An immunochromatographic reagent based on a bifunctional mesoporous SERS nanosen probe

Abstract

The application discloses an immunochromatography reagent based on a bifunctional mesoporous SERS nanolaser probe, wherein the nanolaser probe is a nanolabel for surface modification of a detection antibody, the nanolabel takes dendritic mesoporous SiO2 as a core, a large number of catalytic nanolaser particles Au@Ir are loaded on the surface and the holes of the core, and a layer of high-activity SERS particles is coated on the surface and the holes of the core. The nanolaser probe prepared by the application can realize accurate detection of two important foodborne viruses, Norovirus and enterovirus, in fecal samples, and the detection mode is divided into a SERS mode and a color development mode which can be directly observed. The immunochromatography reagent based on the bifunctional mesoporous SERS nanolaser probe has high flexibility, sensitivity and stability, and can rapidly and accurately detect viruses in various environments.

Description

Technical Field

[0001] This invention relates to the field of immunochromatographic detection technology, and in particular to an immunochromatographic reagent based on a bifunctional mesoporous SERS nanozyme probe. Background Technology

[0002] In the field of rapid food safety testing, there is a widespread demand for on-site screening technologies for foodborne viruses. Norovirus (NoV), adenovirus (AdV), and rotavirus (RV), among others, account for more than half of foodborne illness incidents. These viruses share the following characteristics: strong environmental resistance, surviving for 10 days to 4 weeks under natural conditions; extremely low infectious doses (only 10-100 viral particles are needed to cause disease); rapid human-to-human transmission, easily triggering local outbreaks; and initial symptoms and routes of infection similar to foodborne bacteria, often leading to misdiagnosis. Therefore, early, rapid, and accurate virus diagnosis is crucial for the prevention and control of foodborne viruses. However, existing detection technologies (mainly real-time quantitative PCR and sequencing) are limited by lengthy processes, complex operations, high costs, and reliance on large equipment and clean environments, making it difficult to meet the needs for early real-time screening and rapid, accurate diagnosis of foodborne viruses.

[0003] Immunochromatographic assay (ICA) utilizes antigen-antibody specific reactions to rapidly detect biochemical molecules on test strips. It offers advantages such as simplicity, ease of use, and low cost, making it particularly suitable for on-site screening and home self-testing. However, ICA still faces two major challenges in foodborne virus detection: First, traditional ICA uses gold nanoparticles as colorimetric markers, resulting in low sensitivity (detection limit at the ng / mL level) and a narrow linear range (only 2-3 orders of magnitude), making it difficult to accurately identify low concentrations of pathogens. Second, foodborne virus samples are often food or complex clinical samples (such as feces and vomit), and traditional colloidal nanolabels are prone to aggregation or inactivation in these complex matrices, leading to distorted results. In recent years, novel signal modalities such as fluorescence, magnetic signals, colorimetric enhancement, chemiluminescence, and surface-enhanced Raman scattering (SERS) have been introduced into ICA to improve on-site detection sensitivity. However, SERS-ICA requires the use of a Raman spectrometer to read the signal, which greatly reduces the convenience of ICA in field and home settings. Therefore, relying solely on SERS-ICA cannot meet the needs of rapid field screening for foodborne viruses.

[0004] Nanozymes are a class of nanomaterials with natural enzyme-like activities (such as Fe3O4, Pt, Ir, etc.), possessing characteristics such as high chemical stability, strong environmental adaptability, good durability, and excellent catalytic performance, showing great potential in fields such as biosensing, industrial production, and in vivo therapy. However, the design and application of nanozyme labeling still face several technical challenges: (1) how to increase the number of catalytic sites to further improve sensitivity; (2) how to precisely control the structure and size of nanozymes to reduce non-specific signals on test strips; and (3) how to ensure the material stability and catalytic stability of nanozymes in complex samples. In addition, the quantitative performance of existing nanozyme ICAs is still not ideal. Summary of the Invention

[0005] The present invention aims to at least solve one of the aforementioned technical problems existing in the prior art. Therefore, the object of the present invention is to provide an immunochromatographic reagent based on a bifunctional mesoporous SERS nanozyme probe.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] In a first aspect, the present invention provides a nanotag having a core-shell structure, the core-shell structure comprising a silica nanoparticle as an inner core structure and a noble metal particle as an outer shell structure, wherein a nanocomposite material is filled between the outer shell structure and the inner core structure as a catalytic intermediate layer; the nanocomposite material is composed of at least one of Au, Pt, Pd, Ru and Ir.

[0008] In some embodiments of the present invention, the nanocomposite material is composed of Au and Ir.

[0009] In some embodiments of the present invention, the particle size of the nanocomposite material is 1-50 nm.

[0010] In some embodiments of the present invention, the nanocomposite material is a nanozyme with natural enzyme-like activity.

[0011] In some embodiments of the present invention, the nanozyme with natural enzyme-like activity is Au@Ir.

[0012] In some embodiments of the present invention, the noble metal particles include at least one of Au, Ag and Cu.

[0013] In some embodiments of the present invention, the particle size of the noble metal particles is 20-50 nm.

[0014] In some embodiments of the present invention, the precious metal particles are Au.

[0015] In some embodiments of the present invention, the Au comprises colloidal gold.

[0016] In some embodiments of the present invention, the silica particles include mesoporous silica particles.

[0017] In some embodiments of the present invention, the particle size of the mesoporous silica particles is 50-500 nm.

[0018] In some embodiments of the present invention, the mesoporous silica particles radiate a large number of dendritic pores from the core outward, which can be used to improve probe dispersibility and pack nanozyme particles.

[0019] A second aspect of the invention provides the use of the nanotags described above in the preparation of products for labeling.

[0020] A third aspect of the present invention provides a method for preparing the nanotag probe described above, the method comprising the following steps:

[0021] The mesoporous material is added to a cationic polymer solution and mixed thoroughly. Then, the nanocomposite material is added for adsorption. After adsorption is complete, the cationic polymer is added again and mixed thoroughly. Finally, a solution of noble metal particles is added for adsorption to obtain the nanotag.

[0022] In some embodiments of the present invention, the mesoporous material includes mesoporous silicon dioxide.

[0023] In some embodiments of the present invention, the cationic polymer includes polyethyleneimine, polyglycidylamine, or polyacrylamide.

[0024] In some embodiments of the present invention, the concentration of the nanocomposite material is 0.1-50 mg / mL.

[0025] In some embodiments of the present invention, the adsorption can be repeated multiple times.

[0026] In some embodiments of the invention, thorough mixing includes treatment with ultrasound.

[0027] In some embodiments of the present invention, the duration of the ultrasound is 5-80 minutes.

[0028] In some embodiments of the present invention, the mesoporous material is added to a cationic polymer solution and subjected to ultrasonic treatment for 10-30 minutes.

[0029] In some embodiments of the present invention, the molecular weight of the polyethyleneimine is 600-25000 and the concentration is 0.5-5 mg / mL.

[0030] In some embodiments of the present invention, the preparation method includes the following steps:

[0031] The mesoporous material is added to a cationic polymer solution and ultrasonically mixed for 10-30 minutes. Then, the nanocomposite material is added for adsorption. After adsorption is complete, the cationic polymer is added again and ultrasonically mixed for 10-25 minutes. Finally, a solution of noble metal particles is added for adsorption to obtain the nanotag.

[0032] In some embodiments of the present invention, the preparation method includes the following steps:

[0033] (1) Add triethanolamine to water and stir for 20-40 min. Add dodecyltrimethylammonium bromide and sodium salicylate and stir for 2-5 hours at 70-100℃. Then add tetraethyl silicate and ethanol and stir for 1-3 hours. Separate and extract mesoporous silica.

[0034] (2) Add mesoporous silica to a cationic polymer solution and sonicate for 10-30 minutes. Add the sonicated solution to the nanocomposite solution and sonicate for 10-25 minutes.

[0035] (3) Add the solution obtained by (2) to the cationic polymer solution and sonicate for 10-30 minutes. Add the sonicated solution to the noble metal particle solution and sonicate for 10-25 minutes to obtain the nano-tag probe.

[0036] In some embodiments of the present invention, the separation in step (1) includes separating the solution by centrifugation of mesoporous silica and washing with ethanol.

[0037] In some embodiments of the present invention, the extraction includes three extractions using methanol at 50-80°C, followed by dispersion in ethanol.

[0038] In some embodiments of the present invention, the extraction includes three extractions using methanol at 60°C, followed by redispersing in ethanol.

[0039] A fourth aspect of the present invention provides a kit comprising a nano-labeled probe and a test strip; the nano-labeled probe being the nano-label described above with a surface modified with a detection antibody; the test strip comprising a sample pad, an absorbent pad, and an NC membrane with a detection line.

[0040] In some embodiments of the present invention, the detection line is modified with the detection antibody.

[0041] In some embodiments of the present invention, the concentration of the detection antibody is 0.5-1.5 mg / mL.

[0042] In some embodiments of the present invention, the concentration of the detection antibody is 0.8-1 mg / mL.

[0043] In some embodiments of the present invention, the test strip has two or more detection lines.

[0044] In some embodiments of the present invention, the two or more detection lines are used to detect different viruses.

[0045] In some embodiments of the present invention, the immunochromatographic kit further includes a loading buffer.

[0046] In some embodiments of the present invention, the loading buffer is a PBS solution containing Tween and fetal bovine serum.

[0047] A fifth aspect of the invention provides the use of the kit described above in the preparation of a product for detecting pathogen samples, wherein the pathogen is a virus or a microorganism.

[0048] In some embodiments of the present invention, the pathogen is a virus or a microorganism.

[0049] In some embodiments of the present invention, the sample includes a food sample or a clinical sample.

[0050] In some embodiments of the invention, the clinical sample includes feces, vomit, or urine.

[0051] In some embodiments of the present invention, the virus includes calicivirus, adenovirus, reovirus, or astrovirus.

[0052] In some embodiments of the present invention, the calicivirus includes norovirus.

[0053] A sixth aspect of the present invention provides a method for detecting pathogens for non-diagnostic purposes, the method comprising the following steps:

[0054] Mix the nano-tag probe described above with the sample to be tested, and drop it onto the immunochromatographic test strip for chromatography for 5-20 minutes. After chromatography, drop the catalytic solution onto the detection surface of the immunochromatographic test strip for catalysis. The presence or absence of the detection line indicates whether the sample contains pathogens.

[0055] In some embodiments of the present invention, the sample includes a food sample.

[0056] In some embodiments of the invention, the non-diagnostic purpose includes testing food or environmental samples in a laboratory or field setting.

[0057] In some embodiments of the present invention, the catalyst solution is a sodium acetate solution containing Tween, 3-amino-9-ethylcarbazole and hydrogen peroxide.

[0058] In some embodiments of the present invention, the catalytic time is 2-5 minutes.

[0059] In some embodiments of the present invention, if color bands are generated simultaneously on the detection line and the control line, the sample to be tested contains pathogens.

[0060] In some embodiments of the present invention, if the detection line does not produce a colored band, but the control line produces a colored band, then the sample to be tested does not contain pathogens.

[0061] In some embodiments of the present invention, if the control line does not produce a color band, the test result is invalid regardless of whether the detection line produces a color band.

[0062] A seventh aspect of the present invention provides a method for detecting pathogens for non-diagnostic purposes, the method comprising the following steps:

[0063] The nano-label probe described above is mixed with the sample to be tested and dropped onto the immunochromatographic test strip for chromatography for 5-20 minutes. After chromatography, Raman spectroscopy is performed to detect the pathogen content in the sample to be tested based on the Raman spectral signal intensity.

[0064] In some embodiments of the present invention, determining the content of pathogens in a sample includes comparing the Raman spectral signal intensity with a standard curve.

[0065] In some embodiments of the present invention, the standard curve is prepared by using pathogen solutions of different known concentrations as samples to obtain corresponding Raman spectral signal intensities, establishing the relationship between Raman spectral signal intensity and pathogen concentration, thereby obtaining the standard curve.

[0066] The beneficial effects of this invention are:

[0067] This invention proposes an immunochromatographic reagent based on a bifunctional mesoporous SERS nanozyme probe, which integrates nanozyme colorimetric enhancement and precise SERS quantification into a single nanolabel, enabling immunochromatographic technology to combine the sensitive colorimetric signal for rapid on-site screening with the precise SERS quantification capability for clinical laboratory diagnosis.

[0068] This invention relates to a SERS-nanozyme label (DSAIA) with a three-layer structure of "core-intermediate layer-shell," which is a multifunctional SERS nanozyme tag with monodisperse, strong peroxidase activity, and high-density, high-efficiency SERS hotspots. The dendritic mesoporous SiO2 (dSiO2) core provides abundant internal cavities for high-density catalyst loading and enhances stability in complex samples. The densely packed nanozyme particles in the intermediate layer provide spatial catalytic sites and robust catalytic performance, significantly improving the colorimetric sensitivity of the test strip. The noble metal nanoparticle shell possesses strong SERS activity, forming numerous high-efficiency hotspots, further enhancing the sensitivity of quantitative detection by immunochromatography (ICA).

[0069] This invention combines antibody-modified DSAIA probes with immunochromatography, enabling precise dual-mode detection of norovirus and enteric adenovirus, two important foodborne viruses, in fecal samples. In the post-catalytic colorimetric mode, the immunochromatographic method based on bifunctional mesoporous SERS nanozyme probes exhibits 100-fold higher sensitivity than traditional colloidal gold-based immunochromatography. In SERS mode (Raman spectroscopy analysis), quantitative analysis of the target was performed, with a 500-fold increase in sensitivity. The reagents described in this invention possess high flexibility, sensitivity, and stability, allowing for rapid and accurate detection of foodborne viruses and other highly pathogenic viruses in various environments. They are suitable for diverse applications, including field, home, and clinical laboratory environments, while also meeting the needs of resource-scarce areas and laboratory settings. Attached Figure Description

[0070] Figure 1 This is a schematic diagram illustrating the preparation method for synthesizing the bifunctional mesoporous SERS nanozyme probe of the present invention.

[0071] Figure 2 Electron microscopy characterization of the structure of the bifunctional mesoporous SERS nanozyme probe of the present invention; a is an electron microscopy image of dSiO2; b is an electron microscopy image of DSAI; c is an electron microscopy image of DSAIA; d is a magnified electron microscopy image of a single dSiO2; e is a magnified electron microscopy image of a single DSAI; f is a magnified electron microscopy image of a single DSAIA.

[0072] Figure 3 The following are elemental line scans of the structure of the bifunctional mesoporous SERS nanozyme probe of this invention using electron microscopy: a is an EDS elemental line scan from DSAIA; b is a scan of silicon; c is a scan of oxygen; d is a fused image of the scans of each element; e is a scan of iridium; and f is a scan of gold.

[0073] Figure 4 This is a flowchart illustrating the immunochromatographic technique of the bifunctional mesoporous SERS nanozyme probe of this invention for the detection of norovirus and enteric adenovirus.

[0074] Figure 5 This invention presents the optimized antibody concentration detection results on immunochromatographic test strips based on the bifunctional mesoporous SERS nanozyme probe immunochromatographic technology of this invention.

[0075] Figure 6 This is the result of the chromatography time optimization of the immunochromatographic technique based on bifunctional mesoporous SERS nanozyme probes of this invention.

[0076] Figure 7 The following are the detection results of norovirus and enteric adenovirus using immunochromatographic technology based on bifunctional mesoporous SERS nanozyme probes according to this invention: a) is the detection result before nanozyme catalysis, where (I) represents the detection result in colorimetric mode, with the red star representing the detection limit, and (II) represents the intensity heatmap of SERS on the T line; b) is the detection result after catalysis, where (I) represents the detection result in colorimetric mode, with the red asterisk representing the detection limit, and (II) represents the intensity heatmap of SERS on the T line; c) is the detection result of norovirus in SERS mode, with the red star representing the highest Raman intensity; d) is the detection result of enteric adenovirus in SERS mode, with the red star representing the highest Raman intensity; e) is the calibration curve of T-line SERS signal intensity versus norovirus concentration; f) is the calibration curve of T-line SERS signal intensity versus enteric adenovirus concentration.

[0077] Figure 8 The graph shows the detection results of norovirus and enteric adenovirus using the colloidal gold method of this invention; a) shows the detection result of norovirus; b) shows the detection result of enteric adenovirus; the red star represents the detection limit.

[0078] Figure 9 This is to verify the specificity of the immunochromatographic technique based on the bifunctional mesoporous SERS nanozyme probe of this invention; a is the detection result in the colorimetric mode, where (Ⅰ) is the detection result before catalysis and (Ⅱ) is the detection result after catalysis; b is the detection result in the SERS mode.

[0079] Figure 10 This is a repeatability verification of the immunochromatographic technique based on bifunctional mesoporous SERS nanozyme probes of the present invention; a represents the detection results of the colorimetric mode, where (Ⅰ) represents norovirus at 10 ng / mL and adenovirus at 10 6 The PFU / mL detection results showed that in group a (II), norovirus was 0.1 ng / mL and adenovirus was 10 ng / mL. 4 The PFU / mL detection results show that in group a (IIⅠ), norovirus was 0.001 ng / mL and adenovirus was 10 ng / mL. 2 The results of the PFU / mL test are shown in b; b is the result of the SERS mode test, where (Ⅰ) is the result of the norovirus test and (Ⅱ) is the result of the adenovirus test.

[0080] Figure 11 The images show the analytical results of the immunochromatographic technique based on bifunctional mesoporous SERS nanozyme probes in actual samples. Image a shows the detection results of a lake water sample, where (Ⅰ) represents the detection result before catalysis, (Ⅱ) represents the detection result after catalysis, and (ⅡⅠ) represents the analysis diagram of the SERS mode detection results. Image b shows the detection results of a healthy human fecal sample, where (Ⅰ) represents the detection result before catalysis, (Ⅱ) represents the detection result after catalysis, and (ⅡⅠ) represents the analysis diagram of the SERS mode detection results.

[0081] Figure 12 The images show the analytical results of the immunochromatographic technique based on bifunctional mesoporous SERS nanozyme probes in clinical stool samples; a) shows the detection results of the immunochromatographic technique based on bifunctional mesoporous SERS nanozyme probes; b) shows the detection results of quantitative real-time PCR; c) shows the analysis results of image a; d) shows the analysis results of image b; e) shows the ROC curve for detecting norovirus; f) shows the ROC curve for detecting adenovirus. Detailed Implementation

[0082] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments and comparative examples are all available from conventional commercial sources or can be obtained by existing technical methods. Unless otherwise specified, the experimental or testing methods are conventional methods in the art. In the embodiments, the nanocomposite material is a nanozyme Au@Ir with natural enzyme-like activity. The nanotag probe is a bifunctional mesoporous SERS nanozyme probe carrying a nanozyme with natural enzyme-like activity, referred to simply as a nanozyme probe in the embodiments.

[0083] Example 1

[0084] This embodiment provides a method for preparing the nanotag DSNP-Au@Ir-Au (DSAIA), and observes the prepared DSAIA using electron microscopy. The preparation method is as follows: Figure 1 As shown, the specific steps are as follows.

[0085] (1) Add 100 μL of triethanolamine (TEA) to 10 mL of water and stir at 80 °C for 30 minutes. Add 100 mg of dodecyltrimethylammonium bromide (CTAB) and 289 mg of sodium salicylate and stir for 3 hours. Finally, add 200 μL of tetraethylsilicate (TEOS) and 1.5 mL of ethanol and stir for another 2 hours. Centrifuge at 7200 rpm for 7 minutes to separate the mesoporous silica (dSiO2) nanoparticles from the suspension and wash thoroughly with ethanol to remove residual reactants. Subsequently, extract the mesoporous silica nanoparticles three times (6 hours each time) at 60 °C with acidic methanol (1 mL of 37% hydrochloric acid and 170 mL of anhydrous methanol) and finally disperse them in 200 mL of ethanol.

[0086] (2) 300 μL of dSiO2 nanoparticles (0.5 mg / mL) were uniformly dispersed in 20 mL of polyethyleneimine solution (PEI, 25 mg / mL) and sonicated for 25 minutes to generate polyethyleneimine-coated dSiO2. The aqueous solution of polyethyleneimine-coated dSiO2 particles was added to 120 mL of Au@Ir nanoparticle solution and then sonicated for 15 minutes to obtain DSNP-Au@Ir (DSAI) nanoparticles.

[0087] The preparation steps of Au@Ir particles are as follows: Take 1 mL of 1% chloroauric acid aqueous solution and mix it with 79 mL of distilled water. Then, mix 4 mL of 1% trisodium citrate and 0.7 mL of 1% NaBH4 and quickly add it to the HAuCl4 solution. Stir at room temperature until the solution turns bright red (about 5-10 minutes) to obtain 3 nm AuNPs. Next, heat 200 mL of AuNP solution to 80 °C with continuous stirring in a water bath. Then, gradually add 12 mL of trisodium citrate (14 mM) and 4 mL of Na3IrCl6·H2O (20 mM). After reacting for 30 min, add 2 mL of NaBH4 (250 mM) and stir at 80 °C for another hour. Then stop heating and stir the suspension until it cools to room temperature. Finally, a 1 mg / mL Au@Ir solution is obtained and stored in the dark.

[0088] (3) The obtained DSNP-Au@Ir(DSAI) nanoparticles were uniformly dispersed in 20 mL of polyethyleneimine solution (PEI, 25 mg / mL) and subjected to ultrasonic treatment for 25 minutes. The aqueous solution of polyethyleneimine-coated DSNP-Au@Ir(DSAI) nanoparticles was added to 120 mL of 35 nm colloidal gold solution (AuNPs) and then subjected to ultrasonic treatment for 15 minutes. The final obtained DSNP-Au@Ir-Au(DSAIA) is the nanotag.

[0089] The preparation steps of the 35 nm colloidal gold solution are as follows: 200 mL of HAuCl4 solution (0.01%, mass-volume ratio) was heated to its boiling point and stirred. Then, 1.1 mL of trisodium citrate (1%, mass-volume ratio) was rapidly added to the boiling solution. The suspension was boiled for 15 minutes and cooled to room temperature to obtain gold nanoparticles with a diameter of approximately 35 nm (0.8 mg / mL).

[0090] The morphology of the prepared DSAIA was characterized by transmission electron microscopy (TEM), and elemental line scanning and elemental mapping of DSAIA were performed using energy dispersive spectroscopy.

[0091] Test results as follows Figure 2-3 As shown, Figure 2 The middle af respectively showed dSiO2 ( Figure 2 (a and d), DSAI ( Figure 2 (b and e) and DSAIA ( Figure 2 Typical TEM images of c and f show that the dSiO2 nanoparticles have a uniform size (approximately 260 nm) and a regular dendritic porous structure. Figure 2 (a) Under high-resolution TEM observation, the pore size of dSiO2 is approximately 15 nm. Figure 2 d). After filling the interior of dSiO2 spheres with Au@Ir particles and adsorbing 35 nm AuNPs on the exterior of DSAI nanoparticles, the resulting DSAIA still exhibited excellent dispersibility, with the particle size increasing to approximately 330 nm. Figure 2 (c and f). Figure 2 Images d, e, and f show magnified TEM images of individual dSiO2, DSAI, and the final product DSAIA particles. The images reveal that the surface and interior of DSAI are densely covered with Au@Ir particles, almost filling all the branched pores of dSiO2, while the surface of the prepared DSAIA exhibits a dense AuNP shell.

[0092] EDS element line scan and element mapping results are as follows: Figure 3 As shown, the element distribution within the DSAIA structure is illustrated. Figure 3 (a and d) indicate that Si (blue) Figure 3 (b) O (red) Figure 3 c) and Ir (purple) Figure 3 The (e) element is distributed in the inner layer of the nanocomposite material, while the strong Au element (green) signal is concentrated on the DSAIA surface. Figure 3 (f). Electron microscopy results show that the nanotag prepared in this embodiment has the following structure: a layer of nanocomposite material (nanozyme Au@Ir) catalytic interlayer and a layer of SERS-enhanced particle structure are arranged sequentially on the surface of dendritic mesoporous SiO2 nanoparticles.

[0093] In summary, the nanotag prepared in this embodiment has a dendritic mesoporous silica nanoparticle core, which can load more catalytic particles. It also provides a highly sensitive colorimetric signal that can be observed with the naked eye by electrostatic adsorption of a large number of nanoenzyme particles through the cationic polymer polyethyleneimine (PEI) as a catalytic interlayer. Finally, the surface is covered with a shell of SERS active particles modified with Raman reporter molecules, which provides a highly sensitive SERS signal that can be used for quantitative detection.

[0094] Example 2

[0095] This embodiment provides an optimization of the immunochromatographic technique for nanotag probes (bifunctional mesoporous SERS nanozyme probes), and its detection procedure for norovirus and enteric adenovirus is as follows: Figure 4 As shown, the analysis of the results includes two modes: colorimetric mode and SERS mode. Colorimetric mode involves direct observation of the test strip after chromatography, while SERS mode uses a Raman spectrometer for detection. The LFA test strips used to detect norovirus (NoV) and adenovirus (AdV) mainly consist of a polyvinyl chloride (PVC) backing, a sample pad, an absorbent pad, and an NC membrane with two T lines and one C line. Detection line 1 in the T lines is used to detect adenovirus, and detection line 2 is used to detect norovirus. The norovirus antigen used for the detection was purchased from Zhengzhou Saitukang Biotechnology Co., Ltd., and the enteric adenovirus antigen was purchased from Suzhou Botron Immunotherapy Co., Ltd.

[0096] The preparation steps are as follows:

[0097] The construction of the immunochromatographic system based on nanotag probes (bifunctional mesoporous SERS nanozyme probes) is described below:

[0098] Construction of bifunctional mesoporous SERS nanozyme probe: 5 μL of 5,5'-dithiobis(2-nitrobenzoic acid) (10 μM) was added to 0.5 mL of DSAIA (2 mg / mL), and the mixture was sonicated for 1.5 hours. Centrifuge at 5500 rpm for 8 minutes, discard the supernatant, and resuspend in 0.5 mL of 0.1 M 2-(N-morpholine) ethanesulfonic acid solution (0.1 M, pH 5.5). Then add 100 μL of carbodiimide solution (0.01 M) and 20 μL of N-hydroxysuccinimide solution (0.1 M), and sonicate for 15 min to activate the carboxyl groups on the DSAIA surface. Centrifuge to recover the activated DSAIA, and resuspend in 200 μL of PBST solution (0.01 M, pH 7.4). Add 8 μg of norovirus antibody and enteric adenovirus antibody, respectively, and react with shaking at room temperature for 2 h. Then add 80 μL of BSA (10%), continue the blocking reaction for 1 h, centrifuge to recover the product, wash once with PBST, and resuspend in 200 μL of PBST. The norovirus antibody was purchased from Zhengzhou Saitukang Biotechnology Co., Ltd., and the enteric adenovirus antibody was purchased from Sigma-Aldrich.

[0099] Preparation of Immunochromatographic Test Strips Based on Bifunctional Mesoporous SERS Nanozyme Probes: The LFA test strip for detecting norovirus (NoV) and adenovirus (AdV) mainly consists of a polyvinyl chloride (PVC) backing, a sample pad, an absorbent pad, and an NC membrane with two T lines and one C line. First, 1 mg / mL of adenovirus antibody, 0.8 mg / mL of norovirus antibody, and 0.6 mg / mL of goat anti-mouse antibody IgG were sprayed onto the T1, T2, and C lines of the NC membrane, respectively, at a spraying rate of 1.0 μL / cm. Next, the NC membrane was dried in an oven at 34°C for 3 hours and then assembled with the PVC backing, sample pad, and absorbent pad. After assembly, the immunochromatographic (ICA) test strip was cut into 3.0 mm wide strips, sealed, and stored in a vacuum desiccator at room temperature for future use. The goat anti-mouse antibody IgG was purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0100] The steps for detecting viruses using an immunochromatographic detection platform based on bifunctional mesoporous SERS nanozyme probes are as follows:

[0101] 2 μL of nanozyme probe was mixed thoroughly with 70 μL of loading buffer and test sample at a 1:1 volume ratio, and then applied to the sample pad of the prepared immunochromatographic test strip (T1 line modified with adenovirus antibody; T2 line modified with norovirus antibody) to initiate immunochromatographic detection. The loading buffer consisted of 10 mM PBS buffer containing 2% Tween 20 and 5% FBS. After chromatography, detection in colorimetric and SERS modes could be performed, as shown in the steps below.

[0102] Detection of colorimetric pattern: After the chromatography reaction is completed, 6 μL of catalytic solution is evenly spread onto the chromatography test strip. After catalytic reaction for 3 minutes, the colorimetric results after catalysis are photographed and collected. The catalytic solution is a sodium acetate (10 mM) buffer solution containing 20% ​​(v / v) Tween 20, 4 mM 3-amino-9-ethylcarbazole and 2 mM hydrogen peroxide.

[0103] SERS mode detection: After chromatography, the SERS signal of the T-line of the test strip was recorded using a portable Raman spectrometer (B&WTek, i-Raman Plus BWS465-785H) equipped with a 785nm laser. SERS measurements of the T-line were performed under conditions of 10s exposure time, 5mW laser power, and ~100μm spot size. To reduce experimental error, 20 random measurements were taken at the front of the test area of ​​each sample, and the resulting SERS spectra were averaged for subsequent analysis. To investigate the uniformity of the SERS intensity distribution on the T-line, a Renishaw inVia spectrometer was used. TM A confocal Raman microscopy system with a 785 nm laser excitation source was used to obtain SERS mapping images for each T-line. A computer-controlled xy-stage was used to scan the selected T-region in a 200 μm × 200 μm (400 pixels) area. For SERS mapping, the laser power was set to 1% (~3 mW) and the acquisition time was set to 1 s.

[0104] If adenovirus or norovirus is present in the sample, the nanozyme probe will rapidly capture the target virus and form a virus-DSAIA immune complex, which is then immobilized by specific antibodies coated on the T line, resulting in a colored band and SERS signal on the corresponding T line. If the sample is negative, the virus-DSAIA immune complex cannot form, and no detection signal will appear on the T line. Excess immune nanozyme probes are immobilized by goat anti-mouse antibody (IgG) coated on the control (C) line, producing a visible black band. The absence of a black C line indicates an invalid test.

[0105] Subsequently, the operating conditions of the immunochromatographic technique based on bifunctional mesoporous SERS nanozyme probes were optimized, including antibody coating concentration and immunochromatographic time, to achieve the best detection performance of the platform.

[0106] Using the immunochromatographic assay method described above, test strips coated with different concentrations (0.4, 0.6, 0.8, 1.0, 1.2 mg / mL) of norovirus and enteric adenovirus antibodies were tested in SERS mode. The signal-to-noise ratio (SNR) was calculated to screen for the most suitable antibody coating concentration. The SNR formula is: SNR = Positive SERS signal value ÷ Negative SERS signal value. The positive sample used for the test contained 10... 5 Loading buffer for adenovirus at PFU / mL and norovirus at 1 ng / mL; loading buffer for negative samples is antigen-free.

[0107] After selecting the optimal antibody coating concentration, the chromatography reaction time was optimized. The chromatography reaction time was set to 3, 6, 9, 12, and 15 minutes, and the signal-to-noise ratio was calculated to select the most suitable chromatography reaction time.

[0108] Optimize experimental results as follows Figure 5 and Figure 6 As shown, Figure 5 To optimize the detection of online capture antibody concentrations, the results showed that the concentration of norovirus antibody was 0.8 mg / mL and the concentration of enteric adenovirus antibody was 1 mg / mL. Immunochromatography based on bifunctional mesoporous SERS nanozyme probes achieved the highest signal-to-noise ratio for both norovirus and enteric adenovirus. Figure 6 The results show the optimized chromatography time for immunochromatography based on bifunctional mesoporous SERS nanozyme probes. The results indicate that a 12-minute chromatography reaction time is sufficient to achieve strong colorimetric signals and the highest signal-to-noise ratio in the detection of norovirus and enteric adenovirus. The optimized time also helps the bifunctional mesoporous SERS nanozyme probes and the resulting virus-probe immune complexes exhibit good mobility on NC membranes and ensures stable operation in immunochromatography based on bifunctional mesoporous SERS nanozyme probes.

[0109] Example 3

[0110] This embodiment provides an optimized immunochromatographic technique based on a bifunctional mesoporous SERS nanozyme probe, as described in Example 2, for the detection of different concentrations of norovirus (10-0.0001 ng / mL) and enteroadenovirus (10-0.0001 ng / mL) in both chromogenic and SERS modes. 6 The performance of the (-10PFU / mL) was tested, and the results detected using SERS mode were compared with the results before and after catalysis in display mode, as well as with the results detected by traditional colloidal gold immunochromatography. The specific experimental steps are as follows.

[0111] The detection of norovirus and enteric adenovirus followed the same steps as in Example 2, except that test line 1 in the T-line was used to detect adenovirus, and test line 2 was used to detect norovirus. The sample concentrations used for norovirus were 10, 5, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, 0.0001, and 0 ng / mL, with a sample volume of 70 μL. The concentration used for enteric adenovirus was 10... 6 5×10 5 10 5 5×10 4 10 4 5×10 3 10 3 5×10 2 10 2 50, 10 1 The values ​​of 0 PFU / mL corresponded to logarithmic (base 10) values ​​of 6, 5.7, 5, 4.7, 4, 3.7, 3, 2.7, 2, 1.7, and 1 PFU / mL for the enteroadenovirus concentration and a negative sample, respectively. The sample volume used for detection was 70 μL. Colorimetric and SERS signals were collected before and after catalysis. Calibration curves were established based on the Raman intensity of the obtained SERS signal and the concentrations of norovirus and enteroadenovirus according to the following formula.

[0112]

[0113] Where A1 is the background signal, A2 is the maximum Raman signal intensity, X is the concentration of the sample, X0 is the median concentration, i.e. the concentration value corresponding to the inflection point of the curve, and P is the slope factor.

[0114] Preparation of colloidal gold immunochromatographic reagent: The pH of 4 μg norovirus antibody and enteric adenovirus antibody was adjusted to 9 with 0.2 M K2CO3 and incubated with 1 mL of 30 nm gold nanoparticles (pH 8-9) for 15 min. Next, 50 μL of 10% BSA was added to block unreacted sites on the gold nanoparticles. The prepared immunogold nanoparticles were collected by centrifugation (5000 rpm, 6 min) and resuspended in 200 μL of stock solution (1% BSA, 0.1% PVP, 10% sucrose, and 0.05% Tween-20 in 10 mM PB solution). Finally, the mixture was dropped onto glass fiber paper and dried to prepare the binding pad. The binding pad was then assembled onto an immunochromatographic test strip and cut into 3.0 mm strips for subsequent use. Next, 70 μL of the running buffer and the 1:1 mixture of the test sample were added to the sample pad for immunochromatographic reaction. After the reaction, the red color signal on the test strip was collected to verify the performance of traditional colloidal gold immunochromatographic detection.

[0115] Experimental results are as follows Figure 7 As shown in Figure AF, the experimental results indicate that when the norovirus concentration drops to 0.1 ng / mL and the enteroadenovirus concentration drops to 10... 4 At PFU / mL, the T-line signal was no longer visible to the naked eye. In the post-catalytic colorimetric mode, the limits of detection (vLOD) of immunochromatography based on bifunctional mesoporous SERS nanozyme probes in nanozyme mode were 0.001 ng / mL and 10 ng / mL, respectively. 2 PFU / mL, the sensitivity in the post-catalytic colorimetric mode is 100 times higher than that of colloidal gold immunochromatography. Figure 7 Figures c and d show the results of detecting T-line SERS signals of norovirus and enteric adenovirus in SERS mode, respectively. The detected Raman intensity decreases as the concentration of norovirus and enteric adenovirus decreases. Figure 7 In Figures e and f, the calibration curves of T-line SERS signal intensity versus norovirus and enteric adenovirus concentrations, prepared based on c and d, show that both viruses achieve a wide dynamic range of 6 orders of magnitude, with a goodness of fit R0. 2 The values ​​were 0.985 and 0.983, respectively. According to the IUPAC definition (LOD = blank signal + 3 × blank standard deviation), the detection limits of immunochromatography based on bifunctional mesoporous SERS nanozyme probes in SERS mode for norovirus and enteric adenovirus were 0.14 pg / mL and 11 PFU / mL, respectively. Compared with the pre-catalytic colorimetric mode, the sensitivity was improved by 714 times and 909 times, respectively, and by at least 7 times compared with the post-catalytic colorimetric mode.

[0116] Results of traditional colloidal gold immunochromatographic assays, such as Figure 8 As shown in Figures a and b, the detection limit for norovirus is 0.1 ng / mL, and the detection limit for enteroadenovirus is 5 × 10⁻⁶. 4 PFU / mL, the immunochromatographic technology based on bifunctional mesoporous SERS nanozyme probes of this invention improves the sensitivity of norovirus and adenovirus in nanozyme mode by 100 times and 500 times respectively compared with traditional colloidal gold immunochromatographic assays, and the improvement is more than 700 times in SERS mode.

[0117] In summary, the results highlight the significant advantages of immunochromatographic technology based on bifunctional mesoporous SERS nanozyme probes in rapid on-site detection: the detection time is comparable to that of traditional colloidal gold immunochromatography, while significantly improving the sensitivity of traditional rapid immunoassays, thereby greatly enhancing the current ability to detect viral infections.

[0118] Example 4

[0119] This embodiment provides the specific detection of immunochromatographic technology based on bifunctional mesoporous SERS nanozyme probes, optimized in Example 2. A variety of common pathogenic bacteria were selected for testing, including rotavirus, hepatitis A virus, Shigella, Acinetobacter baumannii, Helicobacter pylori, Enterococcus faecalis, Listeria monocytogenes, Escherichia coli O157:H7, Campylobacter jejuni, Streptococcus pyogenes, and Staphylococcus aureus. Rotavirus and hepatitis A virus antigens were purchased from Changzhou Zhongmei Xinxin Biotechnology Co., Ltd., while other bacterial strains were purchased from BNCC. The specific experimental steps are as follows.

[0120] Immunochromatography using the optimized bifunctional mesoporous SERS nanozyme probe from Example 2 was used to detect samples containing 10... 6 A mixed sample of norovirus and enteric adenovirus at PFU / mL and 10 ng / mL, containing 10 6 PFU / mL rotavirus, hepatitis A virus individual samples and 10 6 Individual samples of Shigella, Acinetobacter baumannii, Helicobacter pylori, Enterococcus faecalis, Listeria monocytogenes, Escherichia coli O157:H7, Campylobacter jejuni, Streptococcus pyogenes, and Staphylococcus aureus were tested at CFU / mL, and the specific steps for testing were the same as those in Example 2.

[0121] Experimental results are as follows Figure 9 Results of (Ⅰ) colorimetric mode before catalysis and results of (ⅠⅠ) after catalysis for a, and Figure 9 As shown in Figure b, the experimental results demonstrate that the immunochromatographic platform based on bifunctional mesoporous SERS nanozyme probes exhibits good selectivity for the target bacteria. Therefore, the specific bacterial antibodies applied to the detection line of the immunochromatographic platform based on bifunctional mesoporous SERS nanozyme probes are sufficient to ensure the high specificity of the detection platform.

[0122] Example 5

[0123] This embodiment provides repeatability detection of immunochromatographic technology based on bifunctional mesoporous SERS nanozyme probes after optimization in Example 2.

[0124] For those containing concentrations of 10 6 10 4 10 2 Seven consecutive tests were performed on mixed samples of norovirus and enteric adenovirus at PFU / mL and concentrations of 10, 0.1, and 0.001 ng / mL. The relative standard deviation (RSD) was calculated for verification. The specific steps of the test were the same as those in Example 2.

[0125] Experimental results are as follows Figure 10As shown in Figures a and b, the SERS signal on the detection line of the test strip changes little, and the RSD values ​​of each test group are all less than 10.80%, indicating that the established immunochromatographic platform based on bifunctional mesoporous SERS nanozyme probes has good reproducibility.

[0126] Example 6

[0127] This embodiment provides a systematic evaluation of the performance of the immunochromatographic technique based on bifunctional mesoporous SERS nanozyme probes optimized in Example 2 for direct detection of foodborne viruses in real complex samples. The specific experimental steps are as follows.

[0128] High concentrations (10 ng / mL) were added to fecal samples from healthy individuals and lake water samples, respectively. 6 PFU / mL), medium concentration (0.1 ng / mL; 10 4 PFU / mL) and low concentrations (0.001 ng / mL; 10 2 Norovirus and adenovirus (PFU / mL) were used to simulate real, complex virus-containing samples, and the detection procedure was the same as in Example 2. The lake water sample was collected from Shuangqing Lake, Baiyun District, Guangzhou City, Guangdong Province.

[0129] Experimental results are as follows Figure 11 As shown in Figures a and b, as the concentration of the target virus in the sample decreases, the intensity of the T-line's color development / SERS correspondingly weakens. The detection results of norovirus and adenovirus in the sample using the color development mode and SERS mode are basically consistent with the detection results under laboratory conditions in Example 3, indicating that the detection method has high accuracy and reliability in complex samples.

[0130] Example 7

[0131] This embodiment demonstrates the diagnostic potential of immunochromatography based on bifunctional mesoporous SERS nanozyme probes for clinical viral foodborne infections, as described in Example 2, and compares it with quantitative real-time PCR. The specific experimental steps are as follows.

[0132] The experimental procedures for immunochromatographic detection based on bifunctional mesoporous SERS nanozyme probes are the same as in Example 2. Quantitative real-time PCR detection was performed using a norovirus nucleic acid detection kit (fluorescent PCR method) (Shanghai Berger Medical Technology Co., Ltd.) and an adenovirus universal and type 41 nucleic acid detection kit (fluorescent PCR method) (Jiangsu Hongweites Pharmaceutical Technology Co., Ltd.). Specific experimental procedures for quantitative real-time PCR are detailed in the kit instructions.

[0133] Experimental results are as follows Figure 12As shown in Figure af, immunochromatography based on bifunctional mesoporous SERS nanozyme probes and quantitative real-time PCR were used to detect 51 norovirus samples, 20 adenovirus samples, and 21 healthy human fecal samples. Figure 12 Both methods (a and b) can significantly distinguish between positive and negative samples. Figure 12 (c and d), and AUC curve analysis showed that the AUC value of the immunochromatographic technique based on bifunctional mesoporous SERS nanozyme probes described in this invention for detecting norovirus can reach 0.993 (c and d), and the AUC curve analysis showed that the AUC value of norovirus detection by the immunochromatographic technique based on bifunctional mesoporous SERS nanozyme probes described in this invention can reach 0.993 (c and d), and the AUC curve analysis showed that Figure 12 In the study of adenovirus, the AUC value reached 0.998 (e). Figure 12 (f) This demonstrates that immunochromatography based on bifunctional mesoporous SERS nanozyme probes, like qPCR, can effectively distinguish between positive and negative samples. Compared to quantitative real-time PCR, the total detection time (including chromatography, catalysis, and SERS reading) of immunochromatography based on bifunctional mesoporous SERS nanozyme probes is approximately 20 minutes, significantly shorter than the 2-3 hours required for quantitative real-time PCR. Furthermore, it is simpler to operate, requires no clean environment, and can flexibly achieve both qualitative and quantitative detection. Therefore, in scenarios such as epidemic prevention and control, immunochromatography based on bifunctional mesoporous SERS nanozyme probes can meet diverse application needs and has enormous practical application potential.

[0134] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A nanotag, characterized in that, The nanotag has a core-shell structure, comprising a silica nanoparticle as the inner core and a noble metal particle as the outer shell. A nanocomposite material is electrostatically adsorbed between the outer shell and the inner core as a catalytic intermediate layer via polyethyleneimine. The nanocomposite material is a nanoenzyme Au@Ir; the noble metal particle is Au; and the silica nanoparticle is dendritic mesoporous silica nanoparticle.

2. The use of the nanotag of claim 1 in the preparation of products for labeling.

3. The method for preparing the nanotag according to claim 1, characterized in that, The preparation method includes the following steps: Dendritic mesoporous silica nanoparticles were added to a polyethyleneimine solution and mixed thoroughly. Then, a nanocomposite material was added for adsorption. After adsorption was completed, polyethyleneimine was added again and mixed thoroughly. Finally, a noble metal particle solution was added for adsorption to obtain the nanotag.

4. An immunochromatographic assay kit, characterized in that, The immunochromatographic kit includes a nano-tag probe and an immunochromatographic test strip; the nano-tag probe is the nano-tag of claim 1 with a surface modified with a detection antibody; the immunochromatographic test strip contains a sample pad, an absorbent pad, and an NC membrane with a detection line; the detection line is modified with the detection antibody.

5. The application of the immunochromatographic reagent kit of claim 4 in the preparation of products for detecting pathogen samples, characterized in that, The pathogen is a virus or a microorganism.

6. The application according to claim 5, characterized in that, The viruses include caliciviruses, adenoviruses, reoviruses, or astroviruses; the caliciviruses include noroviruses.

7. A method for detecting pathogens for non-diagnostic purposes, characterized in that, The method includes the following steps: The nano-tag probe described in claim 4 is mixed with the sample to be tested and dropped onto an immunochromatographic test strip for chromatography for 5-20 minutes. After chromatography, the catalytic solution is dropped onto the detection line of the immunochromatographic test strip for catalysis. The presence or absence of the detection line determines whether the sample to be tested contains pathogens.

8. The method according to claim 7, characterized in that, The catalyst solution is a sodium acetate solution containing Tween, 3-amino-9-ethylcarbazole and hydrogen peroxide.

9. A method for detecting pathogens for non-diagnostic purposes, characterized in that, The method includes the following steps: The nano-tag probe described in claim 4 is mixed with the sample to be tested and dropped onto the immunochromatographic test strip for chromatography for 5-20 minutes. After chromatography, Raman spectroscopy is performed to detect the pathogen content in the sample to be tested based on the Raman spectral signal intensity.

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