An immunosensor using a carbon dot composite as a probe and a coreactant and its applications

Abstract

The present invention provides an immunosensor using a carbon dot composite as a probe and a coreactant, and a preparation method and an application thereof. By using dSiO₂ nanospheres with a large surface area to load a large amount of CDs, a CDs@dSiO₂ nanosphere composite material is prepared as a coreaction reagent for Ru(bpy)₃²⁺, so that the Ru(bpy)₃²⁺-CDs@dSiO₂ system has a significant enhancement in electrochemiluminescence. The present invention constructs an electrochemiluminescence immunosensor based on a carbon dot composite as a Ru(bpy)₃²⁺ coreactant and as a nanoprobe for immobilizing a secondary antibody, for the sensitive detection of human chorionic gonadotropin (HCG). The sensor has the characteristics of high sensitivity, strong specificity, good stability, low detection limit, wide linear range, and simple operation.

Description

Technical Field

[0001] The present invention relates to the field of electrochemiluminescence analysis, and particularly to an immunosensor using a carbon dot complex as a probe and a coreactant, and its application. Background Art

[0002] Currently, there are various immunoassay techniques, such as enzyme-linked immunosorbent assay, capillary electrophoresis combined with chemiluminescence, fluorescence method, electrochemistry method, colorimetry method, etc. Among these immunoassay techniques, electrochemiluminescence immunoassay has attracted much attention due to its high sensitivity and specificity, low detection limit, and rapid detection process.

[0003] The luminescent label is one of the key factors affecting the performance of ECL immunosensors and is crucial for achieving ultrasensitive detection of antigens. In numerous ECL studies, the most widely used ECL reagent to date is ruthenium tris(bipyridine) and its derivatives because they have stable luminescence, low dosage, transparent luminescence mechanism, recoverability, and adaptability at various pH values.

[0004] Although the best combination of luminophore / coreactant, the sensor applicability of Ru(bpy)3 2+ -TPrA is limited because TPrA is toxic, corrosive, and volatile. Therefore, great efforts have been made to use novel coreactants to improve the ECL intensity of Ru(bpy)3 2+

[0005] In recent years, it has been reported that carbon dots are used as coreactants for Ru(bpy)3 2+ This nanomaterial has good solubility, affordability, low toxicity, and strong chemical stability. They are also capable of generating uniform high-energy electron transfer reactions and luminescence of ECL emitters.

[0006] Based on this concept, whether the nanomaterial carbon dots can be added to the classical Ru(bpy)3 2+ electrochemiluminescence system to promote ECL emission and thus improve the sensitivity of immunosensors has become an urgent problem to be solved in this field. Summary of the Invention

[0007] Object of the Invention: Aiming at the deficiencies and defects of the prior art, the present invention provides an immunosensor using a carbon dot complex as a probe and a coreactant, and its preparation method and application. By using dSiO2 NPs with a large surface area to load a large amount of CDs, a CDs@dSiO2 NPs composite material is prepared as a coreaction reagent for Ru(bpy)3 2+ 2+- CDs@dSiO2 system has significant strong electrochemiluminescence. The present invention is based on carbon dot composites as Ru(bpy)3 2+ co-reactants and as nanoprobes for immobilizing the secondary antibody to construct an electrochemiluminescence immunosensor for sensitive detection of human chorionic gonadotropin (HCG). This sensor has the characteristics of high sensitivity, strong specificity, good stability, low detection limit, wide linear range, and simple operation.

[0008] Technical solution:

[0009] I. Aiming at the problems that the electrochemiluminescence intensity of a single ruthenium terpyridine is weak and the traditional co-reaction reagent tripropylamine is toxic, a co-reaction reagent CDs@dSiO2 NPs with good biocompatibility is provided. Due to the enrichment effect of dSiO2 NPs on CDs and the co-reaction promotion effect of CDs@dSiO2 NPs on the Ru(bpy)3 2+ reaction system, the electrochemiluminescence intensity of Ru(bpy)3 2+ is significantly improved.

[0010] The immunosensor of the carbon dot composite of the present invention as a probe and a co-reactant is characterized in that: the immunosensor includes a signal unit Ru(bpy)3 2+ , a co-reactant CDs@dSiO2-Ab2, a Au-Ab1 with non-specific binding sites blocked, and an antigen respectively connected to CDs@dSiO2-Ab2 and Au-Ab1 through antigen-antibody interaction; the CDs@dSiO2-Ab2 is the secondary antibody corresponding to the antigen labeled by CDs@dSiO2 NPs, and the CDs@dSiO2 NPs are dSiO2 NPs loaded with CDs; the Au-Ab1 is a first antibody Au with the antigen corresponding to the surface chemically modified.

[0011] Among them, the preparation method of the CDs@dSiO2-Ab2 includes the following steps:

[0012] 1) Activate the carboxylic acid groups on the surface of CDs@dSiO2 NPs;

[0013] 2) React the secondary antibody with the carboxylic acid groups on the surface and inside of the CDs@dSiO2 NPs treated in step 1) to obtain the secondary antibody corresponding to the antigen labeled by CDs@dSiO N Pss.

[0014] Among them, the preparation method of the CDs@dSiO2 NPs includes the following steps:

[0015] 1) Preparation of CDs: Pour 15 mL - 20 mL of AEAPTMS into a 50 mL three-necked flask. After purging with nitrogen for 10 min - 15 min, heat the solution to 240 °C, and add 0.8 g - 1 g of dehydrated citric acid under stirring. Stop heating after 5 min and let the solution cool naturally. Centrifuge the obtained CDs solution at 10000 rpm for 10 min to remove large particles at the bottom;

[0016] 2) Preparation of dSiO2 nanospheres (dSiO2 NPs): Add triethanolamine with a final concentration of 0.04 mg / mL - 8 mg / mL to water. After stirring for a certain time at 80 ± 5 °C, add cetyltrimethylammonium bromide with a final concentration of 4 mg / mL - 40 mg / mL and sodium salicylate with a final concentration of 0.4 mg / mL - 20 mg / mL, and stir for a certain time to obtain an intermediate solution. Inject tetraethyl orthosilicate into the intermediate solution, stir at 80 ± 5 °C for 0.5 h - 6 h, then dilute with ethanol and collect the product by centrifugation. Wash the precipitate with ethanol several times, and then disperse the precipitate in a HCl / ethanol mixture. Stir at 60 ± 5 °C for a certain time to extract the residual organic template. After repeated extraction, dendritic silica nanospheres are obtained. The particle size of the dSiO2 NPs is 230 nm - 250 nm, preferably 240 nm.

[0017] 3) Preparation of dSiO2 NPs loaded with CDs composite material (CDs@dSiO2 NPs): Add 150 μL - 200 μL of CDs to 50 mL - 60 mL of ethanol containing 50 mg - 60 mg of dendritic SiO2 spheres. After ultrasonic treatment for 5 min, add 1.25 mL - 1.5 mL of ammonia water and stir for 12 h. After centrifugation, wash the CDs@dSiO2 spheres with water and ethanol. Finally, disperse the amino-rich CDs@dSiO2 NPs in ethanol to obtain the final product.

[0018] Among them, the preparation method of the non-specific binding site-blocked Au-Ab1 includes the following steps:

[0019] 1) Immerse a clean electrode into a 1% chloroauric acid solution and electro-deposit at -0.25 V for 30 s to obtain Au NPs;

[0020] 2) React the first antibody with Au NPs and connect the first antibody to Au NPs through Au-S chemistry to obtain Au NPs modified with the first antibody on the surface;

[0021] 3) Use bovine serum albumin to block the non-specific binding sites of the Au NPs modified with the first antibody obtained in step 2) to obtain Au-Ab1 with blocked non-specific binding sites.

[0022] II. Preparation method of immunosensor using carbon dot complex as probe and coreactant, characterized by comprising the following steps:

[0023] 1) Add an aqueous solution of EDC to the dispersion of CDs@dSiO2 NPs to activate the carboxylic acid groups on the surface and inside of CDs@dSiO2 NPs, and obtain an activated dispersion of CDs@dSiO2 NPs;

[0024] 2) Add the secondary antibody to the activated dispersion of CDs@dSiO2 NPs obtained in step 1), react with shaking at 35°C - 37°C for 1.5 h - 2.5 h, and remove the unconnected secondary antibody and by-products by centrifugation to obtain CDs@dSiO2-Ab2;

[0025] 3) Immerse a clean glassy carbon electrode in a 1% chloroauric acid solution, electroplate at -0.25 V for 30 s to deposit Au NPs on the electrode surface;

[0026] 4) Make the primary antibody react with the Au NPs obtained in step 3) by the strong bonding action of the Au-S chemical bond, react at 35°C - 37°C for 1.5 h - 2.5 h, and wash with PBS to remove the unreacted primary antibody to obtain Au NPs modified with the primary antibody on the surface;

[0027] 5) Incubate the Au NPs modified with the primary antibody obtained in step 4) in a PBS buffer solution containing bovine serum albumin at 35°C - 37°C for 0.5 h - 1 h to block non-specific binding sites and obtain Au-Ab1 with non-specific binding sites blocked;

[0028] 6) Incubate the Au-Ab1 obtained in step 5) with the antigen at 35°C - 37°C for 1.5 h - 2.5 h, wash to remove the antigen that has not specifically bound to Au-Ab1, and incubate the obtained product with the CDs@dSiO2-Ab2 obtained in step 2) at 35°C - 37°C for 1.5 h - 2.5 h and then wash to obtain an immunosensor using the carbon dot complex as a probe and a coreactant.

[0029] Wherein, when preparing CDs@dSiO2-Ab2 in steps 1) and 2), take a certain amount of CDs@dSiO2 NPs and dissolve them in distilled water (the concentration of CDs@dSiO2 NPs is 2 mg / mL), weigh 0.05 mM of EDC and add it to the above solution, stir and activate for 0.5 h - 1 h; add 1 mL of the secondary antibody solution corresponding to the antigen to be detected (the concentration of the secondary antibody in the solution is 10 μg / mL), shake in the dark, react at 35°C - 37°C for 1.5 h - 2.5 h, and then remove the unconnected secondary antibody and by-products by centrifugation to obtain CDs@dSiO2-Ab2.

[0030] Among them, when preparing CDs@dSiO2 NPs in step 1), 150 μL of CDs was added to 50 mL of ethanol containing 50 mg of dendritic SiO2 spheres, ultrasonically treated for 5 min, then 1.25 mL of ammonia water was added and stirred for 12 h, centrifuged and washed with water and ethanol to obtain amino-rich CDs@dSiO2 NPs.

[0031] Among them, when preparing Au-Ab1 with blocked non-specific binding sites in step 5), the primary antibody corresponding to the antigen to be detected was connected to Au NPs through the bonding of Au-S chemical bonds. That is, the surface-cleaned glassy carbon electrode was fully immersed in 5 mL - 10 mL of 1% chloroauric acid solution, electro-deposited at -0.25 V for 30 s - 90 s, and then 6 μL - 8 μL of the primary antibody solution with a concentration of 10 μg / mL corresponding to the antigen to be detected was dropped onto the surface of the glassy carbon electrode deposited with Au NPs, and the mixture was incubated at 35 °C - 37 °C for 1.5 h - 2.5 h; the unreacted first antibody was removed by washing with 10 mM PBS with a pH of 7.4 to obtain Au NPs modified with the first antibody on the surface; the PBS solution containing BSA (100 μL - 120 μL, 1.0 wt%) was dropped onto the surface of the modified electrode at 35 °C - 37 °C and incubated for 0.5 h - 1 h to block non-specific binding sites, and after washing with PBS, Au-Ab1 with blocked non-specific binding sites was obtained, that is, Au / Ab1 / BSA, and stored at 4 °C for later use.

[0032] Among them, when preparing the immunosensor with carbon dot complex as the probe and co-reactant in step 6), 6 μL - 8 μL of the PBS solution containing the antigen was dropped onto the surface of the glassy carbon electrode modified with Au / Ab1 / BSA, and incubated at 35 °C - 37 °C for 1.5 h - 2.5 h; the antigen-antibody complex obtained was washed with 1 mL - 2 mL of 10 mM PBS with a pH of 7.4 to remove physically adsorbed antigen; CDs@dSiO2-Ab2 was dropped onto the surface of the glassy carbon electrode modified with the antigen-antibody complex, mixed and incubated at 35 °C - 37 °C for 1.5 h - 2.5 h to construct a sandwich-type immunosensor; the obtained immunosensor was washed with PBS to remove unbound CDs@dSiO2-Ab2, and stored at 4 °C for later use.

[0033] III. Application of the immunosensor with carbon dot complex as the probe and co-reactant in detecting antigens. Based on CDs@dSiO2 NPs, a Ru(bpy)3 2+ -CDs@dSiO2 NPs system can collect strong and stable anodic electrochemiluminescence signals of Ru(bpy)3 2+

[0034] ​Among them, a three-electrode system is adopted for antigen detection, including a working electrode, a counter electrode, and a reference electrode; using the synthesized CDs@dSiO2 NPs as a co-reactant reagent, the electrochemiluminescence signal is collected by an electrochemiluminescence analyzer.

[0035] Among them, the antigen detection includes the following steps:

[0036] 1) Place the working electrode polished on suede containing alumina powder in ethanol and double-distilled water for ultrasonic washing.

[0037] 2) Drop the immunosensor solution onto the surface of the washed working electrode and dry it naturally at room temperature.

[0038] 3) Prepare a PBS buffer solution containing Ru(bpy)3 2+ as the electrolyte of the three-electrode system.

[0039] 4) Set the potential window to 0 - 1.5V, the scan rate to 100mV / s, the high voltage of the photomultiplier tube to 800V, use the electrochemiluminescence analyzer to collect the electrochemiluminescence signal, establish the linear relationship between the electrochemiluminescence intensity and different concentrations of antigens, and obtain the antigen detection result.

[0040] IV. Application of the immunosensor with carbon dot complex as a probe and co-reactant in detecting antigen concentration.

[0041] Among them, the antigen concentration detection includes the following steps:

[0042] 1) Construct an electrochemiluminescence immunosensor containing different known concentrations of human chorionic gonadotropin (HCG) as the electrochemiluminescence signal substance of Ru(bpy)3 2+ In the three-electrode system, use the electrochemiluminescence analyzer to collect the electrochemiluminescence signal, set the potential window at 0 - 1.5V, and establish the linear relationship between the electrochemiluminescence intensity and the HCG concentration. The linear equation is I = 2396.16lg(C HCG ) + 8916.63, and the linear correlation coefficient R is 0.99.

[0043] 2) By replacing the first antibody and the second antibody, the detection of different cancer markers can be achieved; construct the electrochemiluminescence immunosensor containing different known concentrations of different cancer markers, with Ru(bpy)3 2+ as the electrochemiluminescence signal substance. In the three-electrode system, use the electrochemiluminescence analyzer to collect the electrochemiluminescence signal, set the potential window at 0 - 1.5V, and establish the linear relationship between the electrochemiluminescence intensity and the concentration of different cancer markers.

[0044] Among them, in the three-electrode system of step 1), the working electrode is a glassy carbon electrode modified with an electrochemiluminescence immunosensor, the counter electrode is a platinum wire electrode, and the reference electrode is a saturated calomel electrode; the high voltage of the photomultiplier tube of the electrochemiluminescence analyzer is set to 800 V, the applied potential is 0 - 1.5 V, and the scanning rate is 100 mV / s; with Ru(bpy)3 2+ As the electrochemiluminescence signal substance, the specific method for collecting the electrochemiluminescence signal using an electrochemiluminescence analyzer in the three-electrode system is: in a PBS buffer solution with a pH of 7 - 8 containing 50 μM of Ru(bpy)3 2+ to detect the electrochemiluminescence response of the antigen through an electrochemiluminescence analyzer.

[0045] Beneficial effects: Compared with the prior art, the present invention has the following remarkable advantages:

[0046] 1. The present invention loads CDs onto the surface and inside of dSiO2 nanospheres to prepare a CDs@dSiO2 NPs composite material. Due to the enrichment effect of dSiO2 NPs with a large specific surface area on CDs and the co-reaction effect of CDs@dSiO2 NPs on the Ru(bpy)3 2+ system, the electrochemiluminescence intensity of the Ru(bpy)3 2+ material is significantly improved.

[0047] 2. The present invention uses the CDs@dSiO2 NPs composite material as a co-reactant for the classic Ru(bpy)3 2+ to construct a signal-amplified immunosensor, significantly improving the sensitivity of antigen detection.

[0048] 3. The present invention prepares Au NPs and uses them as a carrier for the electrochemiluminescence immunosensor, which is conducive to fixing a large amount of antibodies on its surface and helps in the construction and implementation of the immunosensor.

[0049] 4. The present invention constructs an electrochemiluminescence immunosensor based on the interaction between antigen and antibody. This sensor has the characteristics of high sensitivity, strong specificity, good stability, low detection limit, wide linear range, and simple operation.

[0050] 5. The signal-amplified electrochemiluminescence immunosensor prepared by the present invention has a detection range for human chorionic gonadotropin (HCG) of 0.0005 mIU / mL - 500 mIU / mL, and a detection limit of 0.00019 mIU / mL. Description of the Drawings

[0051] Figure 1 is a schematic diagram of the preparation process of the immunosensor of the present invention;

[0052] Figure 2 TEM images of dSiO2 NPs of the present invention at a lower magnification and the corresponding particle size distribution diagram;

[0053] Figure 3 TEM images of dSiO2 NPs and CDs@dSiO2 NPs of the present invention; A in the figure is the TEM image of dSiO2 NPs; B in the figure is the TEM image of CDs@dSiO2 NPs;

[0054] Figure 4 CDs@dSiO2 NPs, Ru(bpy)3 of the present invention 2+ ,Ru(bpy)3 2+ -ECL diagram of CDs@dSiO2;

[0055] Figure 5 Electrochemiluminescence signal diagram of the immunosensor under different concentrations of HCG of the present invention;

[0056] Figure 6 Linear relationship diagram between the electrochemiluminescence intensity and the logarithm of the HCG concentration of the present invention. Detailed implementation manners

[0057] The technical solutions of the present invention will be further described below in conjunction with the accompanying drawings and specific implementation manners.

[0058] Reagents and instruments used in the present invention: N-[3-(trimethoxysilyl)propyl]ethylenediamine (AEAPTMS), anhydrous citric acid, tetraethyl orthosilicate (TEOS), N,N-dimethylformamide (DMF) and tris(2,2-bipyridine)ruthenium(II) dichloride hexahydrate ([Ru(bpy)3] 2+ ) were purchased from Sigma-Aldrich. Cetyltrimethylammonium bromide (CTAB), hydrochloric acid (HCl), ethanol, ammonia water (chloroauric acid (HAuCl4·4H2O), sodium hydroxide and succinic anhydride were all provided by Sinopharm Chemical Reagent Co., Ltd. N-hydroxysuccinimide (NHS) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) were purchased from AlfaAesar Chemicals Co., Ltd. Bovine serum albumin (BSA) was purchased from Sangon Biotech Co., Ltd. HCG antigen (Ag), HCG primary antibody (Ab1) and HCG secondary antibody (Ab2) were provided by Shanghai Jieyi Biotechnology Co., Ltd. (Shanghai, China). Phosphate buffer solution (PBS, 0.1 M, pH = 7.4) was freshly prepared before use. Chemicals were of analytical grade, purchased directly without further purification, and double-distilled water was used throughout. The MPI-E multi-functional electrochemical and chemiluminescence analysis system was purchased from Xi'an Ruimai Analytical Instrument Co., Ltd.

[0059] Example 1:

[0060] Prepare the second antibody (CDs@dSiO2-Ab2) corresponding to the antigen labeled with dSiO2 nanospheres loaded with CDs dots, including the following steps:

[0061] 1) Prepare CDs:

[0062] Pour 15 mL of AEAPTMS into a 50 mL three-necked flask and purge with nitrogen for 15 min. Then heat the solution to 240 °C and quickly add 1 g of anhydrous citric acid under vigorous stirring. After 5 min, stop heating and let the solution cool naturally. Centrifuge the obtained CDs solution at 10000 rpm for 10 min to remove large particles at the bottom.

[0063] 2) Prepare dSiO2 nanospheres (dSiO2 NPs):

[0064] Add 68 mg of triethanolamine (TEA) to 25 mL of H2O, stir at 80 °C for 30 min, then add 380 mg of cetyltrimethylammonium bromide (CTAB) and 168 mg of sodium salicylate and stir for 1 h. Inject 4 mL of TEOS into the solution and gently stir at 80 °C for 2 h. Dilute the mixture with ethanol and collect by centrifugation. Wash the precipitate with ethanol several times and finally disperse it in a HCl / ethanol mixture and stir at 60 °C for 24 h to extract the residual organic template. Wash the dendritic SiO2 spheres with ethanol and H2O and finally disperse them in ethanol. The TEM images of dSiO2 NPs at lower magnifications and the corresponding particle size distributions are shown in Figure 2 .

[0065] 3) Preparation of dSiO2 nanoloaded CDs composite material (CDs@dSiO2 NPs):

[0066] Add 150 μL of CDs to 50 mL of ethanol containing 50 mg of dendritic SiO2 spheres, sonicate for 5 min, then add ammonia water (1.25 mL) and stir at room temperature for 12 h. Then centrifuge and wash the CDs@dSiO2 NPs spheres with water and ethanol, and finally disperse the amino-rich CDs / dSiO2 NPs in ethanol. The electron microscopy characterization diagrams of dSiO2 NPs and CDs@dSiO2 NPs are shown in Figure 3 .

[0067] 4) Preparation of CDs@dSiO2-Ab2:

[0068] Disperse 5 mg of amino-rich CDs@dSiO2 NPs spheres in 5 mL of DMF, add 25 mg of succinic anhydride and stir for 2 h for carboxylation. After washing three times with H2O, disperse it in 2.5 mL of H2O for further use. Then, couple the carboxyl-rich CDs@dSiO2 NPs spheres with HCG secondary antibody. Briefly, sequentially mix the carboxyl-rich CDs@dSiO2 NPs spheres (500 μL, 2 mg / mL), freshly prepared aqueous EDC solution (30 μL, 10 mg / mL), and HCG-Ab2 antibody (1 mL, 10 μg / mL), and then gently shake at room temperature for 3 h. Centrifuge to collect the CDs@dSiO2-Ab2 conjugate, disperse it in 1 mL of PBS (0.01 M, pH = 7.4, 1% BSA), and store at 4 °C. The enhancement mechanism of electrochemiluminescence of CDs / dSiO2 NPs with Ru(bpy)3 2+ is shown in Figure 2 . Example 2:

[0069] Preparation of the carrier Au / Ab1 / BSA includes the following steps:

[0070] 1) Preparation of Au NPs:

[0071] Ultrasonically wash a glassy carbon electrode (GCE) polished on suede containing 0.3 μm and 0.05 μm alumina powder in ethanol and secondary distilled water; fully immerse the cleaned glassy carbon electrode into a 1% chloroauric acid solution and perform electrodeposition at a voltage of -0.25 V for 30 s, that is, deposit Au NPs on the surface of the glassy carbon electrode.

[0072] 2) Preparation of Au-Ab1:

[0073] Link the primary antibody corresponding to the antigen to be detected to Au NPs through the bonding of Au-S chemical bonds. Briefly, carefully drop 6 μL of the primary antibody solution with a concentration of 10 μg / mL corresponding to the antigen to be detected onto the surface of the glassy carbon electrode deposited with Au NPs, and incubate at 37 °C for 2 h. Wash with 10 mM PBS (pH 7.4) to remove the unreacted first antibody, and obtain Au NPs with the first antibody modified on the surface (Au-Ab1).

[0074] 3) Preparation of Au-Ab1 with non-specific binding sites blocked:

[0075] Incubate Au-Ab1 in a PBS solution containing BSA (100 μL, 1.0 wt%) at 37 °C for 1 h to block non-specific binding sites, wash with PBS, and obtain Au-Ab1 with non-specific binding sites blocked, that is, Au / Ab1 / BSA, and store at 4 °C for later use.

[0076] Example 3:

[0077] Sandwich-type electrochemiluminescence immunosensor based on carbon dots as nanoprobes and coreactants:

[0078] 6 μL of PBS solution containing different concentrations of HCG (concentrations are 0.0005 mIU / mL, 0.001 mIU / mL, 0.01 mIU / mL, 0.1 mIU / mL, 1 mIU / mL, 10 mIU / mL, 100 mIU / mL, 200 mIU / mL, 500 mIU / mL) was dropped onto the surface of the glassy carbon electrode modified with Au-Ab1 and incubated at 37 °C for 2 h. The obtained antigen-antibody complex was carefully washed with 10 mM PBS (pH 7.4) to remove physically adsorbed antigen. Then, the CDs@dSiO2-Ab2 prepared in Example 1 was dropped onto the surface of the electrode incubated with the Ag-Ab1 complex and incubated at 37 °C for 2 h to construct a sandwich-type immunosensor. Finally, the obtained immunosensor was washed with PBS to remove unbound CDs@dSiO2-Ab2 and stored at 4 °C for later use. The schematic diagram of the preparation of this immunosensor is shown in Figure 1 .

[0079] Example 4:

[0080] Detection of different concentrations of HCG by a sandwich-type electrochemiluminescence immunosensor based on carbon dots as nanoprobes and coreactants:

[0081] The detection was carried out using a three-electrode system. The working electrode was the glassy carbon electrode modified with the electrochemiluminescence immunosensor, the counter electrode was a platinum wire electrode, and the reference electrode was a saturated calomel electrode; 50 μM of Ru(bpy)3 2+ was used as the electrochemiluminescence reagent. The high voltage of the photomultiplier tube of the electrochemiluminescence analyzer was set to 800 V, the scanning rate was 100 mV / s, and the potential window was set to 0 - 1.5 V. The electrochemiluminescence signal was collected by the electrochemiluminescence analyzer. The specific steps were as follows: (1) The glassy carbon electrode (GCE) polished on suede containing 0.3 μm and 0.05 μm alumina powder was ultrasonically washed in ethanol and secondary distilled water; (2) The biosensor prepared in Example 3 above was naturally dried at room temperature; (3) A 0.1 M PBS buffer solution containing 50 μM of Ru(bpy)3 2+ was configured as the electrolyte of the three-electrode system. (4) The potential window was set to 0 - 1.5 V, the scanning rate was set to 100 mV / s, and the high voltage of the photomultiplier tube was set to 800 V. The electrochemiluminescence signal was collected by the electrochemiluminescence analyzer to obtain the ECL response of the immunosensor under different concentrations of HCG, as shown in Figure 4 shown, Figure 4The concentrations of HCG were 0.0005 mIU / mL, 0.001 mIU / mL, 0.01 mIU / mL, 0.1 mIU / mL, 1 mIU / mL, 10 mIU / mL, 100 mIU / mL, 200 mIU / mL, and 500 mIU / mL respectively; the test was carried out in 0.1 M PBS buffer containing 50 μM of Ru(bpy)3 2+ . The linear relationship diagram of electrochemiluminescence signal and HCG is shown in Figure 5 , where the linear equation is I = 8916.63 + 2396.16 lg(C HCG ). The linear correlation coefficient R is 0.99, and the detection limit is 0.00019 mIU / mL.

[0082] Effect analysis: In the present invention, CDs were loaded onto the surface and inside of dSiO2 nanospheres to prepare a CDs@dSiO2 NPs composite material. Due to the enrichment effect of dSiO2 NPs with a large specific surface area on CDs and the co-reactant effect of CDs@dSiO2 NPs on the Ru(bpy)3 2+ system, the electrochemiluminescence intensity of the Ru(bpy)3 2+ material was significantly improved. In the present invention, the CDs@dSiO2 NPs composite material was used as a co-reactant for the classical Ru(bpy)3 2+ to construct a signal-amplified immunosensor, which significantly improved the sensitivity of antigen detection.

[0083] In the present invention, Au nanomaterials were prepared and used as carriers for electrochemiluminescence immunosensors, which was beneficial to immobilize a large number of antibodies on their surfaces and contributed to the construction and implementation of immunosensors. In the present invention, an electrochemiluminescence immunosensor was constructed based on the interaction between antigen and antibody. The sensor has the characteristics of high sensitivity, strong specificity, good stability, low detection limit, wide linear range, and simple operation. The detection range of the signal-amplified electrochemiluminescence immunosensor prepared in the present invention for human chorionic gonadotropin (HCG) is 0.0005 mIU / mL - 500 mIU / mL, and the detection limit is 0.00019 mIU / mL.

Claims

1. An immunosensor using a carbon dot complex as a probe and a co-reactant, characterized in that: The immunosensor includes a signal unit Ru(bpy)3 2+ , a coreactant CDs@dSiO2-Ab2, Au-Ab1 with blocked non-specific binding sites, and an antigen respectively connected to CDs@dSiO2-Ab2 and Au-Ab1 through antigen-antibody interactions; the CDs@dSiO2-Ab2 is the second antibody corresponding to the antigen labeled with CDs@dSiO2 NPs, and the CDs@dSiO2 NPs are dSiO2 NPs loaded with CDs; the Au-Ab1 is Au NPs with the first antibody corresponding to the antigen surface chemically modified.

2. The immunosensor using the carbon dot composite as a probe and a coreactant according to claim 1, wherein: The preparation method of the described CDs@dSiO2-Ab2 includes the following steps: 1) Activate the carboxylic acid groups on the surface of CDs@dSiO2 NPs; 2) React the second antibody with the carboxylic acid groups on the surface and inside of the CDs@dSiO2 NPs treated in step 1) to obtain the second antibody corresponding to the antigen labeled with CDs@dSiO2 NPs.

3. The immunosensor using the carbon dot complex according to claim 1 or 2 as a probe and a co-reactant, characterized in that: The particle size of the described CDs@dSiO2 NPs is 230nm - 250nm.

4. The immunosensor using the carbon dot complex according to claim 1 as a probe and a coreactant, characterized in that: The preparation method of the Au-Ab1 with non-specific binding sites blocked includes the following steps: 1) Immerse the clean electrode in a 1% chloroauric acid solution and electro-deposit for 30s at -0.25V to obtain Au NPs; 2) React the first antibody with Au NPs and connect the first antibody to Au NPs through Au-S chemical bonds to obtain Au NPs modified with the first antibody on the surface; 3) Use bovine serum albumin to block the non-specific binding sites of the Au NPs modified with the first antibody obtained in step 2) to obtain Au-Ab1 with non-specific binding sites blocked.

5. Preparation method of immunosensor using carbon dot complex as probe and coreactant, characterized in that: Including the following steps: 1) Add an aqueous solution of EDC to the dispersion of CDs@dSiO2 NPs to activate the carboxylic acid groups on the surface and inside of CDs@dSiO2 NPs to obtain an activated dispersion of CDs@dSiO2 NPs; 2) Add the second antibody to the activated dispersion of CDs@dSiO2 NPs obtained in step 1), react with oscillation at 35°C - 37°C for 1.5h - 2.5h, and remove the unconnected second antibody and by-products by centrifugation to obtain CDs@dSiO2-Ab2; 3) Immerse the clean glassy carbon electrode in a 1% chloroauric acid solution and electro-deposit for 30s at -0.25V to coat Au NPs on the electrode surface; 4) React the first antibody with the Au NPs obtained in step 3) using the strong bonding effect of Au-S chemical bonds, react at 35°C - 37°C for 1.5h - 2.5h, and wash with PBS to remove the unreacted first antibody to obtain Au NPs modified with the first antibody on the surface; 5) Incubate the Au NPs modified with the first antibody obtained in step 4) in a PBS buffer solution containing bovine serum albumin at 35°C - 37°C for 0.5h - 1h to block the non-specific binding sites to obtain Au-Ab1 with non-specific binding sites blocked; 6) Incubate and react the Au-Ab1 obtained in step 5) with the antigen at 35°C - 37°C for 1.5h - 2.5h, wash to remove the antigen that has not specifically bound to Au-Ab1, and incubate the obtained product with the CDs@dSiO2 NPs-Ab2 obtained in step 2) at 35°C - 37°C for 1.5h - 2.5h and then wash to obtain an immunosensor with carbon dot complex as the probe and co-reactant.

6. The preparation method of the immunosensor using the carbon dot composite as a probe and a coreactant according to claim 5, characterized in that: When preparing CDs@dSiO2 NPs in step 1), 150 μL of CDs was added to 50 mL of ethanol containing 50 mg of dendritic SiO2 spheres, sonicated for 5 min, then 1.25 mL of ammonia water was added and stirred for 12 h, centrifuged and washed with water and ethanol to obtain amino-rich CDs@dSiO2 NPs.

7. Use of the carbon dot complex as a probe and a coreactant in an immunosensor for antigen detection for non-diagnostic purposes according to claim 1 or 2.

8. The application according to claim 7, wherein: A three-electrode system including a working electrode, a counter electrode and a reference electrode was used for antigen detection; the synthesized CDs@dSiO2 NPs were used as a coreaction reagent, and the electrochemiluminescence signal was collected by an electrochemiluminescence analyzer.

9. The application according to claim 8, wherein: Antigen detection includes the following steps: 1) The working electrode polished on a chamois leather containing alumina powder was placed in ethanol and doubly distilled water for ultrasonic washing. 2) The immunosensor solution was respectively dropped onto the surface of the washed working electrode and air-dried at room temperature. 3) Configure Ru(bpy)3 2+ in a PBS buffer solution as the electrolyte of the three - electrode system; 4) The potential window was set to 0 - 1.5 V, the scan rate was set to 100 mV / s, the high voltage of the photomultiplier tube was set to 800 V, the electrochemiluminescence signal was collected using an electrochemiluminescence analyzer, the linear relationship between the electrochemiluminescence intensity and different concentrations of antigen was established, and the antigen detection result was obtained.

10. Use of the carbon dot complex as a probe and a coreactant in an immunosensor for antigen concentration detection for non-diagnostic purposes according to claim 1 or 2.

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