Preparation and application of a Cu3(HHTP)2-based nanosheet sandwich electrochemiluminescence immunosensor

Abstract

The invention discloses a preparation and application of a Cu3(HHTP)2-based nanosheet sandwich electrochemiluminescent immunosensor, which relates to the field of immunosensors. The preparation method comprises the following steps: S1: preparing Ru@Cu3(HHTP)2 nanosheets; S2: preparing a BSA-enclosed Ru@Cu3(HHTP)2-Ab1 bioconjugate; S3: preparing a GO-Au nanocomposite; S4: preparing a BSA-enclosed GO-Au-Ab2 bioconjugate; S5: preparing a Cu3(HHTP)2-based nanosheet sandwich electrochemiluminescent immunosensor; and 1 is slowly dripped onto the working electrode; after drying, different concentrations of CRP antigen are placed on the working electrode to recognize and bind to Ab1 coupled with Ru@Cu3(HHTP)2. BSA-blocked GO-Au-Ab2 bioconjugate is added to the working electrode coated with CRP antigen and Ru@Cu3(HHTP)2-Ab1 as an ECL quencher; unbound GO-Au-Ab2 is eluted with a buffer solution to complete the preparation of the working electrode. The prepared working electrode is then assembled on the immunosensor to obtain the Cu3(HHTP)2-based nanosheet sandwich electrochemiluminescence immunosensor. The present invention is used for highly sensitive detection of C-reactive protein.

Description

Technical Field

[0001] The present invention relates to the field of immunosensors, in particular to the preparation and application of a Cu3(HHTP)2-based nanosheet sandwich electrochemiluminescence immunosensor. Background Art

[0002] C-reactive protein (CRP) is a key marker associated with inflammation in the body. Serum CRP levels are closely associated with a variety of complex chronic diseases, making quantitative CRP detection of significant importance. Abnormal CRP expression is closely linked to cardiovascular disease, Alzheimer's disease, lung cancer, and other conditions. Therefore, sensitive measurement of CRP at low abundance levels is crucial for accurate clinical assessment of disease.

[0003] Currently, serum CRP detection is primarily based on immunological principles; commonly used clinical methods include immunoturbidimetry and turbidimetry, as well as enzyme-linked immunosorbent assays, fluorescence, and radioactivity. These methods are generally insensitive, time-consuming, prone to false-negative results, or costly, ineffective, cumbersome, and require bulky equipment, making them unsuitable for on-site screening of individuals at risk for coronary heart disease.

[0004] Immunosensors have the potential for development due to their low cost, flexibility, high sensitivity, speed, and portability. They are increasingly used in clinical sample testing. Currently reported sandwich-based enzyme-linked immunosorbent assays (ELISAs) often use horseradish peroxidase as a marker, but these methods suffer from drawbacks such as easy inactivation, demanding experimental conditions, storage difficulties, difficulty in preparation, and high cost, making them unsuitable for on-site testing.

[0005] This application uses Ru@Cu3(HHTP)2 as a donor and GO-Au as an acceptor to realize electrochemiluminescence resonance energy transfer (ECL-RET), and constructs an electrochemiluminescence immunosensor based on this energy transfer mechanism to achieve highly sensitive analysis of C-reactive protein. Summary of the Invention

[0006] The present invention aims to provide a preparation and application of a Cu3(HHTP)2-based nanosheet sandwich electrochemiluminescence immunosensor for highly sensitive detection of C-reactive protein.

[0007] In order to solve the above technical problems, the specific solution adopted by the present invention is: a method for preparing a Cu3(HHTP)2 nanosheet sandwich electrochemiluminescence immunosensor, comprising the following steps:

[0008] S1: Preparation of Ru@Cu3(HHTP)2 nanosheets; Cu(CO2CH3)2·H2O and HHTP were dissolved in methanol, mixed, and centrifuged to obtain Cu3(HHTP)2 nanosheets, and then Ru(bpy)3Cl2 aqueous solution was added to obtain Ru@Cu3(HHTP)2 nanosheets;

[0009] S2: Preparation of BSA-blocked Ru@Cu3(HHTP)2-Ab1 bioconjugates; the Ru@Cu3(HHTP)2 nanosheets obtained in step S1 are dispersed in water, APTES is added for cross-linking reaction, and Ab1 solution is added. The mixture is centrifuged and BSA solution is added to obtain BSA-blocked Ru@Cu3(HHTP)2-Ab1 bioconjugates for later use;

[0010] S3: Preparation of GO-Au nanocomposite: Au NPs were added to the GO@PEI solution, stirred, and centrifuged to obtain the GO-Au nanocomposite;

[0011] S4: preparing a BSA-blocked GO-Au-Ab2 bioconjugate; dispersing the GO-Au nanocomplex prepared in step S3 in PBS to obtain a GO-Au suspension; adding the Ab2 solution, centrifuging, and then adding the BSA solution to obtain a BSA-blocked GO-Au-Ab2 bioconjugate for later use;

[0012] S5: Prepare a Cu3(HHTP)2-based nanosheet sandwich electrochemiluminescence immunosensor; slowly drop BSA-blocked Ru@Cu3(HHTP)2-Ab1 on the working electrode; after drying, place CRP antigens of different concentrations on the working electrode to recognize and bind to Ab1 coupled with Ru@Cu3(HHTP)2, and add BSA-blocked GO-Au-Ab2 bioconjugate as an ECL quencher to the working electrode coated with CRP antigen and Ru@Cu3(HHTP)2-Ab1; elute the unbound GO-Au-Ab2 with buffer solution to complete the preparation of the working electrode, and then assemble the prepared working electrode on the immunosensor to obtain the Cu3(HHTP)2-based nanosheet sandwich electrochemiluminescence immunosensor.

[0013] As a further optimization of the above technical solution, step S1 is specifically as follows: S101: dissolving Cu(CO2CH3)2·H2O in methanol to obtain solution I; dissolving HHTP in methanol to obtain solution II; mixing solution I and solution II, thermally reacting, and centrifuging to obtain Cu3(HHTP)2 nanosheets; S102: vacuum drying the Cu3(HHTP)2 nanosheets to obtain Cu3(HHTP)2 powder; S103: dispersing the Cu3(HHTP)2 powder in water and injecting Ru(bpy)3Cl2 aqueous solution, centrifuging after oil bath, and freeze-drying to obtain Ru@Cu3(HHTP)2 nanosheets.

[0014] As a further optimization of the above technical solution, in step S101, solution I and solution II were mixed in a round-bottom flask, and after magnetic stirring for 10 min, the mixture was slowly added to a polytetrafluoroethylene autoclave and stored at 65°C for 24 h. After the solvent thermal reaction, the Cu3(HHTP)2 nanosheets were obtained by centrifugation.

[0015] As a further optimization of the above technical solution, step S2 is specifically as follows: S201: dispersing the Ru@Cu3(HHTP)2 nanosheets prepared in step S1 in water, adding APTES, oil bathing, and centrifuging to obtain Ru@Cu3(HHTP)2 nanosheets with APTES loaded on the surface; S203: mixing the Ru@Cu3(HHTP)2 nanosheets with APTES loaded on the surface with GA as a cross-linking agent to activate the APTES amino groups connected to the surface of the Ru@Cu3(HHTP)2 nanosheets; S2 04: Disperse the APTES amino-activated Ru@Cu3(HHTP)2 nanosheets in PBS, add Ab1 solution, and centrifuge to obtain Ru@Cu3(HHTP)2-Ab1 bioconjugate; S205: Dissolve the Ru@Cu3(HHTP)2-Ab1 bioconjugate in PBS, add BSA solution to block the nonspecific active sites remaining on the surface of Ru@Cu3(HHTP)2-Ab1, and obtain BSA-blocked Ru@Cu3(HHTP)2-Ab1.

[0016] As a further optimization of the above technical solution, step S3 is specifically as follows: S301: adding HAuCl4·4H2O solution to water, heating to boiling, adding a reducing agent, stirring, cooling, and centrifuging to obtain Au NPs; S302: adding PEI solution to GO solution, stirring, and centrifuging to obtain GO@PEI; S303: adding Au NPs to the GO@PEI solution, stirring, and centrifuging to obtain GO-Au nanocomplex.

[0017] As a further optimization of the above technical solution, step S302 is specifically as follows: adding the PEI solution to the GO solution, then stirring at 60°C for 12 h, obtaining GO@PEI by centrifugation and washing, and further dispersing it in water to obtain a GO@PEI solution.

[0018] As a further optimization of the above technical solution, step S4 is specifically as follows: S401: dispersing the GO-Au nanocomplex prepared in step S3 in PBS to obtain a GO-Au suspension; S402: adding a coupling solution composed of EDC and NHS to the GO-Au suspension to activate the carboxyl groups distributed on the surface of GO-Au; S403: introducing the Ab2 solution into the carboxyl-activated GO-Au, and centrifuging to obtain a GO-Au-Ab2 bioconjugate; S404: dispersing the GO-Au-Ab2 bioconjugate in PBS, and adding BSA to block the nonspecific active sites on the surface of the GO-Au-Ab2 bioconjugate, thereby obtaining a BSA-blocked GO-Au-Ab2 bioconjugate.

[0019] As a further optimization of the above technical solution, step S5 is specifically as follows: S501: polish the working electrode with a polishing solution composed of alumina powder and water, then rinse with water / ethanol and dry with N2; S502: slowly drop BSA-blocked Ru@Cu3(HHTP)2-Ab1 as an ECL emitter onto the pretreated working electrode; S503: place CRP antigens of different concentrations on the working electrode to recognize and bind to Ab1 coupled with Ru@Cu3(HHTP)2; S504: add BSA-blocked GO-Au-Ab2 bioconjugate as an ECL quencher to the working electrode coated with CRP antigen and Ru@Cu3(HHTP)2-Ab1 for immunization; S505: after the immunization is completed, elute the unbound GO-Au-Ab2 with a buffer solution to obtain an immunosensor.

[0020] As a further optimization of the above technical solution, in step S503, BSA-blocked Ru@Cu3(HHTP)2-Ab1 was slowly dripped onto the pretreated GCE as an ECL emitter and dried at 4°C. Then, different concentrations of CRP antigen were placed on the above GCE to recognize and bind to Ab1 coupled with Ru@Cu3(HHTP)2. After maintaining at 37°C for 30 minutes, unbound CRP was eluted with PBS.

[0021] Application of a Cu3(HHTP)2-based nanosheet sandwich electrochemiluminescence immunosensor in the detection of C-reactive protein.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The Cu3(HHTP)2 nanosheets used in the present invention are an electronically conductive metal organic framework (MOF) having a periodically arranged porous structure with a cavity size of 2 nm, which can not only accommodate a large amount of Ru(bpy)3 2+ , and also limits the spatial diffusion of active substances. Therefore, Ru@Cu3(HHTP)2 can show higher ECL efficiency as an ECL emitter. In the GO-Au composite material prepared by the present invention, GO allows the carrying of a large number of Au particles, avoiding their aggregation in space, thereby effectively quenching the ECL of Ru@Cu3(HHTP)2. A sandwich electrochemiluminescence immunosensor based on the ECL-RET mechanism can be used to target the detection of CRP in human serum samples, with a detection limit as low as 0.26 pg mL -1 .

[0024] Electrochemiluminescence (ECL), an electrically driven luminescence phenomenon, is widely used in the field of biosensing due to its advantages such as low background signal, fast response speed, adjustable potential, and easy signal readout. Based on the high pore density and excellent electrocatalytic performance of Cu3(HHTP)2 nanosheets, a large amount of Ru(bpy)3 2+ Luminophores were constructed to construct Ru@Cu3(HHTP)2ECL complexes, thereby achieving strong ECL in the presence of the co-reactant tripropylamine (TPA). At the same time, the large specific surface area and excellent electron transport properties of graphene oxide nanosheets (GO) can support a large number of Au particles, avoiding their aggregation in space and forming a GO-Au complex. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Middle: (A) Schematic diagram of the preparation process of GO-Au-Ab2; (B) Schematic diagram of the preparation process of Ru@Cu3(HHTP)2-Ab1; (C) Schematic diagram of the assembly process of ECL-RET immunosensor;

[0026] Figure 2 Middle: (A) Transmission electron microscopy image of Cu3(HHTP)2 crystal; (B) Zeta-potential diagrams corresponding to different particles (Cu3(HHTP)2, Ru@Cu3(HHTP)2, Ru@Cu3(HHTP)2-APTES); (C) (a) Ru@Cu3(HHTP)2, (b) Cu3(HHTP)2, (c) Ru(bpy)3 2+ UV-visible absorption spectra; (D) IR spectra of the synthesized Cu3(HHTP)2, Ru@Cu3(HHTP)2, and Ru@Cu3(HHTP)2-APTES;

[0027] Figure 3Medium: different concentrations of CRP (ai: 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50 ng mL -1 ) Signal quenching ECL curve (A) and corresponding calibration curve (B) of the ECL-RET immunoassay system; Stability of the immunosensor (C) (CRP levels were 0.1 and 10 ng mL -1 ) and selectivity (D)(CRP: 5ng mL -1 ; Other interfering agents: 500ng mL -1 ). Error bars represent standard deviation (n=3);

[0028] Figure 4 Middle: (a) SEM image of Cu3(HHTP)2; (b) TEM image of GO; (c) TEM image of GO-Au;

[0029] Figure 5 Middle: UV-visible absorption spectra of (a) GO, (b) GO-Au, and (c) Au;

[0030] Figure 6 Middle: (A) UV-visible absorption spectrum of GO-Au (a) and ECL emission spectrum of Ru@Cu3(HHTP)2 (b); (B) ECL curves of different modified electrodes: (a) GCE / Ru@Cu3(HHTP)2 / Ab1 / BSA / CRP; (b) GCE / Ru@Cu3(HHTP)2 / Ab1 / BSA / CRP / GO-Ab2;

[0031] (c)GCE / Ru@Cu3(HHTP)2 / Ab1 / BSA / CRP / Au-Ab2;

[0032] (d) GCE / Ru@Cu3(HHTP)2 / Ab1 / BSA / CRP / GO-Au-Ab2. DETAILED DESCRIPTION

[0033] The technical solution of the present invention is further elaborated below. Parts not described and disclosed in detail in the following embodiments of the present invention should be understood as existing technologies known or should be known to those skilled in the art.

[0034] The present invention discloses a method for preparing a Cu3(HHTP)2-based nanosheet sandwich electrochemiluminescence immunosensor, comprising the following steps:

[0035] S1: Preparation of Ru@Cu3(HHTP)2 nanosheets; Cu(CO2CH3)2·H2O and HHTP were dissolved in methanol, mixed, and centrifuged to obtain Cu3(HHTP)2 nanosheets, and then Ru(bpy)3Cl2 aqueous solution was added to obtain Ru@Cu3(HHTP)2 nanosheets;

[0036] S101: Dissolve Cu(CO2CH3)2·H2O in methanol to prepare solution I; dissolve HHTP in methanol to prepare solution II; mix solution I and solution II, perform thermal reaction, and centrifuge to obtain Cu3(HHTP)2 nanosheets; specifically, mix solution I and solution II in a round-bottom flask, stir magnetically for 10 minutes, slowly add the mixture into a polytetrafluoroethylene autoclave, store at 65°C for 24 hours, and after solvent thermal reaction, centrifuge to obtain Cu3(HHTP)2 nanosheets.

[0037] S102: vacuum drying the Cu3(HHTP)2 nanosheets to obtain Cu3(HHTP)2 powder;

[0038] S103: Disperse Cu3(HHTP)2 powder in water and inject Ru(bpy)3Cl2 aqueous solution, then centrifuge and freeze-dry in an oil bath to obtain Ru@Cu3(HHTP)2 nanosheets.

[0039] S2: Preparation of BSA-blocked Ru@Cu3(HHTP)2-Ab1 bioconjugates; Ru@Cu3(HHTP)2 nanosheets were dispersed in water, APTES was added for cross-linking reaction, and Ab1 solution was added. The mixture was centrifuged and BSA solution was added to obtain BSA-blocked Ru@Cu3(HHTP)2-Ab1 bioconjugates for later use;

[0040] S201: Disperse the Ru@Cu3(HHTP)2 nanosheets prepared in step S1 in water, add APTES, and perform oil bath and centrifugation to obtain Ru@Cu3(HHTP)2 nanosheets with APTES loaded on the surface;

[0041] S203: mixing the Ru@Cu3(HHTP)2 nanosheets loaded with APTES on the surface with GA as a cross-linking agent to activate the APTES amino groups connected to the surface of the Ru@Cu3(HHTP)2 nanosheets;

[0042] S204: Dispersing Ru@Cu3(HHTP)2 nanosheets activated by APTES amino groups in PBS, adding Ab1 solution, and centrifuging to obtain Ru@Cu3(HHTP)2-Ab1 bioconjugate;

[0043] S205: The Ru@Cu3(HHTP)2-Ab1 bioconjugate is dissolved in PBS, and a BSA solution is added to block the nonspecific active sites remaining on the surface of Ru@Cu3(HHTP)2-Ab1, thereby obtaining BSA-blocked Ru@Cu3(HHTP)2-Ab1.

[0044] S3: Preparation of GO-Au nanocomposite: Au NPs were added to the GO@PEI solution, stirred, and centrifuged to obtain the GO-Au nanocomposite;

[0045] S301: Add HAuCl4·4H2O solution to water, heat to boiling, add reducing agent, stir, cool, and centrifuge to obtain Au NPs;

[0046] S302: adding the PEI solution to the GO solution, stirring, and centrifuging to obtain GO@PEI;

[0047] S303: Adding Au NPs to the GO@PEI solution, stirring, and centrifuging to obtain a GO-Au nanocomposite.

[0048] S4: preparing a BSA-blocked GO-Au-Ab2 bioconjugate; dispersing the GO-Au nanocomplex prepared in step S3 in PBS to obtain a GO-Au suspension; adding the Ab2 solution, centrifuging, and then adding the BSA solution, centrifuging to obtain a BSA-blocked GO-Au-Ab2 bioconjugate for later use;

[0049] S401: dispersing the GO-Au nanocomposite prepared in step S3 in PBS to obtain a GO-Au suspension;

[0050] S402: adding a coupling solution composed of EDC and NHS to the GO-Au suspension to activate the carboxyl groups distributed on the surface of GO-Au;

[0051] S403: introducing the Ab2 solution into the carboxyl-activated GO-Au, and centrifuging to obtain a GO-Au-Ab2 bioconjugate;

[0052] S404: Dispersing the GO-Au-Ab2 bioconjugate in PBS, adding BSA to block the nonspecific active sites on the surface of the GO-Au-Ab2 bioconjugate, thereby preparing a BSA-blocked GO-Au-Ab2 bioconjugate.

[0053] S5: Prepare a Cu3(HHTP)2-based nanosheet sandwich electrochemiluminescence immunosensor; slowly drop BSA-blocked Ru@Cu3(HHTP)2-Ab1 onto the working electrode; place different concentrations of CRP antigen on the working electrode to recognize and bind to Ab1 coupled with Ru@Cu3(HHTP)2, and add BSA-blocked GO-Au-Ab2 bioconjugate as an ECL quencher to the working electrode coated with CRP antigen and Ru@Cu3(HHTP)2-Ab1 for immunofluorescence; elute the unbound GO-Au-Ab2 with buffer solution to prepare the immunosensor.

[0054] S501: polishing the working electrode with a polishing solution consisting of aluminum oxide powder and water, then rinsing with water / ethanol and drying with nitrogen;

[0055] S502: BSA-encapsulated Ru@Cu3(HHTP)2-Ab1 is slowly dropped onto the pretreated working electrode as the ECL emitter;

[0056] S503: CRP antigens at different concentrations were placed on the working electrode to recognize and bind to Ab1 conjugated with Ru@Cu3(HHTP)2. S504: BSA-blocked GO-Au-Ab2 bioconjugates were added as ECL quenchers to the working electrode coated with CRP antigen and Ru@Cu3(HHTP)2-Ab1 for immunofluorescence.

[0057] S505: After the immunization is completed, unbound GO-Au-Ab2 is eluted with a buffer solution, completing the preparation of the working electrode. The prepared working electrode is then assembled on the immunosensor to produce the Cu3(HHTP)2-based nanosheet sandwich electrochemiluminescence immunosensor. It should be noted that the assembly of the working electrode on the immunosensor is a prior art and will not be further described here.

[0058] The technical solution of the present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0059] Example 1

[0060] Preparation of Ru@Cu3(HHTP)2 nanosheets:

[0061] 0.110 g of Cu(CO₂CH₃)₂·H₂O was dissolved in 35 mL of methanol to form a metal precursor solution. 0.087 g of HHTP was added to 42 mL of methanol and ultrasonicated for 5 minutes. The two dispersed precursor solutions were mixed in a 100 mL round-bottom flask. After magnetic stirring for 10 minutes, the mixture was slowly added to a 100 mL polytetrafluoroethylene autoclave and stored at 65°C for 24 hours. After solvothermal reaction, the mixture was centrifuged (9000 rpm for 10 minutes) to obtain black Cu₃(HHTP)₂ nanosheets. The sample was then washed several times with methanol and acetone, respectively. After washing, it was dried in air at 323 K for 6 hours and then in vacuum at 373 K for 12 hours. 50 mg of the prepared Cu₃(HHTP)₂ powder was ultrasonically dispersed in 10 mL of water, and 0.3 mL of a 50 mM Ru(bpy)₃Cl₂ aqueous solution was then injected into the dispersion. After ultrasonic treatment for 15 min, the mixture was placed in an oil bath and stirred at 60 °C for 12 h. Subsequently, Ru(bpy)3 was collected by centrifugation (9000 rpm, 10 min). 2+ The modified Cu3(HHTP)2 nanosheets (Ru@Cu3(HHTP)2) were prepared and washed with water. The collected final precipitate was freeze-dried for 12 h to obtain Ru@Cu3(HHTP)2 nanosheets.

[0062] Preparation of GO-Au nanocomposites:

[0063] 1 mL of HAuCl4·4H2O solution (10 mg mL -1 ) was mixed into 97 mL of water to form a yellow solution, which was heated to boiling. Under vigorous stirring, 3 mL of trisodium citrate (10 mg mL -1 ) was quickly added to the boiling solution as a reducing agent. The resulting mixture was stirred for 15 min until its color changed from bright yellow to wine red. After cooling to room temperature, the mixture was centrifuged (12000 rpm, 12 min), washed with water, and redispersed in 5 mL of water. For the surface modification of GO, 0.5 mL of PEI solution (100 mg mL -1 ) was added to 5 mL of GO solution (5 mg mL -1 ) and then stirred at 60°C for 12 hours. Five PEI-modified GO (GO@PEI) nanoparticles were obtained by centrifugation and washing, and further dispersed in 5 mL of water. For the connection between GO and Au NPs, 0.8 mL of Au NPs were added to 5 mL of the GO@PEI solution and stirred at room temperature for 10 hours. Finally, the GO-Au complex was collected by centrifugation, washed with water, and dispersed in 5 mL of PBS.

[0064] Preparation of Ru@Cu3(HHTP)2-Ab1 bioconjugate:

[0065] Prior to antibody conjugation, amino-functionalized Ru@Cu3(HHTP)2 was synthesized as follows: 4 mg of Ru@Cu3(HHTP)2 solid was dispersed in 5 mL of water and sonicated for 15 min. 100 μL of APTES was added and the solution was stirred in a 55°C oil bath. The reaction lasted for 16 h, and the precipitated sample was obtained after centrifugation / washing. 5 mL of water was then added to the precipitate to redisperse the luminescent complex for subsequent use. Subsequently, 2.5 mL of 0.8 mg mL -1 Ru@Cu3(HHTP)2 was mixed with GA as a crosslinker to activate the APTES amino groups attached to the surface of Ru@Cu3(HHTP)2 nanosheets. After reacting at 4°C for 2 h, the activated product was purified by centrifugation and washed and redispersed in 2.5 mL of PBS. In order to couple the capture antibody to the amino-activated Ru@Cu3(HHTP)2, 100 μL of Ab1 solution (10 μg mL -1 , diluted with 100mM PBS). After incubation at 4°C for 12h, the Ru@Cu3(HHTP)2-Ab1 bioconjugate was collected by centrifugation and washed twice to remove residual Ab1. The purified Ru@Cu3(HHTP)2-Ab1 was redissolved in 2.5mL PBS. 100μL of BSA solution (5%, dissolved in 100mM PBS) was then added to block the remaining nonspecific active sites on the surface of Ru@Cu3(HHTP)2-Ab1. The BSA-blocked Ru@Cu3(HHTP)2-Ab1 was collected by centrifugation and dispersed in 2.5mL PBS.

[0066] Preparation of GO-Au-Ab2 bioconjugate:

[0067] To prepare the GO-Au recognition body modified with the detection antibody (Ab2), 100 μL of the coupling solution consisting of EDC (80 mM) and NHS (40 mM) was added to 2 mL of 1.8 mg mL -1 The GO-Au suspension was added to activate the carboxyl groups distributed on the GO-Au surface. The mixed solution was shaken at room temperature for 30 min. Then, 100 μL of Ab2 solution (10 μg mL -1 , dissolved in 100 mM PBS) was introduced into the carboxyl-activated GO-Au. The conjugation process was performed at 4°C for 12 hours. The GO-Au-Ab2 bioconjugate was obtained by centrifugation, washed with PBS to remove excess Ab2, and redispersed in 2 mL of PBS. To block nonspecific active sites on the surface of the GO-Au-Ab2 bioconjugate, 100 μL of BSA was added. The reaction was allowed to proceed at 4°C for 4 hours. The purified GO-Au-Ab2 / BSA bioconjugate was collected and dispersed in 2 mL of PBS.

[0068] Preparation of a nanoplate sandwich-type electrochemiluminescence immunosensor based on Cu3(HHTP)2

[0069] The ECL-based immunoassay was carried out in a three-electrode system, and its preparation process was as Figure 1 shown. Before assembling the signal probe, the working electrode (GCE, 3 mm) was polished with a polishing solution composed of alumina powder and water, then rinsed with water / ethanol and dried with N2. Then, 8 μL of BSA-blocked Ru@Cu3(HHTP)2-Ab1 as the ECL emitter was slowly dropped onto the pretreated GCE. After drying at 4 °C, different concentrations of CRP antigen were placed on the above-mentioned GCE to recognize and bind to Ab1 that had been conjugated with Ru@Cu3(HHTP)2. After maintaining at 37 °C for 30 min, unbound CRP was eluted with PBS. To immobilize Ab2 on the GCE surface, 8 μL of the GO-Au-Ab2 bioconjugate as the ECL quencher was added to the GCE coated with CRP antigen and Ru@Cu3(HHTP)2-Ab1. After the immunoreaction was completed, unbound GO-Au-Ab2 was eluted with a buffer solution. Finally, the ECL-based immunosensor was stored at 4 °C for the next detection.

[0070] <Characterization of Ru@Cu3(HHTP)2 nanosheets>

[0071] The Cu3(HHTP)2 sheet crystals prepared by the hydrothermal reaction of copper(II) acetate with HHTP served as the support material for the luminescent Ru(bpy)3 2+ in this sandwich-type immunosensor. Scanning electron microscope (SEM) images ( Figure 4 a) showed that the Cu3(HHTP)2 crystals tended to form aggregated spherical particles with an average size of 50 nm. According to transmission electron microscope (TEM) analysis ( Figure 2 A), the Cu3(HHTP)2 crystals consisted of stacked sheet nanostructures. To determine the electrostatic interactions associated with Ru(bpy)3 2+ and Cu3(HHTP)2, the surface potentials of Cu3(HHTP)2 and Ru@Cu3(HHTP)2 were determined using a Zeta potential analyzer. As Figure 2 shown in B, the Zeta potential of Cu3(HHTP)2 was approximately -15.27 mV, and after modification with the positively charged Ru(bpy)3 2+ this value changed to -6.53 mV. The change in the surface potential indicated that Ru(bpy)3 2+ was successfully immobilized in the pores and on the surface of Cu3(HHTP)2 through electrostatic adsorption. Meanwhile, the Ru(bpy)3 2+, the UV-visible spectra of Cu3(HHTP)2 and Ru@Cu3(HHTP)2 demonstrated the binding of Ru(bpy)3 2+ with Cu3(HHTP)2. Cu3(HHTP)2 exhibited distinct absorption peaks at 275, 368, and 650 nm ( Figure 2 C, line b). For the aqueous solution of Ru(bpy)3 2+ , the spectrum showed strong absorption intensities at 243, 285, and 454 nm (line c). In Ru@Cu3(HHTP)2, the absorption peak of Ru(bpy)3 2+ was at 285 nm and that of Cu3(HHTP)2 was at 364 nm, indicating the successful binding of Ru(bpy)3 2+ and Cu3(HHTP)2. To further investigate the surface chemical properties of Ru@Cu3(HHTP)2, the infrared spectra of Cu3(HHTP)2 and Ru@Cu3(HHTP)2 were characterized ( Figure 2 D). The spectrum of Cu3(HHTP)2 showed two strong peaks at 1214 and 1446 cm -1 , corresponding to C-O stretching vibration and C-H shear vibration, respectively. A broad absorption band was observed at 3340 cm -1 , which was attributed to the O-H stretching vibration of hydrogen bond. Compared with the original Cu3(HHTP)2, after loading Ru(bpy)3 2+ , Ru@Cu3(HHTP)2 had a similar infrared spectrum, demonstrating the structural stability of Cu3(HHTP)2. The amino group linked to Ab1 was modified on the surface of Ru@Cu3(HHTP)2. In the spectrum of Ru@Cu3(HHTP)-APTES, Si-O characteristic peaks were found at 1016 and 1103 cm -1 , revealing the presence of APTES. After interacting with APTES, the Zeta potential of Ru@Cu3(HHTP)2 changed from negative to positive (+5.67 mV), confirming the successful functionalization of APTES on the surface of Ru@Cu3(HHTP)2.

[0072] <Characterization of GO-Au Nanocomposites>

[0073] During the ECL-RET process, maintaining a uniform distribution of Au NPs on the surface of the carrier is crucial for promoting ECL-RET between Au NPs and Ru(bpy)3 2+ . Therefore, the morphology of the GO-Au composite was characterized by transmission electron microscopy. GO with a wrinkled sheet structure ( Figure 4 b) was used as the carrier to load Au NPs. The transmission electron microscopy image ( Figure 4c) It is clearly shown that Au NPs with a diameter of 16 nm are well distributed on the surface of graphene oxide.

[0074] <ECL-RET behavior between GO-Au and Ru@Cu3(HHTP)2>

[0075] To confirm the ECL-RET behavior between GO-Au and Ru@Cu3(HHTP)2, the spectral profiles of GO, Au NPs and Ru@Cu3(HHTP)2 were measured. The characteristic peaks of the ultraviolet-visible spectra of graphene oxide flakes and Au NPs ( Figure 5 ) are 230 and 520 nm, respectively. The GO-Au absorption spectrum has two obvious peaks at 230 (GO) and 520 (Au NPs) nm, demonstrating the presence of Au NPs on graphene oxide. The ECL emission peak of Ru@Cu3(HHTP)2 is 620 nm ( Figure 6 A, curve b). These spectra show that in the spectral range of 580 - 680 nm, the GO-Au absorption spectrum has a perfect overlap with the ECL emission of Ru@Cu3(HHTP)2, making the RET process possible. The ECL signals were recorded to evaluate the effects of GO, Au NPs and GO-Au on the ECL-RET process ( Figure 6 B). When antigen-antibody modified Ru@Cu3(HHTP)2 was incubated on the GCE electrode, the intensity-potential plot increased significantly from 1.0 V to 1.3 V (curve a). Injecting Ab2-conjugated graphene oxide into the test system, the ECL intensity decreased sharply at 1.3 V, indicating quenching behavior between Ru(bpy)3 2+ and GO. In addition, after adding Ab2-conjugated Au NPs, the ECL signal decreased from 8200 to 1850 a.u, thus confirming the energy transfer from Au NPs to Ru(bpy)3 2+ . When the same concentration of GO-Au-Ab2 was present, only 5% of the ECL intensity of Ru@Cu3(HHTP)2 could be observed. The results show that GO-Au can effectively quench the ECL of Ru@Cu3(HHTP)2.

[0076] <RET-based ECL immunosensor for C-reactive protein>

[0077] Under the optimal conditions, the intensity-potential plots of ECL obtained by the immunosensor with different CRP concentrations are shown in Figure 3 A. The GCE electrode covered with Ru@Cu3(HHTP)2 was incubated with CRP at 0.005 - 50 ng mL -1 , and then used to capture Ab2-labeled GO-Au, resulting in a gradual decrease in the ECL intensity. Therefore, this application developed a RET-based ECL immunosensor. Figure 3 B shows the correlation between ECL intensity (I) and the logarithm of CRP concentration (log c), with a linear equation of I = 2841-1530.22 lg c (correlation coefficient 0.991). The detection limit was 0.26 pg mL -1 , indicating that the constructed RET-based ECL immunoassay is sufficient to assess CRP levels in clinical samples. Compared with the detection limit and linear range of other representative CRP detection methods, the Ru@Cu3(HHTP)2 / GO-Au system provides good performance for the detection of CRP, as shown in Table 1.

[0078] Table 1

[0079]

[0080] In addition, excellent selectivity and stability are the key to evaluating the accuracy of ECL immunosensor detection results. Figure 3 C. Continuous scanning for 10 cycles and at 0.1 and 10 ng mL -1 The stable ECL response signal was recorded at the CRP of 0.1 ng / mL. The relative standard deviation (RSD) calculated based on the ECL peak intensity was 1.75% (CCRP = 0.1 ng / mL -1 ) and 1.59% (CCRP = 10 ng mL -1 ), indicating that the prepared ECL sensor has excellent stability. In order to verify the selectivity of the sandwich immunosensor, the present application conducted a series of control experiments. Although the concentration of interfering proteins (CA125, AFP, BSA and PSA) was 100 times higher than that of the target CRP (interfering and CRP were 500 and 5 ng / mL, respectively), the obtained ECL intensity was almost the same as that of the blank sample ( Figure 3 D). These results indicate that the GEC did not capture the GO-Au-Ab2 bioconjugate due to the lack of a recognition antigen. However, when CRP was present in the test system, the ECL intensity decreased significantly, indicating that only CRP could bind to Ru@Cu3(HHTP)2-Ab1. In summary, the sandwich immunosensor exhibits excellent stability and selectivity and can be used to determine CRP levels in human serum samples.

[0081] <Detection of C-reactive protein in human serum>

[0082] To demonstrate the applicability of the sandwich immunosensor, it was inserted into human serum samples to detect CRP levels. The accuracy of the immunosensor was verified by adding several standard concentrations of CRP (0.1, 2, and 10 ng mL -1All the results are shown in Table 2, with a recovery range of 98.2% to 99.0% and an RSD (n=3) of less than 3%, confirming the practicality of the sandwich immunosensor in clinical diagnosis.

[0083] Table 2

[0084]

[0085] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing a Cu3(HHTP)2 nanosheet sandwich electrochemiluminescence immunosensor, characterized in that: The following steps are involved: S1: Preparation of Ru@Cu3(HHTP)2 nanosheets; Cu(CO2CH3)2·H2O and HHTP were dissolved in methanol, mixed, and centrifuged to obtain Cu3(HHTP)2 nanosheets, and then Ru(bpy)3Cl2 aqueous solution was added to obtain Ru@Cu3(HHTP)2 nanosheets; S2: Preparation of BSA-blocked Ru@Cu3(HHTP)2-Ab1 bioconjugate; The Ru@Cu3(HHTP)2 nanosheets obtained in step S1 were dispersed in water, APTES was added for cross-linking reaction, and Ab1 solution was added, followed by centrifugation, and BSA solution was added to obtain BSA-blocked Ru@Cu3(HHTP)2-Ab1 bioconjugates for later use; S3: Preparation of GO-Au nanocomposite; Au NPs were added to the GO@PEI solution, stirred, and centrifuged to obtain the GO-Au nanocomposite; S4: Preparation of BSA-blocked GO-Au-Ab2 bioconjugates; The GO-Au nanocomplex prepared in step S3 was dispersed in PBS to obtain a GO-Au suspension; the Ab2 solution was added, the mixture was centrifuged, and the BSA solution was added to obtain a BSA-blocked GO-Au-Ab2 bioconjugate for later use; S5: Preparation of Cu3(HHTP)2-based nanosheet sandwich electrochemiluminescence immunosensor; BSA-encapsulated Ru@Cu3(HHTP)2-Ab1 is slowly dripped onto the working electrode; after drying, different concentrations of CRP antigen are placed on the working electrode to recognize and bind to Ab1 coupled with Ru@Cu3(HHTP)2, and BSA-encapsulated GO-Au-Ab2 bioconjugate is added to the working electrode coated with CRP antigen and Ru@Cu3(HHTP)2-Ab1 as an ECL quencher; unbound GO-Au-Ab2 is eluted with a buffer solution to complete the preparation of the working electrode, and the prepared working electrode is then assembled on the immunosensor to obtain the Cu3(HHTP)2-based nanosheet sandwich electrochemiluminescence immunosensor.

2. The method for preparing a Cu3(HHTP)2 nanosheet sandwich electrochemiluminescence immunosensor according to claim 1, characterized in that: Step S1 is specifically as follows: S101: dissolving Cu(CO2CH3)2·H2O in methanol to prepare solution I; dissolving HHTP in methanol to prepare solution II; mixing solution I and solution II, thermally reacting, and centrifuging to obtain Cu3(HHTP)2 nanosheets; S102: vacuum drying the Cu3(HHTP)2 nanosheets to obtain Cu3(HHTP)2 powder; S103: Disperse Cu3(HHTP)2 powder in water and inject Ru(bpy)3Cl2 aqueous solution, then centrifuge and freeze-dry after oil bath to obtain Ru@Cu3(HHTP)2 nanosheets.

3. The method for preparing a Cu3(HHTP)2-based nanosheet sandwich electrochemiluminescence immunosensor according to claim 2, characterized in that: In step S101, solution I and solution II were mixed in a round-bottom flask, and after magnetic stirring for 10 min, the mixture was slowly added to a polytetrafluoroethylene autoclave and stored at 65°C for 24 h. After solvothermal reaction, the mixture was centrifuged to obtain Cu3(HHTP)2 nanosheets.

4. The method for preparing a Cu3(HHTP)2 nanosheet sandwich electrochemiluminescence immunosensor according to claim 1, characterized in that: Step S2 is specifically as follows: S201: The Ru@Cu3(HHTP)2 nanosheets prepared in step S1 are dispersed in water, APTES is added, and the Ru@Cu3(HHTP)2 nanosheets with APTES loaded on the surface are obtained by oil bath and centrifugation. S203: mixing the Ru@Cu3(HHTP)2 nanosheets loaded with APTES on the surface with GA as a cross-linking agent to activate the APTES amino groups connected to the surface of the Ru@Cu3(HHTP)2 nanosheets; S204: Dispersing Ru@Cu3(HHTP)2 nanosheets activated by APTES amino groups in PBS, adding Ab1 solution, and centrifuging to obtain Ru@Cu3(HHTP)2-Ab1 bioconjugate; S205: The Ru@Cu3(HHTP)2-Ab1 bioconjugate is dissolved in PBS, and a BSA solution is added to block the nonspecific active sites remaining on the surface of Ru@Cu3(HHTP)2-Ab1, thereby obtaining BSA-blocked Ru@Cu3(HHTP)2-Ab1.

5. The method for preparing a Cu3(HHTP)2 nanosheet sandwich electrochemiluminescence immunosensor according to claim 1, characterized in that: Step S3 is specifically as follows: S301: Add HAuCl4·4H2O solution to water, heat to boiling, add reducing agent, stir, cool, and centrifuge to obtain Au NPs; S302: adding the PEI solution to the GO solution, stirring, and centrifuging to obtain GO@PEI; S303: Adding Au NPs to the GO@PEI solution, stirring, and centrifuging to obtain a GO-Au nanocomposite.

6. The method for preparing a Cu3(HHTP)2 nanosheet sandwich electrochemiluminescence immunosensor according to claim 5, characterized in that: Step S302 is specifically as follows: adding the PEI solution to the GO solution, then stirring at 60 °C for 12 h, obtaining GO@PEI by centrifugation and washing, and further dispersing it in water to obtain a GO@PEI solution.

7. The method for preparing a Cu3(HHTP)2 nanosheet sandwich electrochemiluminescence immunosensor according to claim 1, characterized in that: Step S4 is specifically as follows: S401: dispersing the GO-Au nanocomposite prepared in step S3 in PBS to obtain a GO-Au suspension; S402: adding a coupling solution composed of EDC and NHS to the GO-Au suspension to activate the carboxyl groups distributed on the surface of GO-Au; S403: introducing the Ab2 solution into the carboxyl-activated GO-Au, and centrifuging to obtain a GO-Au-Ab2 bioconjugate; S404: Dispersing the GO-Au-Ab2 bioconjugate in PBS, adding BSA to block the nonspecific active sites on the surface of the GO-Au-Ab2 bioconjugate, thereby preparing a BSA-blocked GO-Au-Ab2 bioconjugate.

8. The method for preparing a Cu3(HHTP)2 nanosheet sandwich electrochemiluminescence immunosensor according to claim 1, characterized in that: Step S5 is specifically as follows: S501: polishing the working electrode with a polishing solution consisting of aluminum oxide powder and water, then rinsing with water / ethanol and drying with nitrogen; S502: BSA-encapsulated Ru@Cu3(HHTP)2-Ab1 is slowly dropped onto the pretreated working electrode as the ECL emitter; S503: CRP antigens of different concentrations were placed on the working electrode to recognize and bind to Ab1 coupled with Ru@Cu3(HHTP)2; S504: BSA-blocked GO-Au-Ab2 bioconjugate was added as an ECL quencher to the working electrode coated with CRP antigen and Ru@Cu3(HHTP)2-Ab1 for immunofluorescence; S505: After the immunization is completed, the unbound GO-Au-Ab2 is eluted with a buffer solution to obtain an immunosensor.

9. The method for preparing a Cu3(HHTP)2 nanosheet sandwich electrochemiluminescence immunosensor according to claim 8, characterized in that: In step S503, BSA-blocked Ru@Cu3(HHTP)2-Ab1 was slowly dropped onto the pre-treated GCE as an ECL emitter and dried at 4°C. Then, different concentrations of CRP antigen were placed on the GCE to recognize and bind to the Ab coupled to Ru@Cu3(HHTP)2. 1， After incubation at 37°C for 30 min, unbound CRP was eluted with PBS.

10. Use of the Cu3(HHTP)2-based nanosheet sandwich electrochemiluminescence immunosensor according to claim 1 in the detection of C-reactive protein.

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