Immune MoS < 2 > atTQD fluorescent label as well as preparation method and application thereof

Abstract

The invention provides an immune MoS < 2 > atTQD fluorescent label as well as a preparation method and application thereof, and relates to the technical field of lateral chromatographic detection. The immune MoS < 2 > (at) TQD fluorescent label is a two-dimensional molybdenum sulfide quantum dot-based nano material labeled by an anti-carboxyl fluorescein antibody; the nanometer material based on the two-dimensional molybdenum sulfide quantum dots sequentially comprises a molybdenum disulfide nanosheet, a first coating layer, a second coating layer and a third coating layer from inside to outside, wherein the first coating layer, the second coating layer and the third coating layer are PEI layers for adsorbing CdSe-ZnS quantum dots. Meanwhile, the invention further provides the immune MoS2 (at) TQD fluorescent label, and the immune MoS2 (at) TQD fluorescent label is the quantum dot nanometer material based on two-dimensional molybdenum sulfide, which is marked by adopting an anti-carboxyl fluorescein antibody. The label can be used as an excellent signal label of a nucleic acid on-site rapid detection system, shows more excellent colorimetric and fluorescence signals, and is better in stability.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of lateral chromatographic detection, and particularly relates to an immune MoS2@TQD fluorescent label and a preparation method and application thereof. BACKGROUND

[0002] Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly infectious and pathogenic variant of coronavirus, which is associated with severe respiratory disease. However, the detection and isolation of asymptomatic individuals are often delayed, further accelerating the spread of the epidemic, and diagnostic testing is a key means to combat the spread of this epidemic. At present, for the detection of SARS-CoV-2 virus, there are real-time reverse transcription polymerase chain reaction (RT-PCR), droplet digital polymerase chain reaction (ddPCR), next-generation sequencing (NGS) and other methods for early identification of infected persons. SARS-CoV-2 virus detection based on RT-PCR has become the most powerful and reliable detection method, but this method requires expensive equipment and professional operators, so it cannot be popularized in most places. Therefore, it is particularly important to establish a SARS-CoV-2 virus detection method with high sensitivity, high specificity, simple operation, short time, low cost and portability.

[0003] Clustered regularly interspaced short palindromic repeat-associated protein (CRISPR-Cas) system is an acquired immune mode of bacteria and archaea against phage or virus invasion. Recombinase-aided amplification (RAA) technology is similar to recombinase polymerase amplification (RPA) technology, which uses recombinase and oligonucleotide primers to form a protein-DNA complex that can find homologous sequences in double-stranded DNA. Reverse transcription RAA (RT-RAA) technology is based on the addition of reverse transcriptase to RAA. When different isothermal amplification technologies are used in combination with CRISPR-Cas technology, high sensitivity and specificity amplification of RNA templates can be achieved without thermal cycling steps, and specific target nucleic acids are amplified by trans-cleavage of RNA to confirm positivity.

[0004] Lateral flow assay (LFA) has become a mature and powerful point-of-care testing (POCT) detection tool due to its speed, low cost, portability and ease of use. Quantum dots (QDs) as a new type of fluorescent material have been widely used in LFA due to their high light stability, narrow fluorescence emission spectrum and quantifiable fluorescence intensity. However, the small size, poor biocompatibility and poor stability of quantum dots still pose challenges in biological applications. Therefore, some strategies have been reported for assembling QDs relying on carrier materials such as Fe3O4, SiO2 and two-dimensional materials.

[0005] MoS2 is a two-dimensional transition metal dichalcogenide (TMD). Due to its intrinsic band gap and optoelectronic properties, MoS2 has the characteristics of high sensitivity, fast response, high selectivity, good reliability, fast recovery, good chemical stability, high thermal stability, and high surface activity, and is expected to become an ideal nano-probe. MoS2@quantum dot nanosheets (NSs) assembled by MoS2 and quantum dots have been used for bioimaging, drug delivery and treatment, and biosensors. However, it is worth noting that MoS2 has a strong fluorescence quenching ability. Therefore, it is particularly important to establish a MoS2 quantum dot nanomaterial with excellent light stability, anti-photobleaching and good biocompatibility, and successfully apply it to LFA detection.

[0006] In view of this, the present application is proposed. SUMMARY

[0007] One of the purposes of the present application is to provide a two-dimensional molybdenum sulfide quantum dot nanomaterial. The prepared composite material has good dispersibility and stability, and has a dual mode of colorimetric imaging and fluorescence quantification, and can be used as an excellent signal tag for nucleic acid on-site rapid detection system.

[0008] The second purpose of the present application is to provide a preparation method of the two-dimensional molybdenum sulfide quantum dot nanomaterial.

[0009] The third purpose of the present application is to provide an immune MoS2@TQD fluorescent tag, which is the two-dimensional molybdenum sulfide quantum dot nanomaterial labeled with an anti-carboxyfluorescein antibody.

[0010] The fourth purpose of the present application is to provide a preparation method of the immune MoS2@TQD fluorescent tag.

[0011] The fifth purpose of the present application is to provide an application of the two-dimensional molybdenum sulfide quantum dot nanomaterial or the immune MoS2@TQD fluorescent tag in the preparation of a product for detecting SARS-CoV-2 virus.

[0012] The sixth purpose of the present application is to provide a product for detecting SARS-CoV-2 virus, which comprises: a lateral chromatography test paper, the immune MoS2@TQD fluorescent tag, and an RT-RAA and Cas13a cleavage reaction product.

[0013] In order to achieve the above-mentioned purposes of the present application, the following technical solutions are adopted:

[0014] In a first aspect, the present application provides a two-dimensional molybdenum disulfide quantum dot nanomaterial, which comprises, from inside to outside, a molybdenum disulfide nanosheet, a first coating layer, a second coating layer and a third coating layer; wherein the first coating layer, the second coating layer and the third coating layer are PEI layers adsorbing CdSe-ZnS quantum dots.

[0015] Further, the thickness of the molybdenum disulfide nanosheet is 1-5 nm, and the flake diameter of the molybdenum disulfide nanosheet is 200-5000 nm.

[0016] Further, the thickness of each of the first coating layer, the second coating layer and the third coating layer is independently 7-11 nm.

[0017] In a second aspect, the present application provides a preparation method of the two-dimensional molybdenum disulfide quantum dot nanomaterial according to the first aspect, which comprises the following steps:

[0018] Polyethyleneimine is coated on the surface of the molybdenum disulfide nanosheet to form a first PEI layer, thereby obtaining MoS2-PEI; and CdSe-ZnS quantum dots are electrostatically adsorbed on the surface of the MoS2-PEI to form a first coating layer, thereby obtaining first MoS2@QD.

[0019] The steps of coating polyethyleneimine and electrostatically adsorbing CdSe-ZnS quantum dots are repeated once to form a second coating layer on the surface of the first MoS2@QD, thereby obtaining second MoS2@QD.

[0020] The steps of coating polyethyleneimine and electrostatically adsorbing CdSe-ZnS quantum dots are repeated once again to form a third coating layer on the surface of the second MoS2@QD, thereby obtaining the two-dimensional molybdenum disulfide quantum dot nanomaterial.

[0021] Further, the step of coating the molybdenum disulfide nanosheet with polyethyleneimine comprises:

[0022] The molybdenum disulfide nanosheet, polyethyleneimine and water are mixed and ultrasonically dispersed, the positively charged polyethyleneimine is coated on the surface of the negatively charged molybdenum disulfide nanosheet, and then the excess polyethyleneimine is washed away, thereby obtaining positively charged MoS2-PEI.

[0023] Further, in the preparation of the first coating layer, the mass ratio of the molybdenum disulfide nanosheet to polyethyleneimine is (600-650):1.

[0024] Further, in the preparation of the second coating layer, the mass ratio of the molybdenum disulfide nanosheet to polyethyleneimine is (600-650):1.

[0025] Further, in the process of preparing the third coating layer, the mass ratio of the molybdenum disulfide nanosheet and the polyethyleneimine is (600-650):1.

[0026] Further, the power of the ultrasonic dispersion is 100-300 W, and the time of the ultrasonic dispersion is 20-60 min.

[0027] Further, the step of washing away the excess polyethyleneimine comprises centrifuging to discard the supernatant, the centrifugal force of the centrifugation being 6000-7197 rcf, and the time of the centrifugation being 10-60 min.

[0028] Further, the step of electrostatically adsorbing the CdSe-ZnS quantum dots comprises:

[0029] The MoS2-PEI, the CdSe-ZnS quantum dots and water are mixed and ultrasonically dispersed, the CdSe-ZnS quantum dots are electrostatically adsorbed on the surface of the MoS2-PEI to form a first coating layer, and then the excess CdSe-ZnS quantum dots are washed away to obtain a first MoS2@QD.

[0030] Further, in the process of preparing the first coating layer, the mass ratio of the molybdenum disulfide nanosheet and the CdSe-ZnS quantum dots is (6-7):1.

[0031] Further, in the process of preparing the second coating layer, the mass ratio of the molybdenum disulfide nanosheet and the CdSe-ZnS quantum dots is (6-7):1.

[0032] Further, in the process of preparing the third coating layer, the mass ratio of the molybdenum disulfide nanosheet and the CdSe-ZnS quantum dots is (6-7):1.

[0033] Further, the power of the ultrasonic dispersion is 100-300 W, and the time of the ultrasonic dispersion is 50-60 min.

[0034] Further, the step of washing away the excess quantum dots comprises centrifuging to discard the supernatant, the speed of the centrifugation being 5000-6200 rpm, and the time of the centrifugation being 5-10 min.

[0035] Further, the preparation method further comprises a resuspension step of resuspending the two-dimensional molybdenum disulfide quantum dot nanomaterial in an ethanol solution to obtain an ethanol solution of the MoS2@TQD.

[0036] Further, the concentration of the ethanol solution of the MoS2@TQD is 1-2 g / L.

[0037] In a third aspect, the present invention provides an immune-MoS2@TQD fluorescent label, wherein the immune-MoS2@TQD fluorescent label is a two-dimensional molybdenum sulfide quantum dot nanomaterial as described in the first aspect and is labeled with an anti-carboxyfluorescein antibody.

[0038] In a fourth aspect, the present invention provides a method for preparing the immune MoS2@TQD fluorescent label as described in the third aspect, the preparation method comprising:

[0039] The two-dimensional molybdenum sulfide quantum dot nanomaterial is resuspended in MES buffer containing EDC and NHS, and activated by carboxyl groups to obtain carboxyl-activated MoS2@TQD; the carboxyl-activated MoS2@TQD is then mixed with anti-carboxyfluorescein antibody and incubated; bovine serum albumin is then added for blocking; and after washing, the immune MoS2@TQD fluorescent label is obtained.

[0040] Furthermore, the mass ratio of the two-dimensional molybdenum sulfide quantum dot nanomaterial, EDC and NHS is 1:(0.05-0.1):(0.1-0.5).

[0041] Furthermore, the carboxyl activation is carried out under ultrasonic conditions, the ultrasonic power is 100 to 300 W, and the ultrasonic time is 10 to 20 minutes.

[0042] Furthermore, the mass ratio of the carboxyl-activated MoS2@TQD, anti-carboxyfluorescein antibody and bovine serum albumin is 1:(0.01-0.05):(5-15).

[0043] Furthermore, the incubation time is 2 to 3 hours.

[0044] Furthermore, the washing adopts PBST buffer.

[0045] Furthermore, the immuno-MoS2@TQD fluorescent label was stored in a gold standard diluent with a pH of 7.4.

[0046] In a fifth aspect, the present invention provides an application of the two-dimensional molybdenum sulfide quantum dot nanomaterial or the immune MoS2@TQD fluorescent label in the preparation of a product for detecting SARS-CoV-2 virus.

[0047] In a sixth aspect, the present invention provides a kit for detecting SARS-CoV-2 virus, wherein the product for detecting SARS-CoV-2 virus comprises: the immune MoS2@TQD fluorescent label as described in the third aspect, a CRISPR-Cas13a system for specific detection of SARS-CoV-2 virus, and a lateral flow test paper;

[0048] The CRISPR-Cas13a system for specifically detecting SARS-CoV-2 virus comprises a primer set, wherein the primer set comprises an RT-RAA amplification primer set and nucleotide sequences required by a CRISPR-Cas13a enzyme cutting detection system; the RT-RAA amplification primer set comprises an upstream primer of a SARS-CoV-2 E gene and a downstream primer of the SARS-CoV-2 E gene, the sequence of the upstream primer is shown as SEQ ID No: 1, and the sequence of the downstream primer is shown as SEQ ID No: 2; the nucleotide sequences required by the CRISPR-Cas13a enzyme cutting detection system comprise a specific crRNA, and the sequence of the specific crRNA is shown as SEQ ID No: 3.

[0049] The lateral chromatography test paper comprises a nitrocellulose membrane, a sample pad and an absorption pad on a fixed PVC bottom plate; the nitrocellulose membrane is provided with a test line T and a quality control line C which are spaced apart along the chromatography direction of the sample to be detected; the test line T is coated with streptavidin, and the quality control line C is coated with a goat anti-rabbit IgG antibody.

[0050] Further, the CRISPR-Cas13a system for specifically detecting SARS-CoV-2 virus further comprises a reporter probe, which is a fluorescent reporter probe, one end of which is labeled with biotin and the other end of which is labeled with a fluorescent group FAM; the reporter probe is used for being cut by the activated Cas13a and releasing fluorescence.

[0051] Further, the CRISPR-Cas13a system for specifically detecting SARS-CoV-2 virus further comprises an RT-RAA amplification system and a CRISPR-Cas13a enzyme cutting system; the RT-RAA amplification system comprises a buffer, enzyme-free water and a template RNA; and the CRISPR-Cas13a enzyme cutting system comprises a buffer, enzyme-free water, a ribonucleotide mixed solution, a ribonuclease inhibitor, Cas13a protein, T7 RNA polymerase and magnesium chloride.

[0052] Compared with the prior art, the present application has the following beneficial effects:

[0053] (1) The present application provides an immune MoS2@TQD fluorescent tag, which is labeled with an anti-carboxyfluorescein antibody and based on two-dimensional molybdenum sulfide quantum dot nanomaterials, can be used as an excellent signal tag of a nucleic acid on-site rapid detection system, shows more superior colorimetric and fluorescent signals, and has better stability. It has excellent optical stability and stability in a high-salt environment (10-1000mM NaCl), and the fluorescence remains good within 60 days; and the two-dimensional molybdenum sulfide quantum dot nanomaterials have good dispersion.

[0054] (2) The application provides a product for detecting a SARS-CoV-2 virus, which is based on a CRISPR-Cas13 LFA detection combined with a colorimetric quantitative dual-mode MoS2@TQD fluorescent label, can be used for rapidly and sensitively detecting the SARS-CoV-2 virus in a clinical sample, and can realize rapid colorimetric screening and fluorescent quantification of the SARS-CoV-2 nucleic acid within 35 min; the colorimetric mode can screen out SARS-CoV-2 with a visual detection limit (vLOD) of 500 copies / mL, and the fluorescent signal mode can quantitatively detect SARS-CoV-2 with an LOD as low as 250 copies / mL; the sensitivity is 20 times that of a colloidal gold detection method, and the accuracy of the result is 100%. BRIEF DESCRIPTION OF DRAWINGS

[0055] In order to more clearly illustrate the specific embodiments of the application or the technical solutions in the prior art, the drawings needed in the specific embodiments or the prior art description will be briefly introduced below. Obviously, the drawings in the following description are some embodiments of the application, and other drawings can also be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0056] Figure 1 A synthesis schematic diagram of the immune MoS2@TQD fluorescent label provided by the application.

[0057] Figure 2 A principle diagram of the dual-mode MoS2@TQD fluorescent label combined with the CRISPR-Cas13 LFA detection provided by the application.

[0058] Figure 3A A TEM diagram of the two-dimensional molybdenum sulfide nanosheet provided for Example 1.

[0059] Figure 3B A TEM diagram of the two-dimensional molybdenum sulfide nanosheet coated with a first PEI layer provided for Example 1.

[0060] Figure 3C A TEM diagram (200 nm) of the MoS2@TQD NSs provided for Example 1.

[0061] Figure 3D A TEM enlarged view (50 nm) of the MoS2@TQD NSs provided for Example 1.

[0062] Figure 3E An EDS element analysis diagram of the MoS2@TQD NSs provided for Example 1.

[0063] Figure 3FElemental line scan result of MoS2@TQD NSs provided for Example 1.

[0064] Figure 3G Zeta potential chart of products in each step in the process of preparing MoS2@TQD NSs for Example 1.

[0065] Figure 3H XPS spectrum chart of MoS2@TQD NSs provided for Example 1.

[0066] Figure 3I Fluorescence emission spectrum chart of products in each step in the process of preparing MoS2@TQD NSs for Example 1.

[0067] Figure 3J Fourier infrared spectrum chart of fluorescent label and anti-FAM antibody provided for Example 1.

[0068] Figure 3K Fluorescence chart of fluorescent label MoS2@TQD NSs in high salinity solution provided for Example 1.

[0069] Figure 3L Fluorescence chart of fluorescent label MoS2@TQD NSs in high salinity solution provided for Example 1.

[0070] Figure 4A Screening chart of RT-RAA primers and crRNA provided for Test Example 2; (i) screening of RT-RAA primers; (ii) screening of crRNA of RT-RAA-CRISPR-Cas13a assay, fluorescence value kinetic curve of 4 crRNAs within 30 cycles.

[0071] Figure 4B Confirmatory test negative positive T-line fluorescence intensity chart provided for Test Example 2; error bar indicates standard deviation (n=3).

[0072] Figure 4C Result chart of optimizing probe concentration provided for Test Example 2.

[0073] Figure 4D Result chart of optimizing CRISPR system detection time provided for Test Example 2.

[0074] Figure 4E Result chart of optimizing T-line concentration provided for Test Example 2.

[0075] Figure 4F Result chart of optimizing label addition.

[0076] Figure 5APhotos of CRISPR-assisted dual-mode LFA strips detecting different concentrations of severe acute respiratory syndrome SARS-CoV-2 virus (0-100 copies / µL) under UV light and (ii) natural light provided for Test Example 3.

[0077] Figure 5B Calibration plot of fluorescence intensity on T-line for different concentrations of severe acute respiratory syndrome SARS-CoV-2 virus provided for Test Example 3.

[0078] Figure 5C Fluorescence signal of CRISPR-assisted dual-mode LFA for detecting several respiratory viruses and strip photos under UV light provided for Test Example 3.

[0079] Figure 5D Strips of reproducible MoS2@TQD for detecting severe acute respiratory syndrome SARS-CoV-2 virus RNA at 1 copies / µL (Group 1) and 50 copies / µL (Group 2) provided for Test Example 3; error bars represent standard deviation (n=3).

[0080] Figure 6A Colorimetric photos of throat swab samples using CRISPR-assisted dual-mode LFA strips provided for Test Example 4; numbers above the strips represent 35 positive throat swab samples and 18 negative samples.

[0081] Figure 6B Fluorescence photos of throat swab samples taken using CRISPR-assisted dual-mode LFA strips provided for Test Example 4.

[0082] Figure 6C Photos of colloidal gold test sample results provided for Test Example 4.

[0083] Figure 6D Plot of Ct values of PCR quantification results for 35 positive throat swab samples on the upper coordinates and fluorescence signal intensity of the detection line of the corresponding CRISPR-assisted dual-mode LFA strips on the lower coordinates provided for Test Example 4; error bars represent standard deviation of three independent measurements.

[0084] Figure 6E Severe acute respiratory syndrome SARS-CoV-2 virus detected in healthy clinical samples using CRISPR-assisted dual-mode LFA and RT-PCR kits.

[0085] Figure 6F ROC curve plot of strips for diagnosing patients with severe acute respiratory syndrome coronavirus 2. DETAILED DESCRIPTION

[0086] Unless otherwise defined, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear; however, in the event of any latent ambiguity, definitions provided herein take precedent over any dictionary definition. In this application, the use of "or" means "and / or" unless specifically stated otherwise, e.g., "comprises at least one of A and B" means that the composition can comprise A, or B, or both A and B. Further, the use of "includes" and other forms is intended to be non-exhaustive.

[0087] It should be noted that specific details are set forth in the following description in order to provide a thorough understanding of the application. However, the application can be practiced without many of the details described in this description, many of which are well known in the art. Therefore, the particular implementation described herein is not intended to limit the scope of the application.

[0088] The technical solutions of the present application will be described clearly and completely below in combination with the embodiments. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0089] In order to achieve the above-mentioned purpose of the present application, the following technical solutions are adopted:

[0090] In a first aspect, the present application provides a two-dimensional molybdenum disulfide quantum dot nanomaterial, which comprises, from inside to outside, a molybdenum disulfide nanosheet, a first coating layer, a second coating layer, and a third coating layer; wherein the first coating layer, the second coating layer, and the third coating layer are all PEI layers for adsorbing CdSe-ZnS quantum dots.

[0091] In the present application, molybdenum disulfide nanosheets (hereinafter referred to as MoS2 NSs) are used as the core, which have a folded and stacked film structure and an excellent specific surface area, thereby providing more attachment points for subsequent adsorption of CdSe-ZnS quantum dots (hereinafter referred to as CdSe-ZnS QD), thereby effectively amplifying the detection signal. The two-dimensional molybdenum disulfide quantum dot nanomaterial (hereinafter referred to as MoS2@TQD NSs) relies on the electrostatic adsorption principle of PEI bridging, and utilizes PEI to densely and uniformly distribute CdSe-ZnS QD on the surface of the MoS2 NSs. At the same time, with the increase of the number of coating layers, the loading capacity of CdSe-ZnS QD adsorbed on the MoS2 NSs increases exponentially, thereby significantly enhancing the luminescence capacity of the two-dimensional molybdenum disulfide quantum dot nanomaterial.

[0092] As an optional embodiment, the thickness of the molybdenum disulfide nanosheet is 1 to 5 nm, for example, it can be 1 nm, 1.5 nm, 2 nm, 2.5 nm, 3 nm, 3.5 nm, 4 nm, 4.5 nm, 5 nm, etc., and the diameter of the molybdenum disulfide nanosheet is 200 to 5000 nm, for example, it can be 200 nm, 500 nm, 1000 nm, 1500 nm, 2000 nm, 2500 nm, 3000 nm, 3500 nm, 4000 nm, 4500 nm, 5000 nm, etc.

[0093] As an optional embodiment, the thickness of the first cladding layer, the second cladding layer and the third cladding layer are each independently 7 to 11 nm, for example, 7 nm, 7.5 nm, 8 nm, 8.5 nm, 9 nm, 9.5 nm, 10 nm, 10.5 nm, 11 nm, etc.

[0094] Second, as Figure 1 As shown, the present invention provides a preparation method of the two-dimensional molybdenum sulfide quantum dot nanomaterial as described in the first aspect, the preparation method comprising the following steps:

[0095] Coating polyethyleneimine on the surface of the molybdenum disulfide nanosheet to form a first PEI layer to obtain MoS2-PEI, and then electrostatically adsorbing CdSe-ZnS quantum dots on the surface of the MoS2-PEI to form a first coating layer to obtain a first MoS2@QD;

[0096] Repeat the steps of polyethyleneimine coating and electrostatic adsorption of CdSe-ZnS quantum dots to form a second coating layer on the surface of the first MoS2@QD to obtain a second MoS2@QD;

[0097] Repeat the steps of polyethyleneimine coating and electrostatic adsorption of CdSe-ZnS quantum dots once more to form a third coating layer on the surface of the second MoS2@QD to obtain the two-dimensional molybdenum sulfide quantum dot nanomaterial.

[0098] As an optional embodiment, the preparation method based on two-dimensional molybdenum sulfide quantum dot nanomaterial specifically includes the following steps:

[0099] S1, coating the surface of the molybdenum disulfide nanosheet with polyethyleneimine to form a first PEI layer to obtain a first MoS2-PEI, and then electrostatically adsorbing a first CdSe-ZnS quantum dot on the surface of the first MoS2-PEI to form a first coating layer to obtain a first MoS2@QD;

[0100] S2, polyethyleneimine is coated on the surface of the first MoS2@QD to form a second PEI layer, to obtain a second MoS2-PEI, and then a second CdSe-ZnS quantum dot is electrostatically adsorbed on the surface of the second MoS2-PEI to form a second coating layer, to obtain a second MoS2@QD;

[0101] S3, polyethyleneimine is coated on the surface of the second MoS2@QD to form a third PEI layer, to obtain a third MoS2-PEI, and then a third CdSe-ZnS quantum dot is electrostatically adsorbed on the surface of the third MoS2-PEI to form a third coating layer, to obtain the two-dimensional molybdenum disulfide quantum dot-based nanometer material.

[0102] As an optional implementation, the step of coating the molybdenum disulfide nanosheet with polyethyleneimine includes:

[0103] The molybdenum disulfide nanosheet, polyethyleneimine, and water are mixed and ultrasonically dispersed, the positively charged polyethyleneimine is coated on the surface of the negatively charged molybdenum disulfide nanosheet, and then the excess polyethyleneimine is washed away to obtain the positively charged MoS2-PEI.

[0104] As an optional implementation, the step of coating the first MoS2@QD with polyethyleneimine includes:

[0105] The first MoS2@QD, polyethyleneimine, and water are mixed and ultrasonically dispersed, the polyethyleneimine is coated on the surface of the first MoS2@QD, and then the excess polyethyleneimine is washed away to obtain the second MoS2-PEI.

[0106] As an optional implementation, the step of coating the second MoS2@QD with polyethyleneimine includes:

[0107] The second MoS2@QD, polyethyleneimine, and water are mixed and ultrasonically dispersed, the polyethyleneimine is coated on the surface of the second MoS2@QD, and then the excess polyethyleneimine is washed away to obtain the third MoS2-PEI.

[0108] As an optional implementation, in the process of preparing the first coating layer, the mass ratio of the molybdenum disulfide nanosheet to polyethyleneimine is (600-650):1, which can be 600:1, 605:1, 610:1, 615:1, 620:1, 625:1, 630:1, 635:1, 640:1, 645:1, 650:1, etc.

[0109] As an optional embodiment, in the process of preparing the second coating layer, the mass ratio of the molybdenum disulfide nanosheets and polyethyleneimine is (600-650):1, for example, it can be 600:1, 605:1, 610:1, 615:1, 620:1, 625:1, 630:1, 635:1, 640:1, 645:1, 650:1, etc.

[0110] As an optional embodiment, in the process of preparing the third coating layer, the mass ratio of the molybdenum disulfide nanosheets and polyethyleneimine is (600-650):1, for example, it can be 600:1, 605:1, 610:1, 615:1, 620:1, 625:1, 630:1, 635:1, 640:1, 645:1, 650:1, etc.

[0111] As an optional embodiment, the power of the ultrasonic dispersion is 100-300 W, for example, it can be 100 W, 120 W, 140 W, 160 W, 180 W, 200 W, 220 W, 240 W, 260 W, 280 W, 300 W, etc., and the time of the ultrasonic dispersion is 20-60 min, for example, it can be 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 60 min, etc.

[0112] As an optional embodiment, the step of washing away excess polyethyleneimine includes: centrifuging and discarding the supernatant, the centrifugal force of the centrifugation is 6000-7197rcf, for example, it can be 6000rcf, 6200rcf, 6400rcf, 6800rcf, 7000rcf, 7100rcf, 7197rcf, etc., and the centrifugation time is 10-60min, for example, it can be 10min, 15min, 20min, 25min, 30min, 35min, 40min, 45min, 50min, 55min, 60min, etc.

[0113] As a preferred embodiment, S1 uses polyethyleneimine to coat molybdenum disulfide nanosheets, which specifically includes the following steps:

[0114] (i) dispersing MoS2 NSs in water to obtain an aqueous solution of MoS2 NSs;

[0115] (ii) mixing an aqueous solution of MoS2 NSs and an aqueous solution of PEI, and performing ultrasonic dispersion to obtain a mixed solution;

[0116] (iii) centrifuging the mixture obtained in (ii), discarding the supernatant, and retaining the precipitate;

[0117] (iv) adding water to the precipitate obtained in (iii), ultrasonic dispersion, centrifugation, discarding the supernatant, and retaining the precipitate;

[0118] (v) adding water to the precipitate obtained in (iv), ultrasonic dispersion, centrifugation, discarding the supernatant, and retaining the precipitate;

[0119] (vi) adding water to the precipitate obtained in (v), ultrasonic dispersion, to obtain a first MoS2-PEI nanoparticle aqueous solution.

[0120] As an optional embodiment, the power of the ultrasonic dispersion in steps (ii), (iv), (v), and (vi) is independently 100-300 W, for example, 100 W, 120 W, 140 W, 160 W, 180 W, 200 W, 220 W, 240 W, 260 W, 280 W, 300 W, etc., and the time of the ultrasonic dispersion is independently 30-60 min, for example, 30 min, 35 min, 40 min, 50 min, 55 min, 60 min, etc.

[0121] As an optional embodiment, the centrifugal force of the centrifugation in steps (iii), (iv), and (v) is 7000-7197 rcf, for example, 7000 rcf, 7100 rcf, 7200 rcf, 7300 rcf, 7400 rcf, 7500 rcf, etc., and the time of the centrifugation is 30-60 min, for example, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 60 min, etc.

[0122] As an optional embodiment, S2 adopts polyethyleneimine coating of the first MoS2@QD, which is completely consistent with the above steps (i)-(vi), and the only difference is that the MoS2 NSs are replaced by the first MoS2@QD, and other steps and specific parameter selection are completely consistent.

[0123] As an optional embodiment, S3 adopts polyethyleneimine coating of the second MoS2@QD, which is completely consistent with the above steps (i)-(vi), and the only difference is that the MoS2 NSs are replaced by the second MoS2@QD, and other steps and specific parameter selection are completely consistent.

[0124] As an optional embodiment, the step of electrostatically adsorbing the first CdSe-ZnS quantum dots on the surface of the first MoS2-PEI includes:

[0125] The first MoS2-PEI, the first CdSe-ZnS quantum dots and water are mixed and ultrasonic dispersed, the first CdSe-ZnS quantum dots are electrostatically adsorbed on the surface of the first MoS2-PEI, a first coating layer is formed, and then the excess first CdSe-ZnS quantum dots are washed away to obtain the first MoS2@QD.

[0126] As an optional embodiment, the step of electrostatically adsorbing the second CdSe-ZnS quantum dots on the surface of the second MoS2-PEI comprises:

[0127] The second MoS2-PEI, the second CdSe-ZnS quantum dots and water are mixed and ultrasonic dispersed, the second CdSe-ZnS quantum dots are electrostatically adsorbed on the surface of the second MoS2-PEI, a second coating layer is formed, and then the excess second CdSe-ZnS quantum dots are washed away to obtain the second MoS2@QD.

[0128] As an optional embodiment, the step of electrostatically adsorbing the third CdSe-ZnS quantum dots on the surface of the third MoS2-PEI comprises:

[0129] The third MoS2-PEI, the third CdSe-ZnS quantum dots and water are mixed and ultrasonic dispersed, the third CdSe-ZnS quantum dots are electrostatically adsorbed on the surface of the third MoS2-PEI, a third coating layer is formed, and then the excess third CdSe-ZnS quantum dots are washed away to obtain the third MoS2@QD.

[0130] As an optional embodiment, in the process of preparing the first coating layer, the mass ratio of the MoS2 nanosheet and the CdSe-ZnS quantum dots is (6-7):1, for example, it can be 6:1, 6.1:1, 6.2:1, 6.3:1, 6.4:1, 6.5:1, 6.6:1, 6.7:1, 6.8:1, 6.9:1, 7:1, etc.

[0131] As an optional embodiment, in the process of preparing the second coating layer, the mass ratio of the MoS2 nanosheet and the CdSe-ZnS quantum dots is (6-7):1, for example, it can be 6:1, 6.1:1, 6.2:1, 6.3:1, 6.4:1, 6.5:1, 6.6:1, 6.7:1, 6.8:1, 6.9:1, 7:1, etc.

[0132] As an optional embodiment, in the process of preparing the third coating layer, the mass ratio of the MoS2 nanosheet and the CdSe-ZnS quantum dots is (6-7):1, for example, it can be 6:1, 6.1:1, 6.2:1, 6.3:1, 6.4:1, 6.5:1, 6.6:1, 6.7:1, 6.8:1, 6.9:1, 7:1, etc.

[0133] As an optional embodiment, the power of the ultrasonic dispersion is 100-300 W, for example, it can be 100 W, 120 W, 140 W, 160 W, 180 W, 200 W, 220 W, 240 W, 260 W, 280 W, 300 W, etc., and the time of the ultrasonic dispersion is 50-60 min, for example, it can be 50 min, 52 min, 54 min, 56 min, 58 min, 60 min, etc.

[0134] As an optional embodiment, the step of washing away excess quantum dots includes: centrifuging and discarding the supernatant, the centrifugal speed is 5000-6200 rpm, for example, it can be 5000 rpm, 5200 rpm, 5400 rpm, 5500 rpm, 5600 rpm, 5800 rpm, 6000 rpm, etc., and the centrifugal time is 5-10 min, for example, it can be 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, etc.

[0135] As an optional embodiment, the preparation method further includes a resuspension step: resuspending the two-dimensional molybdenum sulfide quantum dot nanomaterial in an ethanol solution to obtain an ethanol solution of MoS2@TQD.

[0136] As an optional embodiment, S1 electrostatically adsorbing the first CdSe-ZnS quantum dots on the surface of the first MoS2-PEI specifically includes the following steps:

[0137] (a) adding a CdSe-ZnS quantum dot solution to the aqueous solution of the first MoS2-PEI nanoparticles obtained in (vi), and performing ultrasonic dispersion to obtain a mixed solution;

[0138] (b) centrifuging the mixture obtained in (a), discarding the supernatant, and retaining the precipitate;

[0139] (c) adding water to the precipitate obtained in (b), performing ultrasonic dispersion, and then centrifuging, discarding the supernatant, and retaining the precipitate;

[0140] (d) Add ethanol to the precipitate obtained in (c) to resuspend it and perform ultrasonic dispersion to obtain an ethanol solution of the first MoS2@QD nanoparticles.

[0141] As an optional embodiment, the power of the ultrasonic dispersion in steps (a), (c), and (d) is independently 100-300 W, for example, 100 W, 120 W, 140 W, 160 W, 180 W, 200 W, 220 W, 240 W, 260 W, 280 W, 300 W, or the like, and the time of the ultrasonic dispersion is independently 1-60 min, for example, 1 min, 5 min, 10 min, 20 min, 25 min, 30 min, 35 min, 40 min, 50 min, 55 min, 60 min, or the like.

[0142] As an optional embodiment, the centrifugation speed in steps (b) and (c) is 5000-6200 rpm, for example, 5000 rpm, 5200 rpm, 5400 rpm, 5500 rpm, 5600 rpm, 5800 rpm, 6000 rpm, or the like, and the centrifugation time is 5-20 min, for example, 5 min, 10 min, 15 min, 20 min, or the like.

[0143] As an optional embodiment, S2 electrostatically adsorbs the second CdSe-ZnS quantum dots on the surface of the second MoS2-PEI, which is completely consistent with steps (a)-(d) above, and the only difference is that the first MoS2-PEI nanoparticles are replaced by the second MoS2-PEI nanoparticles, and other steps and specific parameter selections are completely consistent.

[0144] As an optional embodiment, S3 electrostatically adsorbs the third CdSe-ZnS quantum dots on the surface of the third MoS2-PEI, which is completely consistent with steps (a)-(d) above, and the only difference is that the first MoS2-PEI nanoparticles are replaced by the third MoS2-PEI nanoparticles, and other steps and specific parameter selections are completely consistent.

[0145] As an optional embodiment, the concentration of the MoS2@TQD ethanol solution is 1-2 g / L, for example, 1 g / L, 1.2 g / L, 1.4 g / L, 1.5 g / L, 1.6 g / L, 1.8 g / L, 2 g / L, or the like.

[0146] In a third aspect, the application provides an immune MoS2@TQD fluorescent tag, which is a carboxyfluorescein antibody-labeled two-dimensional MoS2 quantum dot-based nanomaterial as described in the first aspect.

[0147] In the present application, the carboxyl fluorescein antibody (hereinafter referred to as anti-FAM antibody) is modified on the surface of the two-dimensional molybdenum sulfide quantum dot nanomaterial (hereinafter referred to as MoS2@TQD NSs), obtaining the immunized MoS2@TQD fluorescent tag, which has excellent optical stability, and has excellent stability in a high-salt environment, that is, it indeed exhibits stable fluorescence intensity in a high-salinity solution (10-1000 mM NaCl), and the fluorescence of the fluorescent tag stored in ethanol remains good within 60 days; and the immunized MoS2@TQD fluorescent tag has good dispersibility and stability, and has a colorimetric imaging and fluorescent quantitative dual mode, and can be used as an excellent signal tag for a nucleic acid on-site rapid detection system.

[0148] In a fourth aspect, the present application provides a preparation method of the immunized MoS2@TQD fluorescent tag according to the third aspect, as shown in Figure 1 The preparation method comprises:

[0149] The two-dimensional molybdenum sulfide quantum dot nanomaterial is resuspended in a MES buffer containing EDC and NHS, activated by a carboxyl group, obtaining a carboxyl-activated MoS2@TQD; then the carboxyl-activated MoS2@TQD is mixed with a carboxyl fluorescein antibody (FAM-antibody) and incubated; bovine serum albumin is then added for blocking; and after washing, the immunized MoS2@TQD fluorescent tag is obtained.

[0150] As an optional embodiment, the mass ratio of the two-dimensional molybdenum sulfide quantum dot nanomaterial, EDC and NHS is 1:(0.05-0.1):(0.1-0.5);

[0151] For example, "0.05-0.1" can be 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, etc.

[0152] For example, "0.1-0.5" can be 0.1, 0.2, 0.3, 0.4, 0.5, etc.

[0153] As an optional embodiment, the carboxyl activation is carried out under ultrasonic conditions, the power of the ultrasonic is 100-300 W, for example, it can be 100 W, 120 W, 140 W, 160 W, 180 W, 200 W, 220 W, 240 W, 260 W, 280 W, 300 W, etc., and the time of the ultrasonic is 10-20 min, for example, it can be 10 min, 12 min, 14 min, 15 min, 16 min, 18 min, 20 min, etc.

[0154] As an optional implementation, the mass ratio of the carboxyl-activated MoS2@TQD, the anti-carboxyfluorescein antibody and the bovine serum albumin is 1:(0.01-0.05):(5-15).

[0155] For example, the "0.01-0.05" can be 0.01, 0.02, 0.03, 0.04, 0.05, etc.

[0156] For example, the "5-15" can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, etc.

[0157] As an optional implementation, the incubation time is 1-3h, for example, 1h, 1.5h, 2h, 2.5h, 3h, etc.

[0158] As an optional implementation, the washing uses a PBST buffer.

[0159] As an optional implementation, the immune MoS2@TQD fluorescent tag is stored in a gold standard diluent with a pH value of 7.4.

[0160] In a fifth aspect, the application provides an application of the two-dimensional molybdenum sulfide quantum dot nanomaterial or the immune MoS2@TQD fluorescent tag in preparing a product for detecting SARS-CoV-2 virus.

[0161] In a sixth aspect, the application provides a kit for detecting SARS-CoV-2 virus, and the product for detecting SARS-CoV-2 virus comprises the immune MoS2@TQD fluorescent tag according to the third aspect, a CRISPR-Cas13a system for specifically detecting SARS-CoV-2 virus, and a lateral chromatography test paper.

[0162] The CRISPR-Cas13a system for specifically detecting SARS-CoV-2 virus comprises a primer group, wherein the primer group comprises an RT-RAA amplification primer group and a nucleotide sequence required by a CRISPR-Cas13a enzyme cutting detection system; the RT-RAA amplification primer group comprises an upstream primer of a SARS-CoV-2 E gene and a downstream primer of the SARS-CoV-2 E gene, the sequence of the upstream primer is shown as SEQ ID No: 1, and the sequence of the downstream primer is shown as SEQ ID No: 2; the nucleotide sequence required by the CRISPR-Cas13a enzyme cutting detection system comprises a specific crRNA, and the sequence of the specific crRNA is shown as SEQ ID No: 3.

[0163] The lateral chromatographic test paper comprises a nitrocellulose membrane, a sample pad and an absorption pad fixed on a PVC bottom plate; the nitrocellulose membrane is provided with a test line T and a quality control line C which are spaced apart along the chromatography direction of the sample to be detected; the test line T is coated with streptavidin, and the quality control line C is coated with a goat anti-rabbit IgG antibody.

[0164] As an optional implementation, the CRISPR-Cas13a system for specifically detecting SARS-CoV-2 virus further comprises a reporter probe, which is a fluorescent reporter probe, one end of which is labeled with biotin and the other end of which is labeled with a fluorescent group FAM, the reporter probe being used to be cut by the activated Cas13a and release fluorescence;

[0165] As an optional implementation, the CRISPR-Cas13a system for specifically detecting SARS-CoV-2 virus further comprises an RT-RAA amplification system and a CRISPR-Cas13a enzyme cutting system; the RT-RAA amplification system comprises a buffer, enzyme-free water and a template RNA; and the CRISPR-Cas13a enzyme cutting system comprises a buffer, enzyme-free water, a ribonucleotide mixed solution, a ribonuclease inhibitor, Cas13a protein, T7 RNA polymerase and magnesium chloride.

[0166] In the application, the RT-RAA / CRISPR-Cas13 is combined with the MoS2@TQD LFA to obtain a kit for detecting SARS-CoV-2 virus, which is the first research on the combination of two-dimensional MoS2@TQD composite nanomaterials and LFA for nucleic acid detection based on CRISPR-Cas13; in the process of detecting SARS-CoV-2 virus (the process of detecting SARS-CoV-2 virus using the kit for detecting SARS-CoV-2 virus described in the application), as Figure 2As shown, first, the nucleic acid extracted by thermal cracking is subjected to RT-RAA amplification at 42℃ for 20 min, and the RT-RAA reaction is used to reverse transcribe the RNA into complementary DNA (cDNA), and then amplification is performed to obtain sufficient target nucleic acid. Then the amplification product is specifically identified and cleaved by the CRISPR-Cas13 reaction. The cleavage product is mixed with MoS2@TQD labeled with anti-carboxyfluorescein (FAM) antibody, and is added into the buffer solution with the streptavidin (SA) binding strip on the test line (T line), and is reacted for 10 min. Finally, the T line signal value is obtained by using a fluorescence detector, and a fitting curve is drawn according to the T line fluorescence values of nucleic acids of different concentrations, which can be used for quantitative detection of nucleic acids. In summary, the detection method is simple, high in sensitivity, good in stability, high in repeatability, and strong in specificity, has clinical feasibility and broad application prospect, and can be used for detection and prevention of SARS-CoV-2 virus and infectious pathogens. Finally, the T line signal value is obtained by using a fluorescence detector, and a fitting curve is drawn according to the T line fluorescence values of nucleic acids of different concentrations, which can be used for quantitative detection of nucleic acids. In summary, the detection method is simple, high in sensitivity, good in stability, high in repeatability, and strong in specificity, has clinical feasibility and broad application prospect, and can be used for detection and prevention of SARS-CoV-2 virus and infectious pathogens.

[0167] The application will be further described by the following examples. Unless otherwise specified, the materials in the examples are prepared according to the existing methods or directly purchased from the market.

[0168] Example 1

[0169] The embodiment provides a two-dimensional molybdenum sulfide quantum dot nanomaterial (MoS2@TQD NSs) and an immune MoS2@TQD fluorescent tag.

[0170] The preparation method of the two-dimensional molybdenum sulfide quantum dot nanomaterial (MoS2@TQD NSs) and the immune MoS2@TQD fluorescent tag includes the following steps (as shown in Figure 1

[0171] S1, coating a first PEI layer:

[0172] ​Take 2.5mL MoS2 nanosheets and dissolve them in 7.5mL ultrapure water to obtain an aqueous solution of MoS2 NSs; add 20mg / mL 1 mL of PEI aqueous solution was sealed with a sealing film and ultrasonically dispersed in an ultrasonic instrument at a power of 150 W for 30 minutes to obtain a mixed solution; the mixed solution was centrifuged at 7197 rcf for 15 minutes, the supernatant was discarded, and the precipitate was retained in the above-mentioned centrifuge tube; 5 mL of ultrapure water was taken in the above-mentioned centrifuge tube, ultrasonically dispersed the precipitate at a power of 150 W for 1 minute in an ultrasonic instrument, centrifuged at 7197 rcf for 15 minutes, the supernatant was discarded, and the precipitate was retained in the above-mentioned centrifuge tube; 5 mL of ultrapure water was taken in the above-mentioned centrifuge tube again, ultrasonically dispersed the precipitate at a power of 150 W for 1 minute in an ultrasonic instrument, centrifuged at 7197 rcf for 15 minutes, the supernatant was discarded, and the precipitate was retained in the above-mentioned centrifuge tube; finally, 5 mL of ultrapure water was taken in the above-mentioned centrifuge tube and ultrasonically dispersed at a power of 150 W for 1 minute to obtain an aqueous solution of MoS2-PEI nanoparticles.

[0173] S2. Adsorption of the first CdSe / ZnS quantum dots:

[0174] Take 5mL of the aqueous solution of MoS2-PEI nanoparticles prepared by S1, add 40μL of CdSe / ZnS quantum dots with a concentration of 10mg / mL (purchased from Suzhou Xingshuo Nano CdSe / ZnS-MPA-631QDs), and ultrasonically disperse them in an ultrasonic instrument at a power of 150W for 50min; the ultrasonically dispersed solution is centrifuged at 5600rpm for 6min, and the supernatant is discarded; take 5mL of ultrapure water in the above centrifuge tube, ultrasonically disperse the precipitate in an ultrasonic instrument for 1min, centrifuge at 5600rpm for 6min, and discard the supernatant; take 2mL of anhydrous ethanol to resuspend the precipitate, and obtain the MoS2@QD nanoparticle ethanol solution after ultrasonication; store at 4℃ until used.

[0175] S3, coating the second PEI layer:

[0176] Repeat the steps of S1, except that the MoS2 nanosheets were replaced with 2 mL of S2 to prepare the MoS2@QD nanoparticle ethanol solution.

[0177] S4. Adsorption of the second CdSe / ZnS quantum dots:

[0178] Repeat the steps of S2, except that the aqueous solution of MoS2-PEI nanoparticles prepared in S1 is replaced with 5 mL of the solution prepared in S3.

[0179] S5, coating the third PEI layer:

[0180] The steps of S1 are repeated, with the only difference being that the MoS2 nanosheets are replaced by 2 mL of the solution prepared in S4.

[0181] S6, adsorption of third CdSe / ZnS quantum dots:

[0182] The steps of S2 are repeated, with the only difference being that the aqueous solution of MoS2-PEI nanoparticles prepared in S1 is replaced by 5 mL of the solution prepared in S5, resulting in an ethanol solution of MoS2@TQD NSs with a concentration of 1.25 g / L, which is stored at 4°C for later use.

[0183] S7, binding of anti-FAM antibody (preparation of immunized MoS2@TQD fluorescent tag):

[0184] Take 0.2 mL of MoS2@TQD NSs and resuspend in MES buffer (0.1 M, pH 5.5) containing EDC (10 mM, 10 μL) and NHS (10 mM, 20 μL), activate the carboxyl groups of MoS2@TQD NSs under ultrasonic conditions for 15 min, after purification, mix 0.5 mL of carboxyl-activated MoS2@TQD NSs with 25 μg of anti-FAM antibody, incubate at 37°C for 2 h, then add 100 μL of 10% bovine serum albumin (BSA) to block the unreacted carboxyl sites of MoS2@TQD NSs, finally, wash the immunized MoS2@TQD fluorescent tag-modified anti-FAM antibody 2 times with 0.05% PBST buffer, and store the prepared immunized MoS2@TQD fluorescent tag in gold standard diluent with a pH value of 7.4.

[0185] Example 2

[0186] The present embodiment provides a kit for detecting SARS-CoV-2 virus, and the product for detecting SARS-CoV-2 virus comprises: the immunized MoS2@TQD fluorescent tag provided in Example 1, a CRISPR-Cas13a system for specifically detecting SARS-CoV-2 virus, and a lateral flow test paper.

[0187] The CRISPR-Cas13a system for specifically detecting SARS-CoV-2 virus comprises a primer set, wherein the primer set comprises an RT-RAA amplification primer set and nucleotide sequences required by a CRISPR-Cas13a enzyme cutting detection system; the RT-RAA amplification primer set comprises an upstream primer of a SARS-CoV-2 E gene and a downstream primer of the SARS-CoV-2 E gene, the sequence of the upstream primer is shown as SEQ ID No: 1, and the sequence of the downstream primer is shown as SEQ ID No: 2; the nucleotide sequences required by the CRISPR-Cas13a enzyme cutting detection system comprise a specific crRNA, the sequence of the specific crRNA is shown as SEQ ID No: 3; and the specific CRISPR-Cas13a enzyme cutting detection system is shown in Table 1 as follows:

[0188] Table 1

[0189]

[0190] The CRISPR-Cas13a system for specifically detecting SARS-CoV-2 virus further comprises an RT-RAA amplification system and a CRISPR-Cas13a enzyme cutting system, wherein the RT-RAA amplification system comprises a buffer, enzyme-free water and a template RNA; and the CRISPR-Cas13a enzyme cutting system comprises a buffer, enzyme-free water, a ribonucleotide mixed solution, a ribonuclease inhibitor, a Cas13a protein, a T7 RNA polymerase and magnesium chloride;

[0191] The lateral chromatography test paper is composed of four parts: a nitrocellulose (NC) membrane, a sample pad, an absorbent pad and a PVC base plate. SA (0.6 mg / mL) and goat anti-rabbit IgG antibody (1 mg / mL) are respectively sprayed on the NC membrane as a T line and a control line (C line). After the prepared NC membrane is dried in an oven at 37℃ for 4 h, the NC membrane, the sample pad and the absorbent pad are assembled on the PVC base plate. Two pad plates respectively overlap the NC membrane by 1.5 mm; the assembled card is cut into a 3 mm wide strip, and stored in a desiccator (25℃) for detection;

[0192] The CRISPR-Cas13a system for specifically detecting SARS-CoV-2 virus further comprises a reporter probe, which is a fluorescent reporter probe Bio-RNA-FAM, one end of which is labeled with biotin and the other end of which is labeled with a fluorescent group FAM, and the reporter probe is used for being activated and cut by the active Cas13a and releasing fluorescence;

[0193] Specifically, the use method of the kit for detecting SARS-CoV-2 virus in the embodiment comprises the following steps (as shown in Figure 2 the drawing).

[0194] Use 25 μL of the A buffer of the crowd-test kit, 2 μL of the upstream primer and downstream primer of the SARS-CoV-2E gene, mix with 13.5 μL of enzyme-free water, 5 μL of template RNA and 2.5 μL of the B buffer of the crowd-test kit, and amplify at 42°C for 20 minutes; then, add 5 μL of the amplified product to the CRISPR Cas13a system: 26.5 μL of enzyme-free water, 4 μL of ribonucleotide mixed solution (NTP), 2 μL of ribonuclease inhibitor (RNase inhibitor), 2 μL of Cas13a, 1 μL of T7 RNA polymerase, 1 μL of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, 0.5 μL of magnesium chloride (MgCl2), 3 μL of CRISPR RNA (crRNA) and 5 μL of 100nM reporter probe, and incubate at 37°C for 10 minutes; 8 μL The CRISPR reaction solution and 1 μL of the immune MoS2@TQD fluorescent label prepared in Example 1 were mixed into 72 μL of running buffer (phosphate-buffered saline (1% PBST) containing 1% Tween-20 + 5% fetal bovine serum (FBS) + 5% BSA), and the above-mentioned lateral flow test paper was placed in the mixture; after 5 minutes, the data was read using a fluorometer.

[0195] Test Example 1

[0196] Characterization of morphology, structure and physicochemical properties

[0197] Test sample: Characterization of two-dimensional molybdenum sulfide quantum dot nanomaterials (MoS2@TQD NSs) and immune MoS2@TQD fluorescent labels provided in Example 1.

[0198] Test methods: transmission electron microscopy (TEM), high-resolution transmission electron microscopy (HRTEM), energy dispersive X-ray spectroscopy, and Fourier transform infrared spectroscopy.

[0199] Test results: as shown in Figure 3.

[0200] Figure 3A TEM image of the two-dimensional molybdenum sulfide nanosheet provided in Example 1. Figure 3A As shown, MoS2 NSs present a folded and stacked film structure, and the folding increases the specific surface area, thereby providing more attachment points for quantum dots.

[0201] Figure 3B TEM image of the two-dimensional molybdenum sulfide nanosheet coated with the first PEI layer provided in Example 1. Figure 3B As shown, high-resolution TEM observations magnified by PEI revealed that a transparent thin layer with a thickness of approximately 4 nm was formed on the surface of MoS2 NSs.

[0202] Figure 3C TEM image (200 nm) of MoS2@TQD NSs provided for Example 1. Figure 3D TEM magnified image (50 nm) of MoS2@TQD NSs provided for Example 1. As shown in Figure 3C and 3D High resolution transmission electron microscopy (HRTEM) shows that CdSe / ZnS QDs are densely and uniformly distributed on the surface of MoS2 NSs.

[0203] Figure 3E EDS elemental analysis image of MoS2@TQD NSs provided for Example 1. As shown in Figure 3E Energy dispersive X-ray spectroscopy (EDS) clearly shows the distribution of main elements of MoS2@TQD. S is the common element of QDs and MoS2, while Cd, Se and Zn are densely distributed on the surface of MoS2 (Mo).

[0204] Figure 3F Elemental line scanning result image of MoS2@TQD NSs provided for Example 1. As shown in Figure 3F EDS line scanning proves that CdSe QDs are successfully adsorbed and densely distributed on the surface of MoS2.

[0205] Figure 3G Zeta potential images of products in each step of the process of preparing MoS2@TQD NSs for Example 1. As shown in Figure 3G The preparation of MoS2@TQD relies on the principle of electrostatic adsorption bridged by PEI. The change of zeta potential of each layer of PEI and QD assembly confirms the successful application of this principle in assembling QDs on MoS2.

[0206] Figure 3H XPS spectrum image of MoS2@TQD NSs provided for Example 1. As shown in Figure 3H X-ray photoelectron spectroscopy (XPS) further studies the surface properties of MoS2@TQD NSs. The existence of Mo, S, Cd, Se, Zn and other elements in MoS2@TQD NSs is shown. The high resolution scanning images of Mo 3d, Cd 3d, Zn 2p, S2p and Se 3d are shown respectively. The double peaks at 228.06 and 231.2 eV in Mo 3d spectrum represent Mo 6+The presence of. The cadmium 3d spectrum shows two fitting peaks at 404.6 and 411.32 eV, which belong to cadmium selenide 3d5 / 2 and cadmium selenide 3d3 / 2, respectively. The zinc 2p spectrum shows two fitting peaks at 1021.83 and 1044.99 eV, which belong to ZnS2p3 / 2 and ZnS2p1 / 2, respectively. The sulfur 2p spectrum fits four peaks at 161, 162.3, 161.96 and 163.15 eV, which are attributed to ZnS and MoS2, respectively. The high-resolution spectrum of selenium 3d fits two double peaks. The double peaks at 53.23 and 54.03 eV are the characteristics of CdSe 3d5 / 2 and CdSe 3d3 / 2, respectively. The above results confirm that QDs are assembled layer by layer on the MoS2 surface.

[0207] Figure 3I The fluorescence emission spectra of the products in each step of the preparation process of MoS2@TQD NSs in Example 1 are shown. Figure 3I As shown, the engineered fluorescent nanosheets transfer fluorescence signals and carboxyl groups to the MoS2 surface via QDs. Under excitation with 365nm UV light, the MoS2@QD NSs, MoS2@DQD NSs, and MoS2@TQD NSs solutions emit red light, demonstrating that the MoS2 surface maintains excellent colorimetric properties even after absorbing dense QDs. MoS2@QD NSs, MoS2@DQD NSs, and MoS2@TQDNSs exhibit strong fluorescence signals at a wavelength of 631nm, while MoS2 and MoS2-PEI exhibit no fluorescence. Although the fluorescence intensities of MoS2@QD NSs and MoS2@DQD NSs are lower compared to those of QDs, this evidence confirms that the fluorescence signals of the fluorescent NSs originate entirely from the QDs. When only a single layer of QDs is present on the MoS2 surface, the quenching effect of MoS2 cannot be effectively alleviated. However, after adding the second layer of QDs, the distance between the MoS2 NS and the outermost QDs effectively overcomes the intrinsic fluorescence enhancement (IFE) of the MoS2 NS. Moreover, the loading capacity of QDs adsorbed on MoS2-based NSs increases exponentially with the increase in the number of QD layers, thus significantly enhancing the luminescence ability of the label.

[0208] In addition, from the Fourier transform infrared spectroscopy ( Figure 3J ) It can be seen that the fluorescent label and anti-FAM antibody are at 1650cm -1 There are obvious characteristic absorption peaks at all locations, proving that the antibody is successfully modified on the surface of MoS2@TQD NSs. We further evaluated the optical stability of MoS2@TQDNSs and its stability in a high salt environment.

[0209] like Figure 3KAs shown, MoS2@TQD NSs indeed exhibited stable fluorescence intensity in high-salinity solutions (10-1000 mM NaCl).

[0210] As shown in Figure 3L The fluorescence of MoS2@TQD NSs stored in ethanol remained good within 60 days. The prepared composite material has good dispersity and stability, and has a dual-mode of colorimetric imaging and fluorescence quantification, which can be used as an excellent signal tag for on-site rapid nucleic acid detection system.

[0211] Test Example 2

[0212] Optimization of CRISPR-assisted dual-mode LFA system

[0213] Test principle: RT-RAA isothermal amplification of RNA template, DNA is converted into RNA by T7 transcription, Cas13a-crRNA complex binds to amplified RNA target sequence, triggers Cas13a side-cutting activity, leading to cleavage of fluorescent reporter RNA molecules. Biotin-FAM probe is used for CRISPR-Cas13-assisted dual-mode LFA strip detection. In negative samples, anti-FAM antibody combined with fluorescent label reacts with biotin-FAM probe to form an immune complex. Then, the immune complex is intercepted by SA on the T line. For positive samples, SA fails to intercept FAM antibody when cutting biotin-FAM probe, resulting in invisible T line. In order to achieve the best detection effect, this study selected the conserved sequence of SARS-CoV-2 E gene as the target gene. The upstream and downstream primers of RT-RAA and crRNA were designed according to the full-length fragment of E gene.

[0214] Test results: as shown in Figure 4.

[0215] Figure 4A Screening diagram of RT-RAA primers and crRNA provided for Test Example 2; (i) screening of RT-RAA primers; (ii) screening of crRNA for RT-RAA-CRISPR-Cas13a assay, fluorescence value kinetic curves of 4 crRNAs within 30 cycles.

[0216] As shown in Figure 4A (i), the amplification products of different primers were detected by electrophoresis, and the upstream and downstream primers were screened according to the fluorescence bands. The electrophoresis results of amplification products of different upstream and downstream primers showed that F1R1 band was within the product range, and there was no impurity band in the band, indicating good specificity. Therefore, F1R1 was selected as the amplification primer pair for SARS-CoV-2 E gene.

[0217] As shown in Figure 4A(i) As shown, among the four crRNAs, the fluorescence signal detection dynamic curve of crRNA2 is the highest, showing the fluorescence detection value of CRISPR-Cas13a at 30 min. It can be seen that the fluorescence values of crRNA1, crRNA2, crRNA3 and crRNA4 have statistical differences with the fluorescence value of the negative control (NC), and the fluorescence value of crRNA2 is the highest.

[0218] Based on this, the present application selects crRNA2 as the crRNA for detecting SARS-CoV-2 E gene. The feasibility of CRISPR-assisted dual-mode LFA strip is verified by using SARS-CoV-2 virus with a concentration of 10 3 copies / μL and enzyme-free water (negative control), which not only has a color developing function, but also can be quantified according to the fluorescence signal.

[0219] Figure 4B The confirmation test provided for Test Example 2 is shown in the following table. The error bar represents the standard deviation (n = 3).

[0220] As Figure 4B shown, the negative group has obvious color development and high fluorescence signal value, while the positive group has no obvious color development and low fluorescence signal value, proving the feasibility of the experiment under the conditions of F1R1 and crRNA2.

[0221] In order to obtain the best detection results, the present experiment studies and optimizes the reaction conditions such as biotin-FAM probe concentration, system reaction time, SA concentration on LFA strip, label amount and running buffer. First, the probe concentration in CRISPR-Cas13 is discussed, and the probe concentration is 50nM, 75nM, 100nM, 125nM and 150nM in the presence and absence of target double-stranded DNA (dsDNA). When the probe concentration is too low, the amplified SARS-CoV-2 RNA cannot be completely cut; when the probe concentration is too high, the signal-to-noise ratio (fluorescence intensity of non-target dsDNA / fluorescence intensity of targeted dsDNA) of the fluorescence signal decreases.

[0222] Figure 4C The result graph of the optimized probe concentration provided for Test Example 2 is shown in the following table. When the concentration of the DNA probe is 100nM, the signal-to-noise ratio is the highest. The cutting time determines whether the probe is completely cut. When the cutting time is too short, the Cas13 protein cannot completely cut all the probes in the system.

[0223] Figure 4DThe result graph of the optimized CRISPR system detection time provided for Test Example 2. When the cutting time reached 10 minutes, the signal-to-noise ratio was the highest. As the cutting time was prolonged, the signal-to-noise ratio began to decline, because the fluorescence intensity of the targeted dsDNA reached a plateau, while the fluorescence intensity of the non-targeted dsDNA began to decline. The running buffer was further optimized. FBS and BSA were added in 1% PBST to block the reaction system. Through competitive adsorption, FBS and BSA can inhibit non-specific binding, effectively reducing background interference. Compared with using FBS or BSA alone, the combination of FBS and BSA has a better signal-to-noise ratio. When the concentration of both is 5%, the signal-to-noise ratio reaches a peak. As the concentration increases, the signal-to-noise ratio decreases.

[0224] Figure 4E The result graph of the optimized T-line concentration provided for Test Example 2. As shown in Figure 4E When the SA concentration is low, not all biotin on the probe can be captured, and the fluorescence intensity on the T-line is weak. When the SA concentration on the T-line is 0.6 mg / mL, the signal-to-noise ratio of the fluorescence result is significantly improved. Therefore, 0.6 mg / mL is selected as the spraying concentration of the T-line.

[0225] Figure 4F The result graph of the optimization of label addition. As shown in Figure 4F If the amount of label addition is insufficient, the anti-FAM antibody on the label cannot fully bind to the FAM on the probe. However, excessive labels will cause non-specific binding to the NC membrane, thereby enhancing the background signal. As can be seen from Figure 4F When the amount of label addition is 1 μL, the signal-to-noise ratio is best.

[0226] Test Example 3

[0227] Performance of RT-RAACRISPR-Cas13 fluorescent strips

[0228] Test method: Under the optimal conditions, the performance of the proposed system was evaluated using serially diluted SARS-CoV-2 RNA (100, 50, 10, 5, 1 and 0.5 copies / μL) and enzyme-free water as a negative control.

[0229] Test results: As shown in Figure 5.

[0230] As shown in Figure 5A With the decrease of RNA concentration, the colorimetric intensity gradually increased, reaching a maximum of 0.5 copies / μL, which was still significantly different from the negative control.

[0231] As shown in Figure 5B The calibration curve constructed according to the T-line fluorescence signal is shown, which has a good correlation coefficient (R 2= 0.98). The limit of detection (LOD) of the CRISPR-Cas13 assisted dual-mode LFA strip for detecting SARS-CoV-2 was 0.25 copies / µL, calculated as Yblank+3SDblank (where Yblank represents the average fluorescence signal of the blank control, and 3SDblank represents three times the standard deviation of the blank control), which was 20 times that of the colloidal gold detection system (5 copies / µL). The colloidal gold detection results are shown in Figure.

[0232] In addition, seven respiratory virus standards were also used as interferents, including influenza A (H3N2, H1N1), influenza B (Victoria), human parainfluenza (HPVIs), human rhinovirus (HRVs), respiratory syncytial virus A and respiratory syncytial virus B, to verify the specificity of the CRISPR assisted dual-mode LFA strip. Each sample was tested in triplicate.

[0233] As shown in Figure Figure 5C , all other pathogen test strips showed obvious colorimetric intensity and high fluorescence signal values, while only the SARS-CoV-2 test strip did not show obvious colorimetric intensity and high fluorescence signal values. These results demonstrate the specificity of the CRISPR assisted dual-mode LFA detection strip.

[0234] In addition, the stability of the method was also verified using two sample concentrations, low concentration (1 copies / µL) and medium concentration (50 copies / µL), and the LFA system was tested using 5 different test strips. The colorimetric intensity of each batch of T lines was essentially the same. At a concentration of 1 copies / µL (Group 1), the relative standard deviation (RSD) of the 5 independent strips was 2.6%, and at a concentration of 50 copies / µL (Group 2), the RSD of the 5 independent strips was 6.0% (as shown in Figure Figure 5D ), showing good reproducibility.

[0235] Test Example 4

[0236] Clinical sample verification

[0237] According to the steps of nucleic acid extraction and purification reagents, nucleic acid extraction and purification were performed on clinical samples using a fully automated nucleic acid extractor, and a clinical verification kit meeting the national sensitivity reference value S was used for PCR detection of SARS-CoV-2. According to the instructions of the clinical kit, a cycle threshold (Ct) of less than 40 was positive. All clinical samples were obtained from the Chinese People's Liberation Army Center for Disease Control and Prevention (Ethical Approval Number: Z2022SY013). The extracted nucleic acids were added to the above-mentioned CRISPR assisted dual-mode LFA test strip system for clinical sample test strip detection.

[0238] To further evaluate the clinical feasibility of the CRISPR-assisted dual-mode LFA test strip we proposed, we tested 53 clinical samples, including 35 SARS-CoV-2 patients and 18 healthy people.

[0239] Test results: as shown in Figure 6.

[0240] As Figure 6A and Figure 6B can be seen, for negative and high-concentration positive samples (Ct value less than 36), the system can be directly identified by colorimetric method. For low-concentration samples (Ct value between 36 and 40), the system can be quantitatively detected by fluorescence signal.

[0241] As Figure 6D and Figure 6E can be seen, in addition, the quantitative detection results are consistent with the results of commercial fluorescent quantitative PCR, and the PCR results of clinical samples. Receiver operating characteristic curve (ROC) and confusion matrix are used to evaluate the diagnostic ability of the method.

[0242] As Figure 6F and Table 2 below show that the system has extremely high diagnostic accuracy (area under the curve (AUC) = 1), with sensitivity, specificity and agreement rate all being 100%. There is no missed positive sample, and no false positive result appears in negative samples.

[0243] Table 2

[0244]

[0245] In Table 2 above, the coincidence rate (%) = [(35+18) / (35+18)]x100% = 100%; the sensitivity (%) = [35 / 35]x100% = 100%; the specificity (%) = [18 / 18]x100% = 100%.

[0246] To compare the sensitivity of the CRISPR-assisted dual-mode LFA test strip we proposed with that of commercial colloidal gold test strip, we performed colloidal gold detection.

[0247] As Figure 6C shown, according to the visualization results of colloidal gold test strip, it can be seen that 18 negative samples are detected, but 5 of the 35 positive samples cannot be detected. Therefore, the coincidence rate of colloidal gold detection is 90.56% (see Table 3 below). Compared with the traditional colloidal gold detection method which can only rely on simple colorimetric detection and cannot accurately distinguish low-concentration samples from negative samples, this CRISPR-assisted dual-mode LFA test strip has higher accuracy and sensitivity.

[0248] Table 3

[0249]

[0250] In Table 3 above, the coincidence rate (%) = [(30+18) / (35+18)]x100% = 90.56%; the sensitivity (%) = [30 / 30]x100% = 100%; the specificity (%) = [18 / (5+18)]x100% = 78.26%.

[0251] In summary, the present application designs a colorimetric quantitative dual-mode MoS2@TQD combined CRISPR-Cas13 LFA detection method for rapidly and sensitively detecting SARS-CoV-2 virus in clinical samples. Compared with a single QD, MoS2@TQD shows more superior colorimetric and fluorescent signals, and better stability. Based on CRISPR-assisted MoS2@TQD LFA, rapid colorimetric screening and fluorescent quantification of SARS-CoV-2 nucleic acid can be achieved within 35 minutes. The colorimetric mode can screen SARS-CoV-2 with a visual limit of detection (vLOD) of 500 copies / mL, and the fluorescent signal mode can quantitatively detect SARS-CoV-2 with an LOD as low as 250 copies / mL, which is 20 times more sensitive than colloidal gold detection. In addition, 35 SARS-CoV-2 positive samples and 18 SARS-CoV-2 negative samples were detected by dual-mode LFA, and the results showed that the accuracy was 100%, and the consistency with the quantitative detection results of polymerase chain reaction was 100%. Therefore, our dual-mode LFA based on CRISPR-assisted MoS2@TQD NSs is a very promising POCT, which can be used for rapid visual screening and fluorescent accurate diagnosis of SARS-CoV-2 and other various pathogenic viruses.

[0252] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present application, and not to limit them; although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing embodiments, or make equivalent replacement for part or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. An immune MoS2@TQD fluorescent label, characterized in that The immuno-MoS2@TQD fluorescent label is a two-dimensional molybdenum sulfide quantum dot nanomaterial labeled with an anti-carboxyfluorescein antibody; The two-dimensional molybdenum sulfide quantum dot nanomaterial includes, from the inside out, molybdenum disulfide nanosheets, a first coating layer, a second coating layer, and a third coating layer; wherein the first coating layer, the second coating layer, and the third coating layer are all PEI layers that adsorb CdSe-ZnS quantum dots.

2. The immunoMoS2@TQD fluorescent label according to claim 1, characterized in that The thickness of the molybdenum disulfide nanosheet is 1 to 5 nm, and the diameter of the molybdenum disulfide nanosheet is 200 to 5000 nm; And / or, the thickness of the first cladding layer, the second cladding layer and the third cladding layer are each independently 7 to 11 nm.

3. A method for preparing the immune MoS2@TQD fluorescent label according to claim 1 or 2, characterized in that: The preparation method comprises the following steps: Coating polyethyleneimine on the surface of the molybdenum disulfide nanosheet to form a first PEI layer to obtain MoS2-PEI, and then electrostatically adsorbing CdSe-ZnS quantum dots on the surface of the MoS2-PEI to form a first coating layer to obtain a first MoS2@QD; Repeat the steps of polyethyleneimine coating and electrostatic adsorption of CdSe-ZnS quantum dots to form a second coating layer on the surface of the first MoS2@QD to obtain a second MoS2@QD; Repeat the steps of polyethyleneimine coating and electrostatic adsorption of CdSe-ZnS quantum dots once more to form a third coating layer on the surface of the second MoS2@QD to obtain the two-dimensional molybdenum sulfide quantum dot nanomaterial; The two-dimensional molybdenum sulfide quantum dot nanomaterial is resuspended in MES buffer containing EDC and NHS, and activated by carboxyl groups to obtain carboxyl-activated MoS2@TQD; the carboxyl-activated MoS2@TQD is then mixed with anti-carboxyfluorescein antibody and incubated; bovine serum albumin is then added for blocking; and after washing, the immune MoS2@TQD fluorescent label is obtained.

4. The method for preparing the immune MoS2@TQD fluorescent label according to claim 3, characterized in that: The steps of coating molybdenum disulfide nanosheets with polyethyleneimine include: MoS2 nanosheets, polyethyleneimine and water are mixed and ultrasonically dispersed, and the positively charged polyethyleneimine is coated on the surface of the negatively charged MoS2 nanosheets, and then the excess polyethyleneimine is washed away to obtain positively charged MoS2-PEI; And / or, in the process of preparing the first coating layer, the mass ratio of the molybdenum disulfide nanosheets to polyethyleneimine is (600-650):1; In the process of preparing the second coating layer, the mass ratio of the molybdenum disulfide nanosheets to polyethyleneimine is (600-650):1; In the process of preparing the third coating layer, the mass ratio of the molybdenum disulfide nanosheets to polyethyleneimine is (600-650):1; And / or, the power of the ultrasonic dispersion is 100 to 300 W, and the time of the ultrasonic dispersion is 20 to 60 minutes; And / or, the step of washing away excess polyethyleneimine comprises: centrifuging and discarding the supernatant, the centrifugal force of the centrifugation is 6000-7197 rcf, and the centrifugation time is 10-60 min.

5. The method for preparing the immune MoS2@TQD fluorescent label according to claim 3, characterized in that: The step of electrostatically adsorbing CdSe-ZnS quantum dots comprises: MoS2-PEI, CdSe-ZnS quantum dots and water are mixed and ultrasonically dispersed, and the CdSe-ZnS quantum dots are electrostatically adsorbed on the surface of the MoS2-PEI to form a first coating layer, and then excess CdSe-ZnS quantum dots are washed away to obtain a first MoS2@QD; And / or, in the process of preparing the first coating layer, the mass ratio of the molybdenum disulfide nanosheets to the CdSe-ZnS quantum dots is (6-7):1; In the process of preparing the second coating layer, the mass ratio of the molybdenum disulfide nanosheets and the CdSe-ZnS quantum dots is (6-7):1; In the process of preparing the third coating layer, the mass ratio of the molybdenum disulfide nanosheets to the CdSe-ZnS quantum dots is (6-7):1; And / or, the power of the ultrasonic dispersion is 100 to 300 W, and the time of the ultrasonic dispersion is 50 to 60 minutes; And / or, the step of washing away excess quantum dots comprises: centrifuging and discarding the supernatant, the centrifugal speed is 5000-6200 rpm, and the centrifugal time is 5-10 min. And / or, the preparation method further comprises a resuspending step: resuspending the two-dimensional molybdenum sulfide quantum dot nanomaterial in an ethanol solution to obtain an ethanol solution of MoS2@TQD; And / or, the concentration of the MoS2@TQD ethanol solution is 1-2 g / L.

6. The method for preparing the immune MoS2@TQD fluorescent label according to claim 3, characterized in that: The preparation method comprises: The two-dimensional molybdenum sulfide quantum dot nanomaterial is resuspended in MES buffer containing EDC and NHS, and activated by carboxyl groups to obtain carboxyl-activated MoS2@TQD; the carboxyl-activated MoS2@TQD is then mixed with anti-carboxyfluorescein antibody and incubated; bovine serum albumin is then added for blocking; and after washing, the immuno-MoS2@TQD fluorescent label is obtained; And / or, the mass ratio of the two-dimensional molybdenum sulfide quantum dot nanomaterial, EDC and NHS is 1:(0.05-0.1):(0.1-0.5); And / or, the carboxyl activation is performed under ultrasonic conditions, the ultrasonic power is 100 to 300 W, and the ultrasonic time is 10 to 20 minutes; and / or, the mass ratio of the carboxyl-activated MoS2@TQD, anti-carboxyfluorescein antibody, and bovine serum albumin is 1:(0.01-0.05):(5-15); And / or, the incubation time is 2 to 3 hours; And / or, the washing is performed using PBST buffer; And / or, the immuno-MoS2@TQD fluorescent label is stored in a gold standard diluent at a pH of 7.

4.

7. Use of the immune MoS2@TQD fluorescent label according to claim 1 or 2 in the preparation of a product for detecting SARS-CoV-2 virus.

8. A kit for detecting SARS-CoV-2 virus, characterized in that: The product for detecting SARS-CoV-2 virus includes: the immune MoS2@TQD fluorescent label as described in claim 1 or 2, a CRISPR-Cas13a system for specific detection of SARS-CoV-2 virus, and a lateral flow test paper.

9. The kit for detecting SARS-CoV-2 virus according to claim 8, wherein the CRISPR-Cas13a system for specific detection of SARS-CoV-2 virus comprises: primer sets; Wherein, the primer set includes an RT-RAA amplification primer set and a nucleotide sequence required for the CRISPR-Cas13a enzyme cleavage detection system; the RT-RAA amplification primer set includes an upstream primer of the SARS-CoV-2E gene and a downstream primer of the SARS-CoV-2E gene, the sequence of the upstream primer is shown in SEQ ID No: 1, and the sequence of the downstream primer is shown in SEQ ID No: 2; the nucleotide sequence required for the CRISPR-Cas13a enzyme cleavage detection system includes a specific crRNA, and the sequence of the specific crRNA is shown in SEQ ID No: 3; The lateral flow test paper comprises a nitrocellulose membrane, a sample pad and an absorption pad fixed on a PVC bottom plate; the nitrocellulose membrane is provided with a test line T and a quality control line C spaced apart along the chromatography direction of the sample to be tested; the test line T is coated with streptavidin, and the quality control line C is coated with a goat anti-rabbit IgG antibody.

10. The kit for detecting SARS-CoV-2 virus according to claim 8 or 9, characterized in that The CRISPR-Cas13a system for specific detection of SARS-CoV-2 virus also includes: a reporter probe, wherein the reporter probe is a fluorescent reporter probe, one end of which is labeled with biotin and the other end is labeled with a fluorescent group FAM, and the reporter probe is used to be cleaved by the activated Cas13a and release fluorescence; And / or, the CRISPR-Cas13a system for specific detection of SARS-CoV-2 virus further comprises: RT-RAA amplification system and CRISPR-Cas13a enzyme cleavage system; wherein the RT-RAA amplification system includes a buffer, enzyme-free water and template RNA; the CRISPR-Cas13a enzyme cleavage system includes a buffer, enzyme-free water, a ribonucleotide mixed solution, a ribonuclease inhibitor, Cas13a protein, T7 RNA polymerase and magnesium chloride.

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