A method for constructing a three-enzyme cascade fluorescence immunoassay platform by sequentially integrating ALP, TYR, and HRP

Abstract

The present invention constructs a method for a three-enzyme cascade-triggered fluorescence immunosensing platform by sequentially integrating ALP, TYR, and HRP. This platform relies on the fact that HRP and H2O2 can promote the rapid in-situ fluorescence reaction of dopamine and 1,5-naphthalenediol to produce a strong yellow fluorescent compound (AFC). The molecular formula (C 18 H 15 NO4) and structure of the synthesized AFC were determined by nuclear magnetic resonance mass spectrometry characterization, and based on the fact that ALP can induce the hydrolysis of p-aminophenylethyl phenyl phosphate (PAPP) to produce the intermediate tyramine, and TYR can catalyze the production of DA, which in turn activates HRP, a three-enzyme fluorescence immune system was constructed. The high-sensitivity detection of cardiac troponin I was achieved using this system. This advanced signal transduction scheme can provide a new method for studying multi-enzyme cascade systems and constructing multifunctional biosensors.

Description

Technical Field

[0001] The present invention relates to a method and application for constructing a three-enzyme cascade-triggered fluorescence immunosensor platform by sequentially integrating alkaline phosphatase, tyrosinase, and horseradish peroxidase, which is used for sensitively and selectively detecting cardiac troponin and belongs to the field of biosensing technology. Background Art

[0002] Enzyme cascade reactions can sequentially carry out multiple enzymatic reactions, which are important components of signal transduction and amplification in biological systems and also have broad application prospects in biotechnology. During this reaction process, the product obtained by the catalysis of the first enzyme can directly serve as the reaction substrate of the second enzyme, and the locally high concentration of the catalytic product obtained in situ can reduce the ineffective diffusion of enzyme intermediates. Therefore, compared with single-enzyme catalytic amplification, the enzyme cascade amplification system exhibits higher catalytic efficiency.

[0003] Currently, there is an increasing focus on developing multi-stage enzyme-linked sensing systems with new signal generation that integrate multiple biocatalytic conversions. So far, various methods for multi-enzyme cascade-induced reactions have been developed, such as colorimetry, electrochemistry, chemiluminescence, fluorescence, etc. However, most enzyme cascade sensing systems are based on specific colorimetric methods, in which alkaline phosphatase (ALP) and HRP are commonly used inducing enzymes. In addition, due to the lack of new substrates suitable for the system, the signal output modes of substrates / products in enzyme cascade sensing systems are too few. For example, the Yang group developed an enzyme cascade immunoassay for detecting cTnI based on enzyme-induced fluorescence and colorimetric reactions by coupling ALP and TYR. The Chang group reported a dual-enzyme cascade biosensor based on the self-assembly of copper nanoparticles to enhance the fluorescence signal for ultrasensitive detection of MicroRNA153. However, to our knowledge, most of the reported enzyme cascade sensing systems adopt a dual-enzyme strategy and are in a non-biological environment. But in biological systems, enzyme cascade reactions often require more than two enzymes. In addition, the substrates of enzymes include not only intramolecular interactions but also the interactions of many substances. Therefore, it is very necessary to develop multi-stage enzyme-linked sensing systems with new signal generation mechanisms.

[0004] To overcome these deficiencies, here we constructed a fluorescence immunosensing platform triggered by an ALP-TYR-HRP three-enzyme cascade under mild synthesis conditions and with simple synthesis steps. This detection method has the advantages of rapid response, high selectivity, and high sensitivity. Summary of the Invention

[0005] The technical problem solved by the present invention is: in order to overcome the deficiencies of the prior art, a method for constructing a three-enzyme cascade fluorescence immunosensing platform based on ALP-TYR-HRP for sensitively and selectively detecting cardiac troponin is proposed. HRP and H2O2 can promote the rapid in-situ fluorescence reaction of dopamine and 1,5-naphthalenediol to produce a strong yellow fluorescent compound (AFC). First, ALP can induce the hydrolysis of PAPP to produce the intermediate tyramine. Further introducing TYR can catalyze the production of DA, which in turn activates HRP, thus realizing signal amplification involving three enzymes, ALP, TYR, and HRP. In addition, the cascade catalytic process involving ALP is integrated into ELISA (ALP is used as the labeling enzyme, and TYR and HRP are used as signal reporting enzymes).

[0006] To solve the technical problem of the present invention, the technical solution proposed is: alkaline phosphatase ALP induces the hydrolysis of p-aminophenylethyl phenyl phosphate PAPP to produce the intermediate tyramine. Further introducing TYR can catalyze the production of DA, which in turn activates HRP. HRP and H2O2 can promote the rapid in-situ fluorescence reaction of dopamine and 1,5-naphthalenediol to produce a strong yellow fluorescent compound AFC C 18 H 15 NO4; thus realizing signal amplification involving three enzymes, ALP, TYR, and HRP. The excitation wavelength and emission wavelength of the yellow fluorescent compound AFC are fixed at 490 nm and 550 nm respectively, and AFC is characterized by nuclear magnetic resonance mass spectrometry to determine the molecular formula and structure of AFC; the structural formula of AFC is as follows:

[0007] HRP and H2O2 can promote the rapid in-situ fluorescence reaction of dopamine and 1,5-naphthalenediol to produce a strong yellow fluorescent compound (AFC). ALP can induce the hydrolysis of p-aminophenylethyl phenyl phosphate (PAPP) to produce the intermediate tyramine. Further introducing TYR can catalyze the production of DA, which in turn activates HRP, thus realizing signal amplification involving three enzymes, ALP, TYR, and HRP. In addition, the cascade catalytic process involving ALP is integrated into ELISA (ALP is used as the labeling enzyme, and TYR and HRP are used as signal reporting enzymes). Then the mixture is oscillated at room temperature for 3 minutes, and its fluorescence intensity is detected by a fluorescence spectrometer under excitation at 490 nm.

[0008] The expected ALP catalyzes the cleavage of the phosphate group in PAPP, inducing the conversion of PAPP into tyramine, which is oxidized by TYR to DA. The combined action of HRP and H2O2 promotes the rapid in-situ fluorescence reaction of DA and DHA to generate the fluorescent compound AFC. We used ultraviolet-visible absorption spectroscopy and fluorescence spectroscopy to study the optical properties of AFC. The DA-DHA-HRP-H2O2 mixed solution has a strong emission peak at 550 nm, while the DA-DHA, DA-DHA-HRP, and DA-DHA-H2O2 mixtures have no fluorescence.

[0009] Furthermore, to obtain the optimal conditions for AFC generation, we optimized the pH value, reaction time, and reactant concentrations of the reaction system. First, considering the actual analytical applications, we selected the pH value of the Tris-HCl buffer to be 7.4. The fluorescence intensity of the reaction system can reach stability at 3 min. Therefore, the optimal reaction time for DA and DHA is 3 min. Then, the influence of the reactant concentrations was optimized. As the concentrations of DA, DHA, and H2O2 increased, the value of F1 / F0 increased rapidly. When DA, DHA, and H2O2 were 400 μM, 30 μM, and 60 μM respectively, F1 / F0 tended to be stable. Therefore, the favorable experimental conditions for the concentrations of DA, DHA, and H2O2 are 400 μM, 30 μM, and 60 μM respectively.

[0010] Furthermore, under ultraviolet light irradiation, AFC has an absorption peak at 490 nm, and bright yellow fluorescence can be observed. The excitation wavelength and emission wavelength of AFC are fixed at 490 nm and 550 nm respectively, showing a Stokes shift of about 60 nm. Through theoretical calculations, the generation of the emission peak of AFC near 550 nm can be attributed to the partial charge transfer characteristics of the lowest singlet excited state (S1) exhibited by the natural transition orbit (NTO). Then, the chemical structure of AFC was characterized and confirmed by 1 1H NMR, 13 13C NMR, and high-resolution mass spectrometry.

[0011] Furthermore, first, DA is oxidized to an o-quinone structure in the presence of oxygen. Further intermolecular 1,4-Michael addition occurs between the o-quinone structure and DHA to generate intermediate B. The rearomatization of B forms the coupling product C, but C is easily oxidized to quinone D, and then the furan intermediate E is obtained through intramolecular Michael addition. The enol structure of E can rearrange into the keto form under certain conditions. Finally, an intramolecular condensation reaction occurs between the amino group and the carbonyl group to generate AFC. It should be noted that atmospheric oxygen as an oxidant is a prerequisite for catalyzing the oxidation of catechol to form the strongly emissive fluorophore AFC.

[0012] Furthermore, in the co - presence of HRP and H2O2, an in - situ cyclization reaction occurs between DA and DHA, generating a significant fluorescence signal. In the present invention, a three - enzyme - cascade - triggered fluorescence immunosensing platform is constructed by sequentially integrating ALP, TYR, and HRP. The Yang group developed an enzyme - cascade immunoassay method. Under alkaline conditions, an in - situ cyclization reaction occurs between dopamine and resorcinol. By coupling ALP and TYR, cTnI is detected based on enzyme - induced fluorescence and colorimetric reactions. Compared with the present invention, the reaction conditions in this report are not mild enough. Secondly, a two - enzyme - cascade - triggered fluorescence immunosensing platform is constructed, and the catalytic amplification effect is not as advantageous as that of the present invention. Finally, the emission peak of the reaction product of dopamine and resorcinol in that report is at 460 nm, showing blue emission. In the present invention, the emission peak of the reaction product of dopamine and 1,5 - naphthalenediol is near 550 nm, showing yellow emission.

[0013] Furthermore, the TYR can catalyze the hydroxylation of monophenol to generate catechol. Using tyramine as a substrate, TYR catalyzes the hydroxylation reaction of tyramine to generate DA. In the presence of HRP and H2O2, DA and DHA can produce an obvious fluorescence signal. This enzyme - cascade fluorescence reaction prompted us to construct an in - situ fluorescence sensor for monitoring the activity of TYR.

[0014] Furthermore, a three - enzyme - linked immuno - fluorescence sensor for detecting the activity of ALP using PAPP as a substrate is developed. This sensor is based on the conversion of PAPP to the intermediate tyramine catalyzed by ALP, the hydroxylation reaction of tyramine catalyzed by TYR to generate DA, and the specific reaction of DA and DHA in the HRP - H2O2 system.

[0015] Furthermore, based on the wide application of ALP in conventional ELISA, we constructed a three - enzyme - linked immuno - fluorescence sensor platform by sequentially integrating ALP, TYR, and HRP using cTnI as a model.

[0016] Furthermore, the serum is a highly complex biological fluid containing a large number of proteins and other interfering molecules. Therefore, it is very challenging to identify biomarkers in serum. Due to the good selectivity and sensitivity of the proposed ternary enzyme-linked fluorescence immunosensor, we further applied it to the determination of cTnI in clinical actual serum samples. We measured the cTnI levels of 2 normal adult volunteers and 4 clinical patient samples using an ALP-based ELISA detection system and a commercial standard ELISA kit as a control. Notably, the cTnI concentrations measured by our system were basically consistent with those of the commercial TMB-based standard ELISA kit. These results indicate that the proposed ternary enzyme-linked fluorescence ELISA detection system has a high cTnI evaluation ability in actual clinical samples and has good application prospects in the clinical diagnosis of disease biomarkers.

[0017] Another technical solution proposed to solve the technical problems of the present invention is: to prepare a kit for alkaline phosphatase, tyrosinase, horseradish peroxidase and cardiac troponin according to the described method.

[0018] The following further explains the present invention.

[0019] Add HRP solutions with different concentrations (0 - 4 mU / mL) to Tris-HCl buffer (10 mM, pH 7.4) containing DA (400 μM), DHA (30 μM) and H2O2 (60 μM), then oscillate the mixture at room temperature for 3 minutes, and detect its fluorescence intensity through a fluorescence spectrometer under 490 nm excitation.

[0020] Method for detecting horseradish peroxidase based on the ALP-TYR-HRP ternary enzyme cascade fluorescence sensing platform. Add HRP solution (4 mU / mL) to Tris-HCl buffer (10 mM, pH 7.4) containing DA (400 μM), DHA (30 μM) and H2O2 (60 μM), then oscillate the mixture at room temperature for 3 minutes, and detect its fluorescence intensity through a fluorescence spectrometer under 490 nm excitation. Under the same conditions, the selectivity of a series of competitive enzymes or proteins such as acid phosphatase (ACP), pepsin, cytochrome C (Cyt c), TYR, trypsin (Try), glucose oxidase (GO X ) and lysozyme to HRP was determined.

[0021] Method for detecting tyrosinase based on ALP-TYR-HRP triple-enzyme cascade fluorescence sensing platform. Add TYR at different concentrations (0 - 3.5 U / mL) to Tris-HCl buffer (10 mM, pH = 7.4) containing tyramine (200 μM). Incubate the mixture at 37 °C for 20 minutes, and then add the TYR-tyramine mixed solution to a solution containing DHA (30 μM), H2O2 (60 μM) and HRP (4 mU / mL). After incubating the above mixed solution at room temperature for 3 minutes, measure the fluorescence spectrum with a fluorescence spectrophotometer.

[0022] Method for detecting tyrosinase based on ALP-TYR-HRP triple-enzyme cascade fluorescence sensing platform. Add TYR (3.5 U / mL) to Tris-HCl buffer (10 mM, pH = 7.4) containing tyramine (200 μM). Incubate the mixture at 37 °C for 20 minutes, and then add the TYR-tyramine mixed solution to a solution containing DHA (30 μM), H2O2 (60 μM) and HRP (4 mU / mL). Then shake the mixture at room temperature for 3 minutes, and detect its fluorescence intensity through a fluorescence spectrometer under excitation at 490 nm. Under the same conditions, the selectivity of a series of competitive enzymes or proteins such as GOx, Try, pepsin, lysozyme, Cyt c and ACP for TYR was determined.

[0023] Method for detecting alkaline phosphatase based on the ALP-TYR-HRP triple-enzyme cascade fluorescence sensing platform described above. Add different concentrations of ALP (0 - 1250 mU / mL) to Tris-HCl buffer (10 mM, pH = 9) containing PAPP (1 mM). After incubating the mixture at 37 °C for 30 minutes, add the ALP-PAPP mixed solution to Tris-HCl buffer solution (10 mM, pH = 7.4) containing 3 U / mL TYR. Then add the ALP-PAPP-TYR mixed solution to Tris-HCl buffer solution (10 mM, pH = 7.4) containing 30 μM DHA, 60 μM H2O2 and 4 mU / mL HRP, and then incubate the mixture at 37 °C for 20 minutes. Measure the fluorescence spectrum of the above mixed solution after incubating at room temperature for 3 minutes.

[0024] The method for detecting alkaline phosphatase based on the ALP-TYR-HRP three-enzyme cascade fluorescence sensing platform adds ALP (1250 mU / mL) to Tris-HCl buffer (10 mM, pH = 9) containing PAPP (1 mM). After the mixture is incubated at 37 °C for 30 minutes, the ALP-PAPP mixed solution is added to Tris-HCl buffer solution (10 mM, pH = 7.4) containing 3 U / mL TYR. Then the ALP-PAPP-TYR mixed solution is added to Tris-HCl buffer solution (10 mM, pH = 7.4) containing 30 μM DHA, 60 μM H2O2 and 4 mU / mL HRP, and then the mixture is incubated at 37 °C for 20 minutes. The fluorescence spectrum of the above mixed solution is measured after incubating at room temperature for 3 minutes. Under the same conditions, the selectivity of a series of competitive enzymes or proteins such as GOx, Try, pepsin, ACP, Cytc and lysozyme to ALP was determined.

[0025] The method for detecting cardiac troponin based on the ALP-TYR-HRP three-enzyme cascade fluorescence immunosensing platform, at room temperature, inject 100 μL of mouse anti-cTnI antibody (1 μg / mL) into the wells of an ELISA plate, then cover the plate and incubate overnight at 4 °C. After taking out the wells and washing with washing buffer, add 200 μL of 20% BSA solution to each well and incubate at 37 °C for 1 h to block non-specific adsorption sites. After the removal and washing steps, inject 100 μL of cTnI standards with different concentrations (0 - 150 ng / mL) into the wells, and then incubate at 37 °C for 1 hour. After another removal and washing step, inject 100 μL of sheep monoclonal antibody (2 μg / mL) and incubate at 37 °C for 1 hour. Finally, add 100 μL of ALP-conjugated secondary antibody (1 μg / mL) and incubate after the removal and washing steps. Remove the ALP-conjugated secondary antibody and wash the plate several times. Then, add 300 μL of a mixed solution containing DA (400 μM), DHA (30 μM) and H2O2 (60 μM) to each well of the above plate. Finally, measure the fluorescence spectrum after keeping at room temperature for 3 minutes.

[0026] The method for detecting cardiac troponin based on the ALP-TYR-HRP triple-enzyme cascade fluorescence immunosensing platform. First, a series of monoclonal antibodies are injected into a 96-well plate and incubated overnight at 4°C; after incubation, it is rinsed 4 times with TBST, and 200 μM bovine serum albumin is added to each well to prevent the appearance of non-specific binding sites on the well plate surface; subsequently, it is incubated at 37°C for 1 hour, and the bovine serum albumin is removed with TBST; then 100 μL of cTnI solutions with different concentrations are incubated at 37°C for 1 h, and after removing the unbound cTnI with TBST, anti-goat anti / Ab2 is transferred into the well plate and incubated at 37°C for 1 h; after the incubation ends, it is washed five times with TBST, and then Tris-HCl buffer and ALP-labeled rabbit-goat secondary antibody are added and incubated at 37°C for 1 h to form a sandwich-type sandwich immunity; afterwards, 300 μL of a mixed solution containing DA (400 μM), DHA (30 μM), and H2O2 (60 μM) is added to each well of the above plate. Finally, the fluorescence spectrum is measured after maintaining at room temperature for 3 minutes. Under the same conditions, the selectivity of a series of competitive enzymes or proteins such as AFP, Try, BSA, and Lyso for cTnI was determined.

[0027] The beneficial effects of the present invention:

[0028] The present invention constructs a triple-enzyme cascade-triggered fluorescence immunosensing platform by sequentially integrating ALP, TYR, and HRP. This platform relies on the principle that HRP and H2O2 can promote the rapid in-situ fluorescence reaction of dopamine and 1,5-naphthalenediol to generate strong AFC. First, ALP can induce the hydrolysis of PAPP to produce an intermediate tyramine. Further introducing TYR can catalyze the production of DA, which in turn activates HRP, thus realizing signal amplification involving three enzymes, ALP, TYR, and HRP. In addition, the cascade catalytic process involving ALP is integrated into ELISA (ALP is used as a labeled enzyme, and TYR and HRP are used as signal reporting enzymes). This method has been successfully used to evaluate the cTnI level in clinical serum samples, and the results are satisfactory compared with commercial ELISA. In summary, this advanced signal transduction scheme can provide a new method for studying multi-enzyme cascade systems and constructing multifunctional biosensors.

[0029] (1) The present invention uses DA and DHA as novel substrates for HRP to establish a novel three-stage enzyme-linked immunofluorescence sensing platform, which involves three enzymes, ALP, TYR, and HRP. The integration of the three-enzyme system (ALP, TYR, and HRP) increases the catalytic activity of the cascade reaction by 250.0 times, while the activity of the single-enzyme system (HRP) only increases by 32.0 times. The present invention breaks through the limitations of single enzymes and two enzymes in traditional enzyme cascade reactions and provides a new way for constructing novel enzyme cascade schemes.

[0030] (3) The invention constructs a three - level enzyme - linked immunofluorescence sensing platform based on the in - situ reaction of DA and DHA triggered by HRP, realizing the quantitative analysis of three enzymes, namely HRP, TYR, and ALP, with detection limits of 0.017 mU / mL, 3.33 mU / mL, and 0.017 mU / mL respectively.

[0031] (4) The detection range of the three - level enzyme - linked immunofluorescence analysis developed in this invention for cTnI is 2 - 150 ng / mL, and the detection limit is 0.67 ng / mL. Compared with the traditional commercial fluorescence ELISA labeled with ALP, it has a wider detection range and higher sensitivity. In addition, this method has been successfully applied to the evaluation of cTnI levels in clinical serum samples, with satisfactory results and broad application prospects in clinical diagnosis. Brief Description of the Drawings

[0032] The following further describes the embodiments of the present invention in conjunction with the drawings.

[0033] Figure 1 1H nuclear magnetic resonance spectrum of AFC in Example 1; 1

[0034] Figure 2 13C nuclear magnetic resonance spectrum of AFC in Example 1; 13

[0035] Figure 3 Mass spectrum of AFC in Example 1;

[0036] Figure 4 Absorption spectra of AFC in different situations in Example 2;

[0037] Figure 5 Fluorescence spectra of AFC in different situations in Example 2;

[0038] Figure 6 Kinetic spectra of AFC in Example 2;

[0039] Figure 7 Fluorescence emission spectra of different concentrations of HRP in Example 3;

[0040] Figure 8 Scatter plots of fluorescence intensity of different concentrations of HRP in Example 3;

[0041] Figure 9 Standard curve graphs of different concentrations of HRP in Example 3;

[0042] Figure 10 Bar graphs of fluorescence intensity changes in HRP selectivity in Example 4;

[0043] Figure 11 Fluorescence emission spectra of different concentrations of TYR in Example 5;

[0044] Figure 12 Scatter plot of fluorescence intensity of TYR at different concentrations in Example 5;

[0045] Figure 13 Standard curve of TYR at different concentrations in Example 5;

[0046] Figure 14 Bar chart of fluorescence intensity change of TYR selectivity in Example 6;

[0047] Figure 15 Fluorescence emission spectrum of ALP at different concentrations in Example 7;

[0048] Figure 16 Scatter plot of fluorescence intensity of ALP at different concentrations in Example 7;

[0049] Figure 17 Standard curve of ALP at different concentrations in Example 7;

[0050] Figure 18 Bar chart of fluorescence intensity change of ALP selectivity in Example 8;

[0051] Figure 19 Fluorescence emission spectrum of cardiac troponin I at different concentrations in Example 9;

[0052] Figure 20 Standard curve of cardiac troponin I at different concentrations in Example 9

[0053] Figure 21 Bar chart of fluorescence intensity change of cardiac troponin I selectivity in Example 10

[0054] Figure 22 Schematic diagram of the present invention Detailed implementation manners

[0055] The following is a detailed description of the embodiments of the present invention with reference to the accompanying drawings: These embodiments are implemented on the premise of the technical solution of the present invention, and detailed implementation manners and processes are given, but the protection scope of the present invention is not limited to the following embodiments.

[0056] Example 1: Dissolve 3-hydroxytyramine hydrochloride (189 mg, 1 mmol, 1.0 equivalent), naphthalene-1,5-diol (160 mg, 1 mmol, 1.0 equivalent) and KMnO4 (316 mg, 2 mmol, 2.0 equivalents) in 6 mL of H2O / DMSO (2 / 1 mL). Stir the reaction mixture at room temperature for 5 hours. Then remove the solvent under reduced pressure, and purify the crude product by silica gel column chromatography to obtain a red solid, named AFC (124 mg, 40.1%), and characterize the obtained AFC by NMR and mass spectrometry. AsFigure 1 , Figure 2 and Figure 3 , the accurate molecular formula (C 18 H 15 NO4) and structure of AFC can be obtained.

[0057] Example 2: Add HRP solution (4 mU / mL) to Tris-HCl buffer (10 mM, pH 7.4) containing DA (400 μM), DHA (30 μM) and H2O2 (60 μM), then oscillate the mixture at room temperature for 3 minutes, and detect its fluorescence intensity by a fluorescence spectrometer under excitation at 490 nm. In addition, in three other groups of experiments, HRP + H2O2 / HRP / H2O2 was not added, and other conditions remained unchanged. The optical properties of AFC and kinetic tests were studied by absorption spectroscopy and fluorescence spectroscopy. Only the DA-DHA-HRP-H2O2 mixture showed a strong emission peak at about 550 nm in the fluorescence spectrum, while the other mixtures of DA-DHA, DA-DHA-HRP, and DA-DHA-H2O2 had negligible fluorescence. As Figure 4 , Figure 5 and Figure 6 . It shows that HRP and H2O2 can promote the rapid in-situ fluorescence reaction of DA and DHA to produce a strong yellow fluorescent compound (AFC).

[0058] Example 3: Add HRP solutions with different concentrations (0 - 4 mU / mL) to Tris-HCl buffer (10 mM, pH 7.4) containing DA (400 μM), DHA (30 μM) and H2O2 (60 μM), then oscillate the mixture at room temperature for 3 minutes, and detect its fluorescence intensity by a fluorescence spectrometer under excitation at 490 nm. Fluorescence scanning diagrams, fluorescence intensity scatter diagrams and standard curve diagrams were plotted, as Figure 7 , Figure 8 and Figure 9 . It shows that the detection range of this method for HRP is 0.05 - 3 mU / mL, and the detection limit is 0.017 mU / mL.

[0059] Example 4: Add HRP solution (4 mU / mL) to Tris-HCl buffer (10 mM, pH 7.4) containing DA (400 μM), DHA (30 μM) and H2O2 (60 μM), then oscillate the mixture at room temperature for 3 minutes, and detect its fluorescence intensity by a fluorescence spectrometer under excitation at 490 nm. Under the same conditions, the selectivity of a series of competitive enzymes or proteins such as acid phosphatase (ACP), pepsin, cytochrome C (Cyt c), TYR, trypsin (Try), glucose oxidase (GOx) and lysozyme to HRP was determined, and the concentration of all substances was 4 mU / mL (asFigure 10 ) This indicates that the method is specific for the detection of HRP.

[0060] Example 5: TYR at different concentrations (0 - 3.5 U / mL) was added to Tris-HCl buffer (10 mM, pH = 7.4) containing tyramine (200 μM). The mixture was incubated at 37 °C for 20 minutes, and then the TYR-tyramine mixed solution was added to a solution containing DHA (30 μM), H2O2 (60 μM), and HRP (4 mU / mL). After the above mixed solution was incubated at room temperature for 3 minutes, the fluorescence spectrum was measured using a fluorescence spectrophotometer. Fluorescence scan graphs, fluorescence intensity scatter plots, and standard curves were plotted, as shown in Figure 11 , Figure 12 and Figure 13 . This indicates that the detection range of the method for TYR is 0.01 - 2.5 U / mL, and the detection limit is 3.33 mU / mL.

[0061] Example 6: TYR (3.5 U / mL) was added to Tris-HCl buffer (10 mM, pH = 7.4) containing tyramine (200 μM). The mixture was incubated at 37 °C for 20 minutes, and then the TYR-tyramine mixed solution was added to a solution containing DHA (30 μM), H2O2 (60 μM), and HRP (4 mU / mL). Then the mixture was shaken at room temperature for 3 minutes, and the fluorescence intensity was detected using a fluorescence spectrometer under excitation at 490 nm. Under the same conditions, the selectivity of a series of competitive enzymes or proteins such as GOx, Try, pepsin, lysozyme, Cyt c, and ACP for TYR was determined, and the concentration of all substances was 3.5 U / mL (as shown in Figure 14 ). This indicates that the method is specific for the detection of TYR.

[0062] Example 7: Different concentrations of ALP (0 - 1250 mU / mL) were added to Tris-HCl buffer (10 mM, pH = 9) containing PAPP (1 mM). After the mixture was incubated at 37 °C for 30 minutes, the ALP-PAPP mixed solution was added to Tris-HCl buffer solution (10 mM, pH = 7.4) containing 3 U / mL TYR. Then the ALP-PAPP-TYR mixed solution was added to Tris-HCl buffer solution (10 mM, pH = 7.4) containing 30 μM DHA, 60 μM H2O2, and 4 mU / mL HRP, and then the mixture was incubated at 37 °C for 20 minutes. After incubating at room temperature for 3 minutes, the fluorescence spectrum of the above mixed solution was measured. Fluorescence scan graphs, fluorescence intensity scatter plots, and standard curves were plotted, as shown in Figure 15 , Figure 16 and Figure 17It shows that the detection range of this method for ALP is 0.05 - 1250 mU / mL, and the detection limit is 0.017 mU / mL.

[0063] Example 8: Alkaline phosphatase (ALP) with different concentrations (0 - 1250 mU / mL) was added to Tris-HCl buffer (10 mM, pH = 9) containing p-aminophenylethyl phenyl phosphate (PAPP). After the mixture was incubated at 37 °C for 30 minutes, the ALP-PAPP mixed solution was added to Tris-HCl buffer solution (10 mM, pH = 7.4) containing 3 U / mL TYR, and then the mixture was incubated at 37 °C for 20 minutes. Then the ALP-PAPP-TYR mixed solution was added to Tris-HCl buffer solution (10 mM, pH = 7.4) containing 30 μM DHA, 60 μM H2O2 and 4 mU / mL HRP. After reacting for 3 min, the fluorescence intensity was detected by a fluorescence spectrometer (Ex = 490 nm). Under the same conditions, the selectivity of a series of competitive enzymes or proteins such as GOx, Try, Ppase, ACP, Cyt-c and Lyso for ALP was determined. (Such as Figure 18 ). It shows that the detection of ALP by this method is specific.

[0064] Example 9: The steps of the immunoassay are as follows: First, 100 μL of 1 μg / mL monoclonal antibody (diluted with 10 mM PBS buffer at pH = 7.4) was added to a 96-well plate and incubated overnight at 4 °C. After aspirating the incubation solution in the wells, it was washed three times with 200 μL of TBST and then once with 100 μL of TBST. Then, 200 μL of 20% BSA solution was added to each well and incubated at 37 °C for 1 h. Then the solution was aspirated, washed four times with TBST, and then 100 μL of cTnI solution with different concentrations (0 - 150 ng / mL) was injected into the wells and incubated at 37 °C for 1 hour. After aspirating the incubation solution in the wells, it was washed three times with 200 μL of TBST and then once with 100 μL of TBST. Subsequently, 100 μL of Ab1 (2 μg / mL) was added and incubated at 37 °C for 1 hour. The well plate was rinsed with TBST, and then 100 μL of enzyme-labeled antibody (1 μg / mL) was injected into each well. The solution was incubated at 37 °C for 1 h, and then the solution was aspirated and washed 4 times with TBST washing solution. Finally, a mixed solution of 30 μM DHA, 60 μM H2O2 and 4 mU / mL HRP was added to each well, and Tris-HCl buffer (10 mM, pH = 7.4) was added to control the reaction volume of the mixture to 300 μL. After reacting at room temperature for 3 minutes, the fluorescence spectrum (Ex = 490 nm) was measured. Fluorescence scanning diagrams and standard curve diagrams were plotted, such as Figure 19 and Figure 20It shows that the detection range of this method for cTnI is 2 - 150 ng / mL, and the detection limit is 0.67 ng / mL.

[0065] Example 10: The steps of the immunoassay experiment are as follows: First, add 100 μL of 1 μg / mL monoclonal antibody (diluted with 10 mM PBS buffer at pH = 7.4) to a 96-well plate and incubate overnight at 4°C. After sucking out the incubation solution in the wells, wash three times with 200 μL of TBST, then wash once with 100 μL of TBST. Then add 200 μL of 20% BSA solution to each well and incubate at 37°C for 1 h. Then suck out the solution, wash four times with TBST, and then inject 100 μL of cTnI solutions with different concentrations (0 - 150 ng / mL) into the wells and incubate at 37°C for 1 hour. After sucking out the incubation solution in the wells, wash three times with 200 μL of TBST, then wash once with 100 μL of TBST. Subsequently, add 100 μL of Ab1 (2 μg / mL) and incubate at 37°C for 1 hour. Rinse the well plate with TBST, then inject 100 μL of enzyme-labeled antibody (1 μg / mL) into each well, incubate the solution at 37°C for 1 h, then suck out the solution and wash 4 times with TBST washing solution. Finally, add a mixed solution of 30 μM DHA, 60 μM H2O2, and 4 mU / mL HRP to each well, then add Tris-HCl buffer (10 mM, pH = 7.4), control the reaction volume of the mixture to be 300 μL, and measure the fluorescence spectrum (Ex = 490 nm) after reacting at room temperature for 3 minutes. To verify the selectivity of the sensing system for cTnI, we investigated the effects of some interfering substances, including alpha-fetoprotein (AFP), Lyso, Try, and bovine serum albumin (BSA) on the sensing system. (As Figure 21 ) It shows that this method has specificity for the detection of cTnI.

[0066] Example 11: Under the condition of informed consent, serum samples of four patients with acute myocardial infarction (AMI) and two normal individuals were taken from Jiangsu Provincial People's Hospital and immediately frozen and stored until use after being brought back from the hospital. The detailed procedure of the cascade fluorescence ELISA is similar to the standard sandwich method, except that human serum is used instead of the traditional cTnI solution. Before detection, all serum samples were diluted 20 times and then added to the wells for testing respectively.

[0067] Analysis results of cTnI in human serum samples (normal adults (samples A, B) and AMI patients (samples C, D, E, F) were diluted 0 times and 20 times respectively

[0068]

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

Claims

1. A method for constructing a three-enzyme cascade-triggered fluorescence immunosensing platform for detecting alkaline phosphatase (ALP) by sequentially integrating ALP-TYR-HRP, characterized in that: Alkaline phosphatase (ALP) with different concentrations of 0 - 1250 mU / mL is added to a 10 mM, pH = 9 Tris-HCl buffer containing 1 mM para-aminophenylethyl phenyl phosphate (PAPP); after the mixture is incubated at 37 °C for 30 minutes, the ALP-PAPP mixed solution is added to a 10 mM, pH = 7.4 Tris-HCl buffer solution containing 3 U / mL tyrosinase (TYR); then the ALP-PAPP-TYR mixed solution is added to a 10 mM, pH = 7.4 Tris-HCl buffer solution containing 30 μM 1,5-dihydroxynaphthalene (DHA), 60 μM H2O2 and 4 mU / mL horseradish peroxidase (HRP), and then the mixture is incubated at 37 °C for 20 minutes; the fluorescence spectrum of the above mixed solution is measured after incubating at room temperature for 3 minutes; the excitation wavelength and emission wavelength are 490 nm and 550 nm respectively; the detection range of this method for ALP is 0.05 - 1250 mU / mL, and the detection limit is 0.017 mU / mL.

2. The method for constructing a three-enzyme cascade-triggered fluorescence immunosensing platform for detecting ALP by sequentially integrating ALP-TYR-HRP according to claim 1, characterized in that: Alkaline phosphatase ALP induces the hydrolysis of p-aminophenylethyl phenyl phosphate PAPP to produce the intermediate tyramine. Further introduction of TYR can catalyze the production of dopamine DA, which in turn activates HRP. HRP and H2O2 can promote the rapid in-situ fluorescence reaction of dopamine and 1,5-naphthalenediol to produce a strong yellow fluorescent compound AFC C 18 H 15 NO4; Thus, signal amplification involving three enzymes, ALP, TYR, and HRP, is achieved. The excitation wavelength and emission wavelength of the yellow fluorescent compound AFC are fixed at 490 nm and 550 nm, respectively. AFC is characterized by nuclear magnetic resonance mass spectrometry to determine its molecular formula and structure. The structural formula of AFC is as follows: 。

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