Preparation method and application of ZIF-8 surface space confinement effect-based gold nanocluster emission-enhanced fluorescent composite probe

Abstract

The invention discloses a preparation method and application of a ZIF-8 surface space confinement effect-based gold nanocluster emission-enhanced fluorescent composite probe. The preparation method comprises the following steps: respectively dissolving HAuCl4 and glutathione in water, mixing the obtained two solutions, reacting, and purifying to obtain an AuNCs solution; the preparation method comprises the following steps: respectively dissolving Zn (NO3) 2.6 H2O and 2-methylimidazole in methanol, uniformly mixing the two obtained solutions, reacting, and treating the generated white precipitate to obtain a zeolite imidazole frame-8 solution; mixing the obtained AuNCs solution with a zeolite imidazole framework 8 solution, stirring, self-assembling, and treating to obtain ZIF-8 (Au) nanoparticles; adding zinc nitrate and dimethylimidazole into the nanoparticle solution to react, and treating after the reaction to obtain a sandwich ZIF-8 (at) Au (at) ZIF-8 nano material; the preparation method comprises the following steps: mixing a nano material with an AuNCs solution, and treating to obtain ZIF-8 (at) Au loaded with two layers of AuNCs; and repeating the operation to obtain ZIF-8 (at) (Au (at) ZIF-8) n loaded with n layers of AuNCs. According to the invention, the effective utilization rate of the assembly space and the fluorescence performance are greatly improved, and ultrasensitive detection of fluorescence immunoassay is realized.

Description

1. Technical field:

[0001] The 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 by the spatial confinement effect on the surface of ZIF-8. 2. Background technology:

[0002] Ultrasensitive detection of immunoassays is challenging. Traditional immunological detection methods include enzyme-linked immunosorbent assay, chemiluminescent immunoassay, etc., and the detection sensitivity is mostly limited to the nM-pM level. Although it can effectively diagnose diseases with obvious symptoms, it is difficult to meet the detection needs of markers with concentrations at the fM level or even lower for early screening of major diseases, early diagnosis of neurological disorders, and early detection of cardiovascular diseases, resulting in missed diagnosis, misdiagnosis, and delayed treatment windows of diseases, which seriously threaten the life and health of patients. Therefore, it is urgent to develop ultrasensitive analytical methods to achieve accurate detection of biomarkers.

[0003] Fluorescence immunoassay has been widely used in qualitative, quantitative and imaging of targets, 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 increase the overall fluorescence intensity. However, the incremental luminescence performance of a single fluorophore optimization is limited, and local high-concentration fluorophores are prone to aggregation quenching. Therefore, it is urgent to develop new technologies with high loading capacity and emission efficiency to improve detection sensitivity for fluorescence immunoassay.

[0004] ZIF-8@Au, constructed by loading gold nanoclusters AuNCs on zeolite imidazole framework-8 (ZIF-8) using assembly technology, has been widely used in fluorescence immunoassay fields such as fluorescence detection and bioimaging. It can induce luminescence efficiency enhancement through confinement or aggregation effects, and increase the system loading, with the advantage of dual brightening. However, the current ZIF-8@Au synthesis is mainly based on two methods: late-stage and in-situ synthesis. Both methods embed gold nanoclusters AuNCs in the ZIF-8 structure. Due to internal space limitations and low space utilization, it is impossible to controllably prepare high-loading ZIF-8@Au. Therefore, it is urgent to develop new technologies that can have high loading and promote emission efficiency for ZIF-8@Au to improve detection sensitivity. 3. Summary of the invention:

[0005] The technical problem to be solved by the present invention is: in view of the technical problems of internal space limitation and low space utilization rate of ZIF-8@Au synthesis in the prior art, the present invention provides a preparation method and application of a composite fluorescent probe based on the spatial confinement effect of ZIF-8 surface induced gold nanoclusters emission enhanced. The technical solution of the present invention constructs an ultra-high load of AuNCs on the surface of ZIF-8 through layer-by-layer self-assembly technology, thereby achieving a multiple increase in luminous intensity. The present invention utilizes the surface space binding sites of ZIF-8 and the electrostatic / coordination self-assembly of AuNCs, greatly improving the effective utilization rate of the assembly space and the fluorescence performance, and realizing ultra-sensitive detection of fluorescent immunoassay.

[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 fluorescent probe based on the spatial confinement effect of ZIF-8 surface induced gold nanoclusters to emit enhanced fluorescence, and the preparation method comprises the following steps:

[0008] 1) Synthesis of gold nanoclusters: HAuCl4 and glutathione GSH are fully dissolved in water to obtain HAuCl4 aqueous solution and glutathione GSH solution; the two solutions are mixed evenly at 65-90° C., sodium hydroxide solution is added to the obtained mixed solution to adjust its pH value to 5-8, and the mixture is continuously stirred after adjustment to obtain a gold nanocluster solution; the obtained gold nanocluster solution is purified to obtain a concentration of 0.5-1 mg·mL -1 AuNCs solution;

[0009] 2) Synthesis of zeolite imidazole framework-8: Zn(NO3)2·6H2O and 2-methylimidazole are dissolved in methanol respectively, and after being fully dissolved, a 2-methylimidazole solution and a zinc nitrate solution are obtained; the 2-methylimidazole solution and the zinc nitrate solution are then mixed evenly, and reacted at room temperature for 12 to 24 hours after mixing, and the generated white precipitate is collected; the white precipitate is washed with methanol and water in turn, and dried after washing to obtain zeolite imidazole framework-8; the obtained zeolite imidazole framework-8 is dispersed in water to obtain a concentration of 4 to 8 mg·mL -1 A zeolite imidazolyl framework-8 solution;

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

[0011] 4) Preparation of sandwich ZIF-8@Au@ZIF-8 nanoparticles: Disperse the ZIF-8@(Au) nanoparticles obtained in step 3) into an aqueous solution, and the concentration of the obtained solution is 2-4 mg / mL; then add zinc nitrate and dimethylimidazole to the obtained solution, so that the concentration of zinc nitrate in the solution is 20-50 mM, and the concentration of dimethylimidazole in the solution is 160-400 mM; then react at room temperature for 30-60 minutes, centrifuge and wash in sequence after the reaction, to obtain a sandwich ZIF-8@Au@ZIF-8 nanomaterial;

[0012] 5) The obtained sandwich ZIF-8@Au@ZIF-8 nanomaterial is 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 5 to 10 minutes. After ultrasonication, centrifugal washing is performed to obtain ZIF-8@Au loaded with 2 layers of AuNCs; the above operation is repeated n-2 times to prepare ZIF-8@(Au@ZIF-8) loaded with n layers of AuNCs. n .

[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 5-10mM, and the concentration of the glutathione GSH solution is 7.5-20mM; 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 enhanced fluorescent composite probe induced by gold nanoclusters emission based on the spatial confinement effect of the ZIF-8 surface, the specific process of purifying the gold nanocluster solution in step 1) is: transferring the obtained gold nanocluster solution to a 3000-8000Da 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 on 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 gold nanocluster emission-enhanced fluorescent composite probe based on the spatial confinement effect of the ZIF-8 surface, 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 induced by gold nanoclusters emission based on the spatial confinement effect of ZIF-8 surface, the drying temperature during 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 space confinement effect of ZIF-8 surface induced gold nanoclusters emission enhanced 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, Biotin-Au-ZIF-8@(Au@ZIF-8) was prepared n ;

[0022] b. ELISA avidin modification: First, fix the capture antibody in a 96-well plate and add a concentration of 0.1 to 10 7 fg / mL alpha-fetoprotein AFP sample was incubated for 30 minutes; the 96-well plate was eluted three times with phosphate buffer, and then the biotinylated detection antibody was added and incubated for 30 minutes; it was washed again with phosphate buffer for 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 , the 96-well plate was imaged and analyzed using a fluorescent imager.

[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 carboxyl groups in Biotin-PEG-COOH and the Zn 2+ The coordination effect of ZIF-8@(Au@ZIF-8) n Biotin-ZIF-8@(Au@ZIF-8) was obtained by biotinylation modification. n ;

[0025] b. ELISA avidin modification: First, fix the capture antibody in a 96-well plate and add a concentration of 0.1 to 10 7 fg / mL alpha-fetoprotein AFP sample was incubated for 30 minutes; the 96-well plate was eluted 3 times with phosphate buffer, and then the biotinylated detection antibody was added and incubated for 30 minutes; the phosphate buffer was washed again for 3 times; streptavidin was added and incubated for 30 minutes, and the 96-well plate was eluted 3 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 , the 96-well plate was imaged and analyzed using a fluorescent imager.

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

[0027] 1. The technical solution of the present invention provides a preparation method and application of an enhanced fluorescent composite probe induced by gold nanoclusters emission with controllable outer space. This method increases the loading amount by self-assembling gold nanoclusters on the surface of zeolite imidazole framework-8 (ZIF-8) and improves the fluorescence performance of a single nanogroup based on spatial confinement or aggregation induction effect, effectively overcoming the shortcomings of weak fluorescence intensity, poor stability and poor controllability of gold nanoclusters. Based on the functional modification of the self-assembled fluorescent nanogroup, the generalized application effect of the composite probe in immunoassay (enzyme-linked immunosorbent assay, Elisa) is realized, and the prepared composite probe realizes ultra-sensitive detection of biomarkers such as interleukin-6 (IL-6, LOD reduced by 174 times), C-reactive protein (CRP, LOD reduced by 326 times) and procalcitonin (PCT, LOD reduced by 151 times), and the dynamic range of their detection is expanded 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. 4. 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] Fig. 9 Fluorescence 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] Fig.10 Schematic diagram of the application of high-performance immunofluorescent groups in immunoassay detection;

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

[0040] Fig.12 Comparison of the effects of traditional ELISA method and p-FLISA method using ZIF-8@(Au@ZIF-8)4 probe in detecting interleukin-6 (IL-6);

[0041] Depend on Fig.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] Fig.13 Comparison of the effects 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 Fig.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] Fig.14 Comparison of the effects of traditional ELISA method and p-FLISA method using ZIF-8@(Au@ZIF-8)4 probe in detecting procalcitonin (PCT);

[0045] Depend on Fig.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. V. Specific implementation methods:

[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 do not limit the present invention in any way. Without departing from the technical solution of the present invention, any modification or change made to the present invention that is easily realized by a person of ordinary skill in the art will fall within the scope of protection of the claims of the present invention.

[0047] Embodiment 1:

[0048] The present invention provides a method for preparing a composite probe for enhanced fluorescence emission induced by gold nanoclusters based on the spatial confinement effect on the surface of ZIF-8, 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; the two solutions were mixed evenly at 75-80 °C (when the HAuCl4 aqueous solution and the glutathione GSH solution were mixed, the molar ratio of the active ingredients in the two solutions was 1:2), sodium hydroxide solution was added to the obtained mixed solution to adjust its pH value to 7, and the mixture was stirred continuously after adjustment to obtain a gold nanocluster solution; the obtained gold nanocluster solution was transferred to a 5000 Da dialysis bag and dialyzed for purification at room temperature for 18 h, and a concentration of 0.75 mg mL was obtained after purification. -1The AuNCs solution was prepared by TEM analysis of the particle size, morphology and surface morphology of 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, and after being fully dissolved, a 0.8M 2-methylimidazole solution and a 0.1M 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, and reacted at room temperature for 18 hours, and the generated white precipitate was collected; the white precipitate was washed with methanol and water in turn, and then dried to obtain zeolite imidazole framework-8 (with a particle size of 0.05 to 0.5 μm); the obtained zeolite imidazole framework-8 was dispersed in water to obtain a 6 mg·mL -1 The 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 gold nanocluster AuNCs solution obtained after purification in step 1) and the zeolite imidazole framework-8 solution obtained in step 2) were mixed (when the gold nanocluster AuNCs solution and the zeolite imidazole framework-8 solution were mixed, the mass ratio of the active ingredients in the two solutions was 1:6), and mechanically stirred at room temperature at a speed of 500 rpm for 20 minutes, and the gold nanocluster solution was successfully self-assembled on the zeolite imidazole framework-8, and then washed with ultrapure water to obtain ZIF-8@(Au) nanoparticles, which were freeze-dried and stored for later use (the drying temperature during freeze-drying was -50 to -40°C and the drying time was 32 hours);

[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: Disperse the ZIF-8@(Au) nanoparticles obtained in step 3) into an aqueous solution, and the concentration of the obtained solution is 3 mg / mL; then add zinc nitrate and dimethylimidazole to the obtained solution, so that the concentration of zinc nitrate in the solution is 35 mM, and the concentration of dimethylimidazole in the solution is 220 mM; then react at room temperature for 40 minutes, centrifuge and wash in sequence after the reaction, to obtain a sandwich ZIF-8@Au@ZIF-8 nanomaterial;

[0054] 5) mixing the obtained sandwich ZIF-8@Au@ZIF-8 nanomaterial with the gold nanocluster AuNCs solution obtained in step 1) at a mass ratio of 16:1, ultrasonically treating at 25° C. for 10 min, and centrifugally washing after ultrasonic treatment to obtain ZIF-8@Au loaded with two layers of AuNCs;

[0055] Repeat the above operation 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 operation 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 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 Fig. 9 .

[0057] The specific application of the gold nanocluster emission-enhanced fluorescent composite probe based on the ZIF-8 surface spatial confinement effect-induced emission 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 induced by gold nanoclusters based on 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), and the specific steps are as follows (see the attached schematic diagram for the application) Fig.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 that of ZIF-8@(Au@ZIF-8)4 was 6 mg / mL. The mixture was incubated at room temperature for 24 h. Based on the carboxyl group in Biotin-PEG-COOH and the Zn on the surface of ZIF-8, the Zn 2+The coordination effect of ZIF-8@(Au@ZIF-8)4 was used to realize the biotinylation modification of ZIF-8@(Au@ZIF-8)4 to obtain Biotin-ZIF-8@(Au@ZIF-8)4 (see Appendix Fig.11 );

[0061] b. ELISA avidin modification: 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 the biotinylated IL-6 detection antibody was added and incubated for 30 minutes; it was washed again with phosphate buffer for three times; streptavidin was added and incubated for 30 minutes, and the 96-well plate was washed 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 the unattached Biotin-ZIF-8@(Au@ZIF-8)4, and the 96-well plate was imaged and analyzed using a fluorescent imager. Fig.12 , a comparison of the effects 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 induced by gold nanoclusters based on 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), and the specific steps are as follows (see the attached schematic diagram for the application) Fig.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 that of ZIF-8@(Au@ZIF-8)4 was 6 mg / mL. The mixture was incubated at room temperature for 24 h. Based on the carboxyl group in Biotin-PEG-COOH and the Zn on the surface of ZIF-8, the Zn 2+ The coordination effect of ZIF-8@(Au@ZIF-8)4 was used to realize the biotinylation modification of ZIF-8@(Au@ZIF-8)4 to obtain Biotin-ZIF-8@(Au@ZIF-8)4 (see Appendix Fig.11 );

[0065] b. ELISA avidin modification: First, fix the CRP capture antibody in a 96-well plate and add a 0.1-10 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 the biotinylated CRP detection antibody was added and incubated for 30 minutes; it was washed again with phosphate buffer for three times; streptavidin was added and incubated for 30 minutes, and the 96-well plate was washed 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 the unattached Biotin-ZIF-8@(Au@ZIF-8)4, and the 96-well plate was imaged and analyzed using a fluorescent imager. Fig.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 induced by gold nanoclusters based on 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), and the specific steps are as follows (see the attached schematic diagram for the application) Fig.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 that of ZIF-8@(Au@ZIF-8)4 was 6 mg / mL. The mixture was incubated at room temperature for 24 h. Based on the carboxyl group in Biotin-PEG-COOH and the Zn on the surface of ZIF-8, the Zn 2+ The coordination effect of ZIF-8@(Au@ZIF-8)4 was used to realize the biotinylation modification of ZIF-8@(Au@ZIF-8)4 to obtain Biotin-ZIF-8@(Au@ZIF-8)4 (see Appendix Fig.11 );

[0069] b. Enzyme-linked immunosorbent assay (ELISA) avidin modification: First, fix the PCT capture antibody in a 96-well plate and add a 0.1-10 7fg / mL of procalcitonin PCT sample was incubated for 30 minutes; the 96-well plate was washed three times with phosphate buffer, and then the biotinylated PCT detection antibody was added and incubated for 30 minutes; it was washed again with phosphate buffer for three times; streptavidin was added and incubated for 30 minutes, and the 96-well plate was washed 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 the unattached Biotin-ZIF-8@(Au@ZIF-8)4, and the 96-well plate was imaged and analyzed using a fluorescent imager. Fig.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 spatial confinement effect of ZIF-8 surface-induced 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 are fully dissolved in water to obtain HAuCl4 aqueous solution and glutathione GSH solution; the two solutions are mixed evenly at 65-90° C., sodium hydroxide solution is added to the obtained mixed solution to adjust its pH value to 5-8, and the mixture is continuously stirred after adjustment to obtain a gold nanocluster solution; the obtained gold nanocluster solution is purified to obtain a concentration of 0.5-1 mg·mL -1 AuNCs solution; 2) Synthesis of zeolite imidazole framework-8: Zn(NO3)2·6H2O and 2-methylimidazole are dissolved in methanol respectively, and after being fully dissolved, a 2-methylimidazole solution and a zinc nitrate solution are obtained; the 2-methylimidazole solution and the zinc nitrate solution are then mixed evenly, and reacted at room temperature for 12 to 24 hours after mixing, and the generated white precipitate is collected; the white precipitate is washed with methanol and water in turn, and dried after washing to obtain zeolite imidazole framework-8; the obtained zeolite imidazole framework-8 is dispersed in water to obtain a concentration of 4 to 8 mg·mL -1 A zeolite imidazolyl framework-8 solution; 3) Preparation of ZIF-8@(Au) nanoparticles: The gold nanocluster AuNCs solution obtained after purification in step 1) and the zeolite imidazole framework-8 solution obtained in step 2) were mixed, and mechanically stirred at room temperature at a speed of 400-700 rpm for 10-30 min, and the gold nanocluster solution was successfully self-assembled on the zeolite imidazole framework-8, and then washed with ultrapure water to obtain ZIF-8@(Au) nanoparticles, which were freeze-dried and stored for later use; 4) Preparation of sandwich ZIF-8@Au@ZIF-8 nanoparticles: Disperse the ZIF-8@(Au) nanoparticles obtained in step 3) into an aqueous solution, and the concentration of the obtained solution is 2-4 mg / mL; then add zinc nitrate and dimethylimidazole to the obtained solution, so that the concentration of zinc nitrate in the solution is 20-50 mM, and the concentration of dimethylimidazole in the solution is 160-400 mM; then react at room temperature for 30-60 minutes, centrifuge and wash in sequence after the reaction, to obtain a sandwich ZIF-8@Au@ZIF-8 nanomaterial; 5) The obtained sandwich ZIF-8@Au@ZIF-8 nanomaterial is 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 5 to 10 minutes. After ultrasonication, centrifugal washing is performed to obtain ZIF-8@Au loaded with 2 layers of AuNCs; the above operation is repeated n-2 times to prepare ZIF-8@(Au@ZIF-8) loaded with n layers of AuNCs. n .

2. 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 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 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 specific process of purifying the gold nanocluster solution in step 1) is as follows: transferring the obtained gold nanocluster solution to a 3000-8000 Da dialysis bag, and dialysis purification at room temperature for 12-24 hours.

4. 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 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.

5. 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 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 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. Application of a composite fluorescent probe based on the spatial confinement effect of ZIF-8 surface induced gold nanoclusters emission enhanced 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, Biotin-Au-ZIF-8@(Au@ZIF-8) was prepared n ; b. Avidin modification by ELISA: First, fix the capture antibody in a 96-well plate and add a concentration of 0.1 to 10 7 fg / mL alpha-fetoprotein AFP sample was incubated for 30 minutes; the 96-well plate was eluted three times with phosphate buffer, and then the biotinylated detection antibody was added and incubated for 30 minutes; it was washed again with phosphate buffer for 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 , the 96-well plate was imaged and analyzed using a fluorescent imager.

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 carboxyl groups in Biotin-PEG-COOH and the Zn 2+ The coordination effect of ZIF-8@(Au@ZIF-8) n Biotin-ZIF-8@(Au@ZIF-8) was obtained by biotinylation modification. n ; b. Avidin modification by ELISA: First, fix the capture antibody in a 96-well plate and add a concentration of 0.1 to 10 7 fg / mL alpha-fetoprotein AFP sample was incubated for 30 minutes; the 96-well plate was eluted 3 times with phosphate buffer, and then the biotinylated detection antibody was added and incubated for 30 minutes; the phosphate buffer was washed again for 3 times; streptavidin was added and incubated for 30 minutes, and the 96-well plate was eluted 3 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 , the 96-well plate was imaged and analyzed using a fluorescent imager.

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