A colorimetric-fluorescence-catalysis three-signal microsphere tag, its preparation method and application

Abstract

This invention discloses a colorimetric-fluorescence-catalysis tri-signal microsphere tag, its preparation method, and its applications. The invention uses dendritic silica microspheres (dSiO2) with large pores as a template, first assembling oil-phase quantum dots (rQDs), then assembling oil-phase iron nanoparticles (Fe3O4), followed by coating with a biocompatible microporous ZIF-8 shell, and finally loading platinum nanoparticles (Pt) onto the outer layer to obtain "lychee-like" nanospheres – SQF@ZIF-8 / Pt. The SQF@ZIF-8 / Pt material of this invention can construct a colorimetric-fluorescence-catalysis tri-functional signal probe, with rQDs and Fe3O4 providing fluorescence and colorimetric signals, respectively. The probe of this invention can detect antigen concentration in colorimetric, UV, and catalytic modes. Compared to a single detection mode, the signals in the three detection modes can mutually verify each other, improving accuracy. It offers both visual qualitative and multiple quantitative modes, making it convenient for users.

Description

Technical Field

[0001] This invention relates to a colorimetric-fluorescence-catalysis three-signal microsphere label, its preparation method, and its application. Background Technology

[0002] Lateral chromatographic immunoassay (LFIA), as an in vitro diagnostic tool, has made significant progress over the past few decades due to its compliance with the WHO's ASSURED criteria (affordable, highly sensitive, highly specific, user-friendly, rapid and durable, equipment-free or simple, and deliverable to end users). However, traditional gold nanoparticle (GNP)-labeled LFIA relies on a single signal output, which may face challenges such as insufficient sensitivity, limited detection range, and susceptibility to external interference, limiting its widespread application. Experimental parameters, environmental variations, and batch-to-batch variability inevitably affect the detection performance of traditional single-modality LFIA. In contrast, multimodal LFIA, by integrating multiple signal sensing modes, achieves multi-signal output of the detection results, offering the following advantages: First, by combining the unique advantages of each mode, multimodal LFIA circumvents the inherent limitations of a single signal sensing mode and facilitates high-performance detection. Second, multimodal LFIA can provide multiple signal outputs, which is more attractive than traditional LFIA with only a single signal. Third, the signals generated by multimodal LFIA are independent and do not interfere with each other, allowing for self-validation and self-correction to improve reliability. Fourth, multimodal LFIA integrates visual-based qualitative modes and multiple quantitative modes, allowing users to select the appropriate mode based on available equipment and operating conditions, thereby further enhancing flexibility. Fifth, different signal output modes vary in sensitivity, linear range, signal acquisition, and programming, allowing users to skillfully select the appropriate output signal for specific application scenarios to achieve optimal analytical performance. Therefore, developing multimodal LFIA to ensure reliable and accurate detection performance is highly advisable.

[0003] Multifunctional composite nanomaterials, composed of different functional components, are attracting increasing attention due to their excellent comprehensive properties and broad application prospects in analytical fields. The rational design and synthesis of excellent multifunctional nanohybrid systems can not only combine the inherent properties of different nanomaterials but also enhance physical and chemical properties through the synergistic interactions between various components. Currently, many synthetic methods for multifunctional nanocomposites have been reported, such as in-situ growth strategies, layer-by-layer assembly, template-guided assembly methods, and volatilization-induced self-assembly.

[0004] Seed-mediated growth is used for epitaxial growth on the surface of core nanoparticles, allowing the synthesis of shells with varying thicknesses to modulate the optical properties of multifunctional nanocomposites. Careful control of the precursor concentration is crucial during preparation to avoid additional nucleation. For example, Ju et al. used seed-mediated growth to prepare bimetallic Pd@IrNPs with colorimetric-catalytic dual functionality and evaluated their catalytic performance by adjusting the thickness of the Ir shell.

[0005] In the fabrication of multifunctional composite materials, most nanocomposites that do not require lattice matching and similar chemical properties between substances can be self-assembled layer by layer using organic molecular chains or silica layers. Generally, polymer chains containing groups such as –COOH, –PO3H, –SH, and –NH2 are selected to modify the surface of nanoparticles, and then another functional component is attached through electrostatic adsorption or covalent bonding (such as Au–S bonds) to obtain composite nanoparticles with different functions. For example, Lai et al. utilized the unique surface viscosity of polydopamine (PDA) to uniformly and stably disperse Pd / Pt bimetallic nanocages on the surface of Fe4O3 cores, giving the material both magnetic and catalytic functions. A surface-modifiable SiO2 layer can be used as an intermediate layer. Zeng et al. deposited an iron oxide (Fe3O4) layer on CsPbBr3 perovskite quantum dots through a silica layer to achieve enhanced target enrichment and signal intensity.

[0006] Current multilayer assembly structures are mainly based on rigid templates, such as common non-porous templates like polystyrene latex or silica microspheres. Volatilization-induced self-assembly can co-assemble multifunctional components in micelles or silica nanocarriers to prepare high-load-density composite nanomaterials.

[0007] Although various methods for synthesizing multifunctional nanocomposites have been reported, multilayer assembly structures based on rigid, non-porous templates face a significant challenge in assembling nanounits: the internal space of the template is not effectively utilized, and the number of nanounits that can be assembled on the outer surface of the template is limited. Furthermore, the volatilization-induced self-assembly process is susceptible to localized temperature fluctuations and uneven solvent evaporation rates, which can lead to the random distribution of inorganic compounds with different properties within the synthesized coating structure, thereby affecting the overall performance of the material.

[0008] A key aspect of the development of multimodal lateral flow immunoassay (LFIA) is the rational design of multifunctional signal tags. These tags typically consist of multiple components, each contributing to the output signal of the multimodal LFIA. Therefore, selecting building blocks with different functions and precisely controlling their spatial location and quantity are crucial for achieving efficient multimodal LFIA. Summary of the Invention

[0009] To address the aforementioned technical problems in existing technologies, the purpose of this application is to provide a "colorimetric-fluorescence-catalysis" three-signal microsphere tag, its preparation method, and its application. This invention provides a "lychee-like" nanosphere—SQF@ZIF-8 / Pt—made from dendritic silica microspheres (dSiO2) with large pores as a template. This is achieved by first assembling oil-phase quantum dots (rQDs), then assembling oil-phase iron nanoparticles (Fe3O4), followed by coating with a biocompatible microporous ZIF-8 shell, and finally loading platinum nanoparticles (Pt) onto the outer layer. The large-pore dendritic dSiO2 template enables sufficient assembly of rQDs, improving the material's fluorescence intensity. The Fe3O4 NPs not only provide colorimetric signals but also magnetic properties and peroxidase-like activity. Furthermore, the low molar absorptivity of the Fe3O4 NPs prevents severe quenching of the quantum dots. ZIF-8, as a porous material, has excellent adsorption capacity. The adsorbed PtNPs can synergistically catalyze with the Fe3O4 NPs inside the spheres. As an intermediate layer between Fe3O4 and Pt, it can effectively reduce the magnetic influence of Pt on iron. The synthesis conditions are mild and will not quench the internal quantum dots.

[0010] The technical solution adopted in this invention is as follows:

[0011] A method for preparing a colorimetric-fluorescence-catalysis three-signal microsphere tag involves using dendritic silica microspheres (dSiO2) with large pores as a template. First, the dSiO2 template is thiolized to obtain dSiO2-SH. Then, red fluorescent quantum dots (CdSe / CdS / ZnS, i.e., rQDs) are assembled onto the dSiO2-SH to obtain a thiolized silica sphere / red quantum dot assembly (SQ). Next, oil-phase Fe3O4 nanoparticles with oleic acid ligands on their surface are assembled to obtain the assembly SQF. Finally, a biocompatible microporous ZIF-8 shell is coated onto this shell, and platinum nanoparticles are loaded onto the outer layer to obtain "lychee-like" nanospheres SQF@ZIF-8 / Pt, thus completing the preparation.

[0012] Further, the process of thiolizing the dSiO2 template is as follows: the dSiO2 template is dispersed in ethanol at a concentration of 0.5-1.5 mg / mL, ammonia and 3-mercaptopropyltrimethoxysilane are added to the resulting dSiO2 ethanol solution, the mixture is stirred at room temperature for 10-15 h, the precipitate is collected by centrifugation and washed to obtain the thiolized product dSiO2-SH; wherein, the mass fraction of ammonia is 25-30%, the volume fraction of ammonia in the overall reaction system is 1.0-1.5%, and the volume fraction of 3-mercaptopropyltrimethoxysilane in the overall reaction system is 0.08-0.12%.

[0013] Furthermore, the process of assembling quantum dots rQDs using dSiO2-SH is as follows: dSiO2-SH is added to a solution containing red fluorescent quantum dots CdSe / CdS / ZnS, i.e., rQDs, and sonicated at room temperature for 5-30 minutes. After assembly, the supernatant is removed by centrifugation to obtain the thiolized silicon sphere / red quantum dot assembly SQ; the concentration of the rQDs solution is 5-15 mg / mL, and the mass ratio of dSiO2-SH to rQDs is 1:0.8-1.2.

[0014] Further, the preparation method of the oil phase Fe3O4 nanoparticles includes: adding iron salt, dodecyl glycol, dibenzyl ether, oleic acid and oleylamine into a reaction flask, sonicating to dissolve the solid, purging with nitrogen to completely replace the air in the reaction flask, raising the temperature to 180-200℃ and stirring vigorously for 20-40 minutes, then continuing to raise the temperature to 285-300℃ and stirring for 1-3 hours to end the reaction; after cooling to room temperature, adding methanol to the crude solution to precipitate the magnetic particles generated in the reaction, magnetic separation, removing the supernatant, adding chloroform to the precipitate to dissolve the magnetic particles, adding methanol again to precipitate the particles, and finally dissolving and storing the precipitate in toluene for later use;

[0015] In the preparation method of the oil phase Fe3O4 nanoparticles, the iron salt is acetylacetone iron, and the feeding ratio of the iron salt, dodecanediol, dibenzyl ether, oleic acid and oleylamine is (0.6-0.8)g:(1.8-2.4)g:(8-12)mL:(3-5)mL:(3-5)mL.

[0016] Furthermore, the process of assembling oil-phase Fe3O4 nanoparticles in the SQ assembly is as follows:

[0017] 1) The assembly SQ was dispersed in an ethanol solution of polyethyleneimine (PEI) and stirred at room temperature for 1-3 hours. After the reaction was completed, the product was washed by centrifugation with ethanol. The mass ratio of dSiO2 microsphere template to polyethyleneimine (PEI) in the assembly SQ was 1.2-1.5:1.

[0018] 2) The product of step 1) and the oil phase Fe3O4 nanoparticles were dispersed in toluene, sonicated at room temperature for 5-20 min, and the assembled structure was separated by centrifugation to obtain the product assembly SQF; wherein, the mass ratio of the oil phase Fe3O4 nanoparticles in step 2) to the dSiO2 microsphere template in the assembly SQ in step 1) is 0.8-1.2:1.

[0019] Furthermore, before coating the ZIF-8 shell, the assembly SQF also includes the following steps: the assembly SQF is dispersed in a chloroform solution containing 5-15 mg / mL PVP and stirred at room temperature for 20-30 h to replace the oleic acid ligands on the Fe3O4 surface with PVP. After the reaction is completed, the assembly is precipitated with hexane and then washed to remove excess PVP to obtain the SQF-PVP product.

[0020] The process of coating the ZIF-8 shell with SQF-PVP product is as follows: SQF-PVP, Zn(NO3)2·6H2O and 2-MeIM are dispersed together in methanol and reacted on a shaker at 200-400 rpm for 2-4 h at room temperature; after the reaction, the product is washed by centrifugation with methanol to obtain SQF@ZIF-8; wherein, the molar ratio of Zn(NO3)2·6H2O and 2-MeIM is 1:5-10, and the ratio of the total amount of Zn(NO3)2·6H2O and 2-MeIM to the mass of SQF-PVP is 0.4-0.6:10, the unit of amount of substance is mmol, and the unit of mass is g.

[0021] Furthermore, the load on Pt on SQF@ZIF-8 / Pt is 1%-10%.

[0022] The application of the aforementioned "colorimetric-fluorescence-catalysis" three-signal microsphere tag in the SARS-CoV-2 N protein antigen includes the following steps:

[0023] 1) Preparation of test strips: The test strip consists of three parts: sample pad, NC membrane and absorbent paper. The T line on the NC membrane is coated with the capture antibody Ab2 of the SARS-CoV-2 n protein, and the C line is coated with goat anti-mouse IgG. The strips are then dried and ready for use. After assembling and cutting the test strip components, the test strip product for detection is obtained.

[0024] 2) Preparation of probe SQF@ZIF-8 / Pt-Ab1: The COVID-19 N protein labeled antibody was added to a solution containing "lychee-like" nanospheres SQF@ZIF-8 / Pt. The mass of the COVID-19 N protein labeled antibody was 1-3% of the mass of SQF@ZIF-8 / Pt. The reaction was carried out at room temperature for 2-4 hours, and then blocked with a solution containing 0.5% BSA + 0.1% casein for 2-3 hours. After the reaction was completed, the sample was washed with PBST buffer to obtain probe SQF@ZIF-8 / Pt-Ab1.

[0025] 3) Detection of SARS-CoV-2 N protein antigen concentration and preparation of standard curve:

[0026] S1: Dilute the COVID-19 N protein antigen with loading buffer to prepare a series of antigen standard solutions with different antigen concentrations; add probe SQF@ZIF-8 / Pt-Ab1 to each antigen standard solution for detection, incubate at room temperature for 3-10 min, remove the supernatant after magnetic separation, and resuspend in loading buffer. Add the resulting sample solution to the sample pad of the test strip, perform chromatography for 10-30 min, and then perform signal detection.

[0027] S2: Following the steps in S1, create a standard curve for at least one of the following three modes:

[0028] ① Colorimetric mode: When detecting the signal after 10-30 minutes of chromatography in step S1, take a picture of the test strip under sunlight to obtain the colorimetric signal. Following this process, plot the standard curve in colorimetric mode with the antigen concentration as the x-axis and the gray value of the T line under the colorimetric signal of the test strip as the y-axis.

[0029] ② Fluorescence mode: When detecting the signal after 10-30 minutes of chromatography in step S1, take a fluorescence mode photo under UV light in a dark environment. Following this process, plot the standard curve under the fluorescence mode with the antigen concentration as the x-axis and the R value of the T line under the fluorescence mode photo of the test strip as the y-axis.

[0030] ③ Catalytic mode: When detecting the signal after 10-30 min of chromatography in step S1, add the AEC colorimetric solution and H2O2 solution to the NC membrane of the test strip and react for 20-60 min. After terminating the AEC colorimetric reaction with pure water, record the catalytic mode signal under sunlight using a camera. Following this process, plot the standard curve under the catalytic mode with the antigen concentration as the x-axis and the gray value of the T line under the catalytic mode signal of the test strip as the y-axis.

[0031] 4) When performing actual sample solution testing, refer to the testing process in step 3) to obtain antigen concentration detection results in at least one of the three modes: colorimetric mode, fluorescence mode, and catalytic mode.

[0032] Furthermore, the loading buffer is a pH 7.4 PB buffer containing 1% BSA + 1% Tween-20.

[0033] The key point of this invention is:

[0034] 1. Using dendritic silica microspheres with large pores as a template, rQDs and Fe3O4 were assembled sequentially, which endowed the material with fluorescence properties, as well as magnetic, colorimetric and catalytic properties.

[0035] 2. Fe3O4 itself has low absorbance, and its inherent color and catalytic activity are insufficient, resulting in relatively low detection sensitivity at the μg / mL and ng / mL levels. In order to improve the colorimetric signal brightness of Fe3O4, a layer of 2.5nm PtNPs was adsorbed on the outer layer of ZIF-8.

[0036] 3. Growing a ZIF-8 layer on the outer layer of Fe3O4 can reduce the magnetic shielding effect of the outer PtNPs on Fe3O4, and the pores of ZIF-8 itself can facilitate the diffusion of catalytic substrates inside and outside the ZIF-8 layer.

[0037] 4. Select smaller Pt NPs (2.5 nm). Pt nanoparticles with a size of 2-3 nm have better catalytic activity.

[0038] Compared with the prior art, the beneficial effects achieved by the present invention are:

[0039] 1. Most of the best existing technologies mentioned in Article 2 cover only one or two signal tags, which limits their versatility. This invention prepares a nanosphere with three signal tags at the same time: colorimetric, fluorescence and catalytic, which improves the versatility of practical applications.

[0040] 2. Large-pore dendritic silica can be used as a template to assemble a large number of quantum dots and iron oxide nanoparticles, effectively improving the fluorescence and colorimetric properties of the material while maintaining good dispersibility, which is beneficial for subsequent practical applications.

[0041] 3. A thin layer of ZIF-8 is grown on the outer layer of the nanospheres. The synthesis procedure is mild and does not require high temperatures like other MOFs, thus protecting the fluorescence of the quantum dots. ZIF-8, as a mild intermediate layer, has a microporous structure, providing a good support for the Pt nanoparticles loaded on the outer layer. This facilitates the internal and external dispersion and transfer of the catalytic substrate, thereby achieving a synergistic catalytic effect.

[0042] 4. The SQF@ZIF-8 / Pt material of this invention constructs a three-functional signal probe that integrates colorimetry, fluorescence, and catalysis. rQDs and Fe3O4 provide fluorescence and colorimetric signals, respectively. The probe of this invention can detect antigen concentration in colorimetric, UV, and catalytic modes. The significance of these three modes is that, compared to a single detection mode, the signals can mutually verify each other, improving accuracy. It offers both visual qualitative and multiple quantitative modes, making it convenient for users. Attached Figure Description

[0043] Figure 1 This is a schematic diagram of the SQF@ZIF-8 / Pt synthesis process of the present invention;

[0044] Figure 2These are transmission electron microscope images of the products at each stage of Embodiment 1 of the present invention;

[0045] Figure 3 These are scanning electron microscope images of the products at each stage of Embodiment 1 of the present invention;

[0046] Figure 4 This is the EDS plot of a single SQF@ZIF-8 / Pt in Example 1;

[0047] Figure 5 The results show a comparison of the UV absorption performance of five solutions: dSiO2, rQDs quantum dots, oil-phase nano Fe3O4 particles, Pt NPs, and SQF@ZIF-8 / Pt.

[0048] Figure 6 The results show a comparison of the fluorescence properties of rQDs quantum dots and SQF@ZIF-8 / Pt solutions.

[0049] Figure 7 It is the hysteresis loop of Fe3O4 particles and SQF@ZIF-8 / Pt in Example 1;

[0050] Figure 8 These are the results of the UV absorption curves for different solutions;

[0051] Figure 9a These are the absorbance values ​​of SQF@ZIF-8 / Pt at a wavelength of 500 nm under different mass concentrations;

[0052] Figure 9b The fluorescence intensity results of SQF@ZIF-8 / Pt at different mass concentrations are shown.

[0053] Figure 9c These are the absorbance values ​​at 450 nm for SQF@ZIF-8 / Pt at different mass concentrations after terminating the TMB colorimetric reaction with concentrated sulfuric acid.

[0054] Figure 10 It shows the relationship between the colorimetric mode of the test strip photographed under sunlight and the change of the T-line gray value of the test strip with the concentration of the COVID-19 N protein antigen under the colorimetric signal of the test strip;

[0055] Figure 11 These are fluorescent photographs of the test strip under ultraviolet light in fluorescence mode, and curves showing the relationship between the intensity of the R value of the T line on the test strip and the concentration of the COVID-19 N protein antigen.

[0056] Figure 12 The images show the test strip under sunlight in catalytic mode and the curve showing the relationship between the gray value of the T line under the catalytic mode signal of the test strip and the concentration of the COVID-19 N protein antigen.

[0057] Figure 13These are the results of standard curves prepared in three modes: colorimetric mode, catalytic mode, and fluorescence mode.

[0058] Figure 14 It is the specific detection result of the test strip for different antigens. Detailed Implementation

[0059] In this embodiment of the invention, the mass concentration of ammonia water is 25-28%.

[0060] Example 1:

[0061] 1. Preparation of dendritic mesoporous silica (dSiO2):

[0062] The oil bath temperature was set to 80℃. 25 mL of ultrapure water and 68 mg of triethanolamine (TEA) were added to a 50 mL round-bottom flask, and the mixture was stirred in the oil bath for 30 min. Then, 380 mg of cetyltrimethylammonium bromide (CTAB) and 284 mg of sodium salicylate (NaSaI) were added, and the reaction was continued for one hour. After one hour, 4 mL of tetraethyl orthosilicate (TEOS) was added, and the reaction was continued for another 4 hours. After the reaction was complete, the mixture was washed by centrifugation with anhydrous ethanol, and then stirred for another 6 hours in a mixture of methanol (50 mL) and hydrochloric acid (3 mL, 65% by mass) at 60℃ to remove residual surfactants in the dendritic silica. Finally, the mixture was washed three times by centrifugation with anhydrous ethanol to obtain dendritic mesoporous silica (dSiO2).

[0063] 2. Preparation of dSiO2-SH:

[0064] Dissolve 180 mg dSiO2 in 200 mL anhydrous ethanol, add 2.5 mL ammonia and 200 μL 3-mercaptopropyltrimethoxysilane, stir at room temperature for 12 h, and after the reaction is complete, wash with anhydrous ethanol by centrifugation and store in 30 mL anhydrous ethanol.

[0065] 3. Preparation of oil-phase nano-Fe3O4 particles:

[0066] 700 mg of acetylacetone iron powder, 2.02 g of dodecanediol, 10 mL of dibenzyl ether, 4 mL of oleic acid, and 4 mL of oleylamine were added to a 50 mL three-necked flask and dissolved by sonication. The mixture was heated to 50 °C at a rate of 10 °C / 5 min in an anhydrous and oxygen-free environment, maintaining evacuation until the bubbles on the liquid surface disappeared. Nitrogen gas was then introduced to ensure the reactants were in an anhydrous and oxygen-free environment. The reaction mixture was heated to 200 °C and held at that temperature for 30 min, then further heated to 285 °C and reacted at this temperature for 2 h before the reaction was terminated. After the three-necked flask cooled to room temperature, an appropriate amount of anhydrous methanol solution was added to the crude liquid to precipitate the particles, which were then washed with a magnet. Chloroform solution was added to the precipitate to dissolve the magnetic particles, and the washing process was repeated. Finally, the precipitate was dispersed in toluene.

[0067] 4. Assembly of red quantum dots (rQDs) and Fe3O4:

[0068] Assembly of rQDs: Take 20 mg dSiO2-SH and add 2 mL of toluene solution of rQDs (10 mg / mL). rQDs are red fluorescent quantum dots CdSe / CdS / ZnS. Assemble in an ultrasonic environment at room temperature for 10 min. After assembly, centrifuge to remove the supernatant to obtain the thiolized silicon sphere / red quantum dot assembly SQ.

[0069] Assembly of Fe3O4: The product SQ obtained in the previous step was dispersed in 40 ml of ethanol solution containing 14 mg of polyethyleneimine (PEI) and stirred at room temperature for 2 h. After washing three times with ethanol by centrifugation, it was added to 2 mL of toluene, followed by 20 mg of Fe3O4. Assembly was then carried out in an ultrasonic environment and sonicated at room temperature for 7 min. After assembly, the assembly SQF was obtained. SQF was dispersed in 60 ml of chloroform solution containing 600 mg of PVP (Mw = 10000) (the oleic acid ligand was replaced with PVP to facilitate the next step of ZIF-8 growth). The reaction was stirred at room temperature for 24 h to replace the oleic acid ligand on the Fe3O4 surface with PVP. After the reaction was completed, the assembly was precipitated with hexane, and excess PVP was washed away by centrifugation with a 1:1 (v / v) mixture of n-hexane and chloroform. The product was stored in anhydrous methanol and the final product was labeled SQF-PVP.

[0070] 5. Preparation of PtNPs:

[0071] At room temperature, 533 mg of polyvinylpyrrolidone (PVP, Mw = 24000) was added to 180 mL of anhydrous methanol and an aqueous solution of H₂PtCl₆·6H₂O (6.0 Mm, 20 mL). The mixture was refluxed at 70 °C for 3 h. Methanol was removed by rotary evaporator, and the nanoparticles in the remaining solution were precipitated by acetone. Then, the nanoparticles were washed with a mixture of chloroform and n-hexane (4:1 v / v) at 6000 rpm for 5 min to remove excess PVP. The PtNPs product was stored in 20 mL of anhydrous methanol.

[0072] The PtNPs prepared in step 5 were characterized by TEM and SEM, and their average particle size was 2.5 nm.

[0073] 6. Outer layer growth of ZIF-8 and modification of Pt NPs:

[0074] 10 mg of SQF-PVP was dispersed in 10 mL of anhydrous methanol, and 1 mL of Zn(NO3)2·6H2O (0.0672 mM) methanol solution and 4 mL of 2-MeIM (0.1073 mM) methanol solution were added sequentially. The reaction was carried out at room temperature and shaken at 300 rpm for 3 h. After the reaction was completed, the product was washed three times by centrifugation with methanol to obtain SQF@ZIF-8 product.

[0075] Adsorption of PtNPs: 10 mg of SQF@ZIF-8 was dispersed in 10 mL of anhydrous methanol, and then 0.5 mL of PtNPs solution (containing about 1 mg of PtNPs) was added. The mixture was stirred and adsorbed for 6 h under nitrogen protection. Finally, the mixture was centrifuged and washed to obtain the target product SQF@ZIF-8 / Pt nanospheres with a "lychee-like" appearance.

[0076] Performance tests were performed on the products from each stage of Example 1:

[0077] 1) Transmission electron micrographs of the products at each stage of Example 1 of the present invention are shown below. Figure 2 middle, Figure 2 The sub-images a1-e1 correspond to the transmission electron microscopy (TEM) images of dSiO2, SQ, SQF, SQF@ZIF-8, and SQF@ZIF-8 / Pt, respectively. Figure 2 It can be seen that as rQDs and Fe3O4 are assembled, the pores of the dendritic silicon spheres become smaller and smaller. Figure 2 Subplot d1 shows the growth of ZIF-8. Figure 2 Subplot e1 shows that Pt particles were adsorbed on ZIF-8.

[0078] Scanning electron microscope images of the products at each stage of Example 1 of this invention are shown below. Figure 3 , Figure 3 The sub-images (a2-e2) are scanning electron microscope (SEM) images of dSiO2, SQ, SQF, SQF@ZIF-8, and SQF@ZIF-8 / Pt, respectively, showing the specific morphology of the materials at different synthesis stages. As the building blocks are filled, the pores of the dendritic silicon become smaller, indicating that the internal pore size of the dendritic silicon is gradually occupied.

[0079] 2) See the EDS plot of a single SQF@ZIF-8 / Pt in Example 1. Figure 4 The EDS plot shows the elements contained in SQF@ZIF-8 / Pt.

[0080] 3) Dilute solutions with a concentration of 2 mg / mL were prepared for dSiO2, rQDs quantum dots, oil-phase nano-Fe3O4 particles, PtNPs, and SQF@ZIF-8 / Pt in Example 1. The solvent for the solutions of dSiO2, PtNPs, and SQF@ZIF-8 / Pt was anhydrous methanol, while the solvent for the solutions of rQDs quantum dots and Fe3O4 particles was toluene. The normalized UV absorption curves of the five solutions (dSiO2, rQDs quantum dots, oil-phase nano-Fe3O4 particles, PtNPs, and SQF@ZIF-8 / Pt) are shown below. Figure 5 ,in addition Figure 5 The illustrations in the middle correspond to photographs of the five solutions under sunlight. Figure 5 It characterizes the colorimetric properties of a material, from Figure 5 As can be seen from the data, the ultraviolet absorption spectrum of SQF@ZIF-8 / Pt covers the characteristic ultraviolet absorption peaks of rQDs, Fe3O4, and Pt.

[0081] 4) Dilute solutions of 2 mg / mL were prepared for both rQDs quantum dots and SQF@ZIF-8 / Pt from Example 1. The solvent for the rQDs quantum dot solution was toluene, and the solvent for the SQF@ZIF-8 / Pt solution was anhydrous methanol. The normalized fluorescence intensity curves of the two solutions are shown in [Figure 1]. Figure 6 From Figure 6 As can be seen, SQF@ZIF-8 / Pt possesses excellent fluorescence properties. Figure 6 The illustrations, from left to right, are photographs of rQDs solution under sunlight and ultraviolet light, and photographs of SQF@ZIF-8 / Pt solution under sunlight and ultraviolet light.

[0082] from Figure 6 It can be seen that although SQF@ZIF-8 / Pt assembled iron oxide particles after the quantum dots were assembled, as well as the subsequent ZIF-8 growth and Pt adsorption, it did not affect its fluorescence performance. The fluorescence spectrum of SQF@ZIF-8 / Pt was only slightly blue-shifted compared to pure rQDs, indicating that SQF@ZIF-8 / Pt has excellent fluorescence performance.

[0083] 5) The hysteresis loops of Fe3O4 particles and SQF@ZIF-8 / Pt in Example 1 are shown below. Figure 7 The magnetic properties of the material were characterized. The magnetic saturation intensity of SQF@ZIF-8 / Pt was lower than that of pure Fe3O4 because the amount of Fe3O4 assembled was limited. This proves that the material has excellent magnetic separation function and can use magnetic separation to clean impurities in the sample.

[0084] 6) Prepare the following solutions:

[0085] TMB was dissolved in DMSO to obtain a 1 mM TMB solution. SQF, SQF@ZIF-8, and SQF@ZIF-8 / Pt were each prepared into 0.1 mg / mL solutions using methanol as the solvent.

[0086] ①TMB + H2O2 solution: 1mM TMB solution and 2M H2O2 aqueous solution are mixed in a volume ratio of 1:1.

[0087] ②TMB+H2O2+SQF@ZIF-8 solution: 1mM TMB solution, 2M H2O2 aqueous solution, and 0.1mg / ml SQF@ZIF-8 solution are mixed in a volume ratio of 2:3:1.

[0088] ③TMB+H2O2+SQF solution: 1mM TMB solution, 2M H2O2 aqueous solution, and 0.1mg / ml SQF solution are mixed in a volume ratio of 2:3:1.

[0089] ④TMB+H2O2+SQF@ZIF-8 / Pt solution: 1mM TMB solution, 2M H2O2 aqueous solution, and 0.1mg / ml SQF@ZIF-8 / Pt solution are mixed in a volume ratio of 2:3:1.

[0090] The UV absorption curves of the four solutions ①-④ above are shown in [reference]. Figure 8 , Figure 8 The inset shows photographs of the four solutions after reacting at room temperature for 2 minutes. The insets correspond to the four solutions numbered ①-④ above, from left to right.

[0091] from Figure 8 It can be seen that: when only TMB and H2O2 are present, due to the insufficient oxidation capacity of H2O2, no peak (the characteristic UV absorption peak of TMB oxidation) was observed at 652 nm in the UV spectrum; when SQF was added, a peak was observed at 652 nm, proving that SQF has peroxidase-like activity; when SQF@ZIF-8 was added, the peak at 652 nm weakened because ZIF-8 does not have peroxidase-like activity; after adding SQF@ZIF-8 / Pt, the absorption peak at 652 nm was significantly enhanced, indicating that SQF@ZIF-8 / Pt has excellent peroxidase activity.

[0092] 7) The absorbance values ​​of methanol solutions of different concentrations of SQF@ZIF-8 / Pt at a wavelength of 500 nm are shown in the figure. Figure 9a Fluorescence intensity is shown in Figure 9b .

[0093] A 1 mM TMB solution, a 2 M H2O2 aqueous solution, and methanol solutions of different concentrations of SQF@ZIF-8 / Pt were mixed at a volume ratio of 2:3:1. The mixture was allowed to react at room temperature for 30 min, and the absorbance at 450 nm was obtained after terminating the TMB reaction with concentrated sulfuric acid. Figure 9c .

[0094] Figures 9a-9c The differences in the signal responses of colorimetric, fluorescence, and catalytic signals of SQF@ZIF-8 / Pt in the solution at the same mass concentration can be observed, indicating that SQF@ZIF-8 / Pt has three different sensitivity signals.

[0095] The SARS-CoV-2 N protein capture antibody (Ab2), SARS-CoV-2 N protein marker antibody (Ab1), and SARS-CoV-2 N protein antigen NP used in Example 2 of this invention were all derived from Nanjing Baikang Biotechnology Co., Ltd. Antigens Flu A, Flu B, MP, and HRSV were derived from Feipeng Biotechnology, and Human IgG was derived from Lianke Biotechnology.

[0096] Example 2:

[0097] Antigen detection was performed using the "lychee-like" nanospheres SQF@ZIF-8 / Pt target product finally prepared in Example 1. The experimental steps are as follows:

[0098] 1) The test strip consists of three parts: a sample pad, an NC membrane, and absorbent paper. The sample pad is pre-treated with 20mM PB7.4 buffer (containing 0.5% BSA, 0.1% sodium caseinate, 3% trehalose, 1% Tween-20, and 0.2% PVP24000) and dried for later use. The T line of the NC membrane is coated with the capture antibody (Ab2) of the SARS-CoV-2 N protein, and the C line is coated with goat anti-mouse IgG. The membrane is also dried for later use. After assembling and cutting the test strip assembly, it can be used for the detection of SARS-CoV-2 N protein.

[0099] 2) Preparation of SQF@ZIF-8 / Pt-Ab1: 10 μg of COVID-19 N protein-labeled antibody Ab1 was added to 500 μl of a solution containing 500 μg of SQF@ZIF-8 / Pt. The reaction was carried out at room temperature for 3 h, and then blocked with a final concentration of 0.5% BSA + 0.1% casein for 2.5 h. After the reaction was completed, the solution was washed twice with PBST buffer (i.e., PBS buffer containing 0.05% Tween 20) and stored in PBST at 4°C.

[0100] 3) Testing steps:

[0101] S1: The SARS-CoV-2 N protein antigen (i.e., SARS-CoV-2 NP) was diluted with loading buffer (10mM PB buffer at pH=7.4 + 1% BSA + 1% Tween-20) to dilute the SARS-CoV-2 antigen into a series of concentration gradients (0, 0.025, 0.050, 0.1, 0.25, 0.5, 1, 2.5, 5, 10, 25, 50, 100, 250, 500 ng / ml). 8μg of the probe prepared in step 2) was added, and the mixture was incubated at room temperature for 5 min, followed by magnetic separation for 10 min. The supernatant was removed, and the mixture was resuspended in loading buffer to obtain the sample solution. The sample solution was dropped onto the sample pad of the test strip, and after chromatography for 15 min, the signal was detected.

[0102] S2: Following the steps in S1, create a standard curve for at least one of the following three modes:

[0103] ① Colorimetric mode: When the signal is detected 15 minutes after chromatography in step S1, the colorimetric signal of the test strip is obtained by taking a picture under sunlight. Following this process, a standard curve in colorimetric mode is plotted with the antigen concentration as the x-axis and the gray value of the T line under the colorimetric signal of the test strip as the y-axis.

[0104] ② Fluorescence mode: When detecting the signal 15 minutes after step S1 chromatography, take a fluorescence mode photo under UV light in a dark environment. Following this process, plot the standard curve under the fluorescence mode with the antigen concentration as the x-axis and the R value of the T line under the fluorescence mode photo of the test strip as the y-axis.

[0105] ③ Catalytic mode: When detecting the signal after 15 min of chromatography in step S1, add 30 μl of AEC colorimetric solution and 20 μl of 2MH2O2 solution to the NC membrane of the test strip and react for 30 min. After terminating the AEC colorimetric reaction with pure water, record the catalytic mode signal under sunlight using a camera. Following this process, plot the standard curve under the catalytic mode with the antigen concentration as the x-axis and the gray value of the T line under the catalytic mode signal of the test strip as the y-axis.

[0106] Following the above testing procedure, the relationship between the colorimetric mode of the test strip photographed under sunlight and the change in the gray value of the T line of the test strip under the colorimetric signal as a function of the concentration of the SARS-CoV-2 N protein antigen is shown in the figure. Figure 10 .

[0107] Following the above testing procedure, the fluorescence photographs of the test strips under ultraviolet light in fluorescence mode and the curve showing the relationship between the T-line R-value intensity of the test strips and the concentration of the SARS-CoV-2 N protein antigen are shown below. Figure 11 .

[0108] Following the above testing procedure, the images of the test strip taken under sunlight in catalytic mode and the curve showing the relationship between the gray value of the T line under the catalytic mode signal of the test strip and the concentration of the SARS-CoV-2 N protein antigen are shown below. Figure 12 .

[0109] according to Figure 10-12 The results of the standard curves produced under the three modes are summarized in Figure 13 middle. Figure 13 In the standard curve results, from left to right, these represent the standard curves for catalytic mode, fluorescence mode, and colorimetric mode. From... Figure 3 A comparison of the results from the three standard curves shows that the catalytic mode has the highest sensitivity, followed by the fluorescence mode, while the colorimetric mode is relatively worse.

[0110] In addition, using the probe and test strip of Example 2 of this invention, and following the operation steps 1)-3) of Example 2, different antigen solutions were specifically detected in a catalytic mode. The comparison results of the signal response intensity for different antigen detections are shown in [the figure]. Figure 14 ,from Figure 14 It can be seen that different antigen interference substances were not nonspecifically recognized. The signal response intensity of antigens Flu A, Flu B, MP, HRSV, and Human IgG was much smaller than that of the target antigen SARS-Cov-2 NP, indicating that the test strip constructed in this invention has high specificity.

Claims

1. A method for preparing a "colorimetric-fluorescence-catalysis" three-signal microsphere tag, characterized in that... Using dendritic silica microspheres dSiO2 with large pores as templates, the dSiO2 template was first modified with thiol to obtain dSiO2-SH. Red fluorescent quantum dots CdSe / CdS / ZnS, i.e. rQDs, were then assembled on the dSiO2-SH to obtain the thiolized silica sphere / red quantum dot assembly SQ. Then, oil-phase Fe3O4 nanoparticles with oleic acid ligands on the surface were assembled to obtain the assembly SQF. Finally, a biocompatible microporous ZIF-8 shell was coated on the shell, and platinum nanoparticles were loaded on the outer layer to obtain "lychee-like" nanospheres SQF@ZIF-8 / Pt, thus completing the preparation.

2. The method for preparing a "colorimetric-fluorescence-catalysis" three-signal microsphere tag as described in claim 1, characterized in that... The process of thiolizing the dSiO2 template is as follows: the dSiO2 template is dispersed in ethanol at a concentration of 0.5-1.5 mg / mL. Ammonia and 3-mercaptopropyltrimethoxysilane are added to the resulting dSiO2 ethanol solution. The mixture is stirred at room temperature for 10-15 h. The precipitate is collected by centrifugation and washed to obtain the thiolized product dSiO2-SH. The mass fraction of ammonia is 25-30%, and the volume fraction of ammonia in the overall reaction system is 1.0-1.5%. The volume fraction of 3-mercaptopropyltrimethoxysilane in the overall reaction system is 0.08-0.12%.

3. The method for preparing a "colorimetric-fluorescence-catalysis" three-signal microsphere tag as described in claim 1, characterized in that... The process of assembling quantum dots rQDs using dSiO2-SH is as follows: dSiO2-SH is added to a solution containing red fluorescent quantum dots CdSe / CdS / ZnS, i.e., rQDs, and sonicated at room temperature for 5-30 minutes. After assembly, the supernatant is removed by centrifugation to obtain the thiolized silicon sphere / red quantum dot assembly SQ. The concentration of the rQDs solution is 5-15 mg / mL, and the mass ratio of dSiO2-SH to rQDs is 1:0.8-1.

2.

4. The method for preparing a "colorimetric-fluorescence-catalysis" three-signal microsphere tag as described in claim 1, characterized in that... The preparation method of the oil-phase Fe3O4 nanoparticles includes: adding iron salt, dodecyl glycol, dibenzyl ether, oleic acid and oleylamine into a reaction flask, sonicating to dissolve the solid, purging with nitrogen to completely replace the air in the reaction flask, raising the temperature to 180-200℃ and stirring vigorously for 20-40 minutes, then continuing to raise the temperature to 285-300℃ and stirring for 1-3 hours to end the reaction; cooling to room temperature, adding methanol to the crude solution to precipitate the magnetic particles generated in the reaction, magnetic separation, removing the supernatant, adding chloroform to the precipitate to dissolve the magnetic particles, adding methanol again to precipitate the particles, and finally dissolving the precipitate in toluene for later use; In the preparation method of the oil phase Fe3O4 nanoparticles, the iron salt is acetylacetone iron, and the feeding ratio of the iron salt, dodecanediol, dibenzyl ether, oleic acid and oleylamine is (0.6~0.8) g : (1.8~2.4) g : (8~12) mL : (3~5) mL : (3~5) mL.

5. The method for preparing a "colorimetric-fluorescence-catalysis" three-signal microsphere tag as described in claim 1, characterized in that... The process of assembling oil-phase Fe3O4 nanoparticles using the SQ assembly is as follows: 1) The assembly SQ was dispersed in an ethanol solution of polyethyleneimine (PEI) and stirred at room temperature for 1-3 hours. After the reaction was completed, the product was washed by centrifugation with ethanol. The mass ratio of dSiO2 microsphere template to polyethyleneimine (PEI) in the assembly SQ was 1.2-1.5:

1. 2) The product of step 1) and the oil phase Fe3O4 nanoparticles were dispersed in toluene, sonicated at room temperature for 5-20 min, and the assembled structure was separated by centrifugation to obtain the product assembly SQF; wherein, the mass ratio of the oil phase Fe3O4 nanoparticles in step 2) to the dSiO2 microsphere template in the assembly SQ in step 1) is 0.8-1.2:

1.

6. The method for preparing a "colorimetric-fluorescence-catalysis" three-signal microsphere tag as described in claim 1, characterized in that... Before coating the ZIF-8 shell, the assembly SQF also includes the following steps: the assembly SQF is dispersed in a chloroform solution containing 5-15 mg / mL PVP and stirred at room temperature for 20-30 h to replace the oleic acid ligands on the Fe3O4 surface with PVP. After the reaction is completed, the assembly is precipitated with hexane and then washed to remove excess PVP to obtain the SQF-PVP product. The process of coating the ZIF-8 shell with SQF-PVP product is as follows: SQF-PVP, Zn(NO3)2•6H2O and 2-MeIM are dispersed together in methanol and reacted on a shaker at 200-400 rpm for 2-4 h at room temperature; after the reaction, the product is washed by centrifugation with methanol to obtain SQF@ZIF-8; wherein, the molar ratio of Zn(NO3)2•6H2O and 2-MeIM is 1:5-10, and the ratio of the total amount of Zn(NO3)2•6H2O and 2-MeIM to the mass of SQF-PVP is 0.4-0.6:10, the unit of amount of substance is mmol, and the unit of mass is g.

7. The method for preparing a "colorimetric-fluorescence-catalysis" three-signal microsphere tag as described in claim 1, characterized in that... The load on Pt in SQF@ZIF-8 / Pt is 1%-10%.

8. A "colorimetric-fluorescence-catalysis" three-signal microsphere tag prepared by the method described in any one of claims 1-7.

9. The application of the "colorimetric-fluorescence-catalysis" three-signal microsphere tag as described in claim 8 in the SARS-CoV-2 N protein antigen, characterized in that... The application method includes the following steps: 1) Preparation of test strips: The test strip consists of three parts: sample pad, NC membrane and absorbent paper. The T line on the NC membrane is coated with the capture antibody Ab2 of the SARS-CoV-2 N protein, and the C line is coated with goat anti-mouse IgG. The membrane is then dried for later use. After assembling and cutting the test strip components, the test strip product for detection is obtained. 2) Preparation of probe SQF@ZIF-8 / Pt-Ab1: The COVID-19 N protein labeled antibody was added to a solution containing "lychee-like" nanospheres SQF@ZIF-8 / Pt. The mass of the COVID-19 N protein labeled antibody was 1-3% of the mass of SQF@ZIF-8 / Pt. The reaction was carried out at room temperature for 2-4 hours, and then blocked with a solution containing 0.5% BSA + 0.1% casein for 2-3 hours. After the reaction was completed, the sample was washed with PBST buffer to obtain probe SQF@ZIF-8 / Pt-Ab1. 3) Detection of SARS-CoV-2 N protein antigen concentration and preparation of standard curve: S1: Dilute the COVID-19 N protein antigen with loading buffer to prepare a series of antigen standard solutions with different antigen concentrations; add probe SQF@ZIF-8 / Pt-Ab1 to each antigen standard solution for detection, incubate at room temperature for 3-10 min, remove the supernatant after magnetic separation, and resuspend in loading buffer. Add the resulting sample solution to the sample pad of the test strip, perform chromatography for 10-30 min, and then perform signal detection. S2: Following the steps in S1, create a standard curve for at least one of the following three modes: ① Colorimetric mode: When detecting the signal after 10-30 minutes of chromatography in step S1, take a picture of the test strip under sunlight to obtain the colorimetric signal. Following this process, plot the standard curve in colorimetric mode with the antigen concentration as the x-axis and the gray value of the T line under the colorimetric signal of the test strip as the y-axis. ② Fluorescence mode: When detecting the signal after 10-30 minutes of chromatography in step S1, take a fluorescence mode photo under UV light in a dark environment. Following this process, plot the standard curve under the fluorescence mode with the antigen concentration as the x-axis and the R value of the T line under the fluorescence mode photo of the test strip as the y-axis. ③ Catalytic mode: When detecting the signal after 10-30 min of chromatography in step S1, add the AEC colorimetric solution and H2O2 solution to the NC membrane of the test strip and react for 20-60 min. After terminating the AEC colorimetric reaction with pure water, record the catalytic mode signal under sunlight using a camera. Following this process, plot the standard curve under the catalytic mode with the antigen concentration as the x-axis and the gray value of the T line under the catalytic mode signal of the test strip as the y-axis. 4) When performing actual sample solution testing, refer to the testing process in step 3) to obtain antigen concentration detection results in at least one of the three modes: colorimetric mode, fluorescence mode, and catalytic mode.

10. The application as described in claim 9, characterized in that... The loading buffer is a pH 7.4 PB buffer containing 1% BSA + 1% Tween-20.

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