A spectrally resolved highly monochromatic electrochemiluminescent immunosensor with a radiation band in the blue-green region

Abstract

The present invention relates to a spectrally resolved highly monochromatic electrochemiluminescent immunosensor with a radiation band located in the blue-green light region. The present invention first synthesizes a water-soluble gold nanocluster AuNCs with highly monochromatic electrochemiluminescent radiation, and constructs a spectrally resolved highly monochromatic electrochemiluminescent immunosensor with AuNCs as a marker. The half-peak width of the obtained sensor is 36nm, and the maximum radiation wavelength is 485nm, which is located in the blue-green light region, breaking through the limitation that the maximum radiation wavelength of the spectrally resolved electrochemiluminescent immunosensor with nanoparticles as markers is limited to more than 500nm, and enriching the band range of highly monochromatic ECL immunosensor.

Description

Technical Field

[0001] The invention relates to a spectrally resolved highly monochromatic electrochemiluminescence immunosensor with a radiation band in the blue-green light region, belonging to the field of electrochemiluminescence analysis technology methods. Background Art

[0002] Electrochemiluminescence (ECL) is a technology that combines chemiluminescence and electrochemiluminescence. It has the advantages of fast response, high sensitivity and easy control, and has been widely used in the field of immunoassay. ECL immunosensor is a biosensor that combines immunological methods with analytical chemistry methods. Through the specific binding between antigen and antibody, it has the advantages of high sensitivity, good selectivity, simple operation, easy miniaturization, continuous and rapid automated detection and analysis. Given that conventional commercial ECL equipment usually adopts a structural design that abandons the dispersion device and can only detect the total intensity of ECL radiation, ECL immunosensors are usually total light intensity measurement sensors. ROCHE uses ruthenium bipyridine as a marker and has developed a series of total light intensity measurement ECL immunosensors, monopolizing the in vitro diagnostic application of ECL.

[0003] Zhang used CdZnSe quantum dots as markers to construct the earliest spectrally resolved ECL immunosensor (Anal.Chem.2016,88,6947). The ECL radiation of CdZnSe quantum dots has significant dual-band characteristics, but poor monochromaticity, which limits the application of such spectrally resolved ECL sensors. Zhou found that dual-stabilizer-coated II-VI quantum dots can be used as markers to construct spectrally resolved monochrome single-band ECL immunosensors (Talanta,2017,165,117), and used three dual-stabilizer-coated II-VI quantum dots to construct spectrally resolved multicolor multicomponent ECL immunosensors (Anal.Chem.2018,90,12361), breaking through the limitation that total light intensity measurement ECL sensors can only perform single-index detection.

[0004] At present, the quantum dots that can be used to construct spectrally resolved single-color single-band ECL immunosensors are still relatively limited (Scientia Sinica Chimica, 2021, 51, 679), mainly including dual-stabilizer-coated CdSe (550nm) and CdSe quantum dots (600-780nm), dual-stabilizer-coated Co-CdTe quantum dots (815nm) and methionine-coated gold-silver bimetallic clusters (915nm). So far, only dual-stabilizer-coated CdSe quantum dots can be used to construct spectrally resolved ECL sensors with a half-peak width of less than 40nm (ChemElectroChem 2017, 4, 1714), and the radiation bands of spectrally resolved ECL immunosensors are all in the long-wave region of 550nm and above. There are no reports on spectrally resolved ECL immunosensors with a radiation band in the short-wave region and a maximum radiation wavelength of less than 500nm and related construction technologies.

[0005] Therefore, the development of spectrally resolved highly monochromatic ECL sensors with radiation bands in the shortwave region can further enrich ECL spectroscopy research, expand the information flux of spectrally resolved ECL sensing, and promote the advancement of spectrally resolved ECL immunosensing technology. Summary of the invention

[0006] In view of the shortcomings of the prior art, especially the limitation that the maximum radiation wavelength of spectrally resolved ECL immunosensors using nanoparticles as markers cannot be less than 500nm, one of the objects of the present invention is to provide a spectrally resolved highly monochromatic ECL sensor with a maximum radiation wavelength less than 500nm and a half-peak width less than 40nm. The maximum radiation wavelength of the obtained sensor is 485nm and the half-peak width is 36 nanometers, which breaks through the limitation that the maximum radiation wavelength of the reported spectrally resolved ECL immunosensors is only limited to above 500nm, and has the advantages of good specificity, wide detection range, good stability and low cost in detecting carcinoembryonic antigen.

[0007] The second object of the present invention is to provide a method for constructing the above-mentioned spectrally resolved highly monochromatic ECL immunosensor with a maximum radiation wavelength of less than 500 nm.

[0008] Terminology Note:

[0009] Primary antibody (CEA-Ab1): The primary antibody (Ab1) described in the present invention refers to the antibody corresponding to carcinoembryonic antigen (CEA). The present invention has a better effect on the monoclonal antibody corresponding to the above antigen.

[0010] Secondary antibody (CEA-Ab2): The secondary antibody described in the present invention refers to the corresponding secondary antibody of CEA antigen and primary antibody.

[0011] The present invention is achieved through the following technical solutions:

[0012] A highly monochromatic ECL immunosensor with spectral resolution and a radiation band in the blue-green region, wherein the maximum radiation wavelength of the ECL sensor is less than 500 nm and the half-peak width is less than 40 nm, and the ECL sensor comprises AuNCs-Ab2, CEA-Ab1 with blocked non-specific binding sites, and CEA connected to AuNCs-Ab2 and CEA-Ab1 respectively through antigen-antibody interaction;

[0013] The AuNCs-Ab2 is a second antibody corresponding to the antigen labeled with water-soluble gold nanoclusters AuNCs.

[0014] Preferably, according to the present invention, AuNCs are water-soluble gold nanoclusters AuNCs prepared by using chloroauric acid as a gold source, mercaptopropionic acid as a stabilizer, and zinc acetate as an aggregation inducer.

[0015] According to the preferred embodiment of the present invention, the synthesis steps of AuNCs are:

[0016] (1) Take 35.5 μL of 100 mg / mL HAuCl4·3H2O and add 2.5 mL of deionized water;

[0017] (2) Add 50 μL of mercaptopropionic acid to step (1) and stir for 15 min;

[0018] (3) Add 430 μL 1 M sodium hydroxide to step (2) and adjust the pH to 8.5;

[0019] (4) Add 0.5 mL of 0.1 M zinc acetate to step (3), stir and react at room temperature for 6 h. After the reaction is completed, wash and purify with isopropanol to obtain water-soluble gold nanoclusters AuNCs.

[0020] Preferably, according to the present invention, the synthesis steps of AuNCs-Ab2 are as follows:

[0021] 1) Activate the carboxylic acid groups on the surface of water-soluble gold nanoclusters AuNCs;

[0022] 2) allowing the secondary antibody to react with the carboxylic acid groups on the surface of the water-soluble gold nanoclusters AuNCs treated in step 1) to obtain the secondary antibody corresponding to the antigen labeled by the water-soluble gold nanoclusters AuNCs.

[0023] Preferably, according to the present invention, the specific preparation steps of AuNCs-Ab2 are as follows:

[0024] The purified AuNCs were dissolved in 1 mL of 0.1 M pH 6.0 phosphate buffer solution (PBS) containing 10 mg / mL 1-ethyl-(3-dimethylaminopropionic acid) carbodiimide hydrochloride (EDC) and 10 mg / mL hydroxysuccinimide (NHS), activated for 30 min, purified by centrifugation, and then dispersed in 1 mL of pH 7.4 0.1 M PBS to obtain activated AuNCs; 20 μL of 10 mg / mL secondary antibody CEA-Ab2) was added, incubated at 37°C for 3-5 h, 20 μL of bovine serum albumin (BSA) was added for blocking for 30 min, and the precipitate was collected by centrifugation to obtain AuNCs-Ab2.

[0025] Preferably, according to the present invention, the construction of the highly monochromatic electrochemiluminescent immunosensor with a maximum radiation wavelength of less than 500 nm and a half-peak width of less than 40 nm is carried out by the following steps:

[0026] (1) Using the cleaned and activated Au electrode as the working electrode, labeling the primary antibody CEA-Ab1 on the electrode surface to obtain the primary antibody labeled Au electrode;

[0027] (2) dissolving the purified AuNCs in a phosphate buffer solution containing EDC and NHS to activate the carboxylic acid groups on the surface of water-soluble gold nanoclusters AuNCs;

[0028] (3) adding a secondary antibody (CEA-Ab2) to the activated water-soluble gold nanoclusters AuNCs obtained in step (2), incubating at a constant temperature, adding bovine serum albumin (BSA) for blocking for 30 min, removing unconnected quantum dots and byproducts, and obtaining AuNCs-Ab2;

[0029] (4) adding carcinoembryonic antigen (CEA-Ag) to the surface of the Au electrode labeled with the primary antibody and incubating at room temperature, and then adding the AuNCs-Ab2 aqueous solution obtained in step (3) to the electrode surface and incubating; AuNCs-Ab2 is grafted and fixed to the working electrode surface through antigen-antibody interaction to obtain a highly monochromatic electrochemiluminescent immunosensor.

[0030] A preferred embodiment of the present invention:

[0031] The method for constructing a spectrally resolved highly monochromatic ECL sensor with a maximum radiation wavelength of less than 500 nm and a half-peak width of less than 40 nm is as follows:

[0032] a. Soak the clean Au electrode in 10 mM mercaptopropionic acid overnight to bond MPA to the electrode surface via Au-S bonds.

[0033] b. Add 10 μL of 10 mg / mL EDC and 10 mg / mL NHS to the surface of the modified electrode obtained in a, activate for 30 min, clean the electrode, and remove unreacted EDC and NHS;

[0034] c. 20 μL of 10 mg / mL CEA primary antibody (CEA-Ab1) aqueous solution was added dropwise to the activated electrode surface, incubated for 3 h, BSA was added to block the unreacted active sites on the electrode, and the electrode was washed;

[0035] d. The purified AuNCs were dissolved in 1 mL of 0.1 M pH 6.0 phosphate buffer solution (PBS) containing 10 mg / mL 1-ethyl-(3-dimethylaminopropionic acid) carbodiimide hydrochloride (EDC) and 10 mg / mL hydroxysuccinimide (NHS), activated for 30 min, purified by centrifugation, and then dispersed in 1 mL of pH 7.4 0.1 M PBS to obtain activated AuNCs; 20 μL of 10 mg / mL CEA-Ab2 aqueous solution was added, incubated at 37°C for 3-5 h, 20 μL of bovine serum albumin (BSA) was added for blocking for 30 min, and the precipitate was collected by centrifugation to obtain AuNCs-Ab2;

[0036] e. Carcinoembryonic antigen (CEA-Ag) was added to the electrode surface after treatment in c, incubated at room temperature for 90 minutes, the electrode was cleaned, and then 10 μL of AuNCs-Ab2 was added to the electrode surface and incubated for 1 hour; AuNCs-Ab2 was grafted and fixed to the working electrode surface based on the formation of immune complexes to achieve the preparation of highly monochromatic electrochemiluminescent immunosensor.

[0037] Preferably according to the present invention, the volume fraction of bovine serum albumin in step c and step d is 1%.

[0038] According to a preferred embodiment of the present invention, the flushing solution used for cleaning the electrodes in steps b, c and e is 10 mM pH=7.4 PBS.

[0039] Preferably according to the present invention, the concentration of carcinoembryonic antigen CEA is 0.3 pg / mL to 50 ng / mL.

[0040] Preferably according to the present invention, the carcinoembryonic antigen (CEA-Ag) is dripped onto the electrode surface in the form of an aqueous solution, and the concentration of the carcinoembryonic antigen CEA is 0.3 pg / mL to 50 ng / mL.

[0041] Preferably, according to the present invention, the AuNCs-Ab2 is added dropwise to the electrode surface in the form of an aqueous solution for incubation, and the concentration of AuNCs-Ab2 is 10-20 mg / mL; AuNCs-Ab2 should be sufficient to form an antigen-antibody interaction.

[0042] The method for antigen detection using the above-mentioned spectrally resolved highly monochromatic ECL immunosensor is as follows:

[0043] An ECL immunosensor containing the antigen with a known concentration is constructed, using Hepes as a buffer solution and hydrazine hydrate as a co-reactant. In a three-electrode system, an ECL spectral detection device is used to collect ECL spectral signals, and a linear relationship between the maximum ECL radiation intensity and the antigen concentration is established to obtain the concentration of the antigen to be tested.

[0044] Preferably, according to the present invention, in the above-mentioned three-electrode system, the constructed spectrally resolved highly monochromatic ECL immunosensor is the working electrode, the counter electrode is a platinum wire electrode, and the reference electrode is a saturated calomel electrode; the scanning voltage range is 0 to 1.6 V, the number of scanning circles is 1 to 3 circles, and the scanning speed is 40 to 60 mV / s.

[0045] Specifically, the method for antigen detection using the above-mentioned spectrally resolved highly monochromatic ECL sensor with a maximum radiation wavelength of less than 500nm and a half-peak width of less than 40nm is as follows:

[0046] I: preparing CEA-Ag aqueous solutions of different standard concentrations, using the CEA-Ag aqueous solutions of different standard concentrations, constructing a highly monochromatic ECL sensor according to the construction method of the ECL sensor, using the obtained sensor electrode as the working electrode, the platinum wire as the counter electrode, and the Ag / AgCl electrode as the reference electrode, in a Hepes buffer solution containing 5-20 mM hydrazine hydrate, using cyclic voltammetry to drive electrochemical luminescence;

[0047] II: Collect all photons in the whole ECL process by exposure imaging, and obtain the total spectrum by dispersing all photons radiated by ECL; draw a working curve based on the relationship between the maximum radiation intensity at the maximum radiation wavelength of 485nm on the spectrum curve and the concentration of the standard antigen solution;

[0048] III: A highly monochromatic ECL sensor is constructed using the CEA-Ag to be tested according to the construction method of the ECL sensor; the obtained sensor electrode is used as the working electrode, the platinum wire is used as the counter electrode, and the Ag / AgCl electrode is used as the reference electrode. In a Hepes buffer solution containing 5-20 mM hydrazine hydrate, cyclic voltammetry is used to drive the electrochemiluminescence to be generated; the concentration of the antigen in the sample solution to be tested is detected based on the light intensity signal at the maximum radiation wavelength on the obtained ECL spectrum curve and the working curve.

[0049] A spectrally resolved highly monochromatic ECL immunoassay method is used to detect human carcinoembryonic antigen, characterized in that it is carried out using the above-mentioned highly monochromatic electrochemiluminescence immunosensor.

[0050] The present invention uses mercaptopropionic acid-coated gold nanoclusters as ECL markers; the carboxyl groups on the surface of the gold nanoclusters can be grafted onto the amino groups on the surface of the second antibody after being activated by EDC and NHS, thereby achieving labeling of the second antibody.

[0051] The present invention grafts mercaptopropionic acid onto the surface of a gold electrode of a working electrode in a covalent bonding manner, and grafts the first antibody by further activating the carboxyl group of mercaptopropionic acid on the surface of the gold electrode using EDC and NHS.

[0052] The spectrally resolved highly monochromatic ECL immunosensor and detection method of the present invention are also applicable to alpha-fetoprotein antigen (AFP), carbohydrate antigen (CA125) or prostate-specific antigen (PSA), but have good specificity for carcinoembryonic antigen.

[0053] Beneficial effects of the present invention:

[0054] 1. The present invention can successfully prepare a spectrally resolved highly monochromatic electrochemiluminescence immunosensor with a radiation band in the blue-green light region, a maximum radiation wavelength of less than 500nm, and a half-peak width of less than 40nm. The maximum ECL radiation wavelength of the sensor is 485 nanometers, breaking through the limitation that the maximum radiation wavelength of the reported ECL immunosensor with nano-quantum dots as markers is limited to more than 500nm, and enriching the band range of monochromatic ECL immunoassay.

[0055] 2. The ECL radiation band of the sensor of the present invention does not overlap with the radiation band of the existing spectrally resolved ECL sensor. When combined with the existing spectrally resolved ECL multi-component immunoassay technology, it can further enrich the research content and detection index information of ECL multi-component immunosensor, and provide technical support for the development of high-throughput spectrally resolved multi-color and multi-component ECL immunoassay.

[0056] 3. The ECL immunosensor of the present invention is constructed based on the interaction between antigen and antibody, and has high sensitivity, good specificity, low detection limit, wide linear range, and simple preparation and operation. It adopts the principle of collecting all photons of ECL radiation and dispersing them into a spectrum to carry out immunoanalysis. The electrochemiluminescence signal intensity at the maximum radiation wavelength of 485nm spans more than 3 orders of magnitude, and the detected antigen concentration spans 4 orders of magnitude. It can sensitively detect carcinoembryonic antigen CEA, with a linear range of 1pg / mL to 50ng / mL and a detection limit of 0.3pg / mL. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 This is the infrared spectrum of the gold nanoclusters AuNCs prepared in the example.

[0058] Figure 2This is an electrochemiluminescence intensity-potential (time) curve of the gold nanoclusters AuNCs prepared in the example driven by cyclic voltammetry in 10 mM pH 7.4 Hepes containing 10 mM hydrazine hydrate; the potential window is 0-1.6 V, the scan rate is 50 mV / s; the abscissa is the potential, and the ordinate is the electrochemiluminescence intensity.

[0059] Figure 3 It is an electrochemiluminescence spectrum of the gold nanoclusters AuNCs prepared in the example driven by cyclic voltammetry in 10 mM pH 7.4 Hepes containing 10 mM hydrazine hydrate; the potential window is 0-1.6 V, the scan rate is 50 mV / s; the abscissa is the wavelength, and the ordinate is the electrochemiluminescence intensity.

[0060] Figure 4 This is the electrochemiluminescence spectrum of the gold nanocluster-labeled secondary antibody AuNCs-Ab2 prepared in Example 1 in 10 mM pH 7.4 Hepes containing 10 mM hydrazine hydrate driven by cyclic voltammetry; the potential window is 0-1.6 V, the scanning rate is 50 mV / s; the abscissa is the wavelength, and the ordinate is the electrochemiluminescence intensity.

[0061] Figure 5 This is the electrochemiluminescence spectrum of the electrochemiluminescence immunosensor using gold nanoclusters AuNCs as markers when the concentration of carcinoembryonic antigen CEA is 0.3 pg / mL in Example 3; the abscissa is the wavelength, and the ordinate is the electrochemiluminescence intensity.

[0062] Figure 6 This is the electrochemiluminescence spectrum of the electrochemiluminescence immunosensor using gold nanoclusters AuNCs as markers when the concentration of carcinoembryonic antigen CEA is 1 pg / mL in Example 3; the abscissa is the wavelength, and the ordinate is the electrochemiluminescence intensity.

[0063] Figure 7 This is the electrochemiluminescence spectrum of the electrochemiluminescence immunosensor using gold nanoclusters AuNCs as markers when the concentration of carcinoembryonic antigen CEA is 5 pg / mL in Example 3; the abscissa is the wavelength, and the ordinate is the electrochemiluminescence intensity.

[0064] Figure 8 This is the electrochemiluminescence spectrum of the electrochemiluminescence immunosensor using gold nanoclusters AuNCs as markers when the concentration of carcinoembryonic antigen CEA is 50 pg / mL in Example 3; the abscissa is the wavelength, and the ordinate is the electrochemiluminescence intensity.

[0065] Fig. 9 This is the electrochemiluminescence spectrum of the electrochemiluminescence immunosensor using gold nanoclusters AuNCs as markers when the concentration of carcinoembryonic antigen CEA is 500 pg / mL in Example 3; the abscissa is the wavelength, and the ordinate is the electrochemiluminescence intensity.

[0066] Fig.10 This is the electrochemiluminescence spectrum of the electrochemiluminescence immunosensor using gold nanoclusters AuNCs as markers when the concentration of carcinoembryonic antigen CEA is 3000 pg / mL in Example 3; the abscissa is the wavelength, and the ordinate is the electrochemiluminescence intensity.

[0067] Fig.11 This is the electrochemiluminescence spectrum of the electrochemiluminescence immunosensor using gold nanoclusters AuNCs as markers when the concentration of carcinoembryonic antigen CEA is 10000 pg / mL in Example 3; the abscissa is the wavelength, and the ordinate is the electrochemiluminescence intensity.

[0068] Fig.12 This is the electrochemiluminescence spectrum of the electrochemiluminescence immunosensor using gold nanoclusters AuNCs as markers when the concentration of carcinoembryonic antigen CEA is 50000 pg / mL in Example 3; the abscissa is the wavelength, and the ordinate is the electrochemiluminescence intensity.

[0069] Fig.13 This is the working curve of the immunosensor with gold nanoclusters AuNCs as markers at different concentrations of carcinoembryonic antigen CEA in Example 3; the abscissa is the antigen concentration of the test object, and the ordinate is the electrochemiluminescence intensity.

[0070] Fig.14 This is the specific electrochemiluminescence response diagram of the electrochemiluminescence immunosensor of Experimental Example 1 to the antigen; the abscissa is the type of the analyte, and the ordinate is the electrochemiluminescence intensity.

[0071] Fig.15 This is the electrochemiluminescence spectrum intensity change curve of the AuNCs-Ab2 immune complex stored for different times in 10 mM pH 7.4 Hepes containing 10 mM hydrazine hydrate in Experimental Example 2; the potential window is 0-1.6 V, the scanning rate is 50 mV / s; the horizontal axis is the storage time, and the vertical axis is the electrochemiluminescence intensity.

[0072] Fig.16 It is the electrochemiluminescence spectrum of the bare gold electrode in comparative example 1 driven by cyclic voltammetry in 10 mM pH 7.4 Hepes containing 10 mM hydrazine hydrate; the potential window is 0-1.6 V, the scan rate is 50 mV / s; the abscissa is the wavelength, and the ordinate is the electrochemiluminescence intensity.

[0073] Fig.17 This is the cyclic voltammogram of the bare gold electrode in Example 2 in 0.1M KCl containing 5mM K3Fe(CN)6 / K4Fe(CN)6; the potential window is -0.2 to 0.6V, the scan rate is 50mV / s; the abscissa is the potential, and the ordinate is the current.

[0074] Fig.18 It is the cyclic voltammogram of Au-MPA in 0.1 M KCl containing 5 mM K3Fe(CN)6 / K4Fe(CN)6 in Comparative Example 3; the potential window is -0.2 to 0.6 V, and the scanning rate is 50 mV / s; the abscissa is the potential and the ordinate is the current.

[0075] Fig.19 It is the cyclic voltammogram of Au-MPA-Ab1 in 0.1 M KCl containing 5 mM K3Fe(CN)6 / K4Fe(CN)6 in Comparative Example 4; the potential window is -0.2 to 0.6 V, and the scanning rate is 50 mV / s; the abscissa is the potential and the ordinate is the current.

[0076] Fig. 20 It is the cyclic voltammogram of Au-MPA-Ab1<Ag in 0.1 M KCl containing 5 mM K3Fe(CN)6 / K4Fe(CN)6 in Comparative Example 5; the potential window is -0.2 to 0.6 V, and the scanning rate is 50 mV / s; the abscissa is the potential and the ordinate is the current.

[0077] Fig.21 It is Au|MPA-Ab1 prepared when the carcinoembryonic antigen CEA concentration is 50 pg / mL in Comparative Example 6 <ag>Cyclic voltammogram of Ab2|AuNCs in 0.1M KCl containing 5mM K3Fe(CN)6 / K4Fe(CN)6; the potential window is -0.2~0.6V, the scan rate is 50mV / s; the horizontal axis is the potential and the vertical axis is the current.

[0078] Fig. 22 This is the electrochemiluminescence spectrum of the electrochemiluminescence immunosensor using gold nanoclusters AuNCs as markers when the concentration of carcinoembryonic antigen CEA prepared in Comparative Example 7 is 0; the abscissa is the wavelength, and the ordinate is the electrochemiluminescence intensity. DETAILED DESCRIPTION

[0079] The present invention is further illustrated by the following examples, but is not limited thereto.

[0080] In the embodiment, the electrochemiluminescence spectrum is collected using a detection system that can accurately collect electrochemiluminescence spectrum information in ZL201620300698.3. The electrochemiluminescence spectrum is collected by referring to the spectrum collection method of electrochemiluminescence immunoassay constructed in ZL201610237580.5, the potential window used is 0-1.6V, the scanning speed is 50mV / s, the starting potential is 0V, and the initial scan is positive.

[0081] Preparation of gold nanocluster AuNCs in the embodiment:

[0082] (1) Take 35.5 μL of 100 mg / mL HAuCl4·3H2O and add 2.5 mL of deionized water;

[0083] (2) Add 50 μL of mercaptopropionic acid to step (1) and stir for 15 min;

[0084] (3) Add 430 μL 1 M sodium hydroxide to step (2) and adjust the pH to 8.5;

[0085] (4) Add 0.5 mL of 0.1 M zinc acetate to step (3), stir and react at room temperature for 6 h. After the reaction is completed, wash and purify with isopropanol, and then dissolve the product in deionized water to obtain a monodisperse solution of gold nanoclusters AuNCs.

[0086] The infrared spectrum obtained is Figure 1 As shown by Figure 1 It can be seen that AuNCs contain sufficient carboxyl groups to realize the labeling process.

[0087] The prepared gold nanoclusters AuNCs were diluted to a monodisperse solution of 0.15 mg / mL.

[0088] A gold electrode was used as the working electrode, a platinum wire was used as the counter electrode, an Ag / AgCl electrode was used as the reference electrode, 10 mM Hepes at pH 7.4 was used as the buffer solution, 10 mM hydrazine hydrate was used as the co-reactant, and 0.15 mg / mL gold nanoclusters AuNCs were used as the luminescent reagent.

[0089] Cyclic voltammetry was used for driving, with a potential window of 0 to 1.6 V, a scan rate of 50 mV / s, a starting potential of 0 V, and an initial scan in the positive direction. The electrochemical luminescence intensity-(potential) time curve was obtained as shown in Figure 2 As shown, Figure 2 It can be seen that the gold nanocluster AuNCs / hydrazine hydrate system can produce oxidation-reduction electrochemiluminescence at 0.74V in 10mM pH 7.4 Hepes buffer.

[0090] Cyclic voltammetry was used for driving, with a potential window of 0 to 1.6 V, a scan rate of 50 mV / s, a starting potential of 0 V, and an initial scan in the positive direction. The electrochemical luminescence spectrum obtained is shown in the figure below. Figure 3 As shown, Figure 3 It can be seen that AuNCs can generate a highly monochromatic electrochemiluminescence signal with a maximum emission wavelength at 485 nm and a half-peak width of 36 nm in 10 mM pH 7.4 Hepes buffer containing hydrazine hydrate.

[0091] Example 1

[0092] The specific preparation steps of AuNCs-Ab2 are as follows:

[0093] The purified AuNCs were dissolved in 1 mL of 0.1 M pH 6.0 phosphate buffer solution (PBS) containing 10 mg / mL 1-ethyl-(3-dimethylaminopropionic acid) carbodiimide hydrochloride (EDC) and 10 mg / mL hydroxysuccinimide (NHS), activated for 30 min, purified by centrifugation, and then dispersed in 1 mL of pH 7.4 0.1 M PBS to obtain activated AuNCs; 20 μL of 10 mg / mL secondary antibody (CEA-Ab2) aqueous solution was added, incubated at 37°C for 3-5 h, 20 μL of bovine serum albumin (BSA) was added for blocking for 30 min, and the precipitate was collected by centrifugation to obtain AuNCs-Ab2; it was dispersed in 1 mL of 10 mM pH 7.4 PBS and stored at 4°C for future use.

[0094] A gold electrode was used as the working electrode, a platinum wire as the counter electrode, an Ag / AgCl electrode as the reference electrode, 10 mM pH 7.4 Hepes as the buffer solution, 10 mM hydrazine hydrate as the co-reactant, and 0.15 mg / mL AuNCs-Ab2 as the luminescent reagent.

[0095] Cyclic voltammetry was used for driving, with a potential window of 0 to 1.6 V, a scan rate of 50 mV / s, a starting potential of 0 V, and an initial scan in the positive direction. The electrochemical luminescence spectrum obtained is shown in the figure below. Figure 4 As shown, Figure 4 It can be seen that AuNCs-Ab2 can generate a highly monochromatic electrochemiluminescence signal with a maximum emission wavelength of 485nm and a half-peak width of 36nm in 10mM pH 7.4 Hepes buffer containing hydrazine hydrate. Although the electrochemiluminescence intensity is lower than that of AuNCs, it is attributed to the steric effect of the protein antibody, proving that the secondary antibody has been successfully grafted onto AuNCs.

[0096] Example 2

[0097] Construction of highly monochromatic electrochemiluminescent immunosensor with radiation wavelength below 500 nm:

[0098] a. Soak the clean Au electrode in 10 mM mercaptopropionic acid overnight, add 200 μL MPA and react for 10 h to bond MPA to the electrode surface through Au-S bonds.

[0099] b. Add 10 μL of 10 mg / mL EDC and 10 mg / mL NHS to the surface of the modified electrode obtained in a, activate for 30 min, and wash the electrode with 10 mM pH 7.4 PBS to remove unreacted EDC and NHS;

[0100] c. Add 20 μL of 10 mg / mL primary antibody (CEA-Ab1) to the surface of the activated electrode obtained in step b, incubate for 3 h, add BSA to block the unreacted active sites on the electrode, and wash the electrode with 10 mM pH 7.4 PBS;

[0101] e. 10 μL of Ag-labeled carcinoembryonic antigen (CEA-Ag) was added to the electrode surface after treatment in c, incubated at room temperature for 90 min, the electrode was washed with 10 mM pH 7.4 PBS, and then 10 μL of AuNCs-Ab2 prepared in Example 1 was added to the electrode surface and incubated for 1 h; AuNCs-Ab2 was grafted and fixed to the working electrode surface based on the formation of an immune complex to obtain a highly monochromatic electrochemiluminescent immunosensor.

[0102] Example 3

[0103] Detection of carcinoembryonic antigen using highly monochromatic electrochemiluminescent immunosensor:

[0104] I: Prepare CEA-Ag aqueous solutions of different standard concentrations, and use the CEA-Ag aqueous solutions of different standard concentrations to construct highly monochromatic ECL sensors according to the construction method of the ECL sensor described in Example 2. The CEA antigen concentrations are: 0.3pg / mL, 1pg / mL, 5pg / mL, 50pg / mL, 500pg / mL, 3000pg / mL, 10000pg / mL, 50000pg / mL, to obtain highly monochromatic electrochemiluminescence immunosensors of carcinoembryonic antigens of different concentrations; use the obtained sensor electrode as the working electrode, the platinum wire as the counter electrode, the Ag / AgCl electrode as the reference electrode, 10mM pH 7.4 Hepes as the buffer solution, and 10mM hydrazine hydrate as the co-reactant. Cyclic voltammetry is used for driving, with a potential window of 0-1.6V, a scan rate of 50mV / s, a starting potential of 0V, and an initial scan in the positive direction. The electrochemiluminescence spectra of different concentrations of antigens are as follows: Figure 5-12 As shown;

[0105] II: Collect all photons of the whole ECL process by exposure imaging, and obtain the total spectrum by dispersing all photons radiated by ECL; Draw the working curve based on the relationship between the maximum radiation intensity at the maximum radiation wavelength of 485nm on the spectrum curve and the concentration of the standard antigen solution; The working curve of the electrochemiluminescence immunosensor for antigen is as follows Fig.13 As shown in the figure, it can be seen that with the increase of antigen concentration, the electrochemiluminescence signal gradually increases, and the electrochemiluminescence intensity is proportional to the antigen concentration, indicating that the constructed electrochemiluminescence immunosensor has high sensitivity and great practical application potential;

[0106] III: A highly monochromatic ECL sensor is constructed using the CEA-Ag to be tested according to the construction method of the ECL sensor; the obtained sensor electrode is used as the working electrode, the platinum wire is used as the counter electrode, and the Ag / AgCl electrode is used as the reference electrode. In a Hepes buffer solution containing 5-20 mM hydrazine hydrate, cyclic voltammetry is used to drive the electrochemiluminescence to be generated; the concentration of the antigen in the sample solution to be tested is detected based on the light intensity signal at the maximum radiation wavelength on the obtained ECL spectrum curve and the working curve.

[0107] When the concentration of carcinoembryonic antigen CEA is 0.3 pg / mL, the electrochemiluminescence spectrum of the electrochemiluminescence immunosensor with gold nanoclusters AuNCs as markers is shown in Figure 2. Figure 5 As shown, Figure 5 It can be seen that the electrochemiluminescent immunosensor can generate a highly monochromatic electrochemiluminescent signal with a maximum emission wavelength at 485 nm and a half-peak width of 36 nm in a 10 mM pH 7.4 Hepes buffer containing hydrazine hydrate.

[0108] When the concentration of carcinoembryonic antigen CEA is 1 pg / mL, the electrochemiluminescence spectrum of the electrochemiluminescence immunosensor with gold nanoclusters AuNCs as markers is shown in Figure 2. Figure 6 As shown, Figure 6 It can be seen that the electrochemiluminescent immunosensor can generate a highly monochromatic electrochemiluminescent signal with a maximum emission wavelength at 485 nm and a half-peak width of 36 nm in a 10 mM pH 7.4 Hepes buffer containing hydrazine hydrate.

[0109] When the concentration of carcinoembryonic antigen CEA is 5 pg / mL, the electrochemiluminescence spectrum of the electrochemiluminescence immunosensor with gold nanoclusters AuNCs as markers is shown in Figure 7 As shown, Figure 7 It can be seen that the electrochemiluminescent immunosensor can generate a highly monochromatic electrochemiluminescent signal with a maximum emission wavelength at 485 nm and a half-peak width of 36 nm in a 10 mM pH 7.4 Hepes buffer containing hydrazine hydrate.

[0110] When the concentration of carcinoembryonic antigen CEA is 50 pg / mL, the electrochemiluminescence spectrum of the electrochemiluminescence immunosensor marked by gold nanoclusters AuNCs is shown in Figure 8 As shown, Figure 8 It can be seen that the electrochemiluminescent immunosensor can generate a highly monochromatic electrochemiluminescent signal with a maximum emission wavelength at 485 nm and a half-peak width of 36 nm in a 10 mM pH 7.4 Hepes buffer containing hydrazine hydrate.

[0111] When the concentration of carcinoembryonic antigen CEA is 500 pg / mL, the electrochemiluminescence spectrum of the electrochemiluminescence immunosensor with gold nanoclusters AuNCs as markers is shown in Figure 2. Fig. 9 As shown, Fig. 9 It can be seen that the electrochemiluminescent immunosensor can generate a highly monochromatic electrochemiluminescent signal with a maximum emission wavelength at 485 nm and a half-peak width of 36 nm in a 10 mM pH 7.4 Hepes buffer containing hydrazine hydrate.

[0112] When the concentration of carcinoembryonic antigen CEA is 3000pg / mL, the electrochemiluminescence spectrum of the electrochemiluminescence immunosensor with gold nanoclusters AuNCs as markers is shown in Fig.10 As shown, Fig.10 It can be seen that the electrochemiluminescent immunosensor can generate a highly monochromatic electrochemiluminescent signal with a maximum emission wavelength at 485 nm and a half-peak width of 36 nm in a 10 mM pH 7.4 Hepes buffer containing hydrazine hydrate.

[0113] When the concentration of carcinoembryonic antigen CEA is 10000 pg / mL, the electrochemiluminescence spectrum of the electrochemiluminescence immunosensor with gold nanoclusters AuNCs as markers is shown in Figure 2. Fig.11 As shown, Fig.11 It can be seen that the electrochemiluminescent immunosensor can generate a highly monochromatic electrochemiluminescent signal with a maximum emission wavelength at 485 nm and a half-peak width of 36 nm in a 10 mM pH 7.4 Hepes buffer containing hydrazine hydrate.

[0114] When the concentration of carcinoembryonic antigen CEA is 50000 pg / mL, the electrochemiluminescence spectrum of the electrochemiluminescence immunosensor with gold nanoclusters AuNCs as markers is shown in Figure 2. Fig.12 As shown, Fig.12 It can be seen that the electrochemiluminescent immunosensor can generate a highly monochromatic electrochemiluminescent signal with a maximum emission wavelength at 485 nm and a half-peak width of 36 nm in a 10 mM pH 7.4 Hepes buffer containing hydrazine hydrate.

[0115] The working curve of the electrochemiluminescence immunosensor is shown in Fig.13 As shown in the figure, it can be seen that with the increase of antigen concentration, the electrochemiluminescence signal gradually increases, proving the superior performance of the immunosensor.

[0116] Experimental Example 1

[0117] The same construction method as described in Example 2, except that:

[0118] Carcinoembryonic antigen (Ag(CEA)) was replaced by blank, alpha-fetoprotein antigen, prostate-specific antigen, and carbohydrate antigen 125.

[0119] The electrochemiluminescence responses of electrochemiluminescence immunosensors with different antigens are shown in Figure 2. Fig.14 As shown. Fig.14 As shown, the sensor prepared in this embodiment has good selectivity for carcinoembryonic antigen, and other antigen proteins do not interfere with the target antigen sensing detection of the invention, indicating that the electrochemiluminescence immunosensor has good specificity for carcinoembryonic antigen.

[0120] Experimental Example 2

[0121] The AuNCs-Ab2 immune complex obtained in Example 1 was stored at 4 degrees Celsius for 2, 4, 6, 8, 10, and 12 weeks, respectively.

[0122] Au electrode was used as working electrode, platinum wire as counter electrode, Ag / AgCl electrode as reference electrode, 10mM pH 7.4 Hepes as buffer solution, 10mM hydrazine hydrate as co-reactant, and 0.15mg / mL AuNCs-Ab2 immune complex placed for different time as luminescent material. Cyclic voltammetry was used for driving, with potential window of 0-1.6V, scanning speed of 50mV / s, starting potential of 0V, initial scanning towards positive, and the intensity change curve of electrochemiluminescence spectrum of AuNCs-Ab2 immune complex was obtained.

[0123] The electrochemiluminescence spectrum intensity change curve of AuNCs-Ab2 immune complex as the luminescent substance is shown in the figure below: Fig.15 As shown, Fig.15 It can be seen that the immune complex still has a high electrochemiluminescence intensity after being placed for 12 weeks, indicating that the AuNCs-Ab2 immune complex has excellent stability.

[0124] Comparative Example 1

[0125] A bare gold electrode was used as the working electrode, a platinum wire was used as the counter electrode, an Ag / AgCl electrode was used as the reference electrode, 10 mM Hepes at pH 7.4 was used as the buffer solution, and 10 mM hydrazine hydrate was used as the co-reactant.

[0126] Cyclic voltammetry was used for driving, with a potential window of 0 to 1.6 V, a scan rate of 50 mV / s, a starting potential of 0 V, and an initial scan in the positive direction. The electrochemical luminescence spectrum obtained is shown in the figure below. Fig.16 As shown, Fig.16 It can be seen that the bare gold electrode does not generate an electrochemiluminescence signal in a 10 mM pH 7.4 Hepes buffer containing hydrazine hydrate.

[0127] Comparative Example 2

[0128] The Au electrode was polished with alumina, washed with ultrapure water, and dried with nitrogen gas.

[0129] A gold electrode was used as the working electrode, a platinum wire as the counter electrode, an Ag / AgCl electrode as the reference electrode, and a 0.1 M KCl solution containing 5 mM K3Fe(CN)6 / K4Fe(CN)6 as the electrolyte.

[0130] Cyclic voltammetry was used, with a potential window of -0.2 to 0.6 V, a scan rate of 50 mV / s, a starting potential of -0.2 V, and an initial scan in the positive direction. The cyclic voltammetry curve of a bare gold electrode in potassium ferrocyanide is shown in FIG. Fig.17 As shown by Fig.17 It can be seen that the gold electrode shows a pair of Fe(CN)6 3- / Fe(CN)6 4- The reversible redox peak has a peak potential difference of 70 mV.

[0131] Comparative Example 3

[0132] The Au electrode was polished with alumina, washed with ultrapure water, dried with nitrogen, and immersed in 10 mM mercaptopropionic acid overnight. MPA was bonded to the electrode surface through Au-S bonds to obtain Au-MPA, providing carboxyl groups for fixing antibodies.

[0133] Au-MPA was used as the working electrode, platinum wire as the counter electrode, Ag / AgCl electrode as the reference electrode, and 0.1 M KCl solution containing 5 mM K3Fe(CN)6 / K4Fe(CN)6 as the electrolyte.

[0134] Cyclic voltammetry was used for driving, with a potential window of -0.2 to 0.6 V, a scan rate of 50 mV / s, a starting potential of -0.2 V, and an initial scan in the positive direction. The cyclic voltammetry curve of Au-MPA in potassium ferrocyanide is shown in FIG. Fig.18 As shown by Fig.18 It can be seen that the peak potential of the redox peak becomes larger, which is due to the 3- / Fe(CN)6 4- caused by the electrostatic repulsion between them.

[0135] Comparative Example 4

[0136] 1. After the Au electrode is polished with alumina, it is washed with ultrapure water, dried with nitrogen, and soaked in 10mM mercaptopropionic acid overnight. MPA is bonded to the electrode surface through Au-S bonds to obtain Au-MPA, providing carboxyl groups for fixing antibodies.

[0137] 2. Add 10 μL of 10 mg / mL EDC and 10 mg / mL NHS to the surface of the modified electrode Au-MPA, activate for 30 min, and wash the electrode with 10 mM pH 7.4 PBS to remove unreacted EDC and NHS.

[0138] 3. Add 20 μL of 10 mg / mL CEA primary antibody (CEA-Ab1) to the surface of the activated electrode and incubate for 3 h. Add BSA to block the unreacted active sites on the electrode and wash the electrode with 10 mM pH 7.4 PBS to obtain Au-MPA-Ab1.

[0139] Au-MPA-Ab1 was used as the working electrode, platinum wire as the counter electrode, Ag / AgCl electrode as the reference electrode, and 0.1 M KCl solution containing 5 mM K3Fe(CN)6 / K4Fe(CN)6 as the electrolyte.

[0140] Driven by cyclic voltammetry, the potential window is -0.2 to 0.6 V, the scanning rate is 50 mV / s, the initial potential is -0.2 V, and the initial scan is in the positive direction for scanning. The cyclic voltammogram of Au-MPA-Ab1 in potassium ferricyanide is as Fig.19 shown, and it can be seen from Fig.19 that the peak potential of the redox peak becomes relatively smaller, which is due to the restoration of the redox ability of Fe(CN)6 3- / Fe(CN)6 4- after connecting Ab1.

[0141] Comparative Example 5

[0142] 1. After polishing the Au electrode with alumina, it was washed with ultrapure water, dried with nitrogen, and soaked in 10 mM mercaptopropionic acid overnight. MPA was bonded to the electrode surface through Au-S bonds to obtain Au-MPA, providing carboxyl groups for antibody immobilization.

[0143] 2. 10 μL of 10 mg / mL EDC and 10 mg / mL NHS were dropped onto the surface of the modified electrode Au-MPA and activated for 30 min. The electrode was washed with 10 mM PBS at pH 7.4 to remove unreacted EDC and NHS.

[0144] 3. 20 μL of 10 mg / mL CEA primary antibody (CEA-Ab1) was dropped onto the surface of the activated electrode and incubated for 3 h. BSA was added to block the unreacted active sites on the electrode, and the electrode was washed with 10 mM PBS at pH 7.4 to obtain Au-MPA-Ab1.

[0145] 4. 50 pg / mL of antigen (Ag(CEA)) was dropped onto the surface of Au-MPA-Ab1 and incubated at room temperature for 90 min. The electrode was washed with 10 mM PBS at pH 7.4 to obtain Au-MPA-Ab1<Ag.

[0146] Using Au-MPA-Ab1<Ag as the working electrode, a platinum wire as the counter electrode, and an Ag / AgCl electrode as the reference electrode, a 0.1 M KCl solution containing 5 mM K3Fe(CN)6 / K4Fe(CN)6 was used as the electrolyte.

[0147] Driven by cyclic voltammetry, the potential window is -0.2 to 0.6 V, the scanning rate is 50 mV / s, the initial potential is -0.2 V, and the initial scan is in the positive direction for scanning. The cyclic voltammogram of Au-MPA-Ab1<Ag in potassium ferricyanide is as Fig. 20 shown, and it can be seen from Fig. 20 that the formation of the immune complex leads to a relatively larger peak potential of the redox peak.

[0148] Comparative Example 6

[0149] 1. After polishing the Au electrode with alumina, it was washed with ultrapure water, dried with nitrogen, and soaked overnight in 10 mM mercaptopropionic acid. MPA was bonded to the electrode surface through Au-S bonds to obtain Au-MPA, providing carboxyl groups for antibody immobilization.

[0150] 2. 10 μL of 10 mg / mL EDC and 10 mg / mL NHS were dropped onto the surface of the modified electrode Au-MPA and activated for 30 min. The electrode was washed with 10 mM PBS at pH 7.4 to remove unreacted EDC and NHS.

[0151] 3. 20 μL of 10 mg / mL CEA primary antibody (CEA-Ab1) was dropped onto the surface of the activated electrode and incubated for 3 h. BSA was added to block the unreacted active sites on the electrode, and the electrode was washed with 10 mM PBS at pH 7.4 to obtain Au-MPA-Ab1.

[0152] 4. 50 pg / mL of antigen (Ag(CEA)) was dropped onto the surface of Au-MPA-Ab1 and incubated at room temperature for 90 min. The electrode was washed with 10 mM PBS at pH 7.4 to obtain Au-MPA-Ab1<Ag.

[0153] 5. 10 μL of AuNCs-Ab2 was dropped onto the surface of Au-MPA-Ab1<Ag and incubated for 1 h. AuNCs-Ab2 was grafted and immobilized onto the surface of the working electrode in the form of an immune complex formation, and Au-MPA-Ab1 could be obtained. <ag>Ab2-AuNCs.

[0154] Au-MPA-Ab1 <ag>Ab2-AuNCs was used as the working electrode, platinum wire as the counter electrode, Ag / AgCl electrode as the reference electrode, and 0.1 M KCl solution containing 5 mM K3Fe(CN)6 / K4Fe(CN)6 as the electrolyte.

[0155] Cyclic voltammetry was used for driving, with a potential window of -0.2 to 0.6 V, a scan rate of 50 mV / s, a starting potential of -0.2 V, and an initial scan in the positive direction. Au-MPA-Ab1 <ag>The cyclic voltammetry curves of Ab2-AuNCs in potassium ferrocyanide are shown in Fig.21 As shown. Fig.21 It can be seen that the formation of immune complexes leads to a relative increase in the peak potential of the redox peak.

[0156] Comparative Example 7

[0157] The electrochemiluminescent immunosensor is constructed in the same manner as in Example 2, except that:

[0158] The antigen (Ag(CEA)) added on the surface of Au-MPA-Ab1 was removed to prepare the electrochemiluminescent immunosensor Au-MPA-Ab1 <ag>Ab2-AuNCs。

[0159] Using Au-|MPA-Ab1<Ag as the working electrode, a platinum wire as the counter electrode, and an Ag / AgCl electrode as the reference electrode, 10 mM Hepes with pH 7.4 as the buffer solution, and 10 mM hydrazine hydrate as the coreactant. Driven by cyclic voltammetry, the potential window is 0 to 1.6 V, the scanning speed is 50 mV / s, the initial potential is 0 V, and the initial scan is towards the positive to obtain the electrochemiluminescence spectra at different antigen concentrations.

[0160] When the concentration of carcinoembryonic antigen CEA is 0, the electrochemiluminescence spectrum of the electrochemiluminescence immunosensor using gold nanoclusters AuNCs as the label is as Fig. 22 shown, as Fig. 22 can be seen, this electrochemiluminescence immunosensor does not produce any electrochemiluminescence signal in 10 mM Hepes buffer solution with pH 7.4 containing hydrazine hydrate.< / ag> < / ag> < / ag> < / ag> < / ag>

Claims

1. A spectrally resolved highly monochromatic electrochemiluminescent immunosensor with a maximum radiation wavelength of 485 nanometers and a half-peak width of 36 nanometers, the electrochemiluminescent immunosensor comprising AuNCs-Ab2, CEA-Ab1 with blocked non-specific binding sites, and carcinoembryonic antigens connected to AuNCs-Ab2 and CEA-Ab1 respectively through antigen-antibody interactions; The AuNCs-Ab2 is a second antibody corresponding to the antigen labeled with water-soluble gold nanoclusters AuNCs; AuNCs are water-soluble gold nanoclusters prepared by using chloroauric acid as the gold source, mercaptopropionic acid as the stabilizer, and zinc acetate as the aggregation inducer. The synthesis steps of AuNCs are: (1) Take 35.5 μL of 100 mg / mL HAuCl4∙3H2O and add 2.5 mL of deionized water; (2) Add 50 μL of mercaptopropionic acid to step (1) and stir for 15 min; (3) Add 430 μL 1 M sodium hydroxide to step (2) and adjust the pH to 8.5; (4) Add 0.5 mL of 0.1 M zinc acetate to step (3), stir at room temperature for 6 h, and after the reaction is completed, wash and purify with isopropanol to obtain water-soluble gold nanoclusters AuNCs; The specific preparation steps of AuNCs-Ab2 are as follows: The purified AuNCs were dissolved in 1 mL of 0.1 M pH 6.0 phosphate buffer solution containing 10 mg / mL 1-ethyl-(3-dimethylaminopropionic acid) carbodiimide hydrochloride and 10 mg / mL hydroxysuccinimide, activated for 30 min, purified by centrifugation, and then dispersed in 1 mL of pH 7.4 0.1 M phosphate buffer solution to obtain activated AuNCs; 20 μL of 10 mg / mL CEA-labeled antibody CEA-Ab2 was added, incubated at 37 °C for 3-5 h, and 20 μL of bovine serum albumin was added for blocking for 30 min. The precipitate was collected by centrifugation to obtain AuNCs-Ab2; The method for constructing a spectrally resolved highly monochromatic electrochemiluminescent immunosensor with a maximum radiation wavelength of 485 nanometers and a half-peak width of 36 nanometers is as follows: a. Soak the cleaned Au electrode in 10 mM mercaptopropionic acid overnight to bond MPA to the electrode surface via Au-S bonds; b. Add 10 μL of 10 mg / mL 1-ethyl-(3-dimethylaminopropionic acid) carbodiimide hydrochloride and 10 mg / mL hydroxysuccinimide to the surface of the modified electrode obtained in a, activate for 30 min, clean the electrode to remove unreacted 1-ethyl-(3-dimethylaminopropionic acid) carbodiimide hydrochloride and hydroxysuccinimide; the rinse solution used to clean the electrode is a 10 mM pH = 7.4 phosphate buffer solution, and the concentration of carcinoembryonic antigen CEA is 0.3 pg / mL ~ 50 ng / mL; c. Add 20 μL of 10 mg / mL CEA primary antibody CEA-Ab1 to the activated electrode surface obtained in b, incubate for 3 h, add bovine serum albumin to block the unreacted active sites on the electrode, and wash the electrode; the volume fraction of bovine serum albumin is 1%, the rinse solution used to wash the electrode is 10 mM pH = 7.4 phosphate buffer solution, and the concentration of carcinoembryonic antigen CEA is 0.3 pg / mL ~ 50ng / mL; d. The purified AuNCs were dissolved in a phosphate buffer solution containing 1-ethyl-(3-dimethylaminopropionic acid) carbodiimide hydrochloride and hydroxysuccinimide for activation, and then CEA-labeled antibody CEA-Ab2 was added and incubated at 37°C for 3-5h. Bovine serum albumin was added for blocking for 30 min to obtain AuNCs-Ab2; the volume fraction of bovine serum albumin was 1%; e. Ag-labeled carcinoembryonic antigen CEA-Ag was added to the electrode surface after treatment in c, incubated at room temperature for 90 min, the electrode was cleaned, and then 10 μL AuNCs-Ab2 was added to the electrode surface and incubated for 1 h; AuNCs-Ab2 was grafted and fixed to the working electrode surface in the form of an immune complex to achieve the preparation of a highly monochromatic electrochemiluminescent immunosensor; the rinse solution used to clean the electrode was a 10 mM pH = 7.4 phosphate buffer solution, and the concentration of carcinoembryonic antigen CEA was 0.3 pg / mL ~ 50 ng / mL; The method for antigen detection using the highly monochromatic electrochemiluminescent immunosensor comprises the following steps: I: Using a highly monochromatic electrochemiluminescent immunosensor as the working electrode, a platinum electrode as the counter electrode, and an Ag / AgCl electrode as the reference electrode, cyclic voltammetry was used to drive the gold nanoclusters AuNCs fixed on the surface of the gold electrode to produce electrochemiluminescence in a Hepes buffer solution containing 10 mM hydrazine hydrate; II: Collect all photons of the whole electrochemiluminescence process by exposure imaging, and obtain the total spectrum by dispersing all photons of electrochemiluminescence radiation; draw the working curve according to the relationship between the light intensity at the maximum radiation wavelength on the spectrum curve and the concentration of the antigen standard solution; III: Perform an electrochemiluminescence spectrum test on the sample to be tested according to the method in steps I and II, and detect the antigen concentration in the sample solution according to the light intensity signal at the maximum radiation wavelength on the obtained electrochemiluminescence spectrum curve and the working curve.

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