A mesoporous silica shell nanozyme tag and its preparation method and application

Abstract

The present invention provides a nanozyme label with a mesoporous silica shell, a preparation method thereof and an application thereof, relating to the technical field of chemical detection. The nanozyme label with a mesoporous silica shell includes an Fe3O4 core and a mesoporous silica shell coated on the surface of the Fe3O4 core; wherein, Pt particles are filled in the voids of the mesoporous silica shell, and an influenza A N protein detection antibody is connected to the Pt particles through a carboxyalkyl sulfide. The present invention uses Fe3O4 as the core magnetic core, so that the nanozyme has a rapid magnetic separation ability, significantly simplifies the preparation and operation processes; the thickness of the mesoporous silica is controllable, and it has good stability and a large specific surface area; by increasing the loading amount of Pt particles through the mesoporous structure with a large specific surface area, the peroxidase-like (POD-like) activity is significantly improved, thereby improving the detection sensitivity.

Description

Technical Field

[0001] The present invention relates to the technical field of chemical detection, and in particular to a nanozyme label of a mesoporous silica shell and a preparation method and application thereof. Background Art

[0002] Recurrent epidemics of influenza A pose a major public health challenge, particularly affecting vulnerable groups with impaired immune responses, such as the elderly and children. Therefore, emphasis on timely and accurate viral diagnosis is essential for effective management and control. Over the years, diagnostic methods for viral infections have undergone tremendous progress, including traditional enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay (IFA) and polymerase chain reaction (PCR). Among these methods, real-time reverse transcription polymerase chain reaction (RT-PCR) is generally considered to be the ultimate benchmark for identifying influenza viruses. However, the use of RT-PCR usually requires professional techniques and complex laboratory instruments, limiting its large-scale application in rapid field diagnosis. Therefore, there is increasing interest in the development and implementation of point-of-care testing.

[0003] Lateral flow immunoassay (LFIA) is the most commonly used platform for point-of-care testing (POCT) due to its significant advantages in affordability, real-time analysis, mobility, and user-friendly operation. In particular, colorimetric LFIA (C-LFIA) has attracted considerable interest due to its easy visual identification and suitability for resource-limited settings. However, conventional gold nanoparticle (AuNP)-based colorimetric LFIA has limitations in its sensitivity. In recent years, the emergence of innovative detection methods has improved the sensitivity of LFIA, including fluorescence technology, photothermal technology, and surface-enhanced Raman scattering (SERS) technology. However, these tests often require specific instrumentation, making them infeasible for application in resource-limited settings.

[0004] Nanozymes combine the stability of nanomaterials with the catalytic activity of natural enzymes and are used in colorimetric amplified LFIA (CA-LFIA) through catalytic reactions with specific enzyme substrates. Today, common types of nanozymes include precious metals (Au, Pt, and Cu), metal oxides (Fe 3 O 4 、MnO 2 and TiO 2 ) and carbon materials (such as carbon nanotubes and graphene). Among these nanomaterials, Fe-based 3 O 4 The magnetic separation process of the composite nanozymes can more effectively recover the target analytes.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The object of the present invention is to provide a nanozyme tag with a mesoporous silica shell and a preparation method and application thereof. The nanozyme tag with a mesoporous silica shell further improves the performance of the biosensor and prepares a mesoporous structure to increase the specific surface area of ​​the composite nanozyme.

[0007] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0008] In a first aspect, the present invention provides a nanozyme tag of a mesoporous silica shell, wherein the nanozyme tag of the mesoporous silica shell comprises Fe 3 O 4 The core and the Fe 3 O 4 a mesoporous silica shell on the inner core;

[0009] The voids in the mesoporous silica shell are filled with Pt particles, and antibodies for detecting influenza A N protein are connected to the Pt particles via carboxyl alkane sulfide.

[0010] Preferably, the Fe 3 O 4 The particle size of the core is 95~105 nm.

[0011] Preferably, the thickness of the mesoporous silica shell is 15-25 nm.

[0012] Preferably, the specific surface area of ​​the mesoporous silica shell is 60-62 m 2 ·g -1 .

[0013] Preferably, the average mesopore size of the mesoporous silica shell is 4.8-4.9 nm.

[0014] Preferably, the total pore volume of the mesoporous silica shell is 0.12-0.14 cm 3 ·g -1 .

[0015] Preferably, the particle size of the Pt particles is 3-6 nm.

[0016] Preferably, the carboxyalkane sulfide is 11-mercaptoundecanoic acid (MUA).

[0017] In a second aspect, the present invention provides a method for preparing a nanozyme tag of a mesoporous silica shell as described in the first aspect, the preparation method comprising:

[0018] (1) Fe 3 O 4The nanoparticles are dispersed in an ethanol aqueous solution containing a dispersant, subjected to a first ultrasonic treatment, a catalyst is added, subjected to a second ultrasonic treatment, an organic silicone grease solution is added, subjected to a third ultrasonic treatment, and the Fe 3 O 4 The surface of the nanoparticles is coated with a silica shell to obtain Fe 3 O 4 @SiO 2 ;

[0019] (2) The Fe 3 O 4 @SiO 2 The silica shell is resuspended in an aqueous solution containing a dispersant, and then stirred after adding a base to etch the silica shell to form a mesoporous silica shell. The product is collected by magnetic separation to obtain Fe 3 O 4 @MSiO 2 ;

[0020] (3) The Fe 3 O 4 @MSiO 2 , a dispersant and chloroplatinic acid are mixed in an ethanol aqueous solution, stirred for the first time, a reducing agent is added, and stirred for the second time to deposit Pt particles in the voids of the mesoporous silica shell to obtain Fe 3 O 4 @MSiO 2 @Pt nanozyme;

[0021] (4) The Fe 3 O 4 @MSiO 2 The Pt nanozyme and carboxyl alkane sulfide were mixed in ethanol and subjected to ultrasonic treatment to connect the carboxyl alkane sulfide to the Pt particles to obtain Fe 3 O 4 @MSiO 2 @Pt-MUA seeds;

[0022] (5) The Fe 3 O 4 @MSiO 2 @Pt-MUA seeds were dispersed in MEST buffer, subjected to the first sonication treatment, and added N -hydroxysuccinimide solution and carbodiimide solution, perform a second ultrasonic treatment to obtain a mixture; disperse the mixture in PBS buffer, add influenza A N protein detection antibody, incubate, add bovine serum albumin solution to block non-binding sites, collect the product by a magnet, and obtain the nanozyme label of the mesoporous silica shell.

[0023] Preferably, in step (1), the dispersant is polyvinyl pyrrolidone K40; the catalyst is NH 4 OH; the organic silicone grease is tetraethoxysilane.

[0024] Preferably, in step (1), the Fe 3 O 4 The volume ratio of nanoparticles, ethanol and water is (0.4-0.8):(30-50):(3-7); wherein the Fe 3 O 4 The volume mass ratio of nanoparticles and dispersant is (0.4~0.8) mL:(10~30) mg.

[0025] Preferably, in step (1), the Fe 3 O 4 The volume ratio of the nanoparticles, the catalyst and the silicone grease solution is (1.7-3.7):(3.4-6.4):1; wherein the concentration of the silicone grease solution is 0.9-1.0 g·mL -1 .

[0026] Preferably, in step (1), the power of the first ultrasonic treatment, the second ultrasonic treatment and the third ultrasonic treatment are each independently 60-100 W.

[0027] Preferably, in step (1), the time of the first ultrasonic treatment is 1 to 10 min; the time of the second ultrasonic treatment is 20 to 40 min; and the time of the third ultrasonic treatment is 20 to 40 min.

[0028] Preferably, step (1) further comprises the following post-treatment: washing the Fe 3 O 4 @SiO 2 Afterwards, it is resuspended in ethanol; wherein the Fe 3 O 4 @SiO 2 The concentration is 5~10 µM.

[0029] Preferably, in step (2), the dispersant is polyvinyl pyrrolidone K15; and the alkali is sodium hydroxide solution.

[0030] Preferably, the concentration of the sodium hydroxide solution is 0.25-0.35 g / mL.

[0031] Preferably, in step (2), the Fe 3 O 4 @SiO 2 , the mass ratio of the dispersant to the alkali is 1:(40-60):(300-500); the Fe3 O 4 @SiO 2 The volume ratio of water is 1:(1~5).

[0032] Preferably, in step (2), the stirring speed is 500-550 rpm, and the stirring time is 4-8 h.

[0033] Preferably, step (2) further comprises the following post-treatment: washing the Fe 3 O 4 @MSiO 2 .

[0034] Preferably, in step (3), the dispersant is polyvinyl pyrrolidone K40; and the reducing agent is sodium borohydride.

[0035] Preferably, in step (3), the Fe 3 O 4 @MSiO 2 , the volume ratio of reducing agent and chloroplatinic acid is 100:(7~9):(4~6).

[0036] Preferably, in step (3), the Fe 3 O 4 @MSiO 2 The mass volume ratio of the ethanol and ethanol aqueous solution is (2~4) mg:(40~60) mL; wherein the volume ratio of the ethanol and water is (10~30):(20~40).

[0037] Preferably, in step (3), the rotation speeds of the first stirring and the second stirring are respectively 500-550 rpm.

[0038] Preferably, in step (3), the first stirring time is 10 to 20 min; the second stirring time is 10 to 30 min.

[0039] Preferably, step (3) further comprises the following post-treatment: washing the Fe 3 O 4 @MSiO 2 @Pt nanozyme, and then resuspended in ethanol; wherein the Fe 3 O 4 @MSiO 2 @The volume ratio of Pt nanozyme and ethanol is 1:(1~5).

[0040] Preferably, in step (4), the carboxyalkane sulfide is 11-mercaptoundecanoic acid.

[0041] Preferably, in step (4), the Fe3 O 4 @MSiO 2 @The volume ratio of Pt nanozyme and carboxyl alkane sulfide is (100~300):1.

[0042] Preferably, in step (4), the final concentration of the carboxyl alkane sulfide is 8-12 mM.

[0043] Preferably, in step (4), the power of the ultrasonic treatment is 60-100 W, and the time of the ultrasonic treatment is 0.5-2 h.

[0044] Preferably, in step (5), the Fe 3 O 4 @MSiO 2 @Pt-MUA seeds, N The volume ratio of the -hydroxysuccinimide solution to the carbodiimide solution is (150~250):1:(4~6).

[0045] Preferably, the N The concentration of the hydroxysuccinimide solution is 90~110 mM.

[0046] Preferably, the concentration of the carbodiimide solution is 9-11 mM.

[0047] Preferably, in step (5), the volume ratio of the mixture, influenza A N protein detection antibody and bovine serum albumin is (330-334):1:(90-120).

[0048] Preferably, in step (5), the power of the first ultrasonic treatment and the second ultrasonic treatment are each independently 60-100 W.

[0049] Preferably, in step (5), the time of the first ultrasonic treatment is 20 to 40 s; and the time of the second ultrasonic treatment is 10 to 20 min.

[0050] Preferably, in step (5), the incubation time is 140 to 160 min.

[0051] Preferably, step (5) further comprises the following post-treatment: resuspending the product collected by the magnet in a PBST solution.

[0052] In a third aspect, the present invention provides a use of a nanozyme tag with a mesoporous silica shell as described in the first aspect in preparing a biosensor for detecting influenza A virus.

[0053] Compared with the prior art, the present invention has the following beneficial effects:

[0054] (1) The present invention establishes a lateral flow immunoassay (LFIA) for detecting influenza A (Flu A) using a "three-in-one" multifunctional mesoporous silica shell nanozyme tag; the nanozyme tag is Fe 3 O 4 As the core, it is wrapped by mesoporous silica to form Fe 3 O 4 @MSiO 2 , and Pt particles filled in the voids of the mesoporous silica shell, the advantages of the composite nanozyme are as follows: (a) Fe 3 O 4 As the inner core magnetic core, the nanozyme has a fast magnetic separation ability, which significantly simplifies the preparation and operation process; (b) the thickness of the mesoporous silica is controllable, and it has good stability and a large specific surface area; (c) the loading amount of Pt particles is increased by the mesoporous structure with a large specific surface area, thereby improving the POD-like activity, thereby significantly improving the POD-like activity and thus improving the detection sensitivity.

[0055] (2) This invention is the first to couple a specific antibody to a mesoporous silica shell nanozyme tag, which can be used to construct a CA-LFIA strip to detect influenza A virus. Compared with previously reported catalytically active materials, MSiO 2 With the introduction of , the designed composite nanozyme has a larger specific surface area, which allows more Pt particles to grow on its surface to improve the catalytic performance.

[0056] (3) After the colorimetric catalysis of the nanozyme tag of the mesoporous silica shell of the present invention, the qualitative and quantitative detection results of the nanozyme tag for influenza A N protein were reduced to 0.01 and 0.0089 ng·mL -1 , which is approximately 100 times more sensitive than commercially available colloidal gold nanoparticle (AuNP)-based LFIA strips; for the detection of inactivated H1N1 virus, quantification can be as low as 33 copies·mL -1 .

[0057] (4) The present invention applies the nanozyme tag of the mesoporous silica shell to the analysis of throat swab samples, and shows high accuracy and reproducibility in the throat swab sample simulation experiment. The "three-in-one" multifunctional nanozyme can quickly, specifically and sensitively detect influenza A virus, and has great potential in virus POCT diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0059] Figure 1 The figure is a schematic diagram of the process for preparing the nanozyme tag of the mesoporous silica shell of the present invention.

[0060] Figure 2A The Fe provided in Example 1 3 O 4 @SiO 2 HRTEM image of.

[0061] Figure 2B The Fe provided in Example 1 3 O 4 @MSiO 2 HRTEM image of.

[0062] Figure 2C The Fe provided in Example 1 3 O 4 @MSiO 2 HRTEM image of @Pt.

[0063] Figure 2D The Fe provided in Example 1 3 O 4 @SiO 2 A local enlarged HRTEM image.

[0064] Figure 2E The Fe provided in Example 1 3 O 4 @MSiO 2 A local enlarged HRTEM image.

[0065] Figure 2F The Fe provided in Example 1 3 O 4 @MSiO 2 Locally enlarged HRTEM image of @Pt.

[0066] Figure 2G The Fe provided in Example 1 3 O 4 , Fe 3 O 4 @MSiO 2 and Fe 3 O 4 @MSiO2 Hysteresis curve diagram of @Pt.

[0067] Figure 2H The Fe provided in Example 1 3 O 4 @MSiO 2 Nitrogen adsorption isotherm and pore size distribution analysis diagram.

[0068] Fig.2I The Fe provided in Example 1 3 O 4 @MSiO 2 @Pt's element surface scan.

[0069] Figure 2J The Fe provided in Example 1 3 O 4 , Fe 3 O 4 @MSiO 2 , Fe 3 O 4 @MSiO 2 XRD pattern of @Pt.

[0070] Figure 2K The Fe provided in Example 1 3 O 4 @MSiO 2 High-resolution spectra of Fe, O, Si, and Pt of @Pt.

[0071] Figure 3 The Fe generated by reducing chloroplatinic acid of different concentrations provided in Example 2 3 O 4 @MSiO 2 The corresponding absorbance graph of @Pt.

[0072] Figure 4A The Fe provided in Example 3 3 O 4 @MSiO 2 @Pt catalytic activity diagram in different concentrations of colorimetric solution.

[0073] Figure 4B The Fe provided in Example 3 3 O 4 and Fe 3 O 4 @MSiO 2 Comparison of enzyme kinetic curves of @Pt when TMB is used as substrate.

[0074] Figure 4C The Fe provided in Example 3 3 O 4 and Fe3 O 4 @MSiO 2 @Pt in H 2 O 2 Comparison of enzyme kinetic curves when used as substrate.

[0075] Figure 4D The Fe provided in Example 3 3 O 4 @Pt, Fe 3 O 4 @SiO 2 @Pt and Fe 3 O 4 @MSiO 2 @ESR spectrum of Pt.

[0076] Figure 5 This is an experimental flow chart of the multimodal immunochromatography technology driven by the mesoporous silica shell nanozyme tag provided in Application Example 1 for detecting influenza A.

[0077] Fig. 6A Optimization results of capture antibody concentration provided for Application Example 1.

[0078] Figure 6B Fe provided for Application Example 1 3 O 4 @MSiO 2 @PtOptimization result diagram of the amount of immune label added.

[0079] Figure 6C This is a graph showing the optimization results of the reaction time provided for Application Example 1.

[0080] Fig. 7A Fe provided for Application Example 1 3 O 4 @MSiO 2 @Pt-LFIA platform shows the colorimetric and catalytic enhanced colorimetric results for detecting different concentrations of Flu A-NPs.

[0081] Figure 7B Fe provided for Application Example 1 3 O 4 @SiO 2 @Pt-LFIA platform shows the colorimetric and catalytic enhanced colorimetric results for detecting different concentrations of Flu A-NPs.

[0082] Figure 7C The test results of the commercial AuNP-LFIA test strip provided for Application Example 1.

[0083] Fig.7D Fe provided for Application Example 1 3 O4 @MSiO 2 @Pt-LFIA platform calibration curve for detection of different concentrations of Flu A-NP.

[0084] Fig. 7E Fe provided for Application Example 1 3 O 4 @SiO 2 @Pt-LFIA platform detects different concentrations of Flu A-NPs with bar graph.

[0085] Figure 7F Fe provided for Application Example 1 3 O 4 @MSiO 2 @Pt-LFIA platform specific detection results.

[0086] Figure 7G Fe provided for Application Example 1 3 O 4 @MSiO 2 @Pt-LFIA platform repeatability test results.

[0087] Fig. 8A Fe provided for Application Example 1 3 O 4 @MSiO 2 @Pt-LFIA platform shows the colorimetric and catalytic enhanced colorimetric results for detecting different concentrations of H1N1 inactivated virus.

[0088] Figure 8B Fe provided for Application Example 1 3 O 4 @MSiO 2 @Pt-LFIA platform calibration curve for detecting different concentrations of H1N1 inactivated virus. DETAILED DESCRIPTION

[0089] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear, however, in the case of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. In this application, unless otherwise stated, the use of "or" means "and / or". In addition, the use of the term "including" and other forms is non-limiting.

[0090] It should be noted that specific details are described in the following description to facilitate a full understanding of the present invention. However, the present invention can be implemented in a variety of other ways different from those described herein, and those skilled in the art can make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0091] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0092] In a first aspect, the present invention provides a nanozyme tag with a mesoporous silica shell, such as Figure 1 As shown, the nanozyme tag of the mesoporous silica shell includes Fe 3 O 4 The core and the Fe 3 O 4 a mesoporous silica shell on the inner core;

[0093] The voids in the mesoporous silica shell are filled with Pt particles, and the Pt particles are connected to influenza A N protein detection antibodies (Influenza A N-protein detection antibody, Catalog# 20221124-2) via carboxyl alkane sulfide.

[0094] In the present invention, a "three-in-one" multifunctional mesoporous silica shell nanozyme tag is used; the nanozyme tag is Fe 3 O 4 As the core, it is wrapped by mesoporous silica to form Fe 3 O 4 @MSiO 2 , and Pt particles filled in the voids of the mesoporous silica shell, and then connecting the Pt particles and influenza A N protein detection antibodies through carboxyl alkane sulfide. The advantages of this composite nanozyme are as follows: (a) Fe 3 O 4 As the inner core magnetic core, the nanozyme has a fast magnetic separation ability, which significantly simplifies the preparation and operation process; (b) the thickness of the mesoporous silica is controllable, and it has good stability and a large specific surface area; (c) the loading amount of Pt particles is increased by the mesoporous structure with a large specific surface area, thereby improving the POD-like activity, thereby significantly improving the POD-like activity and thus improving the detection sensitivity.

[0095] As an optional embodiment, the Fe 3 O 4 The particle size of the inner core is 95 to 105 nm, for example, it can be 95 nm, 96 nm, 98 nm, 100 nm, 102 nm, 104 nm, 105 nm, etc.

[0096] As an optional embodiment, the thickness of the mesoporous silica shell is 15-25 nm, for example, it can be 15 nm, 16 nm, 18 nm, 20 nm, 22 nm, 24 nm, 25 nm, etc.

[0097] As an optional embodiment, the specific surface area of ​​the mesoporous silica shell is 60-62 m 2 ·g -1 , for example, it could be 60 m 2 ·g -1 、60.5 m 2 ·g -1 、61 m 2 ·g -1 、61.5 m 2 ·g -1 、62 m 2 ·g -1 wait.

[0098] As an optional embodiment, the average mesopore size of the mesoporous silica shell is 4.8-4.9 nm, for example, 4.8 nm, 4.82 nm, 4.84 nm, 4.86 nm, 4.88 nm, 4.9 nm, etc.

[0099] As an optional embodiment, the total pore volume of the mesoporous silica shell is 0.12-0.14 cm 3 ·g -1 , for example, it can be 0.12 cm 3 ·g -1 , 0.125 cm 3 ·g -1 , 0.13 cm 3 ·g -1 , 0.135 cm 3 ·g -1 , 0.14 cm 3 ·g -1 wait.

[0100] As an optional embodiment, the particle size of the Pt particles is 3 to 6 nm, for example, it can be 3 nm, 3.5 nm, 4 nm, 4.5 nm, 5 nm, 5.5 nm, 6 nm, etc.

[0101] As an optional embodiment, the carboxyalkane sulfide is 11-mercaptoundecanoic acid (MUA).

[0102] In a second aspect, the present invention provides a method for preparing a nanozyme tag having a mesoporous silica shell as described in the first aspect, such as Figure 1 As shown, the preparation method comprises:

[0103] (1) Fe 3 O 4 The nanoparticles are dispersed in an ethanol aqueous solution containing a dispersant, subjected to a first ultrasonic treatment, a catalyst is added, subjected to a second ultrasonic treatment, an organic silicone grease solution is added, subjected to a third ultrasonic treatment, and the Fe 3 O 4 The surface of the nanoparticles is coated with a silica shell to obtain Fe 3 O 4 @SiO 2 ;

[0104] (2) The Fe 3 O 4 @SiO 2 The silica shell is resuspended in an aqueous solution containing a dispersant, and then stirred after adding a base to etch the silica shell to form a mesoporous silica shell. The product is collected by magnetic separation to obtain Fe 3 O 4 @MSiO 2 ;

[0105] (3) The Fe 3 O 4 @MSiO 2 , a dispersant and chloroplatinic acid are mixed in an ethanol aqueous solution, stirred for the first time, a reducing agent is added, and stirred for the second time to deposit Pt particles in the voids of the mesoporous silica shell to obtain Fe 3 O 4 @MSiO 2 @Pt nanozyme;

[0106] (4) The Fe 3 O 4 @MSiO 2 The Pt nanozyme and carboxyl alkane sulfide were mixed in ethanol and ultrasonicated to connect the carboxyl alkane sulfide to the Pt particles to obtain Fe 3 O 4 @MSiO 2 @Pt-MUA seeds;

[0107] (5) The Fe 3 O 4 @MSiO 2 @Pt-MUA seeds were dispersed in MEST buffer, subjected to the first sonication treatment, and added N-hydroxysuccinimide solution and carbodiimide solution, perform a second ultrasonic treatment to obtain a mixture; disperse the mixture in PBS buffer, add influenza A N protein detection antibody, incubate, add bovine serum albumin solution to block non-binding sites, collect the product by a magnet, and obtain the nanozyme label of the mesoporous silica shell.

[0108] As an optional embodiment, in step (1), the dispersant is polyvinyl pyrrolidone K40 (PVP-K40); the catalyst is NH 4 OH; the organic silicone grease is tetraethoxysilane (TEOS).

[0109] As an optional embodiment, in step (1), the Fe 3 O 4 The volume ratio of nanoparticles, ethanol and water is (0.4~0.8):(30~50):(3~7); wherein, "0.4~0.8" can be, for example, 0.4, 0.5, 0.6, 0.7, 0.8, etc.; "30~50" can be, for example, 30, 35, 40, 45, 50, etc.; "3~7" can be, for example, 3, 4, 5, 6, 7, etc.

[0110] As an optional embodiment, in step (1), the Fe 3 O 4 The volume mass ratio of nanoparticles and dispersant is (0.4~0.8) mL:(10~30) mg; wherein, "0.4~0.8" can be, for example, 0.4, 0.5, 0.6, 0.7, 0.8, etc.; "10~30" can be, for example, 10, 15, 20, 25, 30, etc.

[0111] As an optional embodiment, in step (1), the Fe 3 O 4 The volume ratio of nanoparticles, catalyst and silicone grease solution is (0.4~0.8):(1.0~1.5):(0.20~0.25); wherein, "0.4~0.8" can be, for example, 0.4, 0.5, 0.6, 0.7, 0.8, etc.; "1.0~1.5" can be, for example, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, etc.; "0.20~0.25" can be, for example, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, etc.

[0112] As an optional embodiment, the concentration of the organic silicone grease solution is 0.9-1.0 g·mL -1 , for example, it can be 0.9 g·mL -1 , 0.92 g·mL -1 , 0.94 g·mL-1 , 0.96 g·mL -1 , 0.98 g·mL -1 , 1.0 g mL -1 wait.

[0113] As an optional embodiment, in step (1), the power of the first ultrasonic treatment, the second ultrasonic treatment and the third ultrasonic treatment are each independently 60 to 100 W, for example, 60 W, 65 W, 70 W, 75 W, 80 W, 85 W, 90 W, 95 W, 100 W, etc.

[0114] As an optional embodiment, in step (1), the time of the first ultrasonic treatment is 1 to 10 min, for example, it can be 1 min, 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, etc.

[0115] As an optional embodiment, in step (1), the time of the second ultrasonic treatment is 20 to 40 min, for example, it can be 20 min, 25 min, 30 min, 35 min, 40 min, etc.

[0116] As an optional embodiment, in step (1), the duration of the third ultrasonic treatment is 20 to 40 min, for example, 20 min, 25 min, 30 min, 35 min, 40 min, etc.

[0117] As an optional embodiment, step (1) further includes the following post-treatment: washing the Fe 3 O 4 @SiO 2 After that, it was resuspended in ethanol to obtain Fe 3 O 4 @SiO 2 of ethanol solution.

[0118] As an optional embodiment, in step (1), the number of washing times is more than one time, for example, it can be one time, two times, three times, four times, five times, etc.

[0119] As an optional embodiment, in step (1), the Fe 3 O 4 @SiO 2 Fe in ethanol solution 3 O 4 @SiO 2The concentration is 5~10 µM, for example, it can be 5 µM, 6 µM, 7 µM, 8 µM, 9 µM, 10 µM, etc.

[0120] As an optional embodiment, in step (2), the dispersant is polyvinyl pyrrolidone K15 (PVP-K15); and the base is sodium hydroxide solution.

[0121] As an optional embodiment, in step (2), the Fe 3 O 4 @SiO 2 , the mass ratio of dispersant and alkali is 1:(40~60):(300~500); wherein, “40~60” can be, for example, 40, 45, 50, 55, 60, etc.; “300~500” can be, for example, 300, 350, 400, 450, 500, etc.

[0122] As an optional embodiment, the concentration of the sodium hydroxide solution is 0.25~0.35 g / mL, for example, it can be 0.25 g / mL, 0.26 g / mL, 0.27 g / mL, 0.28 g / mL, 0.29 g / mL, 0.30 g / mL, 0.31 g / mL, 0.32 g / mL, 0.33 g / mL, 0.34 g / mL, 0.35 g / mL, etc.

[0123] As an optional embodiment, in step (2), the Fe 3 O 4 @SiO 2 The volume ratio of alcohol to water is 1:(1-5), for example, it can be 1:1, 1:2, 1:3, 1:4, 1:5, etc.

[0124] As an optional embodiment, in step (2), the stirring speed is 500-550 rpm, for example, it can be 500 rpm, 510 rpm, 520 rpm, 530 rpm, 540 rpm, 550 rpm, etc.

[0125] As an optional embodiment, in step (2), the stirring time is 4 to 8 h, for example, it can be 4 h, 4.5 h, 5 h, 5.5 h, 6 h, 6.5 h, 7 h, 7.5 h, 8 h, etc.

[0126] As an optional embodiment, step (2) further includes the following post-treatment: washing the Fe 3 O 4 @MSiO 2 .

[0127] As an optional embodiment, in step (2), the number of washing times is more than one time, for example, it can be one time, two times, three times, four times, five times, etc.

[0128] As an optional embodiment, in step (3), the dispersant is polyvinyl pyrrolidone K40 (PVP-K40); the reducing agent is sodium borohydride (NaBH 4 ).

[0129] As an optional embodiment, in step (3), the Fe 3 O 4 @MSiO 2 , the volume ratio of the reducing agent and chloroplatinic acid is 100:(7~9):(4~6); wherein, the reducing agent "7~9" can be, for example, 7, 7.5, 8, 8.5, 9, etc.; wherein, the chloroplatinic acid "4~6" can be, for example, 4, 4.5, 5, 5.5, 6, etc.

[0130] As an optional embodiment, in step (3), the Fe 3 O 4 @MSiO 2 The mass volume ratio of the ethanol-water solution is (2-4) mg:(40-60) mL; wherein, "2-4" can be, for example, 2, 2.5, 3, 3.5, 4, etc.; wherein, "40-60" can be, for example, 40, 45, 50, 55, 60, etc.

[0131] As an optional embodiment, in step (3), the volume ratio of ethanol to water is (10-30):(20-40); wherein "10-30" can be, for example, 10, 15, 20, 25, 30, etc.; wherein "20-40" can be, for example, 20, 25, 30, 35, 40, etc.

[0132] As an optional embodiment, in step (3), the rotation speeds of the first stirring and the second stirring are each 500-550 rpm, for example, 500 rpm, 510 rpm, 520 rpm, 530 rpm, 540 rpm, 550 rpm, etc.

[0133] As an optional embodiment, in step (3), the first stirring time is 10 to 20 min, for example, it can be 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, 16 min, 17 min, 18 min, 19 min, 20 min, etc.

[0134] As an optional embodiment, in step (3), the second stirring time is 10 to 30 min, for example, it can be 10 min, 12 min, 14 min, 16 min, 18 min, 20 min, 22 min, 24 min, 26 min, 28 min, 30 min, etc.

[0135] As an optional embodiment, step (3) further includes the following post-treatment: washing the Fe 3 O 4 @MSiO 2 @Pt nanozyme, and then resuspended it in ethanol to obtain Fe 3 O 4 @MSiO 2 @Ethanol solution of Pt nanozyme.

[0136] As an optional embodiment, in step (3), the number of washing times is more than one time, for example, it can be one time, two times, three times, four times, five times, etc.

[0137] As an optional embodiment, in step (3), the Fe 3 O 4 @MSiO 2 @Pt nanozyme in ethanol solution, the Fe 3 O 4 @MSiO 2 @The volume ratio of Pt nanozyme and ethanol is 1:(1~5), for example, it can be 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:4, etc.

[0138] As an optional embodiment, in step (4), the carboxyl alkane sulfide is 11-mercaptoundecanoic acid (MUA).

[0139] As an optional embodiment, in step (4), the Fe 3 O 4 @MSiO 2 @The volume ratio of Pt nanozyme and carboxyl chain alkane sulfide is (100~300):1, for example, it can be 100:1, 120:1, 140:1, 160:1, 180:1, 200:1, 220:1, 240:1, 260:1, 280:1, 300:1, etc.

[0140] As an optional embodiment, in step (4), the final concentration of the carboxyl alkane sulfide is 8-12 mM, for example, 8 mM, 8.5 mM, 9 mM, 9.5 mM, 10 mM, 10.5 mM, 11 mM, 11.5 mM, 12 mM, etc.

[0141] As an optional embodiment, in step (4), the power of the ultrasonic treatment is 60-100 W, for example, it can be 60 W, 65 W, 70 W, 75 W, 80 W, 85 W, 90 W, 95 W, 100 W, etc.

[0142] As an optional embodiment, in step (4), the ultrasonic treatment time is 0.5 to 2 h, for example, it can be 0.5 h, 0.6 h, 0.8 h, 1 h, 1.2 h, 1.4 h, 1.6 h, 1.8 h, 2 h, etc.

[0143] As an optional embodiment, in step (5), the Fe 3 O 4 @MSiO 2 @Pt-MUA seeds, N The volume ratio of the -hydroxysuccinimide solution to the carbodiimide solution is (150-250):1:(4-6), for example, it can be 150:1:4, 170:1:4.4, 190:1:4.8, 210:1:5.2, 230:1:5.6, 250:1:6, etc.

[0144] As an optional implementation, the N The concentration of the -hydroxysuccinimide solution is 90-110 M, for example, it can be 90 mM, 92 mM, 94 mM, 96 mM, 98 mM, 100 mM, 102 mM, 104 mM, 106 mM, 108 mM, 110 mM, etc.

[0145] As an optional embodiment, the concentration of the carbodiimide solution is 9~11 M, for example, it can be 9 mM, 9.2 mM, 9.4 mM, 9.5 mM, 9.6 mM, 9.8 mM, 10 mM, 10.2 mM, 10.4 mM, 10.5 mM, 10.6 mM, 10.8 mM, 11 mM, etc.

[0146] As an optional embodiment, in step (5), the volume ratio of the mixture, influenza A N protein detection antibody and bovine serum albumin is (330-334):1:(90-120); wherein "330-334" can be, for example, 330, 331, 332, 333, 334, etc.; wherein "90-120" can be, for example, 90, 96, 102, 108, 114, 120, etc.

[0147] As an optional embodiment, in step (5), before dispersing the mixture in PBS buffer, the mixture needs to be washed with PBST.

[0148] As an optional embodiment, in step (5), the power of the first ultrasonic treatment and the second ultrasonic treatment are each independently 60 to 100 W, for example, 60 W, 65 W, 70 W, 75 W, 80 W, 85 W, 90 W, 95 W, 100 W, etc.

[0149] As an optional embodiment, in step (5), the time of the first ultrasonic treatment is 20 to 40 s, for example, it can be 20 s, 25 s, 30 s, 35 s, 40 s, etc.

[0150] As an optional embodiment, in step (5), the time of the second ultrasonic treatment is 10 to 20 min, for example, it can be 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, 16 min, 17 min, 18 min, 19 min, 20 min, etc.

[0151] As an optional embodiment, in step (5), the incubation time is 140-160 min, for example, it can be 140 min, 145 min, 150 min, 155 min, 160 min, etc.

[0152] As an optional embodiment, step (5) further includes the following post-treatment: resuspending the product collected by the magnet in a PBST solution.

[0153] In a third aspect, the present invention provides a use of a nanozyme tag with a mesoporous silica shell as described in the first aspect in preparing a biosensor for detecting influenza A virus.

[0154] In a fourth aspect, the present invention provides an immunochromatographic test paper for detecting influenza A virus, the immunochromatographic test paper comprising a nitrocellulose membrane, a detection line T line and a quality control line C line being arranged on the nitrocellulose membrane; wherein the detection line T line is coated with an influenza A N-protein capture antibody (Influenza A N-protein capture antibody; Catalog# 20230228-2). wherein the quality control line C line is coated with a goat anti-mouse IgG antibody (Goat Anti-Mouse IgG).

[0155] In a fifth aspect, the present invention provides a method for detecting influenza A virus, the method for detecting influenza A virus comprising the following steps:

[0156] S1、Fe 3 O 4 @MSiO 2 @Pt-Flu A complex formation:

[0157] The nanozyme tag of the mesoporous silica shell of the first aspect and the solution containing influenza A virus are mixed to form Fe 3 O 4 @MSiO 2 @Pt-Flu A complex, the product was collected by magnetic separation.

[0158] S2. Qualitative analysis:

[0159] Fe 3 O 4 @MSiO 2 The Pt-Flu A complex is added to the sample pad of the immunochromatographic test paper described in the fourth aspect, so that the complex moves along the direction of the absorbent pad under the action of capillary tube; in this step, a part of Fe 3 O 4 @MSiO 2 @Pt-Flu A complex binds to influenza A N protein capture antibody on the detection line T to form Fe 3 O 4 @MSiO 2 @Pt-Flu A-mAbs sandwich structure, producing a black band for initial qualitative analysis; excess Fe 3 O 4 @MSiO 2 The Pt-Flu A complex continues to flow forward along the immunolabeling line and is captured on the quality control line.

[0160] S3. Quantitative analysis:

[0161] The colorimetric amplification DAB solution is dropped onto the T line to cause the nanozyme tag to undergo a catalytic reaction to generate oxidized DAB (ox-DAB) with a brown precipitate. In this step, the colorimetric amplification results can be visually identified or quantitatively evaluated through photos taken with a smartphone, and then the grayscale intensity value corresponding to the test line area can be extracted using Image J.

[0162] The present invention is further described below by way of examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or directly purchased from the market.

[0163] Example 1

[0164] This embodiment provides a nanozyme tag with a mesoporous silica shell, and the nanozyme tag with a mesoporous silica shell is prepared by the following steps:

[0165] (1) Fe 3 O 4 @SiO 2 Preparation:

[0166] 600 μL of 100 nm Fe 3 O 4 The nanoparticles were dispersed in a solution containing 40 mL of ethanol, 5 mL of water, and 20 mg of PVPK40 and sonicated at 80 W for 5 min. Then, 1.2 mL of NH 4 OH, and then ultrasonicated at 80 W for 30 min. 220 μL TEOS solution (26 wt%) was added during the ultrasonication and ultrasonicated at 80 W for 30 min. The final product was collected by magnetic separation and washed with ethanol three times. 3 O 4 @SiO 2 Resuspend in 5 mL of ethanol solution.

[0167] (2) Preparation of mesoporous silica shell:

[0168] 3 mg of Fe 3 O 4 @SiO 2 Resuspend in deionized water and add 150 mg PVP K15. Stir briefly and then add 4 mL of sodium hydroxide solution (0.3 g mL -1 ) was added to the mixture and stirred continuously at 26 °C and 500 rpm for 6 h. The final product was then collected by magnetic separation and subsequently rinsed with deionized water and ethanol three times to obtain Fe 3 O 4 @MSiO 2 .

[0169] (3) Loading of Pt particles:

[0170] 3 mg of Fe 3 O 4 @MSiO 2 , 20 mg PVP K40 and 400 μL H 2 PtCl 6 The solution (1 wt%) was mixed in 50 mL of solution (20 mL of ethanol and 30 mL of deionized water), stirred at 500 rpm for 15 min, and then 250 μL of NaBH 4 The solution (0.1 M) was quickly added to the solution and stirred at 500 rpm for 20 min to deposit the Pt particles in the voids of the mesoporous silica shell. During this process, the color of the reaction mixture quickly changed from dark yellow to black. The final product was then collected by magnetic separation. Finally, the product was washed with ethanol and the prepared Fe 3 O 4 @MSiO 2 @Pt nanozyme was resuspended in 5 mL of ethanol solution.

[0171] (4) MUA connection:

[0172] The MUA solution with a final concentration of 10 μM was added to Fe 3 O 4 @MSiO 2 The Pt nanozyme solution was ultrasonicated at 80 W for 1 h to connect the carboxyl alkane sulfide to the Pt particles to obtain Fe 3 O 4 @MSiO 2 @Pt-MUA seeds.

[0173] (5) Preparation of nanozyme tags with mesoporous silica shells:

[0174] Take 2 mL of Fe 3 O 4 @MSiO 2The @Pt-MUA seeds were washed with PBST (0.05%), dispersed in 500 μL of MEST buffer (10 mM, pH 5.5), and sonicated at 80 W for 30 s. Subsequently, 10 μL of freshly prepared NHS (100 mM) and 50 μL of EDC (10 mM) were added, and the carboxyl groups were activated by sonication at 80 W for 15 min to obtain a mixture. Then, the mixture was dispersed in 500 μL of PBS buffer (10 mM, pH 7.4), and 13 μg of influenza A N protein detection antibody was added, and incubated at 26 °C for 150 min. At the same time, in order to block the non-binding sites, 150 μL of BSA solution (10 mg mL -1 ), and finally the product was collected by a magnet and resuspended in 120 μL of PBST (0.05%) solution for further use at 4 °C.

[0175] Test Example 1

[0176] Characterization of Nanozyme Tags with Mesoporous Silica Shells

[0177] Test sample: the test sample provided in Example 1;

[0178] Test methods: High-resolution transmission electron microscopy (HRTEM), X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS) were investigated.

[0179] Test results:

[0180] I. HRTEM test results:

[0181] Fe 3 O 4 @SiO 2 , Fe 3 O 4 @MSiO 2 The morphology of the nanozyme tag with the mesoporous silica shell was determined by HRTEM. Figure 2A , Figure 2B and Figure 2C Describes the core-shell structure of the nanoparticles, Fe 3 O 4 The cores are represented by black spheres with an average size of about 100 nm; meanwhile, these silica shells are about 20 nm thick ( Figure 2D ), this observation confirmed that Fe 3 O 4 NPs were successfully encapsulated in the nonporous silica shell. Figure 2E ), SiO 2After treatment under alkaline conditions, the nonporous silica shell is selectively etched to show an obvious wormhole-like mesoporous structure. The present invention calculates the BET specific surface area of ​​the magnetic mesoporous silica nanoparticles to be 61.46 m 2 ·g -1 , with an average mesopore size of 4.85 nm and a total pore volume of 0.13 cm 3 ·g -1 . Furthermore, from the corresponding HRTEM images ( Figure 2F ) shows that in mesoporous SiO 2 A large number of well-distributed Pt particles are synthesized in situ, and the Pt particles are deposited in the voids of the mesoporous silica shell. The high specific surface area of ​​the mesoporous structure can significantly increase the loading amount of the Pt particles, thereby enhancing the catalytic activity.

[0182] II. Superparamagnetic properties test results:

[0183] The Fe ions were investigated in the magnetic field range of -3000 to +3000 Oe by a vibrating sample magnetometer. 3 O 4 @SiO 2 , Fe 3 O 4 @MSiO 2 and the superparamagnetic properties of the mesoporous silica-shelled nanozyme tags. Figure 2G It shows that the synthesized Fe 3 O 4 , Fe 3 O 4 @MSiO 2 and Fe 3 O 4 @MSiO 2 The saturation magnetization values ​​of @Pt are 64.8, 40.8 and 18.7 emu·g -1 ; Among them, with Fe 3 O 4 In comparison, Fe 3 O 4 @MSiO 2 and Fe 3 O 4 @MSiO 2 The @Pt NZs exhibit a lower saturation magnetization, which may be due to the Fe 3 O 4 Surface SiO 2 The magnetic shielding effect of the shell and Pt particles. 3 O 4 @MSiO 2@Pt can still exhibit a fast response within 45 s under an external magnetic field, thus ensuring rapid and efficient magnetic separation and enrichment of target analytes from complex samples. Figure 2H It shows that Fe 3 O 4 @MSiO 2 The nitrogen adsorption-desorption isotherm and pore size distribution curve of Fe 2+ ... 3 O 4 @MSiO 2 The P / P0 of the sample shows an H3-type hysteresis loop between 0.9 and 1.0, which is a typical characteristic of mesoporous materials. Fig.2I Fe 3 O 4 @MSiO 2 Energy dispersive x-ray spectroscopy (EDS) elemental mapping of @Pt, small-sized Pt (purple) and Si (green) in Fe 3 O 4 (Red) (Blue) Surface overlap distribution, proving that Fe 3 O 4 @MSiO 2 @Pt was successfully prepared.

[0184] III. Crystal structure and crystallinity characterization test results:

[0185] Powder XRD was used to characterize the crystal structure and crystallinity of the material. Figure 2J As shown, Fe 3 O 4 @MSiO 2 The XRD patterns of @PtNZs match well with three standard reference cards, indicating the presence of three main components in the composite: Fe 3 O 4 , Pt and SiO 2 Observation of Fe 3 O 4 @MSiO 2 The diffraction curve of @Pt NZs has a weak diffraction peak at 21.273° corresponding to Fe 3 O 4 There is a weaker peak at 35.072°, which belongs to SiO 2 The strongest peak in the figure appears at 41.395°, which is mainly due to the dominant phase Fe on the (111) crystal plane. 3 O 4The diffraction peak at 46.644° is related to the (111) surface of Pt doping, and the diffraction peaks at 50.460°, 67.245° and 74.144° are related to Fe 3 O 4 Finally, there is a peak at 64.386° that is attributed to SiO 2 (201) plane of the composite material. 3 O 4 @MSiO 2 and Fe 3 O 4 @MSiO 2 @Pt NZs,Fe 3 O 4 The core phase of the mesoporous SiO 2 The introduction of Fe 3 O 4 The relevant XRD peak intensity gradually decreases. Combined with the above HRTEM test results, it is proved that the present invention successfully prepared Fe 3 O 4 @MSiO 2 @Pt nanocomposite.

[0186] XPS was used to further detect the surface elements and their respective valence states. Figure 2K As shown, the high-resolution scan of the Fe 2p spectrum shows that Fe 2p 1 / 2 The peaks of Fe 2p 3 / 2 The peaks are 710.88 eV and 712.98 eV, confirming that Fe 3 O 4 The O 1s orbitals are located at 529.88 eV and 531.98 eV, respectively, which are attributed to -Fe-O- and -O-Si-O-, further proving the presence of Fe in the sample. 3 O 4 and SiO 2 In addition, the Si 2p spectrum peak is decomposed into two main components at 99.58 eV and 102.28 eV. The high-resolution spectrum of Pt shows that Pt 4f 5 / 2 and Pt 4f 7 / 2 The characteristic peaks of the orbitals are 71.58 eV and 74.98 eV, respectively, proving the successful loading of Pt. These results indicate that Fe 3 O 4 @MSiO 2 Successful synthesis of @Pt NZs.

[0187] Test Example 2

[0188] Catalytic performance test

[0189] Test sample: the test sample provided in Example 1;

[0190] Test results:

[0191] from Figure 3 It can be seen that in the range of 0~600 μL, as H 2 PtCl 6 With the addition of , the absorbance gradually increased. It is worth noting that when the amount of Pt precursor added was 600 μL, the catalytic efficiency was the best, but the magnetic recovery rate was the worst. Therefore, the amount of Pt precursor added was selected to be 400 μL in subsequent experiments. The pH value has a significant effect on the activity of nanozyme POD, from Figure 4A It can be seen that Fe 3 O 4 @MSiO 2 @Pt NZs exhibits perfect POD-like activity in the range of pH = 2-6 (highest at pH = 4), and has almost no activity in the range of pH = 7-12. 3 O 4 @MSiO 2 The @Pt NZs exhibited optimal POD-like activity in the DAB colorimetric detection system (pH = 4), making them ultrasensitive nanozyme tags for biosensing.

[0192] Then, under the optimal catalytic conditions, Fe 3 O 4 @MSiO 2 @Pt NZs were subjected to kinetic analysis. Figure 4B , Figure 4C As shown, within the appropriate substrate concentration range, TMB and H 2 O 2 Typical Michaelis-Menten kinetic curves were observed. It is an important indicator reflecting the affinity between an enzyme and its substrate. The lower the value, the stronger the affinity of the enzyme for the substrate. 3 O 4 In comparison, TMB (7.19 mM) and H 2 O 2 (25.35 mM) as substrate, Fe 3 O 4 @MSiO 2 @PT NZs The lower value indicates that Fe 3 O 4 @MSiO 2The affinity of the @Pt composite nanozyme is stronger. The possible reason is that Fe 3 O 4 The active center of Fe 3 O 4 @MSiO 2 The @Pt NZs showed abundant catalytic active sites, a feature enhanced by their porous structure. 2 O 2 Fe3O4@MSiO2@Pt NZs as substrate The value is higher than TMB, indicating that it has a higher affinity for TMB. 2 O 2 of They are 3.51×10 -8 and 2.67×10 -8 M.s -1 These results confirm that Fe 3 O 4 @MSiO 2 @Pt NZs have superior POD-like activity and play a vital role in promoting signal amplification and enhancing the catalytic deposition of nanozymes. 3 O 4 @MSiO 2 The catalytic mechanism of @Pt NZs. The POD-like activity was verified by electron spin resonance (ESR) analysis. The generation of •OH was determined by ESR spectroscopy using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as a spin trap. Figure 4D As shown, Fe 3 O 4 @Pt、Fe 3 O 4 @SiO 2 @Pt、Fe 3 O 4 @MSiO 2 @Pt all showed the characteristic quartet signal of DMPO-•OH (1:2:2:1), and Fe 3 O 4 @MSiO 2 The reactive oxygen species (ROS) (•OH) generated by @Pt in the DMPO system is the largest. Therefore, it is shown that the nano-tag prepared by the present invention has a higher POD-like activity.

[0193] Test Example 3

[0194] LFIA Influenza A Test

[0195] Test sample: the test sample provided in Example 1;

[0196] Test method:

[0197] like Figure 5 As shown, Fe for influenza A detection is given 3 O 4 The structure and workflow of @MSiO2@Pt-LFIA are as follows:

[0198] S1、Fe 3 O 4 @MSiO 2 @Pt-Flu A complex formation: 0.4 μL of the nanozyme tag with mesoporous silica shell prepared in Example 1 was added to different concentrations of Flu A for enrichment, and the Fe 3 O 4 @MSiO 2 @Pt-Flu A complexes were separated by magnet.

[0199] S2. Qualitative analysis: Fe 3 O 4 @MSiO 2 The Pt-Flu A complex is dropped onto the sample pad. Under the action of capillary, the complex moves along the direction of the absorbent pad. 3 O 4 @MSiO 2 The Pt-Flu A complex binds to the capture monoclonal antibodies (mAbs) immobilized on the T line to form Fe 3 O 4 @MSiO 2 @Pt-Flu A-mAbs sandwich structure, producing a black band for initial qualitative analysis; the excess Fe 3 O 4 @MSiO 2 The @Pt immunolabeling continues to flow forward and is captured on the quality control line.

[0200] S3, Quantitative analysis: After the run, the colorimetric amplification DAB solution was dropped onto the T line. Due to its excellent POD-like activity, the synthesized nanotag catalyzed the generation of brown precipitated oxidized DAB (ox-DAB). The colorimetric amplification results can be intuitively identified or quantitatively evaluated through photos taken with a smartphone, and then the grayscale intensity value corresponding to the test line area can be extracted using Image J.

[0201] Test Example 4

[0202] Optimization of LFIA strips

[0203] Test sample: the sample provided in Test Example 3;

[0204] Test method:

[0205] To obtain Fe 3 O 4 @MSiO 2 The optimal color development conditions and sensitivity of the @Pt-LFIA strips were optimized by optimizing several key parameters. First, the antibody concentrations on the T line (0.4, 0.8, 1, 1.5, and 2 mg mL -1 )( Fig. 6A ). It can be observed that the signal intensity corresponding to the test line increases with the increase in the concentration of influenza A detection antibodies. However, too high a concentration of antibody may lead to nonspecific adsorption on the T line. Therefore, the optimal antibody concentration was determined to be 1 mg mL -1 . Discovery of Fe 3 O 4 @MSiO 2 The number of @Pt tags directly affects the amount of antigen binding, thus affecting the sensitivity of the LFIA test strip. Figure 6B As shown in Figure 6C, 0.4 µL of label is the best choice for antigen detection because it reduces T-line nonspecific phenomena and has the highest signal-to-noise ratio. In addition, the time of chromatographic reaction plays an important role in the efficiency of immune response. As shown in Figure 6C, when the time reaches 10 min, the signal-to-noise ratio reaches the highest and the expected detection sensitivity is achieved. Therefore, 10 min is selected as the optimal chromatographic reaction time.

[0206] Test Example 5

[0207] Performance test of LFIA test strips for detecting influenza A virus

[0208] Test conditions: Optimized parameter conditions of Test Example 4.

[0209] Under the optimized conditions, Fe 3 O 4 @MSiO 2 @Pt-LFIA test strips in Flu A-NP (0~100 ng·mL -1 ) was used to evaluate the detection performance in the presence of Fig. 7A As shown in Figure 2, as the concentration of Flu A-NP in C-LFIA decreases, the color of the T line gradually becomes lighter. The results show that without any instrument, the concentration as low as 1 ng mL can be detected visually. -1 ,Compare Figure 7C The commercial colloidal AuNP-based LFIA test strips (10 ng mL -1 ) is 10 times higher, which is due to Fe 3 O 4 @MSiO 2 @Pt has a higher specific surface area.

[0210] Catalytic colorimetry was used to perform qualitative and quantitative analysis of Flu A-NPs. Fig. 7A As shown, the colorimetric signal of CA-LFIA is 0.01 ng·mL -1 , compared with the pre-catalytic (1 ng·mL -1 ) increased by 100 times. 3 O 4 @MSiO 2 @PtNZs sensitivity, a novel mesoporous SiO 2 Layered composite nanozyme (Fe 3 O 4 @SiO 2 @PtNZs), for the detection of influenza A. Figure 7B As shown, Fe 3 O 4 @SiO 2 @Pt-CA-LFIA has a visual value of 1 ng mL-1, which is lower than Fe 3 O 4 @MSiO 2 @Pt-CA-LFIA visual value (0.01 ng·mL -1 ) is 100 times lower and only one order of magnitude higher than the sensitivity before catalysis.

[0211] The above results show that the high specific surface area of ​​the prepared mesoporous structure can increase the loading of Pt particles and improve the Fe 3 O 4 @MSiO 2 @Pt NZs has POD-like activity. The catalytic reaction with DAB produces brown ox-DAB, which significantly amplifies the colorimetric signal intensity of the T line and improves the detection sensitivity. At the same time, Image J software is used to extract the gray value of the area on the T line. Fig.7D As shown in the figure, a calibration curve was established based on the colorimetric signal intensity of the T line and the concentration of Flu A-NP. The correlation coefficient (R 2 ) is 0.999. 3 O 4 @MSiO 2 The limit of quantification of @Pt-LFIA is 0.0089 ng·mL -1 The calculations were performed using the method outlined in the IUPAC protocol (LOD = y blank +3×SD blank , where y blank is the average colorimetric signal intensity of the blank group, SD blank is the standard deviation of these groups). Fig. 7E As shown, the LOD is significantly higher than that of Fe 3 O4 @SiO 2 @Pt-LFIA is 112 times lower.

[0212] To evaluate the specificity of the assay, a range of other respiratory viruses were tested, including RSV, MERS-CoV, SARS-CoV, Flu-B, and COVID-19. Figure 7F As shown, the colorimetric and catalytic colorimetric signal intensities of these viruses on the C-LFIA and CA-LFIA strips were similar to those of the blank group, indicating that Fe 3 O 4 @MSiO 2 @Pt-LFIA strips have high specificity. In order to better evaluate the repeatability of the test strips, the present invention used 5 independent experiments to test influenza A (0.01 and 1 ng·mL -1 ) were tested in parallel. Figure 7G It can be seen that the visualization results of the five groups of C-LFIA and CA-LFIA strips are similar, and the relative standard deviation (RSD) values ​​of the five individual test strips are 2.4% and 1.3%, respectively, confirming that Fe 3 O 4 @MSiO 2 @Pt-LFIA has good repeatability.

[0213] Test Example 6

[0214] Simulated clinical sample test

[0215] Test method: Three concentrations (50, 5 and 0.05 ng·mL) were added to the throat swab samples of healthy people. -1 ) of Flu A-NP to simulate the recovery rate of samples at different target concentrations.

[0216] As shown in Table 1 below (Recovery rate of Flu A-NP in throat swab samples), the average recovery rate of throat swab samples ranged from 95.20% to 106.00%, and the coefficient of variation (CV) fluctuated between 3.62% and 4.32%.

[0217] Table 1

[0218]

[0219] Test results:

[0220] By Fe 3 O 4 @MSiO 2 @Pt-LFIA detected the inactivated H1N1 virus, further verifying the practical analytical capability of the new biosensor. Fig. 8A As shown, the C-LFIA test result is 1×104 copies·mL -1 , CA-LFIA test result is 50 copies·mL -1 , the qualitative sensitivity is increased by 200 times. Figure 8B The results of the fitting curve were used to calculate Fe 3 O 4 @MSiO 2 The LOD of Pt-LFIA for inactivating H1N1 virus particles was 33 copies·mL -1 .

[0221] In summary, the present invention establishes an ultrasensitive catalytic colorimetric sensing platform based on nanozyme tags with mesoporous silica shells. The nanozyme tags with mesoporous silica shells of the present invention have excellent magnetic separation ability and can directly capture and detect target molecules in complex matrices without any pretreatment. 2 The Fe layer increases the specific surface area, making it possible to enhance the POD-like activity of the composite nanozyme. 3 O 4 @MSiO 2 The @Pt-LFIA strips are able to provide conventional colorimetric and colorimetric amplification signals for the detection of influenza A virus and inactivated H1N1 virus in a wide linear range, providing colorimetric results for qualitative analysis, which are 100 times stronger than the commercial LFIA strips based on AuNPs, and have ultra-sensitive quantitative results. It is worth noting that the LFIA test strips of the present invention also showed significant specificity, repeatability and high recovery in influenza virus detection. This "three-in-one" multifunctional mesoporous silica shell nanozyme tag can become a promising pretreatment-free POCT disease diagnostic tool with high sensitivity and accuracy.

[0222] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A nanozyme tag with a mesoporous silica shell, characterized in that: The mesoporous silica shell nanozyme tag comprises a Fe3O4 core and a mesoporous silica shell coated on the surface of the Fe3O4 core; Wherein, the voids of the mesoporous silica shell are filled with Pt particles, and the Pt particles are connected with influenza A N protein detection antibodies via carboxyl alkane sulfide; The thickness of the mesoporous silica shell is 15 to 25 nm, and the specific surface area of ​​the mesoporous silica shell is 60 to 62 m 2 ·g -1 The average mesopore size of the mesoporous silica shell is 4.8-4.9 nm, and the total pore volume of the mesoporous silica shell is 0.12-0.14 cm 3 ·g -1 ; The preparation method of the nanozyme tag of the mesoporous silica shell comprises: (1) Dispersing Fe3O4 nanoparticles in an ethanol aqueous solution containing a dispersant, performing a first ultrasonic treatment, adding a catalyst, performing a second ultrasonic treatment, adding an organic silicone grease solution, performing a third ultrasonic treatment, and then coating the surface of the Fe3O4 nanoparticles with a silica shell to obtain Fe3O4@SiO2; the dispersant is polyvinyl pyrrolidone K40; the catalyst is NH4OH; the organic silicone grease is tetraethoxysilane; the volume ratio of the Fe3O4 nanoparticles, the catalyst and the organic silicone grease solution is (1.7-3.7):(3.4-6.4):1; wherein the concentration of the organic silicone grease solution is 0.9-1.0 g·mL -1 ; (2) resuspending the Fe3O4@SiO2 in an aqueous solution containing a dispersant, adding a base and stirring, etching the silica shell to form a mesoporous silica shell, collecting the product by magnetic separation to obtain Fe3O4@MSiO2; the dispersant is polyvinyl pyrrolidone K15; the base is sodium hydroxide solution; the mass ratio of the Fe3O4@SiO2, the dispersant and the base is 1:(40-60):(300-500); the volume ratio of the Fe3O4@SiO2 and water is 1:(1-5), and the concentration of the sodium hydroxide solution is 0.25-0.35 g / mL; (3) mixing the Fe3O4@MSiO2, dispersant and chloroplatinic acid in an ethanol aqueous solution, performing a first stirring, adding a reducing agent, and performing a second stirring to deposit Pt particles in the voids of the mesoporous silica shell to obtain Fe3O4@MSiO2@Pt nanozyme; (4) mixing the Fe3O4@MSiO2@Pt nanozyme and carboxyl alkane sulfide in ethanol and subjecting them to ultrasonic treatment to connect the carboxyl alkane sulfide to the Pt particles, thereby obtaining Fe3O4@MSiO2@Pt-MUA seeds; (5) dispersing the Fe3O4@MSiO2@Pt-MUA seeds in MEST buffer, performing a first ultrasonic treatment, adding N-hydroxysuccinimide solution and carbodiimide solution, performing a second ultrasonic treatment, and obtaining a mixture; dispersing the mixture in PBS buffer, adding influenza A N protein detection antibody, incubating, and adding bovine serum albumin solution to block non-binding sites, collecting the product by a magnet, and obtaining the nanozyme tag of the mesoporous silica shell.

2. The nanozyme tag of mesoporous silica shell according to claim 1, characterized in that The particle size of the Fe3O4 core is 95-105nm.

3. The nanozyme tag of mesoporous silica shell according to claim 1, characterized in that The particle size of the Pt particles is 3 to 6 nm; And / or, the carboxyalkane sulfide is 11-mercaptoundecanoic acid.

4. A method for preparing a nanozyme tag of a mesoporous silica shell according to any one of claims 1 to 3, characterized in that: The preparation method comprises: (1) Dispersing Fe3O4 nanoparticles in an ethanol aqueous solution containing a dispersant, performing a first ultrasonic treatment, adding a catalyst, performing a second ultrasonic treatment, adding an organic silicone grease solution, performing a third ultrasonic treatment, and then coating the surface of the Fe3O4 nanoparticles with a silica shell to obtain Fe3O4@SiO2; the dispersant is polyvinyl pyrrolidone K40; the catalyst is NH4OH; the organic silicone grease is tetraethoxysilane; the volume ratio of the Fe3O4 nanoparticles, the catalyst and the organic silicone grease solution is (1.7-3.7):(3.4-6.4):1; wherein the concentration of the organic silicone grease solution is 0.9-1.0 g·mL -1 ; (2) resuspending the Fe3O4@SiO2 in an aqueous solution containing a dispersant, adding a base and stirring, etching the silica shell to form a mesoporous silica shell, collecting the product by magnetic separation to obtain Fe3O4@MSiO2; the dispersant is polyvinyl pyrrolidone K15; the base is sodium hydroxide solution; the mass ratio of the Fe3O4@SiO2, the dispersant and the base is 1:(40-60):(300-500); the volume ratio of the Fe3O4@SiO2 and water is 1:(1-5), and the concentration of the sodium hydroxide solution is 0.25-0.35 g / mL; (3) mixing the Fe3O4@MSiO2, dispersant and chloroplatinic acid in an ethanol aqueous solution, performing a first stirring, adding a reducing agent, and performing a second stirring to deposit Pt particles in the voids of the mesoporous silica shell to obtain Fe3O4@MSiO2@Pt nanozyme; (4) mixing the Fe3O4@MSiO2@Pt nanozyme and carboxyl alkane sulfide in ethanol and subjecting them to ultrasonic treatment to connect the carboxyl alkane sulfide to the Pt particles, thereby obtaining Fe3O4@MSiO2@Pt-MUA seeds; (5) dispersing the Fe3O4@MSiO2@Pt-MUA seeds in MEST buffer, performing a first ultrasonic treatment, adding N-hydroxysuccinimide solution and carbodiimide solution, performing a second ultrasonic treatment, and obtaining a mixture; dispersing the mixture in PBS buffer, adding influenza A N protein detection antibody, incubating, and adding bovine serum albumin solution to block non-binding sites, collecting the product by a magnet, and obtaining the nanozyme tag of the mesoporous silica shell.

5. The method for preparing the nanozyme tag of mesoporous silica shell according to claim 4, characterized in that: In step (1), the volume ratio of the Fe3O4 nanoparticles, ethanol and water is (0.4-0.8):(30-50):(3-7); wherein the volume mass ratio of the Fe3O4 nanoparticles and the dispersant is (0.4-0.8) mL:(10-30) mg; And / or, in step (1), the power of the first ultrasonic treatment, the second ultrasonic treatment and the third ultrasonic treatment are each independently 60 to 100 W; And / or, in step (1), the first ultrasonic treatment time is 1 to 10 minutes; the second ultrasonic treatment time is 20 to 40 minutes; the third ultrasonic treatment time is 20 to 40 minutes; And / or, step (1) further includes the following post-treatment: washing the Fe3O4@SiO2 with ethanol and then resuspending it in ethanol; wherein the concentration of the Fe3O4@SiO2 is 5 to 10 μM.

6. The method for preparing the nanozyme tag of mesoporous silica shell according to claim 4, characterized in that: In step (2), the stirring speed is 500-550 rpm, and the stirring time is 4-8 h; And / or, in step (2), the following post-treatment is also included: washing the Fe3O4@MSiO2 with water and ethanol.

7. The method for preparing the nanozyme tag of mesoporous silica shell according to claim 4, characterized in that: In step (3), the dispersant is polyvinyl pyrrolidone K40; the reducing agent is sodium borohydride; And / or, in step (3), the mass ratio of the Fe3O4@MSiO2, the reducing agent and the chloroplatinic acid is 1:(6-7):(6-7); And / or, in step (3), the mass volume ratio of the Fe3O4@MSiO2 and the ethanol aqueous solution is (2-4) mg:(40-60) mL; wherein the volume ratio of the ethanol and water is (10-30):(20-40); And / or, in step (3), the rotation speeds of the first stirring and the second stirring are respectively 500-550 rpm; And / or, in step (3), the first stirring time is 10 to 20 minutes; the second stirring time is 10 to 30 minutes; And / or, in step (3), the following post-treatment is also included: washing the Fe3O4@MSiO2@Pt nanozyme with ethanol and then resuspending it in ethanol; Wherein, the volume ratio of the Fe3O4@MSiO2@Pt nanozyme and ethanol is 1:(1-5).

8. The method for preparing the nanozyme tag of mesoporous silica shell according to claim 4, characterized in that: In step (4), the carboxyl alkane sulfide is 11-mercaptoundecanoic acid; And / or, in step (4), the volume ratio of the Fe3O4@MSiO2@Pt nanozyme to the carboxyl alkane sulfide is (100-300):1; and / or, in step (4), the final concentration of the carboxyl alkane sulfide is 8 to 12 mM; And / or, in step (4), the power of the ultrasonic treatment is 60 to 100 W, and the time of the ultrasonic treatment is 0.5 to 2 h.

9. The method for preparing the nanozyme tag of mesoporous silica shell according to claim 4, characterized in that: In step (5), the volume ratio of the Fe3O4@MSiO2@Pt-MUA seeds, N-hydroxysuccinimide solution and carbodiimide solution is (150-250):1:(4-6); and / or, the concentration of the N-hydroxysuccinimide solution is 90-110 mM; and / or, the concentration of the carbodiimide solution is 9 to 11 mM; And / or, in step (5), the volume ratio of the mixture, influenza A N protein detection antibody and bovine serum albumin is (330-334):1:(90-120); And / or, in step (5), the power of the first ultrasonic treatment and the second ultrasonic treatment are each independently 60 to 100 W; And / or, in step (5), the first ultrasonic treatment time is 20 to 40 seconds; the second ultrasonic treatment time is 10 to 20 minutes; And / or, in step (5), the incubation time is 140 to 160 min; And / or, step (5) further includes the following post-treatment: resuspending the product collected by magnet in PBST solution.

10. Use of the nanozyme tag of the mesoporous silica shell according to claim 1 or 2 in the preparation of a biosensor for detecting influenza A virus.

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