Simultaneous detection of multiple exosomal proteins

Abstract

The present invention provides a method for synchronous detection of multiple exosome proteins, including the steps of forming immunomagnetic beads MBs2@Ab1, forming multiple Ab2-W, forming MBs3 Track, forming a double antibody sandwich complex, forming multiple nucleic acid short chains P, forming a fluorescent liquid to be tested and fluorescence detection. The synchronous detection method mainly uses DNAzyme to convert exosome proteins into nucleic acids, and then uses the CRISPR / Cas system to detect nucleic acids. The combination of the two realizes dual signal amplification, and then distinguishes signals by fluorescence, and finally realizes the synchronous quantitative detection of multiple exosome protein concentrations. The synchronous detection method has high sensitivity, strong specificity, low cost, simple operation, and is conducive to the clinical realization of synchronous quantitative detection of multiple exosome proteins.

Description

Technical Field

[0001] The present invention belongs to the technical field of exosome detection, and specifically relates to a method for synchronously detecting multiple exosome proteins. Background Art

[0002] Exosomes are extracellular vesicles (EVs) with a particle size of 30 to 150 nm that are actively secreted into body fluids by living cells. They not only have a high content in body fluids (up to 1×10 10 particles / mL), a wide source, good stability, strong timeliness, and cell-source specificity, it may be an ideal tumor marker. Studies have shown that exosome proteins are widely involved in epithelial-mesenchymal transition, inducing angiogenesis, promoting the formation of pre-metastatic microenvironment, and immune escape, and play an important role in the occurrence and development of tumors. Currently reported lung cancer protein markers in exosomes include extracellular matrix protein 1, programmed cell death ligand 1, lipopolysaccharide binding protein, MUC1 mucin, leucine-rich α2 glycoprotein 1, epidermal growth factor receptor, and epithelial cell adhesion molecule. However, a specific protein marker that can clearly diagnose lung cancer has not yet been found. The combined detection of multiple protein markers in exosomes is an effective method to improve the accuracy of lung cancer screening.

[0003] At present, the commonly used methods for detecting exosome proteins include enzyme-linked immunosorbent assay, immunoblotting and liquid chromatography-mass spectrometry. Among them, although the enzyme-linked immunosorbent assay (ELISA) can detect a single exosome protein, it is difficult to detect proteins at lower concentrations, and it is not suitable for the simultaneous detection of multiple proteins. Immunoblotting is time-consuming, cannot accurately quantify the protein content, and the test results are interfered by soluble proteins. Liquid chromatography-mass spectrometry requires expensive instruments, relatively complex operations, requires professional technicians, and is difficult to apply clinically. Therefore, it is urgent to develop an exosome analysis method that is conducive to the simultaneous determination of multiple targets for clinical use. Summary of the invention

[0004] In view of this, it is necessary for the present invention to provide a method with high sensitivity, strong specificity, low cost, simple operation and conducive to the simultaneous determination of multiple exosome proteins in clinical practice, so as to solve the above problems.

[0005] To this end, the present invention provides a method for synchronously detecting multiple exosome proteins, comprising the steps of:

[0006] Forming immunomagnetic beads: The capture antibodies Ab1 of multiple exosome proteins to be detected are simultaneously coupled to the carboxyl magnetic beads MBs2 to form immunomagnetic beads MBs2@Ab1;

[0007] Forming multiple Ab2-W: biotinylating the detection antibody Ab2 corresponding to each of the multiple exosome proteins to be detected to form a biotinylated detection antibody Ab2-Bio; coupling each biotinylated detection antibody Ab2-Bio with the corresponding deoxyribozyme W to form a conjugate Ab2-W;

[0008] Forming streptavidin magnetic beads Track: coupling streptavidin magnetic beads MBs3 with the biotinylated substrate chain T of each of the multiple exosome proteins to be detected to form MBs3 Track modified with the multiple biotinylated substrate chains T, and resuspending the MBs3 Track in a shearing reaction solution to form an MBs3 Track suspension, wherein the shearing reaction solution includes a coenzyme factor and an RNase inhibitor;

[0009] Forming a double antibody sandwich complex: mixing and incubating the immunomagnetic beads MBs2@Ab1, the sample to be tested containing the multiple exosome proteins to be tested, and multiple conjugates Ab2-W to form a double antibody sandwich complex;

[0010] Forming a variety of nucleic acid short chains P: adding the MBs3 Track suspension to the double antibody sandwich complex and incubating again, so that each deoxyribozyme W cuts the corresponding biotinylated substrate chain T to generate nucleic acid short chains P, magnetically separating, and collecting the supernatant containing the various nucleic acid short chains P;

[0011] Forming a fluorescent liquid to be tested: first, the supernatant containing a plurality of nucleic acid short chains P is mixed and incubated with a signal solution, wherein the signal solution includes a Cas protein, Cas-crRNA and a fluorescent signal probe corresponding to each nucleic acid short chain P, so that each nucleic acid short chain P is hybridized with its corresponding Cas-crRNA, and the corresponding Cas protein is activated to cut the corresponding fluorescent signal probe so that its fluorescent group recovers fluorescence, thereby forming a fluorescent liquid to be tested;

[0012] Fluorescence detection: A multifunctional microplate reader is used to synchronously detect the fluorescence intensity of each fluorescent group in the fluorescent liquid to be tested, and a corresponding regression equation is constructed using the relationship between the fluorescence intensity of each fluorescent group and the concentration of the corresponding exosome protein to be tested.

[0013] Based on the above, the step of forming the immunomagnetic beads comprises: using the EDC / Sulfo-NHS method to capture the SAA1 antibody Ab1 SAA1 and FV capture antibody Ab1 FV At the same time, the immunomagnetic beads MBs2 are coupled to the carboxyl magnetic beads MBs2 and blocked with a bovine serum albumin solution to obtain the immunomagnetic beads MBs2@Ab1. The immunomagnetic beads MBs2@Ab1 are resuspended in a phosphate buffer solution to form an immunomagnetic bead MBs2@Ab1 suspension.

[0014] Among them, "SAA1" in this article refers to serum amyloid protein A, whose full English name is Serum amyloid A-1protein; "FV" refers to coagulation factor V, whose full English name is Coagulation factor V.

[0015] Based on the above, the steps of forming a plurality of Ab2-Ws include:

[0016] The detection antibody Ab2 of SAA1 SAA1 The solution and Sulfo-NHS-LC-Biotin were evenly mixed and reacted at room temperature to obtain the biotinylated detection antibody Ab2 of SAA1. SAA1 -Bio solution; firstly, the biotinylated detection antibody Ab2 SAA1 -Bio solution and streptavidin solution were reacted at room temperature, and then DNAzyme W1 solution was added and reacted at room temperature to obtain conjugate Ab2 SAA1 -W1 solution;

[0017] The FV detection antibody Ab2 FV The solution and Sulfo-NHS-LC-Biotin were evenly mixed and reacted at room temperature to obtain the biotinylated detection antibody Ab2 of FV. FV -Bio solution; firstly, the biotinylated detection antibody Ab2 FV -Bio solution and streptavidin solution were reacted at room temperature, and then DNAzyme W2 solution was added and reacted at room temperature to obtain conjugate Ab2 FV -W2 solution.

[0018] Based on the above, the SAA1 capture antibody Ab1 SAA1 is a mouse anti-human SAA1 monoclonal capture antibody, the capture antibody Ab1 of FV FV is a rabbit anti-human FV monoclonal capture antibody, and the SAA1 detection antibody Ab2 SAA1 is a mouse anti-human SAA1 monoclonal detection antibody, the FV detection antibody Ab2 FV Sheep anti-human FV polyclonal detection antibody.

[0019] Based on the above, the step of forming the streptavidin magnetic beads Track includes: shaking the mixed solution of substrate chains T1 and T2 with the streptavidin magnetic beads MBs3 at room temperature, so that the substrate chains T1 and T2 are simultaneously modified on the streptavidin magnetic beads MBs3, magnetically separated, and the streptavidin magnetic beads MBs3 Track are obtained; resuspending the streptavidin magnetic beads Track in the shear reaction solution to form an MBs3 Track suspension; wherein the pH of the shear reaction solution is 7.0-8.0, and includes 10-90 mmol / L MgCl 2 , 0.6~1.2mmol / LDTT and 0.8~1.6U / μL RNase inhibitor.

[0020] Based on the above, the deoxyribozyme W1 is the nucleotide sequence shown in SEQ ID No.3, and the deoxyribozyme W2 is the nucleotide sequence shown in SEQ ID No.4; the substrate chain T1 is the nucleotide sequence shown in SEQ ID No.1, and the substrate chain T2 is the nucleotide sequence shown in SEQ ID No.2.

[0021] Based on the above, the step of forming the double antibody sandwich complex comprises: mixing the immunomagnetic bead MBs2@Ab1 suspension, the sample to be tested containing SAA1 and FV, the conjugate Ab2 SAA1 -W1 solution and the conjugate Ab2 FV -W2 solution was mixed and incubated at 37°C and magnetically separated to obtain the double antibody sandwich complex.

[0022] Based on the above, the step of forming multiple nucleic acid short chains P includes: adding the MBs3 Track suspension to the double antibody sandwich complex and shaking the reaction at 37°C, so that the deoxyribozyme W1 cuts the substrate chain T1 and releases the short chain RNA P1, the deoxyribozyme W2 cuts the substrate chain T2 and releases the short chain DNA P2, magnetic separation, and collecting the supernatant containing the short chain RNA P1 and DNA P2.

[0023] Based on the above, the step of forming the fluorescent liquid to be tested includes:

[0024] The signal solution includes 30-180 nmol / L Cas13a, 30-180 nmol / L Cas12a, 150 nmol / L Cas13a-crRNA, 150 nmol / L Cas12a-crRNA, 200-1000 nmol / L SAA1 fluorescent signal probe FQ1 and 200-1000 nmol / L FV fluorescent signal probe FQ2, wherein the nucleic acid sequence of the SAA1 fluorescent signal probe FQ1 from 5′ to 3′ is: Cy5-rUrUrUrUrU-BHQ2, and the nucleic acid sequence of the FV fluorescent signal probe FQ2 from 5′ to 3′ is: FAM-TTATT-BHQ1;

[0025] The signal solution is added to the supernatant containing the short-chain RNA P1 and DNA P2 and incubated at 37° C., so that the short-chain RNA P1 hybridizes with Cas13a-crRNA and activates Cas13a to cut the SAA1 fluorescent signal probe FQ1 so that the Cy5 fluorescent group therein restores fluorescence, and the short-chain DNA P2 hybridizes with Cas12a-crRNA and activates Cas12a to cut the FV fluorescent signal probe FQ2 so that the FAM fluorescent group therein restores fluorescence, thereby forming the fluorescent solution to be tested.

[0026] Based on the above, the step of fluorescence detection includes:

[0027] The shear reaction solution is added to the fluorescent solution to be tested, and the fluorescence intensity value FL of the Cy5 fluorescent group therein is synchronously measured using a fluorescence spectrometer. SAA1 or FL 0(SAA1) , the fluorescence intensity value FL of the FAM fluorescent group FV or FL 0(FV) ;

[0028] In the range of SAA1 concentration of 0.1 to 30 ng / mL, a regression equation for detecting SAA1 concentration was established: Y SAA1 =3682.77C SAA1 +49249.79, of which Y SAA1 is the corresponding fluorescence intensity difference: FL SAA1 -FL 0(SAA1) , multiple correlation coefficient R 2 =0.982, C SAA1 represents the concentration of SAA1;

[0029] In the range of FV concentration of 1 to 50 ng / mL, a regression equation for detecting FV concentration was established: Y FV =20262.48C FV +663842.23, of which Y FVis the corresponding fluorescence intensity difference: FL FV -FL 0(FV) , multiple correlation coefficient R 2 =0.999, C FV Represents the concentration of FV.

[0030] The present invention provides a method for synchronously detecting multiple exosome proteins. The capture antibody Ab1 of each exosome protein is simultaneously coupled to the hydroxy magnetic bead MBs2 to form the immunomagnetic bead MBs2@Ab1, which is used to capture the exosome protein to be detected in the standard sample or the exosome lysate; the detection antibody Ab2 of each exosome protein is connected with the corresponding deoxyribozyme W by utilizing the specific interaction between streptavidin and biotin to form a conjugate Ab2-W, which can bind to the target in the sample to be detected captured by the immunomagnetic bead to form a double antibody sandwich complex; the streptavidin on the surface of the streptavidin magnetic bead MBs3 can bind to biotin, and it is coupled to the biotinylated substrate chain T of each exosome protein to form MBs3 Track; the MBs3 Track is added to the double antibody sandwich complex, and incubated again in the presence of a coenzyme factor, so that each deoxyribozyme W specifically cuts the corresponding biotinylated substrate chain T to produce a nucleic acid short chain P. In this way, the content of each exosome protein is converted into the concentration of the corresponding nucleic acid short chain P, and one protein molecule can be converted into multiple nucleic acids, thereby achieving the first signal amplification; the supernatant containing multiple nucleic acid short chains P is collected by magnetic separation and mixed with a signal solution for incubation, wherein the signal solution includes the Cas protein, Cas-crRNA and fluorescent signal probe corresponding to each nucleic acid short chain P, and the 5′ and 3′ of each fluorescent signal probe are The ends are modified with fluorescent groups and quenching groups respectively, and no fluorescence is emitted under normal circumstances; the CRISPR / Cas system is used to hybridize each nucleic acid short chain P with its corresponding Cas-crRNA to activate the corresponding Cas protein, and the activated Cas protein can continuously cut the corresponding fluorescent signal probe. After the fluorescent signal probe is cut, the fluorescent group therein recovers fluorescence, which can generate a fluorescent signal and form a fluorescent liquid to be tested; in this way, each nucleic acid short chain P can restore the fluorescence of multiple fluorescent groups to achieve a second signal amplification; the linear relationship between the fluorescence intensity of the fluorescent group and the concentration of the exosome protein can be used to achieve quantitative detection of each exosome protein.

[0031] Therefore, the above synchronization detection method provided by the present invention has the following advantages:

[0032] 1) Carboxyl magnetic beads MBs2 were used as separation medium to improve the capture efficiency of the target;

[0033] 2) By simultaneously coupling multiple capture antibodies to MBs2, multifunctional immunomagnetic beads were prepared to achieve the specific capture, separation, and enrichment of multiple exosomal proteins;

[0034] 3) Paired antibodies are used to identify the target. Subsequent detection signals can only be generated when the corresponding capture antibody and detection antibody are bound to the target, thus ensuring the specificity of the detection method;

[0035] 4) DNAzyme and CRISPR / Cas technology are used to achieve dual signal amplification and improve detection sensitivity. At the same time, the detection system is highly universal and can be used not only for the detection of exosome non-membrane proteins, but also for the detection of membrane proteins and exosome proteins that have not yet been clearly located.

[0036] 5) Compared with the common single signal analysis method, the method provided by the present invention has the advantages of small sample size, simple operation steps and high throughput;

[0037] 6) The above-mentioned synchronous detection method can be successfully applied to the detection of SAA1 and FV in plasma exosomes, and the detection results can effectively distinguish lung cancer patients from healthy controls.

[0038] Therefore, the above-mentioned multiple exosome simultaneous detection method provided by the present invention has high sensitivity, strong specificity, low cost, simple operation, and is conducive to the simultaneous quantitative detection of multiple exosome proteins in clinical practice. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is a schematic diagram of the synchronous detection principle of exosome SAA1 and FV concentrations provided in Example 1 of the present invention.

[0040] Figure 2 It is a characterization diagram of the carboxyl magnetic beads MBs2 and the immunomagnetic beads MBs2@Ab1 provided in Example 1 of the present invention; wherein, Figure A in the figure is a transmission electron microscope image of MBs2, and Figure B is a characterization diagram of the hydrated particle size of MBs2@Ab1.

[0041] Figure 3 It is a characterization diagram of MBs3 and MBs3 Track provided in Example 1 of the present invention; wherein, Figure A in the figure is a transmission electron microscope image of MBs3, Figure B is a characterization diagram of the hydrated particle size of MBs3 Track, and Figure C is a characterization diagram of the Zeta potential of MBs3 and MBs3Track.

[0042] Figure 4: This is a gel electrophoresis diagram of W1 and W2 cutting substrate chains T1 and T2 provided in Example 1 of the present invention, wherein lane 1 in the figure: DNA molecular weight standard, the molecular weights from bottom to top are: 50, 100, 150, 200, 250, 300, 400, 500 bp; lane 2: 2 μmol / L W1; lane 3: 2 μmol / L T1; lane 4: 2 μmol / L W1+2 μmol / L T1; lane 5: 2 μmol / L W1+2 μmol / L T1+Mg 2+ ; Lane 6: 2μmol / LW2; Lane 7: 2μmol / L T2; Lane 8: 2μmol / L W2+2μmol / L T2; Lane 9: 2μmol / L W2+2μmol / L T2+Mg 2+ .

[0043] Figure 5 It is a graph showing the results of the trans-cleavage activity investigation of Cas13a and Cas12a provided in Example 1 of the present invention; wherein, Figure A in the figure is a graph showing the results of the trans-cleavage activity investigation of Cas13a, and Figure B is a graph showing the results of the trans-cleavage activity investigation of Cas12a.

[0044] Figure 6 It is a diagram of the mutual interference investigation results of the Cas13a and Cas12a systems provided in Example 1 of the present invention, wherein Group 1 in the figure represents the signal solution; Group 2 represents the signal solution + T1; Group 3 represents the signal solution + T2; Group 4 represents the signal solution + T1 + T2.

[0045] Figure 7 It is a standard curve diagram for simultaneous detection of exosome SAA1 and FV concentrations provided in Example 1 of the present invention, wherein Figure A in the figure is a standard curve diagram for SAA1, and Figure B is a standard curve diagram for FV.

[0046] Figure 8 1 is a characterization diagram of plasma exosomes extracted by the co-precipitation method provided in Example 1 of the present invention, wherein Figure A in the figure is a transmission electron microscopy image of plasma exosomes; Figure B is a WB (Western blotting) characterization image of plasma exosomes.

[0047] Fig. 9 It is a comparison diagram of fluorescence signals when the established method provided in Example 1 of the present invention detects different targets.

[0048] Fig.10 It is a result graph of detecting SAA1 and FV in plasma exosomes using the method provided in Example 1 of the present invention, wherein Figure A is a comparison graph of SAA1 and FV in plasma exosomes of the LC group and the HC group; Figure B is a PCA analysis result graph, and each point in the graph represents a sample.

[0049] Fig.11 This is an optimization diagram of the amount of immunomagnetic beads provided in the experimental condition optimization embodiment of the present invention.

[0050] Fig.12 This is a dilution ratio optimization diagram of the conjugate Ab2-W provided in the experimental condition optimization example of the present invention.

[0051] Fig.13 It is a MBs3 Track dilution ratio optimization diagram provided in the experimental condition optimization embodiment of the present invention.

[0052] Fig.14 The Mg in the shear reaction solution provided in the experimental condition optimization embodiment of the present invention is 2+ Concentration optimization plot.

[0053] Fig.15 It is a concentration optimization result diagram of Cas13a and Cas12a in the signal solution provided in the experimental condition optimization embodiment of the present invention.

[0054] Fig.16 This is a graph showing the concentration optimization results of the FQ1 and FQ2 fluorescent probes in the signal solution provided in the experimental condition optimization example of the present invention.

[0055] Among them, in the sequence list:

[0056] SEQ ID No. 1 is the nucleotide sequence of Track 1 used in the embodiment of the present invention;

[0057] SEQ ID No. 2 is the nucleotide sequence of Track 2 used in the embodiment of the present invention;

[0058] SEQ ID No. 3 is the nucleotide sequence of DNAzyme Walker 1 used in the embodiment of the present invention;

[0059] SEQ ID No. 4 is the nucleotide sequence of DNAzyme Walker 2 used in the embodiment of the present invention;

[0060] SEQ ID No.5 is the nucleotide sequence of Cas12a-crRNA used in the embodiment of the present invention;

[0061] SEQ ID No.6 is the nucleotide sequence of Cas13a-crRNA used in an embodiment of the present invention. DETAILED DESCRIPTION

[0062] The technical scheme of the present invention is further described in detail below through specific implementation methods. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art.

[0063] The present invention provides a method for synchronous detection of multiple exosome proteins, including the steps of forming immunomagnetic beads MBs2@Ab1, forming multiple Ab2-W, forming MBs3 Track, forming a double antibody sandwich complex, forming multiple nucleic acid short chains P, forming a fluorescent liquid to be tested and fluorescence detection. The synchronous detection method mainly uses DNAzyme to convert exosome proteins into nucleic acids, and then uses the CRISPR / Cas system to detect nucleic acids. The combination of the two realizes dual signal amplification, and then distinguishes signals through fluorescence, and finally realizes the synchronous quantitative detection of the concentrations of multiple exosome proteins.

[0064] Taking SAA1 and FV as an example, a method for synchronously detecting SAA1 and FV is provided below. The reagents and materials used in the embodiment of the present invention are as follows:

[0065] Carboxyl magnetic beads (Magnetic beads 2, MBs2) and streptavidin magnetic beads (Magnetic beads 3, MBs3) were purchased from Baiyun Nanotechnology Co., Ltd.; FV standard, SAA1 standard, rabbit anti-human FV monoclonal capture antibody (Ab1 FV ) were purchased from Abcam; mouse anti-human SAA1 monoclonal capture antibody (Ab1 SAA1 ), mouse anti-human SAA1 monoclonal detection antibody (Ab2 SAA1 ) were purchased from Shanghai Meddis Medical Technology Co., Ltd.; sheep anti-human FV polyclonal detection antibody (Ab2 FV ), Sulfo-NHS-LC-Biotin, and total exosome isolation kit (from plasma) were purchased from Thermo Fisher Scientific Inc., USA; RNase inhibitors were purchased from Beyotime Biotechnology; Streptavidin (SA) was purchased from Merck Life Sciences; 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), N-hydroxysulfosuccinimide sodium salt ( sodiumsalt, Sulfo-NHS), and dithiothreitol (DTT) were purchased from Shanghai Jingchun Biochemical Technology Co., Ltd.; ELISA colorimetric solution was purchased from Shenggong Bioengineering Co., Ltd.; 96-well ELISA plate was purchased from Guangzhou Jiete Biofiltration Co., Ltd.; 96-well all-black microplate was purchased from Shanghai Jingan Biotechnology Co., Ltd.; LbuCas13a protein was purchased from Guangzhou Bolais Biotechnology Co., Ltd.; LbaCas12a protein ( 20Lba Cas12a) and NEBuffer r2.1 (10×) were purchased from NEB ENGLAND, UK; Neuron specific enolase (NSE) was purchased from Sigma-Aldrich, USA; SAA1 ELISA kit was purchased from Wuhan Elaruite Biotechnology Co., Ltd.; FVELISA kit was purchased from Wuhan Huamei Bioengineering Co., Ltd.; Carcinoembryonic antigen (CEA) was purchased from Beijing Keyue Zhongkai Biotechnology Co., Ltd.; Immunoglobulin G (IgG) was purchased from Solebao Technology Co., Ltd.; and exosome lysis buffer was purchased from Shanghai Yumeibo Biotechnology Co., Ltd.

[0066] The nucleic acids used were purchased from Sangon Biotechnology Co., Ltd., and the nucleic acid sequences are shown in SEQ ID No. 1 to 6 and Table 1. The experimental water was Milli-Q water (resistivity greater than 18.2 MΩ·cm).

[0067] Table 1 Names and base sequences of nucleic acids used in the experiment

[0068]

[0069] Note: The underlined part is Mg 2+ DNAzyme cleavage site; the bold part is Mg 2+ The catalytic core of a DNAzyme.

[0070] The main instruments used in the embodiments of the present invention are: transmission electron microscope (JEM-2100, Japan); magnetic measurement system (MPMS3, Quantum Design, USA); multifunctional microplate reader (SpectraMax M2e, SpectraMaxi3x, Meigu Molecular Instrument Co., Ltd.); ultra-micro-ultraviolet spectrophotometer (Thermo Nanodrop2000, Thermo Fisher Scientific, USA), gel imaging system (Amersham Imager 600, GE, USA), gel image analysis system (Gel Doc XR+, BIO-RAD, USA), ultraviolet-visible spectrophotometer (UV 1601, Shimadzu, Japan); nanoparticle size and Zeta potential analyzer (Zetasizer Nano-zs 90, Malvern, UK); ultrapure water device (Millipore, USA).

[0071] The preparation solutions mainly used in various embodiments of the present invention are as follows:

[0072] 1) 0.01 mol / L PBS (Phosphate buffer solution, pH 7.4): Dissolve commercially available PBS powder in 2 L of Milli-Q water, mix well, and sterilize by high pressure before use.

[0073] 2) PBST (Phosphate Tween Buffer, pH 7.4): Contains 0.01 mol / L PBS, 0.05% Tween 20, pH 7.4. Add 250 μL Tween 20 to 500 mL 0.01 mol / L PBS buffer and mix thoroughly.

[0074] 3) Shear reaction solution (pH 7.5): containing 20mmol / L Tris-HCl, 150mmol / L NaCl, 5mmol / L KCl, 30mmol / L MgCl 2 , 1mmol / L DTT and 1U / μL RNase inhibitor.

[0075] 4) 0.01 mol / L 4-Morpholineethanesulfonic acid (MES) buffer: weigh 1.0 g of MES monohydrate and dissolve it in Milli-Q water, dilute to 500 mL, and adjust the pH to 6 with KOH.

[0076] 5) Signal solution: containing 150nmol / L Cas13a, 150nmol / L Cas12a, 150nmol / L Cas13a-crRNA, 150nmol / L Cas12a-crRNA, 0.8μmol / L FQ1, 0.8μmol / L FQ2, 1×NE Buffer r2.1.

[0077] Embodiment 1

[0078] A synchronous detection method for SAA1 and FV is provided in the first embodiment of the present invention, and its synchronous detection principle is as follows: Figure 1 shown.

[0079] First, two capture antibodies Ab1 SAA1 and Ab1 FV At the same time, it is coupled to MBs2 to form the immunomagnetic beads MBs2@Ab1, which are used to capture SAA1 and FV in standard samples or exosome lysates;

[0080] Secondly, using the specific interaction between streptavidin and biotin, the detection antibody Ab2 of SAA1 and FV was SAA1 and Ab2 FVConjugate with two DNAzymes W1 and W2 to form conjugate Ab2 SAA1 -W1 and Ab2 FV -W2;

[0081] Again, the conjugate Ab2 SAA1 -W1 and Ab2 FV -W2 binds to the targets SAA1 and FV captured by the immunomagnetic beads, respectively, to form a double antibody sandwich complex;

[0082] Then, the streptavidin on the surface of MBs3 is coupled with two biotinylated substrate chains T1 and T2 by utilizing the feature that the streptavidin can bind to biotin, forming MBs3 Track and adding it to the double antibody sandwich complex;

[0083] Subsequently, the coenzyme factor Mg 2+ In the presence of , the DNAzyme W1 in the double antibody sandwich complex can specifically cut the substrate chain T1 and release the short-chain RNA P1, and the DNAzyme W1 chain then cuts the other substrate chain T1; similarly, the DNAzyme W2 will cyclically cut the substrate chain T2 to produce the short-chain DNA P2; in this way, the content of SAA1 and FV is converted into the concentration of the nucleic acid short chains P1 and P2, and one protein molecule can be converted into multiple nucleic acids, realizing the first signal amplification;

[0084] Next, the supernatant containing short-chain nucleic acids RNA P1 and DNA P2 is collected by magnetic separation, and a signal solution is added to the solution, wherein the signal solution contains Cas12a, Cas13a, Cas12a-crRNA, Cas13a-crRNA, SAA1 fluorescent signal probe FQ1, and FV fluorescent signal probe FQ2; the 5′ and 3′ ends of the fluorescent signal probes FQ1 and FQ2 are modified with fluorescent groups Cy5 and FAM and quenching groups BHQ2 and BHQ1, respectively, and do not emit fluorescence under normal circumstances. When RNA P1 and DNA P2 hybridize with Cas13a-crRNA and Cas12a-crRNA, respectively, the two Cas proteins Cas13a and Cas12a can be activated; the activated Cas13a can continuously cut the SAA1 fluorescent signal probe FQ1 in the solution, and the activated Cas12a can cut the FV fluorescent signal probe FQ2 in the solution. After the fluorescent signal probes FQ1 and FQ2 are cut, the fluorescence of the fluorescent groups Cy5 and FAM is restored, and a fluorescent signal is generated to form a fluorescent liquid to be tested. In this way, the presence of a P1 / P2 line can restore the fluorescence of multiple Cy5 / FAM fluorescent groups, achieving a second signal amplification;

[0085] Finally, the linear relationship between the fluorescence intensity of the Cy5 / FAM fluorescent group and the SAA1 / FV concentration was used to construct a linear regression equation to achieve quantitative detection of SAA1 and FV.

[0086] The synchronous detection method of SAA1 and FV provided in the first embodiment of the present invention will be specifically explained in further detail below.

[0087] Step 1: Formation of immunomagnetic beads MBs2@Ab1

[0088] The preparation method of the immunomagnetic beads MBs2@Ab1 comprises the following steps:

[0089] (1) Buffer solution replacement: Ab1 was replaced by ultrafiltration SAA1 The buffer was replaced with PBS; a certain volume of Ab1 SAA1 Add 50KDa ultrafiltration tube, add PBS to its volume of 0.5mL. Centrifuge at 4℃12000g for 7min, discard the filtrate; add PBS to a total volume of 0.5mL and centrifuge for the second time. The centrifugation conditions are the same as the first time. Ultrafiltration is performed 4 times in total to obtain Ab1 after replacing the buffer. SAA1 ;

[0090] (2) Washing the magnetic beads: Take 800 μL of 10 mg / mL MBs2 and add it to a low-adsorption centrifuge tube. Perform magnetic separation and discard the supernatant. Wash the magnetic beads three times with MES buffer, 1 mL each time.

[0091] (3) Activating magnetic beads: Add 200 μL of 0.6 mol / L Sulfo-NHS solution and 0.3 mol / L EDC solution to the low adsorption centrifuge tube in step (2) above, shake at room temperature for 15 min, and then magnetically separate and discard the supernatant, wherein the Sulfo-NHS solution and the solution both use MES buffer as solvent;

[0092] (4) Adding capture antibody: Add 400 μL of capture antibody solution to the low adsorption centrifuge tube in step (3) above, and shake at 37°C for 2 h, wherein the capture antibody solution includes 0.01 mol / L PBS, 0.05 mg / mL Ab1 SAA1 and 0.05 mg / mL Ab1 FV Composition: magnetic separation to obtain immunomagnetic beads, and the supernatant is stored separately; the immunomagnetic beads are washed twice with 400 μL PBS;

[0093] (5) Blocking: Add 2 mL of 5% bovine serum albumin (BSA) blocking solution to the low adsorption centrifuge tube in step (4) above, and shake at room temperature for 1 h. After the reaction, discard the supernatant by magnetic separation, and wash the immunomagnetic beads with PBS three times to obtain BSA-blocked immunomagnetic beads, which are represented by MBs2@Ab1.

[0094] (6) Storage: Add 4 mL of PBS to resuspend the immunomagnetic beads to form an immunomagnetic bead MBs2@Ab1 suspension, and store it at 4°C for later use.

[0095] Characterization of immunomagnetic beads MBs2@Ab1

[0096] 1) The morphology of MBs2 was characterized by TEM. Figure 2 As shown in A. It can be seen from the figure that MBs2 is approximately spherical with a diameter of about 300nm. The entire magnetic bead consists of two layers, the dark part in the middle is the magnetic core, and the lighter part at the edge may be related to the polymer material on the surface of MBs2.

[0097] 2) Using hydration particle size and Zeta potential instrument to measure MBs2 and coupled Ab1 SAA1 and Ab1 FV The particle size of the formed immunomagnetic beads MBs2@Ab1 was measured, and the results were as follows Figure 2 As shown in B. The hydrated particle size of MBs2 is 369.3 nm. After coupling with antibodies, the hydrated particle size increases to 488.3 nm, which proves that the immunomagnetic beads MBs2@Ab1 were successfully prepared.

[0098] Step 2: Formation of multiple Ab2-W

[0099] In this embodiment, there are two types of Ab2-W, namely Ab2 SAA1 -W1 and Ab2FV-W2, the specific preparation method is as follows:

[0100] 2.1Ab2 SAA1 and Ab2 FV Biotinylation

[0101] (1) Replacement of antibody buffer solution: Ab2 SAA1 The buffer was replaced with PBS and 40 μL of 5.1 mg / mL Ab2 SAA1 Add to a 50KDa ultrafiltration tube, add PBS buffer to its volume of 0.5mL; centrifuge at 4℃12000g for 7min, discard the filtrate; add PBS to the ultrafiltration tube to a total volume of 0.5mL and perform a second centrifugation. The centrifugation conditions are the same as the first time, and ultrafiltration is performed 4 times in total; collect the antibody after replacing the buffer, add PBS to make the antibody Ab2 SAA1 The final concentration was 1 mg / mL;

[0102] (2) Add 180 μL of Milli-Q water to 1 mg of Sulfo-NHS-LC-Biotin and mix well to make the final concentration of Sulfo-NHS-LC-Biotin to 10 mmol / L;

[0103] (3) Add the antibody Ab2 at a concentration of 1 mg / mL to the above-mentioned antibody Ab2 at a volume ratio of 1000:66.5 SAA1 Sulfo-NHS-LC-Biotin at a concentration of 10 mmol / L was added to the solution, vortexed to mix, and oscillated at room temperature for 1 h;

[0104] At the same time, take 40 μL of 9.9 mg / mL Ab2 FV PBS was added to make Ab2 FV The final concentration was 1 mg / mL, and the volume ratio was 1000:66.5 to 1 mg / mL Ab2 FV Sulfo-NHS-LC-Biotin was added to the solution, vortexed to mix, and oscillated at room temperature for 1 h;

[0105] (4) Referring to the ultrafiltration method in step (1) above, unreacted Sulfo-NHS-LC-Biotin in step (3) was removed, and the ultrafiltration solution was collected to obtain Ab2 SAA1 -Bio and Ab2 FV -Bio storage solution.

[0106] (5) Storage: Ab2 SAA1 -Bio and Ab2 FV -Bio stock solution was stored at -20℃ until use.

[0107] 2.2 Biotinylated detection antibody Ab2 SAA1 / Ab2 FV Conjugated with DNAzyme W1 / W2

[0108] (1) Preparation of Ab2 SAA1 -W1 stock solution: take 6.4 μL Ab2 SAA1 -Bio storage solution was placed in a low adsorption centrifuge tube, 16 μL of 20 μmol / L SA solution was added and reacted at room temperature for 30 min, then 8 μL of 200 μmol / L W1 solution was added and reacted at room temperature for 30 min, and then 1.6 μL of PBS was added to obtain Ab2 SAA1 -W1 mother liquor;

[0109] (2) Preparation of Ab2 FV -W2 mother solution: Prepare Ab2 according to the above step (1) SAA1-W1 mother solution was prepared by the same steps as Ab2 FV -W2 mother liquor.

[0110] Step 3: Create MBs3 Track

[0111] The preparation method of MBs3 Track in this embodiment includes the following steps:

[0112] (1) Washing the magnetic beads: Take 510 μL of 2 mg / mL MBs3 in a low-adsorption centrifuge tube, separate by magnetic separation, discard the supernatant, and wash the magnetic beads MBs3 three times with PBS buffer;

[0113] (2) Adding a mixture of substrate chains T1 and T2: Add 1062.5 μL of a 500 nmol / L mixture containing T1 and T2 to the above-mentioned low adsorption centrifuge tube, mix thoroughly, and react at room temperature for 30 min to allow T1 and T2 to couple to the magnetic beads MBs3 to form MBs3 Track, and then perform magnetic separation; wash the magnetic beads MBs3 Track twice with PBS, each time with 1062.5 μL; wherein the mixture containing T1 and T2 includes 250 nmol / L of substrate chain T1, 250 nmol / L of substrate chain T2, and 1 U / μL of RNase inhibitor;

[0114] (3) Resuspension: Resuspend MBs3 Track in 1020 μL of the shearing reaction solution to form an MBs3 Track suspension.

[0115] MBs3 Track Characterization

[0116] TEM results are as follows Figure 3 As shown in A, MBs3 is approximately spherical with a particle size of about 300 nm. It consists of a black magnetic core in the middle and a light-colored portion at the edge. The light-colored portion may be related to the polymer material and streptavidin on the surface of the magnetic beads.

[0117] In order to characterize whether T1 and T2 were successfully modified on the surface of MBs3, the hydrated particle size and Zeta potential of MBs3 before and after coupling with T1 and T2 were measured respectively. Figure 3 B and 3C.

[0118] from Figure 3 It can be seen from B that the hydrated particle size of MBs3 is 398.2nm. After combining with T1 and T2, the hydrated particle size increases to 494.1nm. Figure 3 As can be seen in C, the Zeta potentials of MBs3 and MBs3 Track are 6.58 mV and -33.4 mV, respectively. After T1 and T2 were modified on the surface of the magnetic beads, the potential deviated to the negative direction, indicating that T1 and T2 were successfully coupled to the surface of MBs3.

[0119] Step 4: Formation of double antibody sandwich complex

[0120] In this embodiment, the double antibody sandwich complex is mainly prepared by the following steps:

[0121] (1) Adding immunomagnetic beads MBs2@Ab1: Add 35 μL of the above immunomagnetic beads MBs2@Ab1 suspension to each well of a 96-well plate, and wash the immunomagnetic beads MBs2@Ab1 once with PBST;

[0122] (2) Adding the test sample containing SAA1 and FV: Add the mixed solution containing SAA1 and FV or the exosome lysate into a 96-well plate, 100 μL per well;

[0123] (3) Add Ab2 SAA1 -W1 and Ab2 FV -W2: Ab2 SAA1 -W1 stock solution was diluted with PBS at a volume ratio of 1:4 to form Ab2 SAA1 -W1 dilution, Ab2 FV -W2 stock solution was diluted with PBS at a volume ratio of 1:8 to form Ab2 SAA1 -W1 diluent; the Ab2 SAA1 -W1 dilution and the Ab2 FV -W2 dilution solution was mixed to form a mixed solution; the mixed solution was added to the above-mentioned 96-well plate, 5 μL of the above-mentioned mixed solution was added to each well, and incubated at 37°C for 90 min to form a double antibody sandwich complex; magnetic separation was performed, the supernatant was discarded, and the mixture was washed 3 times with PBST, 300 μL each time; and then washed once with 300 μL of the shear reaction solution to obtain the double antibody sandwich complex.

[0124] Step 5: Formation of multiple nucleic acid short chains P

[0125] In this embodiment, the multiple nucleic acid short chains P are composed of short-chain RNA P1 and short-chain DNA P2. The specific preparation method of this step is as follows: add the above-mentioned MBs3 Track suspension to the 96-well plate formed with the double antibody sandwich complex in "Step 4, forming a double antibody sandwich complex", add 30 μL of MBs3 Track suspension to each well, and shake the reaction at 37° C. for 60 minutes, so that the DNAzyme W1 and W2 in the double antibody sandwich complex are in the coenzyme factor Mg in the MBs3 Track suspension. 2+ Under the action of the coenzyme factor Mg, the substrate chains T1 and T2 on the MBs3 track are cut respectively to form a mixture containing short nucleic acid chains. 2+Under the action of , DNAzyme W1 specifically and cyclically cuts substrate chain T1 and releases short-chain RNA P1, and DNAzyme W2 cyclically cuts substrate chain T2 to produce short-chain DNA P2, forming a mixture containing nucleic acid short chains P1 and P2.

[0126] DNAzyme cleavage activity test for substrate strand T

[0127] Gel electrophoresis was used to characterize the cleavage activity of DNAzyme on substrate chain T. The specific test method is as follows:

[0128] Prepare the solution according to the following groups, place each group of samples at 37℃ for 1 hour of shaking reaction, and analyze the product using polyacrylamide gel experiment. Prepare 15% polyacrylamide gel, and load 12μL sample per well, specifically add 10μL reaction product and 2μL Loading buffer to each well; the electrophoresis voltage is 110V, and the electrophoresis time is 2.5h. After the electrophoresis, take out the gel, immerse it in the nucleic acid dye Gold View staining solution, shake it at room temperature for 30 minutes, and use the gel imaging analysis system to image the gel. The results are as follows: Figure 4 shown.

[0129] The groups are as follows: 1) 2μmol / L W1; 2) 2μmol / L T1; 3) 2μmol / L W1 + 2μmol / L T1; 4) 2μmol / L W1 + 2μmol / L T1 + 30mmol / L MgCl 2 ;5)2μmol / L W2;6)2μmol / L T2;7)2μmol / L W2+2μmol / LT2;8)2μmol / L W2+2μmol / L T2+30mmol / L MgCl 2 .

[0130] from Figure 4 It can be seen that lane 4 shows that when there is no Mg 2+ When W1 and T1 were mixed and incubated at 37°C, no hybridization between W1 and T1 and cleavage of T1 were observed; Lane 5 shows that when Mg 2+ When Mg is present, no complete T1 band is observed, and short-chain products after T1 cleavage can be seen below the original T1 band. This result shows that in Mg 2+ When Mg is present, the W1 chain can effectively cleave the T1 chain. Similarly, lane 8 shows that when Mg is absent, 2+ When W2 and T2 were mixed and incubated at 37°C, no hybridization between W2 and T2 and cleavage of T2 were observed; Lane 9 shows that when Mg 2+ When Mg is present, no complete T2 band is observed, and short-chain products of T2 cleavage can be seen below the original T2 band. This result shows that in Mg2+ When present, the W2 chain can effectively cleave the T2 chain. Figure 4 It can be seen that the W1 / W2 chain can effectively cut the T1 / T2 chain.

[0131] Step 6: Form the fluorescent liquid to be tested

[0132] In this embodiment, this step uses a Cy5 fluorescent probe as a probe for detecting short-chain RNA P1, and a FAM fluorescent probe as a probe for detecting short-chain DNA P2, and the Cy5 and FAM fluorescent groups are processed by the CRISPR / Cas system to restore fluorescence. The specific implementation steps are as follows:

[0133] The mixture containing the nucleic acid short chains P1 and P2 obtained by the above "Step 5, forming a plurality of nucleic acid short chains P" is subjected to magnetic separation treatment to obtain a supernatant containing the nucleic acid short chains P1 and P2, and 20 μL of the supernatant of the nucleic acid short chains P1 and P2 is drawn into another new 96-well all-black microplate, 10 μL of the signal solution is added to each well, and incubated at 37 ° C for 60 min, so that the short chain RNA P1 is hybridized with Cas13a-crRNA and Cas13a is activated to cut the SAA1 fluorescent signal probe FQ1 so that the multiple Cy5 fluorescent groups therein restore fluorescence, and the short chain DNA P2 is hybridized with Cas12a-crRNA and Cas12a is activated to cut the FV fluorescent signal probe FQ2 so that the multiple FAM fluorescent groups therein restore fluorescence, thereby forming a fluorescent liquid to be tested.

[0134] 6.1 Activity investigation of Cas12a and Cas13a

[0135] (1) Prepare a Cas12a reaction system and a Cas13a reaction system, wherein the Cas12a reaction system comprises 150nmol / L Cas12a, 150nmol / L Cas12a-crRNA and 0.8μmol / L FQ2, and the Cas13a reaction system comprises 150nmol / L Cas13a, 150nmol / L Cas13a-crRNA and 0.8μmol / L FQ1.

[0136] (2) Add 20 μL of different concentrations of T2 or T1 to a 96-well all-black microplate, then add 10 μL of Cas12a reaction system or Cas13a reaction system to each well, and incubate at 37°C for 60 min.

[0137] (3) Add 70 μL of shear reaction solution to each well and measure the fluorescence intensity using a multi-function microplate reader. The test results are as follows: Figure 5 shown.

[0138] from Figure 5 It can be seen that: with Figure 5 T1 in A and Figure 5 As the concentration of T2 in B increases, the fluorescence intensity gradually increases, indicating that both Cas13a and Cas12a have strong trans-cleavage activity.

[0139] 6.2 Investigation of mutual interference between Cas12a and Cas13a reaction systems

[0140] Whether the Cas12a and Cas13a systems interfere with each other directly affects the results of subsequent experiments, so it is necessary to investigate. First, add 20 μL of 5nmol / L T2, T1 or T1+T2 solution to a 96-well all-black microplate, then add 10 μL of signal solution to each well and incubate at 37°C for 60 minutes, then add 70 μL of shear reaction solution to each well, and use a multi-function microplate reader to detect the fluorescence intensity of FQ1 and FQ2 at 658nm and 522nm, respectively. The results are as follows: Figure 6 shown.

[0141] from Figure 6 It can be seen from the figure that when there is no target, FL 658 and FL 522 When only T1 was present, an obvious fluorescence signal could be detected at 658 nm, while FL 522 There is no obvious fluorescence signal, indicating that T1 can effectively activate Cas13a but not Cas12a. Similarly, from group 3, it can be seen that T2 can only activate the activity of Cas12a but has no obvious effect on Cas13a. Group 4 shows that when T1 and T2 are present at the same time, FL 658 and FL 522 Both were strong and compared with FL in group 2 658 and FL in Group 3 522 The signal intensities are comparable, indicating that there is no obvious mutual interference between the Cas12a and Cas13a systems.

[0142] Step 7: Fluorescence detection

[0143] First, add the shear reaction solution to the fluorescent solution to be tested, and then use a fluorescence spectrometer to synchronously measure the fluorescence intensity value FL or FL of the fluorescent signal therein. 0 , and then the corresponding regression equation is established using the relationship between fluorescence intensity and exosome protein concentration. The main purpose of adding the shear reaction solution in this step is to increase the volume of the solution to be detected, reduce the detection error caused by the small volume of the solution to be detected, and affect the accuracy and sensitivity of the detection.

[0144] Since the FQ fluorescent probe used in this embodiment is composed of 5nt nucleic acid and its 5' and 3' fluorescent signals and quenching groups, it is difficult to directly measure its fluorescence spectrum, so this embodiment uses the trans-cleavage activity of Cas12a and Cas13a to cut FQ2 and FQ1 probes, and then measure its fluorescence spectrum. The maximum excitation wavelength and maximum emission wavelength of the fluorescent group Cy5 in the FQ1 fluorescent probe are 643nm and 658nm respectively, and the maximum excitation wavelength and maximum emission wavelength of the fluorescent group FAM in the FQ2 probe are 499nm and 522nm. Preferably, the present embodiment selects 633nm and 658nm as the excitation wavelength and emission wavelength when measuring the FQ1 signal, and selects 480nm and 522nm as the excitation wavelength and emission wavelength when measuring the FQ2 probe fluorescence signal.

[0145] Specifically, the shear reaction solution is first added to the 96-well all-black microplate containing the fluorescent solution to be tested in "Step 6, forming the fluorescent solution to be tested", and 70 μL of the shear reaction solution is added to each well; then the fluorescence intensity FL of FQ1 is detected at 658 nm using a multifunctional microplate reader. SAA1 or FL 0(SAA1) , detect the fluorescence intensity FL of FQ2 at 522nm FV or FL 0(FV) ; Then, the corresponding regression equations were established using the relationship between the concentration of SAA1 and the fluorescence intensity of FQ1, and the concentration of FV and the fluorescence intensity of FQ2.

[0146] 7.1 Establishment of standard curve

[0147] SAA1 and FV standards were prepared into different concentrations using PBS buffer solution and fluorescence intensity was detected according to the method provided in this example. SAA1 , the concentration of FV FV As the independent variable, the corresponding fluorescence signal intensity FL SAA1 and FL FV As the dependent variable, a standard curve was established, such as Figure 7 As shown. Among them, FL 0(SAA1) and FL 0(FV) are the concentrations of SAA1, C SAA1 , the concentration of FV FV The fluorescence intensity corresponding to the blank test.

[0148] from Figure 7 It can be seen from A that in the range of 0.1 to 30 ng / mL of the concentration of SAA1, the detection C SAA1 The linear regression equation for Y SAA1 =3682.77C SAA1 +49249.79, of which YSAA1 is the corresponding fluorescence intensity difference: FL SAA1 -FL 0(SAA1) , multiple correlation coefficient R 2 =0.982.

[0149] from Figure 7 It can be seen from B that in the range of FV concentration of 1 to 50 ng / mL, the detection C FV The linear regression equation for Y FV =20262.48C FV +663842.23, of which Y FV is the corresponding fluorescence intensity difference: FL FV -FL 0(FV) , multiple correlation coefficient R 2 =0.999.

[0150] 7.2 Detection Limit

[0151] Prepare 4 low-concentration standard solutions, including mixed solutions with SAA1 and FV concentrations of 5, 30, 200, and 400 pg / mL; then perform the experiment according to the method steps provided in the embodiment of the present invention; and simultaneously measure the fluorescence intensity of the solvent blank to calculate value( is the fluorescence intensity of the solvent blank, SD is the standard deviation), and the fluorescence intensity in the mixed solution is greater than The target concentration at 50°C was the detection limit of the established method. The results are shown in Table 2.

[0152] Table 2 Detection results of low concentration SAA1 and FV

[0153] <![CDATA[C SAA1 / FV (pg / mL)]]> 5 30 200 400 <![CDATA[FL SAA1 ]]> 421754 452308 482874 491862 <![CDATA[FL FV ]]> 2008688 2337192 2598477 2607197

[0154] It can be seen from Table 2 that when the SAA1 concentration is 0, the blank solution When the concentration of SAA1 is 30pg / mL, its corresponding fluorescence intensity is >448406.74, so the detection limit of this method for SAA1 is 30pg / mL. Similarly, the detection limit of FV protein is 200pg / mL.

[0155] 7.3 Precision and spike recovery

[0156] Plasma exosomes were extracted and lysed using a co-precipitation kit. The specific steps are as follows:

[0157] 1) Thawing plasma: Take the plasma out of the -80°C freezer and place it in a 37°C water bath until it is completely thawed;

[0158] 2) Plasma pretreatment: 200 μL of plasma was placed in a 1.5 mL centrifuge tube and centrifuged at 2000 g for 20 min at room temperature; the supernatant was placed in a new centrifuge tube and centrifuged at 10000 g for 20 min at room temperature, and the supernatant was taken;

[0159] 3) Add 100 μL PBS buffer to the plasma and vortex to mix, add 60 μL coprecipitation reagent to the centrifuge tube and vortex to mix;

[0160] 4) After reacting at room temperature for 10 min, centrifuge at 10,000 g for 5 min and discard the supernatant;

[0161] 5) Centrifuge at 10,000 g for 30 seconds at room temperature and discard the supernatant again;

[0162] 6) Add 50 μL PBS buffer to resuspend the precipitate to obtain an exosome solution, which was stored at -80°C for future use. The exosome solution was characterized by transmission electron microscopy and WB as shown in the following figure. Figure 8 shown.

[0163] Figure 8 A shows that vesicles with sizes between 30 and 150 nm can be observed under the TEM field of view. Figure 8 It can be seen from B that the extracted exosomes contain proteins such as Alix, HSP70, TSG101, CD63, etc. Therefore, plasma exosomes were successfully extracted in this example.

[0164] Take 6.5 μL of the above exosome solution, add 6.5 μL of exosome lysis solution, lyse on ice for 10 minutes to obtain exosome lysate. When determining SAA1 in exosome lysate, dilute the exosome lysate 50 times with PBS. Add different amounts of SAA1 and FV to the diluted exosome lysate, and the final concentrations of SAA1 are 5 ng / mL and 10 ng / mL, respectively, and the final concentrations of FV are 15 ng / mL and 30 ng / mL, respectively. Each concentration is paralleled for 3 times, and the experiment is carried out according to the method steps provided in the embodiment of the present invention. The RSD and spike recovery rate are calculated according to the standard curve, and the results are shown in Table 3.

[0165] It can be seen from Table 3 that the recoveries of SAA1 and FV in spiked samples detected by the method established in this embodiment range from 93.20% to 95.80% and 94.20% to 98.67%, respectively, and the corresponding RSDs are 7.10% to 9.33% and 7.16% to 9.41%, respectively.

[0166] Table 3 Precision and spike recovery of SAA1 and FV (n=3)

[0167]

[0168] 7.4 Specificity

[0169] IgG, CEA and NSE, which may coexist with SAA1 and FV in actual samples, were selected as negative controls. The method established in this example was used for detection. SAA1 and FV were measured simultaneously. The detection results were compared to evaluate the specificity of the method. The results are shown in FIG. Fig. 9 shown.

[0170] from Fig. 9 It can be seen that when only one target, SAA1, is added, a clear fluorescence signal can be detected at 658nm, but no obvious signal at 522nm. Similarly, when only FV is added, FL 522 The signal is obvious, and FL 658 No obvious signal. When SAA1 and FV are present at the same time, there are obvious fluorescence signals at 658nm and 522nm. In addition, there are no obvious fluorescence signals in CEA, NSE and IgG groups. Therefore, the method established in this example has strong specificity.

[0171] 7.5 Methodological comparison

[0172] To further investigate the accuracy of the method established in this example, the results were compared with those of ELISA. The specific steps are as follows:

[0173] 1) The co-precipitation method was used to extract exosomes from 9 plasma samples, and the method and steps were the same as "7.3 Precision and spike recovery".

[0174] 2) Detection by the established method: Take 6.5 μL of exosome solution, add 6.5 μL of exosome lysis buffer, and lyse on ice for 10 min to obtain exosome lysis buffer. Dilute the exosome lysis buffer at a volume ratio of 1:50, and detect SAA1 and FV using the method established in this example. The test results are as follows: Fig.10 shown.

[0175] 3) ELISA detection: ELISA kit was used to detect SAA1 and FV in exosome lysate. The test results are as follows Fig.10 shown.

[0176] Fig.10 The results showed that: correlation analysis showed that there was a correlation between the results of SAA1 detected by the established method and ELISA (r=0.960, P<0.001); there was also a correlation between the results of FV detected by the two methods (r=0.981, P<0.001). Therefore, the accuracy of the method established in this example was relatively strong.

[0177] The established method and the ELISA kit were compared in detail in terms of the types of detection targets, detection time, etc., and the results are shown in Table 4. Table 4 shows that the established method in this example can simultaneously detect two proteins (SAA1 and FV) within about 3.5 hours, while ELISA can only detect one protein within the corresponding time. In addition, the detection limit of the established method is significantly lower than that of the ELISA kit, indicating that its sensitivity is higher than that of the ELISA method.

[0178] Table 4 Comparison of the established method with ELISA for detection of SAA1 and FV

[0179]

[0180] Note: The time, detection limit and linear range of ELISA for SAA1 and FV in the table refer to the SAA1 or FV commercial kits produced by Wuhan Elaruite Biotechnology Co., Ltd. and Wuhan Huamei Bioengineering Co., Ltd.

[0181] 7.6 Simultaneous detection of SAA1 and FV in plasma exosomes

[0182] After approval by the Ethics Committee and informed consent, plasma samples were collected from 43 lung cancer patients (LC group) in the respiratory department of a hospital and 49 healthy controls (HC group) in the health examination center for experiments. Plasma exosomes were extracted by coprecipitation method, and the extraction steps were the same as "7.3 Precision and spike recovery rate". The exosomes were lysed with lysis buffer to release the contents, and the lysis steps were the same as step 2 in "7.5 Methodology Comparison". SAA1 and FV in the exosome lysate were detected using the method steps provided in this example. The results are as follows Fig.10 shown.

[0183] Fig.10 A shows that the SAA1 signal intensity of the LC group was higher than that of the HC group, but the difference was not statistically significant (Z=-1.498, P=0.134); the fluorescence signal intensity of FV in the LC group was stronger than that in the HC group, and the difference was statistically significant (t=-2.664, P=0.009). Fig.10 The PCA results of B showed that the levels of SAA1 and FV could effectively distinguish lung cancer patients from healthy people, indicating that SAA1 and FV may be potential markers for lung cancer.

[0184] Therefore, the synchronous detection method of SAA1 and FV in exosomes provided in this embodiment is mainly based on DNAzyme and CRISPR-Cas12a / Cas13a system, which can achieve quantitative high-sensitivity detection. This method has the following advantages: 1) Carboxyl magnetic beads are used as separation media to improve the capture efficiency of the target; 2) By coupling two capture antibodies to MBs2 at the same time, multifunctional immunomagnetic beads are prepared to achieve specific capture, separation and enrichment of SAA1 and FV; 3) Paired antibodies are used to identify the target, and subsequent detection signals can only be generated on the basis that the corresponding capture antibody and detection antibody are bound to the target, ensuring the specificity of the detection method; 4) DNAzyme and CRISPR-Cas12a / Cas13a are used to achieve dual signal amplification and improve detection sensitivity; 5) The detection system is highly universal and can be used not only for the detection of non-membrane proteins in exosomes, but also for the detection of membrane proteins and exosome proteins that have not yet been clearly located; 6) Compared with the common single signal analysis method, the strategy adopted in this study has the advantages of small sample size, simple operation steps and high throughput. Based on the above advantages, the detection limits of the established method for SAA1 and FV were as low as 30pg / mL and 200pg / mL, respectively. In addition, the established method can be successfully applied to the detection of SAA1 and FV in plasma exosomes, and the detection results can effectively distinguish lung cancer patients from healthy controls.

[0185] Experimental Condition Optimization Example

[0186] According to the method for simultaneous detection of exosomal proteins SAA1 and FV provided in Example 1 of the present invention, in particular, the steps of "step 4, forming a double antibody sandwich complex", "step 5, forming a plurality of nucleic acid short chains P", and "step 6, forming a fluorescent liquid to be tested", the amount of the immunomagnetic beads MBs2@Ab1, Ab2 SAA1 -W1 and Ab2 FV -W2 dilution ratio, MBs3 Track dosage, Mg 2+ The concentration of , as well as the concentration of Cas13a / Cas12a and FQ1 / FQ2 in the signal solution, have an important influence on the fluorescence intensity. The optimal conditions of the above influencing factors are selected according to the fluorescence intensity.

[0187] 1. Dosage optimization test of immunomagnetic beads MBs2@Ab1

[0188] If the amount of immunomagnetic beads MBs2@Ab1 is too small, a large number of exosomes cannot be captured, thus affecting the detection sensitivity; if the amount is too large, it will cause waste. Therefore, the single-factor method was used to optimize the amount of immunomagnetic beads MBs2@Ab1.

[0189] The method used was as follows: 5 μL, 15 μL, 25 μL, 35 μL, and 45 μL of the immunomagnetic bead MBs2@Ab1 suspension were added to each well of the 96-well plate in "Step 4, forming a double antibody sandwich complex", and the other steps of the method provided in Example 1 of the present invention were followed. The results are shown in FIG. Fig.11 shown.

[0190] from Fig.11 It can be seen that when the amount of immunomagnetic beads is less than 35 μL, the fluorescence signal gradually increases with the increase in the amount of immunomagnetic beads, which may be related to the increase in the capture efficiency of the target. When the amount of immunomagnetic beads is further increased, the signal intensity decreases slightly; the reason may be that the high concentration of immunomagnetic beads precipitates significantly, affecting the capture of the target by the immunomagnetic beads. Therefore, the optimal amount of MBs2@Ab1 suspension is 35 μL.

[0191] 2. Ab2 SAA1 -W1 and Ab2 FV -W2 dilution ratio optimization test

[0192] Ab2 SAA1 -W1 dilution ratio optimization test method is: "Step 4, forming a double antibody sandwich complex" "add Ab2 SAA1 -W1 and Ab2 FV -W2" step, PBS was used as solvent, Ab2 SAA1 The dilution ratios of -W1 were 1:32, 1:16, 1:8, 1:4, and 1:2 respectively. The other experiments were carried out according to the method steps provided in Example 1 of the present invention. The results are as follows: Fig.12 Medium FL 658 -FL 0 As shown in the curve.

[0193] Ab2 FV -W2 dilution ratio optimization test method is: step "four, forming a double antibody sandwich complex" "add Ab2 SAA1 -W1 and Ab2 FV -W2" step, PBS was used as solvent, Ab2 FV The dilution ratios of -W2 were 1:64, 1:32, 1:16, 1:8, and 1:4, respectively. The other experiments were carried out according to the method steps provided in Example 1 of the present invention. The results are as follows: Fig.12 Medium FL 522 -FL 0 As shown in the curve.

[0194] from Fig.12 It can be seen that when Ab2 SAA1 -W1 dilution ratio is 1:4, FL 658 -FL 0Maximum; when Ab2 FV -W2 dilution ratio is 1:8, FL 522 -FL 0 The signal intensity is strong. When the dilution ratio continues to decrease, the corresponding fluorescence signals of the two do not change significantly. SAA1 The optimal dilution ratio of -W1 is 1:4, and Ab2 FV -The optimal dilution ratio of W2 is 1:8.

[0195] 3. MBs3 Track dosage optimization test

[0196] The method used in the experiment: In "Step 3, forming MBs3 Track", the amount of 2 mg / mL MBs3 and the amount of the shear reaction solution used to resuspend MBs3 Track were 1:1, 1:2, 1:4, 1:8, and 1:16, respectively. The other steps of the method provided in Example 1 of the present invention were used for the experiment. The results are as follows Fig.13 shown.

[0197] When the amount of MBs3 Track is too low, it will affect the cutting efficiency of W1 and W2, while too high a concentration will cause reagent waste. Fig.13 It can be seen that when the dilution ratio of MBs3 Track is 1:2, FL 522 -FL 0 Maximum, and FL 658 -FL 0 Therefore, the optimal dilution ratio of MBs3 Track is 1:2.

[0198] 4. Mg 2+ Concentration optimization selection test

[0199] Since DNAzyme W1 / W2 cleavage of substrate strands T1 / T2 depends on Mg 2+ The presence of MgCl in the shear solution was optimized. 2 The concentrations of the samples were 10, 30, 50, 70, and 90 mmol / L, respectively. The other experiments were carried out according to the method steps provided in Example 1 of the present invention. The results are as follows Fig.14 shown.

[0200] from Fig.14 It can be seen that when MgCl 2 When the concentration increased from 10mmol / L to 30mmol / L, FL 522 -FL 0 Enhanced, while FL 658 -FL 0 Slightly decreased. 2+As the concentration increases further, the fluorescence signal decreases significantly. 2+ The optimal concentration is 30mmol / L.

[0201] 5. Signal solution optimization test

[0202] The concentrations of Cas13a, Cas12a, FQ1, and FQ2 in the signal solution directly affect the fluorescence signal intensity, so they are optimized. The molar ratio of Cas13a and Cas12a to the corresponding crRNA in the signal solution used in the following optimization experiment is 1:1.

[0203] 5.1 Cas13a / Cas12a concentration optimization experiment

[0204] The method used in the concentration optimization test of Cas13a and Cas12a: 1) The concentrations of Cas13a in the signal solution were 30, 60, 90, 120, 150, and 180 nmol / L, respectively. The other methods and steps provided in Example 1 of the present invention were used for the experiment. The results are as follows Fig.15 Medium FL 658 -FL 0 2) The concentrations of Cas12a in the signal solution were 30, 60, 90, 120, 150, and 180 nmol / L, respectively. The other experiments were carried out according to the method steps provided in Example 1 of the present invention. The results are shown in FIG. Fig.15 Medium FL 522 -FL 0 As shown in the curve.

[0205] from Fig.15 It can be seen that when the concentrations of Cas13a and Cas12a are both 150nmol / L, FL 658 -FL 0 Larger, and FL 522 -FL 0 The strongest, therefore, the optimal concentrations of Cas12a and Cas13a in the signal solution are both 150nmol / L.

[0206] 5.2 Concentration optimization experiment of FQ1 and FQ2 probes

[0207] The method used in the concentration optimization test of FQ1 and FQ2 is as follows: 1) the concentration of FQ1 in the signal solution is 30, 60, 90, 120, 150, 180 nmol / L respectively, and the other steps are carried out according to the method steps provided in Example 1 of the present invention. The results are as follows Fig.16 Medium FL 658 -FL 02) The concentrations of FQ2 in the signal solution were 30, 60, 90, 120, 150, and 180 nmol / L, respectively. The other steps were followed in the experiment according to the method of Example 1 of the present invention. The results are shown in FIG. Fig.16 Medium FL 522 -FL 0 As shown in the curve.

[0208] from Fig.16 It can be seen that as the concentration of FQ1 and FQ2 probes increases, the fluorescence signal intensity gradually increases. Among them, when the concentration of FQ1 and FQ2 probes is greater than 800nmol / L, the signal intensity increases slightly. Therefore, in order to save costs, the optimal concentration of FQ1 and FQ2 probes is 800nmol / L.

[0209] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, ordinary technicians in the field should understand that the specific implementation methods of the present invention can still be modified or some technical features can be replaced by equivalents without departing from the spirit of the technical solution of the present invention, which should be included in the scope of the technical solution for protection of the present invention. Sequence Listing <110> Zhengzhou University <120> Simultaneous detection of multiple exosomal proteins <130> 2021.10.9 <160> 6 <170> SIPOSequenceListing 1.0 <210> 1 <211> 45 <212> DNA <213> Artificial Sequence() <220> <221> rRNA <222> (15)..(45) <400> 1 ttttttttttttttucuagg aagguugcgu cacuauacgu cggca 45 <210> 2 <211> 50 <212> DNA <213> Artificial Sequence() <220> <221> rRNA <222> (19) <400> 2 ttttttttttttttaccgac tataggttgt gttactcgtt gactgaaact 50 <210> 3 <211> 63 <212> DNA <213> Artificial Sequence() <400> 3 tttttttttt tttttttttt tttttttttt gtgacgcaag gctagctaca acgacttcct 60 aga 63 <210> 4 <211> 62 <212> DNA <213> Artificial Sequence() <400> 4 tttttttttt tttttttttt tttttttttt aacacaactc cgagccggtc gaaatagtcg 60 gt 62 <210> 5 <211> 41 <212> RNA <213> Artificial Sequence() <400> 5 uaauuucuac uaaguguaga uagucaacga guaacacaac c 41 <210> 6 <211> 51 <212> RNA <213> Artificial Sequence() <400> 6 gaccacccca aaaaugaagg ggacuaaaac agccgacgua uagugacgca a 51

Claims

1. A method for synchronously detecting multiple exosome proteins for non-diagnostic purposes, comprising the steps of: Forming immunomagnetic beads: The capture antibodies Ab1 of multiple exosome proteins to be detected are simultaneously coupled to the carboxyl magnetic beads MBs2 to form immunomagnetic beads MBs2@Ab1; Forming multiple Ab2-Ws: biotinylating the detection antibody Ab2 corresponding to each of the multiple exosome proteins to be detected to form a biotinylated detection antibody Ab2-Bio; Each biotinylated detection antibody Ab2-Bio is coupled to the corresponding deoxyribozyme W to form a conjugate Ab2-W; Forming streptavidin magnetic beads Track: coupling streptavidin magnetic beads MBs3 with the biotinylated substrate chain T of each of the multiple exosome proteins to be detected to form MBs3 Track modified with the multiple biotinylated substrate chains T, and resuspending the MBs3 Track in a shearing reaction solution to form an MBs3 Track suspension, wherein the shearing reaction solution includes a coenzyme factor and an RNase inhibitor; Forming a double antibody sandwich complex: mixing and incubating the immunomagnetic beads MBs2@Ab1, the sample to be tested containing the multiple exosome proteins to be tested, and multiple conjugates Ab2-W to form a double antibody sandwich complex; Forming a variety of nucleic acid short chains P: adding the MBs3 Track suspension to the double antibody sandwich complex and incubating again, so that each deoxyribozyme W cuts the corresponding biotinylated substrate chain T to generate nucleic acid short chains P, magnetically separating, and collecting the supernatant containing the various nucleic acid short chains P; Forming a fluorescent liquid to be tested: first, the supernatant containing a plurality of nucleic acid short chains P is mixed and incubated with a signal solution, wherein the signal solution includes a Cas protein, Cas-crRNA and a fluorescent signal probe corresponding to each nucleic acid short chain P, and each nucleic acid short chain P is hybridized with its corresponding Cas-crRNA, and the corresponding Cas protein is activated to cut the corresponding fluorescent signal probe to restore the fluorescence of its fluorescent group, thereby forming a fluorescent liquid to be tested, wherein the Cas protein is Cas12a or Cas13a, and the Cas-crRNA is Cas12a-crRNA or Cas13a-crRNA; Fluorescence detection: A multifunctional microplate reader is used to synchronously detect the fluorescence intensity of each fluorescent group in the fluorescent liquid to be tested, and a corresponding regression equation is constructed using the relationship between the fluorescence intensity of each fluorescent group and the concentration of the corresponding exosome protein to be tested.

2. The synchronous detection method according to claim 1, It is characterized in that The step of forming the immunomagnetic beads comprises: using the EDC / Sulfo-NHS method to capture the SAA1 antibody Ab1 SAA1 and FV capture antibody Ab1 FV At the same time, the immunomagnetic beads MBs2 are coupled to the carboxyl magnetic beads MBs2 and blocked with a bovine serum albumin solution to obtain the immunomagnetic beads MBs2@Ab1. The immunomagnetic beads MBs2@Ab1 are resuspended in a phosphate buffer solution to form an immunomagnetic bead MBs2@Ab1 suspension.

3. The synchronous detection method according to claim 2, It is characterized in that The steps of forming a plurality of Ab2-Ws include: The detection antibody Ab2 of SAA1 SAA1 The solution and Sulfo-NHS-LC-Biotin were evenly mixed and reacted at room temperature to obtain the biotinylated detection antibody Ab2 of SAA1. SAA1 -Bio solution; firstly, the biotinylated detection antibody Ab2 SAA1 -Bio solution and streptavidin solution were reacted at room temperature, and then DNAzyme W1 solution was added and reacted at room temperature to obtain conjugate Ab2 SAA1 -W1 solution; The FV detection antibody Ab2 FV The solution and Sulfo-NHS-LC-Biotin were evenly mixed and reacted at room temperature to obtain the biotinylated detection antibody Ab2 of FV. FV -Bio solution; firstly, the biotinylated detection antibody Ab2 FV -Bio solution and streptavidin solution were reacted at room temperature, and then DNAzyme W2 solution was added and reacted at room temperature to obtain conjugate Ab2 FV -W2 solution.

4. The synchronous detection method according to claim 3, It is characterized in that The SAA1 capture antibody Ab1 SAA1 is a mouse anti-human SAA1 monoclonal capture antibody, the capture antibody Ab1 of FV FV is a rabbit anti-human FV monoclonal capture antibody, and the SAA1 detection antibody Ab2 SAA1 is a mouse anti-human SAA1 monoclonal detection antibody, the FV detection antibody Ab2 FV Sheep anti-human FV polyclonal detection antibody.

5. The synchronous detection method according to claim 3 or 4, It is characterized in that The step of forming the streptavidin magnetic beads Track comprises: shaking a mixture of substrate chains T1 and T2 with the streptavidin magnetic beads MBs3 at room temperature to modify the substrate chains T1 and T2 on the streptavidin magnetic beads MBs3 at the same time, magnetically separating to obtain the streptavidin magnetic beads MBs3 Track; resuspending the streptavidin magnetic beads Track in the shear reaction solution to form an MBs3Track suspension; wherein the pH of the shear reaction solution is 7.0 to 8.0 and includes 10 to 90 mmol / L MgCl 2 , 0.6-1.2 mmol / L DTT and 0.8-1.6 U / µL RNase inhibitor.

6. The synchronization detection method according to claim 5, It is characterized in that The deoxyribozyme W1 is the nucleotide sequence shown in SEQ ID No.3, and the deoxyribozyme W2 is the nucleotide sequence shown in SEQ ID No.4; the substrate chain T1 is the nucleotide sequence shown in SEQ ID No.1, and the substrate chain T2 is the nucleotide sequence shown in SEQ ID No.

2.

7. The synchronization detection method according to claim 6, It is characterized in that The step of forming a double antibody sandwich complex comprises: mixing the immunomagnetic bead MBs2@Ab1 suspension, the sample to be tested containing SAA1 and FV, the conjugate Ab2 SAA1 -W1 solution and the conjugate Ab2 FV -W2 solution was mixed and incubated at 37°C and magnetically separated to obtain the double antibody sandwich complex.

8. The synchronization detection method according to claim 7, It is characterized in that The step of forming multiple nucleic acid short chains P includes: adding the MBs3 Track suspension to the double antibody sandwich complex and shaking the reaction at 37°C, so that the deoxyribozyme W1 cuts the substrate chain T1 and releases the short chain RNA P1, the deoxyribozyme W2 cuts the substrate chain T2 and releases the short chain DNA P2, magnetic separation, and collecting the supernatant containing the short chain RNA P1 and DNA P2.

9. The synchronous detection method according to claim 8, It is characterized in that The step of forming the fluorescent liquid to be tested comprises: The signal solution includes 30-180 nmol / L Cas13a, 30-180 nmol / L Cas12a, 150 nmol / LCas13a-crRNA, 150 nmol / L Cas12a-crRNA, 200-1000 nmol / L SAA1 fluorescent signal probe FQ1 and 200-1000 nmol / L FV fluorescent signal probe FQ2, wherein the nucleic acid sequence of the SAA1 fluorescent signal probe FQ1 from 5′ to 3′ is: Cy5-rUrUrUrUrU-BHQ2, and the nucleic acid sequence of the FV fluorescent signal probe FQ2 from 5′ to 3′ is: FAM-TTATT-BHQ1; The signal solution is added to the supernatant containing the short-chain RNA P1 and DNA P2 and incubated at 37° C., so that the short-chain RNA P1 hybridizes with Cas13a-crRNA and activates Cas13a to cut the SAA1 fluorescent signal probe FQ1 so that the Cy5 fluorescent group therein restores fluorescence, and the short-chain DNA P2 hybridizes with Cas12a-crRNA and activates Cas12a to cut the FV fluorescent signal probe FQ2 so that the FAM fluorescent group therein restores fluorescence, thereby forming the fluorescent solution to be tested.

10. The synchronization detection method according to claim 9, It is characterized in that The step of fluorescence detection comprises: The shear reaction solution is added to the fluorescent solution to be tested, and the fluorescence intensity value FL of the Cy5 fluorescent group therein is synchronously measured using a multifunctional microplate reader. SAA1 or FL 0(SAA1) , the fluorescence intensity value FL of the FAM fluorescent group FV or FL 0(FV) ; In the range of SAA1 concentration of 0.1 to 30 ng / mL, a regression equation for detecting SAA1 concentration was established: Y SAA1 =3682.77 C SAA1 + 49249.79, of which Y SAA1 is the corresponding fluorescence intensity difference: FL SAA1 -FL 0(SAA1) , multiple correlation coefficient R 2 =0.982, C SAA1 represents the concentration of SAA1; In the range of FV concentration of 1 to 50 ng / mL, a regression equation for detecting FV concentration was established: Y FV =20262.48 C FV + 663842.23, of which Y FV is the corresponding fluorescence intensity difference: FL FV -FL 0(FV) , multiple correlation coefficient R 2 =0.999, C FV Represents the concentration of FV.

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