Double-antibody sandwich immunoassay method for fibrinogen based on up-conversion nanoparticles and magnetic beads

Abstract

The invention provides a method for immunoassay of fibrinogen based on up-conversion nanoparticle and magnetic bead double antibody sandwich. The UCNPs labeled antibody and the magnetic bead labeled capture antibody are jointly used for fibrinogen detection, the detection sensitivity and specificity are remarkably improved, the operation steps are simple, and the detection time is remarkably shortened. Experimental results show that the sensitivity of the detection method is improved by several times compared with that of a traditional ELISA method, and the lower detection limit is achieved, so that the application of the detection method in complex biological samples is more practical. In conclusion, the method shows a good clinical application prospect and is expected to play an important role in future clinical diagnosis and treatment.

Description

Technical Field

[0001] The invention belongs to biotechnology and relates to fibrinogen detection, in particular to a method for immunoassaying fibrinogen based on upconversion nanoparticles and magnetic bead double antibody sandwich immunoassay. Background Art

[0002] In the field of biomedical testing, the detection of fibrinogen plays an important role in disease diagnosis, disease monitoring and prognosis assessment. Fibrinogen is a glycoprotein synthesized by hepatocytes. As a key factor in the blood coagulation process, it can be converted into fibrin in the coagulation cascade reaction, thereby forming a coagulation network structure, promoting wound healing and hemostasis. Therefore, abnormal changes in the level of fibrinogen in plasma are closely related to a variety of thrombotic and inflammatory diseases, and its accurate detection is crucial for early diagnosis, risk assessment and monitoring of treatment effects. Chinese patent CN 103946391A discloses a fibrinogen determination method, which is complex to operate, has low specificity and sensitivity. Chinese patent CN 105548562 A discloses a fibrinogen immunoturbidimetric detection kit, which is complex to operate and has low specificity. At present, the enzyme-linked immunosorbent assay (ELISA) method is widely used in clinical practice. However, although the enzyme-linked immunosorbent assay (ELISA) is sensitive, it still has problems such as long time consumption, complex operation, and the specificity and sensitivity need to be improved, which makes it difficult to meet the clinical demand for rapid and reliable detection. Therefore, developing a rapid, highly sensitive and specific fibrinogen detection method has important clinical application value. Summary of the invention

[0003] In order to solve the problems in the prior art, the present invention provides a method for double antibody sandwich immunoassay of fibrinogen based on upconversion nanoparticles (UCNPs) and magnetic beads (MBs). The present invention combines UCNPs-labeled antibodies with magnetic bead-labeled capture antibodies for the detection of fibrinogen, which not only significantly improves the sensitivity and specificity of the detection, but also has simple operation steps and significantly shortens the detection time. The experimental results of the present invention show that the sensitivity of the detection method is several times higher than that of the traditional ELISA method, and a lower detection limit is achieved, which makes its application in complex biological samples more practical. In summary, the method of the present invention shows good clinical application prospects and is expected to play an important role in future clinical diagnosis and treatment.

[0004] To achieve the above object, the technical solution of the present invention is:

[0005] A method for immunoassaying fibrinogen based on upconversion nanoparticles and magnetic beads double antibody sandwich, characterized in that: fibrinogen standard solutions of different concentrations are mixed with magnetic beads MBs-Ab bound to anti-fibrinogen antibodies, incubated in a buffer at 30-50°C, then the MBs-Ab-fibrinogen complex is separated by magnetic separation, and then UCNPs-labeled anti-fibrinogen antibodies UCNPs-Ab are added to the complex, and incubated in a buffer at 30-50°C to allow UCNPs-Ab to bind to the MBs-Ab-fibrinogen complex to form a double antibody sandwich structure complex; unbound UCNPs-Ab is removed by magnetic separation, the double antibody sandwich structure complex is redispersed in a buffer, and the double antibody sandwich structure complex fluorescence signal is measured under 900-1100nm laser excitation, the fluorescence intensity is proportional to the fibrinogen concentration, and a standard curve is established; the fluorescence intensity obtained by immunoassaying the sample to be tested with the double antibody sandwich structure complex is calculated by the established standard curve to obtain the fibrinogen concentration.

[0006] According to one embodiment of the present invention, the above-mentioned buffer solution is a conventional buffer solution in the art, such as PBS buffer, Tris-HCl buffer, HEPES buffer, MES buffer, TAPS buffer, etc. Studies have found that the fluorescence intensity of the double antibody sandwich structure complex in the system of the present invention is different in different buffer solutions. Preferably, the buffer solution of the present invention is selected from one or more of PBS buffer, Tris-HCl buffer, and HEPES buffer; more preferably, the buffer solution of the present invention is PBS buffer.

[0007] The study also found that the pH value in the reaction system will seriously affect the fluorescence intensity of the complex of the double antibody sandwich structure in the system of the present invention. The present invention examined the fluorescence intensity under five different pH values ​​(pH = 6.6, 7.0, 7.4, 7.8, 8.2). The results showed that when the pH value was 6.6-7.0, the fluorescence intensity gradually increased; when the pH value was 7.4, the fluorescence intensity dropped sharply. Therefore, the pH value in the reaction system of the present invention is 6.6-7.0.

[0008] The study also found that the antibody concentration has an important influence on the fluorescence intensity of the complex of the double antibody sandwich structure in the system of the present invention. As the antibody concentration increases, the amount of antibody bound to the upconversion nanoparticles (UCNPs) and magnetic beads (MBs) also increases. However, when the antibody concentration reaches about 7μg / μL (UCNPs) and 6μg / μL (MBs), respectively, the fluorescence intensity tends to be stable. Even if more antibodies are added, it is difficult to bind to the surface of the nanoparticles. The concentration of the upconversion nanoparticles (UCNPs) of the present invention is 1-8μg / μL, preferably 5-8μg / μL, and more preferably 7-8μg / μL. The concentration of the magnetic beads (MBs) of the present invention is 1-8μg / μL, preferably 5-8μg / μL, and more preferably 6-8μg / μL.

[0009] The study also found that the incubation time of the reaction between UCNPs-Ab and fibrinogen also has an important influence on the fluorescence intensity of the complex of the double antibody sandwich structure in the system of the present invention. The present invention investigated the incubation time of UCNPs-Ab and fibrinogen (from 10 minutes to 60 minutes), and the results showed that the fluorescence intensity gradually increased with the increase of incubation time and reached the highest value at 40 minutes. The incubation time of the reaction between UCNPs-Ab and fibrinogen of the present invention is 10-60min, preferably 30-50min, and more preferably 40-50min.

[0010] According to one embodiment of the present invention, a method for immunoassaying fibrinogen based on upconversion nanoparticles and magnetic beads double antibody sandwich is characterized in that: a fibrinogen standard solution of different concentrations is mixed with magnetic beads MBs-Ab bound to anti-fibrinogen antibodies, incubated in a buffer at 30-50° C. for 40-50 minutes, the MBs-Ab-fibrinogen complex is separated from the solution by magnetic separation, an anti-fibrinogen antibody UCNPs-Ab labeled with UCNPs is added to the complex, and then incubated in a buffer at 30-50° C. for 40-50 minutes to allow the UCNPs-Ab to bind to the MBs-Ab-fibrinogen complex. The invention discloses a method for preparing a double antibody sandwich structure comprising the steps of: removing unbound UCNPs-Ab by magnetic separation, re-dispersing the complex forming the double antibody sandwich structure in a buffer solution, and measuring the fluorescence signal under 980 nm laser excitation, wherein the fluorescence intensity is proportional to the concentration of fibrinogen, and establishing a standard curve; calculating the fibrinogen concentration by using the fluorescence intensity obtained by double antibody sandwich immunoassay of the sample to be tested through the established standard curve; the buffer solution is PBS buffer solution, and the pH value in the reaction system is 6.6-7.0; the concentration of the upconversion nanoparticles (UCNPs) is 7-8 μg / μL, and the concentration of the magnetic beads (MBs) is 6-8 μg / μL.

[0011] According to one embodiment of the present invention, the preparation method of the above-mentioned UCNPs-Ab is: dissolving PEG-modified UCNPs in PBS buffer, adding EDC·HCl and Sulfo-NHS to mix evenly, and reacting at room temperature for 30-50 minutes; after the reaction is completed, centrifuging and collecting the precipitate, washing with PBS, dispersing the precipitate in PBS buffer, adding fibrinogen mouse monoclonal antibody, and cross-linking reaction at room temperature overnight to obtain UCNPs-Ab.

[0012] According to one embodiment of the present invention, the preparation method of the above-mentioned MBs-Ab is as follows: COOH-MB is washed with PBS buffer, EDC·HCl and Sulfo-NHS are added and mixed, and the mixture is reacted at room temperature for 20-40 minutes; after the reaction is completed, the precipitate is collected by centrifugation, washed with PBS, and the precipitate is dispersed in PBS buffer, and fibrinogen mouse monoclonal antibody is added and mixed, and the mixture is continuously stirred at room temperature for 3-5 hours, and unbound antibodies are removed by magnetic separation to obtain MBs-Ab capture probes.

[0013] According to one embodiment of the present invention, the preparation method of the above-mentioned PEG-modified UCNPs is as follows: dissolving oleic acid-coated β-NaYF4:Yb (20%), Tm (0.5%) UCNPs in chloroform, adding DSPE-PEG-COOH, stirring until the chloroform is completely evaporated, adding ultrapure water and then ultrasonically dispersing them fully, and centrifuging to remove unbound PEG and other impurities to obtain PEG-modified UCNPs; the preparation method of the oleic acid-coated β-NaYF4:Yb (20%), Tm (0.5%) UCNPs is as follows: dissolving YCl3·6H2O, YbCl3·6H2O and TmCl3·6H2O in deionized water, and then mixing with oleic acid and 1-octadecene, and mixing. The liquid is stirred at room temperature while excluding oxygen with argon, and the reaction is carried out for 0.5-1.5 hours; then it is heated to 150-160°C and maintained for 0.5-1.5 hours to obtain a transparent yellow solution; then the reaction system is cooled, and oxygen is continuously excluded with argon; after the reaction system is cooled to room temperature, a methanol solution containing ammonium fluoride and sodium hydroxide is added, and the reaction system is heated to 60-70°C and maintained for 10-30 minutes to evaporate methanol, and is further heated to 280-300°C and maintained for 1-2 hours, and then cooled to room temperature, ethanol is added to precipitate nanoparticles, and the precipitate is collected by centrifugation, washed with cyclohexane, and the precipitate is dispersed in cyclohexane, and freeze-dried to obtain oleic acid-coated β-NaYF4:Yb (20%), Tm (0.5%) UCNPs.

[0014] Beneficial effects:

[0015] The present invention provides a method for double antibody sandwich immunoassay of fibrinogen based on upconversion nanoparticles (UCNPs) and magnetic beads (MBs). The present invention combines UCNPs-labeled antibodies with magnetic bead-labeled capture antibodies for the detection of fibrinogen, which not only significantly improves the sensitivity and specificity of the detection, but also has simple operation steps and significantly shortens the detection time. The experimental results show that the sensitivity of the detection method is several times higher than that of the traditional ELISA method, and a lower detection limit is achieved, which makes its application in complex biological samples more practical. In summary, the method of the present invention shows good clinical application prospects and is expected to play an important role in future clinical diagnosis and treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Schematic diagram of the double antibody sandwich immunoassay based on upconversion nanoparticles (UCNPs) and magnetic beads (MBs);

[0017] Figure 2 TEM characterization of UCNPs;

[0018] Figure 3 TEM characterization of PEG-UCNPs;

[0019] Figure 4 SEM characterization of UCNPs;

[0020] Figure 5 HRTEM characterization of UCNPs;

[0021] Figure 6 HAADF-STEM characterization of UCNPs;

[0022] Figure 7 XRD characterization of UCNPs;

[0023] Figure 8 EDS characterization of UCNPs;

[0024] Fig. 9 FT-IR characterization images of UCNPs and PEG-UCNPs;

[0025] Fig.10 Fluorescence images of UCNPs;

[0026] Fig.11 Fluorescence intensity graphs of UCNPs from different synthesis batches;

[0027] Fig.12 Fluorescence intensity graph of UCNPs for 7 consecutive days in a week;

[0028] Fig.13 UV image of capture probe;

[0029] Fig.14 Fluorescent probe UV images;

[0030] Fig.15 MBs and capture probe particle size diagram;

[0031] Fig.16 Particle size diagram of UCNPs and fluorescent probe;

[0032] Fig.17 Zeta potential diagram of MBs and capture probes;

[0033] Fig.18 Zeta potential diagram of UCNPs and fluorescent probe;

[0034] Fig.19 Optimization diagram of antibody coupling amount of UCNPs;

[0035] Fig. 20 Optimization diagram of antibody coupling amount of MBs;

[0036] Fig.21 Buffer optimization diagram;

[0037] Fig. 22 Incubation time optimization diagram;

[0038] Fig.23 Reaction pH optimization diagram;

[0039] Fig.24 Feasibility diagram of fibrinogen detection;

[0040] Fig.25 Fluorescence intensity graph of fibrinogen at different concentrations;

[0041] Fig.26 Linear graph of different fibrinogen concentrations;

[0042] Fig. 27 Validation specificity plot;

[0043] Fig.28 Linearity graph of fibrinogen detected by ELISA;

[0044] Fig.29 Comparison of serum spiked samples using two methods. DETAILED DESCRIPTION

[0045] In the following examples, the present invention is only exemplarily described, but those skilled in the art can make various modifications to the present invention without departing from the spirit and scope of the present invention after reading this patent application. Unless otherwise specified, the parts described in the present invention are all parts by weight, and the percentages are all weight percentages.

[0046] The raw materials and reagents described in the present invention are all commercially available products. Rare earth chloride, ammonium fluoride (NH4F), EDC·HCl and Sulfo-NHS were purchased from Aladdin Reagent Co., Ltd. (Shanghai, China). Fibrinogen (Fbg), fibrin (Fbn), thrombin (Thr), immunoglobulin G (IgG), bovine serum albumin (BSA), recombinant albumin (Alb), myoglobin (Myo) and MagnaBind carboxyl-derivatized magnetic beads were purchased from Thermo Fisher Scientific (Pittsburgh, Pennsylvania, USA). Fibrinogen mouse monoclonal antibody (11-B3-D8, 1-A7, 2 mg / mL) was purchased from BGI (Hangzhou, China). All aqueous solutions were prepared using ultrapure water (Millipore, USA). Phosphate buffer (PBS, pH 7.4, 0.01 M) was purchased from Shanghai Sangon Biotechnology Co., Ltd. (Shanghai, China). Enzyme-linked immunosorbent assay (ELISA) kit was purchased from Shanghai Sangon Biotechnology Co., Ltd. (Shanghai, China).

[0047] Example 1

[0048] The preparation of UCNPs-Ab and MBs-Ab was carried out according to the following steps:

[0049] (1) Synthesis of β-NaYF4:Yb(20%),Tm(0.5%)UCNPs

[0050] First, YCl3·6H2O (482 mg), YbCl3·6H2O (155 mg) and TmCl3·6H2O (2.8 mg) were dissolved in deionized water (2 mL), and the solution was added to a 100 mL flask containing oleic acid (15 mL) and 1-octadecene (30 mL). The mixture was stirred at room temperature while excluding oxygen with argon and reacted for about 1 hour. Next, the mixture was slowly heated to 120°C to remove moisture and maintained at 156°C for about 1 hour until a uniform transparent yellow solution was obtained. Subsequently, the reaction system was cooled to room temperature, and oxygen was continuously excluded with argon during the period. After cooling, a methanol solution (10 mL) containing ammonium fluoride (NH4F, 296 mg) and sodium hydroxide (NaOH, 200 mg) was added to the system. The mixture was heated to 70°C and maintained for 20 minutes to evaporate methanol. Then, it was continued to be heated to 290°C and maintained for 1.5 hours, and then cooled to room temperature. After the reaction, ethanol (20 mL) was added to precipitate the nanoparticles, and the precipitate was collected by centrifugation (9000 rpm, 10 min). After washing four times, the final product was redispersed in cyclohexane (20 mL) and freeze-dried for later use. Figure 2 ) and scanning electron microscopy (SEM) (Attached Figure 4) observed that the synthesized UCNPs had good dispersion and spherical morphology, with an average particle size of about 33 nm. In addition, the clear lattice spacing was well consistent with the (100) crystal plane (Appendix Figure 5 ). At the same time, elemental analysis (HAADF-STEM) confirmed the presence of F, Na, Tm and Y elements, proving the successful synthesis of the nanoparticles (Attached Figure 6 ). X-ray diffraction (XRD) (Attached Figure 7 ) The results showed that UCNPs had a hexagonal phase structure, which was consistent with the standard crystal data, and energy dispersive spectrometry (EDS) (Appendix Figure 8 ) also shows the presence of four elements.

[0051] (2) Preparation of PEG-modified UCNPs

[0052] First, the oleic acid-coated β-NaYF4:Yb (20%), Tm (0.5%) UCNPs (40 mg) synthesized in step (1) were dissolved in chloroform (4 mL). Then, 0.8 mL of the mixed solution was added to a sample bottle and DSPE-PEG-COOH (15 mg) was added. Next, chloroform (4.2 mL) was added and stirred until the chloroform was completely evaporated. During the evaporation of chloroform, the surface of UCNPs was modified with PEG to form more hydrophilic nanoparticles. Subsequently, 3 mL of ultrapure water was added to the container and ultrasonic treatment was performed to fully disperse the UCNPs. After the treatment was completed, centrifugation (10000 rpm, 20 minutes) was performed to obtain COOH-modified UCNPs (COOH-UCNPs). This step will remove unbound PEG and other impurities to ensure the purity of the final product. Through PEG modification, the water solubility of UCNPs is significantly improved, enhancing its application potential in the biomedical field. After PEG modification, the size of the nanoparticles increased to about 35 nm (appended Figure 3 In addition, FT-IR spectroscopy showed that PEG modification was successful (see Fig. 9 ). Under spectral analysis, UCNPs produced significant fluorescence signals under 980nm laser excitation (Appendix Fig.10 ), and the fluorescence intensity remained consistent in different batches and 7-day continuous measurements, indicating that it has excellent stability (Appendix Fig.11 , 12 ).

[0053] (3) Preparation of UCNPs-Ab

[0054] Using EDC·HCl and Sulfo-NHS as crosslinkers, the fibrinogen mouse monoclonal antibody was covalently bound to UCNPs. The specific steps are as follows: 1 mg UCNPs was dissolved in 1 mL PBS buffer (10 mM, pH 7.4) and sonicated for 10 minutes. Then, 0.5 mg EDC·HCl and 1 mg Sulfo-NHS were added to activate the carboxyl groups of UCNPs, and the mixture was gently shaken at room temperature for 40 minutes. After the reaction, the activated UCNPs were collected by centrifugation and washed three times with PBS. Subsequently, the obtained precipitate was dispersed in 1 mL PBS buffer, and the fibrinogen mouse monoclonal antibody was added, and the crosslinking reaction was allowed to proceed overnight at room temperature. The final UCNPs-Ab conjugate was dispersed in 1% BSA-PBS (1 mg / mL) and stored at 4°C for later use.

[0055] (4) Preparation of MBs-Ab

[0056] First, 100 μL of COOH-MB (concentration 5 mg / mL) was washed twice with PBS buffer. Then, 0.5 mg of EDC·HCl and 1 mg of Sulfo-NHS were added to activate the carboxyl groups of MBs and reacted for 30 minutes. After activation, the MBs were washed three times with PBS again to remove unbound reagents. Next, the activated COOH-MB was mixed with a fibrinogen mouse monoclonal antibody (100 μL) contained in PBS buffer and stirred continuously at room temperature for 4 hours. Afterwards, the unbound antibody was removed by magnetic separation to obtain the MBs-Ab capture probe. Finally, the obtained MBs-Ab conjugate was also dispersed in 1% BSA-PBS (5 mg / mL) and stored at 4°C for later use. Ultraviolet-visible spectroscopy (UV-Vis) showed that the absorbance at 280 nm decreased significantly after antibody fixation, indicating that the antibody was successfully bound to the MBs (attached Fig.13 ) and UCNPs (with Fig.14 At the same time, dynamic light scattering (DLS) results showed that the capture probe (attached Fig.15 ) and fluorescent probes (attached Fig.16 ) increased significantly, while the capture probe (attached Fig.17 ) and fluorescent probes (attached Fig.18 ) became more negative than before, further confirming the successful immobilization of the antibody.

[0057] Example 2 Detection of fibrinogen by immunoassay based on UCNPs-MBs

[0058] The principle of double antibody sandwich immunoassay based on upconversion nanoparticles (UCNPs) and magnetic beads (MBs) is as follows Figure 1 shown.

[0059] First, the standard solution of fibrinogen with different concentrations was mixed with magnetic beads bound to anti-fibrinogen antibodies (MBs-Ab, 5 μL) and incubated in PBS buffer at 37°C for 40 minutes to ensure that fibrinogen was fully bound to the capture antibody. After the incubation, the MBs-Ab-fibrinogen complex was separated from the solution by magnetic separation and washed three times with fresh PBS buffer to remove unbound components and reduce background interference. Then, UCNPs-labeled anti-fibrinogen antibodies (UCNPs-Ab, 10 μL) were added to the above complex and incubated in PBS buffer at 37°C for 40 minutes to allow UCNPs-Ab to bind to the MBs-Ab-fibrinogen complex to form a double antibody sandwich structure. After the incubation, the unbound UCNPs-Ab was removed by magnetic separation and washed again with PBS buffer three times to ensure the purity of the sample. In order to quantitatively detect the concentration of fibrinogen, the final complex was redispersed in 200 μL PBS buffer and the upconversion fluorescence signal of UCNPs was measured under 980 nm laser excitation. The fluorescence intensity is proportional to the concentration of fibrinogen. By establishing a standard curve, the fibrinogen concentration in the sample can be quantitatively analyzed.

[0060] The study found that the amount of antibody bound to the nanoparticle surface has an important impact on the present invention. As the antibody concentration increases, the amount of antibody bound to upconversion nanoparticles (UCNPs) and magnetic beads (MBs) also increases. When the antibody concentration reaches about 7μg / μL (UCNPs) and 6μg / μL (MBs), respectively, the fluorescence intensity tends to be stable. Even if more antibody is added, steric hindrance will prevent it from further binding to the nanoparticle surface. Therefore, the optimized antibody binding amount is 7μg / μL (UCNPs) and 6μg / μL (MBs). Fig.19 ), MBs was 6μg / μL (attached Fig. 20 ).

[0061] The type of buffer solution also has an important impact on the present invention. The present invention evaluated five different buffers. The fluorescence was strongest in PBS buffer, which may be due to the suitable ion concentration in PBS that is conducive to antigen-antibody recognition. Therefore, PBS buffer was selected as the reaction buffer for subsequent experiments (see Appendix). Fig.21 The present invention also studied the different incubation times (from 10 minutes to 60 minutes) of the reaction between UCNPs-Ab and fibrinogen. The fluorescence intensity gradually increased with the increase of incubation time and reached the highest value at 40 minutes (see Figure 2). Fig. 22). Therefore, the optimal incubation time of UCNPs-Ab and fibrinogen is 40 minutes. The study also found that pH value also has an important influence on the present invention, so five different pH values ​​were tested for optimization. At pH 7.0, the fluorescence intensity was the highest, which may be because this pH value can maintain the protein structure and promote specific antigen-antibody recognition (see Appendix Fig.23 ). Therefore, all subsequent reactions used a buffer with a pH of 7.0.

[0062] Firstly, the feasibility of the fluorescent immunosensor based on upconversion nanoparticles (UCNPs) and magnetic beads (MBs) in the detection of fibrinogen was verified (see Appendix). Fig.24 ). Next, the developed method was used to study the relationship between the fluorescence intensity of UCNPs and fibrinogen concentration under the optimized conditions. To determine the detection limit of the fluorescent immunosensor, the inventors analyzed fibrinogen concentrations of 5, 10, 25, 50, 100, 200, and 400 ng / mL (see Appendix Fig.25 The experimental results showed that the fluorescence intensity and fibrinogen concentration showed a good linear relationship in the range of 5 to 100 ng / mL, the calibration equation was Y = 0.0034X + 0.0023, and the correlation coefficient R 2 The detection limit was 0.9977, and the detection limit reached 2ng / mL, with a significant improvement in sensitivity (see Appendix Fig.26 ).

[0063] In order to verify the specificity of the detection method, a series of selectivity tests were performed. Other biomolecules (such as fibrin, thrombin, immunoglobulin G, etc.) were introduced under the same experimental conditions to ensure that the detected signal was caused only by fibrinogen. In addition, fibrinogen detection was performed in serum samples diluted 10 times, and the accuracy and sensitivity of the method were verified by standard spiking experiments. In the selective experiments, the specificity of the detection platform was further verified by adding potential interferents such as immunoglobulin G (IgG), bovine serum albumin (BSA), recombinant albumin (Alb) and myoglobin (Myo). The results showed that the fluorescence signal of fibrinogen was significantly higher than the background signal induced by other substances, and no obvious nonspecific reaction was observed (Appendix Fig. 27 ). These optimization and validation experiments confirmed the excellent performance of the detection platform in terms of sensitivity and specificity, providing a reliable method for the rapid and accurate detection of fibrinogen.

[0064] In order to verify the fibrinogen detection platform of the present invention in terms of sensitivity ( Fig.28 ) and accuracy (with Fig.29), the inventors conducted a comparative experiment with the traditional enzyme-linked immunosorbent assay (ELISA) kit. The experimental results show that the UCNPs-MBs double antibody sandwich immunoassay of the present invention is significantly superior to the standard ELISA method in terms of detection limit and response time. Specifically, the detection limit of the ELISA kit is about 10ng / mL, while the detection limit of this method is as low as 2ng / mL, and the sensitivity is increased by 5 times. In addition, under the same sample processing conditions, the detection platform of the present invention can complete the detection within 1 hour, while the ELISA kit usually requires more than 2 hours of incubation and washing steps. This time advantage is particularly important for clinical emergency testing, which helps to obtain test results faster and support clinical decision-making.

[0065] The detection platform of the present invention realizes rapid and sensitive detection of fibrinogen through the combination of UCNPs and MBs, and is suitable for clinical application scenarios requiring high specificity and sensitivity. The method is easy to operate and has a short detection time. It not only reduces the complexity of sample preparation and processing, but also improves the detection efficiency. It provides an effective detection tool for the early diagnosis of thrombotic and hemorrhagic diseases, and shows a wide range of application prospects in biomedical research and clinical diagnosis.

Claims

1. A method for immunoassay of fibrinogen based on upconversion nanoparticles and magnetic beads double antibody sandwich immunoassay, characterized in that: The fibrinogen standard solution of different concentrations was mixed with the magnetic beads MBs-Ab bound to the anti-fibrinogen antibody, and incubated in a buffer at 30-50°C. Then, the MBs-Ab-fibrinogen complex was separated from the solution by magnetic separation. The anti-fibrinogen antibody UCNPs-Ab labeled with UCNPs was added to the complex, and then incubated in a buffer at 30-50°C to allow the UCNPs-Ab to bind to the MBs-Ab-fibrinogen complex to form a complex with a double antibody sandwich structure. The unbound UCNPs-Ab was removed by magnetic separation, and the complex with the double antibody sandwich structure was redispersed in the buffer. The method comprises the following steps: adding the fluorescent signal of the double antibody sandwich structure complex to the washing liquid, and measuring the fluorescent signal of the double antibody sandwich structure complex under 900-1100nm laser excitation, wherein the fluorescence intensity is proportional to the concentration of fibrinogen, and a standard curve is established; the fluorescence intensity obtained by immunoassaying the sample to be tested with the double antibody sandwich structure complex is calculated by the established standard curve to obtain the fibrinogen concentration; the buffer solution is selected from PBS buffer, Tris-HCl buffer, HEPES buffer, MES buffer or TAPS buffer; the pH value in the reaction system is 6.6-7.0; the incubation time of the reaction between UCNPs-Ab and fibrinogen is 10-60min; The preparation method of the UCNPs-Ab is as follows: dissolving PEG-modified UCNPs in a PBS buffer, adding EDC·HCl and Sulfo-NHS to mix evenly, and reacting at room temperature for 30-50 minutes; after the reaction is completed, collecting the precipitate by centrifugation, washing with PBS, dispersing the precipitate in a PBS buffer, adding a fibrinogen mouse monoclonal antibody, and cross-linking reaction at room temperature overnight to obtain a UCNPs-Ab conjugate; the concentration of UCNPs is 1-8 μg / μL; The preparation method of the MBs-Ab is as follows: COOH-MB is washed with PBS buffer, EDC·HCl and Sulfo-NHS are added and mixed, and the mixture is reacted at room temperature for 20-40 minutes; after the reaction is completed, the precipitate is collected by centrifugation, the precipitate is washed with PBS, dispersed in PBS buffer, fibrinogen mouse monoclonal antibody is added and mixed, and the mixture is continuously stirred at room temperature for 3-5 hours, and unbound antibodies are removed by magnetic separation to obtain the MBs-Ab capture probe; the concentration of MBs is 1-8 μg / μL.

2. The method according to claim 1, characterized in that: The buffer solution is selected from one or more of PBS buffer, Tris-HCl buffer, and HEPES buffer.

3. The method according to claim 1, characterized in that: The buffer solution is PBS buffer.

4. The method according to claim 1, characterized in that: The concentration of UCNPs was 5-8 μg / μL.

5. The method according to claim 1, characterized in that: The concentration of MBs was 5-8 μg / μL.

6. The method according to claim 1, characterized in that: The incubation time of UCNPs-Ab and fibrinogen reaction was 30-50 min.

7. The method according to claim 1, characterized in that: The buffer solution is PBS buffer, the concentration of UCNPs is 7-8 μg / μL, the concentration of MBs is 6-8 μg / μL, and the incubation time of the reaction between UCNPs-Ab and fibrinogen is 40-50 min.

8. The method according to any one of claims 1 to 7, characterized in that: The fibrinogen standard solution of different concentrations was mixed with the magnetic beads MBs-Ab bound to the anti-fibrinogen antibody, incubated in a buffer at 30-50°C for 40-50 minutes, and the MBs-Ab-fibrinogen complex was separated from the solution by magnetic separation. The anti-fibrinogen antibody UCNPs-Ab labeled with UCNPs was added to the complex, and then incubated in a buffer at 30-50°C for 40-50 minutes to allow the UCNPs-Ab to bind to the MBs-Ab-fibrinogen complex to form a double antibody sandwich structure; the unbound UCNPs were removed by magnetic separation. Ps-Ab, the complex forming a double antibody sandwich structure is redispersed in a buffer, and the fluorescence signal is measured under 980nm laser excitation, the fluorescence intensity is proportional to the concentration of fibrinogen, and a standard curve is established; the fluorescence intensity obtained by the double antibody sandwich immunoassay of the sample to be tested is calculated by the established standard curve to obtain the fibrinogen concentration; the buffer solution is PBS buffer, and the pH value in the reaction system is 6.6-7.0; the concentration of upconversion nanoparticles (UCNPs) is 7-8μg / μL, and the concentration of magnetic beads (MBs) is 6-8μg / μL.

9. The method according to claim 8, characterized in that: The standard curve equation of fluorescence intensity and fibrinogen concentration is Y=0.0034X+0.0023, where X is the fibrinogen concentration and Y is the fluorescence intensity.

10. The method according to claim 1, characterized in that: The preparation method of the PEG-modified UCNPs is as follows: dissolving oleic acid-coated β-NaYF4:Yb (20%), Tm (0.5%) UCNPs in chloroform, adding DSPE-PEG-COOH, stirring until the chloroform is completely evaporated, adding ultrapure water and then ultrasonically dispersing them fully, and centrifuging to remove unbound PEG and other impurities to obtain PEG-modified UCNPs; the preparation method of the oleic acid-coated β-NaYF4:Yb (20%), Tm (0.5%) UCNPs is as follows: dissolving YCl3·6H2O, YbCl3·6H2O and TmCl3·6H2O in deionized water, and then mixing with oleic acid and 1-octadecene, and stirring the mixture at room temperature , while excluding oxygen with argon, react for 0.5-1.5 hours; then heat to 150-160°C and maintain for 0.5-1.5 hours to obtain a transparent yellow solution; then cool the reaction system, during which oxygen is continuously excluded with argon; after the reaction system is cooled to room temperature, a methanol solution containing ammonium fluoride and sodium hydroxide is added, heated to 60-70°C and maintained for 10-30 minutes to evaporate methanol, continued to heat to 280-300°C and maintained for 1-2 hours, then cooled to room temperature, ethanol was added to precipitate nanoparticles, and the precipitate was collected by centrifugation, washed with cyclohexane, dispersed in cyclohexane, and freeze-dried to obtain oleic acid-coated β-NaYF4:Yb (20%), Tm (0.5%) UCNPs.

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