Electrochemiluminescence detection method and system

Abstract

The invention provides an electrochemical luminescence detection method and system.The electrochemical luminescence detection method comprises the following steps that S1, polystyrene microspheres are dispersed in ethyl alcohol, amino silane is dropwise added into the ethyl alcohol, then polypyrrole, a carbon nanomaterial and a metal nanomaterial are sequentially added, and composite polystyrene microspheres are obtained; s2, loading the ruthenium complex combined with the detection antibody on the composite polystyrene microspheres; s3, dispensing the composite polystyrene microspheres on the surface of an electrode, drying the electrode, and fixing the electrode in an electrochemical reaction tank; s4, adding a standard target antigen and a capture antibody aiming at the standard target antigen into the electrochemical reaction tank; and S5, adding an electron donor into the electrochemical reaction tank, applying a voltage, detecting a luminescence reaction, and collecting and sorting data. According to the invention, through the interaction of the polypyrrole, the carbon nanomaterial, the metal nanomaterial and the ruthenium complex, the detection stability is ensured while the sensitivity and the accuracy of electrochemical luminescence detection are improved.

Description

Technical Field

[0001] The present invention relates to the technical field of optical detection, in particular to an electrochemiluminescence detection method and system. Background Art

[0002] Electrochemiluminescence (ECL), as an advanced analytical detection technology, combines the advantages of electrochemistry and chemiluminescence and has attracted much attention in recent years in the fields of scientific research and practical applications. Its core principle is based on the excited-state substances generated during the electrochemical reaction process. When these substances return to the ground state, they release energy in the form of light radiation, generating detectable luminescence signals. Compared with traditional optical detection methods, electrochemiluminescence detection has many excellent characteristics. First of all, it has extremely high sensitivity and accuracy, and can accurately detect analytes in the picomole or even femtomole level. This is because the electrochemical reaction can efficiently initiate the luminescence process in the local area of the electrode surface, effectively avoiding a large amount of background noise interference and significantly amplifying the signal. Secondly, electrochemiluminescence detection has strong selectivity. By cleverly designing the electrode material, electrochemical reaction system, and labeling substances, highly specific recognition and detection of specific target molecules can be achieved, reducing the misjudgment interference of non-target substances. Moreover, the dynamic range of this technology is wide, and it can quantitatively determine analytes across multiple orders of magnitude of concentration ranges, meeting the detection requirements of different content substances in complex sample systems and providing accurate results from trace to relatively high concentration levels.

[0003] With the booming development of the life science field, the demand for rapid and accurate detection of biomolecules (such as nucleic acids, proteins, polypeptides, hormones, etc.) is becoming increasingly urgent. In clinical diagnosis, disease early biomarkers often exist in body fluids at extremely low concentrations. The electrochemiluminescence detection technology can keenly capture these weak signal changes in the background of complex blood, urine, or saliva samples, providing key evidence for the early screening, diagnosis, and disease condition monitoring of diseases. For example, in the detection of tumor markers, for the accurate quantification of markers such as carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP), electrochemiluminescence detection helps doctors achieve earlier and more accurate cancer diagnosis and improve the cure rate of patients.

[0004] Although the electrochemiluminescence detection technology has significant advantages, it still faces a series of technical challenges in the actual application and promotion process. The luminescent substances often need to be combined with a carrier and then fixed on the electrode surface. However, in this case, the carrier often introduces interfacial resistance, reduces the electron transfer efficiency, and easily leads to insufficient sensitivity and accuracy of the electrochemical reaction of the luminescent substances; and the luminescent substances are often combined with the carrier in a simple way, with insufficient loading amount and easy to fall off, and are also easily damaged, resulting in poor detection stability; the current technology cannot solve the above problems simultaneously. Summary of the Invention

[0005] In order to solve the technical problems of insufficient sensitivity and accuracy in existing electrochemical luminescence detection and poor detection stability, the electrochemical luminescence detection method proposed by the present invention includes the following steps:

[0006] S1. Disperse polystyrene microspheres in ethanol, drop aminopropylsilane into ethanol, and then sequentially add polypyrrole, carbon nanomaterials, and metal nanomaterials, stir at room temperature for 1-3 h, and obtain composite polystyrene microspheres after drying;

[0007] S2. Load the ruthenium complex conjugated with the detection antibody onto the composite polystyrene microspheres through the biotin-streptavidin mediated method;

[0008] S3. Drop the composite polystyrene microspheres loaded with the ruthenium complex onto the surface of the electrode, and fix the dried electrode in the electrochemical reaction cell;

[0009] S4. Add the standard target antigen and the capture antibody against the standard target antigen into the electrochemical reaction cell to form a capture antibody-antigen-detection antibody complex;

[0010] S5. Add an electron donor into the electrochemical reaction cell and apply a voltage to trigger the luminescence reaction of the ruthenium complex on the electrode, detect the luminescence reaction, and collect and collate the data.

[0011] Preferably, the aminopropylsilane is any one of 3-aminopropyltrimethoxysilane, 3-aminopropyltriethoxysilane, phenylaminomethyltrimethoxysilane, and phenylaminomethyltriethoxysilane.

[0012] Preferably, the carbon nanomaterial is any one of carbon nanotubes and graphene.

[0013] Preferably, the metal nanomaterial is any one of gold nanoparticles, silver nanoparticles, and copper nanoparticles.

[0014] Preferably, the ruthenium complex is any one of tris(bipyridine)ruthenium(II), ruthenium(II) chloride hexahydrate, tris(4,4'-dimethyl-2,2'-bipyridine)ruthenium(II) hexafluorophosphate, and tris(2,2'-bipyridine)ruthenium(II) NHS ester.

[0015] Preferably, the detection antibody is any one of anti-PSA monoclonal antibody, anti-CEA monoclonal antibody, and anti-AFP monoclonal antibody.

[0016] Preferably, the capture antibody is an anti-PSA capture antibody, an anti-CEA capture antibody, or an anti-AFP capture antibody corresponding to the detection antibody.

[0017] Preferably, the standard target antigen is PSA, CEA, or AFP corresponding to the detection antibody and the capture antibody.

[0018] Preferably, the electron donor is any one of tripropylamine, 2-dibutylaminoethanol, triethanolamine and triisopropanolamine.

[0019] The electrochemiluminescence detection system proposed in the present invention includes a sample injection system, an electrochemiluminescence system directly connected to the sample injection system, an optical detection system facing the electrochemiluminescence system, and a data acquisition and processing system directly connected to the optical detection system, which can implement the above-mentioned electrochemiluminescence detection method.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] 1) The π-π conjugated system of polypyrrole, the π-π conjugated system of carbon nanomaterials, and the π-π conjugated system of the terpyridine ligand in the ruthenium complex have a structural synergistic effect, which greatly improves the conductivity of the composite polystyrene microspheres, accelerates the transfer of electrons from the electrode to the ruthenium complex, reduces the response time of the electrochemical reaction of the ruthenium complex, and improves its sensitivity and accuracy;

[0022] 2) Metal nanomaterials can amplify the electrochemiluminescence intensity of ruthenium complexes through the surface plasmon resonance effect, further improving its sensitivity and accuracy;

[0023] 3) The polypyrrole structure contains a large number of NH bonds, which can easily generate hydrogen bonds with nitrogen atoms in the ruthenium complex, thereby enhancing the binding between the ruthenium complex and the composite polystyrene microspheres and reducing shedding. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is a schematic flow chart of the electrochemiluminescence detection method;

[0025] Figure 2 It is a structural diagram of the electrochemiluminescence detection system.

[0026] Figure numerals: 1. Sampling system; 2. Electrochemiluminescence system; 3. Optical detection system; 4. Data acquisition and processing system. DETAILED DESCRIPTION

[0027] The technical solution of the present invention is described in detail below with reference to the accompanying drawings and specific implementation methods.

[0028] like Figure 1 As shown, the electrochemiluminescence detection method proposed by the present invention includes the following steps:

[0029] S1. Disperse polystyrene microspheres in ethanol, drop aminopropylsilane into the ethanol, and then successively add polypyrrole, carbon nanomaterials, and metal nanomaterials, stir at room temperature for 1 - 3 h, and obtain composite polystyrene microspheres after drying. The carbon nanomaterials are any one of carbon nanotubes and graphene. The mass ratio of polystyrene microspheres, ethanol, polypyrrole, carbon nanomaterials, metal nanomaterials, and aminopropylsilane is: (30 - 40):(25 - 35):(10 - 15):(10 - 15):(5 - 10):(1 - 3). The diameter of the polystyrene microspheres is 2 - 5 μm.

[0030] S2. Load the ruthenium complex conjugated with the detection antibody onto the composite polystyrene microspheres by the biotin - streptavidin mediated method.

[0031] S3. Drop - coat the composite polystyrene microspheres loaded with the ruthenium complex onto the surface of the electrode, and fix the dried electrode in the electrochemical reaction cell.

[0032] S4. Add the standard target antigen and the capture antibody against the standard target antigen into the electrochemical reaction cell to form a capture antibody - antigen - detection antibody complex.

[0033] S5. Add an electron donor into the electrochemical reaction cell and apply a voltage to trigger the luminescence reaction of the ruthenium complex on the electrode, detect the luminescence reaction, and collect and collate the data.

[0034] Example 1: In S1, the aminopropylsilane is 3 - aminopropyltrimethoxysilane, the metal nanomaterials are gold nanoparticles, stir at room temperature for 2 h, and the mass ratio of polystyrene microspheres, ethanol, polypyrrole, carbon nanomaterials, metal nanomaterials, and aminopropylsilane is: 35:28:15:15:5:2; in S2, the ruthenium complex conjugated with the detection antibody is tris(bipyridine)ruthenium conjugated with anti - PSA monoclonal antibody; in S4, add 0.5 ng / mL of the standard target antigen PSA and anti - PSA capture antibody into the electrochemical reaction cell to form a capture antibody - antigen - detection antibody complex; in S5, add tripropylamine into the electrochemical reaction cell and apply a voltage of 1.0 V to trigger the luminescence reaction of the ruthenium complex.

[0035] Example 2: In S1, the aminosilane is 3-aminopropyltriethoxysilane, the metal nanomaterial is silver nanoparticles, stirred at room temperature for 1 h, and the mass ratio of polystyrene microspheres, ethanol, polypyrrole, carbon nanomaterials, metal nanomaterials and aminosilane is: 35:30:12:15:7:1; in S2, the ruthenium complex conjugated with the detection antibody is tris(bipyridine)ruthenium(III) hexahydrate conjugated with anti-CEA monoclonal antibody; in S4, 1 ng / mL of the standard target antigen CEA and anti-CEA capture antibody are added into the electrochemical reaction cell to form a capture antibody-antigen-detection antibody complex; in S5, 2-dibutylaminoethanol is added into the electrochemical reaction cell and a voltage of 1.0 V is applied to trigger the luminescence reaction of the ruthenium complex.

[0036] Example 3: In S1, the aminosilane is phenylaminomethyltrimethoxysilane, the metal nanomaterial is copper nanoparticles, stirred at room temperature for 1.5 h, and the mass ratio of polystyrene microspheres, ethanol, polypyrrole, carbon nanomaterials, metal nanomaterials and aminosilane is: 32:30:15:12:8:3; in S2, the ruthenium complex conjugated with the detection antibody is tris(4,4'-dimethyl-2,2'-bipyridine)ruthenium(III) hexafluorophosphate conjugated with anti-AFP monoclonal antibody; in S4, 5 ng / mL of the standard target antigen AFP and anti-AFP capture antibody are added into the electrochemical reaction cell to form a capture antibody-antigen-detection antibody complex; in S5, triethanolamine is added into the electrochemical reaction cell and a voltage of 1.0 V is applied to trigger the luminescence reaction of the ruthenium complex.

[0037] Example 4: In S1, the aminosilane is phenylaminomethyltriethoxysilane, the metal nanomaterial is gold nanoparticles, stirred at room temperature for 2.5 h, and the mass ratio of polystyrene microspheres, ethanol, polypyrrole, carbon nanomaterials, metal nanomaterials and aminosilane is: 36:28:14:15:5:2; in S2, the ruthenium complex conjugated with the detection antibody is tris(2,2'-bipyridine)ruthenium(III) NHS ester conjugated with anti-PSA monoclonal antibody; in S4, 10 ng / mL of the standard target antigen PSA and anti-PSA capture antibody are added into the electrochemical reaction cell to form a capture antibody-antigen-detection antibody complex; in S5, triisopropanolamine is added into the electrochemical reaction cell and a voltage of 1.0 V is applied to trigger the luminescence reaction of the ruthenium complex.

[0038] Example 5: In S1, the aminosilane is 3-aminopropyltrimethoxysilane, and the metal nanomaterial is silver nanoparticles. The mixture is stirred at room temperature for 3 hours. The mass ratio of polystyrene microspheres, ethanol, polypyrrole, carbon nanomaterial, metal nanomaterial and aminosilane is 38:32:12:10:7:1; in S2, the ruthenium complex bound to the detection antibody is terpyridine ruthenium chloride hexahydrate bound to an anti-CEA monoclonal antibody; in S4, 50 ng / mL of the standard target antigen CEA and the anti-CEA capture antibody are added to the electrochemical reaction cell to form a capture antibody-antigen-detection antibody complex; in S5, tripropylamine is added to the electrochemical reaction cell and a voltage of 1.0 V is applied to trigger the luminescence reaction of the ruthenium complex.

[0039] Example 6: In S1, the aminosilane is selected from phenylaminomethyltrimethoxysilane, and the metal nanomaterial is selected from copper nanoparticles. The mixture is stirred at room temperature for 1.5 hours. The mass ratio of polystyrene microspheres, ethanol, polypyrrole, carbon nanomaterial, metal nanomaterial and aminosilane is 40:25:12:13:8:2; in S2, the ruthenium complex bound to the detection antibody is tris(4,4'-dimethyl-2,2'-bipyridyl)ruthenium hexafluorophosphate bound to the anti-AFP monoclonal antibody; in S4, 100 ng / mL of the standard target antigen AFP and the anti-AFP capture antibody are added to the electrochemical reaction cell to form a capture antibody-antigen-detection antibody complex; in S5, 2-dibutylaminoethanol is added to the electrochemical reaction cell and a voltage of 1.0 V is applied to trigger the luminescence reaction of the ruthenium complex.

[0040] Comparative Example 1: The electrochemiluminescence detection method comprises the following steps:

[0041] 1. Disperse polystyrene microspheres in ethanol, add 3-aminopropyltrimethoxysilane dropwise to the ethanol, then add carbon nanotubes and gold nanoparticles in sequence, stir at room temperature for 2 h, and dry to obtain composite polystyrene microspheres;

[0042] The mass ratio of polystyrene microspheres, ethanol, carbon nanotubes, gold nanoparticles, and 3-aminopropyltrimethoxysilane is 50:28:15:5:2;

[0043] 2. The terpyridine ruthenium bound to the anti-PSA monoclonal antibody was loaded onto the composite polystyrene microspheres through a biotin-streptavidin-mediated method;

[0044] 3. The composite polystyrene microspheres loaded with ruthenium complexes are drop-coated on the electrode surface, and the electrode is fixed in the electrochemical reaction cell after drying;

[0045] 4. Add 0.5 ng / mL standard target antigen PSA and anti-PSA capture antibody to the electrochemical reaction cell to form a capture antibody-antigen-detection antibody complex;

[0046] 5. Add tripropylamine into the electrochemical reaction cell and apply a voltage of 1.0 V to trigger the luminescence reaction of the ruthenium complex; detect the luminescence reaction and collect and collate the data.

[0047] Comparative Example 2: The electrochemiluminescence detection method comprises the following steps:

[0048] 1. Disperse polystyrene microspheres in ethanol, drop phenylaminomethyltrimethoxysilane into ethanol, and then sequentially add polypyrrole and copper nanoparticles, stir at room temperature for 1.5 h, and obtain composite polystyrene microspheres after drying;

[0049] The mass ratio of polystyrene microspheres, ethanol, polypyrrole, copper nanoparticles and phenylaminomethyltrimethoxysilane is: 44:30:15:8:3;

[0050] 2. Load tris(4,4'-dimethyl-2,2'-bipyridine) ruthenium hexafluorophosphate combined with anti-AFP monoclonal antibody onto the composite polystyrene microspheres by biotin-streptavidin mediation method;

[0051] 3. Drop the composite polystyrene microspheres loaded with ruthenium complex onto the electrode surface, and fix the electrode on the electrochemical reaction cell after drying;

[0052] 4. Add 5 ng / mL standard target antigen AFP and anti-AFP capture antibody into the electrochemical reaction cell to form a capture antibody-antigen-detection antibody complex;

[0053] 5. Add triethanolamine into the electrochemical reaction cell and apply a voltage of 1.0 V to trigger the luminescence reaction of the ruthenium complex; detect the luminescence reaction and collect and collate the data.

[0054] Comparative Example 3: The electrochemiluminescence detection method comprises the following steps:

[0055] 1. Disperse polystyrene microspheres in ethanol, drop 3-aminopropyltrimethoxysilane into ethanol, and then sequentially add polypyrrole and carbon nanotubes, stir at room temperature for 3 h, and obtain composite polystyrene microspheres after drying;

[0056] The mass ratio of polystyrene microspheres, ethanol, polypyrrole, carbon nanotubes and 3-aminopropyltrimethoxysilane is: 45:32:12:10:1;

[0057] 2. Load tris(2,2'-bipyridyl) ruthenium(III) chloride hexahydrate combined with anti-CEA monoclonal antibody onto the composite polystyrene microspheres by biotin-streptavidin mediation method;

[0058] 3. Drop the composite polystyrene microspheres loaded with ruthenium complex onto the electrode surface, and fix the electrode on the electrochemical reaction cell after drying;

[0059] 4. Add 50 ng / mL of the standard target antigen CEA and the anti-CEA capture antibody into the electrochemical reaction cell to form a capture antibody-antigen-detection antibody complex;

[0060] 5. Add tripropylamine into the electrochemical reaction cell and apply a voltage of 1.0 V to trigger the luminescence reaction of the ruthenium complex; Detect the luminescence reaction and collect and collate the data.

[0061] Comparative Example 4: The electrochemiluminescence detection method includes the following steps:

[0062] 1. Disperse polystyrene microspheres in ethanol, drop phenylaminomethyltrimethoxysilane into ethanol, stir at room temperature for 1.5 h, and obtain composite polystyrene microspheres after drying;

[0063] The mass ratio of polystyrene microspheres, ethanol, and amino silane is: 73:25:2;

[0064] 2. Load tris(4,4'-dimethyl-2,2'-bipyridine) ruthenium hexafluorophosphate conjugated with anti-AFP monoclonal antibody onto the composite polystyrene microspheres through the biotin-streptavidin mediated method;

[0065] 3. Drop the composite polystyrene microspheres loaded with the ruthenium complex onto the electrode surface, and fix the electrode on the electrochemical reaction cell after drying;

[0066] 4. Add 100 ng / mL of the standard target antigen AFP and the anti-AFP capture antibody into the electrochemical reaction cell to form a capture antibody-antigen-detection antibody complex;

[0067] 5. Add 2-dibutylaminoethanol into the electrochemical reaction cell and apply a voltage of 1.0 V to trigger the luminescence reaction of the ruthenium complex; Detect the luminescence reaction and collect and collate the data.

[0068] Perform performance tests on the above-mentioned examples and comparative examples. Refer to the standard YY / T 1175-2010, test and collate the data when the concentration of the detection antibody is 1 μg / mL and the concentration of the capture antibody is 2 μg / mL, and obtain the accuracy and coefficient of variation of the standard target antigen concentration. The specific results are as follows:

[0069] Table 1 Detection results of the electrochemiluminescence detection methods of the examples and comparative examples

[0070]

[0071] As can be seen from Table 1, all the data of Examples 1 to 6 are overall better than those of Comparative Examples 1 to 4. This is because in Examples 1 to 6, both polypyrrole and carbon nanomaterials have good electrical conductivity, and there is a structural synergistic effect among the π-π conjugate systems of polypyrrole, the π-π conjugate system of carbon nanomaterials, and the terpyridine ligand in the ruthenium complex. They are combined with each other through intermolecular π-π stacking, making the π-electron systems of the three components no longer isolated but forming a continuous electron transport network, thus greatly improving the electrical conductivity of the composite polystyrene microspheres, accelerating the transfer of electrons from the electrode to the ruthenium complex, reducing the response time of the electrochemical reaction of the ruthenium complex, and improving its sensitivity and accuracy. At the same time, through the surface plasmon resonance effect, the metal nanomaterials can amplify the electrochemiluminescence intensity of the ruthenium complex, further improving its sensitivity and accuracy. Finally, a large number of N-H bonds are contained in the polypyrrole structure of the composite polystyrene microspheres, which easily form hydrogen bonds with the nitrogen atoms in the ruthenium complex, thereby enhancing the binding between the ruthenium complex and the composite polystyrene microspheres and reducing the shedding situation. In summary, the components in the composite polystyrene microspheres cooperate with each other, making the electrochemiluminescence detection method and system provided by this application have high detection sensitivity and accuracy and good stability. In Comparative Example 1, polypyrrole is not contained, so it cannot produce a structural synergistic effect with the π-π conjugate system of carbon nanomaterials and the terpyridine ligand in the ruthenium complex; nor can it form hydrogen bonds with the nitrogen atoms in the ruthenium complex to enhance the binding between the ruthenium complex and the composite polystyrene microspheres; finally, the accuracy and coefficient of variation data are both poor. In Comparative Examples 2 and 3, carbon nanomaterials and metal nanomaterials are not contained respectively, but since polypyrrole is still contained, the overall accuracy and coefficient of variation are better than those of Comparative Example 1. In Comparative Example 4, neither polypyrrole nor carbon nanomaterials and metal nanomaterials are contained, so the accuracy and coefficient of variation are the worst.

[0072] As Figure 2 shown, the electrochemiluminescence detection system proposed by the present invention includes a sampling system 1, an electrochemiluminescence system 2 directly connected to the sampling system 1, an optical detection system 3 facing the electrochemiluminescence system 2, and a data acquisition and processing system 4 directly connected to the optical detection system 3.

[0073] In the electrochemiluminescence system 2, an electrochemical reaction cell is used as the electrochemical reaction container, and a mixed solution of sodium perchlorate solution and Tris-HCl buffer solution is used as the electrolyte. The pH range of the electrolyte is 7 to 9, the concentration of the sodium perchlorate solution is 0.1 to 1.0 mM, the concentration of the Tris-HCl buffer solution is 0.1 to 1.0 mM, and the mass ratio of the sodium perchlorate solution to the Tris-HCl buffer solution is (60 to 70):(30 to 40). A glassy carbon electrode is used as the working electrode, a platinum metal is used as the counter electrode, and an Ag / AgCl electrode is used as the reference electrode.

[0074] The above-disclosed are only the preferred embodiments of the present invention, and of course, the scope of the rights of the present invention cannot be limited thereby. It should be noted that for those skilled in the art of this technology, any equivalent changes made to the present invention without departing from the design structure and principle of the present invention shall be regarded as the protection scope of the present invention.

Claims

1. An electrochemiluminescence detection method, characterized in that, The electrochemiluminescence detection method includes the following steps: S1. Dispersing polystyrene microspheres in ethanol, dropping amino silane into the ethanol, and then successively adding polypyrrole, carbon nanomaterials and metal nanomaterials, stirring at room temperature for 1 - 3 h, and obtaining composite polystyrene microspheres after drying; S2. Loading ruthenium complex conjugated with detection antibody onto the composite polystyrene microspheres through biotin-streptavidin mediated method; S3. Drop-coating the composite polystyrene microspheres loaded with ruthenium complex onto the electrode surface, and fixing the dried electrode in the electrochemical reaction cell; S4. Adding standard target antigen and capture antibody against the standard target antigen into the electrochemical reaction cell to form a capture antibody-antigen-detection antibody complex; S5. Adding an electron donor into the electrochemical reaction cell and applying a voltage to trigger the luminescence reaction of the ruthenium complex on the electrode, detecting the luminescence reaction and collecting and sorting out data.

2. The electrochemiluminescence detection method according to claim 1, wherein The amino silane is any one of 3-aminopropyltrimethoxysilane, 3-aminopropyltriethoxysilane, phenylaminomethyltrimethoxysilane and phenylaminomethyltriethoxysilane.

3. The electrochemiluminescence detection method according to claim 1, wherein The carbon nanomaterials are any one of carbon nanotubes and graphene.

4. The electrochemiluminescence detection method according to claim 1, characterized in that, The metal nanomaterials are any one of gold nanoparticles, silver nanoparticles and copper nanoparticles.

5. The electrochemiluminescence detection method according to claim 1, characterized in that, The ruthenium complex is any one of tris(bipyridine)ruthenium(II), ruthenium(II) chloride hexahydrate tris(bipyridine), tris(4,4'-dimethyl-2,2'-bipyridine)ruthenium(II) hexafluorophosphate and tris(2,2'-bipyridine)ruthenium(II) NHS ester.

6. The electrochemiluminescence detection method according to claim 1, wherein The detection antibody is any one of anti-PSA monoclonal antibody, anti-CEA monoclonal antibody and anti-AFP monoclonal antibody.

7. The electrochemiluminescence detection method according to claim 6, wherein The capture antibody is anti-PSA capture antibody, anti-CEA capture antibody or anti-AFP capture antibody corresponding to the detection antibody.

8. The electrochemiluminescence detection method according to claim 7, wherein The standard target antigen is PSA, CEA or AFP corresponding to the detection antibody and the capture antibody.

9. The electrochemiluminescence detection method according to claim 1, wherein The electron donor is any one of tripropylamine, 2-dibutylaminoethanol, triethanolamine and triisopropanolamine.

10. An electrochemiluminescence detection system, characterized in that, The electrochemiluminescence detection system includes a sampling system, an electrochemiluminescence system directly connected to the sampling system, an optical detection system facing the electrochemiluminescence system, and a data acquisition and processing system directly connected to the optical detection system, and can implement the electrochemiluminescence detection method described in any one of claims 1 - 9.

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