dimensional representation of the particle size distribution of double-particle-size latex microspheres according to an embodiment of the present application;

[0018] Figure 3 A dynamic light scattering correlation function diagram of double-particle-size latex microspheres according to an embodiment of the present application; Figure 4 A schematic diagram of the nuclear magnetic resonance hydrogen spectrum of the chemical structure of a graft copolymer according to an embodiment of the present application;

[0019] Figure 5 A schematic diagram of a standard curve according to an embodiment of the present application. DETAILED DESCRIPTION

[0020] Various exemplary embodiments of the present application will now be described in detail with reference to the accompanying drawings. Note that the relative arrangement, numerical expressions, and numerical values of the components and steps set forth in these embodiments are not limiting to the scope of the present application unless otherwise specifically stated.

[0021] Those skilled in the art can understand 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, and do not represent any specific technical meaning, nor do they represent a necessary logical sequence between them. It should also be understood that in the embodiments of the present application, "a plurality of" can mean two or more, and "at least one" can mean one, two, or more. It should also be understood that for any component, data, or structure mentioned in the embodiments of the present application, unless specifically limited or given a contrary implication by the context, it can generally be understood as one or more. In addition, the term "and / or" in the present application is only a description of the association relationship between the associated objects, which means that there can be three relationships, for example, A and / or B can mean that A exists alone, A and B exist together, and B exists alone. In addition, the character " / " in the present application generally represents an "or" relationship between the front and rear associated objects. It should also be understood that the description of each embodiment of the present application emphasizes the differences between the embodiments, and the same or similar parts can be referred to each other, and for the sake of brevity, they will not be repeated.

[0022] It is to be understood that the figures illustrated in the drawings are shown by way of example, and not by way of limitation. It is to be understood that the same or equivalent parts appearing on different drawings are designated by the same reference characters, and that the drawings are not necessarily to scale.

[0023] In order to make the purposes, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative effort should fall within the scope of the present application.

[0024] Figure 1 A flow chart of a method for detecting oxidized low density lipoprotein based on immunoturbidimetry disclosed in the embodiments of the present application. The method described in the embodiments of the present application involves the synthesis of polyethylene glycol-b-poly(methyl methacrylate) block copolymer, the preparation and surface modification of latex microspheres, and the detection of the concentration of oxidized low density lipoprotein (ox-LDL) in serum using the modified latex microspheres.

[0025] As Figure 1As 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 radical grafting reaction. The synthesis of polyethylene glycol-b-poly(methacrylic acid) block copolymer is the basis of the whole process. This synthesis usually adopts controlled / living 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 need to be strictly controlled, including temperature, time and inert atmosphere, etc. Specifically, the reaction system needs to be incubated at a preset temperature for a predetermined time, and nitrogen is usually used as an inert gas to protect the reaction system and prevent oxygen from interfering with the radical polymerization process. After the reaction is completed, the reaction product is poured into a precipitant for precipitation, which can effectively separate the target product. Commonly used precipitants include but are not limited to non-polar solvents such as diethyl ether and n-hexane. Subsequently, the precipitate is collected by centrifugation, which 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 residual solvents and moisture, and the purified polyethylene glycol-b-poly(methacrylic acid) block copolymer is obtained.

[0026] Specifically, it includes: coupling polyethylene glycol with bromoalkyl compound by active esterification reaction to prepare macromolecular initiator; preparing block copolymer by atom transfer radical polymerization reaction with macromolecular initiator to initiate methacrylic acid monomer polymerization; grafting dimethylaminoethyl methacrylate monomer on the block copolymer skeleton by radical grafting reaction to prepare graft copolymer.

[0027] Among them, the macromolecular initiator is prepared by coupling polyethylene glycol with bromoalkyl compound by active esterification reaction, which includes: dissolving polyethylene glycol in anhydrous tetrahydrofuran, adding triethylamine, stirring at low temperature for a predetermined time to form a reaction solution; slowly adding α-bromoisobutyryl bromide tetrahydrofuran solution to 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.

[0028] The method comprises the following steps: adding a monomer, a catalyst, and a ligand into a reaction vessel in a predetermined proportion, 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 vessel, wherein the macromolecular initiator is polyethylene glycol and the organic solvent is tetrahydrofuran; subjecting the reaction system to multiple cycles of vacuuming and inert gas filling, 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 precipitate to obtain a block copolymer containing the target product, polyethylene glycol-b-polymethacrylic acid.

[0029] 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%.

[0030] Next, the PEG-b-PMAA block copolymer was synthesized: 5 g of PEG was dissolved in 50 mL of anhydrous THF, 1.1 equivalents of TEA was added, and the mixture was stirred at 0°C for 30 minutes. 1.1 equivalents of BIBB in THF was slowly added dropwise and allowed to react 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 distillation under reduced pressure to obtain the PEG macroinitiator. A 50 mL round-bottom flask was charged with 10 mL of MAA, 0.1 g of CuBr, and 0.2 mL of PMDETA. The PEG macroinitiator was dissolved in 10 mL of THF and added to the reaction flask. The mixture was vacuum-filled with nitrogen three times and heated in an oil bath at 70°C for 4 hours. The reaction mixture was poured into methanol to precipitate, which was collected by centrifugation and dried under vacuum to obtain PEG-b-PMAA.

[0031] Next, PDMAEMA grafting was performed: 1 g of PEG-b-PMAA was dissolved in 20 mL of DMF, and 2 g of DMAEMA and 0.05 g of AIBN were added. The reaction was repeated three times with vacuum and nitrogen filling cycles, and then heated in an oil bath at 70°C for 6 hours. The reaction mixture was poured into diethyl ether for precipitation, which was then collected by centrifugation and dried under vacuum to obtain the final product.

[0032] It should be noted that product characterization can use gel permeation chromatography (GPC) to determine molecular weight and molecular weight distribution, nuclear magnetic resonance hydrogen spectrum (1H NMR) to determine chemical structure (such as Figure 2 The functional groups are analyzed by Fourier transform infrared spectroscopy (FT-IR).

[0033] It should be noted that all glassware needs to be pre-dried, water and oxygen should be strictly avoided during the reaction, BIBB is irritating, and protective equipment should be worn during operation in a fume hood, and the polymerization temperature should be controlled within ± 1°C to ensure the consistency of the degree of polymerization.

[0034] At step S102, the surface of the latex microspheres of different particle sizes is modified by graft copolymer, and then the microspheres of different particle sizes are coupled with different OxLDL monoclonal antibodies respectively to prepare double-particle-size latex microsphere markers.

[0035] It should be understood that latex microspheres are polymer particles with uniform particle size and surface properties, which are widely used in biomedical, material science and other fields. In an embodiment, the particle size of the latex microspheres can be controlled by adjusting the amount of surfactant and initiator. The role of the surfactant is to stabilize the monomer emulsion and prevent particle agglomeration during polymerization; and the initiator is used to start the polymerization reaction. Commonly used surfactants include sodium dodecyl sulfate (SDS), sodium dodecyl benzene sulfonate (SDBS), etc.; the initiator can be selected from potassium persulfate (KPS), ammonium persulfate (APS), etc. By accurately controlling the proportion of the amounts of the two substances, latex microspheres of different particle sizes can be obtained.

[0036] In order to further improve the stability and functionality of the latex microspheres, surface modification is needed. The embodiments of the present application use a stabilizer containing graft copolymer to modify the surface of the latex microspheres. This modification can change the surface properties of the 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 adding the latex microsphere dispersion, and fully combining the stabilizer with the latex microspheres under stirring. In this process, the hydrophobic segment of the graft copolymer will adsorb on the surface of the latex microspheres, while the hydrophilic segment will stretch into the aqueous phase, forming a stable "brush-like" structure. Finally, membrane separation technology (such as ultrafiltration or dialysis) is used to remove the unbound stabilizer, and the surface-modified latex microspheres are obtained.

[0037] Specifically, the method comprises: mixing an aqueous solution with a surfactant, purging inert gas to remove oxygen, and heating to a polymerization temperature; adding a graft copolymer and latex microspheres of different particle sizes to the mixture, stirring to uniformly disperse; adding an aqueous solution of an initiator, and performing a polymerization reaction at the polymerization temperature for a preset time; cooling to room temperature, and filtering to obtain a dispersion of latex microspheres of different particle sizes; coupling the modified large target particle size latex microspheres with a low-activity oxidized low-density lipoprotein monoclonal antibody under the action of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride coupling agent; coupling the modified small target particle size latex microspheres with a low-activity oxidized low-density lipoprotein monoclonal antibody under the action of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride coupling agent; and mixing the two coupled latex microsphere markers in a preset ratio to obtain a dual-particle-size latex microsphere marker.

[0038] In one embodiment, the preparation of 100 nm latex microspheres is first performed: 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 purged for 15 minutes to remove oxygen, and the temperature is raised to 70°C. 15 g of freshly distilled styrene monomer is added and stirred uniformly. An aqueous solution of 0.1 g of potassium persulfate (KPS) initiator is added, and the reaction is performed for 4 hours. The temperature is cooled to room temperature, and a 0.22 μm filter membrane is used for filtration to obtain a dispersion of latex microspheres.

[0039] Then the preparation of 500 nm latex microspheres is performed: the above steps are followed, but the amount of SDS is reduced to 0.05 g, and the amount of KPS is increased to 0.15 g.

[0040] Next, the surface modification of the latex microspheres is performed: 0.1 g of a stabilizer is dissolved in 50 mL of deionized water and stirred uniformly. 10 mL of a dispersion of latex microspheres (5% solid content) is slowly added dropwise, and stirred at room temperature for 2 hours. Ultrafiltration centrifuge tubes (100 kDa molecular weight cut-off) are used for centrifugal purification three times to remove unbound stabilizers.

[0041] In addition, the characterization of the modified latex microspheres can be performed by dynamic light scattering (DLS) to determine the particle size distribution (using a Malvern Zetasizer Nano ZS instrument), transmission electron microscopy (TEM) to observe the morphology of the microspheres (using a JEOL JEM-2100F microscope), and thermogravimetric analysis (TGA) to determine the grafting amount of the stabilizer (using a TA Instruments Q500 instrument)

[0042] It should be noted that the styrene monomer needs to be purified by vacuum distillation, the stirring speed needs to be strictly controlled (400-500 rpm) during the emulsion polymerization process to ensure the uniformity of the particle size, and vigorous stirring should be avoided during surface modification to prevent the aggregation of the latex microspheres.

[0043] At step S103, the serum sample is mixed with the double-particle-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 the standard curve established based on the standard solution.

[0044] After the preparation and modification of the latex microspheres are completed, the final 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 of 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, an ultrafiltration centrifuge device can be used to concentrate the serum sample, and then diluted with buffer to the target concentration.

[0045] The method comprises the following steps: obtaining a serum sample, separating the serum by centrifugation, and concentrating the serum sample using an ultrafiltration centrifuge device to dilute it to the target concentration with a buffer; configuring a tris-hydroxymethyl aminomethane hydrochloride buffer reaction solution containing sodium chloride, disodium ethylenediaminetetraacetate, a surfactant, polyethylene glycol, a stabilizer, and a blocking agent, and the pH value of the reaction solution is 4.0-6.5; preparing a plurality of concentrations of oxidized low-density lipoprotein standard solution, incubating the standard solution with the reaction solution at 37°C for 5 min, then adding the double-particle-size latex microsphere marker for mixing, and continuing to incubate at 37°C for 3 min; using a biochemical analyzer to measure the absorbance at the target wavelength by the two-point endpoint method, and calculating the difference between the absorbance values at the beginning and end of the reaction to establish a standard curve according to the absorbance difference values measured for each concentration of standard solution; taking the serum sample to be tested, processing it according to the same operation steps as establishing the standard curve, measuring and calculating the difference between the absorbance values at the target wavelength of the sample, and calculating the concentration of oxidized low-density lipoprotein in the sample according to the standard curve.

[0046] In one embodiment, human serum samples are collected and centrifuged at 3000 rpm for 10 minutes to separate serum. The serum samples are concentrated using ultrafiltration centrifuge tubes (100 kDa molecular weight cut-off). The samples are diluted to the appropriate concentration with PBST buffer (pH 7.4). A series of standard ox-LDL solutions (0-150 U / L) are prepared. 3 μL of the standard solution is incubated with 180 μL of the reaction solution at 37°C for 5 minutes, then 60 μL of the double-size latex microsphere marker mixture is added, and the incubation reaction is continued at 37°C for 3 minutes. The absorbance at 570 nm is measured throughout the incubation reaction using a fully automatic biochemical analyzer (such as Hitachi 7180). Then the absorbance value A0 immediately after the modified double-size latex microspheres are added is recorded, and the absorbance value A1 after 3 minutes of incubation is recorded. The absorbance value difference (DA) of each concentration standard solution is calculated as DA = A1-A0. The standard curve of ox-LDL concentration versus absorbance difference (DA) is plotted. 3 μL of the sample to be tested is taken and the above steps are performed. The concentration of ox-LDL in the sample is calculated according to the standard curve.

[0047] wherein the standard curve (as shown in Figure 5 ) can include: , wherein, represents the absorbance value difference measured at 570 nm wavelength, represents the absorbance value difference offset, , , , is an equation parameter, which is solved according to the test results of each concentration standard, represents the concentration of ox-LDL.

[0048] In summary, the detection method based on double-size modified latex microspheres has multiple advantages. First, the use of two different sizes of latex microspheres can improve the sensitivity and specificity of the detection and expand the detection range. The larger size microspheres mainly improve the detection sensitivity of low values, while the smaller size microspheres improve the upper limit of detection. Second, the antibody-labeled latex microspheres after surface modification have good dispersibility and stability, which can reduce non-specific adsorption and improve the accuracy of detection. Finally, this method is simple to operate and does not require complex instruments and equipment, making it suitable for routine use in clinical laboratories.

[0049] Figure 3 is a three-dimensional representation of the particle size distribution of a double-size latex microsphere disclosed in the embodiments of the present application, Figure 3 is a dynamic light scattering correlation function graph of a double-size latex microsphere disclosed in the embodiments of the present application. As Figure 2The graph shows the dynamic size distribution of two different latex microspheres. The horizontal axis represents the size range from 0 to 1000 nm, the vertical axis is the time span from 0 to 10 min, and the Z-axis shows the relative intensity. Two distinct peak regions can be observed in the three-dimensional space: the first peak region is located near 100 nm, with a peak intensity of about 0.8-1.0, and the peak shape exhibits a normal distribution characteristic; the second peak region is located near 500 nm, with a relatively low peak intensity of about 0.6-0.8. The half-peak width of the two peaks in this distribution is about 16 nm and 50 nm, respectively, indicating the uniformity of the size distribution. In the time dimension, the intensity distribution shows a weak fluctuation, which is consistent with the fluctuation in particle size measurement caused by Brownian motion. The entire three-dimensional surface is marked with the viridis color scale, which transitions from blue to yellow, with higher intensity and brighter color.

[0050] Figure 3 The graph shows the decay process of the autocorrelation function of 100 nm and 500 nm latex microspheres with delay time. The horizontal axis is the delay time, ranging from 0 to 300 μs, and the vertical axis is the correlation function g2(τ). The correlation function of 100 nm microspheres (blue curve) shows a rapid exponential decay, decaying to the baseline level at about 100 μs; the 500 nm microspheres (red curve) show a slower decay process, which 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.

[0051] Figure 4 A schematic diagram of the nuclear magnetic resonance hydrogen spectrum of the chemical structure of a graft copolymer disclosed in the embodiments of the present application. The embodiments of the present application disclose a graft copolymer stabilizer, which is composed of three blocks of polyethylene glycol (PEG), polymethacrylic acid (PMAA) and polydimethylaminoethyl methacrylate (PDMAEMA) connected by chemical bonds. The main chain is composed of polyethylene glycol (PEG) and methacrylic acid (MAA) block copolymer PEG-b-PMAA formed by controlled radical polymerization. On the main chain, the third monomer dimethylaminoethyl methacrylate (DMAEMA) is grafted by free radical grafting reaction to form PDMAEMA side chain.

[0052] The synthesis process can be divided into three main steps: preparation of PEG macromolecular initiator, first, using 2-bromo-2-methylpropionyl bromide (BIBB) to chemically modify PEG, introducing bromoalkyl at one end of PEG as a macromolecular initiator for controlled radical polymerization. Synthesis of PEG-b-PMAA block copolymer, using copper-catalyzed controlled radical polymerization (ATRP) method, taking PEG macromolecular initiator as initiator and MAA as monomer, controlled polymerization was carried out under copper (I) bromide / PMDETA catalytic system, and PEG-b-PMAA block copolymer was prepared. Grafting of PDMAEMA side chain, taking PEG-b-PMAA as macromolecular initiator and DMAEMA as monomer, PDMAEMA side chain was grafted and grown on the main chain through free radical grafting reaction, so as to obtain the final graft copolymer product.

[0053] In the structure of the graft copolymer, the hydrophobic PEG main chain endows the product with good hydrophilicity and biocompatibility; PMAA endows the product with acidic functional groups, which can be used for pH response; and the PDMAEMA side chain has quaternary ammonium salt groups with positive charge, which can interact with negative charge. Therefore, the graft copolymer can better couple with antibodies.

[0054] From the schematic diagram of nuclear magnetic resonance hydrogen spectrum, it can be observed that there is a obvious strong peak at about 3.6 ppm, which can be attributed to the methylene protons (-CH2CH2O-) on the PEG main chain. The peak appearing at about 2.8 ppm can be attributed to the methylene protons (-CH2-) in the PMAA block. And the peak at about 1.2 ppm is from the methyl protons (-N(CH3)2) in the PDMAEMA side chain.

[0055] Notably, in the range of 0.8-1.8 ppm, multiple small peaks can also be observed, which correspond to the methyl protons (-CH3) on the PMAA and PDMAEMA backbone. The presence of these peaks further confirms the successful introduction of the three polymer blocks.

[0056] In addition, by integrating the relative intensity of each peak, the relative proportion of PEG, PMAA and PDMAEMA three blocks can be quantitatively calculated, so as to determine the accurate composition of the stabilizer. This is of great significance for optimizing the performance and application of the stabilizer.

[0057] Figure 5A schematic diagram of a standard curve disclosed in embodiments of the present application. It should be understood that the standard curve shown in the figure is exemplary and not limiting. 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 means of expression to clearly describe the relevant 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 have various forms according to the fitting of different data, and has flexibility and diversity, and the purpose is to disclose an exemplary description, not a restrictive provision of a specific form.

[0058] As shown in Figure 5 , the standard curve comprises: , wherein, represents the difference in absorbance value measured at a wavelength of 570 nm, represents the difference in absorbance value offset, , , , is an equation parameter, which is solved according to the test results of each concentration standard, represents the concentration of ox-LDL.

[0059] The standard curve covers the ox-LDL concentration range of 0-150 U / L, which is very practical for clinical application. The curve shows a high slope in the low concentration region (0-150 U / L), indicating that the method has good sensitivity for low concentration ox-LDL. However, when the concentration continues to increase, the curve gradually flattens.

[0060] The main curve is composed of experimental data points and a fitted curve, where the red scattered points represent experimental data points, and each data point is labeled with an error bar to reflect the systematic and random errors 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 x 1 / (1 + exp[-(a + b x lnC + c x C)]), where the fitting parameter values are: ΔA0 = 85.3, K = 1832.6, a = -2.31, b = 0.827, c = -0.00456. The light pink area on both sides of the curve represents the 95% confidence interval range. Within the ox-LDL concentration range of 0-150 U / L, the curve shows a clear upward trend with a large slope; when the concentration approaches 150 U / L, the curve slope gradually decreases and tends to be flat, showing typical immune analysis reaction kinetics characteristics.

[0061] A standardized residual analysis plot is provided below the main curve graph to assess the accuracy and reliability of the fitted curve. The horizontal axis maintains the same ox-LDL concentration range as the main graph (0-150 U / L), and the vertical axis represents the standardized residual values. The blue scattered dots in the residual plot indicate the degree of deviation between the experimental measurements and the theoretical fit values, and the zero line (dashed line) represents the ideal state of perfect fit. The residual distribution shows that across the entire concentration range, the standardized residual values ​​are generally distributed within ±2 and are randomly distributed on both sides of the zero line, exhibiting no significant systematic deviations.

[0062] 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.

[0063] Furthermore, an embodiment of the present application also discloses a computer program product, which, when running on a terminal device, enables the terminal device to execute any of the above methods.

[0064] Through the description of the above embodiments, it can be seen that those skilled in the art can clearly understand that all or part of the steps in the above-mentioned embodiment methods can be implemented by means of software plus the necessary general hardware platform. Based on this understanding, the technical solution of the present application, or the part that contributes to the prior art, can be embodied in the form of a software product, which can be stored in a storage medium such as ROM / RAM, a magnetic disk, an optical disk, etc., and includes a number of 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 each embodiment of the present application or certain parts of the embodiments.

[0065] It should be noted that the various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. Reference can be made to the common and similar parts between the various embodiments. For the devices disclosed in the embodiments, since they correspond to the methods disclosed in the embodiments, the description is relatively simple, and the relevant parts can be referred to the description of the methods.

[0066] It should also be noted that, in the embodiments of the present application, the terms such as first and second, etc. are used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between such entities or operations. Moreover, the terms "comprising", "containing", or any other variant thereof are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements in the list, but can also include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without more limitations, an element defined by the statement "comprising a" does not exclude the existence of additional identical elements in the process, method, article, or apparatus that includes the element.

[0067] The above description of disclosed embodiments enables a person skilled in the art to implement or use the present application. Various modifications to these embodiments will be apparent to those skilled in the art, 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 the 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 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, wherein the one or more programs include instructions, and when the instructions are executed by the processor, the processor executes a method for detecting oxidized low-density lipoprotein based on immunoturbidimetry, characterized by comprising: Polyethylene glycol and alkyl bromide compounds were coupled by active esterification reaction to prepare macroinitiators; Through atom transfer radical polymerization, methacrylic acid monomers are polymerized with a macroinitiator to prepare polyethylene glycol-b-polymethacrylic acid block copolymers; A free radical grafting reaction is adopted to prepare a graft copolymer by grafting dimethylaminoethyl methacrylate monomer onto a block copolymer as a skeleton; The surfaces of latex microspheres of different sizes were modified with graft copolymers, and then the microspheres of different sizes were coupled with different OxLDL monoclonal antibodies to prepare double-size latex microsphere markers. The method of using graft copolymer to modify the surface of latex microspheres of different particle sizes includes: The graft copolymer is dissolved in an aqueous solution, and then the latex microsphere dispersion is slowly added dropwise. Under stirring conditions, the stabilizer containing the graft copolymer is fully combined with the latex microspheres. The hydrophobic segment of the graft copolymer is adsorbed on the surface of the latex microspheres, while the hydrophilic segment extends into the aqueous phase, forming a stable "brush-like" structure. The unbound stabilizer is removed using membrane separation technology to obtain surface-modified latex microspheres. 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 based on a standard curve established based on the standard solution.

2. The device according to claim 1, characterized in that in, The surface of latex microspheres of different particle sizes is modified by graft copolymers, and then the microspheres of different particle sizes are coupled with different OxLDL monoclonal antibodies to prepare dual-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 to the mixture, stirring to uniformly disperse them; 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 latex microsphere dispersions of different particle sizes; The modified latex microspheres with large target particle size were coupled with a low-activity oxidized low-density lipoprotein monoclonal antibody in the presence of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride as a coupling agent. The modified latex microspheres with small target particle size were coupled with a low-activity oxidized low-density lipoprotein monoclonal antibody in the presence of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride as a coupling agent. The two coupled latex microsphere markers are mixed in a preset ratio to obtain a double-size latex microsphere marker.

3. The device 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 based on a standard curve established based on a standard solution, including: Obtaining a serum sample, separating the serum by centrifugation, and concentrating the serum sample using an ultrafiltration centrifuge device, and then diluting the serum sample to a target concentration using a buffer; preparing a tris (hydroxymethyl)aminomethane hydrochloride buffered reaction solution comprising sodium chloride, disodium edetate, a surfactant, polyethylene glycol, a stabilizer, and a blocking agent, 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, mix, and continue incubating at 37°C for 3 minutes; The absorbance at the target wavelength is measured using a biochemical analyzer using a two-point endpoint method, and the difference between the absorbance values ​​at the start and end of the reaction is calculated to establish a standard curve based on 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 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.

4. The device according to claim 1, characterized in that in, The macroinitiator is prepared by coupling polyethylene glycol with a brominated alkyl compound using an active esterification reaction, including: Dissolve polyethylene glycol in anhydrous tetrahydrofuran, add triethylamine, and stir at low temperature for a predetermined time to form a reaction solution; Slowly adding a tetrahydrofuran solution of α-bromoisobutyryl bromide dropwise 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 distillation under reduced pressure to obtain a polyethylene glycol macromolecular initiator.

5. The device according to claim 1, characterized in that in, The method comprises the following steps: preparing a polyethylene glycol-b-polymethacrylic acid block copolymer by atom transfer radical polymerization and initiating polymerization of methacrylic acid monomer with a macromolecular initiator; 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.

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