A method for detecting horseradish peroxidase by forming fluorescent copolymer at room temperature and an enzyme-linked immunosorbent assay for detecting cardiac troponin type I

Abstract

The present invention relates to a method for detecting horseradish peroxidase by forming a fluorescent copolymer at room temperature and an enzyme-linked immunosorbent assay for detecting cardiac troponin type I, and belongs to the field of nano-biosensor technology. The method comprises: generating a green fluorescent copolymer based on p-phenylenediamine and polyethyleneimine under the action of horseradish peroxidase to detect horseradish peroxidase. In addition, in view of the wide application of the enzyme in immunoassay, we use it as a marker enzyme to construct an immunofluorescence sensing platform for detecting cardiac troponin type I. The fluorescent copolymer nanoparticles based on p-phenylenediamine and polyethyleneimine in the present invention have the advantages of room temperature synthesis, simple steps, strong fluorescence, etc. The constructed immunosensor platform successfully realizes the detection of cardiac troponin type I in the serum samples of patients with acute myocardial infarction. The experimental results are consistent with those of the standard test kit, which shows that the immunofluorescence test kit designed by us has great potential in disease diagnosis.

Description

Technical Field

[0001] The invention relates to a method for detecting horseradish peroxidase by forming fluorescent copolymer at room temperature, an enzyme-linked immunosorbent assay method for detecting cardiac troponin based on a novel fluorescent substrate with the action of horseradish peroxidase and its application, and belongs to the field of nanotechnology. Background Art

[0002] Enzyme-linked immunosorbent assay (ELISA) is mainly based on the specific reaction of antigen and antibody and the efficient biocatalytic properties of enzyme. Due to its good accuracy and practicality, low cost and high throughput, it has been widely used in food safety analysis, environmental monitoring and biomedical diagnosis. The method usually uses an enzyme, usually horseradish peroxidase (HRP) to catalyze the conversion of the corresponding substrate into a colorful or highly fluorescent quantitative product. In addition, horseradish peroxidase is also the most widely studied member of the peroxidase family. Due to its extremely high stability in aqueous solution, it has a wide range of applications in diagnosis, biosensing and biotechnology. Therefore, in this regard, it remains necessary and important to develop a convenient strategy to measure HRP activity.

[0003] At present, the methods for detecting HRP activity include fluorescence, colorimetry, electrochemistry and chemiluminescence. Correspondingly, some immunoassays have been established based on these analytical methods. Among these immunoassays, colorimetric immunoassay is a conventional method for detecting target analytes in laboratories and industry / clinical settings. However, traditional colorimetric ELISA has the problem of high detection limit. To solve this dilemma, Shiddiky et al. studied a new type of colorimetric immunosensor in Analytical Chemistry in 2017. The sensor couples gold nanoporous iron oxide nanoparticles and horseradish peroxidase, uses 3,3,5,5-tetramethylbenzidine as the enzyme substrate, and performs colorimetric detection of autoantibodies in patients with ovarian epithelial cancer in the presence of hydrogen peroxide. It is even expected to be extended to further detect biomarkers of other diseases. Zhang et al. reported an ultrasensitive colorimetric biosensor for human chorionic gonadotropin (hCG) detection in Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy in 2019. The sensor co-immobilizes HRP and mouse anti-hcg monoclonal antibody (gh-submit, mAb1) on the surface of Au@Pt NP to improve catalytic efficiency and specificity, forming an Au@Pt-HRP dual-functionalized probe, which greatly improves the detection limit of the colorimetric method. However, the synthesis and modification of these expensive nanomaterials require a lot of time. In addition, precious metal nanomaterials are easily affected by external factors. At the same time, fluorescent immunoassay as an alternative or supplement to colorimetric immunoassay has attracted more and more attention. However, it is regrettable that there are currently few reports on fluorescent reactions triggered by horseradish peroxidase. Therefore, it is of great significance to explore new fluorescent substrates and fluorescent reactions of horseradish peroxidase in immunoassays labeled with horseradish peroxidase.

[0004] Here, we found in the preliminary experimental screening that polyethyleneimine and p-phenylenediamine can quickly form fluorescent copolymer nanoparticles (FCNPs) at room temperature under the action of horseradish peroxidase and hydrogen peroxide, which indicates that these two substances can serve as new fluorescent substrates for horseradish peroxidase. Interestingly, we also synthesized fluorescent copolymer nanoparticles (FCNPs) with different wavelengths by introducing groups with different electron push-pull capabilities based on the p-phenylenediamine structure. In addition, given the characteristic that horseradish peroxidase is often used as a marker enzyme in immunoassay, we used cardiac troponin type I as a target antigen and constructed a fluorescent immunosensor platform based on this, and successfully detected cardiac troponin type I in serum samples of patients with acute myocardial infarction and compared it with the standard kit. The results show that the immunosensor we developed has the advantages of simple operation, low cost, and simple signal generation, and has great potential in disease diagnosis. Summary of the invention

[0005] The purpose of the present invention is to find a new HRP-catalyzed fluorescent substrate, namely p-phenylenediamine and polyethyleneimine, in order to overcome the shortcomings of the prior art. On the one hand, HRP is used to catalyze these two substances to generate fluorescent copolymer nanoparticles (FCNPs) to detect the activity of HRP; on the other hand, the property of HRP as a marker enzyme in enzyme-linked immunosorbent assay is used to construct a fluorescent immunosensor with cardiac troponin type I as the target antigen.

[0006] One of the technical schemes of the present invention is a method for detecting horseradish peroxidase by forming a fluorescent copolymer in situ at room temperature. Under room temperature conditions, a certain amount of polyethyleneimine, p-phenylenediamine, hydrogen peroxide and horseradish peroxidase of different concentrations are added to a Tris-HCl buffer solution, and strong green FCNPs are generated after reaction, and the fluorescence intensity of the FCNPs gradually increases with the increase of the concentration of horseradish peroxidase, thereby realizing the quantitative detection of horseradish peroxidase.

[0007] When constructing a sensing platform for detecting horseradish peroxidase based on a fluorescent copolymer formed in situ at room temperature, the pH of the Tris-HCl buffer solution is 7.4, the concentration of Tris-HCl is 10 mM, and the concentrations of polyethyleneimine, p-phenylenediamine and hydrogen peroxide are 5 mg / mL, 60 μM and 500 μM, respectively.

[0008] Take 500 μL Tris-HCl buffer solution, first incubate 500 μM hydrogen peroxide and different concentrations of horseradish peroxidase on a shaker at 25°C, then draw 10 μL and 3 μL from 250 mg / mL polyethyleneimine and 10 mM p-phenylenediamine mother solutions into centrifuge tubes, shake well, react for several minutes, and then measure the fluorescence intensity using a fluorescence spectrometer to draw a graph.

[0009] The second technical solution of the present invention is a method for detecting cardiac troponin type I based on an immunofluorescence sensor that induces in situ formation of fluorescent copolymers using horseradish peroxidase. In a 96-well plate, the capture antibody is combined with the antigen, and then the antigen is connected to the primary antibody, which is then connected to the HRP-labeled secondary antibody to construct a sandwich immune system. Finally, a fluorescent substrate is added to achieve quantitative detection of cardiac troponin type I by changing the fluorescence intensity of the fluorescent copolymer.

[0010] When constructing a fluorescent immunosensor platform triggered by horseradish peroxidase, the pH of the Tris-HCl buffer solution is 7.4, the concentration of Tris-HCl is 10 mM, the concentration of polyethyleneimine is 5 mg / mL, the concentration of p-phenylenediamine is 60 μM, the concentration of hydrogen peroxide is 500 μM, the incubation temperature of horseradish peroxidase and hydrogen peroxide is 25°C, the concentration of bovine serum albumin is 20 mg / mL, the concentration of capture antibody is 1 μg / mL, the concentration of primary antibody is 2 μg / mL, and the concentration of HRP-labeled secondary antibody is 0.5 μg / mL.

[0011] First, a series of monoclonal antibodies were injected into 4 wells of a 96-well plate and incubated overnight. After incubation, the plate was washed with TBST five times, and 200 μM bovine serum albumin was added to each well to prevent nonspecific binding sites on the surface of the plate. Subsequently, the plate was incubated at 37°C for 1 hour, and the bovine serum albumin was removed with TBST. Then, 100 μL of cTnI solution with different concentrations was incubated at 37°C for 1 hour, and after removing unbound cTnI with TBST, anti-goat anti / Ab2 was transferred to the plate and incubated at 37°C for 1 hour. After the incubation, the plate was washed with TBST five times, and then Tris-HCl buffer, HRP-labeled rabbit goat secondary antibody and hydrogen peroxide were added to incubate at 25°C for 1 hour to form a sandwich immunoassay. Finally, the plate was washed with TBST, and p-phenylenediamine and polyethyleneimine were added to the plate. Fluorescence spectroscopy was performed after reacting at room temperature for several minutes.

[0012] The third technical solution of the present invention is the application of an immunofluorescent sensor based on horseradish peroxidase-induced in situ formation of fluorescent copolymers to detect cardiac troponin type I in serum. After sandwich immunization is completed, a certain amount of diluted human serum is added to a Tris-HCl buffer solution at room temperature, and then a certain amount of polyethyleneimine and p-phenylenediamine are added. After reacting for several minutes, strong green FCNPs are generated, and the fluorescence intensity of FCNPs gradually increases with the increase of cardiac troponin type I, thereby realizing quantitative detection of cardiac troponin type I in serum.

[0013] Preferably, when constructing a fluorescent immunosensor platform triggered by horseradish peroxidase, the pH of the Tris-HCl buffer solution is 7.4, the concentration of Tris-HCl is 10 mM, the concentration of polyethyleneimine is 5 mg / mL, the concentration of p-phenylenediamine is 60 μM, the concentration of hydrogen peroxide is 500 μM, the incubation temperature of horseradish peroxidase and hydrogen peroxide is 25°C, the concentration of bovine serum albumin is 20 mg / mL, the concentration of capture antibody is 1 μg / mL, the concentration of primary antibody is 2 μg / mL, and the concentration of HRP-labeled secondary antibody is 0.5 μg / mL.

[0014] First, a series of monoclonal antibodies were injected into a 96-well plate and incubated overnight at 4°C. After incubation, the plate was rinsed with TBST five times, and 200 μM bovine serum albumin was added to each well to prevent nonspecific binding sites on the plate surface. Subsequently, the plate was incubated at 37°C for 1 hour, and the bovine serum albumin was removed with TBST. Then, 100 μL of cTnI solution with different concentrations was incubated at 37°C for 1 hour, and after removing unbound cTnI with TBST, anti-goat anti / Ab2 was transferred to the plate and incubated at 37°C for 1 hour. After the incubation, the plate was washed with TBST five times, and then Tris-HCl buffer, HRP-labeled rabbit goat secondary antibody and hydrogen peroxide were added for incubation at 25°C for 1 hour to form a sandwich immunoassay. Finally, the plate was rinsed with TBST, and human diluted serum was added to the plate, followed by p-phenylenediamine and polyethyleneimine, and fluorescence spectroscopy was performed at room temperature.

[0015] The fourth technical solution of the present invention is the application of an immunofluorescent sensor based on horseradish peroxidase-induced in situ formation of fluorescent copolymers to the preparation of a kit for detecting cardiac troponin type I in real patient serum samples. After the bovine serum albumin blocking is completed, 100 μL of patient serum samples of different concentrations are added, and then the sandwich immunization is completed according to the above steps. Finally, a certain amount of polyethyleneimine and p-phenylenediamine are added. After reacting for several minutes, strong green FCNPs are generated, and the fluorescence intensity of FCNPs gradually increases with the increase of cardiac troponin type I, thereby realizing the quantitative detection of cardiac troponin type I in patient serum samples.

[0016] The present invention is further described below.

[0017] When constructing a fluorescent immunosensor platform triggered by horseradish peroxidase, the pH of the Tris-HCl buffer solution is 7.4, the concentration of Tris-HCl is 10 mM, the concentration of polyethyleneimine is 5 mg / mL, the concentration of p-phenylenediamine is 60 μM, the concentration of hydrogen peroxide is 500 μM, the incubation temperature of horseradish peroxidase and hydrogen peroxide is 25°C, the concentration of bovine serum albumin is 20 mg / mL, the concentration of capture antibody is 1 μg / mL, the concentration of primary antibody is 2 μg / mL, the concentration of HRP-labeled secondary antibody is 0.5 μg / mL, and the patient serum sample is diluted 10 times with PBS.

[0018] First, a series of monoclonal antibodies were injected into a 96-well plate and incubated overnight at 4°C. After incubation, the plate was washed with TBST five times, and 200 μM bovine serum albumin was added to each well to prevent nonspecific binding sites on the plate surface. Subsequently, the plate was incubated at 37°C for 1 hour, and the bovine serum albumin was removed with TBST. Then, 100 μL of patient serum samples of different concentrations were added to the plate and incubated at 37°C for 1 hour. After removing unbound cTnI with TBST, anti-goat anti / Ab2 was transferred to the plate and incubated at 37°C for 1 hour. After the incubation, the plate was washed with TBST five times, and then Tris-HCl buffer, HRP-labeled rabbit goat secondary antibody and hydrogen peroxide were added and incubated at 25°C for 1 hour to form a sandwich immunoassay. Finally, the plate was washed with TBST, and human diluted serum was added to the plate, followed by p-phenylenediamine and polyethyleneimine. Fluorescence spectroscopy was performed after reacting at room temperature for several minutes.

[0019] Beneficial effects of the present invention:

[0020] The first method of the present invention is to use horseradish peroxidase to catalyze p-phenylenediamine and polyethyleneimine to form green fluorescent copolymer FCNPs at room temperature in the presence of hydrogen peroxide. The formation of the fluorescent copolymer is inseparable from horseradish peroxidase, so that high-selective detection of horseradish peroxidase can be achieved; in addition, the fluorescence intensity of FCNPs increases with the increase of the concentration of horseradish peroxidase, so quantitative detection of horseradish peroxidase can be achieved. Polyethyleneimine itself is alkaline when dissolved in water, and the fluorescence of FCNPs will be stronger under alkaline conditions. The second method of the present invention is that polyethyleneimine itself is alkaline when dissolved in water. In the subsequent tests, we uniformly use hydrochloric acid to adjust the pH of the alkaline solution after adding polyethyleneimine back to 7.4 for better biological applications. The results show that subsequent tests can still be performed in a neutral environment. The third method of the present invention is to use the characteristics of horseradish peroxidase as a marker enzyme in enzyme-linked immunosorbent assay, and use cardiac troponin type I as the targeting antigen to design an immunofluorescence sensor. P-phenylenediamine and polyethyleneimine are used as fluorescent substrates for enzyme-labeled secondary antibodies, and fluorescent signals of cardiac troponin type I are output by forming fluorescent copolymer nanoparticles, and then quantitative analysis is performed. It is worth mentioning that the detection of cardiac troponin type I by previous immune methods in complex environments is limited to spiked experiments, and it is difficult to achieve quantitative analysis. Here, the sensing platform we constructed can not only be applied to a complex environment such as human diluted serum to achieve quantitative detection of cardiac troponin type I, but also successfully achieve detection of cardiac troponin type I in serum samples of patients with acute myocardial infarction. The detection limit is calculated according to the formula 3σ / k, where σ is the standard deviation of 11 blank samples, and k is the slope. The detection limit is as low as 20pg / mL, which is much better than the detection of cardiac troponin type I reported in the past. The preparation of fluorescent copolymer nanoparticles in the present invention has the advantages of reaction at room temperature, mild conditions, simple steps, strong fluorescence and good biocompatibility, indicating that our immunofluorescence kit has great potential in the diagnosis of related diseases in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The embodiments of the present invention will be further described below in conjunction with the accompanying drawings.

[0022] Figure 1 is the absorption spectrum of the green FCNPs prepared in Example 1;

[0023] Figure 2 The fluorescence excitation spectrum of the green FCNPs prepared in Example 1;

[0024] Figure 3 is the fluorescence emission spectrum of the green FCNPs prepared in Example 1;

[0025] Figure 4These are photos of the green FCNPs prepared in Example 1 under sunlight and ultraviolet light;

[0026] Figure 5 TEM characterization image of FCNPs prepared in Example 2;

[0027] Figure 6 is the particle size distribution diagram of FCNPs prepared in Example 2;

[0028] Figure 7 This is a force microscopy characterization image of FCNPs prepared in Example 2;

[0029] Figure 8 It is a force microscopy height characterization image of FCNPs prepared in Example 2;

[0030] Fig. 9 is a force microscopy height distribution diagram of FCNPs prepared in Example 2;

[0031] Fig.10 The time scan diagram of FCNPs prepared in Example 3 under different conditions;

[0032] Fig.11 The fluorescence emission spectra of FCNPs prepared in Example 4 before and after nitrogen protection

[0033] Fig.12 The fluorescence emission spectra of FNCPs prepared in 5 under the action of ascorbic acid at different concentrations are shown in FIG.

[0034] Fig.13 Fluorescence emission spectra of different FCNPs prepared by the reaction of three kinds of o-p-phenylenediamine with polyethyleneimine in implementation 6

[0035] Fig.14 The bar graph of FCNPs prepared by reacting polyethyleneimine with p-phenylenediamine of different molecular weights in Example 7 is shown in FIG.

[0036] Fig.15 Fluorescence histogram of FNCPs prepared by using 8 different alkaline substances and p-phenylenediamine

[0037] Fig.16 The structural formula of the substance similar to the p-phenylenediamine structure according to the different electron donating abilities in implementation 9 is

[0038] Fig.17 Normalized fluorescence emission spectra of FCNPs prepared from a substance with a structure similar to p-phenylenediamine and polyethyleneimine in Example 9

[0039] Fig.18 Fluorescence emission spectra of green FCNPs prepared by using horseradish peroxidase at different concentrations in 10 experiments

[0040] Fig.19 The standard curve spectrum of green FCNPs prepared by 10 different concentrations of horseradish peroxidase

[0041] Fig. 20 Photographs of green FCNPs prepared by using horseradish peroxidase at different concentrations under UV light

[0042] Fig.21 A histogram of fluorescence intensity changes for 11 different horseradish peroxidase interfering substances

[0043] Fig. 22 Fluorescence emission spectra of the immunofluorescence kit for the implementation of 12 different concentrations of cardiac troponin I type

[0044] Fig.23 The relative fluorescence intensity standard curve of cardiac troponin I type 1 at different concentrations is shown in Figure 13.

[0045] Fig.24 For the implementation of 14 immunofluorescence kits for the selective fluorescence emission spectrum of cardiac troponin type I

[0046] Fig.25 Standard curve for 15 dilutions of human serum cardiac troponin I

[0047] Fig.26 Schematic diagram of a fluorescent immunoassay kit for detecting cardiac troponin type I based on horseradish peroxidase triggering DETAILED DESCRIPTION

[0048] The following is a detailed description of an embodiment of the present invention in conjunction with the accompanying drawings: This embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation method and process are given, but the protection scope of the present invention is not limited to the following embodiment.

[0049] Example 1: At room temperature, take 500 μL Tris-HCl buffer solution, first add 500 μM hydrogen peroxide and 120 mU / mL horseradish peroxidase and incubate at 25°C for several minutes, then add 5 mg / mL polyethyleneimine and 60 μM p-phenylenediamine, then adjust the pH of the solution back to 7.4 with hydrochloric acid and shake well. After reacting for several minutes, green fluorescence was observed under ultraviolet light, indicating that green FCNPs were successfully prepared. The absorption intensity of green FCPNs was detected by ultraviolet absorption spectrometer, and the absorption spectrum was drawn, as shown in FIG. Figure 1 The fluorescence intensity of green FCNPs was detected by fluorescence spectrometer, with an excitation wavelength of 370 nm and an emission wavelength of 510 nm, and the fluorescence excitation spectrum and fluorescence emission spectrum were plotted, as shown in Figure 2 , Figure 3 , Figure 4 Photos of green FCNPs under sunlight and UV light.

[0050] Example 2: At room temperature, take 500 μL Tris-HCl buffer solution, first add 500 μM hydrogen peroxide and 120 mU / mL horseradish peroxidase and incubate at 25°C, then add 5 mg / mL polyethyleneimine and 60 μM p-phenylenediamine, then adjust the pH of the solution back to 7.4 with hydrochloric acid, and shake well. After reacting for a few minutes, take a certain amount of FCNPs and drop them on a copper plate, and characterize them using transmission electron microscopy (TEM) and force microscopy to obtain TEM images and corresponding particle size distribution images, force microscopy images and height images of nanoparticles and corresponding height distribution images. The experimental results show that the green FCNPs we prepared are nanoparticles with an irregular spherical structure with a diameter of about 3 nm and a height of about 2 nm, such as Figure 5 and Figure 6 , Figure 7 , Figure 8 and Fig. 9 .

[0051] Example 3: At room temperature, take 2mL Tris-HCl buffer solution and divide it into four tubes, i.e., four groups. The first group only adds 5mg / mL polyethyleneimine and 60μM p-phenylenediamine; the second group adds 500μM hydrogen peroxide, and then adds 5mg / mL polyethyleneimine and 60μM p-phenylenediamine; the third group first adds 120mU / mL horseradish peroxidase, and then adds 5mg / mL polyethyleneimine and 60μM p-phenylenediamine; the fourth group first adds 500μM hydrogen peroxide and 120mU / mL horseradish peroxidase and incubates at 25℃, and then adds 5mg / mL polyethyleneimine and 60μM p-phenylenediamine. The above groups then use hydrochloric acid to adjust the pH of the solution back to 7.4 and shake well. The FCNPs were time scanned by fluorescence spectrometer, with an excitation wavelength of 370nm and an emission wavelength of 510nm. The time scan graph was drawn. The experimental results show that the reaction can only occur rapidly to produce fluorescence in the presence of horseradish peroxidase and hydrogen peroxide, as shown in FIG. Fig.10 .

[0052] Example 4: Take 1 mL of Tris-HCl buffer solution at room temperature and divide it into two tubes, i.e., 500 μL in each tube, add 5 mg / mL polyethyleneimine and 60 μM p-phenylenediamine to each tube, one group is protected by nitrogen, and the other group is exposed to reaction, and then the pH of the above two groups of solutions is adjusted back to 7.4 with hydrochloric acid. One day later, the FCNPs are time scanned by a fluorescence spectrometer, with an excitation wavelength of 370 nm and an emission wavelength of 510 nm, and the fluorescence emission spectrum is plotted, as shown in FIG. Fig.11This result strongly proves that the mechanism of polyethyleneimine and p-phenylenediamine to form such a fluorescent copolymer nanoparticle is oxidative polymerization.

[0053] Example 5: Take 3 tubes of 500 μL Tris-HCl buffer solution and divide them into 3 groups. Add 500 μM hydrogen peroxide to all three groups. The first group is the control group. The second and third groups are added with 500 μM and 800 μM ascorbic acid respectively and incubated at room temperature. After the end, the three groups are uniformly added with 120 mU / mL horseradish peroxidase and incubated at room temperature. Then, 5 mg / mL polyethyleneimine and 60 μM p-phenylenediamine are added for reaction. Then, the pH of the solution is adjusted back to 7.4 with hydrochloric acid. Then, the fluorescence intensity is detected by fluorescence spectrometer, with an excitation wavelength of 370 nm and an emission wavelength of 510 nm. The fluorescence emission spectrum is drawn. The results show that the rapid reaction is caused by the catalysis of hydroxyl radicals produced by the action of horseradish peroxidase and hydrogen peroxide. When ascorbic acid is used as a scavenger of hydroxyl radicals, the fluorescence of the system decreases significantly, as shown in FIG. Fig.12 .

[0054] Example 6: Take 3 tubes of 500μL Tris-HCl buffer solution and divide them into 3 groups. All three groups are first added with 500μM hydrogen peroxide and 120mU / mL horseradish peroxidase and incubated at 25°C, and then 5mg / mL polyethyleneimine and 60μM o- / m- / p-phenylenediamine are added respectively, where 1: o-phenylenediamine, 2: m-phenylenediamine, 3: p-phenylenediamine. The pH of the solution of the above three groups is then adjusted back to 7.4 with hydrochloric acid. After the reaction, the fluorescence intensity is detected by a fluorescence spectrometer with an excitation wavelength of 370nm and an emission wavelength of 510nm. The fluorescence emission spectrum is plotted, and the results show that only p-phenylenediamine can form green fluorescent nanoparticles with polyethyleneimine in the presence of horseradish peroxidase and hydrogen peroxide, as shown in FIG. Fig.13 , The inset is a photograph of nanoparticles formed by three types of phenylenediamines and polyethyleneimine under ultraviolet light.

[0055] Example 7: Take 5 tubes of 500μL Tris-HCl buffer solution and divide them into 5 groups. These five groups are first added with 500μM hydrogen peroxide and 120mU / mL horseradish peroxidase and incubated at 25°C, and then 60μM p-phenylenediamine and 5mg / mL of different alkaline substances are added respectively, among which a: polyethyleneimine, b: ethanolamine, c: ethylenediamine, d: phenylethylamine, and e: sodium hydroxide. The above groups then adjusted the pH of the solution back to 7.4 with hydrochloric acid. After the reaction, the fluorescence intensity was detected by a fluorescence spectrometer with an excitation wavelength of 370nm and an emission wavelength of 510nm. The fluorescence emission spectrum was drawn, and the results showed that only ethylenediamine and polyethyleneimine could form fluorescent nanoparticles with p-phenylenediamine under the action of horseradish peroxidase and hydrogen peroxide, such as Fig.14The reason can be attributed to the fact that the two substances have similar structures, and ethylenediamine is the monomer of polyethyleneimine.

[0056] Example 8: Take 5 tubes of 500μL Tris-HCl buffer solution and divide them into 5 groups. These five groups are first added with 500μM hydrogen peroxide and 120mU / mL horseradish peroxidase and incubated at 25℃, and then the same amount of polyethyleneimine with different molecular weights and 60μM p-phenylenediamine are added respectively. After reacting for several minutes, the fluorescence intensity is detected by fluorescence spectrometer. Among them, a does not add polyethyleneimine, b: molecular weight is 600, c: molecular weight is 1800, d: molecular weight is 10000, e: molecular weight is 70000. The above groups then use hydrochloric acid to adjust the pH of the solution back to 7.4, the excitation wavelength is 370nm, and the emission wavelength is 510nm. The fluorescence emission spectrum is drawn, and the results show that as the molecular weight increases, the degree of polymerization deepens and the fluorescence intensity becomes stronger. Among them, the polyethyleneimine side chain with a molecular weight of 70,000 has the best effect, such as Fig.15 .

[0057] Example 9: Take 6 tubes of 500 μL Tris-HCl buffer solution and divide them into 6 groups. These 6 groups are first added with 500 μM hydrogen peroxide and 120 mU / mL horseradish peroxidase for incubation at 25°C, and then add substances with a structure similar to p-phenylenediamine (different substituent groups are connected on the basis of the p-phenylenediamine structure) and polyethyleneimine respectively. After reacting for several minutes, the fluorescence intensity is detected by a fluorescence spectrometer, and the fluorescence emission spectrum is plotted, which is then normalized to achieve a shift from 470 nm to 512 nm in the emission wavelength. The results show that as the electron-withdrawing ability of the electron-withdrawing group increases, the wavelength blue shifts more significantly; the electron-donating ability of the electron-donating group increases, the wavelength red shifts more significantly, such as Fig.16 , Fig.17 .

[0058] Example 10: Take 500 μL Tris-HCl buffer solution at room temperature, add 500 μM hydrogen peroxide and 0-150 mU / mL horseradish peroxidase respectively and incubate at room temperature, then add 5 mg / mL polyethyleneimine and 60 μM p-phenylenediamine and shake well, then adjust the pH of the solution back to 7.4 with hydrochloric acid, react for several minutes, then observe green fluorescence under ultraviolet light, indicating that green FCNPs are successfully prepared. The fluorescence intensity of green FCNPs is detected by fluorescence spectrometer, with an excitation wavelength of 370 nm, and the fluorescence emission spectrum and standard curve are plotted. The results show that the fluorescence intensity of green FCNPs increases with the increase of horseradish peroxidase concentration, and presents a two-stage linear relationship within 0-150 mU / mL, with a detection limit as low as 130 μU / mL, such as Fig.18 , Fig.19 and Fig. 20 .

[0059] Example 10: At room temperature, take 10 portions of 500 μL Tris-HCl buffer solution, add 500 μM hydrogen peroxide in advance, and then add 120 mU / mL of different substances respectively: 0, blank; 1, acid phosphohydrolase; 2, alkaline phosphohydrolase; 3, inorganic pyrophosphatase; 4, bovine serum albumin; 5, lysosomal zymolysin; 6, cytochrome c; 7, glucose oxidase; 8, trypsin; 9, tyrosinase; 10, pepsin, incubate at room temperature, then add 5 mg / mL polyethyleneimine and 60 μM p-phenylenediamine and shake well, then adjust the pH of the solution back to 7.4 with hydrochloric acid, and after the reaction, use a fluorescence spectrometer to detect the fluorescence intensity, with an excitation wavelength of 370 nm and an emission wavelength of 510 nm. Then, 120 mU / mL horseradish peroxidase was added to each centrifuge tube, incubated at room temperature, and reacted for several minutes. The fluorescence intensity was detected again by fluorescence spectrometer, with the excitation wavelength at 370 nm and the emission wavelength at 510 nm. The fluorescence intensity bar graph before and after the addition of horseradish peroxidase was drawn. The experimental results showed that the system had strong anti-interference ability, such as Fig.21 .

[0060] Example 11: 100 μL of 1 μg / mL monoclonal antibody was injected into a 96-well plate and incubated overnight at 4°C. After incubation, it was rinsed with TBST 5 times, and 200 μL of bovine serum albumin was added to each well to prevent the appearance of non-specific binding sites on the surface of the well plate. Subsequently, it was incubated at 37°C for 1 hour, and the bovine serum albumin was removed with TBST. Then 100 μL of cTnI solution of different concentrations was incubated at 37°C for 1 hour, and after removing unbound cTnI with TBST, anti-goat anti / Ab2 was transferred to the well plate and incubated at 37°C for 1 hour. After the incubation, TBST was washed five times, and then Tris-HCl buffer, HRP-labeled rabbit goat secondary antibody and hydrogen peroxide were added and incubated at 25°C for 1 hour, thereby forming a sandwich immunization. Next, rinse with TBST, then add 500 μM hydrogen peroxide and incubate at room temperature for 15 minutes. After that, add 5 mg / mL polyethyleneimine and 60 μM p-phenylenediamine to the well plate, and then adjust the pH of the solution back to 7.4 with hydrochloric acid. After a few minutes at room temperature, perform fluorescence spectrum analysis with an excitation wavelength of 370 nm and an emission wavelength of 510 nm, and draw the fluorescence emission spectrum and standard curve of the immunofluorescence kit, as shown in the figure. Fig. 22 , Fig.23 The experimental results showed that the fluorescence intensity was positively correlated with the concentration of added cardiac troponin type I. The detection limit was calculated to be as low as 20pg / mL according to the formula 3σ / k, where σ is the standard deviation of 11 blank samples and k is the slope, which is much better than the detection of cardiac troponin type I reported previously.

[0061] Example 12: 100 μL of 1 μg / mL monoclonal antibody was injected into a 96-well plate and incubated overnight at 4°C. After incubation, it was rinsed with TBST 5 times, and 200 μL of bovine serum albumin was added to each well to prevent the appearance of non-specific binding sites on the surface of the well plate. Subsequently, it was incubated at 37°C for 1 hour, and the bovine serum albumin was removed with TBST. Then, 200 ng / mL of cardiac troponin type I, alpha-fetoprotein, lysosomal zymolysin, pepsin, and trypsin 100 μL were added to each well, incubated at 37°C for 1 hour, and after removing the unbound substances with TBST, anti-goat / Ab2 was transferred to the well plate and incubated at 37°C for 1 hour. After the incubation was completed, TBST was washed five times, and then Tris-HCl buffer, HRP-labeled rabbit goat secondary antibody and hydrogen peroxide were added and incubated at 25°C for 1 hour, thereby forming a sandwich immunization. Next, rinse with TBST and then add 500 μM hydrogen peroxide. After incubation at room temperature, add 5 mg / mL polyethyleneimine and 60 μM p-phenylenediamine to the well plate, and then adjust the pH of the solution back to 7.4 with hydrochloric acid. After reacting at room temperature for several minutes, perform fluorescence spectrum analysis with an excitation wavelength of 370 nm and an emission wavelength of 510 nm, and draw the fluorescence emission spectrum of the immunofluorescence kit. The experimental results show that the kit has good selectivity, such as Fig.24 .

[0062] Example 13: 100 μL of 1 μg / mL monoclonal antibody was injected into a 96-well plate and incubated overnight at 4°C. After incubation, it was rinsed with TBST 5 times, and 200 μL of bovine serum albumin was added to each well to prevent the appearance of non-specific binding sites on the surface of the well plate. Subsequently, it was incubated at 37°C for 1 hour, and the bovine serum albumin was removed with TBST. Then, 100 μL of cardiac troponin type I of different concentrations was added to each well, incubated at 37°C for 1 hour, and after removing the unbound substances with TBST, anti-goat anti / Ab2 was transferred to the well plate and incubated at 37°C for 1 hour. After the incubation was completed, TBST was washed five times, and then Tris-HCl buffer, HRP-labeled rabbit goat secondary antibody and hydrogen peroxide were added and incubated at 25°C for 1 hour, thereby forming a sandwich immunization. Next, rinse with TBST, then add 500μM hydrogen peroxide and incubate at room temperature. After the end, add a certain amount of diluted human serum to the well plate, then add 5mg / mL polyethyleneimine and 60μM p-phenylenediamine, and then adjust the pH of the solution back to 7.4 with hydrochloric acid. After reacting at room temperature for several minutes, perform fluorescence spectrum analysis with an excitation wavelength of 370nm and an emission wavelength of 510nm, and draw a standard curve of the immunofluorescence kit. The experimental results show that compared with previous reports, this kit can still quantify cardiac troponin I type even in a complex environment such as serum. Fig.25 .

[0063] Example 14: At room temperature, take 6 portions of 500 μL Tris-HCl buffer solution, first add 500 μM hydrogen peroxide and 0-150 mU / mL horseradish peroxidase and incubate at room temperature, then add 5 mg / mL polyethyleneimine and a series of substances with similar structures to p-phenylenediamine and shake well. After reacting at room temperature for several minutes, perform fluorescence spectrum analysis, and fill in the table with the chemical structure of each substance, the excitation wavelength and emission wavelength of different FCNPs formed, the relative quantum yield and photos under ultraviolet light. The experimental results show that the wavelength adjustment of FCNPs is achieved by connecting different groups with electron donating capabilities on the basis of the p-phenylenediamine structure, and the relevant properties are shown in Table 1.

[0064] Table 1 shows the spectral properties of FCNPs formed by substances with similar structures to p-phenylenediamine and polyethyleneimine

[0065]

[0066] Example 15: The concentration of cardiac troponin type I contained in each patient serum sample was calculated in advance using a standard kit, and then according to the routine operation of immunoassay, after blocking with bovine serum albumin, 100 μL of patient serum containing different concentrations of cardiac troponin type I was added, and then incubated at 37°C for 1 hour. After removing the unbound patient serum sample with TBST, anti-goat anti- / Ab2 was transferred to the well plate and incubated at 37°C for 1 hour. After the incubation, TBST was washed five times, and then Tris-HCl buffer, HRP-labeled rabbit goat secondary antibody and hydrogen peroxide were added to incubate at 25°C for 1 hour, thereby forming a sandwich immunoassay. Next, TBST was used for washing, and then 500 μM hydrogen peroxide was added for incubation at room temperature, followed by 5 mg / mL polyethyleneimine and 60 μM p-phenylenediamine, and then the pH of the solution was adjusted back to 7.4 with hydrochloric acid. After reacting for several minutes at room temperature, fluorescence spectrum analysis was performed with an excitation wavelength of 370 nm and an emission wavelength of 510 nm. The recovery rate of cardiac troponin I was calculated and compared with that of the standard kit, where A and B are normal human serum samples, and C, D, E, and F are different patient samples. The experimental results show that the data of the two are basically consistent, suggesting that the immunosensor we designed has great potential in disease diagnosis, as shown in Table 2.

[0067] Table 2 shows the recovery rate of cardiac troponin I detected in patient serum samples compared with the standard kit

[0068]

[0069] The present invention is not limited to the specific technical solutions described in the above embodiments, and all technical solutions formed by equivalent replacement are within the protection scope required by the present invention.

Claims

1. A method for detecting horseradish peroxidase by forming a fluorescent copolymer at room temperature, characterized in that: Under room temperature, horseradish peroxidase HRP and hydrogen peroxide H2O2 were first added to a Tris-HCl buffer solution and incubated on a shaker, followed by the addition of polyethyleneimine PEI and p-phenylenediamine, which were mixed evenly to generate strong green fluorescent copolymer FCNPs. The fluorescence intensity of the synthesized FCNPs was detected by a fluorescence spectrometer; the detection of horseradish peroxidase was achieved through the change in fluorescence intensity; The synthesis conditions of FCNPs were as follows: the pH of Tris-HCl buffer solution was 7.4, the concentration of Tris-HCl was 10 mM, the concentration of horseradish peroxidase was 120 mU / mL, the concentration of hydrogen peroxide was 500 μM, the concentration of p-phenylenediamine was 60 μM, and the concentration of polyethyleneimine was 5 mg / mL.

2. A method for preparing a detection kit for detecting cardiac troponin type I based on an enzyme-linked immunosorbent assay sensor constructed using a fluorescent copolymer formed at room temperature as a substrate, characterized in that: First, a series of monoclonal antibodies were injected into a 96-well plate and incubated overnight at 4°C. After incubation, the plate was washed with TBST five times, and 200 μM bovine serum albumin was added to each well to prevent the appearance of nonspecific binding sites on the surface of the plate. Subsequently, the plate was incubated at 37°C for 1 hour, and the bovine serum albumin was removed with TBST. Then, 100 μL of cardiac troponin type I solution of different concentrations was incubated at 37°C for 1 hour, and after removing the unbound cardiac troponin type I with TBST, anti-goat anti- / Ab2 was transferred to the plate and incubated at 37°C for 1 hour. After the incubation, the plate was washed with TBST five times, and then Tris-HCl buffer, HRP-labeled rabbit goat secondary antibody and hydrogen peroxide were added to incubate at 25°C for 1 hour, thus forming a sandwich immunization. After washing with TBST, 500 μM hydrogen peroxide was added and incubated at room temperature. After the end, a certain amount of human diluted serum was added to the plate, followed by 5 mg / mL polyethyleneimine and 60 μM p-phenylenediamine was added, and then the pH of the solution was adjusted back to 7.4 with hydrochloric acid. After reacting at room temperature for several minutes, fluorescence spectroscopy analysis was performed. The pH of the Tris-HCl buffer solution was 7.4, the concentration of Tris-HCl was 10 mM, the concentration of bovine serum albumin was 20 mg / mL, the concentration of monoclonal antibody was 1 μg / mL, the concentration of goat antibody was 2 μg / mL, and the concentration of HRP-labeled rabbit and goat secondary antibody was 0.5 μg / mL.

3. The method for preparing a detection kit for detecting cardiac troponin type I based on an enzyme-linked immunosorbent assay sensor constructed using a fluorescent copolymer formed at room temperature as a substrate according to claim 2, characterized in that: The volume of each substance added to the 96-well plate was 100 μL, and the incubation conditions for the antigen and antibody were 37°C for 1 hour; the amount of Tris-HCl buffer solution was 300 μL, and the amounts of p-phenylenediamine and polyethyleneimine in the fluorescent substrate were 1.2 μL 10 mM stock solution and 6 μL 250 mg / mL stock solution, respectively.

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