A three-dimensional molybdenum disulfide sheet-shaped composite nanomaterial, a preparation method thereof and application thereof in immunochromatography

Abstract

The application provides a three-dimensional molybdenum disulfide sheet-shaped composite nanomaterial, a preparation method thereof and application thereof in immunochromatography, and belongs to the technical field of nanomaterial biological detection. The MoS2 nanosheet is used as an inner layer substrate, two layers of gold nanoparticles are loaded by using layer-by-layer self-assembly of polyethylene imine, and controllable SERS hot spots are provided. From the structure, the MoS2 nanosheet has a large surface area and can provide more reaction sites, so that the colorimetric capacity and SERS activity are improved. The two layers of dense AuNPs enhance the colorimetric capacity and provide a large number of SERS hot spots. The material has good stability, dispersity, excellent SERS performance and colorimetric capacity, is obviously superior to traditional SERS labels, can realize stable and sensitive detection of target analytes in complex actual samples, has great application potential, can replace traditional immunochromatographic detection methods, and meets detection requirements in different situations.

Description

Technical Field

[0001] This invention relates to the field of nanomaterial biodetection technology, and in particular to a three-dimensional molybdenum disulfide sheet-like composite nanomaterial, its preparation method, and its application in immunochromatography. Background Technology

[0002] Immunochromatographic assay (ICA), with its advantages of speed, simplicity, structural simplicity, and low cost, has become one of the most promising rapid on-site detection methods, widely used in biomarker diagnosis, infectious disease pathogen screening, food safety, and environmental monitoring. Traditional ICA uses colorimetric tags (such as gold nanoparticles and latex microspheres) as reporter molecules, performing qualitative or semi-quantitative analysis based on color changes on the test strip's detection line. However, it generally suffers from low sensitivity, poor stability, and inaccurate quantification. Therefore, there is an urgent need to develop novel nanosignal tags to improve the performance of immunochromatography. Summary of the Invention

[0003] In view of this, the purpose of this invention is to provide a three-dimensional molybdenum disulfide sheet-like composite nanomaterial, its preparation method, and its application in immunochromatography. The three-dimensional molybdenum disulfide sheet-like composite nanomaterial provided by this invention has extremely strong colorimetric-SERS dual signal and stable detection capability.

[0004] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0005] This invention provides a three-dimensional molybdenum disulfide sheet-like composite nanomaterial, comprising a two-dimensional MoS2 inner layer and two layers of gold nanoparticle shells encapsulating the surface of the two-dimensional MoS2 inner layer. The single-layer gold nanoparticle shell includes a polyethyleneimine layer and gold nanoparticles distributed on the surface of the polyethyleneimine layer.

[0006] Preferably, the sheet diameter of the two-dimensional MoS2 inner layer is 400–800 nm;

[0007] The thickness of the single-layer polyethyleneimine layer is 1–10 nm;

[0008] The gold nanoparticles have a particle size of 20–40 nm.

[0009] This invention provides a method for preparing the above-mentioned three-dimensional molybdenum disulfide sheet-like composite nanomaterial, comprising the following steps:

[0010] (1) Provide a two-dimensional nanosheet dispersion of MoS2;

[0011] (2) The MoS2 two-dimensional nanosheet dispersion was ultrasonically mixed with the first polyethyleneimine aqueous solution to obtain MoS2@PEI nanosheets;

[0012] (3) The MoS2@PEI nanosheets were ultrasonically mixed with the first colloidal gold solution to obtain MoS2@Au nanosheets;

[0013] (4) The MoS2@Au sheet-like composite nanoparticles were ultrasonically mixed with a second polyethyleneimine aqueous solution to obtain MoS2@Au@PEI nanosheets;

[0014] (5) The MoS2@Au@PEI nanosheets were ultrasonically mixed with the second colloidal gold solution to obtain a three-dimensional molybdenum disulfide sheet-like composite nanomaterial.

[0015] Preferably, the concentrations of the first polyethyleneimine aqueous solution and the second polyimide aqueous solution are independently 0.1–5 mg / mL;

[0016] The mass ratio of the MoS2 two-dimensional nanosheets to the first polyethyleneimine and the second polyethyleneimine is 100:1 to 10:1 to 10.

[0017] The mass ratio of the MoS2 two-dimensional nanosheets to the first colloidal gold and the second colloidal gold is 1:10-30:10-30.

[0018] This invention provides the application of the above-mentioned three-dimensional molybdenum disulfide sheet-like composite nanomaterials in immunochromatographic analysis for non-diagnostic purposes.

[0019] This invention provides a three-dimensional molybdenum disulfide sheet-like composite nanomaterial SERS immune tag, comprising the above-mentioned three-dimensional molybdenum disulfide sheet-like composite nanomaterial, a Raman molecule chemically coupled to the three-dimensional molybdenum disulfide sheet-like composite nanomaterial, and a viral antibody chemically coupled to the Raman reporter molecule;

[0020] The Raman reporter molecule is 5,5′-dithiobis(dinitrobenzic acid).

[0021] Preferably, the viral antibody is a monkeypox virus antibody.

[0022] This invention provides a method for preparing the above-mentioned three-dimensional molybdenum disulfide sheet-like composite nanomaterial SERS immune tag, comprising the following steps:

[0023] The three-dimensional molybdenum disulfide sheet-like composite nanomaterial was ultrasonically mixed with Raman reporter molecules to obtain MoS2@Au-Au-DTNB composite nanosheets;

[0024] The MoS2@Au-Au-DTNB composite nanosheets were mixed with a carboxyl activator to activate the carboxyl groups, thereby obtaining carboxyl-activated MoS2@Au-Au-DTNB composite nanosheets.

[0025] The activated carboxyl group MoS2@Au-Au-DTNB composite nanosheets were mixed with viral antibodies and incubated to block unbound sites, resulting in a three-dimensional molybdenum disulfide sheet-like composite nanomaterial SERS immune tag.

[0026] This invention provides a monkeypox virus detection test strip, comprising a nitrocellulose membrane, a conjugate pad, a sample pad, an absorbent pad, and a base plate. The nitrocellulose membrane is characterized by having a T-line and a C-line, the surface of the T-line being coated with a monkeypox virus capture antibody, and the surface of the C-line being coated with a goat anti-mouse antibody.

[0027] The surface of the conjugate pad is coated with the above-mentioned three-dimensional molybdenum disulfide sheet-like composite nanomaterial SERS immune tag, and the viral antibody in the three-dimensional molybdenum disulfide sheet-like composite nanomaterial SERS immune tag is monkeypox virus antibody.

[0028] This invention provides a non-diagnostic method for detecting monkeypox virus using a colorimetric-SERS dual-signal mode, comprising the following steps:

[0029] The sample to be tested is mixed with a buffer solution to obtain the test solution;

[0030] The test solution is added to the sample pad of the monkeypox virus test strip. After standing, the color of the T line is observed. The color intensity of the T line is compared with the colorimetric card to obtain the content of monkeypox virus in the test sample. The colorimetric card is a colorimetric image of the T line of the test strip for different concentrations of monkeypox virus protein.

[0031] Alternatively, the solution to be tested is added to the sample pad of the test strip, and after standing, the Raman signal intensity of the T line is measured. The content of monkeypox virus in the sample to be tested is obtained according to the Raman signal intensity and a predetermined standard curve. The standard curve is a linear relationship curve between the monkeypox virus content and the Raman signal intensity.

[0032] This invention provides a three-dimensional molybdenum disulfide (MoS2@Au-Au) sheet-like composite nanomaterial, comprising a two-dimensional MoS2 inner layer and two layers of gold nanoparticle shells encapsulating the surface of the two-dimensional MoS2 inner layer. Each gold nanoparticle shell comprises a polyethyleneimine layer and gold nanoparticles distributed on the surface of the polyethyleneimine layer. Molybdenum disulfide (MoS2) nanosheets are typical two-dimensional film-like transition metal chalcogenides, possessing unique physical and chemical properties, including strong catalytic activity, excellent colorimetric ability, large specific surface area, high band gap tunability, and excellent dispersibility. Importantly, the molybdenum disulfide nanostructure exhibits a unique surface-enhanced Raman scattering (SERS) chemical enhancement effect, which can significantly improve the SERS activity of the noble metal on its surface. This invention uses MoS2 nanosheets as the inner substrate and utilizes the layer-by-layer self-assembly of polyethyleneimine (PEI) to load two layers of gold nanoparticles (AuNPs), providing controllable SERS hotspots. Structurally, MoS2 nanosheets have a large surface area, providing more reaction sites, thereby improving colorimetric ability and SERS activity. The two dense layers of AuNPs enhance colorimetric ability while providing a large number of SERS hotspots. This material exhibits good stability, dispersibility, and excellent SERS performance and colorimetric ability, significantly superior to traditional SERS tags. It can achieve stable and sensitive detection of target analytes in complex real-world samples, possessing great application potential and can replace traditional immunochromatographic detection methods to meet detection needs in various situations.

[0033] This invention provides a method for preparing the above-mentioned three-dimensional molybdenum disulfide sheet-like composite nanomaterials. This invention utilizes the PEI layer-by-layer self-assembly method to prepare three-dimensional molybdenum disulfide sheet-like composite nanomaterials. The preparation method is simple and can achieve mass production.

[0034] This invention provides the application of the aforementioned three-dimensional molybdenum disulfide sheet-like composite nanomaterials in non-diagnostic immunochromatographic analysis. The three-dimensional molybdenum disulfide sheet-like composite nanomaterials provided by this invention have broad application prospects, including biomarker diagnosis, infectious disease prevention, food safety, and environmental monitoring. By modifying the surface of this composite nanomaterial with different detection antibodies, it can serve as a high-performance SERS immunotag for the rapid detection of various targets.

[0035] This invention provides a three-dimensional molybdenum disulfide sheet-like composite nanomaterial SERS immunotag, comprising the aforementioned three-dimensional molybdenum disulfide sheet-like composite nanomaterial, a Raman molecule chemically coupled to the three-dimensional molybdenum disulfide sheet-like composite nanomaterial, and a viral antibody chemically coupled to the Raman reporter molecule; the Raman reporter molecule is 5,5′-dithiobis(dinitrobenzoic acid) (DTNB). This invention modifies the surface of the three-dimensional molybdenum disulfide sheet-like composite nanomaterial with a carboxyl-containing Raman reporter molecule. The surface carboxyl groups, after activation, can be used for antibody conjugation, allowing for very simple surface functionalization of the nanomaterial, thereby achieving colorimetric-SERS dual-signal mode detection of the virus. This tag combines the chemical enhancement of MoS2 and the electromagnetic enhancement generated by the bilayer dense AuNPs, exhibiting extremely strong SERS performance and excellent colorimetric ability.

[0036] This invention provides a non-diagnostic method for detecting monkeypox virus using a colorimetric-SERS dual-signal mode. This method can directly output a colorimetric signal similar to colloidal gold immunochromatography, and also provide a highly sensitive SERS signal, achieving stable and sensitive detection of target analytes in complex real-world samples. Example results show that the colorimetric sensitivity for detecting monkeypox virus antigen is 0.2 ng / mL; the sensitivity (LOD) of SERS immunochromatography is 0.002 ng / mL. Attached Figure Description

[0037] Figure 1 The preparation process of three-dimensional molybdenum disulfide sheet-like composite nanomaterials;

[0038] Figure 2 The preparation process of SERS immune tags for three-dimensional molybdenum disulfide sheet-like composite nanomaterials;

[0039] Figure 3 Schematic diagram of monkeypox virus colorimetric-SERS dual-signal mode detection;

[0040] Figure 4 Transmission electron microscope images of each component during the preparation of three-dimensional molybdenum disulfide sheet-like composite nanomaterials;

[0041] Figure 5 The results show the stability characterization of the three-dimensional molybdenum disulfide sheet-like composite nanomaterial.

[0042] Figure 6 The results of the immunochromatographic assay for detecting monkeypox virus based on a three-dimensional molybdenum disulfide sheet-like composite nanomaterial are shown in the figure, and the comparison with other immunoassay methods are also presented.

[0043] Figure 7 The results of testing actual samples using three-dimensional molybdenum disulfide sheet-like composite nanomaterials;

[0044] Figure 8 Figure 1 shows the detailed colorimetric analysis results of an immunochromatographic system for detecting monkeypox virus based on three-dimensional molybdenum disulfide sheet-like composite nanomaterials. Detailed Implementation

[0045] This invention provides a three-dimensional molybdenum disulfide sheet-like composite nanomaterial, comprising a two-dimensional MoS2 inner layer and two layers of gold nanoparticle shells encapsulating the surface of the two-dimensional MoS2 inner layer. The single-layer gold nanoparticle shell includes a polyethyleneimine layer and gold nanoparticles distributed on the surface of the polyethyleneimine layer.

[0046] In this invention, the sheet diameter of the two-dimensional MoS2 inner layer is preferably 400-800 nm, more preferably 500-700 nm; the thickness of the two-dimensional MoS2 inner layer is preferably 1-5 nm, more preferably 2-4 nm.

[0047] In this invention, the molecular weight of the polyethyleneimine is preferably 5,000 to 80,000, more preferably 10,000 to 60,000; the thickness of the monolayer polyethyleneimine layer is preferably 1 to 10 nm, more preferably 1 to 5 nm.

[0048] In this invention, the particle size of the gold nanoparticles is preferably 20–40 nm, more preferably 30 nm. In this invention, the mass ratio of the two-dimensional MoS2 to the total mass of the gold nanoparticles is preferably 1:20–50, more preferably 1:30.

[0049] This invention provides a method for preparing the above-mentioned three-dimensional molybdenum disulfide sheet-like composite nanomaterial, comprising the following steps:

[0050] (1) Provide a two-dimensional nanosheet dispersion of MoS2;

[0051] (2) The MoS2 two-dimensional nanosheet dispersion was ultrasonically mixed with the first polyethyleneimine aqueous solution to obtain MoS2@PEI nanosheets;

[0052] (3) The MoS2@PEI nanosheets were ultrasonically mixed with the first colloidal gold solution to obtain MoS2@Au nanosheets;

[0053] (4) The MoS2@Au sheet-like composite nanoparticles were ultrasonically mixed with a second polyethyleneimine aqueous solution to obtain MoS2@Au@PEI nanosheets;

[0054] (5) The MoS2@Au@PEI nanosheets were ultrasonically mixed with the second colloidal gold solution to obtain a three-dimensional molybdenum disulfide sheet-like composite nanomaterial.

[0055] The present invention first provides a MoS2 two-dimensional nanosheet dispersion. In the present invention, the concentration of MoS2 two-dimensional nanosheets in the MoS2 two-dimensional nanosheet dispersion is preferably 0.25-2 mg / mL, more preferably 1-1.5 mg / mL.

[0056] In this invention, the method for preparing the MoS2 two-dimensional nanosheet dispersion preferably includes the following steps:

[0057] Monolayer MoS2 two-dimensional nanosheets were ultrasonically mixed with water, and nanosheets with a diameter of <300 nm in the supernatant were discarded. The remaining precipitate was then resuspended in water.

[0058] In this invention, the water is preferably deionized water. In this invention, the ultrasonic mixing power is preferably 500W, and the time is preferably 10 minutes.

[0059] In this invention, the MoS2 two-dimensional nanosheet dispersion is ultrasonically mixed with a first polyethyleneimine aqueous solution to obtain MoS2@PEI nanosheets. In this invention, the concentration of the first polyethyleneimine aqueous solution is preferably 0.1–5 mg / mL, more preferably 1 mg / mL; the mass ratio of the MoS2 two-dimensional nanosheets to the first polyethyleneimine is preferably 100:1–10, more preferably 100:5–10.

[0060] In this invention, the ultrasonic mixing power is preferably 500W, and the time is preferably 10-60 min, more preferably 40 min. During the ultrasonic mixing process, PEI self-assembles on the MoS2 surface to form positively charged MoS2@PEI nanosheets.

[0061] In this invention, after ultrasonic mixing, the resulting ultrasonic mixture is preferably centrifuged and washed to remove excess polyethyleneimine, and the residue is resuspended in water. Preferably, the centrifugation and washing are performed twice.

[0062] After obtaining the MoS2@PEI nanosheets, the present invention ultrasonically mixes the MoS2@PEI nanosheets with a first colloidal gold solution to obtain MoS2@Au nanosheets. In this invention, the concentration of the first colloidal gold solution is preferably 20 nM; the mass ratio of the MoS2 two-dimensional nanosheets to the first colloidal gold is preferably 1:20–50, more preferably 1:30. In this invention, the particle size of the first colloidal gold is preferably 20–40 nm, more preferably 30 nm.

[0063] In this invention, the ultrasonic mixing power is preferably 500W, and the time is preferably 10-60 min, more preferably 30 min. In this invention, the colloidal gold solution carries a negative charge, and during the ultrasonic mixing process, a dense layer of AuNPs is adsorbed onto the surface of the MoS2@PEI nanosheets.

[0064] In this invention, after ultrasonic mixing, the resulting ultrasonic mixture is preferably centrifuged and washed to remove excess gold nanoparticles (AuNPs), and the remaining material is resuspended in ethanol for later use. Preferably, the washing after centrifugation is performed once.

[0065] After obtaining the MoS2@Au nanosheets, the present invention ultrasonically mixes the MoS2@Au sheet-like composite nanoparticles with a second polyethyleneimine aqueous solution to obtain MoS2@Au@PEI nanosheets. In the present invention, the concentration of the second polyethyleneimine aqueous solution is preferably 0.1-5 mg / mL, more preferably 0.1 mg / mL; the mass ratio of the MoS2 two-dimensional nanosheets to the second polyethyleneimine is preferably 100:1-10, more preferably 100:5-10.

[0066] In this invention, the power of the ultrasonic mixing is preferably 500W, and the time is preferably 10-60 min, more preferably 15 min.

[0067] In this invention, after ultrasonic mixing, the resulting ultrasonic mixture is preferably centrifuged and washed to remove excess polyethyleneimine, and the residue is resuspended in water. Preferably, the centrifugation and washing are performed twice.

[0068] After obtaining the MoS2@Au@PEI nanosheets, the present invention ultrasonically mixes the MoS2@Au@PEI nanosheets with a second colloidal gold solution to obtain a three-dimensional molybdenum disulfide sheet-like composite nanomaterial. In this invention, the concentration of the second colloidal gold solution is preferably 20 nM; the mass ratio of the MoS2 two-dimensional nanosheets to the second colloidal gold is preferably 1:20–50, more preferably 1:30. In this invention, the particle size of the second colloidal gold is preferably 20–40 nm, more preferably 30 nm.

[0069] In this invention, the power of the ultrasonic mixing is preferably 500W, and the time is preferably 10 to 60 minutes, more preferably 30 minutes.

[0070] In this invention, after ultrasonic mixing, the resulting ultrasonic mixture is preferably centrifuged and washed to remove excess gold nanoparticles (AuNPs), and the remaining material is resuspended in ethanol for later use. Preferably, the washing after centrifugation is performed once.

[0071] This invention provides the application of the aforementioned three-dimensional molybdenum disulfide sheet-like composite nanomaterials in non-diagnostic immunochromatographic analysis. The three-dimensional molybdenum disulfide sheet-like composite nanomaterials provided by this invention have broad application prospects, including biomarker diagnosis, infectious disease prevention, food safety, and environmental monitoring. By modifying the surface of this composite nanomaterial with different detection antibodies, it can serve as a high-performance SERS immunotag for the rapid detection of various targets.

[0072] This invention provides a three-dimensional molybdenum disulfide sheet-like composite nanomaterial SERS immune tag, comprising the aforementioned three-dimensional molybdenum disulfide sheet-like composite nanomaterial, a Raman molecule chemically coupled to the three-dimensional molybdenum disulfide sheet-like composite nanomaterial, and a viral antibody chemically coupled to the Raman reporter molecule; wherein the Raman reporter molecule is 5,5′-dithiobis(dinitrobenzoic acid).

[0073] In this invention, the 5,5′-dithiobis(dinitrobenic acid) and the three-dimensional molybdenum disulfide sheet-like composite nanomaterial are chemically coupled via Au-S bonds, and the amino group of the viral antibody is chemically coupled to the carboxyl group of 5,5′-dithiobis(dinitrobenic acid).

[0074] In this invention, the viral antibody is preferably a monkeypox virus antibody. Specifically, the anti-monkeypox virus antibody is a commercially available antibody, specifically a monoclonal anti-MPXV A29 protein antibody, catalog number 40891-M001740891-M0027, purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd.

[0075] This invention provides a method for preparing the above-mentioned three-dimensional molybdenum disulfide sheet-like composite nanomaterial SERS immune tag, comprising the following steps:

[0076] The three-dimensional molybdenum disulfide sheet-like composite nanomaterial was ultrasonically mixed with Raman reporter molecules to obtain MoS2@Au-Au-DTNB composite nanosheets;

[0077] The MoS2@Au-Au-DTNB composite nanosheets were mixed with a carboxyl activator to activate the carboxyl groups, thereby obtaining carboxyl-activated MoS2@Au-Au-DTNB composite nanosheets.

[0078] The activated carboxyl group MoS2@Au-Au-DTNB composite nanosheets were mixed with viral antibodies and incubated to block unbound sites, resulting in a three-dimensional molybdenum disulfide sheet-like composite nanomaterial SERS immune tag.

[0079] This invention involves ultrasonically mixing the aforementioned three-dimensional molybdenum disulfide sheet-like composite nanomaterial with a Raman reporter molecule to obtain MoS2@Au-Au-DTNB composite nanosheets. In this invention, the Raman reporter molecule is preferably provided in solution form, and the concentration of the Raman reporter molecule solution is preferably 10–100 μM, more preferably 10 μM. In this invention, the ultrasonic mixing power is preferably 30–90 min, more preferably 60 min.

[0080] In this invention, after ultrasonic mixing, the resulting ultrasonic mixture is preferably washed to remove excess Raman reporter molecules; the washing is preferably ethanol washing. The resulting MoS2@Au-Au-DTNB composite nanosheets are preferably dispersed in a 10 mM 2-(N-morpholine)ethanesulfonic acid solution with a pH of 5.5.

[0081] After obtaining the MoS2@Au-Au-DTNB composite nanosheets, the present invention mixes the MoS2@Au-Au-DTNB composite nanosheets with a carboxyl activator to activate the carboxyl groups, thereby obtaining carboxyl-activated MoS2@Au-Au-DTNB composite nanosheets. In the present invention, the carboxyl activator is preferably a carbodiimide solution and an N-hydroxysuccinimide solution, the concentration of the carbodiimide solution is preferably 0.1M, the concentration of the N-hydroxysuccinimide solution is preferably 0.1M, and the volume ratio of the carbodiimide solution to the N-hydroxysuccinimide solution is preferably 1:2.

[0082] In this invention, the carboxyl activation is preferably performed under ultrasonic conditions, and the carboxyl activation time is preferably 15 min.

[0083] After obtaining MoS2@Au-Au-DTNB composite nanosheets with activated carboxyl groups, this invention mixes the MoS2@Au-Au-DTNB composite nanosheets with viral antibodies and incubates them to block unbound sites, thereby obtaining a three-dimensional molybdenum disulfide sheet-like composite nanomaterial SERS immune tag. In this invention, the concentration of the viral antibody solution is preferably 5–20 μg / mL, more preferably 8 μg / mL.

[0084] In this invention, the incubation is preferably carried out under shaking conditions, the incubation temperature is preferably 30°C, and the incubation time is preferably 0.5–3 hours, more preferably 2 hours. This invention preferably uses BSA to seal unbound sites.

[0085] After incubation, the present invention preferably collects the three-dimensional molybdenum disulfide sheet-like composite nanomaterial SERS immune tags by centrifugation and stores them in a buffer solution.

[0086] This invention provides a monkeypox virus detection strip, comprising a nitrocellulose membrane, a conjugate pad, a sample pad, an absorbent pad, and a base plate. The nitrocellulose membrane has T-lines and C-lines; the surface of the T-lines is coated with monkeypox virus capture antibodies, and the surface of the C-lines is coated with goat anti-mouse antibodies. The surface of the conjugate pad is coated with the aforementioned three-dimensional molybdenum disulfide sheet-like composite nanomaterial SERS immunotag, wherein the viral antibody in the three-dimensional molybdenum disulfide sheet-like composite nanomaterial SERS immunotag is a monkeypox virus antibody.

[0087] In this invention, the method for preparing the monkeypox virus test strip preferably includes the following steps:

[0088] Monkeypox virus capture antibody and goat anti-mouse antibody were sprayed onto nitrocellulose membrane to form T lines and C lines, respectively, to obtain nitrocellulose membrane coated with antibodies.

[0089] A three-dimensional molybdenum disulfide sheet-like composite nanomaterial SERS immunotag was coated onto the conjugate pad to obtain a conjugate pad coated with an immunotag.

[0090] The antibody-coated nitrocellulose membrane, the immunolabeled conjugate pad, the sample pad, the absorbent pad, and the base plate are assembled to obtain a monkeypox virus test strip.

[0091] In this invention, the concentration of the goat anti-mouse antibody is preferably 0.8 mg / mL; the concentration of the monkeypox virus capture antibody is 0.5–1.1 mg / mL, preferably 0.9 mg / mL. In this invention, the monkeypox virus capture antibody is a commercially available antibody, catalog number 40891-M0027, purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd.; the goat anti-mouse antibody is a commercially available antibody, purchased from Sangon Biotech (Shanghai) Co., Ltd., catalog number D111024.

[0092] The present invention does not have any special requirements for the assembly method; any assembly method known to those skilled in the art can be used.

[0093] After obtaining the monkeypox virus test strip, the present invention preferably cuts the obtained monkeypox virus test strip to obtain monkeypox virus test strips; the width of the monkeypox virus test strips is preferably 3.5 mm.

[0094] This invention provides a non-diagnostic method for detecting monkeypox virus using a colorimetric-SERS dual-signal mode, comprising the following steps:

[0095] The sample to be tested is mixed with a buffer solution to obtain the test solution;

[0096] The test solution is added to the sample pad of the monkeypox virus test strip. After standing, the color of the T line is observed. The color intensity of the T line is compared with the colorimetric card to obtain the content of monkeypox virus in the test sample. The colorimetric card is a colorimetric image of the T line of the test strip for different concentrations of monkeypox virus protein.

[0097] Alternatively, the solution to be tested is added to the sample pad of the test strip, and after standing, the Raman signal intensity of the T line is measured. The content of monkeypox virus in the sample to be tested is obtained according to the Raman signal intensity and a predetermined standard curve. The standard curve is a linear relationship curve between the monkeypox virus content and the Raman signal intensity.

[0098] This invention involves mixing the sample to be tested with a buffer solution to obtain the test solution. In this invention, the sample to be tested is preferably a saliva sample or an environmental sample, such as river water; and the buffer solution preferably comprises 10 wt% PBST and 20 wt% BSA.

[0099] In this invention, the volume ratio of the sample to the buffer solution is preferably 10:1.

[0100] In this invention, the test solution is added to the sample pad of a monkeypox virus test strip, and after standing, the color of the T line is observed. The color intensity of the T line is compared with a predetermined colorimetric card to obtain the content of monkeypox virus in the test sample. The colorimetric card is a colorimetric image of the T line of a monkeypox virus protein test strip with different concentrations.

[0101] In this invention, the sample volume of the test solution is preferably 50-100 μL; the settling time is preferably 15 min.

[0102] In this invention, if the sample contains monkeypox virus, the three-dimensional molybdenum disulfide sheet-like composite nanomaterial SERS immunotag in the binding pad rapidly binds to the target antigen to form a virus-tagged immune complex, which is then fixed by a capture antibody pre-coated on the T line, producing a visible black band. If the sample is negative, no monkeypox virus-labeled immune complex can be formed for T line capture. Therefore, no visible colorimetric signal appears in the T line region. Excess immunosignal tags are fixed on the C line with goat anti-mouse antibody. Therefore, a black C line always appears on the nitrocellulose membrane, indicating the validity of the test.

[0103] Alternatively, in this invention, the test solution is added to the sample pad of the test strip, and after standing, the Raman signal intensity of the T line is measured. The content of monkeypox virus in the test sample is obtained based on the Raman signal intensity and a predetermined standard curve; the standard curve is a linear relationship curve between the monkeypox virus content and the Raman signal intensity. In this invention, the preferred amount of the test solution added is 50–100 μL; the preferred standing time is 15 min.

[0104] The method for obtaining the standard curve preferably includes the following steps:

[0105] Provide monkeypox virus antigen solutions with known concentrations at varying gradients;

[0106] A test solution was prepared using monkeypox virus antigen solutions of known gradient concentrations as the test sample. The test solution was added to the sample pad of the test strip, and after standing, the Raman signal intensity of the T line was measured to obtain the Raman signal intensity corresponding to different concentrations of monkeypox virus antigen solutions. A standard curve was plotted with the concentration of monkeypox virus antigen solution as the abscissa and the Raman signal intensity as the ordinate.

[0107] In this invention, the preparation process of the three-dimensional molybdenum disulfide sheet-like composite nanomaterial is as follows: Figure 1 As shown, the preparation process of the three-dimensional molybdenum disulfide sheet-like composite nanomaterial SERS immune tag is as follows: Figure 2 As shown in the diagram, the detection method of monkeypox virus using colorimetric-SERS dual-signal mode is as follows: Figure 3 As shown.

[0108] The following detailed description, in conjunction with embodiments, illustrates the three-dimensional molybdenum disulfide sheet-like composite nanomaterials provided by the present invention, their preparation method, and their application in immunochromatography. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0109] Example 1: Preparation of three-dimensional molybdenum disulfide sheet-like composite nanomaterials

[0110] (1) Treatment of MoS2 nanosheets:

[0111] 10 mL (1 mg / mL) of purchased monolayer MoS2 nanosheets with a size <800 nm were sonicated for 10 min. MoS2 with a diameter <300 nm was removed by centrifugation. The remaining relatively uniform MoS2 nanosheets were then redispersed in 40 mL of deionized water.

[0112] (2) Preparation of MoS2@PEI nanosheets:

[0113] 1 mL of 1 mg / mL PEI aqueous solution was added to the above molybdenum disulfide suspension, and the mixture was sonicated for 40 min to form PEI-coated molybdenum disulfide nanosheets (MoS2@PEI). The MoS2@PEI was collected by centrifugation, washed twice, and resuspended in 10 mL of deionized water for later use.

[0114] (3) Preparation of MoS2@Au composite nanosheets:

[0115] 5 mL of the prepared MoS2@PEI nanosheets were mixed with 40 mL of AuNPs with a particle size of 30 nm, and the mixture was sonicated for 30 min to allow the AuNPs to adsorb onto the PEI surface. Excess AuNPs were washed away, and the MoS2@Au composite nanosheets were collected by centrifugation and stored in 10 mL of deionized water for later use.

[0116] (4) Preparation of MoS2@Au-PEI composite nanosheets:

[0117] 10 mL of MoS2@Au nanosheets were added to 40 mL of 0.1 mg / mL PEI aqueous solution. After sonication for 15 min, a second layer of PEI was formed to coat the surface of MoS2@Au, creating MoS2@Au-PEI. The nanosheets were washed twice by centrifugation and then resuspended in 10 mL of deionized water for later use.

[0118] (5) Preparation of MoS2@Au-Au composite nanosheets:

[0119] The prepared MoS2@Au-PEI composite nanosheet solution was mixed with 40 mL of colloidal gold solution with a particle size of 30 nm and incubated under ultrasound. After ultrasound treatment for 30 min, the resulting MoS2@Au-Au composite nanosheets were collected by centrifugation, washed with deionized water, and resuspended in 10 mL of ethanol for subsequent use.

[0120] Figure 4 The images show transmission electron microscopy (HRTEM) images of the components involved in the preparation of the three-dimensional molybdenum disulfide sheet-like composite nanomaterial. Image a shows MoS2 nanosheets, image b shows MoS2@Au nanosheets, image c shows MoS2@Au-Au nanosheets, image d shows a magnified view of a portion of MoS2@Au, image e shows a magnified view of a portion of MoS2@Au-PEI, and image f shows a magnified view of a portion of MoS2@Au-Au. The HRTEM results demonstrate that the MoS2@Au-Au composite nanomaterial possesses a controllable hotspot structure, a large specific surface area, and excellent dispersibility.

[0121] The stability characterization results of the three-dimensional molybdenum disulfide sheet-like composite nanomaterials prepared in this embodiment are as follows: Figure 5 As shown. Figure 5 In the figures, a represents the color changes and corresponding Raman intensities of MoS2@Au-Au at different pH values ​​(pH = 2–13); b represents the color changes and corresponding Raman intensities of MoS2@Au-Au stored for different times (0–60 days); and c represents the color changes and corresponding UV-Vis spectra of colloidal gold and MoS2@Au-Au at different salt concentrations (0–1000 mM NaCl). It can be seen that the composite nanomaterials provided by this invention exhibit excellent stability.

[0122] Example 2: Preparation of SERS immunotags and monkeypox virus detection strips using three-dimensional molybdenum disulfide sheet-like composite nanomaterials.

[0123] 10 μL of Raman reporter molecule DTNB (10 mM) was added to 10 mL of MoS2@Au-Au ethanol solution. After sonication for 1 h, the DTNB-labeled MoS2@Au-Au nanosheets were washed with ethanol, collected by centrifugation, and redispersed in 0.5 mL of 2-(N-morpholine)ethanesulfonic acid solution (10 mM, pH 5.5). Then, 5 μL of carbodiimide solution (0.1 M) and 10 μL of N-hydroxysuccinimide solution (0.1 M) were added, and the mixture was sonicated for 15 min to activate the carboxyl groups on the surface of MoS2@Au-Au. The carboxyl groups were recovered by centrifugation and resuspended in 200 μL of PBS buffer (10 mM, pH 7.4). 8 μg of anti-monkeypox virus antibody was added to the MoS2@Au-Au-DTNB solution, and the mixture was shaken at 30 °C for 2 h. Then, 80 μL of BSA (10%) was added to block any unreacted sites on the nanosheets. Finally, the SERS immune tags were collected by centrifugation and redispersed in 0.5 mL of storage buffer.

[0124] Anti-monkeypox virus antibodies were sprayed onto the test line (T line) to capture the monkeypox virus-SERS tag immune complex. Goat anti-mouse IgG was sprayed onto the control line (C line) to capture excess SERS tags. The SERS immune tags were applied to the conjugate pad and lyophilized. The strip was then assembled with the absorbent pad, sample pad, nitrocellulose membrane, and base plate to form a test strip for later use.

[0125] Example 3

[0126] The molybdenum disulfide sheet-like composite nanomaterials provided by this invention, after surface modification with monkeypox virus antibodies, can be used as high-performance SERS tags for detection in immunochromatographic systems. In this embodiment, a monkeypox virus antibody-modified SERS tag is used as an immunochromatographic system tag to detect virus samples containing different concentrations (100–0.01 ng / mL).

[0127] Figure 6 The results of the immunochromatographic analysis system for detecting monkeypox virus based on three-dimensional molybdenum disulfide sheet-like composite nanomaterials are presented. Figure 6 Image a shows a photograph of the MoS2@Au-Au immunochromatographic test strip and the corresponding SERS mapping image; Figure 6 In Figure b, the SERS spectrum is shown at the corresponding concentration. As the viral concentration decreases, the color intensity of the T line and the SERS intensity on the test strip gradually weaken. Three points are randomly measured on each T line, and the obtained SERS spectra are averaged to obtain a repeatable SERS signal. Figure 6Figure c shows the calibration curve for the SERS signal intensity of monkeypox virus detected by the SERS immunochromatographic test strip. Based on this, the sensitivity (LOD) of the SERS immunochromatographic assay was calculated to be 0.002 ng / mL. Figure 6 Figure d shows the linear fitting results for low concentrations of monkeypox virus; Figure 6 Figures e and f show the results of colloidal gold immunochromatography and ELISA. The sensitivity of colloidal gold immunochromatography for detecting monkeypox virus was 1 ng / mL, while the sensitivity of the commercial ELISA kit was 0.01 ng / mL. The comparison shows that the established MoS2@Au-Au immunochromatographic test strip has approximately 500 times higher sensitivity for detecting monkeypox virus than the colloidal gold test strip and approximately 5 times higher sensitivity than the ELISA kit.

[0128] Figure 7 In Figures a and b, the performance of the MoS2@Au-Au immunochromatographic test strip in clinical throat swabs and environmental lake water samples, respectively, is demonstrated. It can be seen that the colorimetric and SERS signal results of the MoS2@Au-Au immunochromatographic test strip in detecting complex samples are very close to the detection results of PBS samples, proving that the established method has good practical sample detection capabilities.

[0129] Figure 8 Detailed colorimetric images of monkeypox virus detection based on MoS2@Au-Au (a) and conventional immunochromatography (b) are shown. The colorimetric sensitivity of the MoS2@Au-Au test strip is 0.2 ng / mL, which is 5 times higher than that of colloidal gold immunochromatography.

[0130] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A three-dimensional molybdenum disulfide sheet-like composite nanomaterial SERS immune tag, comprising a three-dimensional molybdenum disulfide sheet-like composite nanomaterial, a Raman reporter molecule chemically coupled to the three-dimensional molybdenum disulfide sheet-like composite nanomaterial, and a viral antibody chemically coupled to the Raman reporter molecule; The three-dimensional molybdenum disulfide sheet-like composite nanomaterial includes a two-dimensional MoS2 inner layer and two gold nanoparticle shells wrapped around the surface of the two-dimensional MoS2 inner layer. The single gold nanoparticle shell includes a polyethyleneimine layer and gold nanoparticles distributed on the surface of the polyethyleneimine layer. The Raman reporter molecule is 5,5′-dithiobis(dinitrobenzoic acid); The sheet diameter of the two-dimensional MoS2 inner layer is 400–800 nm; The thickness of the polyethyleneimine layer is 1–10 nm; The gold nanoparticles have a particle size of 20–40 nm; The preparation method of the three-dimensional molybdenum disulfide sheet-like composite nanomaterial includes the following steps: (1) Provide a two-dimensional nanosheet dispersion of MoS2; (2) The MoS2 two-dimensional nanosheet dispersion was ultrasonically mixed with the first polyethyleneimine aqueous solution to obtain MoS2@PEI nanosheets; (3) The MoS2@PEI nanosheets were ultrasonically mixed with the first colloidal gold solution to obtain MoS2@Au nanosheets; (4) The MoS2@Au sheet-like composite nanoparticles were ultrasonically mixed with a second polyethyleneimine aqueous solution to obtain MoS2@Au@PEI nanosheets; (5) The MoS2@Au@PEI nanosheets were ultrasonically mixed with the second colloidal gold solution to obtain a three-dimensional molybdenum disulfide sheet-like composite nanomaterial.

2. The three-dimensional molybdenum disulfide sheet-like composite nanomaterial SERS immune tag according to claim 1, characterized in that, The viral antibody is a monkeypox virus antibody.

3. The three-dimensional molybdenum disulfide sheet-like composite nanomaterial SERS immune tag according to claim 1, characterized in that, The concentrations of the first polyethyleneimine aqueous solution and the second polyimide aqueous solution are independently 0.1–5 mg / mL; The mass ratio of the MoS2 two-dimensional nanosheets to the first polyethyleneimine and the second polyethyleneimine is 100:1 to 10:1 to 10. The mass ratio of the MoS2 two-dimensional nanosheets to the first colloidal gold and the second colloidal gold is 1:10-30:10-30.

4. A method for preparing the three-dimensional molybdenum disulfide sheet-like composite nanomaterial SERS immune tag according to claim 1 or 2, comprising the following steps: The three-dimensional molybdenum disulfide sheet-like composite nanomaterial was ultrasonically mixed with Raman reporter molecules to obtain MoS2@Au-Au-DTNB composite nanosheets; The MoS2@Au-Au-DTNB composite nanosheets were mixed with a carboxyl activator to activate the carboxyl groups, thereby obtaining carboxyl-activated MoS2@Au-Au-DTNB composite nanosheets. The activated carboxyl group MoS2@Au-Au-DTNB composite nanosheets were mixed with viral antibodies and incubated to block unbound sites, resulting in a three-dimensional molybdenum disulfide sheet-like composite nanomaterial SERS immune tag.

5. A monkeypox virus detection test strip, comprising a nitrocellulose membrane, a conjugate pad, a sample pad, an absorbent pad, and a base plate, characterized in that, The nitrocellulose membrane has T lines and C lines. The surface of the T lines is coated with monkeypox virus capture antibodies, and the surface of the C lines is coated with goat anti-mouse antibodies. The surface of the conjugate pad is coated with the three-dimensional molybdenum disulfide sheet-like composite nanomaterial SERS immune tag as described in claim 1 or 2, wherein the viral antibody in the three-dimensional molybdenum disulfide sheet-like composite nanomaterial SERS immune tag is a monkeypox virus antibody.

6. A colorimetric-SERS dual-signal mode detection method for monkeypox virus for non-diagnostic purposes, comprising the following steps: The sample to be tested is mixed with a buffer solution to obtain the test solution; The test solution is added to the sample pad of the monkeypox virus test strip according to claim 5, and after standing, the color of the T line is observed. The color intensity of the T line is compared with the colorimetric card to obtain the content of monkeypox virus in the test sample. The colorimetric card is a colorimetric image of the T line of the monkeypox virus protein test strip at different concentrations. Alternatively, the solution to be tested is added to the sample pad of the test strip, and after standing, the Raman signal intensity of the T line is measured. The content of monkeypox virus in the sample to be tested is obtained according to the Raman signal intensity and a predetermined standard curve. The standard curve is a linear relationship curve between the monkeypox virus content and the Raman signal intensity.

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