Preparation method and application of a composite probe based on ZIF-8 surface spatial confinement effect-induced gold nanocluster emission-enhanced fluorescence

Abstract

The present invention discloses a preparation method and application of a composite probe for gold nanoclusters emitting enhanced fluorescence induced by the spatial confinement effect of ZIF-8 surface. HAuCl4 and glutathione are dissolved in water, the two solutions are mixed and reacted, and purified to obtain an AuNCs solution; Zn(NO3)2·6H2O and 2-methylimidazole are dissolved in methanol, the two solutions are mixed and reacted, and the generated white precipitate is treated to obtain a zeolite imidazole framework-8 solution; the obtained AuNCs solution and the zeolite imidazole framework-8 solution are mixed, stirred, self-assembled, and treated to obtain ZIF-8@(Au) nanoparticles; zinc nitrate and dimethylimidazole are added to the nanoparticle solution for reaction, and after treatment, a sandwich ZIF-8@Au@ZIF-8 nanomaterial is obtained; the nanomaterial is mixed and treated with the AuNCs solution to obtain a ZIF-8@Au loaded with two layers of AuNCs; the operation is repeated to obtain a ZIF-8@(Au@ZIF-8) loaded with n layers of AuNCs. n The present invention significantly improves the effective utilization rate of assembly space and fluorescence performance, and realizes ultra-sensitive detection of fluorescence immunoassay.

Description

Technical Field

[0001] The present invention belongs to the technical field of fluorescence sensing, and specifically relates to a preparation method and application of a composite fluorescence probe based on the induction of gold nanoclusters emission-enhanced fluorescence by the spatial confinement effect on the surface of ZIF-8. Background Art

[0002] Ultrasensitive immunoassays are challenging. Traditional immunological detection methods, including enzyme-linked immunosorbent assays (ELISAs) and chemiluminescent immunoassays, often have sensitivity limited to the nanomolar to picoliter range. While they can effectively diagnose diseases with obvious symptoms, they struggle to meet the demands of early screening for major diseases, early diagnosis of neurological disorders, and early detection of cardiovascular disease, where marker concentrations are at the femtoliter level or even lower. This can lead to missed diagnoses, misdiagnoses, and delayed treatment windows, posing a serious threat to patients' lives and health. Therefore, there is an urgent need to develop ultrasensitive analytical methods for the precise detection of biomarkers.

[0003] Fluorescence immunoassays have been widely used in the qualitative, quantitative, and imaging of target objects, but the detection sensitivity is often limited by the intensity of the fluorescence signal. At present, fluorescence signal enhancement methods are mainly divided into two categories: one is the optimization of fluorophores, such as improving the chemical structure and skeleton to enhance the luminescence efficiency of a single fluorophore. The other is a local "enzyme-free" method to aggregate a large number of fluorophores in a carrier to enhance the overall fluorescence intensity. However, the incremental luminescence performance of optimized single fluorophores is limited, and local high-concentration fluorophores are prone to aggregation quenching. Therefore, it is urgent to develop new technologies for fluorescence immunoassays that have high loading capacity and can promote emission efficiency to improve detection sensitivity.

[0004] ZIF-8@Au, constructed by zeolite imidazole framework-8 (ZIF-8) using assembly techniques to load gold nanoclusters (AuNCs) onto the ZIF-8 framework, has been widely used in fluorescence immunoassays such as fluorescence detection and bioimaging. It can enhance luminescence efficiency through confinement or aggregation effects and increase the system loading, offering dual advantages. However, current ZIF-8@Au synthesis methods primarily rely on post-processing and in situ synthesis. Both methods embed gold nanoclusters (AuNCs) within the ZIF-8 structure. Due to internal space limitations and low space utilization, it is difficult to controllably prepare high ZIF-8@Au loadings. Therefore, new technologies are urgently needed to develop ZIF-8@Au that can achieve both high loading and enhanced emission efficiency to improve detection sensitivity. Summary of the Invention

[0005] The present invention addresses the technical problems of internal space limitations and low space utilization in prior art ZIF-8@Au synthesis. The present invention provides a method for preparing and applying a composite fluorescent probe that utilizes the spatial confinement effect of the ZIF-8 surface to induce enhanced emission from gold nanoclusters. This approach utilizes layer-by-layer self-assembly technology to construct an ultra-high loading of AuNCs on the ZIF-8 surface, thereby achieving a multi-fold increase in luminescence intensity. This approach utilizes the electrostatic / coordinate self-assembly of spatial binding sites on the ZIF-8 surface with AuNCs, significantly improving the effective utilization of the assembly space and fluorescence performance, enabling ultrasensitive detection in fluorescent immunoassays.

[0006] In order to solve the above problems, the technical solution adopted by the present invention is:

[0007] The present invention provides a method for preparing a composite probe based on the spatial confinement effect of ZIF-8 surface-induced gold nanoclusters to emit enhanced fluorescence, the preparation method comprising the following steps:

[0008] 1) Synthesis of gold nanoclusters: HAuCl4 and glutathione (GSH) were fully dissolved in water to obtain a HAuCl4 aqueous solution and a glutathione (GSH) solution, respectively. The two solutions were mixed at 65-90°C. Sodium hydroxide solution was added to the resulting mixed solution to adjust the pH to 5-8. After adjustment, the mixture was stirred continuously to obtain a gold nanocluster solution. The resulting gold nanocluster solution was purified to obtain a concentration of 0.5-1 mg·mL. -1 Gold nanocluster AuNCs solution;

[0009] 2) Synthesis of Zeolite Imidazole Framework-8: Zn(NO3)2·6H2O and 2-methylimidazole were dissolved in methanol, respectively, and after sufficient dissolution, a 2-methylimidazole solution and a zinc nitrate solution were obtained. The 2-methylimidazole solution and the zinc nitrate solution were then mixed evenly, and the mixture was reacted at room temperature for 12-24 hours. The resulting white precipitate was collected. The white precipitate was washed with methanol and water in sequence, and then dried to obtain zeolite imidazole framework-8. The obtained zeolite imidazole framework-8 was dispersed in water to obtain a concentration of 4-8 mg·mL -1 Zeolite imidazolyl framework-8 solution;

[0010] 3) Preparation of ZIF-8@(Au) Nanoparticles: The purified gold nanocluster AuNCs solution obtained in step 1) and the zeolite imidazole framework-8 solution obtained in step 2) were mixed and mechanically stirred at 400-700 rpm at room temperature for 10-30 minutes. The gold nanocluster solution successfully self-assembled on the zeolite imidazole framework-8. The mixture was then washed with ultrapure water to obtain ZIF-8@(Au) nanoparticles, which were then freeze-dried and stored for later use.

[0011] 4) Preparation of sandwich ZIF-8@Au@ZIF-8 nanoparticles: The ZIF-8@(Au) nanoparticles obtained in step 3) were dispersed in an aqueous solution to a concentration of 2 to 4 mg / mL. Zinc nitrate and dimethylimidazole were then added to the resulting solution to a concentration of 20 to 50 mM zinc nitrate and 160 to 400 mM dimethylimidazole, respectively. The mixture was then reacted at room temperature for 30 to 60 minutes, followed by centrifugation and washing to obtain a sandwich ZIF-8@Au@ZIF-8 nanomaterial.

[0012] 5) The obtained sandwich ZIF-8@Au@ZIF-8 nanomaterial was mixed with the gold nanocluster AuNCs solution obtained in step 1) at a mass ratio of 16:1. After mixing, the mixture was ultrasonicated at 25°C for 5-10 minutes. After ultrasonication, the mixture was centrifuged and washed to obtain ZIF-8@(Au@ZIF-8)2 loaded with two layers of AuNCs. The above operation was repeated n-2 times to prepare ZIF-8@(Au@ZIF-8) loaded with n layers of AuNCs. n ; where n=2, 3, or 4.

[0013] According to the above-mentioned preparation method of the gold nanocluster emission-enhanced fluorescent composite probe based on the spatial confinement effect induced by the ZIF-8 surface, the concentration of the HAuCl4 aqueous solution obtained in step 1) is 5-10 mM, and the concentration of the glutathione GSH solution is 7.5-20 mM; when the HAuCl4 aqueous solution and the glutathione GSH solution are mixed, the molar ratio of the active ingredients in the two solutions is 1:1.5-2.

[0014] According to the above-mentioned preparation method of the composite probe based on the spatial confinement effect of ZIF-8 surface induced gold nanoclusters to emit enhanced fluorescence, the specific process of purifying the gold nanocluster solution in step 1) is: transferring the obtained gold nanocluster solution to a 3000-8000 Da dialysis bag and dialyzing and purifying it at room temperature for 12-24 hours.

[0015] According to the above-mentioned preparation method of the gold nanocluster emission-enhanced fluorescent composite probe based on the spatial confinement effect induced by the ZIF-8 surface, the concentration of the 2-methylimidazole solution obtained in step 2) is 0.5-1M, and the concentration of the zinc nitrate solution is 0.05-0.25M; when the 2-methylimidazole solution and the zinc nitrate solution are mixed, the volume ratio between the two is 1:1.

[0016] According to the above-mentioned preparation method of the composite fluorescent probe based on the surface spatial confinement effect of ZIF-8 induced gold nanoclusters emission enhancement, the particle size of the zeolite imidazole framework-8 obtained in step 2) is 0.05 to 0.5 μm.

[0017] According to the above-mentioned preparation method of the gold nanocluster emission-enhanced fluorescent composite probe based on the spatial confinement effect of the ZIF-8 surface, when the gold nanocluster AuNCs solution and the zeolite imidazole framework-8 solution are mixed in step 3), the mass ratio of the active ingredients in the two solutions is 1:4~10.

[0018] According to the above-mentioned preparation method of the enhanced fluorescence composite probe based on the gold nanoclusters emission induced by the spatial confinement effect on the ZIF-8 surface, the drying temperature during the freeze-drying in step 3) is -70 to -20°C and the drying time is 24 to 48 hours.

[0019] In addition, a composite fluorescent probe based on the ZIF-8 surface spatial confinement effect-induced gold nanoclusters emission-enhanced fluorescence in immunoassay analysis is provided.

[0020] According to the above application, the specific steps of the application are:

[0021] a. ZIF-8@(Au@ZIF-8) n Biotinylation modification of AuNCs: 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were used to activate the carboxyl groups of AuNCs ligands. Biotin was then modified onto the surface of AuNCs based on the covalent coupling reaction between the amino group and the activated carboxyl group to obtain biotinylated Biotin-AuNCs. Finally, the Biotin-AuNCs were modified onto ZIF-8@(Au@ZIF-8) by electrostatic self-assembly. n surface, and Biotin-Au-ZIF-8@(Au@ZIF-8) was prepared n ;

[0022] b. Avidin modification of enzyme-linked immunosorbent assay: First, the capture antibody was fixed in a 96-well plate and a concentration of 0.1 to 10 7 fg / mL alpha-fetoprotein (AFP) sample was incubated for 30 minutes; the 96-well plate was washed three times with phosphate buffer, and then biotinylated detection antibody was added and incubated for 30 minutes; the plate was washed again with phosphate buffer three times; streptavidin was added and incubated for 30 minutes, and the 96-well plate was eluted three times with phosphate buffer; finally, Biotin-Au-ZIF-8@(Au@ZIF-8) was added to the 96-well plate. n Incubate for 1 h and wash 3 times with buffer solution to remove unattached Biotin-Au-ZIF-8@(Au@ZIF-8) n , and a fluorescence imager was used to image and analyze the 96-well plate.

[0023] According to the above application, the specific steps of the application are:

[0024] a. ZIF-8@(Au@ZIF-8) n Biotinylation modification of ZIF-8: Biotin-PEG-COOH was added to ZIF-8@(Au@ZIF-8) n The mixture was mixed, and the concentration of Biotin-PEG-COOH in the obtained mixture was 0.01-0.1 mg / mL, ZIF-8@(Au@ZIF-8) n The concentration of Biotin-PEG-COOH was 4-8 mg / mL; after mixing, the mixture was incubated at room temperature for 24 h. Based on the interaction between the carboxyl group in Biotin-PEG-COOH and the Zn 2+ The coordination effect of ZIF-8@(Au@ZIF-8) is realized n Biotin-ZIF-8@(Au@ZIF-8) was obtained by biotinylation modification. n ;

[0025] b. Avidin modification of enzyme-linked immunosorbent assay: First, the capture antibody was fixed in a 96-well plate and a concentration of 0.1 to 10 7 fg / mL alpha-fetoprotein (AFP) sample was incubated for 30 minutes; the 96-well plate was washed three times with phosphate buffer, and then biotinylated detection antibody was added and incubated for 30 minutes; the plate was washed again with phosphate buffer three times; streptavidin was added and incubated for 30 minutes, and the 96-well plate was eluted three times with phosphate buffer; finally, Biotin-ZIF-8@(Au@ZIF-8) was added to the 96-well plate. n Incubate for 1 h and wash 3 times with buffer solution to remove unattached Biotin-ZIF-8@(Au@ZIF-8) n , and a fluorescence imager was used to image and analyze the 96-well plate.

[0026] The positive beneficial effects of the present invention are:

[0027] 1. The present invention provides a method for preparing and applying a composite fluorescent probe featuring enhanced emission from gold nanoclusters induced by spatially controlled lateral arrangement. This method increases the loading of gold nanoclusters by self-assembling them onto the surface of zeolite imidazole framework-8 (ZIF-8) and, through spatial confinement or aggregation-induced effects, enhances the fluorescence performance of individual nanoclusters. This effectively overcomes the shortcomings of gold nanoclusters, such as weak fluorescence intensity, poor stability, and poor preparation controllability. Functionalization of the self-assembled fluorescent nanoclusters enables the composite probe to be used in immunoassays (enzyme-linked immunosorbent assays, ELISAs). The prepared composite probe achieves ultrasensitive detection of biomarkers such as interleukin-6 (IL-6, LOD reduced by 174-fold), C-reactive protein (CRP, LOD reduced by 326-fold), and procalcitonin (PCT, LOD reduced by 151-fold), extending the detection dynamic range by more than two orders of magnitude.

[0028] 2. The novel fluorescent composite probe obtained in the present invention has the advantages of simple and mild preparation method, short time consumption and high sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Schematic diagram of the preparation process of composite nanomaterials with self-assembled gold nanoclusters on the surface of self-assembled zeolite imidazole framework-8 (ZIF-8@AuNCs);

[0030] Figure 2 Transmission electron microscopy (TEM) image of gold nanoparticles AuNCs;

[0031] Figure 3 Transmission electron microscopy (TEM) image of ZIF-8;

[0032] Figure 4 Zeta potential diagrams of AuNCs, ZIF-8, and Au-ZIF-8;

[0033] Figure 5 TEM characterization of ZIF-8@Au@ZIF-8 nanoparticles;

[0034] Figure 6 TEM characterization of ZIF-8@(Au@ZIF-8)2 nanoparticles;

[0035] Figure 7 TEM characterization of ZIF-8@(Au@ZIF-8)3 nanoparticles;

[0036] Figure 8 TEM characterization of ZIF-8@(Au@ZIF-8)4 nanoparticles;

[0037] Figure 9Fluorescence emission spectra of ZIF-8@Au@ZIF-8, ZIF-8@(Au@ZIF-8)2, ZIF-8@(Au@ZIF-8)3, and ZIF-8@(Au@ZIF-8)4;

[0038] Figure 10 Schematic diagram of the application of high-performance immunofluorescent groups in immunoassay detection;

[0039] Figure 11 UV resonance absorption spectrum of ZIF-8@AuNCs functionalized with avidin;

[0040] Figure 12 Comparison of the detection of interleukin-6 (IL-6) by traditional ELISA and p-FLISA using ZIF-8@(Au@ZIF-8)4 probes;

[0041] Depend on Figure 12 It can be seen that compared with ELISA, the LOD of p-FLISA is 174 times lower and the dynamic range is more than two orders of magnitude larger.

[0042] Figure 13 Comparison of the effectiveness of traditional ELISA method and p-FLISA method using ZIF-8@(Au@ZIF-8)4 probe in detecting C-reactive protein (CRP);

[0043] Depend on Figure 13 It can be seen that compared with ELISA, the LOD of p-FLISA is 326 times lower and the dynamic range is more than two orders of magnitude larger.

[0044] Figure 14 Comparison of the effects of traditional ELISA and p-FLISA using ZIF-8@(Au@ZIF-8)4 probe in detecting procalcitonin (PCT);

[0045] Depend on Figure 14 It can be seen that compared with ELISA, the LOD of p-FLISA is 151 times lower and the dynamic range is more than two orders of magnitude larger. DETAILED DESCRIPTION

[0046] The technical solution of the present invention is further described by the following examples. The following are only preferred embodiments of the present invention, which are used to illustrate the present invention and are not intended to limit the present invention in any way. Without departing from the technical solution of the present invention, any changes or modifications that are easily implemented by a person of ordinary skill in the art will fall within the scope of protection of the claims of the present invention.

[0047] Example 1:

[0048] The present invention is based on the preparation method of the ZIF-8 surface spatial confinement effect-induced gold nanocluster emission-enhanced fluorescent composite probe, and the detailed steps are as follows:

[0049] 1) Synthesis of gold nanoclusters: HAuCl4 and glutathione (GSH) were fully dissolved in water to obtain a 7 mM HAuCl4 aqueous solution and a 12.5 mM glutathione (GSH) solution, respectively. The two solutions were mixed at 75-80°C (the molar ratio of the active ingredients in the HAuCl4 aqueous solution and the glutathione (GSH) solution was 1:2). Sodium hydroxide solution was added to the resulting mixed solution to adjust its pH to 7, and the mixture was stirred continuously to obtain a gold nanocluster solution. The resulting gold nanocluster solution was transferred to a 5000 Da dialysis bag and dialyzed for purification at room temperature for 18 h to obtain a gold nanocluster solution with a concentration of 0.75 mg mL -1 The AuNCs solution was prepared by TEM analysis of the particle size, morphology and surface morphology of the AuNCs. Figure 2 , observe the shape of AuNCs);

[0050] 2) Synthesis of zeolite imidazole framework-8: Zn(NO3)2·6H2O and 2-methylimidazole were dissolved in methanol, respectively. After sufficient dissolution, a 0.8 M 2-methylimidazole solution and a 0.1 M zinc nitrate solution were obtained. The 2-methylimidazole solution and the zinc nitrate solution were then mixed evenly in a volume ratio of 1:1. After mixing, the mixture was reacted at room temperature for 18 h, and the resulting white precipitate was collected. The white precipitate was washed with methanol and water in sequence, and then dried to obtain zeolite imidazole framework-8 (with a particle size of 0.05-0.5 μm). The obtained zeolite imidazole framework-8 was dispersed in water to obtain a concentration of 6 mg·mL -1 Zeolite imidazolyl framework-8 solution (TEM was used to analyze the particle size, morphology and surface morphology of ZIF-8, as shown in the attached Figure 3 , observe the shape of ZIF-8);

[0051] 3) Preparation of ZIF-8@(Au) Nanoparticles: The purified gold nanocluster (AuNCs) solution obtained in step 1) was mixed with the zeolite imidazole framework-8 solution obtained in step 2) (the mass ratio of the active ingredients in the AuNCs solution and the zeolite imidazole framework-8 solution was 1:6). The mixture was mechanically stirred at 500 rpm at room temperature for 20 minutes to allow the gold nanocluster solution to self-assemble on the zeolite imidazole framework-8. The particles were then washed with ultrapure water to obtain ZIF-8@(Au) nanoparticles, which were then lyophilized at -50 to -40°C for 32 hours for future use.

[0052] Zeta potential was used to analyze the potential of AuNCs, ZIF-8 and ZIF-8@(Au), as shown in the attached figure. Figure 4 TEM was used to analyze the particle size, morphology and surface morphology of ZIF-8@(Au). Figure 5 , observe the shape of ZIF-8@(Au);

[0053] 4) Preparation of sandwich ZIF-8@Au@ZIF-8: The ZIF-8@(Au) nanoparticles obtained in step 3) were dispersed in an aqueous solution to a concentration of 3 mg / mL. Zinc nitrate and dimethylimidazole were then added to the resulting solution to a concentration of 35 mM zinc nitrate and 220 mM dimethylimidazole, respectively. The mixture was then reacted at room temperature for 40 minutes, followed by centrifugation and washing to obtain a sandwich ZIF-8@Au@ZIF-8 nanomaterial.

[0054] 5) The obtained sandwich ZIF-8@Au@ZIF-8 nanomaterial was mixed with the gold nanocluster AuNCs solution obtained in step 1) at a mass ratio of 16:1, and then ultrasonicated at 25°C for 10 minutes. After ultrasonication, the mixture was centrifuged and washed to obtain ZIF-8@(Au@ZIF-8)2 loaded with two layers of AuNCs;

[0055] Repeat the above steps to prepare ZIF-8@(Au@ZIF-8)3 loaded with 3 layers of AuNCs and ZIF-8@(Au@ZIF-8)4 loaded with 4 layers of AuNCs. Repeat the above steps n-2 times to prepare ZIF-8@(Au@ZIF-8) loaded with n layers of AuNCs. n .

[0056] TEM analysis of ZIF-8@(Au@ZIF-8) n The particle size, morphology and surface morphology characteristics of Figure 6-8 , using the attached Figure 6-8 TEM image of ZIF-8@(Au@ZIF-8) n In addition, the fluorescence intensity spectrum shows the difference in fluorescence intensity enhancement caused by different numbers of assembly layers, such as Figure 9 .

[0057] The specific application of the composite fluorescent probe based on the ZIF-8 surface spatial confinement effect-induced gold nanoclusters emission-enhanced fluorescence in immunoassay analysis prepared in the embodiment of the present invention is as follows:

[0058] Application Example 1:

[0059] The composite probe for enhanced fluorescence emission of gold nanoclusters induced by the spatial confinement effect of ZIF-8 surface prepared in Example 1 of the present invention was applied to the detection of interleukin-6 (IL-6) by enzyme-linked immunosorbent assay (ELISA). The specific steps are as follows (see the attached schematic diagram for the application) Figure 10 ):

[0060] a. Biotinylation modification of ZIF-8@(Au@ZIF-8)4: Biotin-PEG-COOH was mixed with ZIF-8@(Au@ZIF-8)4. The concentration of Biotin-PEG-COOH in the mixed solution was 0.05 mg / mL and the concentration of ZIF-8@(Au@ZIF-8)4 was 6 mg / mL. After mixing, the mixture was incubated at room temperature for 24 h. Based on the carboxyl group in Biotin-PEG-COOH and the Zn 2+ The coordination effect of ZIF-8@(Au@ZIF-8)4 was used to achieve biotinylation modification of ZIF-8@(Au@ZIF-8)4 to obtain Biotin-ZIF-8@(Au@ZIF-8)4 (see Appendix Figure 11 );

[0061] b. Avidin modification of enzyme-linked immunosorbent assay: First, the IL-6 capture antibody was fixed in a 96-well plate, and a concentration of 0.1 to 10 7 fg / mL alpha-fetoprotein (AFP) sample was incubated for 30 minutes; the 96-well plate was washed three times with phosphate buffer, and then biotinylated IL-6 detection antibody was added and incubated for 30 minutes; the plate was washed again with phosphate buffer three times; streptavidin was added and incubated for 30 minutes, and the 96-well plate was eluted three times with phosphate buffer; finally, Biotin-ZIF-8@(Au@ZIF-8)4 was added to the 96-well plate and incubated for 1 hour, and washed three times with buffer solution to remove unattached Biotin-ZIF-8@(Au@ZIF-8)4, and the 96-well plate was imaged and analyzed using a fluorescence imager. Figure 12 , a comparison of the effectiveness of traditional ELISA method and p-FLISA method using ZIF-8@(Au@ZIF-8)4 probe in detecting IL-6; compared with ELISA, the LOD of p-FLISA is 174 times lower and the dynamic range is more than two orders of magnitude larger.

[0062] Application Example 2:

[0063] The composite probe for enhanced fluorescence emission of gold nanoclusters induced by the spatial confinement effect of ZIF-8 surface prepared in Example 1 of the present invention was applied to the detection of C-reactive protein (CRP) by enzyme-linked immunosorbent assay (ELISA). The specific steps are as follows (see the attached schematic diagram for the application) Figure 10 ):

[0064] a. Biotinylation modification of ZIF-8@(Au@ZIF-8)4: Biotin-PEG-COOH was mixed with ZIF-8@(Au@ZIF-8)4. The concentration of Biotin-PEG-COOH in the mixed solution was 0.05 mg / mL and the concentration of ZIF-8@(Au@ZIF-8)4 was 6 mg / mL. After mixing, the mixture was incubated at room temperature for 24 h. Based on the carboxyl group in Biotin-PEG-COOH and the Zn 2+ The coordination effect of ZIF-8@(Au@ZIF-8)4 was used to achieve biotinylation modification of ZIF-8@(Au@ZIF-8)4 to obtain Biotin-ZIF-8@(Au@ZIF-8)4 (see Appendix Figure 11 );

[0065] b. Avidin modification of enzyme-linked immunosorbent assay: First, fix the CRP capture antibody in a 96-well plate and add avidin at a concentration of 0.1 to 10 7 fg / mL C-reactive protein (CRP) sample was incubated for 30 minutes; the 96-well plate was washed three times with phosphate buffer, and then biotinylated CRP detection antibody was added and incubated for 30 minutes; the plate was washed again with phosphate buffer three times; streptavidin was added and incubated for 30 minutes, and the 96-well plate was eluted three times with phosphate buffer; finally, Biotin-ZIF-8@(Au@ZIF-8)4 was added to the 96-well plate and incubated for 1 hour, and washed three times with buffer solution to remove unattached Biotin-ZIF-8@(Au@ZIF-8)4, and the 96-well plate was imaged and analyzed using a fluorescence imager. Figure 13 , a comparison of the effects of traditional ELISA method and p-FLISA method using ZIF-8@(Au@ZIF-8)4 probe in detecting CRP; compared with ELISA, the LOD of p-FLISA is 326 times lower and the dynamic range is more than two orders of magnitude larger.

[0066] Application Example 3:

[0067] The composite probe for enhanced fluorescence emission of gold nanoclusters induced by the spatial confinement effect of ZIF-8 surface prepared in Example 1 of the present invention was applied to the detection of procalcitonin (PCT) by enzyme-linked immunosorbent assay (ELISA). The specific steps are as follows (see the attached schematic diagram for the application) Figure 10 ):

[0068] a. Biotinylation modification of ZIF-8@(Au@ZIF-8)4: Biotin-PEG-COOH was mixed with ZIF-8@(Au@ZIF-8)4. The concentration of Biotin-PEG-COOH in the mixed solution was 0.05 mg / mL and the concentration of ZIF-8@(Au@ZIF-8)4 was 6 mg / mL. After mixing, the mixture was incubated at room temperature for 24 h. Based on the carboxyl group in Biotin-PEG-COOH and the Zn 2+ The coordination effect of ZIF-8@(Au@ZIF-8)4 was used to achieve biotinylation modification of ZIF-8@(Au@ZIF-8)4 to obtain Biotin-ZIF-8@(Au@ZIF-8)4 (see Appendix Figure 11 );

[0069] b. Avidin modification of enzyme-linked immunosorbent assay: First, fix the capture antibody of PCT in a 96-well plate and add avidin at a concentration of 0.1 to 10 7 fg / mL of procalcitonin PCT sample was incubated for 30 minutes; the 96-well plate was washed three times with phosphate buffer, and then biotinylated PCT detection antibody was added and incubated for 30 minutes; the plate was washed again with phosphate buffer three times; streptavidin was added and incubated for 30 minutes, and the 96-well plate was eluted three times with phosphate buffer; finally, Biotin-ZIF-8@(Au@ZIF-8)4 was added to the 96-well plate and incubated for 1 hour, and washed three times with buffer solution to remove unattached Biotin-ZIF-8@(Au@ZIF-8)4, and the 96-well plate was imaged and analyzed using a fluorescence imager. Figure 14 , a comparison of the effects of traditional ELISA method and p-FLISA method using ZIF-8@(Au@ZIF-8)4 probe in detecting PCT; compared with ELISA, the LOD of p-FLISA is 151 times lower and the dynamic range is more than two orders of magnitude larger.

Claims

1. A method for preparing a composite probe based on the surface spatial confinement effect of ZIF-8 to induce gold nanoclusters to emit enhanced fluorescence, characterized in that: The preparation method comprises the following steps: 1) Synthesis of gold nanoclusters: HAuCl4 and glutathione (GSH) were fully dissolved in water to obtain a HAuCl4 aqueous solution and a glutathione (GSH) solution, respectively. The two solutions were mixed at 65-90°C. Sodium hydroxide solution was added to the resulting mixed solution to adjust the pH to 5-8. After adjustment, the mixture was stirred continuously to obtain a gold nanocluster solution. The resulting gold nanocluster solution was purified to obtain a concentration of 0.5-1 mg·mL. -1 Gold nanocluster AuNCs solution; 2) Synthesis of Zeolite Imidazole Framework-8: Zn(NO3)2·6H2O and 2-methylimidazole were dissolved in methanol, respectively, and after sufficient dissolution, a 2-methylimidazole solution and a zinc nitrate solution were obtained. The 2-methylimidazole solution and the zinc nitrate solution were then mixed evenly, and the mixture was reacted at room temperature for 12-24 hours. The resulting white precipitate was collected. The white precipitate was washed with methanol and water in sequence, and then dried to obtain zeolite imidazole framework-8. The obtained zeolite imidazole framework-8 was dispersed in water to obtain a concentration of 4-8 mg·mL -1 Zeolite imidazolyl framework-8 solution; 3) Preparation of ZIF-8@(Au) Nanoparticles: The purified gold nanocluster AuNCs solution obtained in step 1) and the zeolite imidazole framework-8 solution obtained in step 2) were mixed and mechanically stirred at 400-700 rpm at room temperature for 10-30 minutes. The gold nanocluster solution successfully self-assembled on the zeolite imidazole framework-8. The mixture was then washed with ultrapure water to obtain ZIF-8@(Au) nanoparticles, which were then freeze-dried and stored for later use. 4) Preparation of sandwich ZIF-8@Au@ZIF-8 nanoparticles: The ZIF-8@(Au) nanoparticles obtained in step 3) were dispersed in an aqueous solution to a concentration of 2 to 4 mg / mL. Zinc nitrate and dimethylimidazole were then added to the resulting solution to a concentration of 20 to 50 mM zinc nitrate and 160 to 400 mM dimethylimidazole, respectively. The mixture was then reacted at room temperature for 30 to 60 minutes, followed by centrifugation and washing to obtain a sandwich ZIF-8@Au@ZIF-8 nanomaterial. 5) The obtained sandwich ZIF-8@Au@ZIF-8 nanomaterial was mixed with the gold nanocluster AuNCs solution obtained in step 1) at a mass ratio of 16:

1. After mixing, the mixture was ultrasonicated at 25°C for 5-10 minutes. After ultrasonication, the mixture was centrifuged and washed to obtain ZIF-8@(Au@ZIF-8)2 loaded with two layers of AuNCs. The above operation was repeated n-2 times to prepare ZIF-8@(Au@ZIF-8) loaded with n layers of AuNCs. n ; where n=2, 3, or 4.

2. The method for preparing a composite probe based on ZIF-8 surface spatial confinement effect-induced gold nanoclusters emission-enhanced fluorescence according to claim 1, characterized in that: The concentration of the HAuCl4 aqueous solution obtained in step 1) is 5-10 mM, and the concentration of the glutathione GSH solution is 7.5-20 mM; when the HAuCl4 aqueous solution and the glutathione GSH solution are mixed, the molar ratio of the active ingredients in the two solutions is 1:1.5-2.

3. The method for preparing a composite probe based on ZIF-8 surface spatial confinement effect-induced gold nanoclusters emission-enhanced fluorescence, according to claim 1, characterized in that: The specific process of purifying the gold nanocluster solution in step 1) is as follows: transferring the obtained gold nanocluster solution into a 3000-8000 Da dialysis bag and dialysis purification at room temperature for 12-24 hours.

4. The method for preparing a composite probe based on the ZIF-8 surface spatial confinement effect-induced gold nanocluster emission-enhanced fluorescence according to claim 1, characterized in that: The concentration of the 2-methylimidazole solution obtained in step 2) is 0.5-1 M, and the concentration of the zinc nitrate solution is 0.05-0.25 M; when the 2-methylimidazole solution and the zinc nitrate solution are mixed, the volume ratio between the two is 1:

1.

5. The method for preparing a composite probe based on the ZIF-8 surface spatial confinement effect-induced gold nanocluster emission-enhanced fluorescence according to claim 1, characterized in that: The particle size of the zeolite imidazole framework-8 obtained in step 2) is 0.05 to 0.5 μm.

6. The method for preparing a composite fluorescent probe based on the ZIF-8 surface spatial confinement effect-induced gold nanoclusters emission-enhanced fluorescence according to claim 1, characterized in that: In step 3), when the gold nanocluster AuNCs solution and the zeolite imidazole framework-8 solution are mixed, the mass ratio of the active ingredients in the two solutions is 1:4-10.

7. The method for preparing a composite fluorescent probe based on the ZIF-8 surface spatial confinement effect-induced gold nanoclusters emission-enhanced fluorescence according to claim 1, characterized in that: The drying temperature during freeze-drying in step 3) is -70 to -20°C, and the drying time is 24 to 48 hours.

8. Use of a composite fluorescent probe based on the enhanced emission of gold nanoclusters induced by the spatial confinement effect on the surface of ZIF-8, prepared according to the preparation method of any one of claims 1 to 7, in immunoassay analysis.

9. The use according to claim 8, characterized in that The specific steps of the application are: a. ZIF-8@(Au@ZIF-8) n Biotinylation modification of AuNCs: 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were used to activate the carboxyl groups of AuNCs ligands. Biotin was then modified onto the surface of AuNCs based on the covalent coupling reaction between the amino group and the activated carboxyl group to obtain biotinylated Biotin-AuNCs. Finally, the Biotin-AuNCs were modified onto ZIF-8@(Au@ZIF-8) by electrostatic self-assembly. n surface, and Biotin-Au-ZIF-8@(Au@ZIF-8) was prepared n ; b. Avidin modification of enzyme-linked immunosorbent assay: First, the capture antibody was fixed in a 96-well plate and a concentration of 0.1 to 10 7 fg / mL alpha-fetoprotein (AFP) sample was incubated for 30 minutes; the 96-well plate was washed three times with phosphate buffer, and then biotinylated detection antibody was added and incubated for 30 minutes; the plate was washed again with phosphate buffer three times; streptavidin was added and incubated for 30 minutes, and the 96-well plate was eluted three times with phosphate buffer; finally, Biotin-Au-ZIF-8@(Au@ZIF-8) was added to the 96-well plate. n Incubate for 1 h and wash 3 times with buffer solution to remove unattached Biotin-Au-ZIF-8@(Au@ZIF-8) n , and a fluorescence imager was used to image and analyze the 96-well plate.

10. The use according to claim 8, characterized in that The specific steps of the application are: a. ZIF-8@(Au@ZIF-8) n Biotinylation modification of ZIF-8: Biotin-PEG-COOH was added to ZIF-8@(Au@ZIF-8) n The mixture was mixed, and the concentration of Biotin-PEG-COOH in the obtained mixture was 0.01-0.1 mg / mL, ZIF-8@(Au@ZIF-8) n The concentration of Biotin-PEG-COOH was 4-8 mg / mL; after mixing, the mixture was incubated at room temperature for 24 h. Based on the interaction between the carboxyl group in Biotin-PEG-COOH and the Zn 2+ The coordination effect of ZIF-8@(Au@ZIF-8) is realized n Biotin-ZIF-8@(Au@ZIF-8) was obtained by biotinylation modification. n ; b. Avidin modification of enzyme-linked immunosorbent assay: First, the capture antibody was fixed in a 96-well plate and a concentration of 0.1 to 10 7 fg / mL alpha-fetoprotein (AFP) sample was incubated for 30 minutes; the 96-well plate was washed three times with phosphate buffer, and then biotinylated detection antibody was added and incubated for 30 minutes; the plate was washed again with phosphate buffer three times; streptavidin was added and incubated for 30 minutes, and the 96-well plate was eluted three times with phosphate buffer; finally, Biotin-ZIF-8@(Au@ZIF-8) was added to the 96-well plate. n Incubate for 1 h and wash 3 times with buffer solution to remove unattached Biotin-ZIF-8@(Au@ZIF-8) n , and a fluorescence imager was used to image and analyze the 96-well plate.

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