Method for detecting monkey pox virus by establishing immunochromatography based on MoS2 (at) Au nanosheet

Abstract

The invention discloses a method for establishing immunochromatography for detecting monkey pox virus based on a MoS2 (at) Au nanosheet. The method comprises the following steps: 1, sequentially adsorbing a Raman report molecule and a monkey pox virus specific antibody on the surface of a MoS2 (at) Au nanosheet to prepare a nanoprobe, and dispersing the nanoprobe in PBST to obtain a nanoprobe dispersion liquid; 2, preparing an immunochromatography test strip; 3, mixing a to-be-detected sample with the nanoprobe to obtain a to-be-detected solution, and applying the to-be-detected solution to the immunochromatography test strip for a chromatographic reaction; and 4, after the chromatographic reaction is completed, signal acquisition is performed on the detection line on the immunochromatographic test strip, and an adopted signal reading mode comprises at least one of visual colorimetry, surface enhanced Raman scattering and photo-thermal detection. According to the present invention, the multi-mode detection can be achieved, the sensitivity is high, and the detection flexibility and the result reliability are substantially enhanced through the design, such that the rapid screening requirement of the base site can be met, and the accurate quantification requirement of the laboratory can be met.

Description

Technical Field

[0001] This invention relates to the field of analytical detection technology. Specifically, it relates to a method for immunochromatographic detection of monkeypox virus based on MoS2@Au nanosheets. Background Technology

[0002] Monkeypox virus (MPXV), a double-stranded DNA virus belonging to the orthopoxvirus genus, has become a major global health threat. MPXV is highly transmissible from person to person, and its clinical symptoms (such as fever and characteristic rash) are highly similar to those of smallpox, measles, and chickenpox, posing a significant challenge to early and accurate diagnosis. MPXV enters the body through mucous membranes or broken skin, initially infecting dendritic cells and macrophages, and spreading to local lymph nodes within 48 hours. Most MPXV patients die from multiple organ failure and acute respiratory distress. Therefore, there is an urgent need to develop a rapid and sensitive detection method to directly identify MPXV in different clinical samples and improve the accuracy of early diagnosis.

[0003] Currently, several methods exist for detecting MPXV, but each has its limitations. Nucleic acid amplification technologies, particularly quantitative polymerase chain reaction (qPCR) and gene sequencing, are fundamental to the clinical diagnosis of MPXV infection. However, the requirements for specialized, pollution-free laboratories, sophisticated equipment, and well-trained personnel significantly limit the accessibility of these technologies in resource-constrained areas. ELISA, as a commonly used immunoassay technique, is widely applied in clinical diagnosis and research testing. However, its complex procedures, reliance on specialized equipment, and difficulty in rapid field application make it challenging. Immunochromatographic analysis (ICA) for MPXV detection offers advantages such as rapid response and ease of operation, making it one of the most promising point-of-care testing (POCT) methods. By rapidly and sensitively detecting MPXV in various clinical specimens, ICA is crucial for early diagnosis, timely isolation, and ultimately, more effective control of MPXV outbreaks. However, traditional colloidal gold (AuNPs)-based ICA only provides visual colorimetric signals for rapid qualitative identification, exhibiting low sensitivity and limited quantitative capabilities. Therefore, a rapid, convenient, and highly sensitive detection method is urgently needed. In recent years, surface enhanced Raman scattering (SERS) has attracted increasing attention from researchers due to its high sensitivity, plasmon resonance, and significant amplification when combined with noble metals. Summary of the Invention

[0004] Therefore, the technical problem to be solved by the present invention is to provide a method for immunochromatographic detection of monkeypox virus based on MoS2@Au nanosheets, which can realize multi-mode detection and has high sensitivity.

[0005] To solve the above-mentioned technical problems, the present invention provides the following technical solution:

[0006] The method for immunochromatographic detection of monkeypox virus based on MoS2@Au nanosheets includes the following steps: Step 1: Preparation of nanoprobes: Raman reporter molecules and monkeypox virus-specific antibodies are sequentially adsorbed on the surface of MoS2@Au nanosheets to prepare nanoprobes, and the nanoprobes are dispersed in PBST to obtain nanoprobe dispersion.

[0007] Step 2: Prepare the immunochromatographic test strip;

[0008] Step 3: Perform immunochromatographic detection: Mix the sample to be tested with the nanoprobe dispersion prepared in Step 1 to obtain the test solution. Apply the test solution to the immunochromatographic test strip and perform the chromatographic reaction.

[0009] Step 4: Multimodal signal detection: After the chromatography reaction is completed, the signal on the detection line of the immunochromatographic test strip is acquired. The signal readout mode used includes at least one of visual colorimetry, surface-enhanced Raman scattering, and photothermal detection.

[0010] This invention is based on synthesized MoS2@Au nanoprobes. These MoS2@Au nanoprobes, when applied to test strips, can generate signals for visual colorimetric analysis, as well as surface-enhanced Raman scattering (SERS) and photothermal signals for instrumental quantitative analysis. A multifunctional immunochromatographic assay (M-ICA) platform integrating visual, SERS, and photothermal conversion has been developed and applied to the detection of monkeypox virus. This platform enables rapid and highly sensitive detection of monkeypox through multiple modes, including visual, SERS, and photothermal methods. Users can perform rapid on-site screening by observing color changes in the test line, or conduct precise quantitative analysis using a portable Raman spectrometer or infrared laser irradiation to obtain digital test results. The M-ICA platform has a visual limit of detection (LOD) of 0.5 ng / mL for MPXV antigen (MPXV A29L protein), while the LODs for SERS and photothermal detection modes are 4.2 pg / mL and 3 pg / mL, respectively, which are 238 times and 333 times higher than those of conventional Au NPs ICA. The clinical applicability of this M-ICA platform for inactivated MPXV was tested. The LOD of SERS for inactivated MPXV was 225 copies / mL, and the LOD of photothermal inactivation for inactivated MPXV was 210 copies / mL. Experimental results indicate that the M-ICA platform formed using MoS2@Au nanoprobes is a highly efficient, stable, and multifunctional flexible high-performance sensor for MPXV detection, providing a new approach for the clinical diagnosis of monkeypox virus.

[0011] The above-mentioned method for immunochromatographic detection of monkeypox virus based on MoS2@Au nanosheets involves the preparation of MoS2@Au nanosheets by assembling Au NPs on the surface of MoS2 nanosheets using a polyethyleneimine-mediated seed growth strategy to form MoS2@Au nanosheets; including the following steps:

[0012] S1. Mix the monolayer MoS2 dispersion with ultrapure water at a volume ratio of (2~4):(4~6). After ultrasonic treatment and centrifugation, remove the supernatant, collect the bottom MoS2 nanosheet precipitate, and disperse the MoS2 nanosheet precipitate in ultrapure water to obtain a MoS2 nanosheet dispersion. It can be quickly dispersed in water, which is convenient for subsequent operations. The bottom MoS2 nanosheet precipitate has a size of 600-900nm. Using larger MoS2 particles can load more Au particles and improve detection sensitivity.

[0013] S2. Mix the MoS2 nanosheet dispersion with a polyethyleneimine aqueous solution at a volume ratio of (8~10):1, wherein the polyethyleneimine concentration in the polyethyleneimine aqueous solution is 20 mg / mL; after ultrasonic treatment, washing, and centrifugation, collect the MoS2@PEI nanosheet precipitate, and disperse the MoS2@PEI nanosheet precipitate in ultrapure water to obtain a MoS2@PEI nanosheet dispersion; it can be rapidly dispersed in water, which facilitates subsequent operations;

[0014] S3. The obtained MoS2@PEI nanosheet dispersion was mixed with Au NPs solution at a volume ratio of 1: (4~6). After ultrasonic treatment, centrifugation and washing, the MoS2@Au seed precipitate was collected and dispersed in anhydrous ethanol to obtain MoS2@Au seed dispersion, which is convenient for long-term storage.

[0015] S4. Add the MoS2@Au seed dispersion, hydroxylamine hydrochloride, and polyvinylpyrrolidone sequentially to the aqueous solution and mix. After ultrasonic treatment, add 150 μL of chloroauric acid solution, and after ultrasonic treatment again, centrifugation, and washing, collect the MoS2@Au nanosheet precipitate. Disperse the MoS2@Au nanosheet precipitate in anhydrous ethanol to obtain the MoS2@Au nanosheet dispersion for later use and long-term storage.

[0016] The above-mentioned method for immunochromatographic detection of monkeypox virus based on MoS2@Au nanosheets uses DTNB as the Raman reporter molecule. DTNB is adsorbed on the surface of MoS2@Au nanosheets to form MoS2@Au / DTNB nanosheets. The monkeypox virus-specific antibody is linked to the MoS2@Au / DTNB nanosheets through a coupling reaction between carboxyl and amino groups to form a nanoprobe.

[0017] The above-mentioned method for immunochromatographic detection of monkeypox virus based on MoS2@Au nanosheets involves the following steps: adding DTNB ethanol solution to MoS2@Au nanosheets dispersed in ethanol, followed by ultrasonic treatment and washing, collecting the MoS2@Au / DTNB nanosheet precipitate, and storing the precipitate in 2 ml of ethanol for later use.

[0018] The above-mentioned method for immunochromatographic detection of monkeypox virus based on MoS2@Au nanosheets uses DTNB ethanol solution with a concentration of 40~120μM.

[0019] The above-mentioned method for immunochromatographic detection of monkeypox virus based on MoS2@Au nanosheets uses DTNB at a concentration of 80 μM.

[0020] The above-mentioned method for immunochromatographic detection of monkeypox virus based on MoS2@Au nanosheets involves the following steps for linking monkeypox virus-specific antibodies to MoS2@Au / DTNB: MoS2@Au / DTNB nanosheets are washed with ultrapure water to obtain a suspension. MES buffer is added to the suspension, followed by EDC solution and NHS solution, which are mixed thoroughly. After sonication, the supernatant is discarded to obtain a precipitate. The precipitate is resuspended in PBST solution, monkeypox virus-specific antibodies are added and thoroughly mixed, and the mixture is incubated at room temperature with shaking. BSA solution is then added for incubation. Finally, the mixture is centrifuged, washed with PBST solution, and the nanoprobe precipitate is obtained. The nanoprobe precipitate is then resuspended in PBST for storage. This process maintains pH stability to prevent material aggregation and denaturation, and provides a suitable ionic environment to reduce non-specific adsorption.

[0021] In the above-described method for immunochromatographic detection of monkeypox virus based on MoS2@Au nanosheets, step two involves an immunochromatographic test strip comprising a PVC base plate, a sample pad, an NC membrane, and an absorbent pad. A detection line and a control line are positioned on the NC membrane. The detection line is coated with an antibody for capturing monkeypox virus antigen, and the control line is coated with goat anti-mouse polyclonal IgG antibody.

[0022] In the above-mentioned method for immunochromatographic detection of monkeypox virus based on MoS2@Au nanosheets, in step three, the sample to be tested is the elution buffer of a biological sample collected by swab after soaking in the running buffer. The running buffer is a phosphate buffer containing 1% Tween-20 and 10% BSA, and the amount of nanoprobe used is 1uL.

[0023] In the above method for immunochromatographic detection of monkeypox virus based on MoS2@Au nanosheets, step four involves visual colorimetric detection, which is determined by visually observing whether there are black bands on the detection line.

[0024] Surface-enhanced Raman scattering (SERS) detection involves illuminating the detection line with a 785 nm Raman spectrometer to collect and analyze DTNB at 1331 cm⁻¹. -1 The characteristic Raman peak intensity at the location was quantitatively analyzed based on a pre-established calibration curve of Raman intensity versus antigen concentration.

[0025] Photothermal detection uses a wavelength of 808 nm and a power density of 1.5 W / cm². 2 The near-infrared laser irradiates the detection line, and the temperature rise value ΔT is captured and recorded in real time by an infrared thermal imager. Quantitative analysis is performed based on a pre-established temperature change versus antigen concentration calibration curve.

[0026] The technical solution of the present invention achieves the following beneficial technical effects:

[0027] 1. This invention is based on synthesized MoS2@Au nanoprobes. These MoS2@Au nanoprobes on test strips can generate signals for visual colorimetric analysis, as well as surface-enhanced Raman scattering (SERS) and photothermal signals for instrumental quantitative analysis. A multifunctional immunochromatographic assay (M-ICA) platform integrating visual, SERS, and photothermal conversion has been developed. Applied to the detection of monkeypox virus, it enables rapid and highly sensitive detection of monkeypox through multiple modes including visual, SERS, and photothermal methods. Users can perform rapid on-site screening by observing color changes in the test line, or perform precise quantitative analysis using a portable Raman spectrometer or infrared laser irradiation to obtain digital test results. The limit of detection (LOD) for MPXV antigen (MPXV A29L protein) using the M-ICA platform is 0.5 ng / mL, while the limits of detection for SERS and photothermal detection modes are 4.2 pg / mL and 3 pg / mL, respectively, which are 238 times and 333 times that of conventional Au NPs ICA. The clinical applicability of this M-ICA platform for inactivated MPXV was tested. The LOD of SERS for inactivated MPXV was 225 copies / mL, and the LOD of photothermal inactivation for inactivated MPXV was 210 copies / mL. Experimental results indicate that the M-ICA platform formed using MoS2@Au nanoprobes is a highly efficient, stable, and multifunctional flexible high-performance sensor for MPXV detection, providing a new approach for the clinical diagnosis of monkeypox virus.

[0028] 2. The MoS2@Au nanoprobe used in this invention is a typical core-shell structure material, with a monolayer of MoS2 as the core and Au nanoparticles as the outer shell. Monolayer molybdenum disulfide nanosheets are a class of two-dimensional transition metal disulfide materials with unique physicochemical properties. These atomic-level nanostructures possess outstanding performance, such as excellent catalytic activity, ultra-high specific surface area, tunable bandgap structure, efficient photothermal conversion capability, and excellent dispersibility in various solutions. MoS2 nanosheets exhibit a significant chemical enhancement effect, and coating them with a layer of Au nanoparticles can improve the sensitivity of SERS detection. In this structure, MoS2 not only promotes strong near-infrared light absorption and converts it into heat, but also provides attachment sites for the reduction of Au nanoparticles. Au nanoparticles enhance photothermal performance through plasmon resonance and significantly amplify the Raman signal through localized surface plasmon resonance (LSPR). It can achieve both efficient photothermal conversion and SERS function.

[0029] 3. This detection method exhibits high specificity for varicella-zoster virus, vaccinia virus, measles virus, and herpes simplex virus, maintaining good batch-to-batch reproducibility across all detection modalities. These results demonstrate that the immunochromatographic method based on MoS2@Au nanosheets is a highly sensitive and accurate diagnostic tool for monkeypox virus, providing a potential approach for immunodiagnosis of monkeypox virus in various application scenarios. This design significantly enhances the flexibility and reliability of the detection, meeting both the rapid screening needs of grassroots fieldwork and the precise quantitative requirements of laboratories.

[0030] In summary, this application utilizes specific nanoprobe materials (MoS2@Au and sequentially modified Raman reporter molecules and MPXV specific antibodies) to apply them to a specific detection target (monkeypox virus), achieving a monkeypox virus test that is both highly sensitive and easy to operate. It also enables visual visualization and surface-enhanced Raman scattering, photothermal multi-signal output modes, and the detection method exhibits excellent sensitivity, specificity, and repeatability. Attached Figure Description

[0031] Figure 1 a represents the synthesis process of the MoS2@Au nanoprobe; Figure 1 b represents the detection of MPXV using colorimetric, SERS, and photothermal methods;

[0032] Figure 2 a, b, and c are high-resolution transmission electron microscopy (HR-TEM) images of MoS2 nanosheets, MoS2@Au seed (molybdenum disulfide@gold seed nanosheets), and MoS2@Au (molybdenum disulfide@gold nanosheets), respectively. Figure 2d and e are magnified HR-TEM images of MoS2@Auseed and MoS2@Au, respectively; Figure 2 f is the elemental mapping image of MoS2@Au; Figure 2 g represents the zeta potential diagram of the products obtained at different stages; Figure 2 h represents the corresponding UV-Vis absorption spectra of MoS2, MoS2@Au seed, and MoS2@Au; Figure 2 i represents the energy-dispersive X-ray (EDX) spectrum of MoS2@Au; Figure 2 j represents the wide-scan XPS spectrum of MoS2@Au and the corresponding high-resolution Mo 3d, S 2p, and Au 4f spectra.

[0033] Figure 3 a represents the SERS signal intensity of MoS2@Au modified with different concentrations of DTNB; Figure 3 b represents the SERS signal intensity of MoS2@Au products at different stages modified with 80uMDTNB; Figure 3 c represents the SERS signal intensity of MoS2@Au stored in ethanol for different times; Figure 3 d represents the temperature change of different materials over 5 minutes under the same irradiation (808 nm, 1.5 W / cm²); Figure 3 e represents the temperature change of different concentrations of MoS2@Au within 5 minutes under the same irradiation (808 nm, 1.5 W / cm²); Figure 3 f represents the temperature change of MoS2@Au solution (400 μg / mL) under irradiation with different power densities; Figure 3 g shows thermal images of different materials after 5 minutes under the same irradiation (808 nm, 1.5 W / cm²); Figure 3 h represents the photothermal heating curves for 5 cycles under the laser on / off cycle; Figure 3 i represents the heating and cooling curves used for calculating photothermal conversion efficiency;

[0034] Figure 4 a shows the visual and thermal images of MoS2@Au-M-ICA stripes used to detect different concentrations of MPXV; Figure 4 b is the average SERS spectrum of the T-line; Figure 4 c represents the temperature of test strips with different concentrations under an infrared camera; Figure 4 d represents the calibration curve of Raman intensity versus antigen concentration; Figure 4 e is the calibration curve of photothermal signal (ΔT) versus antigen concentration; Figure 4 f shows photographs based on commercial colloidal gold detection at different MPXV concentrations; Figure 4 g represents the visual and thermal images specific to MPXV detected by ICA; Figure 4h represents the specificity test of the SERS mode for non-target viral proteins; Figure 4 i represents the average temperature of the T series; Figure 4 j represents the visual and thermal images of MoS2@Au-M-ICA reproducibility; Figure 4 k represents the reproducibility of MoS2@Au-M-ICA.

[0035] Figure 5 a shows the visual and thermal images of M-ICA bands for MPXV inactivation at different concentrations; Figure 5 b represents the average SERS signal strength corresponding to the T-line; Figure 5 c is the calibration curve of Raman intensity versus inactivated MPXV; Figure 5 d represents the average temperature of line T; Figure 5 e represents the calibration curve of the photothermal signal (ΔT) and the inactivated MPXV. Detailed Implementation

[0036] Materials used in this invention: MoS2 was purchased from Jiangsu Pioneer Nanomaterials Technology Co., Ltd. Chloroauric acid tetrahydrate (HAuCl4·4H2O), hydroxylamine hydrochloride (NH2OH·HCl), and ethanol (99.5%) were purchased from Shanghai Chemical Reagent Co., Ltd. (China). Phosphate buffered saline (PBS, pH=7.4, 10 mM), polyethyleneimine (PEI), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), polyvinylpyrrolidone (PVP), 2-(N-morpholino)ethanesulfonic acid (MES), N-hydroxysuccinimide (NHS), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) were purchased from Sigma-Aldrich (USA). Bovine serum albumin (BSA, pH 7.0) was purchased from GPC Biotechnology. Fetal bovine serum (FBS) was purchased from Thermo Fisher Scientific Chemicals. All aqueous solutions were prepared using deionized water obtained through the Millipore Milli-Q system. Of the components of the chromatography test strip, all except the nitrocellulose membrane (purchased from Sartorius, Spain) were purchased from JEY Biotech Ltd. (Shanghai, China). Goat anti-mouse IgG was purchased from Sangon Biotech Co., Ltd. (Shanghai, China). MPXV A29L protein and anti-MPXV A29L protein monoclonal antibody were purchased from China National Biotec Group Co., Ltd. (Beijing).

[0037] Rapid and highly sensitive identification of viral antigens is crucial for timely diagnosis and effective epidemic management. However, traditional gold nanoparticle-based immunochromatographic assays (ICA) face limitations due to low detection sensitivity. This study designed a multifunctional molybdenum disulfide-gold (MoS2@Au) nanoprobe and used it to fabricate ICA test strips. This platform combines SERS detection, photothermal properties, and visual colorimetric output. MoS2@Au nanosheets provide a significant photothermal conversion efficiency (48.3%). Based on these properties, we proposed a multifunctional immunochromatographic assay (M-ICA) platform for monkeypox virus (MPXV) detection, providing three signal readout methods. The overall design, synthesis, and detection process of the MoS2@Au-M-ICA of this invention are as follows: Figure 1 As shown. Figure 1 As shown in Figure a, the multifunctional MoS2@Au nanoprobes were prepared using a PEI-mediated seed growth strategy. First, PEI was coated onto the surface of MoS2 nanosheets to form a positively charged surface. This facilitated the electrostatic adsorption of 13 nm Au NPs to form MoS2@Au seeds. Then, continuous Au NPs were grown on the surface by reduction with HAuCl4·4H2O to form MoS2@Au. These seed-coated particles were then functionalized using a 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) Raman reporter to obtain MoS2@Au / DTNB. Finally, a fully functionalized MoS2@Au nanoprobe was obtained by chemically conjugating an MPXV-specific antibody, specifically an anti-MPXV A29L protein monoclonal antibody, via EDC / NHS. Figure 1The detection protocol described in section b involves mixing a fixed volume of supernatant (sample and running buffer) with MoS2@Au SERS nanoprobes to form a mixture, which is then dropped onto the sample pad of an ICA test strip. After capillary flow is initiated, the mixture migrates along the test strip. If viral antigens are present, they specifically bind to the MoS2@Au nanoprobes and are captured on the test (T) line via antigen-antibody interaction. Unbound or excess probes continue to flow and are subsequently captured on the control (C) line as an internal control. Signal acquisition is then performed after the MoS2@Au nanoprobes have completed the chromatographic flow process. For sensitive and quantitative detection, 785 nm SERS or photothermal conversion based on an 808 nm laser-induced temperature rise can be used. These two detection modes can be used independently and can be flexibly selected according to specific detection requirements. Furthermore, since the MoS2@Au nanoprobes are inherently black, their accumulation on the T and C lines produces a clearly visible signal, making intuitive visual detection similar to traditional gold nanoparticle-based detection. This trimodal readout strategy enables multifunctional signal acquisition and is suitable for qualitative screening and quantitative analysis in different diagnostic environments.

[0038] 1.1 Preparation of MoS2@Au nanosheets

[0039] MoS2@Au nanosheets were synthesized by densely immobilizing Au NPs (colloidal gold) on the surface of MoS2 using PEI (polyethyleneimine)-assisted electrostatic assembly. The specific steps are as follows:

[0040] First, 3 mL of a monolayer MoS2 dispersion (MoS2 concentration 1 mg / mL) was mixed with 5 mL of ultrapure water, and then subjected to high-power sonication (100 W, 10 min), followed by centrifugation (centrifugal force 7197 rcf, 10 min) to systematically remove smaller MoS2 particles from the supernatant to ensure homogeneity. The resulting dispersion was then redispersed with 9 mL of ultrapure water and centrifuged again to obtain a MoS2 nanosheet dispersion containing larger, uniformly shaped MoS2 nanosheets (600-900 nm in size).

[0041] Next, 1 mL of PEI aqueous solution (PEI concentration of 20 mg / mL) was added to the above MoS2 nanosheet dispersion, mixed, and subjected to vigorous sonication (100 W) for 40 min to form PEI-coated MoS2 (MoS2@PEI) nanosheet precipitate. The MoS2@PEI nanosheet precipitate was washed twice with ultrapure water, centrifuged (7197 rcf, 10 min), and redispersed in 2 mL of ultrapure water to obtain the MoS2@PEI dispersion. In addition, to promote the adsorption of Au NPs (13 nm) on the MoS2@PEI nanosheets, 2 mL of the prepared MoS2@PEI dispersion was combined with 10 mL of Au NPs solution (Au NPs concentration of 3.94 mg / mL) and subjected to vigorous sonication (100 W) for 40 min. Then, the MoS2@Au nanosheets were separated by centrifugation (3500 rpm, 8 min) to remove excess Au NPs. The collected nanosheets were washed sequentially with ultrapure water and ethanol, and then redispersed in 2 mL of ethanol for later use to obtain the MoS2@Au seed dispersion.

[0042] Finally, 2 mL of the prepared MoS2@Au seed dispersion, 120 mg PVP, and 15 mg NH2OH·HCl were sequentially added to 30 mL of an aqueous solution to obtain a mixture. The aqueous solution was prepared using ultrapure water. The mixture was subjected to vigorous sonication (100 W) for 8 min to promote uniform dispersion. Subsequently, 150 μL of a 1% (w / w) HAuCl4·4H2O solution was added, and the mixture was sonicated vigorously for 6 min. The resulting solution was then centrifuged (3200 rpm, 8 min). The solution was washed once with ultrapure water, and then washed with ethanol to remove excess PVP, thus preparing MoS2@Au nanosheets. The nanosheets were then redispersed in 2 mL of ethanol for later use, yielding a MoS2@Au nanosheet dispersion.

[0043] 1.2 Preparation of MoS2@Au nanoprobes

[0044] To improve sensitivity in SERS-based quantitative analysis, superior tag SERS activity is essential. We chose DTNB as the optimal Raman reporter protein because it has the following advantages: DTNB can easily bind to noble metal surfaces via cleavable disulfide bonds, and its carboxyl group facilitates antibody conjugation. MoS2@Au nanoprobes were prepared by sequentially modifying the Raman reporter molecule DTNB and an anti-MPXV A29L protein monoclonal antibody (via carboxyl-amino group conjugation) onto nanosheets. Details are as follows:

[0045] First, 8 μL of the prepared DTNB ethanol solution (DTNB concentration of 80 μmol / L) was added to the MoS2@Au nanosheet dispersion prepared in section 1.1 above, and the mixture was subjected to strong ultrasonic treatment (100 W) for 60 min. After centrifugation (3200 rpm, 8 min), the solution was washed with anhydrous ethanol to remove excess DTNB, yielding DTNB-modified MoS2@Au, which was then stored in 2 mL of ethanol for later use, thus obtaining the MoS2@Au / DTNB dispersion.

[0046] Next, after centrifuging the MoS2@Au / DTNB dispersion and discarding the supernatant, the resulting precipitate was washed with 1 mL of ultrapure water to obtain a suspension. Then, 500 μL of MES buffer (pH 5.8, MES concentration 10 μmol / L) was added to the suspension, followed by 5 μL of freshly prepared EDC solution (EDC concentration 100 μmol / L) and 10 μL of freshly prepared NHS solution (NHS concentration 100 μmol / L). The mixture was thoroughly vortexed and sonicated for 15 min to complete the activation process. After discarding the supernatant, the precipitate was resuspended in 200 μL of 0.05% PBST solution (Tween concentration 0.05%). 8 μg of MPXV specific antibody was added, mixed thoroughly, and incubated at room temperature for 2 hours with shaking. Subsequently, 100 μL of 10% BSA solution was added to block the remaining carboxyl sites, and the mixture was incubated for 1 hour. Centrifuge the solution (3200 rpm, 6 min), wash with 0.05% PBST solution to remove excess BSA. Finally, resuspend the precipitate in 200 μL of 0.05% PBST for storage.

[0047] 1.3 Preparation of Immunochromatographic Test Strips

[0048] The immunochromatographic test strip mainly consists of four parts: a PVC base plate, a sample pad, an NC membrane, and an absorbent pad. First, goat anti-mouse polyclonal IgG antibody and MPXV antibody were diluted to 1.2 mg / mL and 1.2 mg / mL, respectively. Using a membrane scrubbing instrument, they were sprayed onto the NC membrane as the control line (C line) and the detection line (T line), and dried in a 37°C incubator for 2 hours. Then, the sample pad, the dried NC membrane, and the absorbent pad were sequentially assembled onto the PVC base plate, overlapping each other by 2 mm. After assembly, the strips were cut into 3 mm wide test strips, sealed, and stored in a vacuum drying oven at room temperature.

[0049] 1.4 Evaluation of the photothermal properties of MoS2@Au nanosheets

[0050] Quantitative characterization of the photothermal conversion efficiency and photothermal stability of MoS2@Au nanosheets under near-infrared (NIR) laser irradiation. First, a laser with a wavelength of 808 nm and a power density of 1.5 W / cm² was used. 2 MoS2@Au nanosheets were irradiated with a NIR laser (400 μg / mL) for 5 minutes. The resulting temperature rise (ΔT = ΔT1 - ΔT0) was then captured in real-time by an infrared thermal imager, and the value after subtracting the background was used for quantitative analysis; where ΔT1 and ΔT0 represent the temperature changes of the sample group and the blank control (water) before and after irradiation, respectively. Finally, the temperature was measured by five consecutive NIR laser irradiation cycles (808 nm, 1.5 W / cm²). 2 The photothermal durability of MoS2@Au nanosheets was systematically evaluated. Each cycle consisted of a 300-second heating phase followed by a 300-second cooling phase, and real-time temperature change curves were monitored and recorded using infrared thermal imaging.

[0051] 1.5 Detection process using MoS2@Au-M-ICA

[0052] The MPXV test strip was prepared according to the procedure described in 1.3. MPXV antigen (MPXV A29L protein) was diluted with running buffer (PBS containing 1% Tween-20 and 10% BSA) to prepare standard solutions with concentrations ranging from 0.005 to 100 ng / mL; 0 ng / mL served as a negative control. Subsequently, 70 μL of standard solution was retained for each concentration gradient, and 1 μL of MoS2@Au nanoprobe dispersion was added to obtain a mixed solution. The mixed solution was loaded onto the sample pad of the ICA test strip. After 15 minutes, the T-line spectrum was recorded using a portable 785 nm Raman spectrometer (10 mW, 10 s), and the signal average of five randomly selected points was taken. Quantitative analysis was performed using a calibration curve plotted with known antigen concentrations.

[0053] In photothermal mode, an 808nm near-infrared laser is used at 1.5 W / cm². 2 The sample was irradiated with T-rays for 10 seconds. The resulting temperature changes were captured in real time using an infrared thermal imager and quantitatively analyzed.

[0054] In addition, inactivated MPXV was diluted with running buffer to prepare concentrations ranging from 2.25 × 10⁻⁶. 7 Up to 2.25×10 2 The standard solution was prepared in 10 copies / mL format, with 0 copies / mL used as a negative control. The specific detection procedure was the same as above.

[0055] 1.6 Detection of Simulated Samples

[0056] First, pharyngeal swab samples were soaked in 0.5 mL of running buffer (PBS containing 1% Tween-20 and 10% BSA) and thoroughly mixed to obtain the eluent. 63 μL of the eluent was then added to 7 μL of MPXV antigen at different concentrations (0.1, 1, and 10 ng / mL), followed by 1 μL of MoS2@Au nanoprobe dispersion to form the spiked sample solution. The spiked sample solution was then dropped onto the sample pad. After chromatographic reaction for 15 min, the colorimetric signal was observed visually. The detection steps for SERS and photothermal modes are shown in Figure 1.5. The recovery rate of the target protein in the pharyngeal swab samples was calculated using a pre-established standard curve, and three tests were performed to improve accuracy.

[0057] 2. Results and Discussion

[0058] 2.1 Synthesis and Characterization of MoS2@Au Nanosheets

[0059] High-resolution transmission electron microscopy (HR-TEM) images of MoS2, MoS2@Au seeds, and MoS2@Au nanosheets (Figs. 2a-c). 13 nm Au NPs are densely adsorbed on the MoS2 nanosheets. Figs. 2d-e are magnified images of the MoS2@Au seeds and MoS2@Au nanosheets, respectively. It can be seen that the size of the Au NPs in Fig. 2e is significantly larger than that in Fig. 2d. Furthermore, energy-dispersive spectroscopy elemental mapping (Fig. 2f) further confirms the elemental composition of the MoS2@Au nanosheets, where green represents S, blue represents Au nanoparticles, and red represents Mo. Figure 2 As shown in g, the surface charge properties of MoS2@Au nanosheets were compared and analyzed by zeta potential measurement. After PEI adsorption, the zeta potential of MoS2 changed from -30.3 mV to +44.2 mV, indicating that the cationic PEI layer was successfully coated on the electronegative MoS2 nanosheets. After assembly with AuNPs, the zeta potential of MoS2@PEI decreased to -13.7 mV, forming MoS2@Au seeds. Au in HAuCl4·4H2O was then added to the zeta potential. 3+Upon reduction to Au, the zeta potential of the MoS2@Au seeds further decreased to -20.1 mV, forming MoS2@Au. These results indicate that AuNPs were successfully assembled on the MoS2@PEI surface via electrostatic interactions. As shown in Figure 2h, the UV-Vis spectrum provides further evidence for the successful formation of MoS2@Au nanosheets. The initial MoS2 exhibits a broad, featureless absorption spectrum that gradually decreases at longer wavelengths. In contrast, the MoS2@Au nanosheets show significantly enhanced light absorption in the visible to near-infrared region. Notably, a distinct shoulder peak appears at approximately 570 nm, corresponding to the characteristic localized surface plasmon resonance (LSPR) of the Au nanostructure. This optical property confirms the dual compatibility of this material in 785 nm SERS and 808 nm photothermal applications.

[0060] As shown in Figure 2i, EDX spectroscopy confirmed the presence of Mo, S, and Au elements on the surface of the MoS2@Au nanosheets, indicating that Au NPs were successfully bonded to the MoS2 nanosheets. The surface properties of the MoS2@Au nanosheets were characterized and analyzed using X-ray photoelectron spectroscopy (XPS). Figure 2 As shown in Figure j, in the wide-scan 22XPS spectra of MoS2@Au nanosheets, Au 4f, S 2p, Mo 3d, C 1s, Au 4d, O 1s, and Au 4p exhibit distinct peaks at 84.01, 162.20, 228.99, 285.40, 353.31, 531.96, and 547.00 eV, respectively. Furthermore, the high-resolution XPS spectra of Mo 3d (Mo 3d5 / 2 and Mo 3d3 / 2), S2p (S2p3 / 2 and S2p1 / 2), and Au 4f (Au 4f7 / 2 and Au 4f5 / 2) are completely consistent with literature values. These results indicate that the surface composition of the film-like signal tag consists only of Au, Mo, S, O, and C (C from PEI), with no other impurities introduced into the nanosheets.

[0061] 2.2 Material and photothermal properties of MoS2@Au nanosheets

[0062] DTNB-modified MoS2@Au nanosheets (MoS2@Au / DTNB) can serve as immunotags for processing complex samples. DTNB acts as a Raman reporter molecule, providing a SERS signal within the SERS tag. The concentration of DTNB modified on the nanosheets directly affects the SERS intensity. To obtain a high-intensity SERS signal, different concentrations of DTNB molecules were immobilized on the surface of MoS2@Au nanosheets. As shown in Figure 3a, the SERS signal intensity of MoS2@Au increases with increasing DTNB concentration, reaching its strongest at 80 μM DTNB modification. When the DTNB concentration exceeds 80 μM, the SERS signal intensity remains essentially unchanged, indicating that the DTNB modification concentration has reached saturation. Figure 3 As shown in b, no characteristic peak of DTNB was detected after direct modification of MoS2, indicating that pure MoS2 does not have a SERS enhancement effect. However, after modifying MoS2@Au with 80 μM DTNB, the SERS signal was significantly enhanced, approximately twice the intensity of the SERS signal of MoS2@Au seed.

[0063] Secondly, the time stability of MoS2@Au / DTNB stored in ethanol was evaluated. As shown in Figure 3c, the SERS signal intensity of MoS2@Au / DTNB was at 1331 cm⁻¹. -1 The time stability was assessed by performing seven repeated measurements on the same batch of samples within 30 days.

[0064] As mentioned earlier, the extensive absorption of MoS2@Au in the near-infrared region indicates that it can serve as a superior photothermal conversion labeling probe to improve the detection performance of the photothermal MoS2@Au-M-ICA format. Therefore, this study systematically investigated the photothermal conversion performance of MoS2@Au nanosheets. Ultrapure water was used as a control, and a power density of 1.5 W / cm² was employed. 2 MoS2, AuNPs, MoS2@Au seeds, and MoS2@Au solution were irradiated with an 808 nm laser for 5 minutes. As shown in Figure 3d, within the same irradiation time, the heating rate of the MoS2@Au solution was faster than that of other groups. This indicates that the MoS2@Au nanosheets possess excellent photothermal properties, which is also confirmed by the infrared thermal imaging results in Figure 3g. At a power density of 1.5 W / cm²... 2MoS2@Au solutions of different concentrations (100-500 µg / mL) were irradiated with laser light 808 times for 5 min. To investigate the concentration-dependent photothermal effect, nanoparticle suspensions with concentrations of 100 to 400 μg / mL were irradiated under the same laser conditions. Figure 3 e). The results showed that, within the same time period, the temperature gradually increased with increasing concentration. This temperature increase was closely related to the gradual darkening of the T line on the bar graph. A concentration of 400 µg / mL was determined to be sufficient for detection. The MoS2@Au solution (400 μg / mL) was then exposed to an 808 nm laser at different laser power densities (0.5, 1, 1.5, and 2 W / cm²). 2 The effect of laser power density on temperature was investigated. As shown in Figure 3f, at 1.5 W / cm²... 2 At this power, the solution reached its maximum temperature after 5 minutes of irradiation, indicating that 1.5 W / cm²... 2 It is the optimal power for studying photothermal conversion performance.

[0065] To evaluate photothermal stability, the MoS2@Au solution was subjected to 5 on / off cycles under 808 nm irradiation. Figure 3 As shown in Figure 3i, the maximum temperature did not decrease significantly during the five cycles, indicating that MoS2@Au possesses good photothermal stability. Furthermore, the SERS signal of MoS2@Au before and after photothermal treatment was characterized. The SERS intensity remained almost unchanged before and after 808 nm irradiation, indicating that the effect of photothermal treatment on the SERS performance of the MoS2@Au tag is negligible. In addition, the photothermal heating-cooling curve was also detected. As shown in Figure 3i, the heat transfer time constant τs of MoS2@Au is 155.21 s, and the photothermal conversion efficiency (η) is 48.3%.

[0066] 2.3 Performance Evaluation of MoS2@Au-M-ICA

[0067] Based on a series of optimized experimental parameters, including running buffer, antibody concentration on the T line, and number of tags, a rapid quantitative detection system for MPXV combining SERS and photothermal ICA was successfully constructed. The specific optimization process is as follows: Figure 4As shown in Figure 4a (Ⅰ), when the sample contains a high concentration of antigen, a distinct black band can be observed on the T line. As the antigen concentration decreases, the color of the T line gradually fades. From the test strip image, it can be seen that the visual detection sensitivity is 0.5 ng / mL. The average SERS spectrum on the T line corresponding to different concentrations of MPXV is shown in Figure 4b. As the antigen concentration decreases, the corresponding SERS signal intensity gradually weakens. When the antigen concentration is as low as 0.005 ng / mL, the SERS intensity is still significantly different from the blank control. The calibration curve shown in Figure 5d shows that R 2 The method exhibits a strong correlation coefficient of 0.98 and a wide dynamic detection range spanning multiple orders of magnitude. Furthermore, the LOD for MPXV antigen in SERS mode is 0.0042 ng / mL, calculated as the mean SERS intensity at 1331 cm⁻¹ (3 times the standard deviation of the blank group), which is 238 times higher than that of AuNP-based LFA. Figure 4 f). The photothermal detection results are shown in Figure 4a (II). As the MPXV antigen concentration increases, the temperature on the T line gradually rises, and the image gradually changes from light green to red. The test strip image shows that the visual detection sensitivity is 0.1 ng / mL. The average temperature on the T line corresponding to different concentrations of MPXV is shown (each test strip was tested 5 times, and the average value was calculated). Figure 4 As shown in Figure c, the temperature gradually decreases as the antigen concentration decreases. The calibration curve in Figure 5e, obtained by plotting the relationship between temperature and antigen concentration, shows a strong linear correlation (Rc). 2 = 0.99). The LOD of this method for MPXV antigen in photothermal mode was calculated to be 0.003 ng / mL.

[0068] To verify the specificity of this method, this study used varicella-zoster virus (VZV), vaccinia virus (VACV), measles virus (MV), and herpes simplex virus (HSV) at concentrations of 100 ng / mL as interfering antigens. Visual and thermal imaging were used for detection. Figure 4 As shown in g, when the sample contains MPXV antigen (1 ng / mL), a black band can be seen on the corresponding T line at 1331 cm⁻¹. -1 A strong SERS signal intensity was observed at this location. Figure 4h). Other interfering viruses showed lower SERS signal intensities on their respective T lines, consistent with the negative control. Figure 4i shows the different temperatures corresponding to the T lines measured by an infrared camera in photothermal mode. It can be clearly seen that the T line temperature of the sample containing MPXV antigen was higher than that of the other control groups.

[0069] These results demonstrate that the proposed MoS2@Au SERS-ICA exhibits high specificity. Next, the reproducibility of MoS2@Au-M-ICA was assessed by detecting high / medium / low concentrations (100, 10, and 1 ng / mL) of MPXV antigen. The experiment was repeated five times, and the results are shown in Figure 4j. Black bands were visible on the T-line for all three groups of MPXV antigen. Within each group, the SERS signal intensity and photothermal temperature were almost identical for every five test strips. Furthermore, from... Figure 4 As can be seen from k, the relative standard deviations of the SERS signals on the three T lines are all less than 9.62%, further demonstrating the high stability and repeatability of our method.

[0070] Detection of inactivated MPXV (2.25×10⁻⁶) 7 The feasibility of the MoS2@Au-M-ICA platform was more effectively and intuitively verified by plotting different concentrations of inactivated MPXV (2.25 × 10⁻⁶ copies / mL). As shown in Figure 5a, the T line gradually faded in color and the temperature decreased, changing from red to yellow. For SERS quantification mode, different concentrations of inactivated MPXV (2.25 × 10⁻⁶ copies / mL) were plotted. 7 -2.25×10 2 copies / mL) and the corresponding 1331 cm -1 The SERS intensity at the location (Figure 5b) decreases as the viral concentration decreases. As shown in Figure 5c, a high correlation coefficient (R0) is obtained. 2 The calibration curve ( = 0.961) shows that the LOD of MPXV inactivation in SERS mode is 225 copies / mL. As shown in Figure 5d, in photothermal quantitative mode, the temperature corresponding to different concentrations of inactivated MPXV was recorded using an infrared camera, resulting in Figure 5e with a high correlation coefficient (R). 2 The calibration curve (=0.998) showed that the LOD of MPXV inactivation in photothermal mode was 210 copies / mL. These results indicate that MoS2@Au-M-ICA has multiple detection modes, high sensitivity, and high accuracy, making it suitable for point-of-care testing (POCT) of viruses in clinical samples.

[0071] 2.4 Application in pharyngeal swab samples

[0072] The actual clinical application validation of ICA was as follows: different concentrations (10, 1, and 0.1 ng / mL) of MPXV were added to throat swab samples to simulate clinical samples. Figure 5 As shown in Figure a, the color of the T-line gradually fades as the protein concentration decreases. Table 1 lists the recoveries calculated from the calibration curves. The recoveries for Raman detection ranged from 92.45% to 96.48%, with coefficients of variation (CVs) remaining below 8%. Furthermore, photothermal readings exhibited greater stability, with recoveries between 92.36% and 102.42%, demonstrating higher accuracy and repeatability (CVs < 5%). These results collectively validate the reliable quantitative performance of this method in sample matrices. Both Raman and photothermal modes demonstrated good recoveries and reproducibility within a concentration range of 0.1–10 ng / mL.

[0073] Table 1. MPXV recovery rate in throat swab samples

[0074]

[0075] In summary, this invention successfully synthesized MoS2@Au nanosheets with visualization capabilities, surface-enhanced Raman scattering (SERS), and photothermal properties via a PEI-mediated seed growth method. Based on MoS2@Au nanosheets, we constructed a multi-mode lateral flow immunochromatographic test strip integrating visualization detection, SERS detection, and photothermal signal readout, enabling ultrasensitive detection of MPXV and inactivated MPXV within 15–20 minutes. AuNPs coated on the MoS2 surface enhance Raman scattering and near-infrared absorption, supporting sensitive signal acquisition in both Raman and photothermal modes. Importantly, the MoS2@Au nanosheets achieved a high photothermal conversion efficiency of 48.3% under 808 nm laser irradiation, demonstrating strong photothermal transduction capabilities. The detection limits for MPXV using this method were 4.2 pg / mL in Raman mode and 3 pg / mL in photothermal mode, representing improvements of approximately 238-fold and 333-fold, respectively, compared to commercial gold nanoparticle-based detection methods. The limit of detection (LOD) for inactivated MPXV was 225 copies / mL in SERS mode and 210 copies / mL in photothermal mode. Furthermore, the specificity, reproducibility, and detection of real respiratory viruses in pharyngeal swab samples were validated. MoS2@Au-M-ICA offers a wider range of applications through the complementarity of its three output signals and allows for flexible switching of detection modes according to scenario requirements. Therefore, this method shows significant potential as a diagnostic platform for rapid and accurate detection of pathogenic microorganisms in field conditions.

[0076] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of the claims of this patent application.

Claims

1. A method for the establishment of an immunochromatographic detection of monkeypox virus based on MoS2@Au nanosheets, characterized by, Comprising the following steps: Step one, preparation of nanoprobes: adsorbing Raman reporter molecules and monkeypox virus specific antibodies on the surface of MoS2@Au nanosheets in turn to prepare nanoprobes, and dispersing the nanoprobes in PBST to obtain a nanoprobe dispersion; Step two, preparation of an immunochromatographic test strip; Step three, immunochromatographic detection: mixing the sample to be tested with the nanoprobe dispersion prepared in step one to obtain a test solution, applying the test solution to the immunochromatographic test strip, and performing chromatographic reaction; Step four, multi-modal signal detection: after the chromatographic reaction is completed, the detection line on the immunochromatographic test strip is subjected to signal acquisition, and the signal reading mode adopted includes at least one of visual colorimetry, surface-enhanced Raman scattering and photothermal detection.

2. The method for establishing an immunochromatographic detection of monkeypox virus based on MoS2@Au nanosheets according to claim 1, characterized in that, The preparation method of MoS2@Au nanosheets is to assemble Au NPs on the surface of MoS2 nanosheets through a polyethyleneimine-mediated seed growth strategy to form MoS2@Au nanosheets; Comprising the following steps: S1, mixing single-layer MoS2 dispersion liquid with ultrapure water at a volume ratio of (2-4):(4-6), after ultrasonic treatment and centrifugation, removing the supernatant, collecting the MoS2 nanosheet precipitate at the bottom, and dispersing the MoS2 nanosheet precipitate in ultrapure water to obtain a MoS2 nanosheet dispersion liquid; S2, mixing the MoS2 nanosheet dispersion liquid with a polyethyleneimine aqueous solution at a volume ratio of (8-10):1, after ultrasonic treatment, washing, and centrifugation, collecting MoS2@PEI nanosheet precipitate, and dispersing the MoS2@PEI nanosheet precipitate in ultrapure water to obtain a MoS2@PEI nanosheet dispersion liquid; S3, mixing the obtained MoS2@PEI nanosheet dispersion liquid with Au NPs solution at a volume ratio of 1:(4-6), after ultrasonic treatment, centrifugation, and washing, collecting MoS2@Au seed precipitate, and dispersing the MoS2@Au seed precipitate in anhydrous ethanol to obtain a MoS2@Au seed dispersion liquid; S4, adding MoS2@Au seed dispersion liquid, hydroxylamine hydrochloride, and polyvinylpyrrolidone into water in sequence, after ultrasonic treatment, adding chloroauric acid solution, again after ultrasonic treatment, centrifugation, and washing, collecting MoS2@Au nanosheet precipitate, and dispersing the MoS2@Au nanosheet precipitate in anhydrous ethanol to obtain a MoS2@Au nanosheet dispersion liquid for standby use.

3. The method for establishing an immunochromatographic detection of monkeypox virus based on MoS2@Au nanosheets according to claim 1, characterized in that, The Raman reporter molecule is DTNB, which is adsorbed on the surface of MoS2@Au nanosheets to form MoS2@Au / DTNB nanosheets, and the monkeypox virus specific antibody is connected to the MoS2@Au / DTNB nanosheets through carboxyl-amino coupling reaction to form nanoprobes.

4. The method for establishing an immunochromatographic detection of monkeypox virus based on MoS2@Au nanosheets according to claim 3, characterized in that, The synthesis method of MoS2@Au / DTNB nanosheets is to add DTNB ethanol solution into MoS2@Au nanosheets dispersed in ethanol, after ultrasonic treatment and washing, collecting MoS2@Au / DTNB nanosheet precipitate, and storing the MoS2@Au / DTNB nanosheet precipitate in ethanol for standby use.

5. The method for establishing an immunochromatographic detection of monkeypox virus based on MoS2@Au nanosheets according to claim 4, characterized in that, The concentration of DTNB in the ethanol solution is 40-120 μM.

6. The method for establishing an immunochromatographic detection of monkeypox virus based on MoS2@Au nanosheets according to claim 5, characterized in that, The concentration of DTNB is 80 μM.

7. The method for establishing an immunochromatographic detection of monkeypox virus based on MoS2@Au nanosheets according to claim 3, characterized in that, The specific method for connecting the monkeypox virus specific antibody to MoS2@Au / DTNB is as follows: the MoS2@Au / DTNB nanosheet is washed with ultrapure water to obtain a suspension, MES buffer is added to the suspension, and then EDC solution and NHS solution are sequentially added and uniformly mixed, the supernatant is discarded after ultrasonic treatment, the precipitate is resuspended in PBST solution, the monkeypox virus specific antibody is added and mixed thoroughly, and the mixture is cultured by shaking at room temperature, BSA solution is added for incubation, and finally the precipitate is obtained by centrifugal treatment and washing with PBST solution, and the nanoprobes are resuspended in PBST for storage and standby use.

8. The method for establishing an immunochromatographic detection of monkeypox virus based on MoS2@Au nanosheets according to claim 1, characterized in that, In step two, the immunochromatography test strip includes a PVC bottom plate, a sample pad, an NC membrane, and a water absorption pad, the NC membrane is provided with a detection line and a quality control line, the detection line is coated with an antibody for capturing monkeypox virus antigens, and the quality control line is coated with a goat anti-mouse polyclonal IgG antibody.

9. The method for establishing an immunochromatographic detection of monkeypox virus based on MoS2@Au nanosheets according to claim 1, characterized in that, In step three, the sample to be tested is an eluate obtained by soaking a swab-collected biological sample in a running buffer, the running buffer is a phosphate buffer containing 1% tween-20 and 10% BSA, and the amount of nanoprobes used is 1 uL.

10. The method for establishing an immunochromatographic detection of monkeypox virus based on MoS2@Au nanosheets according to claim 1, characterized in that, In step four, visual colorimetric detection is performed by observing whether there is a black band on the detection line with the naked eye. Surface-enhanced Raman scattering detection is to use a Raman spectrometer with a wavelength of 785 nm to irradiate the detection line, collect and analyze the characteristic Raman peak intensity of DTNB at 1331 cm -1 , and quantitative analysis is carried out according to the pre-established Raman intensity and antigen concentration calibration curve; Photothermal detection is to use near-infrared laser with wavelength of 808 nm and power density of 1.5 W / cm 2 to irradiate the detection line, capture and record the temperature rise value ΔT in real time through the infrared thermal imager, and conduct quantitative analysis according to the pre-established temperature change and antigen concentration calibration curve.

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