Double-signal immunoassay method based on core-shell structure composite metal nano enzyme

Abstract

According to the double-signal immunoassay method based on the core-shell structure composite metal nano enzyme, the good peroxidase activity of Au (at) CeO2 (at) Pt NPs and the characteristic that oxidized TMB can quench AuAg NCs red fluorescence are combined, the Au (at) CeO2 (at) Pt NPs is used for marking a target molecule monoclonal antibody and then serves as a marking probe, colorimetric and fluorescent double-signal recognition detection on a target molecule is effectively achieved, and the detection sensitivity is high. The accuracy and the anti-interference capability of immunodetection can be improved. Immunoassay of biomacromolecules, pathogens and the like such as proteins, viruses and bacteria can be realized by replacing recognition elements such as antibodies, and the method has a wide application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of immunoassay analysis, and specifically relates to a colorimetric-fluorescent dual-signal immunoassay method based on the peroxidase activity of composite metal nanoparticles and the quenching effect of oxidized TMB fluorescence signals, including the preparation of Au@CeO2@Pt nanoparticles with a core-shell structure, and coupling Au@CeO2@Pt nanoparticles with antibodies. Background Art

[0002] Enzyme-linked immunosorbent assay (ELISA) is a commonly used immunological method in biomedical diagnosis, environmental monitoring and food safety. It is widely popular because of its simple operation, rapidity and high specificity. In the sandwich ELISA test, a quantitative coated antibody is fixed on the surface of the ELISA plate by physical adsorption, and then the sample to be tested is added. After adding horseradish peroxidase (HRP)-labeled antibodies, the color is developed with TMB substrate. The depth of color in the ELISA plate is positively correlated with the concentration of the analyte and can be observed or further measured by the naked eye, spectrophotometer or microplate reader. Due to its simple process, high sensitivity, strong specificity, and observation by the naked eye, it has become an effective and convenient analytical method for clinical diagnostic biomarkers. Although colorimetric analysis has some advantages in convenience and visual detection, the sensitivity and specificity of the single-signal immunoassay based on colorimetry may cause inaccurate results, which ultimately limits its application in practical environments. Therefore, dual-mode immunoassays have emerged. Since the two signals can be self-corrected, they can provide more reliable results compared with single-signal detection. They have higher accuracy and can reduce possible false signals.

[0003] Nanozymes with peroxidase (POD) and oxidase (OXD)-like activities can play the role of HRP as a signal marker. The construction of nanozyme-based ELISA based on the specific binding of antigen-antibody can efficiently analyze biomarkers and pathogens. The excellent performance of nanozymes has broadened the application of enzyme-linked immunosorbent assay in biomarker detection. Various nanozymes are mainly composed of inorganic nanomaterials, because the metal active center can effectively simulate the catalytic redox process of natural enzymes. Moreover, compared with single metal nanoparticles, multi-component metals often have novel or enhanced physical or chemical properties due to various synergistic effects. Therefore, exploring composite metal nanozymes has a relatively broad development and application prospect.

[0004] In summary, based on the need to develop highly sensitive and convenient ELISA detection methods, it is a problem that needs to be solved to explore the preparation of simple and catalytically active composite metal nanozymes, and to combine their good peroxidase activity with the excellent fluorescent activity of other nanoparticles to establish a sensitive and convenient ELISA detection method. Summary of the invention

[0005] 1. Technical issues to be resolved

[0006] The present invention proposes a dual-signal immunoassay method based on a core-shell structure composite metal nanozyme. While utilizing the catalytic activity of the nanozyme to realize colorimetric determination, combined with the property that oxidized TMB can quench the red fluorescence of gold and silver nanoclusters, a new immunoassay method is established to solve the technical problem of how to improve the accuracy and anti-interference ability of immunoassay through colorimetric-fluorescence dual signal recognition and detection, thereby realizing sensitive and convenient detection of the target molecules to be measured.

[0007] (II) Technical solution

[0008] In order to solve the above technical problems, the present invention proposes a dual-signal immunoassay method based on a core-shell structure composite metal nanozyme, which comprises the following steps: preparing Au@CeO2@PtNPs and gold-silver nanoclusters AuAg NCs; coupling and labeling the target molecule monoclonal antibody Ab2 with Au@CeO2@Pt NPs to prepare a signal probe Au@CeO2@Pt-Ab2; coating the target molecule monoclonal antibody Ab1 on an ELISA plate; after the target molecule combines with Ab1 and Au@CeO2@Pt-Ab2 in sequence to form an immune sandwich complex, TMB is added, and Au@CeO2@PtNPs catalyzes the oxidation of TMB into oxidized TMB, generating a visible colorimetric signal at 652nm, and at the same time, due to the fluorescence inner filter effect, the oxidized TMB quenches the red fluorescence emitted by AuAgNCs, thereby realizing a colorimetric-fluorescence dual-mode immunoassay.

[0009] Furthermore, the preparation of Au@CeO2@PtNPs includes the following steps:

[0010] (1) Preparation of AuNPs: Ultrapure water was placed in a round-bottom flask, and chloroauric acid aqueous solution was added during vigorous stirring. When the temperature rose to 100°C, sodium citrate aqueous solution was added and the reaction was continued until the solution turned wine red. The solution was naturally cooled to room temperature and then stored at 4°C for later use.

[0011] (2) Preparation of Au@CeO2NPs: The prepared AuNPs and Na2CO3 solution were mixed together and magnetically stirred. Ce(NO3)3·6H2O was added dropwise to the mixture at a constant rate. The temperature was immediately raised to 90°C, and the color of the solution changed from wine red to purple. After reflux, the solution was naturally cooled to room temperature and stored at 4°C for later use.

[0012] (3) Preparation of Au@CeO2@PtNPs: H2PtCl6 was added dropwise to the Au@CeO2NPs prepared above during stirring. After continued stirring, the temperature was rapidly raised to 80°C. Subsequently, an aqueous solution of ascorbic acid was added dropwise under vigorous stirring. The color of the mixture changed from purple to dark brown. The product was ultrasonically washed with ultrapure water for multiple times and centrifuged until the supernatant was clear and transparent. The target product collected by centrifugation was freeze-dried and stored for later use.

[0013] Furthermore, the preparation of Au@CeO2@PtNPs includes the following steps:

[0014] (1) Preparation of AuNPs: 99 mL of ultrapure water was placed in a 250 mL round-bottom flask. 1 mL of chloroauric acid aqueous solution was added during vigorous stirring. When the temperature rose to 100 °C, 2.25 mL of 10 mg / mL sodium citrate aqueous solution was added and the reaction was continued until the solution turned wine red. The solution was cooled naturally to room temperature and then stored at 4 °C for later use.

[0015] (2) Preparation of Au@CeO2NPs: 50mL of prepared AuNPs and 210mL of 5mM Na2CO3 solution were mixed together and magnetically stirred for 10min. 50mL of 10mM Ce(NO3)3·6H2O was added dropwise to the mixture at a constant rate. The temperature was immediately raised to 90℃, and the color of the solution changed from wine red to purple. After reflux for 12h, the mixture was naturally cooled to room temperature and stored at 4℃ for later use.

[0016] (3) Preparation of Au@CeO2@PtNPs: 2.5 mL of H2PtCl6 was dropped into 150 mL of the Au@CeO2NPs sol prepared above during stirring. After stirring for 15 min, the temperature was quickly raised to 80°C. Subsequently, 11.2 mL of 20 mM AA aqueous solution was added dropwise under vigorous stirring. The color of the mixture changed from purple to dark brown. The product was ultrasonically washed several times with ultrapure water and centrifuged until the supernatant was clear and transparent. The target product collected by centrifugation was freeze-dried and stored for later use.

[0017] Furthermore, the preparation of AuAg NCs includes the following steps: adding HAuCl4 to BSA solution, adding AgNO3 solution to the mixture, stirring vigorously at 37°C, adding NaOH solution, continuing to stir at a constant temperature, and then collecting and storing for later use.

[0018] Furthermore, the preparation of AuAgNCs included the following steps: adding 4 mL of 10 mM HAuCl4 to 5 mL of 50 mg / mL BSA solution, adding 1.0 mL of 10 mM AgNO3 solution to the mixture, stirring vigorously at 37 °C for 5 min, adding 1 mL of 1 M NaOH solution, continuing to stir at a constant temperature for 12 h, and then collecting and storing for later use.

[0019] Furthermore, the preparation of Au@CeO2@Pt-Ab2 includes the following steps: dispersing Au@CeO2@PtNPs in PBS and sonicating to make them uniformly dispersed; adding Ab2 to the mixture, incubating on a vertical mixer at room temperature, and then adding BSA for incubation to block nonspecific binding sites; centrifuging the Au@CeO2@Pt-Ab2 conjugate, dispersing it in a PBS solution containing BSA, and storing it at 4°C for future use.

[0020] Furthermore, the preparation of Au@CeO2@Pt-Ab2 includes the following steps: dispersing 6 mg of Au@CeO2@PtNPs in 6 mL of PBS with pH = 8 and ultrasonicating for 20 min to make it evenly dispersed; adding 150 μL of 1.2 mg / mL Ab2 to the mixture, incubating on a vertical mixer at room temperature for 1 h, adding 600 μL of 10 wt % BSA and incubating for another 30 min to block nonspecific binding sites; centrifuging the Au@CeO2@Pt-Ab2 conjugate at 6000 rpm for 5 min, and dispersing it in 6 mL of PBS solution containing 20 mg BSA, and storing it at 4°C for future use.

[0021] (III) Beneficial effects

[0022] The present invention proposes a dual-signal immunoassay method based on a core-shell structure composite metal nanozyme, which combines the good peroxidase activity of Au@CeO2@Pt NPs with the property that oxidized TMB can quench the red fluorescence of AuAg NCs, and uses Au@CeO2@PtNPs to label the target molecule monoclonal antibody as a labeled probe, effectively realizing the colorimetric and fluorescent dual-signal recognition and detection of the target molecule, which can improve the accuracy and anti-interference ability of the immunoassay. By replacing the recognition elements such as antibodies, the immunoassay of biological macromolecules such as proteins, viruses and bacteria and pathogens can be realized, which has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 TEM images, EDS spectra, element mapping and particle size distribution of AuNPs, Au@CeO2, Au@CeO2@Pt NPs, etc., obtained in the examples of the present invention;

[0024] In the figure: (a) is the TEM image of Au NPs; (b) is the TEM image of Au@CeO2; (c) is the TEM image of Au@CeO2@PtNPs (multiple particle fields of view); (d) is the TEM image of Au@CeO2@Pt NPs (local single particle field of view); (e) is the Au element mapping of Au@CeO2@PtNPs; (f) is the Ce element mapping of Au@CeO2@PtNPs; (g) is the O element mapping of Au@CeO2@PtNPs; (h) is the Pt element mapping of Au@CeO2@PtNPs; (i) is the composite element mapping of Au@CeO2@PtNPs; (j) is the EDS spectrum of Au@CeO2@Pt NPs; (k) is the particle size distribution of Au@CeO2@PtNPs;

[0025] Figure 2 TEM images, high-resolution images and average particle size distribution of AuAgNCs obtained in the examples of the present invention;

[0026] In the figure: (a) is a TEM image; (b) is a high-resolution image; (c) is the average particle size distribution;

[0027] Figure 3 The Zeta potential and EPR spectrum of the product obtained in the embodiment of the present invention;

[0028] In the figure: (a) is the Zeta potential of Au, Au@CeO2, Au@CeO2@Pt(ACP)NPs, Au@CeO2@Pt-Ab2 (pH=8.0); (b) is the EPR spectra of Au@CeO2 NPs and Au@CeO2@PtNPs (oxygen vacancy characterization);

[0029] Figure 4 XPS spectrum of Au@CeO2@PtNPs;

[0030] In the figure: (a) is the full spectrum; (b) is the Ce 3d spectrum; (c) is the O1s spectrum; (d) is the Pt4f spectrum;

[0031] Figure 5 The effects of different factors on the peroxidase properties of Au@CeO2@PtNPs;

[0032] In the figure: (a) is pH; (b) is temperature; (c) is H2O2 concentration; (d) is TMB concentration;

[0033] Figure 6 is the enzymatic kinetic curve of Au@CeO2@PtNPs;

[0034] In the figure: (a) and (b) are the Michaelis-Menten curves and corresponding Lineweaver-Burk curves of different TMB concentrations under a fixed H2O2 concentration (15mM); (c) and (d) are the Michaelis-Menten curves and corresponding Lineweaver-Burk curves of different H2O2 concentrations under a fixed TMB concentration (0.4mM);

[0035] Figure 7 This is a correlation analysis diagram based on Au@CeO2@PtNPs enzyme reaction;

[0036] In the figure: (a) is the EPR spectrum of Au@CeO2@PtNPs (hydroxyl radical capture test); (b) is the catalytic properties of Au@CeO2@PtNPs and Au@CeO2@Pt-Ab2 conjugates; (c) is the UV-absorption spectrum of different systems; (d) is the UV-visible spectrum of different peroxidase substrates; (e) is the UV-visible absorption spectrum of oxTMB and the fluorescence emission (Em) spectrum of AuAg NCs; (f) is the fluorescence image of AuAgNCs quenched by oxTMB;

[0037] Figure 8 The basic principle of dual-signal immunoassay for detecting SARS-CoV-2N-protein based on core-shell structure composite metal nanozyme Au@CeO2@PtNPs is shown;

[0038] Fig. 9 The colorimetric-fluorescence dual-mode detection response and standard curve of SARS-CoV-2N-protein based on the core-shell structure composite metal nanozyme Au@CeO2@Pt NPs;

[0039] In the figure: (a) and (b) are the UV-visible spectrum intensity and the corresponding linear relationship when the concentration of SARS-CoV-2N-protein is 0.1-200 ng / mL; (c) and (d) are the fluorescence intensity and the corresponding linear relationship when the concentration range of SARS-CoV-2N-protein is 0.1-100 ng / mL;

[0040] Fig.10 To test the specificity, stability and reproducibility of the colorimetric signal for the colorimetric-fluorescence dual-mode detection of SARS-CoV-2N-protein based on Au@CeO2@PtNPs;

[0041] In the figure: (a) is the specificity of the fluorescence signal; (b) is the specificity of the colorimetric signal (the blank sample is PBS buffer); (c) is the stability of the immunoassay; and (d) is the reproducibility. DETAILED DESCRIPTION

[0042] In order to make the purpose, content and advantages of the present invention more clear, the specific implementation methods of the present invention are further described in detail below in conjunction with the drawings and examples.

[0043] This embodiment proposes a dual-signal immunoassay method based on a core-shell structure composite metal nanozyme, and the dual-signal immunoassay method specifically includes the following steps:

[0044] 1. Preparation of Au@CeO2@PtNPs

[0045] (1) Preparation of AuNPs: 99 mL of ultrapure water was placed in a 250 mL round-bottom flask. 1 mL of chloroauric acid aqueous solution (1 wt%) was added during vigorous stirring. When the temperature rose to 100 °C, 2.25 mL of sodium citrate aqueous solution (10 mg / mL) was added and the reaction was continued until the solution turned wine red. The solution was cooled naturally to room temperature and then stored at 4 °C for later use.

[0046] (2) Preparation of Au@CeO2NPs: 50mL of prepared Au NPs and 210mL of Na2CO3 solution (5mM) were mixed together and magnetically stirred for 10min. 50mL of Ce(NO3)3·6H2O (10mM) was added dropwise to the mixture at a constant rate. The temperature was immediately raised to 90℃, and the color of the solution changed from wine red to purple. After reflux for 12h, the solution was naturally cooled to room temperature and stored at 4℃ for later use.

[0047] (3) Preparation of Au@CeO2@PtNPs: 2.5 mL of H2PtCl6 (1 wt%) was dropped into 150 mL of the Au@CeO2NPs sol prepared above during stirring. After stirring for 15 min, the temperature was quickly raised to 80°C. Subsequently, 11.2 mL of ascorbic acid (AA) (20 mM) aqueous solution was added dropwise under vigorous stirring. The color of the mixture changed from purple to dark brown. The product was ultrasonically washed with ultrapure water for multiple times and centrifuged until the supernatant was clear and transparent. The target product collected by centrifugation was freeze-dried and stored for later use.

[0048] 2. Preparation of AuAgNCs

[0049] Add 4 mL of HAuCl4 (10 mM) to 5 mL of BSA (50 mg / mL) solution, add 1.0 mL of AgNO3 (10 mM) solution to the mixture, stir vigorously at 37 °C for 5 min, add 1 mL of NaOH (1 M) solution, continue stirring at constant temperature for 12 h, and then collect and store for later use.

[0050] 3. Preparation of Au@CeO2@Pt-Ab2 signal probe

[0051] 6 mg Au@CeO2@PtNPs were redispersed in 6 mL of PBS (pH = 8) and sonicated for 20 min to make it evenly dispersed; 150 μL of SARS-CoV-2N-protein Ab2 (1.2 mg / mL) was added to the mixture, incubated on a vertical mixer at room temperature for 1 h, and then 600 μL of BSA (10 wt %) was added and incubated for another 30 min to block nonspecific binding sites; the Au@CeO2@Pt-Ab2 conjugate was centrifuged at 6000 rpm for 5 min and redispersed in 6 mL of PBS solution containing 20 mg BSA and stored at 4 °C for future use.

[0052] 4. Characterization of Au@CeO2@PtNPs, AuAgNCs, etc.

[0053] The above-obtained products such as Au@CeO2@PtNPs were characterized by TEM, EDS spectroscopy, elemental mapping, XPS and EPR.

[0054] (1) TEM, EDS spectroscopy, elemental mapping, and Zeta potential characterization

[0055] The microstructure and morphology of Au@CeO2@PtNPs and AuAgNCs were characterized by TEM. Figure 1 As shown in (a)-(c), Au@CeO2@PtNPs were successfully synthesized via a layer-by-layer assembly strategy. Figure 1 (a) shows that the average particle size of the prepared AuNPs is about 16 nm; Figure 1 As shown in (b), the surrounding CeO2 shell is evenly wrapped around the gold nanoparticles. Figure 1 (c), (d) and (k) show the average particle size of Au@CeO2@PtNPs, which is about 60nm. Figure 1 (c) and (d) show that the shell of CeO2@PtNPs is composed of tiny nanoparticles, which increases the specific surface area of ​​the material and thus provides more catalytic sites. Figure 1 (e)-(i) show the elemental distribution of Au, Ce, O and Pt in Au@CeO2@PtNPs, as well as the elemental distribution of energy dispersive X-ray spectroscopy (EDS), confirming the successful preparation of Au@CeO2@PtNPs. The size and morphology of AuAg NCs were characterized by HR-TEM. Figure 2 As shown in Figure 2, the average hydrodynamic diameter of AuAgNCs is mainly between 2.0 and 3.0 nm, and the overall size distribution is narrow. Figure 3 As shown in (a), Zeta potential analysis further confirmed the successful preparation of Au@CeO2@PtNPs.

[0056] (2) XPS and EPR characterization

[0057] X-ray photoelectron spectroscopy (XPS) further confirmed the surface composition and valence state of Au@CeO2@PtNPs. Figure 4 (a) shows the coexistence of Ce, O, and Pt elements, and the TEM image has shown that the CeO2@Pt shell layer of the gold core is about 20-25nm, which is much thicker than the thickness that can be measured by XPS. Therefore, the characteristic binding energy of Au4f is not tested. Figure 4 As shown in (b), the characteristic peaks of Ce 3d coexist with Ce 3+ and Ce 4+ And there are 8 characteristic peaks in total. If v and u represent 3d 5 / 2 and 3D 3 / 2 The spin-orbit coupling of the orbital is 881.9eV(v), 888.6eV(v′), 898.1eV(v″), 900.2eV(u 0 The characteristic peaks at 906.4eV (u′) and 916.3eV (u″) are Ce 4+ The characteristic peak of 883.4eV (v 0 ), and the other two peaks at 901.4 eV (u) correspond to Ce 3+ Oxidation state. By normalizing the peak area, Ce can be calculated 3+ The relative content is 28.37%. In addition, Figure 4 The XPS spectrum of O1s shown in (c) can be decomposed into three peaks: the peak at 530.8 eV corresponds to Ce 4+ The surrounding lattice oxygen O 2+ The peak at 531.9 eV corresponds to the oxygen vacancies in the material, while the peak at 532.6 eV corresponds to the oxygen ions adsorbed on the surface of the oxygen vacancies. XPS is a surface characterization technique, while electron paramagnetic resonance (EPR) can reflect the single-electron oxygen vacancies on the surface and inside the material. Figure 3 (b) can further confirm the existence of surface oxygen vacancies. Under the same conditions, the EPR signal intensity of Au@CeO2@PtNPs at g≈2.002 is relatively stronger than that of Au@CeO2, indicating that the addition of Pt increases the content of surface oxygen vacancies. Figure 4 As shown in (d), the XPS spectrum of Pt4f is divided into four peaks. 7 / 2 At 71.7 eV and Pt4f 5 / 2 The peak at 75.1 eV is attributed to Pt 0 , while the peaks at 73.5 eV and 76.9 eV are attributed to metallic Pt 2+ In summary, Au@CeO2@PtNPs were successfully prepared.

[0058] 5. Peroxidase activity and mechanism analysis of Au@CeO2@PtNPs

[0059] The peroxidase activity of Au@CeO2@PtNPs was investigated through a series of comparative experiments, and the optimal colorimetric analysis characteristics depend on the concentration of the substrate, temperature, and pH. Figure 5 As shown in (a), when the pH of the system is 4.0, the UV-visible absorption value of the reaction solution reaches the maximum. Figure 5 (b) shows that the UV-visible absorption value remains at a high level in the range of 0-40°C, and the optimal catalytic activity is around 30°C. All experiments were carried out at room temperature for ease of operation. Figure 5 (c) and (d) show that when the concentrations of TMB and H2O2 are 15mM and 0.4mM, respectively, the UV-visible absorption value basically reaches the maximum.

[0060] To further elucidate the steady-state kinetics and catalytic mechanism of Au@CeO2@PtNPs, the concentration of TMB was varied while keeping the concentration of H2O2 constant. max ) is higher, indicating that the enzyme has a stronger ability to catalyze the substrate, while the Michaelis constant (K m ) is smaller, the stronger the affinity between the enzyme and the substrate. m and V max The steady-state kinetic parameters were calculated using the Lineweaver-Burk double reciprocal curve of the Michaelis-Menten equation ( Figure 6 As shown in the following table, compared with the kinetic parameters of other enzymes, the K of Au@CeO2@PtNPs with TMB as substrate is m The values ​​were smaller or comparable to those of other enzymes, indicating that they had a better affinity for TMB.

[0061]

[0062] V of Au@CeO2@Pt NPs with TMB as substrate max They are about 7.58, 3.33, and 3.09 times that of Au@CeO2NPs, Fe3O4 NPs, and HRP, respectively, indicating that Au@CeO2@PtNPs has a stronger catalytic ability for TMB, proving that it has an outstanding ability to oxidize TMB similar to peroxidase.

[0063] The EPR spectrum was used to test the capture of hydroxyl radicals (·OH). In this test, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as a spin trap material can capture ·OH to form a stable DMPO / ·OH complex, and the relative intensity of its characteristic spectrum is 1:2:2:1. Figure 7As shown in (a), the signal intensity of the DMPO / ·OH composite is much higher than that of the blank group without the addition of Au@CeO2@PtNPs, proving that Au@CeO2@PtNPs act as peroxidase to catalyze the generation of ·OH.

[0064] Figure 7 (b) shows that the catalytic activity of Au@CeO2@Pt-Ab2 conjugate has no significant change compared with Au@CeO2@PtNPs. Figure 7 As shown in (c), compared with Au NPs or Au@CeO2 NPs, the absorbance of TMB changes significantly when Au@CeO2@PtNPs are added in the presence of H2O2 under acidic conditions (pH = 4), indicating the formation of a charge transfer complex oxTMB. The synergistic effect of Pt and CeO2 may be the reason for the improved peroxide catalytic activity of Au@CeO2@Pt NPs. The introduction of dopants reduces the formation energy of oxygen vacancies, thereby generating additional oxygen vacancies, which can accelerate the catalytic oxidation of H2O2. Figure 7 (d) shows that Au@CeO2@PtNPs can also catalyze other colorants (ABTS and OPD) in the presence of H2O2, indicating that the peroxidase mimetic activity of Au@CeO2@PtNPs is independent of the substrate. The above results show that Au@CeO2@PtNPs is expected to become an ideal peroxidase mimetic enzyme.

[0065] like Figure 7 As shown in (e), due to the fluorescence internal rate effect, the blue oxTMB can quench the fluorescence of AuAgNCs, resulting in a decrease in fluorescence intensity. Figure 7 As shown in (f), after AuAg NCs were quickly added to the reaction solution and reacted for 2 minutes, its fluorescence intensity at a wavelength of 670 nm was greatly reduced compared with when Au@CeO2@Pt NPs were not added, and the degree of fluorescence quenching of AuAg NCs increased with the increase of oxTMB concentration.

[0066] 6. Establishment of the standard curve for colorimetric-fluorescence dual-mode detection of SARS-CoV-2 N-protein based on Au@CeO2@Pt peroxidase

[0067] The principle of colorimetric-fluorescence dual-mode detection of SARS-CoV-2 N-protein based on Au@CeO2@Pt is as follows Figure 8As shown in the figure, Au@CeO2@PtNPs and AuAg NCs were prepared, and on this basis, Au@CeO2@PtNPs and SARS-CoV-2N-protein antibody Ab2 were coupled to prepare the signal probe Au@CeO2@Pt-Ab2; the monoclonal antibody Ab1 of SARS-CoV-2N-protein was coated on the ELISA plate, and the non-specific binding sites were blocked with BSA; when SARS-CoV-2N-protein combined with Ab1 and Au@CeO2@Pt-Ab2 in turn to form an immune sandwich complex, the simulated peroxidase activity of Au@CeO2@PtNPs oxidized the colorless TMB into blue oxidized TMB (oxTMB). At the same time, the blue oxTMB can also quench the fluorescence of AuAg NCs at a wavelength of 670nm through the inner filter effect, thereby realizing the specific and sensitive detection of SARS-CoV-2N-protein in complex samples in a colorimetric and fluorescent dual signal mode.

[0068] Under the optimal experimental conditions, a standard curve between the absorbance value, fluorescence intensity value and SARS-CoV-2N-protein concentration was established. Fig. 9 As shown in (a), as the concentration of the target analyte increases, the absorbance of the blue oxTMB gradually increases, indicating that the absorbance is positively correlated with the concentration of the target N-protein. Fig. 9 As can be seen in (b), the absorbance increases linearly with the increase of SARS-CoV-2 N-protein concentration (0.1-200 ng / mL), and the fitted linear equation is as follows: Y = 0.129 + 0.0041X (R 2 =0.9953, X represents the concentration of SARS-CoV-2N-protein, Y represents the absorbance at 652nm wavelength), the detection limit (LOD) is calculated to be 0.062ng / mL (S / N=3). In addition, Fig. 9 (c) shows that as the concentration of SARS-CoV-2N-protein increases, the degree of fluorescence quenching of AuAg NCs at 670nm wavelength gradually increases. Fig. 9 As shown in (d), the linear equation between the fluorescence intensity and the logarithm of the concentration of SARS-CoV-2N-protein (0.1-100 ng / mL) is Y=918.64–70.52lgC N (R 2 =0.9923, C NRepresents the concentration of SARS-CoV-2N-protein, Y represents the fluorescence value at 670nm), and the calculated LOD is 0.036ng / mL (S / N=3). The above results confirm that the dual-mode immunoassay has the characteristics of a wide detection range and low LOD, and can achieve sensitive quantitative detection of SARS-CoV-2N-protein.

[0069] 7. Specificity, detection stability and reproducibility of SARS-CoV-2 N-protein colorimetric-fluorescence dual-mode detection based on Au@CeO2@Pt peroxidase

[0070] Specificity is the basis for the feasibility of dual-mode immunoassay. Five proteins (S-protein, FluB, FluA, RSV, MERS) with a concentration of 100 ng / mL were selected as interfering substances. Fig.10 (a) and (b) show that the interfering agent's response is similar to the blank and significantly different from the target protein (10 ng / mL), indicating that the dual-mode immunoassay has a high specificity.

[0071] Stability is an important indicator of immunoassay methods, such as Fig.10 As shown in (c), after Ab1 was coated into the wells of a 96-well plate and stored at 4°C for 18 days, the dual-mode signal intensity did not change significantly, indicating that this method also has good stability.

[0072] Repeatability is another important factor in immunoassay performance. The reproducibility of the immunoassay was examined by 9 sets of parallel experiments when detecting the same concentration of target protein (10 ng / mL). Fig. 9 As shown in (d), the relative standard deviations (RSDs) of the colorimetric signal and the fluorescence signal were 3.59% and 4.88%, respectively, indicating that the method has good reproducibility.

[0073] 8. Simulation sample testing

[0074] The feasibility of the dual-mode immunoassay was demonstrated using simulated urine and simulated saliva. The results are shown in the table below. The recovery rates of this method for simulated urine and simulated saliva were 87.37%-108.78% (colorimetric signal) and 94.43%-108.12% (fluorescence signal), respectively, indicating that the dual-mode immunoassay method has good applicability in detecting target objects in actual samples.

[0075]

[0076]

[0077] According to the embodiment, the good peroxidase activity of Au@CeO2@PtNPs of the present invention is combined with the property that oxidized TMB can quench the red fluorescence of AuAgNCs, and Au@CeO2@PtNPs are labeled with target molecule monoclonal antibodies as labeling probes, which effectively realizes the colorimetric and fluorescent dual signal recognition and detection of the target molecule SARS-CoV-2N-protein, with the advantages of high sensitivity, simple operation, good reproducibility, strong anti-interference, etc. By replacing recognition elements such as antibodies, immunoassays of biological macromolecules such as proteins, viruses, and bacteria and pathogens can be realized, which has broad application prospects.

[0078] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the technical principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A dual-signal immunoassay method based on a core-shell structure composite metal nanozyme, characterized in that: The dual-signal immunoassay method comprises the following steps: preparing Au@CeO2@PtNPs and gold-silver nanoclusters AuAgNCs; coupling and labeling the target molecule monoclonal antibody Ab2 with Au@CeO2@PtNPs to prepare the signal probe Au@CeO2@Pt-Ab2; coating the target molecule monoclonal antibody Ab1 on an ELISA plate; after the target molecule combines with Ab1 and Au@CeO2@Pt-Ab2 in sequence to form an immune sandwich complex, TMB is added, and Au@CeO2@PtNPs catalyzes the oxidation of TMB into oxidized TMB, generating a visible colorimetric signal at 652 nm, and simultaneously due to the fluorescence inner filter effect, the oxidized TMB quenches the red fluorescence emitted by AuAgNCs, thereby realizing a colorimetric-fluorescence dual-mode immunoassay.

2. The dual-signal immunoassay method based on core-shell structure composite metal nanozyme according to claim 1, characterized in that: The preparation of Au@CeO2@PtNPs includes the following steps: (1) Preparation of AuNPs: Ultrapure water was placed in a round-bottom flask, and chloroauric acid aqueous solution was added during vigorous stirring. When the temperature rose to 100°C, sodium citrate aqueous solution was added and the reaction was continued until the solution turned wine red. The solution was naturally cooled to room temperature and then stored at 4°C for later use. (2) Preparation of Au@CeO2NPs: The prepared AuNPs and Na2CO3 solution were mixed together and magnetically stirred. Ce(NO3)3·6H2O was added dropwise to the mixture at a constant rate. The temperature was immediately raised to 90°C, and the color of the solution changed from wine red to purple. After reflux, the solution was naturally cooled to room temperature and stored at 4°C for later use. (3) Preparation of Au@CeO2@PtNPs: H2PtCl6 was added dropwise to the Au@CeO2NPs prepared above during stirring. After continued stirring, the temperature was rapidly raised to 80°C. Subsequently, an aqueous solution of ascorbic acid was added dropwise under vigorous stirring. The color of the mixture changed from purple to dark brown. The product was ultrasonically washed with ultrapure water for multiple times and centrifuged until the supernatant was clear and transparent. The target product collected by centrifugation was freeze-dried and stored for later use.

3. The dual-signal immunoassay method based on core-shell structure composite metal nanozyme according to claim 2, characterized in that: The preparation of Au@CeO2@PtNPs includes the following steps: (1) Preparation of AuNPs: 99 mL of ultrapure water was placed in a 250 mL round-bottom flask. 1 mL of chloroauric acid aqueous solution was added during vigorous stirring. When the temperature rose to 100 °C, 2.25 mL of 10 mg / mL sodium citrate aqueous solution was added and the reaction was continued until the solution turned wine red. The solution was cooled naturally to room temperature and then stored at 4 °C for later use. (2) Preparation of Au@CeO2NPs: 50mL of prepared AuNPs and 210mL of 5mM Na2CO3 solution were mixed together and magnetically stirred for 10min. 50mL of 10mM Ce(NO3)3·6H2O was added dropwise to the mixture at a constant rate. The temperature was immediately raised to 90℃, and the color of the solution changed from wine red to purple. After reflux for 12h, the mixture was naturally cooled to room temperature and stored at 4℃ for later use. (3) Preparation of Au@CeO2@PtNPs: 2.5 mL of H2PtCl6 was dropped into 150 mL of the Au@CeO2NPs sol prepared above during stirring. After stirring for 15 min, the temperature was quickly raised to 80 °C. Subsequently, 11.2 mL of 20 mM AA aqueous solution was added dropwise under vigorous stirring. The color of the mixture changed from purple to dark brown. The product was ultrasonically washed several times with ultrapure water and centrifuged until the supernatant was clear and transparent. The target product collected by centrifugation was freeze-dried and stored for later use.

4. The dual-signal immunoassay method based on core-shell structure composite metal nanozyme according to claim 1, characterized in that: The preparation of AuAgNCs includes the following steps: adding HAuCl4 to BSA solution, adding AgNO3 solution to the mixture, stirring vigorously at 37°C, adding NaOH solution, continuing constant temperature stirring, and then collecting and storing for later use.

5. The dual-signal immunoassay method based on core-shell structure composite metal nanozyme according to claim 4, characterized in that: The preparation of AuAgNCs includes the following steps: 4 mL of 10 mM HAuCl4 is added to 5 mL of 50 mg / mL BSA solution, 1.0 mL of 10 mM AgNO3 solution is added to the mixture, and after vigorous stirring at 37 °C for 5 min, 1 mL of 1 M NaOH solution is added, and the mixture is continuously stirred at a constant temperature for 12 h before being collected and stored for later use.

6. The dual-signal immunoassay method based on core-shell structure composite metal nanozyme according to claim 1, characterized in that: The preparation of Au@CeO2@Pt-Ab2 includes the following steps: dispersing Au@CeO2@PtNPs in PBS and sonicating to make them uniformly dispersed; adding Ab2 to the mixture, incubating on a vertical mixer at room temperature, and then adding BSA to incubate to block nonspecific binding sites; centrifuging the Au@CeO2@Pt-Ab2 conjugate and dispersing it in a PBS solution containing BSA, and storing it at 4°C for future use.

7. The dual-signal immunoassay method based on core-shell structure composite metal nanozyme according to claim 6, characterized in that: The preparation of Au@CeO2@Pt-Ab2 includes the following steps: 6 mg Au@CeO2@Pt NPs were dispersed in 6 mL PBS with pH = 8 and ultrasonicated for 20 min to make it uniformly dispersed; 150 μL 1.2 mg / mL Ab2 was added to the mixture, incubated on a vertical mixer at room temperature for 1 h, and then 600 μL 10 wt % BSA was added and incubated for another 30 min to block nonspecific binding sites; The Au@CeO2@Pt-Ab2 conjugate was centrifuged at 6000 rpm for 5 min and dispersed in 6 mL of PBS solution containing 20 mg BSA and stored at 4 °C until use.

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