Raman-enhanced nano-label based on polydopamine core-shell structure, preparation method and application of Raman-enhanced nano-label in immunochromatography

Abstract

The invention provides a Raman enhanced nano-label based on a polydopamine core-shell structure, a preparation method and application of the Raman enhanced nano-label in immunochromatography, and relates to the technical field of immunochromatography. The Raman enhanced nano-label based on the polydopamine core-shell structure sequentially comprises a PDA microsphere core, a PEI layer and a gold and silver nano-particle layer from inside to outside, the gold and silver nanoparticle layer is gold and silver nanoparticles with a core-shell structure; the gold and silver nanoparticle layer is connected with a Raman signal molecule DTNB, and a terminal carboxyl group of the Raman signal molecule DTNB is coupled with a capture antibody of an H1N1 virus or a capture antibody of an SARS-CoV-2 virus. The nano-label has colorimetric and SERS enhanced activity at the same time, has the advantages of rich hot spots, high stability, excellent optical performance and the like, is used for an immunochromatography sensor to detect two respiratory viruses at the same time, adopts qualitative and quantitative dual-mode detection, and improves the detection flexibility, sensitivity and accuracy.

Description

Technical Field

[0001] The present invention relates to the technical field of immunochromatography, and in particular to a Raman-enhanced nano-label based on a polydopamine core-shell structure, a preparation method thereof, and application in immunochromatography. Background Art

[0002] In recent decades, sudden respiratory infections caused by the rapid spread of airborne droplets, such as coronaviruses, influenza viruses, and respiratory syncytial virus, have posed a serious threat to public health. Early clinical symptoms of respiratory infections (such as sore throat, cough, fever, and headache) are relatively similar, posing a significant challenge to the existing healthcare system during peak influenza season. Therefore, the development of convenient and cost-effective point-of-care testing (POCT) strategies to enable timely differential diagnosis of infection sources to guide clinical treatment and prevention is urgent.

[0003] Based on the specific binding of antibodies and antigens and the principle of chromatography, lateral flow immunochromatography (LFA) offers advantages such as ease of use, low cost, and portability, making it a well-established point-of-care (POCT) tool for disease diagnosis and personal health self-assessment. Traditional LFIA relies on colloidal gold nanoparticles (AuNPs) to provide visible colorimetric results in the test strip's detection zone. However, the insufficient colorimetric signal intensity generated by AuNPs alone leads to limitations in colorimetric LFIA, including low sensitivity, poor reproducibility, and insufficient quantitative capability. To enhance the analytical performance of LFIA, researchers are integrating more sensitive signal readout technologies into LFIA systems, including surface-enhanced Raman spectroscopy (SERS), fluorescence, catalytic amplification, and magnetic signals. These technologies have successfully achieved highly sensitive detection of biomarkers, pathogens, and nucleic acids. SERS offers advantages over other methods, such as non-destructiveness, resistance to photobleaching, robustness, ultrasensitivity, and fingerprint recognition, enabling quantitative detection at the single-molecule level. Furthermore, SERS nanotags, fabricated from noble metal nanoparticles coated with specific Raman reporter molecules, can generate both a strong Raman signal (with a narrow characteristic peak) and colorimetric visualization on LFIA test strips. This dual-signal mode makes the SERS-LFIA method both flexible and practical for diverse application scenarios.

[0004] The design of the SERS tag determines the detection performance of the LFIA. However, SERS tags based on single plasmonic nanoparticles (gold / silver and anisotropic nanoparticles) generally suffer from poor stability in complex samples, insufficient SERS activity, and susceptibility to environmental interference, which can easily lead to false detections in clinical samples using SERS-LFIA. Recent studies have demonstrated that composite core-shell nanotags based on SiO2, Fe3O4, and two-dimensional materials can be applied to SERS-LFIA systems, further improving SERS response and stability. However, existing studies have either suffered from complex preparation processes or failed to achieve simultaneous improvements in SERS and colorimetric sensitivity.

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

[0006] One objective of the present invention is to provide a Raman-enhanced nanotag based on a polydopamine core-shell structure. The nanotag comprises, from the inside out, a PDA microsphere core, a PEI layer, and a gold-silver nanoparticle layer. This PDA core-shell Raman-enhanced nanotag exhibits both colorimetric and SERS-enhancing activity. When combined with immunochromatographic detection techniques, it can be used for multiplexed detection of respiratory viruses, enabling rapid, accurate, and highly sensitive qualitative and quantitative detection of H1N1 and SARS-CoV-2.

[0007] The second object of the present invention is to provide a method for preparing the Raman-enhanced nanotag based on the polydopamine core-shell structure.

[0008] The third object of the present invention is to provide an application of the Raman-enhanced nanolabel based on the polydopamine core-shell structure in the preparation of products for detecting multiple respiratory viruses.

[0009] A fourth object of the present invention is to provide a product for detecting multiple respiratory viruses, wherein the product for detecting multiple respiratory viruses comprises: a Raman-enhanced nanotag and an immunochromatographic test paper; wherein the Raman-enhanced nanotag comprises the Raman-enhanced nanotag based on a polydopamine core-shell structure.

[0010] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are adopted: In a first aspect, the present invention provides a Raman-enhanced nanotag based on a polydopamine core-shell structure, wherein the Raman-enhanced nanotag based on the polydopamine core-shell structure comprises, from the inside to the outside, a PDA microsphere core, a PEI layer, and a gold and silver nanoparticle layer; Wherein, the gold-silver nanoparticle layer is gold-silver nanoparticles with a core-shell structure; The gold and silver nanoparticle layer is connected to a Raman signal molecule DTNB, and the terminal carboxyl group of the Raman signal molecule DTNB is coupled to a capture antibody for the H1N1 virus or a capture antibody for the SARS-CoV-2 virus.

[0011] Furthermore, the core of the PDA microsphere is a nano-microsphere structure generated by oxidative self-polymerization.

[0012] Furthermore, the particle size of the PDA microsphere core is 150-250 μm.

[0013] Furthermore, the PEI layer is self-assembled on the surface of the PDA microspheres.

[0014] Furthermore, the thickness of the PEI layer is 1-5 μm.

[0015] Furthermore, the gold and silver nanoparticle layer is formed by adsorption on the PEI layer through electrostatic force.

[0016] Furthermore, the gold-silver nanoparticles with a core-shell structure include, from the inside to the outside, a gold core and a silver shell.

[0017] Furthermore, the particle size of the gold-silver nanoparticles with a core-shell structure is 20-30 nm.

[0018] Furthermore, the particle size of the gold core is 10-25 nm, and the thickness of the silver shell is 0-10 nm.

[0019] In a second aspect, the present invention provides a method for preparing a Raman-enhanced nanotag based on a polydopamine core-shell structure as described in the first aspect, the preparation method comprising: (1) Dopamine undergoes oxidative self-polymerization to obtain the PDA microsphere core; (2) mixing the PDA microspheres and the PEI solution, performing ultrasonic treatment, and self-assembling the PEI layer on the surface of the PDA microspheres to obtain PDA@PEI nanoparticles; (3) mixing the PDA@PEI nanoparticles and the gold-silver nanoparticle solution, and performing ultrasonic treatment to form the gold-silver nanoparticle layer on the surface of the PDA@PEI nanoparticles to obtain PAu@Ag nanoparticles; (4) mixing the PAu@Ag nanoparticles and the DTNB solution and performing ultrasonic treatment to obtain PAu@Ag-DTNB nanoparticles; (5) The PAu@Ag-DTNB nanoparticles, carbodiimide solution and N -hydroxysuccinimide solution to perform activation reaction to obtain carboxyl-activated PAu@Ag nanoparticles; (6) Mixing the carboxyl-activated PAu@Ag nanoparticles with a capture antibody and performing a coupling reaction to obtain the Raman-enhanced nanotag based on the polydopamine core-shell structure; wherein the capture antibody includes a capture antibody for the H1N1 virus or a capture antibody for the SARS-CoV-2 virus.

[0020] Furthermore, in step (1), the dopamine undergoes an oxidative self-polymerization reaction in a solvent, and the concentration of dopamine in the prepared dopamine solution is 5-10 mg / mL.

[0021] Furthermore, in step (1), the temperature of the oxidative self-polymerization reaction is 30-35° C., and the time of the oxidative self-polymerization reaction is 15-20 h.

[0022] Furthermore, in step (2), the mass ratio of the PDA microspheres to PEI is 1:(1-5).

[0023] Furthermore, in step (2), the concentration of PEI in the PEI solution is 15-20 mg / mL.

[0024] Furthermore, in step (2), the power of the ultrasonic treatment is 60-100 W, and the time of the ultrasonic treatment is 10-30 min.

[0025] Furthermore, in step (3), the mass ratio of the PDA@PEI nanoparticles to the gold and silver nanoparticles is 1:(0.5~1).

[0026] Furthermore, in step (3), the power of the ultrasonic treatment is 60-100 W, and the time of the ultrasonic treatment is 10-30 min.

[0027] Furthermore, in step (4), the concentration of the DTNB solution is 10-50 µM.

[0028] Furthermore, in step (4), the power of the ultrasonic treatment is 60-100 W, and the time of the ultrasonic treatment is 60-90 min.

[0029] Furthermore, in step (5), the concentration of the carbodiimide solution is 5-15 μM.

[0030] Furthermore, in step (5), the N The concentration of hydroxysuccinimide solution is 50~150 μM.

[0031] Furthermore, in step (5), the activation reaction time is 10 to 30 minutes.

[0032] Furthermore, in step (6), the coupling reaction specifically includes the following steps: The carboxyl-activated PAu@Ag nanoparticles are resuspended in a buffer solution, a capture antibody is added, and the reaction is carried out for 1 to 3 hours; bovine serum albumin is then added, and the reaction is continued for 0.5 to 1.5 hours. After centrifugation, the Raman-enhanced nanotag based on the polydopamine core-shell structure is obtained; wherein the capture antibody includes a capture antibody for the H1N1 virus or a capture antibody for the SARS-CoV-2 virus.

[0033] Furthermore, in step (5), the mass ratio of the carboxyl-activated PAu@Ag nanoparticles, the H1N1 virus capture antibody and the bovine serum albumin is 1:(0.01-0.02):(5-15).

[0034] Furthermore, in step (5), the mass ratio of the carboxyl-activated PAu@Ag nanoparticles, the SARS-CoV-2 virus capture antibody, and bovine serum albumin is 1:(0.01-0.02):(5-15). Furthermore, in step (6), the buffer solution includes a PBST solution.

[0035] Furthermore, in step (6), the rotation speed of the centrifugal treatment is 4000-4500 r / min, and the time of the centrifugal treatment is 5-10 min.

[0036] In a third aspect, the present invention provides a use of the Raman-enhanced nanotag based on a polydopamine core-shell structure in the preparation of a product for detecting multiple respiratory viruses.

[0037] In a fourth aspect, the present invention provides a product for detecting multiple respiratory viruses, wherein the product for detecting multiple respiratory viruses comprises: a Raman-enhanced nanotag and an immunochromatographic test paper; Wherein, the Raman enhanced nanotag includes the Raman enhanced nanotag based on the polydopamine core-shell structure as described in the first aspect; The immunochromatographic test paper includes a sample pad, an NC membrane, and a water-absorbing pad sequentially arranged on a fixed bottom plate along the chromatographic direction of the sample to be tested; the NC membrane is provided with a detection line T1, a detection line T2, and a quality control line C spaced apart along the chromatographic direction of the sample to be tested; the detection line T1 is coated with an H1N1 antibody, the detection line T2 is coated with a SARS-CoV-2 antibody, and the quality control line C is coated with a goat anti-mouse IgG antibody.

[0038] Compared with the prior art, the present invention has the following beneficial effects: (1) The Raman-enhanced nanotag based on the PDA core-shell structure proposed in this paper has superior performance: First, the self-assembly mediated by electrostatic forces enables a large number of Au@AgNPs to attach to the surface of the PDA microspheres, enhancing the colorimetric performance of the PAu@Ag tag. Second, the LSPR effect of the Au@Ag NPs and the large number of interstitial "hot spots" on the PAu@Ag surface double enhance the SERS activity.

[0039] (2) The Raman-enhanced nanotag based on the PDA core-shell structure proposed in the present invention can be used for immunochromatographic detection, and by analyzing the colorimetric and SERS intensity results of the detection line area within 16 minutes, it can achieve ultrasensitive qualitative and quantitative detection of H1N1 and SARS-CoV-2 simultaneously.

[0040] (3) The colorimetric / SERS dual-enhanced LFIA test strip proposed in the present invention has both colorimetric and SERS signal modes, which provides application prospects for improving the flexibility and practicality of LFIA technology in different application scenarios. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0042] Figure 1 The figure is a schematic flow chart of the preparation method of the Raman-enhanced nano-tag based on the PDA core-shell structure according to the present invention.

[0043] Figure 2A HRTEM images of the PDA microspheres provided in Example 1 and Example 2.

[0044] Figure 2B HRTEM images of PAu@Ag provided in Example 1 and Example 2.

[0045] Figure 2C Elemental surface scanning images of PAu@Ag provided in Example 1 and Example 2.

[0046] Figure 2D XRD patterns of PDA and PAu@Ag provided in Examples 1 and 2.

[0047] Figure 2E XPS spectra of PAu@Ag provided in Example 1 and Example 2.

[0048] Figure 3A Schematic diagram of the principle of combining DTNB and Au@Ag provided in Examples 1 and 2.

[0049] Figure 3B UV-visible absorption spectra and SERS signal diagrams of Au@Ag generated by reducing silver nitrate with different concentrations provided by the tags in Example 1 and Example 2.

[0050] Figure 3C Electromagnetic field simulation of the PAu@Ag tags provided in Examples 1 and 2.

[0051] Figure 3D The SERS signal diagrams of PAu@Ag at different salt concentrations provided in Example 1 and Example 2.

[0052] Figure 3EThe SERS signal diagrams of PAu@Ag at different pH values provided in Example 1 and Example 2.

[0053] Figure 4 Schematic diagram of the PAu@Ag-LFA detection principle provided for Application Example 1.

[0054] Figure 5A The cross-detection results of PAu@Ag-LFA provided for Application Example 1.

[0055] Figure 5B SEM image of the T-line area of the PAu@Ag-LFA test strip provided for Application Example 1.

[0056] Figure 6A The colorimetric results of the PAu@Ag-LFA platform provided for Application Example 1 for simultaneous detection of different concentrations of H1N1 and SARS-CoV-2 N proteins.

[0057] Figure 6B The Au@Ag-LFA test strip provided for Application Example 1 simultaneously detects different concentrations of H1N1 and SARS-CoV-2 N proteins.

[0058] Figure 6C The commercial Au-LFA test strips provided for Application Example 1 show the test results of different concentrations of H1N1 and SARS-CoV-2 N proteins.

[0059] Figure 6D Average SERS spectra of the PAu@Ag-LFA platform provided for Application Example 1 for simultaneous detection of H1N1 and SARS-CoV-2 N proteins at different concentrations.

[0060] Figure 6E Calibration curves for simultaneous detection of H1N1 and SARS-CoV-2 N proteins at different concentrations using the PAu@Ag-LFA platform provided for Application Example 1.

[0061] Figure 6F Calibration curve of the Au@Ag-LFA test strip provided for Application Example 1 for simultaneous detection of different concentrations of H1N1 and SARS-CoV-2 N proteins.

[0062] Figure 6G The results of the commercial ELISA kit provided in Application Example 1 for detecting different concentrations of H1N1 and SARS-CoV-2 N proteins.

[0063] Figure 6H Specificity detection results of the PAu@Ag-LFA platform provided for Application Example 1.

[0064] Figure 7AColorimetric results of the PAu@Ag-LFA platform provided for Application Example 1 for simultaneous detection of different concentrations of inactivated H1N1 and SARS-CoV-2 viruses.

[0065] Figure 7B The commercial Au-LFA test strips provided for Application Example 1 show the results of detecting different concentrations of H1N1 and SARS-CoV-2 inactivated viruses.

[0066] Figure 7C Average SERS spectra of the PAu@Ag-LFA platform provided for Application Example 1 for simultaneous detection of inactivated H1N1 and SARS-CoV-2 viruses at different concentrations.

[0067] Figure 7D Calibration curves for simultaneous detection of different concentrations of H1N1 and SARS-CoV-2 inactivated viruses using the PAu@Ag-LFA platform provided in Application Example 1. DETAILED DESCRIPTION

[0068] Unless otherwise defined herein, scientific and technological 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.

[0069] It should be noted that the following description sets forth specific details to facilitate a full understanding of the present invention. However, the present invention can be implemented in a variety of other ways than those described herein, and those skilled in the art may make similar generalizations without departing from the scope of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0070] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0071] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are adopted: In a first aspect, the present invention provides a Raman-enhanced nanotag based on a polydopamine core-shell structure, wherein the Raman-enhanced nanotag based on the polydopamine core-shell structure comprises, from the inside to the outside, a PDA microsphere core, a PEI layer, and a gold and silver nanoparticle layer; Wherein, the gold-silver nanoparticle layer is gold-silver nanoparticles with a core-shell structure; The gold and silver nanoparticle layer is connected to a Raman signal molecule DTNB, and the terminal carboxyl group of the Raman signal molecule DTNB is coupled to a capture antibody for the H1N1 virus or a capture antibody for the SARS-CoV-2 virus.

[0072] In the present invention, the Raman-enhanced nanotag based on the PDA core-shell structure comprises, from the inside out, a PDA core, a polyethyleneimine layer (PEI self-assembled layer), an Au@Ag shell connected to DTNB, and an antibody coupled after encoding DTNB. The Raman-enhanced nanotag based on the PDA core-shell structure has excellent performance.

[0073] First, polydopamine (PDA), a multimolecular polymer formed by the self-polymerization of dopamine (DA) in alkaline solution, offers a simple preparation process, excellent dispersibility and biocompatibility, and strong colorimetric performance, making it an ideal label carrier for immunochromatographic detection. Second, based on an electrostatic interaction-mediated self-assembly strategy, the optical properties of PDA microspheres and gold-silver nanoparticles (hereafter referred to as Au@Ag NPs) were integrated to significantly enhance the colorimetric detection performance of the label. Third, Au@Ag bimetallic nanoparticles exhibit a higher SPR effect than pure noble metal materials, enhancing SERS activity. Finally, the high-density interstitial arrangement of Au@Ag on the PDA surface creates numerous "hotspots," further enhancing SERS activity.

[0074] Based on this, the present invention has developed a core-shell composite nanotag (PAu@Ag) with a polydopamine (PDA) core coated with a large number of Au@Ag NPs. This nanotag not only enhances colorimetric performance but also significantly improves SERS performance by generating numerous "hotspots." The multifunctional LFIA platform constructed based on the PAu@Ag dual-signal tag enables simultaneous qualitative colorimetric analysis and quantitative SERS analysis, potentially serving as a universal method for pathogen immunodiagnosis. This PDA-based core-shell Raman-enhanced nanotag combines both colorimetric and SERS enhancement, offering advantages such as abundant hotspots, strong stability, and excellent optical properties.

[0075] As an optional embodiment, the core of the PDA microsphere is a nano-microsphere structure generated by oxidative self-polymerization.

[0076] Furthermore, the particle size of the PDA microsphere core is 150-250 μm, for example, it can be 150 μm, 160 μm, 170 μm, 180 μm, 190 μm, 200 μm, 210 μm, 220 μm, 230 μm, 240 μm, 250 μm, etc.

[0077] As an optional embodiment, the PEI layer is self-assembled on the surface of the PDA microspheres.

[0078] As an optional embodiment, the thickness of the PEI layer is 1-5 μm, for example, 1 μm, 1.5 μm, 2 μm, 2.5 μm, 3 μm, 3.5 μm, 4 μm, 4.5 μm, 5 μm, etc.

[0079] As an optional embodiment, the gold and silver nanoparticle layer is formed by adsorption on the PEI layer through electrostatic force.

[0080] As an optional embodiment, the gold-silver nanoparticles with a core-shell structure include, from the inside to the outside, a gold core and a silver shell.

[0081] As an optional embodiment, the particle size of the gold and silver nanoparticles with a core-shell structure is 20-30 nm, for example, it can be 20 nm, 22 nm, 24 nm, 26 nm, 28 nm, 30 nm, etc.

[0082] As an optional embodiment, the particle size of the gold core is 10~25 nm, for example, it can be 10 nm, 12 nm, 14 nm, 16 nm, 18 nm, 20 nm, 22 nm, 24 nm, 25 nm, etc., and the thickness of the silver shell is 0~10 nm, for example, it can be 0.1 nm, 0.5 nm, 1 nm, 2 nm, 4 nm, 6 nm, 8 nm, 10 nm, etc.

[0083] In a second aspect, the present invention provides a method for preparing a Raman-enhanced nanotag based on a polydopamine core-shell structure as described in the first aspect, the preparation method comprising: (1) Dopamine undergoes oxidative self-polymerization to obtain the PDA microsphere core; (2) mixing the PDA microspheres and the PEI solution, performing ultrasonic treatment, and self-assembling the PEI layer on the surface of the PDA microspheres to obtain PDA@PEI nanoparticles; (3) mixing the PDA@PEI nanoparticles and the gold-silver nanoparticle solution, and performing ultrasonic treatment to form the gold-silver nanoparticle layer on the surface of the PDA@PEI nanoparticles to obtain PAu@Ag nanoparticles; (4) mixing the PAu@Ag nanoparticles and the DTNB solution and performing ultrasonic treatment to obtain PAu@Ag-DTNB nanoparticles; (5) The PAu@Ag-DTNB nanoparticles, carbodiimide solution and N -hydroxysuccinimide solution to perform activation reaction to obtain carboxyl-activated PAu@Ag nanoparticles; (6) Mixing the carboxyl-activated PAu@Ag nanoparticles with a capture antibody and performing a coupling reaction to obtain the Raman-enhanced nanotag based on the polydopamine core-shell structure; wherein the capture antibody includes a capture antibody for the H1N1 virus or a capture antibody for the SARS-CoV-2 virus.

[0084] The nanotags described in this invention are based on the oxidative self-polymerization of dopamine and electrostatic self-assembly mediated by the cationic polymer PEI. They exhibit both colorimetric and SERS enhancement, resulting from a simple, efficient, and reproducible preparation process. The invention also provides a dual-mode immunochromatographic technique using the PAu@Ag nanotags for the detection of multiple respiratory viruses.

[0085] As an optional embodiment, in step (1), the dopamine is subjected to an oxidative self-polymerization reaction in a solvent, and the concentration of dopamine in the prepared dopamine solution is 5 to 10 mg / mL, for example, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, etc.

[0086] As an optional embodiment, in step (1), the temperature of the oxidative self-polymerization reaction is 30-35°C, for example, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, etc.

[0087] As an optional embodiment, in step (1), the time of the oxidative self-polymerization reaction is 15 to 20 hours, for example, it can be 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, etc.

[0088] As an optional embodiment, in step (2), the mass ratio of the PDA microspheres to PEI is 1:(1-5), for example, it can be 1:1, 1:2, 1:3, 1:4, 1:5, etc.

[0089] As an optional embodiment, in step (2), the concentration of PEI in the PEI solution is 15-20 mg / mL, for example, 15 mg / mL, 16 mg / mL, 17 mg / mL, 18 mg / mL, 19 mg / mL, 20 mg / mL, etc.

[0090] As an optional embodiment, in step (2), 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.

[0091] As an optional embodiment, in step (2), the ultrasonic treatment time is 10 to 30 min, for example, it can be 10 min, 12 min, 14 min, 15 min, 16 min, 18 min, 20 min, 22 min, 24 min, 26 min, 28 min, 30 min, etc.

[0092] As an optional embodiment, in step (3), the mass ratio of the PDA@PEI nanoparticles to the gold and silver nanoparticles is 1:(0.5-1), for example, it can be 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, or 1:1.

[0093] As an optional embodiment, in step (3), 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.

[0094] As an optional embodiment, in step (3), the ultrasonic treatment time is 10 to 30 min, for example, it can be 10 min, 12 min, 14 min, 15 min, 16 min, 18 min, 20 min, 22 min, 24 min, 26 min, 28 min, 30 min, etc.

[0095] As an optional embodiment, in step (4), the concentration of the DTNB solution is 10-50 µM, for example, 10 µM, 15 µM, 20 µM, 25 µM, 30 µM, 35 µM, 40 µM, 45 µM, 50 µM, etc.

[0096] 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.

[0097] As an optional embodiment, in step (4), the ultrasonic treatment time is 60 to 90 min, for example, it can be 60 min, 65 min, 70 min, 75 min, 80 min, 85 min, 90 min, etc.

[0098] As an optional embodiment, in step (5), the concentration of the carbodiimide solution is 5-15 µM, for example, 5 µM, 6 µM, 8 µM, 10 µM, 12 µM, 14 µM, 15 µM, etc.

[0099] As an optional implementation, in step (5), the N The concentration of the hydroxysuccinimide solution is 50-150 µM, for example, 50 µM, 60 µM, 80 µM, 100 µM, 120 µM, 140 µM, 150 µM, etc.

[0100] As an optional embodiment, in step (5), the activation reaction time is 10 to 30 min, for example, it can be 10 min, 12 min, 14 min, 15 min, 16 min, 18 min, 20 min, 22 min, 24 min, 26 min, 28 min, 30 min, etc.

[0101] As an optional embodiment, in step (6), the coupling reaction specifically includes the following steps: The carboxyl-activated PAu@Ag nanoparticles are resuspended in a buffer solution, and capture antibodies for H1N1 and SARS-CoV-2 are added, and the reaction is carried out for 1 to 3 hours (for example, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, etc.); bovine serum albumin is then added, and the reaction is continued for 0.5 to 1.5 hours (for example, 0.5 hours, 0.6 hours, 0.8 hours, 1 hour, 1.2 hours, 1.4 hours, 1.5 hours, etc.), and the reaction is carried out by centrifugation to obtain the Raman-enhanced nanotag based on the polydopamine core-shell structure; wherein the capture antibody includes a capture antibody for the H1N1 virus or a capture antibody for the SARS-CoV-2 virus.

[0102] As an optional embodiment, in step (5), the mass ratio of the carboxyl-activated PAu@Ag nanoparticles, the H1N1 virus capture antibody and the bovine serum albumin is 1:(0.01-0.02):(5-15); Among them, "0.01~0.02" can be, for example, 0.01, 0.012, 0.014, 0.015, 0.016, 0.018, 0.02, etc.; among them, "5~15" can be, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, etc.

[0103] As an optional embodiment, in step (5), the mass ratio of the carboxyl-activated PAu@Ag nanoparticles, the capture antibody of the SARS-CoV-2 virus, and the bovine serum albumin is 1:(0.01-0.02):(5-15); Among them, "0.01~0.02" can be, for example, 0.01, 0.012, 0.014, 0.015, 0.016, 0.018, 0.02, etc.; among them, "5~15" can be, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, etc.

[0104] As an optional embodiment, in step (6), the buffer solution includes PBST solution.

[0105] As an optional embodiment, in step (6), the rotation speed of the centrifugal treatment is 4000~4500 r / min, for example, it can be 4000 r / min, 4100 r / min, 4200 r / min, 4300 r / min, 4400 r / min, 4500 r / min, etc.

[0106] As an optional embodiment, in step (6), the centrifugal treatment time is 5 to 10 min, for example, it can be 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, etc.

[0107] In a third aspect, the present invention provides a use of the Raman-enhanced nanotag based on a polydopamine core-shell structure in the preparation of a product for detecting multiple respiratory viruses.

[0108] In the present invention, the Raman-enhanced nanotag-driven colorimetric / SER dual-enhanced immunoassay technology based on the PDA core-shell structure is used as a detection product for ultrasensitive detection of respiratory viruses, which can achieve simultaneous, rapid, accurate, and highly sensitive qualitative and quantitative detection of H1N1 and SARS-CoV-2 in untreated biological samples.

[0109] In a fourth aspect, the present invention provides a product for detecting multiple respiratory viruses, wherein the product for detecting multiple respiratory viruses comprises: a Raman-enhanced nanotag and an immunochromatographic test paper; Wherein, the Raman enhanced nanotag includes the Raman enhanced nanotag based on the polydopamine core-shell structure as described in the first aspect; The immunochromatographic test paper includes a sample pad, an NC membrane, and a water-absorbing pad sequentially arranged on a fixed bottom plate along the chromatographic direction of the sample to be tested; the NC membrane is provided with a detection line T1, a detection line T2, and a quality control line C spaced apart along the chromatographic direction of the sample to be tested; the detection line T1 is coated with an H1N1 antibody, the detection line T2 is coated with a SARS-CoV-2 antibody, and the quality control line C is coated with a goat anti-mouse IgG antibody.

[0110] In a fifth aspect, the present invention provides a method for detecting multiple respiratory viruses, comprising the following steps: The Raman-enhanced nanotag based on the PDA core-shell structure as described in the first aspect is added to a solution containing both H1N1 and SARS-CoV-2, and after brief mixing, a PAu@Ag immune tag-target complex is obtained; wherein the solution containing H1N1 and SARS-CoV-2 includes a buffer containing N protein and / or a throat swab sample containing H1N1 and SARS-CoV-2 viruses; The PAu@Ag immunolabel-target complex is dropped onto the sample pad of the immunochromatographic test strip, and the colorimetric results at the detection line of the immunochromatographic test strip are then observed for rapid qualitative detection. A Raman spectrometer is used in the detection line area of the immunochromatographic test strip to read the Raman signal at the detection line, thereby achieving quantitative detection of H1N1 and SARS-CoV-2.

[0111] In the present invention, the respiratory virus detection method adopts the Raman-enhanced nanotag based on the PDA core-shell structure as described in the first aspect for detection. This method directly and specifically binds the target analyte from a complex sample, and through the colorimetric visual observation results and SERS intensity analysis of the detection line area in a relatively short time, it achieves simultaneous ultrasensitive qualitative and quantitative detection of H1N1 and SARS-CoV-2.

[0112] It should be noted that the proposed colorimetric and SERS-enhancing PAu@Ag nanotag combines the optical properties of PDA microspheres and Au@Ag NPs, significantly enhancing the system's colorimetric detection performance. The high-density interstitial arrangement of Au@Ag bimetallic nanoparticles on the PDA surface, combined with the bimetallic surface plasmon resonance (SPR) effect, generates numerous Raman "hotspots," further enhancing SERS activity. The colorimetric / SERS dual-enhanced LFIA test strip, constructed based on the PAu@Ag nanocomposite, achieves rapid qualitative screening of targets in colorimetric mode and ultrasensitive quantitative analysis in SERS mode, successfully expanding the application of existing LFIA detection technology to clinical scenarios such as early infection identification and dynamic viral load monitoring.

[0113] The present invention is further described below by way of examples. Unless otherwise specified, the materials in the examples were prepared according to existing methods or directly purchased from the market.

[0114] Preparation Example 1 This preparation example provides an Au@Ag solution, which is prepared by the following steps: (1) Preparation of 20 nm colloidal gold: Wash three times with deionized water and three times with ethanol, then dry a 200 mL conical flask. Add 100 mL of deionized water, 1 mL of 1% HAuCl4 solution, and a stirring bar. Place the flask on a magnetic stirrer and heat until the liquid boils. Maintain a stirring rate of 680 r / min and add 4 mL of 1% TSC solution at once. Continue heating and stirring for 15 minutes, then stop heating and continue stirring to cool to room temperature. Finally, use deionized water to re-adjust the volume to 100 mL to obtain a colloidal gold solution.

[0115] (2) Preparation of 25 nm Au@Ag: Take another 200 mL conical flask that has been washed three times with deionized water and ethanol, then dried. Add 150 mL of deionized water, 30 mL of 20 nm colloidal gold solution, 2.5 mL of 1% TSC solution, and a stirrer. Place on a magnetic stirrer and heat until the liquid boils. Maintaining a stirring rate of 680 r / min, quickly add AgNO3 solution with a final concentration of 150 µM. Continue heating and stirring for 25 min, then stop heating and continue stirring until cooled to room temperature to obtain the Au@Ag solution.

[0116] Example 1 This embodiment provides a Raman-enhanced nanotag based on a PDA core-shell structure. The Raman-enhanced nanotag based on the PDA core-shell structure comprises, from the inside to the outside, a PDA core, a PEI layer, and an Au@Ag shell layer.

[0117] The Au@Ag shell is connected to a Raman signal molecule DTNB; the Raman signal molecule DTNB is connected to the Au@Ag shell via an Ag-S bond; and the terminal carboxyl group of the Raman signal molecule DTNB is coupled to an H1N1 virus capture antibody.

[0118] like Figure 1 As shown, the Raman enhanced nanotag based on the PDA core-shell structure described in this embodiment is prepared by the following steps: (1) Preparation of PDA nanoparticles: Wash three times with deionized water and three times with ethanol, then dry a 50 mL Erlenmeyer flask. Add 22 mL of deionized water, 1 mL of 28% ammonia, 10 mL of anhydrous ethanol, and a stirring bar. Seal the flask with Parafilm and stir at 680 rpm in a 30°C oil bath for 10 minutes. Once the reagents are evenly mixed, add 150 mg of DA powder to the flask at once. Seal the flask with Parafilm and continue stirring for 16 hours. After the reaction is complete, centrifuge at 10,000 rpm for 6 minutes, discard the supernatant, and collect the brown precipitate at the bottom. Wash the precipitate twice with deionized water and resuspend it in 20 mL of ethanol to obtain PDA microspheres.

[0119] (2) Preparation of PDA@PEI nanoparticles: 1 mL of prepared PDA microspheres was added to 20 mL of deionized water and subjected to vigorous sonication for 20 minutes to ensure uniform dispersion of the PDA microspheres in the aqueous solution. Under sonication, 2 mL of a 10 mg / mL PEI solution was added at once. After continuous sonication for 25 minutes, the solution was centrifuged at 7800 rpm for 10 minutes, the supernatant discarded, and the precipitate collected. The precipitate was washed twice with deionized water and resuspended in 10 mL of deionized water to obtain PDA@PEI nanoparticles.

[0120] (3) Preparation of PAu@Ag nanoparticles: 30 mL of Au@Ag solution was added to the aqueous PDA@PEI nanoparticle solution. After continuous sonication for 15 minutes, the mixture was centrifuged at 4200 rpm for 5 minutes. The supernatant was discarded and the precipitate was collected. After washing twice with deionized water, the precipitate was resuspended in 6 mL of ethanol to obtain PAu@Ag nanoparticles.

[0121] (4) Preparation of PAu@Ag-DTNB nanoparticles: An ethanol solution of PAu@Ag nanoparticles was mixed with 30 μM DTNB and subjected to vigorous sonication (100 W) for 1 hour. The mixture was then centrifuged at 4200 rpm for 5 minutes, the supernatant discarded, and the precipitate collected. After washing twice with ethanol, the precipitate was resuspended in 6 mL of ethanol to obtain a PAu@Ag-DTNB SERS tag.

[0122] (5) Preparation of carboxyl-activated MS@Pt-DTNB nanoparticles: 1 mL of the prepared PAu@Ag-DTNB SERS tag was taken, centrifuged at 4200 r / min for 5 min, the supernatant was removed, and then resuspended in 500 μL of MEST buffer (0.1 M, pH 5.8, 0.01% Tween-20). 10 μL of EDC (100 mM) and 10 μL of NHS (100 mM) aqueous solution were added, and the reaction was carried out under vigorous ultrasonic conditions for 15 min to obtain carboxyl-activated MS@Pt-DTNB nanoparticles.

[0123] (6) Preparation of Raman-enhanced nanolabels based on PDA core-shell structure The PAu@Ag-DTNB with surface carboxyl groups activated was centrifuged at 4200 rpm for 5 minutes, the supernatant removed, and the cell was resuspended in 0.4 mL of PBST buffer (pH 7.4, 0.05% Tween-20). 15 μg of H1N1 virus capture antibody was added, and the cell was incubated with shaking on a thermomixer at room temperature for 2 hours. Finally, 200 μL of 10% BSA (w / v) was added to the solution, and the reaction was continued with shaking for 1.5 hours. The supernatant was then centrifuged and washed twice with 0.5 mL of PBST buffer to obtain the antibody-conjugated PAu@Ag immunolabel. Finally, the cell was resuspended in 0.4 mL of PBST buffer containing 0.01% NaN₃ (w / v) for later use.

[0124] in, Figure 2A This is the HRTEM image of the PDA microspheres prepared in step (1) of this example. Figure 2B This is the HRTEM image of the PAu@Ag nanoparticles prepared in step (3) of this example. Figure 2C This is an elemental surface scan of the PAu@Ag nanoparticles prepared in step (3) of this embodiment. Figure 2D These are the XRD patterns of the PDA nanoparticles and PAu@Ag nanoparticles prepared in this example. Figure 2E This is the XPS spectrum of the PAu@Ag nanoparticles prepared in step (3) of this example.

[0125] Depend on Figure 2A to Figure 2E The above characterization results show that the PAu@Ag tag provided by the present invention has a uniform structure, distinct layers, and excellent monodispersity and stability.

[0126] Example 2 This embodiment provides a Raman-enhanced nanotag based on a PDA core-shell structure. The Raman-enhanced nanotag based on the PDA core-shell structure comprises, from the inside to the outside, a PDA core, a PEI layer, and an Au@Ag shell layer.

[0127] The Au@Ag shell is connected to a Raman signal molecule DTNB; the Raman signal molecule DTNB is connected to the Au@Ag shell through an Ag-S bond; and the terminal carboxyl group of the Raman signal molecule DTNB is coupled to a capture antibody for the SARS-CoV-2 virus.

[0128] like Figure 1 As shown, the Raman enhanced nanotag based on the PDA core-shell structure described in this embodiment is prepared by the following steps: (1) Preparation of PDA nanoparticles: Wash three times with deionized water and three times with ethanol, then dry a 50 mL Erlenmeyer flask. Add 22 mL of deionized water, 1 mL of 28% ammonia, 10 mL of anhydrous ethanol, and a stirring bar. Seal the flask with Parafilm and stir at 680 rpm in a 30°C oil bath for 10 minutes. Once the reagents are evenly mixed, add 150 mg of DA powder to the flask at once. Seal the flask with Parafilm and continue stirring for 16 hours. After the reaction is complete, centrifuge at 10,000 rpm for 6 minutes, discard the supernatant, and collect the brown precipitate at the bottom. Wash the precipitate twice with deionized water and resuspend it in 20 mL of ethanol to obtain PDA microspheres.

[0129] (2) Preparation of PDA@PEI nanoparticles: 1 mL of prepared PDA microspheres was added to 20 mL of deionized water and subjected to vigorous sonication for 20 minutes to ensure uniform dispersion of the PDA microspheres in the aqueous solution. Under sonication, 2 mL of a 10 mg / mL PEI solution was added at once. After continuous sonication for 25 minutes, the solution was centrifuged at 7800 rpm for 10 minutes, the supernatant discarded, and the precipitate collected. The precipitate was washed twice with deionized water and resuspended in 10 mL of deionized water to obtain PDA@PEI nanoparticles.

[0130] (3) Preparation of PAu@Ag nanoparticles: 30 mL of Au@Ag solution was added to the aqueous PDA@PEI nanoparticle solution. After continuous sonication for 15 minutes, the mixture was centrifuged at 4200 rpm for 5 minutes. The supernatant was discarded and the precipitate was collected. After washing twice with deionized water, the precipitate was resuspended in 6 mL of ethanol to obtain PAu@Ag nanoparticles.

[0131] (4) Preparation of PAu@Ag-DTNB nanoparticles: An ethanol solution of PAu@Ag nanoparticles was mixed with 30 μM DTNB and subjected to vigorous sonication (100 W) for 1 hour. The mixture was then centrifuged at 4200 rpm for 5 minutes, the supernatant discarded, and the precipitate collected. After washing twice with ethanol, the precipitate was resuspended in 6 mL of ethanol to obtain a PAu@Ag-DTNB SERS tag.

[0132] (5) Preparation of carboxyl-activated MS@Pt-DTNB nanoparticles: 1 mL of the prepared PAu@Ag-DTNB SERS tag was taken, centrifuged at 4200 r / min for 5 min, the supernatant was removed, and then resuspended in 500 μL of MEST buffer (0.1 M, pH 5.8, 0.01% Tween-20). 10 μL of EDC (100 mM) and 10 μL of NHS (100 mM) aqueous solution were added, and the reaction was carried out under vigorous ultrasonic conditions for 15 min to obtain carboxyl-activated MS@Pt-DTNB nanoparticles.

[0133] (6) Preparation of Raman-enhanced nanolabels based on PDA core-shell structure The PAu@Ag-DTNB with surface carboxyl groups activated was centrifuged at 4200 rpm for 5 minutes, the supernatant removed, and the cell was resuspended in 0.4 mL of PBST buffer (pH 7.4, 0.05% Tween-20). 15 μg of SARS-CoV-2 capture antibody was added, and the cell was incubated with shaking on a thermomixer at room temperature for 2 hours. Finally, 200 μL of 10% BSA (w / v) was added to the solution, and the reaction was continued with shaking for 1.5 hours. The supernatant was then centrifuged and washed twice with 0.5 mL of PBST buffer to obtain the antibody-conjugated PAu@Ag immunolabel. Finally, the cell was resuspended in 0.4 mL of PBST buffer containing 0.01% NaN₃ (w / v) for later use.

[0134] in, Figure 2A This is the HRTEM image of the PDA microspheres prepared in step (1) of this example. Figure 2B This is the HRTEM image of the PAu@Ag nanoparticles prepared in step (3) of this example. Figure 2C This is an elemental surface scan of the PAu@Ag nanoparticles prepared in step (3) of this embodiment. Figure 2D These are the XRD patterns of the PDA nanoparticles and PAu@Ag nanoparticles prepared in this example. Figure 2E This is the XPS spectrum of the PAu@Ag nanoparticles prepared in step (3) of this example.

[0135] Depend on Figure 2A to Figure 2E The above characterization results show that the PAu@Ag tag provided by the present invention has a uniform structure, distinct layers, and excellent monodispersity and stability.

[0136] Test Example 1 SERS performance test Test sample: the sample obtained in step (4) provided in Example 1 and Example 2.

[0137] Test method: Take 200 μL of the sample obtained in step 4, centrifuge at 4200 r / min for 5 min, discard the supernatant, add the sediment obtained by enrichment at the bottom to the surface of a clean silicon wafer, and then use a portable Raman spectrometer to collect the 1333 cm -1 The SERS signal at .

[0138] Test results: Figure 3A Schematic diagram of the principle of combining DTNB and Au@Ag provided in this embodiment. Figure 3B The UV-visible absorption spectra and SERS signal diagrams of Au@Ag generated by reducing silver nitrate with different concentrations provided in this example. Figure 3C This is the electromagnetic field simulation of the PAu@Ag tag provided in this embodiment. Figure 3D The SERS signal diagrams of PAu@Ag at different salt concentrations provided in this example. Figure 3E The SERS signal diagrams of PAu@Ag at different pH values provided in this example.

[0139] Depend on Figure 3A to Figure 3E The above characterization results demonstrate that the controlled synthesis of the Ag shell maximizes the SERS intensity of the PAu@Ag tag. Furthermore, the resulting MS@Pt product exhibits high salt tolerance, a stable SERS signal in the pH range of >3, and reproducible production. These results demonstrate that the colorimetric and SERS-enhanced PAu@Ag tag prepared by this invention exhibits a strong SERS response and high stability, making it suitable for use with complex samples.

[0140] Application Example 1 This application example provides a method for detecting multiple respiratory viruses, which includes the following steps: The PDA core-shell structure-based SERS tags prepared in Examples 1 and 2 were added to a solution containing both H1N1 and SARS-CoV-2, and after brief mixing, a PAu@Ag immune tag-target complex was obtained; wherein the solution containing H1N1 and SARS-CoV-2 included a buffer containing N protein and / or a throat swab sample containing H1N1 and SARS-CoV-2 viruses; The PAu@Ag immunolabel-target complex is dropped onto the sample pad of the immunochromatographic test strip, and the colorimetric results at the detection line of the immunochromatographic test strip are then observed for rapid qualitative detection. A Raman spectrometer is used in the detection line area of the immunochromatographic test strip to read the Raman signal at the detection line, thereby achieving quantitative detection of H1N1 and SARS-CoV-2.

[0141] in, Figure 4Schematic diagram of the PAu@Ag-LFA detection principle provided for Application Example 1. Figure 5A The cross-detection results of PAu@Ag-LFA provided for Application Example 1. Figure 5B This is an SEM image of the T-line region of the PAu@Ag-LFA test strip provided in Application Example 1. The results show that the PAu@Ag-LFA does not cross-bind when simultaneously detecting H1N1 and SARS-CoV-2, and quantitative detection can be achieved through interception of the PAu@Ag tag on the T-line.

[0142] in, Figure 6A Colorimetric results for simultaneous detection of H1N1 and SARS-CoV-2 N protein at varying concentrations using the PAu@Ag-LFA platform, provided for Application Example 1. As shown in Figure 6A, the visualization of the T line gradually decreases with decreasing target concentration. The visual sensitivity of the PAu@Ag-LFA for H1N1 and SARS-CoV-2 N proteins is 0.5 ng / mL and 0.05 ng / mL, respectively.

[0143] in, Figure 6B The Au@Ag-LFA test strips provided for Application Example 1 show the simultaneous detection of H1N1 and SARS-CoV-2 N protein at different concentrations. As shown in Figure 6B, the visual sensitivity of Au@Ag-LFA for H1N1 and SARS-CoV-2 N proteins is 1 ng / mL and 0.1 ng / mL, respectively, which is two times lower than the colorimetric sensitivity of PAu@Ag-LFA.

[0144] in, Figure 6C The commercial Au-LFA test strips provided for Application Example 1 show the results of different concentrations of H1N1 and SARS-CoV-2 N proteins. As shown in Figure 6C, the commercial Au-LFA test strips have visual sensitivity of 0.5 ng / mL for H1N1 and 0.5 ng / mL for SARS-CoV-2 N proteins. While consistent with the colorimetric sensitivity of PAu@Ag-LFA for H1N1, their sensitivity for SARS-CoV-2 is still 10 times lower.

[0145] in, Figure 6D Average SERS spectra of the PAu@Ag-LFA platform used in Application Example 1, simultaneously detecting different concentrations of H1N1 and SARS-CoV-2 N proteins. Figure 6D shows a positive correlation between the SERS signal and the N protein, and even at concentrations as low as 0.005 ng / mL for H1N1 and 0.0025 ng / mL for SARS-CoV-2 N proteins, the samples can still be distinguished from the blank control.

[0146] in, Figure 6E Calibration curves for simultaneous detection of H1N1 and SARS-CoV-2 N protein at varying concentrations using the PAu@Ag-LFA platform, provided for Application Example 1. As shown in Figure 6E, the detection limits for quantitative detection of H1N1 and SARS-CoV-2 were calculated to be 1.132 ng / mL and 1.027 ng / mL, respectively.

[0147] in, Figure 6F Calibration curves for the Au@Ag-LFA test strips used in Application Example 1 for simultaneous detection of H1N1 and SARS-CoV-2 N protein at varying concentrations. As shown in Figure 6F, the limits of detection for quantitative detection of H1N1 and SARS-CoV-2 are 253.068 ng / mL and 24.1 ng / mL, respectively, representing 223.6 and 22.5 times lower sensitivities than the PAu@Ag-LFA in quantitative mode.

[0148] in, Figure 6G The commercial ELISA kit provided in Application Example 1 detects H1N1 and SARS-CoV-2 N protein at different concentrations. As shown in Figure 6G, the quantitative sensitivity of the ELISA kit for detecting H1N1 and SARS-CoV-2 is 0.177 ng / mL and 0.035 ng / mL, respectively, which is 34 times and 156 times lower than the sensitivity of PAu@Ag-LFA, respectively.

[0149] in, Figure 6H Figure 6H shows the specificity of the PAu@Ag-LFA platform for application example 1. As shown in Figure 6H, PAu@Ag-LFA is highly specific in the detection of respiratory viruses.

[0150] in, Figure 7A The colorimetric results of the PAu@Ag-LFA platform provided for Application Example 1 for simultaneous detection of different concentrations of H1N1 and SARS-CoV-2 inactivated viruses. Figure 7A It can be seen that the visual sensitivity of PAu@Ag-LFA to inactivate H1N1 and SARS-CoV-2 viruses is 3.3×10 5 and 1.23×10 5 copies / mL.

[0151] in, Figure 7B The commercial Au-LFA test strips provided for Application Example 1 were used to test different concentrations of H1N1 and SARS-CoV-2 inactivated viruses. Figure 7B It can be seen that the naked eye sensitivity of the commercial Au-LFA test strips for H1N1 and SARS-CoV-2 inactivated viruses is 1.65×10 6and 2.46×10 5 copies / mL, which are 5 times and 2 times lower than the colorimetric sensitivity of PAu@Ag-LFA, respectively.

[0152] in, Figure 7C The average SERS spectra of the PAu@Ag-LFA platform provided for Application Example 1 for simultaneous detection of different concentrations of H1N1 and SARS-CoV-2 inactivated viruses. Figure 7C It can be seen that the SERS signal is positively correlated with the virus concentration, and the H1N1 and SARS-CoV-2 viruses are at 1.65×10 4 and 2.46×10 3 At a lower concentration of 10 copies / mL, there was still a significant difference compared with the blank control group.

[0153] in, Figure 7D The calibration curve of the PAu@Ag-LFA platform for simultaneous detection of different concentrations of H1N1 and SARS-CoV-2 inactivated viruses provided in Application Example 1. Figure 7D It can be seen that the detection limits of H1N1 and SARS-CoV-2 quantitative detection are as low as 6.63×10 3 and 624 copies / mL.

[0154] These results demonstrate the high sensitivity and multiplexed detection capabilities of the colorimetric / SERS dual-enhanced LFA detection system, making it suitable for the detection of pathogenic microorganisms in complex samples. Furthermore, the PAu@Ag-LFA offers rapid qualitative and precise quantitative capabilities, and can flexibly switch detection modes based on scenario requirements, broadening its application scope.

[0155] In summary, the PAu@Ag tag invented in this application, prepared by loading a large number of bimetallic Au@Ag NPs on the PDA surface, combines the advantages of large specific surface area, colorimetric enhancement, and SERS enhancement, significantly improving the sensitivity and detection range of existing LFIA systems. The LFA detection system based on this tag achieves simultaneous ultrasensitive qualitative and quantitative detection of H1N1 and SARS-CoV-2 through colorimetric and SERS dual-signal detection modes, providing a more efficient and stable point-of-care (POCT) strategy for the detection of multiple pathogens using multimodal LFA.

[0156] 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 above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above 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 Raman enhanced nanotag based on a polydopamine core-shell structure, characterized in that: The Raman enhanced nano-tag based on the polydopamine core-shell structure comprises, from the inside to the outside, a PDA microsphere core, a PEI layer, and a gold and silver nanoparticle layer; Wherein, the gold-silver nanoparticle layer is gold-silver nanoparticles with a core-shell structure; The gold and silver nanoparticle layer is connected to a Raman signal molecule DTNB, and the terminal carboxyl group of the Raman signal molecule DTNB is coupled to a capture antibody for the H1N1 virus or a capture antibody for the SARS-CoV-2 virus.

2. The Raman enhanced nanotag based on polydopamine core-shell structure according to claim 1, characterized in that: The core of the PDA microsphere is a nano-microsphere structure generated by oxidative self-polymerization; And / or, the particle size of the PDA microsphere core is 150-250 μm.

3. The Raman enhanced nanotag based on polydopamine core-shell structure according to claim 1, characterized in that: The PEI layer is self-assembled on the surface of the PDA microspheres; And / or, the thickness of the PEI layer is 1-5 μm.

4. The Raman enhanced nanotag based on polydopamine core-shell structure according to claim 1, characterized in that: The gold and silver nanoparticle layer is formed by adsorption on the PEI layer through electrostatic force; And / or, the gold-silver nanoparticles with a core-shell structure comprise, from the inside to the outside, a gold core and a silver shell; And / or, the particle size of the gold and silver nanoparticles having a core-shell structure is 20-30 nm; And / or, the particle size of the gold core is 10-25 nm, and the thickness of the silver shell is 0-10 nm.

5. A method for preparing a Raman enhanced nanotag based on a polydopamine core-shell structure according to any one of claims 1 to 4, characterized in that: The preparation method comprises: (1) Dopamine undergoes oxidative self-polymerization to obtain the PDA microsphere core; (2) mixing the PDA microspheres and the PEI solution, performing ultrasonic treatment, and self-assembling the PEI layer on the surface of the PDA microspheres to obtain PDA@PEI nanoparticles; (3) mixing the PDA@PEI nanoparticles and the gold-silver nanoparticle solution, and performing ultrasonic treatment to form the gold-silver nanoparticle layer on the surface of the PDA@PEI nanoparticles to obtain PAu@Ag nanoparticles; (4) mixing the PAu@Ag nanoparticles and the DTNB solution and performing ultrasonic treatment to obtain PAu@Ag-DTNB nanoparticles; (5) The PAu@Ag-DTNB nanoparticles, carbodiimide solution and N -hydroxysuccinimide solution to perform activation reaction to obtain carboxyl-activated PAu@Ag nanoparticles; (6) Mixing the carboxyl-activated PAu@Ag nanoparticles with a capture antibody and performing a coupling reaction to obtain the Raman-enhanced nanotag based on the polydopamine core-shell structure; wherein the capture antibody includes a capture antibody for the H1N1 virus or a capture antibody for the SARS-CoV-2 virus.

6. The method for preparing a Raman enhanced nanotag based on a polydopamine core-shell structure according to claim 5, characterized in that: In step (1), the dopamine is subjected to an oxidative self-polymerization reaction in a solvent, and the concentration of dopamine in the prepared dopamine solution is 5-10 mg / mL; And / or, in step (1), the temperature of the oxidative self-polymerization reaction is 30-35° C., and the time of the oxidative self-polymerization reaction is 15-20 h; And / or, in step (2), the mass ratio of the PDA microspheres to PEI is 1:(1-5); and / or, in step (2), the concentration of PEI in the PEI solution is 15-20 mg / mL; And / or, in step (2), the power of the ultrasonic treatment is 60-100 W, and the time of the ultrasonic treatment is 10-30 min.

7. The method for preparing a Raman enhanced nanotag based on a polydopamine core-shell structure according to claim 5, characterized in that: In step (3), the mass ratio of the PDA@PEI nanoparticles to the gold and silver nanoparticles is 1:(0.5-1); And / or, in step (3), the power of the ultrasonic treatment is 60-100 W, and the time of the ultrasonic treatment is 10-30 min; and / or, in step (4), the concentration of the DTNB solution is 10-50 µM; And / or, in step (4), the power of the ultrasonic treatment is 60-100 W, and the time of the ultrasonic treatment is 60-90 min.

8. The method for preparing a Raman enhanced nanotag based on a polydopamine core-shell structure according to claim 5, characterized in that: In step (5), the concentration of the carbodiimide solution is 5-15 μM; And / or, in step (5), the N -The concentration of hydroxysuccinimide solution is 50~150 μM; And / or, in step (5), the activation reaction time is 10 to 30 min; And / or, in step (6), the coupling reaction specifically includes the following steps: The carboxyl-activated PAu@Ag nanoparticles are resuspended in a buffer solution, a capture antibody is added, and the reaction is carried out for 1 to 3 hours; bovine serum albumin is then added, and the reaction is continued for 0.5 to 1.5 hours. The Raman-enhanced nanotag based on the polydopamine core-shell structure is obtained after centrifugation; wherein the capture antibody includes a capture antibody for the H1N1 virus or a capture antibody for the SARS-CoV-2 virus; And / or, in step (5), the mass ratio of the carboxyl-activated PAu@Ag nanoparticles, the H1N1 virus capture antibody, and bovine serum albumin is 1:(0.01-0.02):(5-15); And / or, in step (5), the mass ratio of the carboxyl-activated PAu@Ag nanoparticles, the SARS-CoV-2 virus capture antibody, and bovine serum albumin is 1:(0.01-0.02):(5-15); and / or, in step (6), the buffer solution comprises PBST solution; And / or, in step (6), the rotation speed of the centrifugal treatment is 4000-4500 r / min, and the time of the centrifugal treatment is 5-10 min.

9. Use of a Raman-enhanced nanotag based on a polydopamine core-shell structure according to any one of claims 1 to 4 in the preparation of a product for detecting multiple respiratory viruses.

10. A product for detecting multiple respiratory viruses, characterized in that: The products for detecting multiple respiratory viruses include: Raman-enhanced nanotags and immunochromatographic test strips; Wherein, the Raman enhanced nanotag comprises the Raman enhanced nanotag based on the polydopamine core-shell structure according to any one of claims 1 to 4; The immunochromatographic test paper includes a sample pad, an NC membrane, and a water-absorbing pad sequentially arranged on a fixed bottom plate along the chromatographic direction of the sample to be tested; the NC membrane is provided with a detection line T1, a detection line T2, and a quality control line C spaced apart along the chromatographic direction of the sample to be tested; the detection line T1 is coated with an H1N1 antibody, the detection line T2 is coated with a SARS-CoV-2 antibody, and the quality control line C is coated with a goat anti-mouse IgG antibody.

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