An antibody complex, a biosensor, an ECL detection platform for SARS-CoV-2 and a detection method

Abstract

The present invention discloses an antibody complex, a biosensor, an ECL detection platform and a detection method for SARS-CoV-2, relating to the technical field of electrochemiluminescence detection. A nanocomposite comprises the following raw materials: β-cyclodextrin, a coreactant and metal nanoparticles, wherein the metal nanoparticles grow in-situ on the β-cyclodextrin, the metal nanoparticles are covalently connected to the amino group of the coreactant through a metal-N bond, and at the same time, the coreactant is also enriched in the hydrophobic inner cavity of the β-cyclodextrin. The metal nanoparticles are covalently connected to the coreactant through a metal-N bond, and the coreactant is also enriched in the hydrophobic cavity of the β-cyclodextrin, thereby realizing a high solid loading amount of the coreactant on the electrode surface, thus greatly improving the signal intensity of the subsequent ECL emitter and being beneficial to improving the detection sensitivity for the analyte.

Description

Technical Field

[0001] The present invention relates to the technical field of electrochemiluminescence detection, and in particular, to an antibody complex, a biosensor, an ECL detection platform and a detection method for SARS-CoV-2. Background Art

[0002] Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused many deaths worldwide. As one of the four main structural proteins of SARS-CoV-2, the nucleocapsid protein has the richest structural features and is generally considered as a biomarker of SARS-CoV-2. Recently, a variety of methods for detecting SARS-CoV-2 nucleocapsid protein (ncovNP) have been developed, such as enzyme-linked immunosorbent assay (ELISA), luciferase IP system (LIPS) and lateral flow assay (LFA). However, these methods have defects such as low sensitivity and complex operation, and there are application limitations in existing detection methods. Therefore, it is crucial to develop a sensitive and simple ncovNP detection method.

[0003] Electrochemiluminescence (ECL) technology has characteristics such as low background, high sensitivity and simple operation, and has received wide attention in the fields of clinical analysis, environmental monitoring and food safety. It has been reported to use the ECL method for SARS-CoV-2-related nucleic acid detection. Although the detection sensitivity of this method is relatively high, it has disadvantages such as a cumbersome extraction process and false negatives in results. Compared with nucleic acid detection, protein detection has the characteristics of simpler operation, higher specificity and higher accuracy. So far, there are few reports on ECL protein detection of ncovNP.

[0004] Excellent ECL emitters are one of the keys to achieving highly sensitive ECL detection. Among many ECL emitters such as organic polymers, inorganic complexes, organic small molecules, inorganic nanomaterials such as metal nanoclusters and various quantum dots, organic polymers have attracted much attention due to their advantages such as high photostability, low cytotoxicity and easy functionalization. The organic polymers reported as ECL signal probes mainly include polyfluorene and polyfluorene derivatives, poly(phenylene vinylene) and heterocyclic-containing polymers. For example, polyfluorene derivatives PFBT and PFO were developed as ECL emitters to achieve true-color ECL imaging for multiplex detection and ratio detection of microRNA-155 respectively. The Cui research group used a heterocyclic thiophene conjugated microporous polymer with strong bipolar ECL emission as an emitter to detect rhodamine B (Cui, L, 2021). The Ju research group prepared polymer dots containing poly(phenylene vinylene) with high ECL efficiency to construct a detection for Fe 3+The ECL biosensor (Feng, Y.Q. 2018). However, the types of organic polymers reported in the literature are limited. Therefore, it is of great significance to develop new ECL polymer emitters.

[0005] In addition, the introduction of coreactants is also crucial for highly sensitive ECL detection. The most common method of introducing coreactants is to directly add them to the detection bottom solution. Although this method is simple, there are problems with the stability and repeatability of the ECL system. In situ generation of coreactants by enzyme-catalyzed reactions faces complex operations and harsh enzyme usage conditions, so its application is greatly limited.

[0006] In view of this, the present invention is specifically proposed. Summary of the Invention

[0007] The purpose of the present invention is to provide an antibody complex, a biosensor, an ECL detection platform and a detection method for SARS-CoV-2 to solve the above technical problems.

[0008] The inventors found that immobilizing coreactants on the electrode surface can overcome the disadvantages of the methods of introducing coreactants, such as covalent bonding and crosslinking. However, it is still technically challenging to achieve a high loading amount of coreactants on the electrode surface, and how to achieve a high loading amount of coreactants on the electrode surface is a technical problem that urgently needs to be solved.

[0009] Since hydrophobic local enrichment can significantly increase the concentration of coreactants on the electrode surface, enriching coreactants through a material with a hydrophobic cavity may be an ideal choice to achieve a high loading of coreactants on the electrode surface. Research shows that if the local hydrophobic enrichment of coreactants is aimed at the coreactants present in the detection solution, this will still lead to problems with the stability and repeatability of the ECL system.

[0010] Based on this, it is of great value to develop a material with an internal hydrophobic cavity for enriching coreactants to achieve their immobilization on the electrode surface. β-cyclodextrin (β-CD) has an internal hydrophobic cavity and a large number of hydroxyl groups, and is mainly widely used in material functionalization, host-guest recognition and metal ion coordination in the ECL field. For example, β-CD-functionalized carbon nanohorns are used to detect naringin because naringin can enter the cavity of β-CD to form an inclusion complex. There are also reports on using the host-guest recognition between aptamers and Ru(bpy)3 2+ -β-CD for the detection of thrombin. There are also reports on using the metal-binding sites of β-CD to bind to Pb 2+ to synthesize metal-organic frameworks (MOFs).

[0011] The present invention is implemented as follows:

[0012] The present invention provides a nanocomposite, which comprises the following raw materials: β-cyclodextrin, a co-reactant, and metal nanoparticles. The metal nanoparticles grow in-situ on β-cyclodextrin. The metal nanoparticles are covalently linked to the amino group of the co-reactant through a metal-N bond, and the co-reactant is located in the hydrophobic inner cavity of β-cyclodextrin. The co-reactant is a hydrophobic compound with an amino group.

[0013] The above-mentioned co-reactant being located in the hydrophobic inner cavity of β-cyclodextrin includes, but is not limited to, the following situations: the co-reactant is enriched in the hydrophobic inner cavity of β-cyclodextrin.

[0014] Based on the confinement effect of the nano-cavity of β-cyclodextrin and the β-CD metal ion binding sites, the inventors found that it can provide two completely different forces for fixing the co-reactant at the same time, namely hydrophobic local enrichment and covalent bonding through metal nanoparticles grown in-situ on β-CD. On the one hand, the internal hydrophobic cavity of β-cyclodextrin binds to the hydrophobic co-reactant through hydrophobic interaction in a hydrophobic environment, which can perform hydrophobic local enrichment on the co-reactant. On the other hand, β-cyclodextrin can coordinate with metal ions through the metal ion coordination sites arranged on the surface of β-cyclodextrin. After a reduction reaction, the metal ions are reduced to metal nanoparticles and grow in-situ on the surface of β-cyclodextrin. In addition, the metal nanoparticles are covalently linked to the amino group of the co-reactant through a metal-N bond, enabling more co-reactants to be enriched (fixed) on the surface of the electrode (the working electrode on the subsequent biosensor). Thus, a high loading amount of the co-reactant on the electrode surface can be achieved, thereby greatly improving the signal intensity of the subsequent ECL emitter, which is beneficial to improving the detection sensitivity for the analyte. The proposed nanocomposite provides a strong clue for achieving ultrasensitive detection of the analyte protein.

[0015] The analyte includes, but is not limited to, serum, blood, and environmental samples.

[0016] The inventors verified through experiments that the hydrophobic local enrichment strategy of the co-reactant can significantly improve ECL emission and detection sensitivity by utilizing the hydrophobic cavity of β-CD.

[0017] The inventors found that fixing the co-reactant to the working electrode through a β-cyclodextrin-metal nanoparticle composite can improve the stability of the ECL signal.

[0018] In a preferred embodiment of the application of the present invention, the metal nanoparticles include, but are not limited to: platinum, lead, zinc, tin, copper, gold, or silver.

[0019] In one embodiment, the above-mentioned coreactant is 3-(dibutylamino)propylamine, N,N-dimethylethylenediamine, N,N-diethylethylenediamine, N,N-diisopropylethylenediamine, tripropylamine, triethylamine or tetramethylethylenediamine. The inventors found that TDBA can be used as a coreactant to enhance the ECL signal of ECL emitters (such as PDP PNPs).

[0020] Specifically, the hydrophobic cavity of β-CD can effectively enrich TDBA, generating more TDBA ·+ , thereby improving the ECL emission of the ECL emitter. The inventors utilized the internal hydrophobic cavity of β-CD for hydrophobic local enrichment of 3-(dibutylamino)propylamine (TDBA). On the other hand, the coordination sites of β-CD metal ions enable metal nanoparticles (such as Pt nanoparticles (PtNPs)) to grow in situ on β-CD more easily, which can fix TDBA and subsequent antibodies (Ab2) through Pt-N bonds.

[0021] The present invention also provides a method for preparing a nanocomposite, which includes: mixing a metal ion-containing solution with β-cyclodextrin, and through reduction, reacting to obtain a β-cyclodextrin-metal nanoparticle composite, and then mixing and reacting the β-cyclodextrin-metal nanoparticle composite with a coreactant to obtain a nanocomposite combined with the coreactant.

[0022] After the reduction reaction, the metal ions bound to the coordination sites of β-cyclodextrin are reduced to metal nanoparticles, so that the metal nanoparticles grow in situ on the surface of β-cyclodextrin. The β-cyclodextrin-metal nanoparticle composite and the coreactant achieve the immobilization of the coreactant through metal-N bonds. With the synergistic effect of the hydrophobic local enrichment of TDBA by β-cyclodextrin and the covalent bonding of metal-N bonds, a high immobilization amount of TDBA on the electrode surface is achieved.

[0023] In a preferred embodiment of the application of the present invention, the metal ion-containing solution is mixed with β-cyclodextrin, and the volume-mass ratio when the metal ion-containing solution is mixed with β-cyclodextrin is: 10-100 μL: 1-3 mg. And the volume concentration of the metal ion-containing solution is 1-1.2%.

[0024] Under the above mixing ratio, the metal ions can be better distributed on the metal ion coordination sites of β-cyclodextrin.

[0025] In an alternative embodiment, the solution obtained by mixing the metal nanoparticle-containing solution with β-cyclodextrin is stirred, a reducing agent is added for reaction, and then sodium citrate is added to obtain a β-cyclodextrin-metal nanoparticle composite. Sodium citrate serves as a stabilizer to prevent the aggregation of metal nanoparticles.

[0026] In an alternative embodiment, the reducing agent described above is NaBH4.

[0027] In an alternative embodiment, before mixing with the co-reactant, it further includes washing the β-cyclodextrin-metal nanoparticle complex with water to obtain a dispersion of the β-cyclodextrin-metal nanoparticle complex; the stirring time is 12 h - 14 h. Washing with water is used to collect the β-cyclodextrin-metal nanoparticle complex and remove water-soluble impurities such as the reaction buffer. In an alternative embodiment, it can be washed multiple times as needed. Stirring, in one embodiment, can be carried out for an overnight reaction.

[0028] In a preferred embodiment of the application of the present invention, the mixing volume ratio of the β-cyclodextrin-metal nanoparticle complex to the co-reactant is 70 - 200:1, and the mixing reaction time of the β-cyclodextrin-metal nanoparticle complex and the co-reactant is 10 - 18 h, and the reaction temperature is 0 - 5 °C. The inventors found that good results can also be achieved when the volume of the co-reaction reagent is small.

[0029] At the above mixing volume ratio, it can satisfy that the co-reactant binds better in the hydrophobic inner cavity of the β-cyclodextrin-metal nanoparticle complex. The low-temperature reaction is beneficial to promoting the reaction to proceed better.

[0030] The present invention also provides an antibody complex, which includes: a nanocomplex or a nanocomplex prepared by the above preparation method, and an antibody, and the metal nanoparticles of the nanocomplex are covalently bonded to the amino group of the antibody through a metal-N bond.

[0031] This antibody complex can be placed on an electrode or other solid-phase carriers for detection and analysis.

[0032] In an alternative embodiment, the antibody is an antibody against the SARS-CoV-2 nucleocapsid protein. In addition, in other embodiments, the above antibody can also be selected as an antibody that can recognize other antigens to be detected, so as to achieve precise detection of other proteins.

[0033] The present invention also provides a preparation method of an antibody complex, which includes: mixing and reacting the β-cyclodextrin-metal nanoparticle complex, the co-reactant, and the antibody;

[0034] Or, directly mixing and reacting the above-mentioned nanocomplex with the antibody.

[0035] Both of the above two methods can achieve the preparation of the antibody complex and realize the binding of the antibody and the nanocomplex.

[0036] In a preferred embodiment of the application of the present invention, the antibody added in the mixing reaction is added in the form of an antibody solution, and the concentration of the antibody solution is 45 - 200 μg / mL. Preferably, it is 50 μg / mL. At the above antibody addition concentration, the ECL response signal can be increased, the ECL signal intensity can be enhanced, thereby improving the signal sensitivity of detection.

[0037] In an alternative embodiment, the mixing volume ratio of the β-cyclodextrin-metal nanoparticle complex, the co-reactant, and the antibody solution is 1:70 - 200:10.

[0038] In an alternative embodiment, the solution after the mixing reaction is blocked, centrifuged, and dispersed; the dispersion is carried out in a PBS solution. Blocking is used to block the remaining active sites of the antibody complex. Centrifugation is used to remove the blocking agent.

[0039] In an alternative embodiment, the pH of the PBS is 7 - 8; in an alternative embodiment, the pH of the PBS is 7.4.

[0040] The inventors found that the dispersion of the antibody complex has a certain influence on the ECL signal intensity. The ECL signal increases as the pH increases from 4.0 to 7.4 and reaches a maximum at pH 7.4. As the pH value exceeds 7.4, the ECL intensity gradually decreases. Physiological pH is beneficial to maintaining the biological activities of antibodies and antigens.

[0041] The present invention also provides a biosensor, which includes: a working electrode, an ECL emitter is modified on the working electrode, and an antibody is coupled to the ECL emitter; the ECL emitter is selected from carboxyl-functionalized poly[2,5-dioctyl-1,4-phenylene] nanoparticles (PDP PNPs). By detecting the ECL signal on the working electrode, accurate qualitative and quantitative determination of antigens can be achieved.

[0042] In other embodiments, the above ECL emitter can also be selected from other emitters.

[0043] In an alternative embodiment, the antibody is an antibody against the SARS-CoV-2 nucleocapsid protein.

[0044] The present invention also provides a method for preparing a biosensor, which includes: activating the working electrode modified with the ECL emitter, and then incubating and blocking the activated working electrode with the antibody. The blocking step is used to block the remaining binding sites of the PDP PNPs.

[0045] The above activation is achieved by incubating the working electrode with EDC / NHS (v / v = 4:1, 10 μL) at room temperature for 1.0 hour to activate the carboxyl groups of PDP PNPs. In an alternative embodiment, the above working electrode is incubated with the primary antibody.

[0046] In an alternative embodiment, the preparation method further includes modifying the ECL emitter on the working electrode, which includes: coating a nano-dispersion of carboxyl-functionalized poly[2,5-dioctyl-1,4-phenylene] on the working electrode and drying.

[0047] In an alternative embodiment, the preparation method further includes the preparation of a carboxyl-functionalized poly[2,5-dioctyl-1,4-phenylene] nano-dispersion, which includes mixing PDP with PSMA, adding water and then evaporating to obtain a carboxyl-functionalized poly[2,5-dioctyl-1,4-phenylene] nano-dispersion.

[0048] The present invention also provides an ECL detection platform for SARS-CoV-2, which includes a biosensor and an antibody complex.

[0049] Compared with other methods such as enzyme-linked immunosorbent assay (ELISA), luciferase IP system (LIPS), and lateral flow assay (LFA), the ECL detection platform provided by the present invention has a wider linear range and a lower detection limit. The ECL detection platform provided by the present invention has good intra-assay and inter-assay reproducibility.

[0050] The present invention also provides a method for detecting SARS-CoV-2 using the above ECL detection platform for SARS-CoV-2. The detection method is for non-diagnostic purposes of diseases and includes: mixing and incubating the biosensor with the sample to be tested, then adding the antibody complex and incubating, and recording the ECL signal through the electrode system.

[0051] The ECL detection platform can detect environmental samples and dead animal bodies.

[0052] In an alternative embodiment, the mixing and incubation time of the biosensor with the sample to be tested is 55 - 120 min; preferably 60 min. The response signal gradually increases with the increase of the incubation time and reaches the maximum value at 60 minutes. When the incubation time exceeds 60 minutes, the ECL intensity slightly decreases and gradually stabilizes.

[0053] In an alternative embodiment, the incubation time after adding the antibody complex is 55 - 80 min; preferably 60 min. Under the above incubation time, the biosensor has a good signal intensity.

[0054] In an alternative embodiment, the ECL signal detection is carried out in PBS, the scanning potential is from 0 V to +1.5 V, and the scanning rate and the photomultiplier tube are set to 300 mV / s and 800 V, respectively.

[0055] The present invention also provides an ECL emitter, and the ECL emitter is selected from carboxyl-functionalized poly[2,5-dioctyl-1,4-phenylene] nanoparticles (PDP PNPs).

[0056] The present invention also provides a preparation method of an ECL emitter, and the preparation method includes: mixing PDP with PSMA, adding water and then evaporating to obtain a carboxyl-functionalized poly[2,5-dioctyl-1,4-phenylene] nano-dispersion;

[0057] In an alternative embodiment, the mixing mass ratio of the above PDP to PSMA is 5-8:1.

[0058] The novel PDP PNPs open up a promising ECL polymeric emitter. The combination of TDBA-β-CD-Pt nanocomposite with PDP PNPs enables the sensitive detection of ncovNP, providing a new detection method for ncovNP.

[0059] The present invention has the following beneficial effects:

[0060] The present invention provides a nanocomposite. On the one hand, the internal hydrophobic cavity of β-cyclodextrin can bind to the hydrophobic coreactant through hydrophobic interaction in a hydrophobic environment, enabling the hydrophobic local enrichment of the coreactant. On the other hand, β-cyclodextrin can coordinate with metal ions through the metal ion coordination sites arranged on the surface of β-cyclodextrin. After a reduction reaction, the metal ions are reduced to metal nanoparticles and in-situ grow on the surface of β-cyclodextrin. In addition, the metal nanoparticles are covalently connected to the amino group of the coreactant through a metal-N bond, enabling more coreactants to be enriched (fixed) on the surface of the electrode (the working electrode on the subsequent biosensor). Thus, a high solid loading of the coreactant on the electrode surface can be achieved, thereby significantly increasing the signal intensity of the subsequent ECL emitter and facilitating the improvement of the detection sensitivity for the analyte. The proposed nanocomposite provides a strong clue for the ultrasensitive detection of the target protein. The synergistic effect of the hydrophobic local enrichment of the coreactant and the covalent binding of the metal-N bond can significantly enhance the ECL emission and improve the detection sensitivity.

[0061] The present invention also provides an antibody complex, and the antibody complex can be placed on an electrode or other solid-phase carriers for the detection and analysis of antigens.

[0062] The present invention also provides a biosensor and an ECL detection platform. By detecting the ECL signal on the working electrode, accurate qualitative and quantitative determination of antigens can be achieved. Compared with other methods such as enzyme-linked immunosorbent assay (ELISA), luciferase IP system (LIPS), and lateral flow assay (LFA), the ECL detection platform provided by the present invention has a wider linear range and lower detection limit. The ECL detection platform provided by the present invention has good within-batch and between-batch reproducibility.

[0063] In addition, the present invention also provides an ECL polymer luminophore. Through the nanocomposite material and the ECL polymer luminophore, hypersensitive detection of the target substance can be realized. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required for the embodiments. It should be understood that the following drawings only show some embodiments of the present invention, and therefore should not be regarded as limiting the scope. For those of ordinary skill in the art, other related drawings can be obtained based on these drawings without creative efforts.

[0065] Figure 1 For the preparation schematic diagram;

[0066] Figure 2 For the morphology and elemental characterization diagram of the nanomaterial;

[0067] Figure 3 For the XPS spectrum of PDP PNPs;

[0068] Figure 4 For the FT-IR spectra of (A)(a) PDP and (b) PDP PNPs; (B) the ultraviolet-visible absorption spectrum of PDP PNPs; (C) the fluorescence emission spectrum (FL), fluorescence excitation spectrum (FLE), and ECL emission spectrum of PDP PNPs; (D) the three-dimensional ECL surface image of PDP PNPs;

[0069] Figure 5 For the ECL response diagrams of different modified electrodes and the experimental results of ECL stability verification;

[0070] Figure 6 For the ECL mechanism diagram;

[0071] Figure 7 For the CV and EIS characterization result diagrams;

[0072] Figure 8 For the ECL intensity of the biosensor under different conditions;

[0073] Figure 9Graphs showing the quantitative detection results and stability verification results of the biosensor;

[0074] Figure 10 Graph showing the reproducibility verification results of the biosensor with 100 pg / mL ncovNP. Detailed implementation manners

[0075] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be described clearly and completely below. For those not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are followed. For reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0076] The features and performance of the present invention will be further described in detail below in conjunction with the embodiments.

[0077] Drugs and reagents

[0078] Poly[2,5-dioctyl-1,4-phenylene], polystyrene maleic anhydride, SARS-CoV-2 nucleocapsid protein (ncovNP), and mouse anti-SARS-CoV-2 nucleocapsid protein monoclonal antibody, tetrahydrofuran, 3-(dibutylamino)propylamine, β-cyclodextrin, N-hydroxysuccinimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide salt, bovine serum albumin, sodium borohydride, sodium citrate, phosphate buffer solution, chloroplatinic acid.

[0079] Instruments

[0080] Transmission electron microscope, MPI-A electrochemiluminescence analyzer, Fourier transform infrared spectrometer, CHI 760D electrochemical workstation, ultraviolet spectrophotometer, electron paramagnetic resonance (EPR) instrument, X-ray photoelectron spectrometer, fluorescence spectrophotometer.

[0081] Example 1

[0082] This example provides a β-CD-Pt nanocomposite and its preparation method.

[0083] Dissolve 1.0 mg of β-CD in deionized water and perform sonication to form a 1.0 mg / mL β-CD solution. Then, add 10 μL of H2PtCl6 (1%) to the β-CD solution and stir vigorously overnight. Subsequently, slowly add 25 μL of NaBH4 (0.01 M) to the above mixed solution and react for 20 min under stirring conditions. Continue to add 10 μL of sodium citrate (0.01 M) and continue stirring for 30 min to form the β-CD-Pt nanocomposite, which is centrifuged at 10000 rpm and washed three times with water for collection. Finally, disperse the collected β-CD-Pt in 1.0 mL of water to obtain its dispersion.

[0084] As a control, in the absence of β-CD, Pt nanoparticles (PtNPs) were prepared according to the same reduction method as above.

[0085] Example 2

[0086] This example provides an antibody complex and a preparation method thereof.

[0087] The preparation of the TDBA-β-CD-Pt@Ab2 complex was carried out with reference to Figure 1 shown in B of

[0088] The preparation process of the secondary antibody complex (TDBA-β-CD-Pt@Ab2) is as shown in principle Figure 1 B. Briefly, mix 4.0 μL of TDBA, 400 μL of β-CD-Pt dispersion, and 40 μL of Ab2 (50 μg / mL) and react at 4.0 °C for 14 hours to form the TDBA-β-CD-Pt@Ab2 nanocomposite. Then, block the remaining active sites of TDBA-β-CD-Pt@Ab2 by incubating BSA (1%, 100 μL) with the nanocomposite for 40 minutes. Finally, centrifuge the obtained TDBA-β-CD-Pt@Ab2 at 8000 rpm, wash it three times with 0.10 M PBS (pH 7.4), and further disperse it in 400 μL of 0.10 M PBS (pH 7.4) and store it at 4.0 °C for later use.

[0089] Example 3

[0090] This example provides a preparation method of PDP PNPs.

[0091] First, dissolve 5.0 mg of PDP and 1.0 mg of PSMA in tetrahydrofuran (THF) to prepare their respective solutions (1.0 mg / mL). Subsequently, mix the obtained PDP and PSMA solutions thoroughly for 2.0 h and further inject into deionized water (10 mL). Next, heat the resulting mixed solution at 80 °C in an air atmosphere until THF is completely evaporated to obtain a PDP PNPs dispersion. Principle Figure 1 Figure A depicts the preparation process of PDP PNPs.

[0092] Example 4

[0093] This example provides a method for preparing an ECL sensor (ECL detection platform).

[0094] After polishing with alumina (0.3 and 0.05 μm) and alternately ultrasonic cleaning with pure water and ethanol, the GCE (Φ = 4.0 mm) is covered with the PDP PNPs dispersion (10 μL) and dried naturally at room temperature. Then, activate the carboxyl groups of PDP PNPs by incubating the electrode with EDC / NHS (v / v = 4:1, 10 μL) at room temperature for 1.0 h. Thereafter, incubate the primary antibody (Ab1, 10 μL) on the modified electrode at 4.0 °C for 14 h. Continue to incubate BSA (1%, 10 μL) on the modified electrode for a further 1.0 h to block the residual binding sites of PDP PNPs, obtaining an ECL immunosensor (Ab1 / PDP PNPs / GCE). In each modification step, the electrode is washed with 0.10 M PBS (pH 7.4). The preparation process is as shown in Principle Figure 1 Figure C.

[0095] The measurement method of the ECL sensor is as follows:

[0096] The constructed immunosensor (Ab1 / PDP PNPs / GCE) is incubated with different concentrations of ncovNP at 37 °C for 1.0 h, and then a sandwich structure is constructed by capturing the TDBA-β-CD-Pt@Ab2 biocomplex on the electrode surface at 37 °C for 1.0 h. The modified GCE serves as the working electrode, a platinum wire counter electrode, and an Ag / AgCl reference electrode form a three-electrode system to record the ECL signal. The ECL detection is carried out in 0.10 M PBS (3.0 mL, pH 7.4). The scanning potential is from 0 V to +1.5 V, and the scanning rate and photomultiplier tube (PMT) are set to 300 mV / s and 800 V, respectively.

[0097] Experimental Example 1

[0098] This experimental example conducts the morphology and elemental characterization of nanomaterials.

[0099] A scanning electron microscope (SEM) and a transmission electron microscope (TEM) were used to characterize the morphology of the nanomaterials prepared in the above examples. Figure 2 A in presents the TEM image of PDP PNPs, which shows spherical shapes with an average diameter of 103 nm. Figure 2 B in shows the SEM image of Pt NPs, in which free Pt NPs with an average size of about 30 nm are shown. Figure 2 C in is the TEM image of β-CD-Pt nanocomposites, and a large number of Pt NPs are observed. The assembly and arrangement of Pt NPs clearly reveal the circular cavity structure of β-CD, indicating that Pt NPs grow in situ on the ligand sites of β-CD metal ions. In addition, energy dispersive scanning (EDS) mapping was used to ensure the elemental distribution of β-CD-Pt. Figure 2 D in shows the EDS mapping of C, N, O, and Pt elements. The results show that Pt NPs are assembled on β-CD molecules.

[0100] X-ray photoelectron spectroscopy (XPS) of PDP PNPs and β-CD-Pt was explored to further determine their elemental distributions. Figure 2 E clearly shows the characteristic peaks of C(1s) and O(1s) from PDP PNPs. Figure 2 F is the XPS spectrum of β-CD-Pt, showing the XPS characteristic peaks of C, O, N, and Pt elements from β-CD-Pt.

[0101] Figure 3 A in and Figure 3 B in respectively show the corresponding XPS spectra of each element with various bonds. As shown, the binding energies of C-C, C-O, and C=O in C(1s) are 284.88 eV, 286.29 eV, and 287.6 eV ( Figure 3 A in ), and the binding energies of C-O and C=O in O(1s) are 532.49 eV and 533.68 eV ( Figure 3 B in ). The presence of the C=O covalent bond indicates that PDPPNPs are successfully carboxyl-functionalized. The corresponding bonding characteristic peaks of N(1s) and Pt(4f) are shown in Figure 3 C and Figure 3 D. The binding energies of Pt-N, C-N, and N-H in N(1s) are 398.28 eV, 399.56 eV, and 401.37 eV ( Figure 3 C in ), and the binding energies of Pt-N4f 7 / 2 and Pt-N 4f 5 / 2 can be observed at 72.53 eV and 76.5 eV respectively ( Figure 3In D). The XPS characteristic peaks of β-CD-Pt proved the successful preparation of β-CD-Pt.

[0102] Fourier transform infrared spectroscopy (FT-IR) was used to study the composite materials of PDP PNPs. As Figure 4 shown in -1 A. For the original PDP, two absorption peaks appeared near 2855 cm -1 and 2922 cm -1 (curve a), which were attributed to the stretching vibration of C-H bonds on the alkyl chain of PDP. The peak at 788 cm -1 was due to the alkyl C–H rocking mode. Subsequently, the peak at 1471 cm -1 was attributed to the skeletal vibration of the benzene ring. Then, the stretching vibration peak of the C-O-C bond was observed at 1064 cm -1 . Compared with the original PDP, a new FT-IR peak (curve b) appeared at 1778.4 cm

[0103] for PDP PNPs, which was coordinated with –COOH. The FT-IR spectrum indicated the successful preparation of PDP PNPs. Figure 4 For the ultraviolet-visible absorption and fluorescence (FL) spectra of PDP PNPs, characterizations were carried out, and the results are shown in Figure 4 B and Figure 4 3C respectively. As shown in the figure, the characteristic absorption peak of PDP PNPs was located at 347 nm ( Figure 4 B), and the excitation and emission spectra of PDP PNPs reached obvious peaks at 337 nm ( Figure 4 C, black line) and 448 nm ( Figure 4 C, red line) respectively. In addition, the ECL spectra and ECL three-dimensional surface images of the synthesized PDP PNPs were measured in 3.0 mL PBS (pH 7.4) containing 10 μL TDBA, and the results are shown in Figure 4 C and

[0104] 3D. The ECL emission wavelength of PDP PNPs was located at 633 nm (

[0105] C, blue line and Figure 5 D), showing a significant red shift (△λ = 190 nm) compared with the fluorescence emission spectrum, which may be attributed to the energy separation of the surface states of PDP PNPs. Therefore, all characterizations confirmed the successful preparation of PDP PNPs.

[0104] Experimental Example 2

[0105] This experimental example explored the ECL mechanism. The ECL behaviors of different materials, including PDP PNPs, β-CD-Pt, β-CD, and Pt NPs, were studied, which were modified on the corresponding electrodes and tested in 0.10 M PBS (pH 7.4). Figure 5A and 4B depict the ECL signals in PBS without and with 5.7 mM TDBA, respectively. As shown, regardless of the presence of the coreactant TDBA, almost no ECL signal was detected at β-CD-Pt / GCE (curve b), β-CD / GCE (curve c), and Pt NPs / GCE (curve d). However, PDPPNPs / GCE exhibited a weak ECL signal without TDBA ( Figure 5 A, curve a), and showed an intense ECL response at +1.5 V in the presence of TDBA ( Figure 5 B, curve a), indicating that the detected ECL emission originated from PDP PNPs rather than β-CD or Pt NPs. In addition, it was revealed that TDBA could act as a coreactant to enhance the ECL signal of PDP PNPs.

[0106] Figure 5 In (A), the ECL responses of different modified electrodes (A) without and (B) with 5.7 mM TDBA: (a) PDP PNPs / GCE, (b) β-CD-Pt / GCE, (c) β-CD / GCE, and (d) Pt NPs / GCE.

[0107] We compared the stability of the ECL signal with the addition of the coreactant TDBA to the detection solution and that immobilized on the electrode surface. Figure 5 C shows the corresponding results. For TDBA (5.7 mM) in PBS, the ECL signal was recorded for 10 consecutive potential scans at PDP PNPs / GCE, and the results are shown in Figure 5 C (curve a). For the case where TDBA was immobilized on GCE, the ECL signal was detected on the biosensor TDBA-β-CD-Pt@Ab2 / ncovNP / Ab1 / PDP PNPs / GCE, and the results are as shown in Figure 4 C (curve b). As observed, with the increase in the number of cyclic scans, curve a showed a decreasing ECL signal. However, curve b showed stable ECL emission under the same number of cyclic scans, indicating the superiority of the β-CD-Pt nanocomposite in immobilizing TDBA on the electrode.

[0108] Figure 5 In C, the ECL stabilities of (a) PDP PNPs / GCE with 5.7 mM TDBA and (b) TDBA-Pt@Ab2 / ncovNP / Ab1 / PDPPNPs / GCE.

[0109] The ECL emission amplification effect of β-CD on PDP PNPs was further explored. As a comparison, TDBA-Pt@Ab2 / ncovNP / Ab1 / PDP PNPs / GCE without β-CD was also constructed using the same assembly method as the biosensor (TDBA-β-CD-Pt@Ab2 / ncovNP / Ab1 / PDP PNPs / GCE), with TDBA-Pt replacing TDBA-β-CD-Pt. From Figure 5 As can be seen from D, the ECL signal intensity without β-CD was approximately 1978 a.u (curve b). However, the ECL signal intensity of the biosensor with β-CD was approximately 9760 a.u., which was nearly 5 times stronger than that of the biosensor without β-CD. The results indicate that the coreactant TDBA hydrophobic local enrichment strategy we developed using the hydrophobic cavity of β-CD can significantly enhance ECL emission and improve detection sensitivity.

[0110] Figure 5 In D, the ECL signals of (a) TDBA-β-CD-Pt@Ab2 / ncovNP / Ab1 / PDP PNPs / GCE and (b) TDBA-Pt@Ab2 / ncovNP / Ab1 / PDP PNPs / GCE.

[0111] Differential pulse voltammetry (DPV) was used to explore the electrochemical oxidation of the coreactant TDBA on different modified electrodes. First, with TDBA added to PBS, the DPV curve was explored using a bare GCE, and the results are plotted in Figure 5 E (curve a). A strong oxidation peak of TDBA was detected at +0.90 V, which was attributed to the generation of TDBA during the electrochemical reduction process. ·+ Subsequently, the DPV curve was explored using TDBA-β-CD-Pt@Ab2 / ncovNP / Ab1 / PDP PNPs / GCE under the condition that TDBA was hydrophobically locally enriched and covalently fixed, and the results are plotted in Figure 5 E (curve b), showing a relatively strong oxidation peak at +0.90 V. Finally, the DPV curve of TDBA fixed only by covalent bond (Pt-N bond) was also explored using TDBA-Pt@Ab2 / ncovNP / Ab1 / PDP PNPs / GCE, and the results are plotted in Figure 5 E (curve c), showing a significant reduction in the oxidation peak in the absence of β-CD. The above experiments confirmed that the hydrophobic cavity of β-CD can effectively enrich TDBA, generating more TDBA ·+ and thus enhancing the ECL emission of PDP PNPs.

[0112] Figure 5In E, anodic DPV curves of (a) bare GCE containing 5.7 mM TDBA, (b) TDBA-β-CD-Pt@Ab2 / ncovNP / Ab1 / PDP PNPs / GCE, and (c) TDBA-Pt@Ab2 / ncovNP / Ab1 / PDP PNPs / GCE.

[0113] In addition, 5,5-dimethyl-1-pyrroline N-oxide (DMPO) was used as a TDBA radical scavenger for electron paramagnetic resonance (EPR) characterization to further confirm the hydrophobic local enrichment of TDBA by β-CD. As can be seen from Figure 5 F, under light irradiation, the EPR signal from the DMPO-TDBA adduct is stronger in the presence of β-CD than in the absence of β-CD, indicating that β-CD with an internal hydrophobic cavity can effectively enrich TDBA. Figure 5 In F, EPR spectra of (a) TDBA-β-CD-Pt and (b) TDBA-Pt under light irradiation. The detection solution was PBS (3.0 mL, 0.10 M, pH 7.4), the ECL scan rate was 300 mV / s, and the scan rate of DPV was 100 mV / s.

[0114] The possible ECL mechanism of the biosensor can be speculated as follows. First, PDP PNPs and the coreactant TDBA are electro-oxidized to generate PDP PNPs ·+ and TDBA ·+ , respectively. In the absence of β-CD (principle Figure 6 , without β-CD), when TDBA is only fixed by a covalent bond (Pt-N bond), less TDBA ·+ is generated, and then less TDBA · will generate by losing a proton (H + ), thus generating a less excited species PDP PNPs ＊ . It should be noted that in the presence of β-CD (principle Figure 2 , with β-CD), due to the hydrophobic interaction between TDBA and β-CD, more TDBA ·+ is generated, and then TDBA ·+ will lose H + to generate more TDBA · , which will then react with PDP PNPs ·+ to form a large amount of PDP PNPs ＊ , thus significantly promoting the ECL emission of the PDP PNPs / TDBA system.

[0115] Experimental Example 3

[0116] Characterization of the Biosensor Assembly Process

[0117] Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were performed in [Fe(CN)6] 3- / 4- solution (5.0 mM) to characterize the assembly of the biosensor. Figure 7 A The CV results were plotted. As shown, the bare GCE showed the reversible redox current of [Fe(CN)6] 3- / 4- (curve a). PDP PNPs / GCE showed a decreased peak current (curve b) because the conductivity of PDP PNPs was very poor. When PDP PNPs / GCE was continuously incubated with Ab1 and BSA, the obtained electrode (Ab1 / PDP PNPs / GCE) showed a further decreased peak current (curve c). With the incubation of ncovNP (curve d), ncovNP / Ab1 / PDP PNPs / GCE showed a smaller peak current than Ab1 / PDP PNPs / GCE. Meanwhile, the assembly of the biosensor was characterized using EIS technology, and the results are shown in Figure 7 B. The electron transfer resistance (Ret) of the probe [Fe(CN)6] 3- / 4- at the electrode interface was related to the semicircle part of the EIS diagram. As shown, the bare GCE showed a smaller Ret (curve a). With the continuous immobilization of Ab1, BSA, and ncovNP on the electrode, the semicircle of the EIS diagram gradually decreased due to the continuously expanding Ret of [Fe(CN)6] 3- / 4- on different modified electrodes (curves b - d). Overall, CV and EIS confirmed the successful assembly of the ECL biosensor.

[0118] Figure 7 In, (a) bare GCE, (b) PDP PNPs / GCE, (c) Ab1 / PDP PNPs / GCE, (d) ncovNP / Ab1 / PDPPNPs / GCE in [Fe(CN)6] 3- / 4- solution (5.0 mM); CV measurement: working voltage -0.2 to +0.6 V, scan rate 100 mV / s; working voltage was 0.22 V, amplitude was 5.0 mV, frequency was 10 -1 ~10 5 Hz, for EIS measurement.

[0119] Experimental Example 4

[0120] This experimental example explored the effects of different experimental conditions on the biosensor.

[0121] Optimization of experimental conditions is necessary for the analysis and application of biosensors. In this work, the pH value of PBS, the concentration of Ab2, and the incubation time of ncovNP and Ab2 were optimized using the biosensor TDBA-β-CD-Pt@Ab2 / ncovNP / Ab1 / PDP PNPs / GCE with 1.0 ng / mL ncovNP. Figure 8 A shows the variation of the response signal with the pH value. It was observed that the ECL signal increased as the pH increased from 4.0 to 7.4 and reached a maximum at pH 7.4. As the pH value exceeded 7.4, the ECL intensity gradually decreased. This phenomenon can be explained by the fact that physiological pH values are conducive to maintaining the biological activities of antibodies and antigens.

[0122] Figure 8 B and 8C show the variation of the response signal with the incubation time of ncovNP and Ab2, respectively. As can be seen, the response signal gradually increased with the increase of the incubation time and reached a maximum at 60 minutes. When the incubation time exceeded 60 minutes, the ECL intensity decreased slightly and gradually tended to be stable. Therefore, in this work, an incubation time of 60 minutes was considered the optimal incubation time for ncovNP and Ab2.

[0123] Figure 8 D shows the variation of the response signal with the concentration of Ab2. A graph of the ECL response relationship of the biosensor incubated with Ab2 at a concentration of 1.0 ng / mL ncovNP, conditions: in PBS (3.0 mL, 0.10 M, pH 7.4); scan rate: 300 mV / s. As can be seen, the ECL intensity increased as the concentration of Ab2 increased in the range of 1.0 to 50 μg / mL. When the concentration exceeded 50 μg / mL, the ECL intensity decreased slightly and gradually tended to be stable. Therefore, in this work, a concentration of 50 μg / mL was regarded as the optimized concentration of Ab2.

[0124] Experimental Example 5

[0125] Analytical performance of immunosensor

[0126] The application of the ECL biosensor was first explored through the quantitative bioassay of the target ncovNP, and the results are plotted in Figure 9 A and 7B. As the concentration range of ncovNP was from 50 fg / mL to 1.0 ng / mL, the ECL response in PBS (0.10 M, pH 7.4) gradually expanded. In this concentration range, the ECL intensity showed a good linear relationship with the logarithm of the concentration. At a signal-to-noise ratio (S / N) of 3, compared with other works for detecting ncovNP, the proposed ECL biosensor had a wider linear range and a lower detection limit. The comparison is shown in Table 1.

[0127] Table 1 Comparison of the analytical performance of different ncovNP detection strategies

[0128]

[0129] The selectivity of the biosensor was further investigated in the presence of BSA, carcinoembryonic antigen (CEA), and alpha-fetoprotein (AFP) as possible interfering substances. The ECL responses of the modified electrode to the target ncovNP (1.0 ng / mL), blank samples, selected interfering substances at 100-fold the concentration of ncovNP, and their mixtures were measured in 3.0 mL (0.10 M, pH 7.4). The results are as Figure 9 shown in C. Strong ECL signals were observed in the case of using ncovNP (1.0 ng / mL) alone and the mixture containing ncovNP (1.0 ng / mL) and interfering substances. Moreover, the reactions in both cases were almost the same. The responses to the interfering substances were negligible compared to the response to ncovNP (1.0 ng / mL), indicating that the biosensor has good selectivity for ncovNP.

[0130] Stability is very important for the biosensor. The ECL signals of the biosensor incubated with 100 pg / mL ncovNP were recorded within 10 consecutive scan cycles, Figure 9 and the results are shown in D. It can be seen that the ECL response did not change significantly, and a relative standard deviation (RSD) of 2.95% was obtained, indicating that the proposed ECL biosensor has acceptable stability.

[0131] In addition, this experimental example also provided a reproducibility experiment. Four biosensors constructed with the same batch and different batches were used for within-batch and between-batch tests, and the results are as Figure 10 shown. The RSDs for within-batch and between-batch determinations were 3.79% and 3.32%, respectively, indicating that the proposed ECL biosensor has good reproducibility.

[0132] Experimental Example 6

[0133] Analysis of real serum samples

[0134] To test the practicality of the biosensor we constructed, a standard addition method was used to perform a spike recovery experiment in serum. The concentration of ncovNP in serum diluted 50-fold was evaluated. Subsequently, three different concentrations of ncovNP were added to the diluted serum to detect the recovery rate. As shown in Table 2, the recovery rates were in the range of 98.0 - 99.2%, indicating that the ECL platform can be used to detect ncovNP in real serum samples.

[0135] Table 2 Recovery results of ncovNP in human serum samples

[0136]

[0137] The present invention innovatively develops β-cyclodextrin (β-CD) with an internal hydrophobic cavity to achieve the hydrophobic local enrichment of the co-reactant TDBA. Meanwhile, the ligand sites of β-CD metal ions enable Pt NPs to grow in situ on β-CD, which can also immobilize TDBA through Pt-N bonds. The synergistic effect of the two forces, namely hydrophobic local enrichment and covalent bonding, achieves a high immobilization amount of TDBA on the electrode surface. Meanwhile, the novel PDP PNPs open up a promising ECL polymer emitter. The combination of TDBA-β-CD-Pt nanocomposites and PDP PNPs enables the sensitive detection of ncovNP, providing a new detection method for ncovNP. More importantly, β-CD provides the inspiration for the hydrophobic local enrichment of co-reactants to improve the sensitivity of ECL detection.

[0138] [1] Eissa, S.; Zourob, M. Development of a Low-Cost Cotton-Tipped Electrochemical Immunosensor for the Detection of SARS-CoV-2. Anal. Chem. 2021, 93, 1826 - 1833.

[0139] [2] Grant, B. D.; Anderson, C. E.; Williford, J. R.; Alonzo, L. F.; Glukhova, V. A.; Boyle, D. S.; Weigl, B. H.; Nichols, K. P. SARS-CoV-2 Coronavirus Nucleocapsid Antigen-Detecting Half-Strip Lateral Flow Assay Toward the Development of Point of Care Tests Using Commercially Available Reagents. Anal. Chem. 2020, 92, 11305 - 11309.

[0140] [3]Eissa, S.; Alhadrami, H. A.; Al-Mozaini, M.; Hassan, A. M.; Zourob, M. Voltammetric-based immunosensor for the detection of SARS-CoV-2 nucleocapsid antigen. Microchimica Acta. 2021, 188.

[0141] [4]Liu, J. H.; Ruan, G. T.; Ma, W. L.; Sun, Y. J.; Yu, H. D.; Xu, Z. H.; Yu, C. M.; Li, H.; Zhang, C. W.; Li, L. Horseradish peroxidase-triggered direct in situ fluorescent immunoassay platform for sensing cardiac troponin I and SARS-CoV-2 nucleocapsid protein in serum. Biosens. Bioelectron. 2021, 113823.

[0142] [5]Liang, C. L.; Liu, B. C.; Li, J. F.; Lu, J. H.; Zhang, E. H.; Deng, Q. T.; Zhang, L.; Chen, R. A.; Fu, Y. S.; Li, C. Y.; Li, T. T. A nanoenzyme linked immunochromatographic sensor for rapid and quantitative detection of SARS-CoV-2 nucleocapsid protein in human blood. Sens. Actuators B Chem. 2021, 349, 130718.

[0143] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. An ECL detection platform, characterized in that, It includes a biosensor and an antibody complex; The biosensor includes: a working electrode, on which an ECL emitter is modified; an antibody is conjugated to the ECL emitter; The antibody complex includes: a nanocomplex, and an antibody, and the metal nanoparticles of the nanocomplex are covalently bonded to the amino group of the antibody through a metal-N bond; The nanocomplex includes the following raw materials: β-cyclodextrin, a coreactant, and metal nanoparticles, the metal nanoparticles are in-situ grown on the β-cyclodextrin, the metal nanoparticles are covalently linked to the amino group of the coreactant through a metal-N bond, and the coreactant is located in the hydrophobic inner cavity of the β-cyclodextrin, and the coreactant is a hydrophobic compound with an amino group.

2. The ECL detection platform according to claim 1, wherein, The metal nanoparticles are selected from platinum, lead, zinc, tin, copper, gold or silver; The coreactant is 3-(dibutylamino)propylamine, N,N-dimethylethylenediamine, N,N-diethylethylenediamine, N,N-diisopropylethylenediamine, tripropylamine, triethylamine or tetramethylethylenediamine.

3. The ECL detection platform according to claim 1, characterized in that, The ECL emitter is selected from carboxyl-functionalized poly[2,5-dioctyl-1,4-phenylene] nanoparticles.

4. The ECL detection platform according to claim 3, wherein The antibody conjugated to the ECL emitter is an antibody against the SARS-CoV-2 nucleocapsid protein; The antibody in the antibody complex is an antibody against the SARS-CoV-2 nucleocapsid protein.

5. The ECL detection platform according to claim 1, characterized in that, The nanocomplex is prepared by the following method, and its preparation method includes: mixing a solution containing metal ions with β-cyclodextrin, and after reduction, reacting to obtain a β-cyclodextrin-metal nanoparticle complex, and then mixing the β-cyclodextrin-metal nanoparticle complex with a coreactant and reacting to obtain a nanocomplex combined with the coreactant.

6. The ECL detection platform according to claim 5, characterized in that, Mix a solution containing metal ions with β-cyclodextrin, and the volume-mass ratio when mixing the solution containing metal ions with β-cyclodextrin is: 10-100 μL: 1-3 mg; and the volume concentration of the solution containing metal ions is 1-1.2%.

7. The ECL detection platform according to claim 6, characterized in that, Stir the solution after mixing the solution containing the metal nanoparticles with β-cyclodextrin, add a reducing agent to react, and then add sodium citrate to obtain a β-cyclodextrin-metal nanoparticle complex.

8. The ECL detection platform according to claim 5, wherein Before mixing with the coreactant, it also includes washing the β-cyclodextrin-metal nanoparticle complex to obtain a dispersion of the β-cyclodextrin-metal nanoparticle complex; the stirring time is 12 h-14 h; The mixing volume ratio of the β-cyclodextrin-metal nanoparticle complex to the coreactant is 70-200:1, and the mixing reaction time of the β-cyclodextrin-metal nanoparticle complex and the coreactant is 10-18 h, and the reaction temperature is 0-5 °C.

9. The ECL detection platform according to claim 1, characterized in that, The preparation method of the biosensor includes: activating the working electrode modified with the ECL emitter, and then incubating and blocking the activated working electrode with the antibody.

10. The ECL detection platform according to claim 9, wherein The preparation method also includes modifying the ECL emitter on the working electrode, which includes: drop-coating a nanodispersion of carboxyl-functionalized poly[2,5-dioctyl-1,4-phenylene] on the working electrode and drying.

11. The ECL detection platform according to claim 10, wherein, The preparation method further includes the preparation of a carboxyl-functionalized poly[2,5-dioctyl-1,4-phenylene] nano-dispersion, which includes mixing PDP with PSMA, adding water and then evaporating to obtain the carboxyl-functionalized poly[2,5-dioctyl-1,4-phenylene] nano-dispersion.

12. The ECL detection platform according to claim 1, wherein The preparation method of the antibody complex includes the following preparation methods (1) or (2): (1) Mixing and reacting a β-cyclodextrin-metal nanoparticle complex, a co-reactant, and an antibody; (2) Directly mixing and reacting the nano-complex with the antibody.

13. The ECL detection platform according to claim 12, wherein, In the mixing reaction described in the preparation method (1) or (2), the added antibody is added in the form of an antibody solution, and the concentration of the antibody solution is 45-200 μg / mL.

14. The ECL detection platform according to claim 13, wherein In the mixing reaction described in the preparation method (1) or (2), the added antibody is added in the form of an antibody solution, and the concentration of the antibody solution is 50 μg / mL.

15. The ECL detection platform according to claim 12, characterized in that, In the preparation method (1), the mixing volume ratio of the β-cyclodextrin-metal nanoparticle complex, the co-reactant, and the antibody solution is 1:70-200:

10.

16. The ECL detection platform according to claim 12, wherein After the mixing reaction in the preparation method (1) or (2), the solution is blocked, centrifuged, and dispersed; The dispersion is carried out in a PBS solution; the pH of the PBS is 7-8.

17. The ECL detection platform according to claim 16, wherein, The pH of the PBS is 7.

4.

18. A method for detecting SARS-CoV-2 using the ECL detection platform for SARS-CoV-2 according to any one of claims 1-17, wherein the detection method is for non-diagnostic purposes of diseases, and is characterized in that, It includes: Mixing and incubating the biosensor with the sample to be tested, then adding the antibody complex and incubating, and recording the ECL signal through the electrode system.

19. The detection method according to claim 18, wherein, The mixing and incubation time of the biosensor with the sample to be tested is 55-120 min.

20. The detection method according to claim 19, wherein The mixing and incubation time of the biosensor with the sample to be tested is 60 min.

21. The detection method according to claim 18, wherein The incubation time after adding the antibody complex is 55-80 min.

22. The detection method according to claim 21, wherein The incubation time after adding the antibody complex is 60 min.

23. The detection method according to claim 18, characterized in that, The ECL signal detection is carried out in PBS, the scanning potential is 0 V to +1.5 V, and the scanning rate and photomultiplier tube are set to 300 mV / s and 800 V, respectively.

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