Composite nanometer luminescent material, preparation method and application thereof, nanometer detection label and immunochromatographic test strip

Abstract

The application belongs to the technical field of immunochromatographic detection, and particularly relates to a composite nanoluminescent material, a preparation method and application thereof, a nanodetection label and an immunochromatographic test strip. The composite nanoluminescent material comprises molybdenum disulfide nanosheets and a fluorescent layer loaded on the surface of the molybdenum disulfide nanosheets, and the fluorescent layer is composed of a plurality of fluorescent unit layers, and each fluorescent unit layer comprises a cationic polymer self-assembly layer and a negative quantum dot layer which are arranged in a stack. The application utilizes the molybdenum disulfide nanosheets to load quantum dots to generate a co-enhanced colorimetric fluorescence signal, and exhibits a powerful ability of flexible, super-sensitive and multiplex detection of common agricultural drugs in competitive lateral flow immunoassay (LFA). The application can flexibly and simultaneously monitor two pesticides (clothianidin and carbendazim) and two veterinary drugs (kanamycin and chloramphenicol) in complex food and experimental samples, and widens the application range of the existing LFA technology.

Description

Technical Field

[0001] This invention belongs to the field of immunochromatographic detection technology, specifically involving composite nanoluminescent materials and their preparation methods and applications, nano-detection tags, and immunochromatographic test strips. Background Technology

[0002] Pesticides and veterinary drugs are used in global agricultural production to effectively prevent plant and animal diseases and increase food yields. The dosage of these chemicals is generally strictly limited because most commonly used insecticides (e.g., thiamethoxam [CLO] and carbendazim [CBZ]) and veterinary drugs (e.g., kanamycin [KAN] and chloramphenicol [CAP]) are toxic small molecules that are difficult to biodegrade and can cause serious illnesses in humans or animals, such as nephrotoxicity, hepatotoxicity, reproductive disorders, myelotoxicity, and even cancer. However, the overuse and inappropriate use of these drugs remains widespread and difficult to control in practice. They not only directly contaminate food, soil, and water but also harm human health through bioaccumulation and the food chain. Therefore, simple, rapid, and sensitive monitoring of pesticide residues in agricultural products and environmental samples is crucial for ensuring food and public safety.

[0003] Lateral flow immunoassay (LFA) combines the advantages of direct visual recognition, specific immune reactions, and rapid chromatographic separation, and has become one of the most mature on-site detection technologies, widely used in clinical diagnosis, personal health monitoring, and rapid screening of agricultural pesticide residues. However, traditional colorimetric immunochromatography has inherent drawbacks such as low sensitivity and poor quantitative ability, severely limiting its application in trace substance detection, and also suffers from insufficient brightness of colloidal gold colorimetric signals. In recent years, efforts have been made to develop sensitive optical signals for LFA applications. Quantum dot (QD)-based strategies have significant advantages over methods based on other signal modes (such as chemiluminescence, surface-enhanced Raman scattering, and fluorescent dyes). Furthermore, due to the unique small size effect of quantum dots, slightly adjusting the particle size or shell thickness can yield quantum dots with different fluorescence emission wavelengths, thereby generating fluorescent signals of different colors. Different colored fluorescent tags can correspond to multiple analytes in a single detection, thus improving the multi-target analytical capability of LFA. Secondly, quantum dot-based composite nanomaterials with high stability and high luminescence can be easily prepared by encapsulating or coating quantum dot particles onto a nanostructure (such as silicon spheres, magnetic beads, polymers, and graphene oxide). However, current quantum dot materials require specialized ultraviolet equipment or reading devices to acquire fluorescence signals, and because they cannot generate sufficient colorimetric signals, they cannot be directly read with the naked eye. This characteristic of quantum dot tags reduces the simplicity and user-friendliness of the LFA method, limiting its application in resource-scarce areas. Summary of the Invention

[0004] The purpose of this invention is to provide composite nanoluminescent materials, their preparation methods and applications, nano-detection tags, and immunochromatographic test strips. The nano-detection tags obtained from the composite nanoluminescent materials provided by this invention generate colorimetric / fluorescent activity in lateral flow immunoassay methods, enabling dual-mode analysis and detection of colorimetry and fluorescence, greatly expanding the application range of traditional colorimetric or fluorescence immunochromatographic analysis, and improving detection sensitivity.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] This invention provides a composite nanoluminescent material, comprising molybdenum disulfide nanosheets and a fluorescent layer loaded on the surface of the molybdenum disulfide nanosheets. The fluorescent layer is composed of several fluorescent unit layers, each fluorescent unit layer comprising a cationic polymer self-assembled layer and a negatively charged quantum dot layer stacked together. The cationic polymer self-assembled layer and the negatively charged quantum dot layer in the fluorescent layer are spaced apart, and the cationic polymer self-assembled layer in the fluorescent layer is in contact with the surface of the molybdenum disulfide nanosheets.

[0007] Preferably, the molybdenum disulfide nanosheets are single-layer molybdenum disulfide nanosheets, and the sheet size of the molybdenum disulfide nanosheets is 200~2000 nm.

[0008] Preferably, the cationic polymer self-assembled layer is a polyethyleneimine self-assembled layer, and the weight-average molecular weight of polyethyleneimine is 5000~80000; the negatively charged quantum dot layer is a carboxylated quantum dot self-assembled layer.

[0009] This invention provides a method for preparing the composite nanoluminescent material described above, comprising the following steps:

[0010] A plurality of fluorescent unit layers are prepared on the surface of the molybdenum disulfide nanosheets, and a fluorescent layer is obtained on the surface of the molybdenum disulfide nanosheets to obtain the composite nanoluminescent material; the preparation method of the single fluorescent unit layer includes: mixing a carrier aqueous dispersion and a cationic polymer aqueous solution for self-assembly to obtain a pre-coated carrier; mixing the aqueous dispersion of the pre-coated carrier and a negatively charged quantum dot aqueous dispersion for modification to obtain a single fluorescent unit layer; the carrier aqueous dispersion is an aqueous dispersion of molybdenum disulfide nanosheets or an aqueous dispersion of molybdenum disulfide nanosheets with a plurality of fluorescent unit layers loaded on the surface.

[0011] This invention provides the application of the composite luminescent nanomaterials described in the above technical solutions or the composite luminescent nanomaterials prepared by the preparation methods described in the above technical solutions in the preparation of immunochromatographic components, equipment or products.

[0012] This invention provides a nano-detection tag, comprising the composite nanoluminescent material described in the above technical solution or the composite nanoluminescent material prepared by the preparation method described in the above technical solution, and a detection antibody modified on the surface of the composite nanoluminescent material.

[0013] This invention provides an immunochromatographic test strip comprising a nitrocellulose membrane, a sample pad, an absorbent pad, and a base plate. The nitrocellulose membrane is loaded with four detection lines and one control line. The four detection lines are respectively coated with KAN-BSA binding antigen, CAP-BSA binding antigen, CLO-BSA binding antigen, and CBZ-BSA binding antigen. The control line is modified with goat anti-mouse IgG.

[0014] This invention provides a qualitative detection method for thiamethoxam, carbendazim, kanamycin, and chloramphenicol, comprising the following steps:

[0015] The nano-detection tag described in the above technical solution is resuspended in the sample solution, and the immunochromatographic test strip described in the above technical solution is inserted. The colorimetric signals of the four detection lines on the immunochromatographic test strip are visually observed to obtain the qualitative detection results of thiamethoxam, carbendazim, kanamycin and chloramphenicol in the sample solution.

[0016] This invention provides a quantitative detection method for thiamethoxam, carbendazim, kanamycin, and chloramphenicol, comprising the following steps:

[0017] The nano-detection tag described in the above technical solution is resuspended in the sample solution and inserted into the immunochromatographic test strip described in the above technical solution. After being excited by ultraviolet light, the fluorescence intensity of the sample to be tested is obtained.

[0018] The fluorescence intensity of the sample solution is input into the standard linear curve or standard linear equation of the content of thiamethoxam, carbendazim, kanamycin and chloramphenicol versus fluorescence intensity to obtain the quantitative detection results of thiamethoxam, carbendazim, kanamycin and chloramphenicol in the sample solution; the standard linear curve or standard linear equation of the content of thiamethoxam, carbendazim, kanamycin and chloramphenicol versus fluorescence intensity is a standard linear curve or standard linear equation with the content of thiamethoxam, carbendazim, kanamycin and chloramphenicol as independent variables and the corresponding fluorescence intensity as dependent variables.

[0019] This invention provides a composite luminescent nanomaterial comprising molybdenum disulfide nanosheets and a fluorescent layer loaded on the surface of the molybdenum disulfide nanosheets. The fluorescent layer consists of several fluorescent unit layers, each of which includes a cationic polymer self-assembled layer and a negatively charged quantum dot layer stacked on top of each other. The cationic polymer self-assembled layer and the negatively charged quantum dot layer are spaced apart in the fluorescent layer, and the cationic polymer self-assembled layer in the fluorescent layer is in contact with the surface of the molybdenum disulfide nanosheets. This invention uses molybdenum disulfide nanosheets as a colorimetric material in the luminescent composite material. Compared with common colorimetric materials such as colloidal gold, molybdenum disulfide (MoS2) nanosheets exhibit better stability, dispersibility, and colorimetric ability. However, molybdenum disulfide nanosheets also exhibit strong fluorescence quenching ability. This invention, by stacking several layers of the negatively charged quantum dot layer on the molybdenum disulfide nanosheets, effectively overcomes the fluorescence quenching ability and internal filtration effect of molybdenum disulfide, greatly improving the specificity of the composite material for multi-objective analysis in LFA systems. Meanwhile, the composite nanoluminescent material provided by this invention utilizes molybdenum disulfide (MoS2) nanosheets loaded with several layers of quantum dots (QDs) to generate a co-enhanced colorimetric fluorescence signal. Figure 19 The results demonstrate its powerful ability to flexibly, ultrasensitively, and multiplex detect common agricultural pesticides in competitive lateral flow immunoassay (LFA). Furthermore, this invention employs a dual-mode detection approach combining colorimetric and fluorescence detection using molybdenum disulfide nanosheets and quantum dot layers. This dual-mode LFA can flexibly and simultaneously monitor two pesticides (thiamethoxam and carbendazim) and two veterinary drugs (kanamycin and chloramphenicol) in complex food and experimental samples. The colorimetric detection limit is as low as 0.1 ng / mL, and the fluorescence detection sensitivity is at least 46 times higher than the colorimetric sensitivity. In summary, the composite nanoluminescent material provided by this invention, when applied to LFA, significantly expands the application scope of existing LFA technology without increasing detection time, and has broad application prospects for the real-time detection of small molecule pollutants under various conditions.

[0020] This invention provides a method for preparing the composite luminescent nanomaterials described in the above technical solution. The preparation method provided by this invention employs a layer-by-layer self-assembly method mediated by a cationic polymer (specifically PEI). Through the assembly and loading of multiple layers of cationic polymer (specifically PEI), a composite luminescent nanomaterial (film-like blank signal tag) with several quantum dot layers is successfully prepared.

[0021] This invention provides an immunochromatographic test strip comprising a nitrocellulose membrane, a sample pad, an absorbent pad, and a base plate. The nitrocellulose membrane is loaded with four detection lines and one control line. The four detection lines are respectively coated with KAN-BSA binding antigen, CAP-BSA binding antigen, CLO-BSA binding antigen, and CBZ-BSA binding antigen. The control line is modified with goat anti-mouse IgG. In this invention, there are four detection lines, which are used to detect thiamethoxam, carbendazim, kanamycin, and chloramphenicol. The immunochromatographic test strip provided by this invention can flexibly and simultaneously monitor two pesticides (thiamethoxam and carbendazim) and two veterinary drugs (kanamycin and chloramphenicol) in complex food and experimental samples. The detection limit for colorimetric signal observation is as low as 0.1 ng / mL, and the fluorescence intensity sensing sensitivity is at least 46 times higher than the colorimetric sensitivity. The immunochromatographic test strip provided by this invention greatly expands the application scope of existing LFA technology without increasing the detection time, and shows great application prospects in the targeted detection of small molecule pollutants under different conditions. Attached Figure Description

[0022] Figure 1 This is a schematic diagram illustrating the preparation method of the synthesis of the two-color molybdenum disulfide composite nanofilm in Example 1 of the present invention;

[0023] Figure 2 Electron microscopy characterization of the structure of the colorimetric fluorescent molybdenum disulfide composite nanofilm of Example 1 of the present invention;

[0024] Figure 3 This is the membrane concentration optimization result of immunochromatography based on molybdenum disulfide composite nanomembrane in Example 2 of the present invention;

[0025] Figure 4 This is the result of the chromatography time optimization for immunochromatography based on molybdenum disulfide composite nanomembrane in Example 2 of the present invention;

[0026] Figure 5 This describes the signal detection principle of the dual fluorescence reading system in Embodiment 2 of the present invention.

[0027] Figure 6 This is an evaluation of the detection performance of red / green molybdenum disulfide composite nanofilm tags with different quantum dot shells in the immunochromatographic system of Example 2 of the present invention;

[0028] Figure 7 This is an evaluation of the detection performance of red / green molybdenum disulfide composite nanofilm tags with different quantum dot shells in the immunochromatographic system of Example 2 of the present invention;

[0029] Figure 8 This is an evaluation of the detection performance of red / green molybdenum disulfide composite nanofilm tags with different quantum dot shells in the immunochromatographic system of Example 2 of the present invention;

[0030] Figure 9 This is a flowchart of the experimental procedure for simultaneous detection of carbendazim, thiamethoxam, chloramphenicol and kanamycin using immunochromatography based on molybdenum disulfide composite nanomembrane in Example 3 of the present invention.

[0031] Figure 10 The image shows the detection results of carbendazim, thiamethoxam, chloramphenicol and kanamycin simultaneously detected by immunochromatography based on molybdenum disulfide composite nanomembrane in Example 3 of the present invention, and a comparison with other immunoassay methods.

[0032] Figure 11 The image shows the detection results of carbendazim, thiamethoxam, chloramphenicol and kanamycin simultaneously detected by immunochromatography based on molybdenum disulfide composite nanomembrane in Example 3 of the present invention, and a comparison with other immunoassay methods.

[0033] Figure 12 The image shows the detection results of carbendazim, thiamethoxam, chloramphenicol and kanamycin simultaneously detected by immunochromatography based on molybdenum disulfide composite nanomembrane in Example 3 of the present invention, and a comparison with other immunoassay methods.

[0034] Figure 13 The results of immunochromatography based on molybdenum disulfide composite nanomembrane in Example 3 of this invention were obtained in actual environmental and food samples.

[0035] Figure 14 The results of immunochromatography based on molybdenum disulfide composite nanomembrane in Example 3 of this invention were obtained in actual environmental and food samples.

[0036] Figure 15 The results of immunochromatography based on molybdenum disulfide composite nanomembrane in Example 3 of this invention were obtained in actual environmental and food samples.

[0037] Figure 16 The results of immunochromatography based on molybdenum disulfide composite nanomembrane in Example 3 of this invention were obtained in actual environmental and food samples.

[0038] Figure 17 This serves as a repeatability verification of the molybdenum disulfide composite nanomembrane-based immunochromatography in Example 3 of the present invention.

[0039] Figure 18 This serves as a specificity verification for the molybdenum disulfide composite nanomembrane-based immunochromatography in Example 3 of the present invention.

[0040] Figure 19 This image shows a schematic diagram of the nanoprobe and its multilayer quantum dot shell structure, as well as the fluorescence spectrum of the nanofilm. Detailed Implementation

[0041] This invention provides a composite nanoluminescent material, comprising molybdenum disulfide nanosheets and a fluorescent layer loaded on the surface of the molybdenum disulfide nanosheets. The fluorescent layer is composed of several fluorescent unit layers, each fluorescent unit layer comprising a cationic polymer self-assembled layer and a negatively charged quantum dot layer stacked together. The cationic polymer self-assembled layer and the negatively charged quantum dot layer in the fluorescent layer are spaced apart, and the cationic polymer self-assembled layer in the fluorescent layer is in contact with the surface of the molybdenum disulfide nanosheets.

[0042] In this invention, unless otherwise specified, all raw materials / components used in the preparation are commercially available products well known to those skilled in the art.

[0043] In this invention, the fluorescent layer is preferably composed of 1, 2, 3 or 4 fluorescent unit layers, and more preferably 3 layers.

[0044] In a specific embodiment of the present invention, the composite nanoluminescent material includes molybdenum disulfide nanosheets, and three cationic polymer self-assembled layers and three negatively charged quantum dot layers loaded on the surface of the molybdenum disulfide nanosheets; the three cationic polymer self-assembled layers and the three negatively charged quantum dot layers are spaced apart from each other, and one cationic polymer self-assembled layer is in contact with the surface of the molybdenum disulfide nanosheets.

[0045] The composite luminescent nanomaterial provided by this invention includes molybdenum disulfide nanosheets. In this invention, the molybdenum disulfide nanosheets serve as a carrier for the negatively charged quantum dot layer. Simultaneously, the molybdenum disulfide nanosheets possess excellent colorimetric capabilities, allowing for direct visual reading of detection results, thus realizing a colorimetric detection mode for the composite luminescent nanomaterial. In this invention, the molybdenum disulfide nanosheets are preferably monolayer molybdenum disulfide nanosheets, and the sheet size of the molybdenum disulfide nanosheets is preferably 200~2000 nm, more preferably 500~1500 nm.

[0046] The composite luminescent nanomaterial provided by this invention comprises three cationic polymer self-assembled layers and three negatively charged quantum dot layers loaded on the surface of molybdenum disulfide nanosheets; the three cationic polymer self-assembled layers and the three negatively charged quantum dot layers are spaced apart from each other, and one cationic polymer self-assembled layer is in contact with the surface of the molybdenum disulfide nanosheets. Specifically, the arrangement of the three cationic polymer self-assembled layers and the three negatively charged quantum dot layers on the surface of the molybdenum disulfide nanosheets is as follows: a first cationic polymer self-assembled layer, a first negatively charged quantum dot layer, a second cationic polymer self-assembled layer, a second negatively charged quantum dot layer, a third cationic polymer self-assembled layer, and a third negatively charged quantum dot layer are sequentially arranged on the surface of the molybdenum disulfide nanosheets. The cationic polymer self-assembled layer is preferably a polyethyleneimine (PEI) self-assembled layer, and the weight-average molecular weight of polyethyleneimine is preferably 5000-80000, more preferably 6000-70000. The negatively charged quantum dot layer is preferably a carboxylated quantum dot layer. In this invention, the ZnS shell of carboxyl-modified quantum dots serves to protect against fluorescence quenching, and the carboxyl modification can be used as an active site for antibody conjugation. In this invention, the negatively charged quantum dot layer is preferably a carboxyl-modified quantum dot layer. In a specific embodiment of this invention, the carboxyl-modified quantum dots (CdSe / ZnS-COOH) are designated as CdSe / ZnS-625-MPA (QD). 625 (red) and CdSe / ZnS-525-MPA (QD 525 (Green), purchased from Mesolight Inc (Suzhou, China).

[0047] The composite luminescent nanomaterial provided by this invention is a film-like colorimetric-fluorescence dual-enhanced nanomaterial composed of molybdenum disulfide (MoS2) nanosheets and quantum dots. Continuously coating the surface of two-dimensional molybdenum disulfide nanosheets with three layers of quantum dots can effectively overcome the fluorescence quenching ability and internal filtering effect of molybdenum disulfide, and generate a colorimetric / fluorescence dual signal mode in the immunochromatographic method.

[0048] The composite luminescent nanomaterial provided by this invention loads red or green quantum dots onto the surface of molybdenum disulfide nanosheets. The resulting thin-film MTQD consists of a highly stable MoS2 core and a multilayer shell containing thousands of small quantum dots. The former provides a broad reaction interface and a strong colorimetric signal, while the latter greatly enhances luminescence.

[0049] This invention provides a method for preparing the composite nanoluminescent material described above, comprising the following steps:

[0050] A plurality of fluorescent unit layers are prepared on the surface of the molybdenum disulfide nanosheets, and a fluorescent layer is obtained on the surface of the molybdenum disulfide nanosheets to obtain the composite nanoluminescent material; the preparation method of the single fluorescent unit layer includes: mixing a carrier aqueous dispersion and a cationic polymer aqueous solution for self-assembly to obtain a pre-coated carrier; mixing the aqueous dispersion of the pre-coated carrier and a negatively charged quantum dot aqueous dispersion for modification to obtain a single fluorescent unit layer; the carrier aqueous dispersion is an aqueous dispersion of molybdenum disulfide nanosheets or an aqueous dispersion of molybdenum disulfide nanosheets with a plurality of fluorescent unit layers loaded on the surface.

[0051] In a specific embodiment of the present invention, when the fluorescent layer is composed of three fluorescent unit layers, the preparation method of the composite nanoluminescent material preferably includes the following steps:

[0052] (1) The aqueous dispersion of molybdenum disulfide nanosheets and the aqueous solution of cationic polymer were mixed and self-assembled to obtain the first pre-coated molybdenum disulfide nanosheets; the aqueous dispersion of the first pre-coated molybdenum disulfide nanosheets and the aqueous dispersion of negatively charged quantum dots were mixed and modified to obtain the first coated molybdenum disulfide nanosheets.

[0053] (2) The first coated molybdenum disulfide nanosheets were mixed with an aqueous dispersion of a cationic polymer and self-assembled to obtain a second pre-coated molybdenum disulfide nanosheets; the second pre-coated molybdenum disulfide nanosheets were mixed with an aqueous dispersion of negatively charged quantum dots and modified to obtain a second coated molybdenum disulfide nanosheets.

[0054] (3) The second coated molybdenum disulfide nanosheets were mixed with an aqueous dispersion of cationic polymer and self-assembled to obtain a third pre-coated molybdenum disulfide nanosheets; the third pre-coated molybdenum disulfide nanosheets were mixed with an aqueous dispersion of negatively charged quantum dots and modified to obtain the composite nanoluminescent material.

[0055] This invention involves self-assembling a molybdenum disulfide nanosheet aqueous dispersion with a cationic polymer aqueous solution to obtain a first pre-coated molybdenum disulfide nanosheet (MoS2@PEI). The first pre-coated molybdenum disulfide nanosheet aqueous dispersion is then modified with a negatively charged quantum dot aqueous dispersion to obtain a first coated molybdenum disulfide nanosheet (MQDs). The molybdenum disulfide nanosheets are dispersed in water to obtain a molybdenum disulfide nanosheet aqueous dispersion. The water is preferably deionized water. The mass concentration of the molybdenum disulfide nanosheet aqueous dispersion is preferably 0.33 mg / mL. The mass concentration of the cationic polymer aqueous solution is 0.1~10 mg / mL, more preferably 0.2 mg / mL. The volume ratio of the molybdenum disulfide nanosheet aqueous dispersion to the cationic polymer aqueous solution is preferably 3:2. The self-assembly is performed under ultrasonic conditions, and the ultrasonic time is preferably 0~60 min, but not 0, more preferably 30 min. The ultrasonic power is preferably 200 W, and the frequency is preferably 40 kHz. After the self-assembly is completed, the present invention preferably separates the self-assembly reaction solution into solid and liquid phases. The obtained solid product is washed with water to obtain the first pre-coated molybdenum disulfide nanosheets. The solid-liquid separation is preferably centrifugal separation. The water washing is preferably deionized water washing, and the number of water washings is preferably 2. The present invention preferably removes excess PEI from the surface of the solid product by water washing. The present invention preferably resuspends the obtained first pre-coated molybdenum disulfide nanosheets in deionized water to obtain an aqueous dispersion of the first pre-coated molybdenum disulfide nanosheets. The molar concentration of the negatively charged quantum dot aqueous dispersion is preferably 0.1-1 mol / L, more preferably 0.1-0.8 mol / L. The volume ratio of the negatively charged quantum dot aqueous dispersion to the cationic polymer aqueous solution is preferably 0.1:20. The modification is carried out under ultrasonic conditions, and the ultrasonic time is preferably 0-60 min, and not 0, more preferably 40 min. The ultrasonic power is preferably 200 W, and the frequency is preferably 40 kHz. After the modification is completed, the present invention preferably separates the modified reaction solution into solid and liquid phases, and washes the obtained solid product with water to obtain the first coated molybdenum disulfide nanosheets. The solid-liquid separation is preferably centrifugal separation. The water washing is preferably deionized water washing, and the number of water washings is preferably two. The present invention preferably removes excess negatively charged quantum dots from the surface of the solid product by water washing. The present invention preferably resuspends the obtained first coated molybdenum disulfide nanosheets in deionized water to obtain an aqueous dispersion of the first coated molybdenum disulfide nanosheets.

[0056] After obtaining the first coated molybdenum disulfide nanosheet aqueous dispersion, the present invention mixes the first coated molybdenum disulfide nanosheet aqueous dispersion with a cationic polymer aqueous solution for self-assembly to obtain a second pre-coated molybdenum disulfide nanosheet; the second pre-coated molybdenum disulfide nanosheet aqueous dispersion is then mixed with a negatively charged quantum dot aqueous dispersion for modification to obtain the second coated molybdenum disulfide nanosheet. The volume of the first coated molybdenum disulfide nanosheet aqueous dispersion is preferably 10 mL. The mass concentration of the cationic polymer aqueous solution is 0.1~10 mg / mL, more preferably 0.2 mg / mL. The volume ratio of the first coated molybdenum disulfide nanosheet aqueous dispersion to the cationic polymer aqueous solution is preferably 1:1. The self-assembly is carried out under ultrasonic conditions, and the ultrasonic time is preferably 0~60 min, but not 0, more preferably 30 min. The ultrasonic power is preferably 200 W, and the frequency is preferably 40 kHz. After the self-assembly is completed, the present invention preferably separates the self-assembly reaction solution into solid and liquid phases, and the obtained solid product is washed with water to obtain the second pre-coated molybdenum disulfide nanosheet. The solid-liquid separation is preferably centrifugal separation. The water washing is preferably deionized water washing, and the number of water washings is preferably 2. This invention preferably removes excess PEI from the surface of the solid product by water washing. This invention preferably resuspends the obtained second pre-coated molybdenum disulfide nanosheets in deionized water to obtain a second pre-coated molybdenum disulfide nanosheet aqueous dispersion. The molar concentration of the negatively charged quantum dot aqueous dispersion is preferably 0.1~1 mol / L, more preferably 0.1~0.8 mol / L. The volume ratio of the negatively charged quantum dot aqueous dispersion to the cationic polymer aqueous solution is preferably 0.1:10. The modification is carried out under ultrasonic conditions, and the ultrasonic time is preferably 0~60 min, and not 0, more preferably 40 min. The ultrasonic power is preferably 200 W, and the frequency is preferably 40 kHz. After the modification is completed, the present invention preferably separates the modified reaction solution into solid and liquid phases. The obtained solid product is washed with water to obtain a second coated molybdenum disulfide nanosheet (MDQDs). The solid-liquid separation is preferably centrifugation. The water washing is preferably deionized water washing, and the number of water washings is preferably 2. The present invention preferably removes excess negatively charged quantum dots on the surface of the solid product by water washing. The present invention preferably resuspends the obtained second coated molybdenum disulfide nanosheets in deionized water to obtain an aqueous dispersion of the second coated molybdenum disulfide nanosheets.

[0057] After obtaining the second coated molybdenum disulfide nanosheet aqueous dispersion, the second coated molybdenum disulfide nanosheet aqueous dispersion and the cationic polymer aqueous solution are mixed for self-assembly to obtain the third pre-coated molybdenum disulfide nanosheets; the third pre-coated molybdenum disulfide nanosheet aqueous dispersion is then mixed with the negatively charged quantum dot aqueous dispersion for modification to obtain the composite nanoluminescent material. The volume of the second coated molybdenum disulfide nanosheet aqueous dispersion is preferably 10 mL. The mass concentration of the cationic polymer aqueous solution is 0.1~10 mg / mL, more preferably 0.2 mg / mL. The volume ratio of the second coated molybdenum disulfide nanosheet aqueous dispersion to the cationic polymer aqueous solution is preferably 1:1. The self-assembly is carried out under ultrasonic conditions, and the ultrasonic time is preferably 0~60 min, and not 0, more preferably 30 min. The ultrasonic power is preferably 200 W, and the frequency is preferably 40 kHz. After the self-assembly is completed, the present invention preferably performs solid-liquid separation of the self-assembly reaction solution, and the obtained solid product is washed with water to obtain the third pre-coated molybdenum disulfide nanosheets. The solid-liquid separation is preferably centrifugal separation. The water washing is preferably deionized water washing, and the number of water washings is preferably 2. This invention preferably removes excess PEI from the surface of the solid product by water washing. This invention preferably resuspends the obtained third pre-coated molybdenum disulfide nanosheets in deionized water to obtain a third pre-coated molybdenum disulfide nanosheet aqueous dispersion. The molar concentration of the negatively charged quantum dot aqueous dispersion is preferably 0.1~1 mol / L, more preferably 0.1~0.8 mol / L. The volume ratio of the negatively charged quantum dot aqueous dispersion to the cationic polymer aqueous solution is preferably 0.1:10. The modification is carried out under ultrasonic conditions, and the ultrasonic time is preferably 0~60 min, and not 0, more preferably 40 min. The ultrasonic power is preferably 200 W, and the frequency is preferably 40 kHz. After the modification is completed, this invention preferably separates the modification reaction solution into solid and liquid phases, and the obtained solid product is washed with water to obtain composite nanoluminescent materials (MTQDs). The solid-liquid separation is preferably centrifugal separation. The water washing is preferably deionized water washing, and the number of water washings is preferably 2. The present invention preferably removes excess negatively charged quantum dots from the surface of the solid product by washing with water.

[0058] In this invention, the obtained composite nanoluminescent material is preferably resuspended in ethanol for storage and later use.

[0059] The preparation method provided by this invention is based on electrostatic self-assembly, in which CdSe / ZnS-COOH QDs or CdSe / ZnS-MPA QDs are modified layer by layer on the surface of molybdenum disulfide nanosheets. The preparation process is efficient, reproducible, and suitable for industrial production.

[0060] This invention provides the application of the composite luminescent nanomaterials described in the above technical solutions or the composite luminescent nanomaterials prepared by the preparation methods described in the above technical solutions in the preparation of immunochromatographic components, equipment or products.

[0061] This invention provides a nano-detection tag, comprising the composite nanoluminescent material described in the above technical solution or the composite nanoluminescent material prepared by the preparation method described in the above technical solution, and a detection antibody modified on the surface of the composite nanoluminescent material.

[0062] In this invention, the content (modification amount) of the detection antibody in the nano-detection tag is preferably 5~10 mg / mL.

[0063] This invention provides a method for preparing the nano-detection tag described above, preferably comprising the following steps:

[0064] The composite luminescent nanomaterial, activating reagent, and buffer solution are mixed and activated to obtain the activated composite luminescent nanomaterial.

[0065] The activated composite luminescent nanomaterial, specific antibody, and buffer solution were mixed and coupled, and then bovine serum albumin (BSA) solution was added for blocking to obtain the nano-detection tag.

[0066] This invention involves activating the composite luminescent nanomaterial by mixing and combining an activating reagent with a buffer solution to obtain an activated composite luminescent nanomaterial. In this invention, the activating reagent preferably includes 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS). The buffer solution is preferably a PSBT buffer solution, with a preferred mass content of 0.05%. This invention does not have specific requirements for the amount of the activating reagent and buffer solution, as long as the floating-point groups connected to the quantum dots in the third negatively charged quantum dot layer on the surface of the composite luminescent nanomaterial are fully activated. The activation is performed under ultrasonic conditions, with the ultrasonication time preferably being 15 minutes. After activation, this invention preferably obtains the activated composite luminescent nanomaterial by centrifugation.

[0067] After obtaining the activated composite luminescent nanomaterial, the present invention mixes the activated composite luminescent nanomaterial, a specific antibody, and a buffer solution for a coupling reaction, and then adds bovine serum albumin (BSA) solution for blocking to obtain the nano-detection tag. In this invention, the specific antibody is preferably a specific antibody recognizing thiamethoxam (CLO) (CLO-antibody), a specific antibody recognizing carbendazim (CBZ-antibody), a specific antibody recognizing kanamycin (KAN) (KAN-antibody), or a specific antibody recognizing chloramphenicol (CAP) (CAP-antibody). The buffer solution is preferably PSBT buffer, and the mass content of the PSBT buffer is preferably 0.05%. The coupling reaction is carried out at room temperature under shaking conditions, and the coupling reaction time is preferably 2 hours. The mass content of the BSA solution is preferably 10%. The blocking reaction time is preferably 1 hour. After blocking, the present invention preferably washes the obtained reaction product with PSBT buffer to obtain the nano-detection tag, and the mass content of the PSBT buffer is preferably 0.05%.

[0068] This invention obtains a functionalized dual-color film-like fluorescent tag (immuno-MTQD) by directly coupling specific antibodies to numerous negatively charged groups (carboxyl groups or 3-mercaptopropionic acid) on the third layer of the QD shell of the composite nanoluminescent material. The negatively charged quantum dots in the nanodetection tags that form the two detection lines for detecting thiamethoxam and carbendazim are of different types than those in the nanodetection tags that form the two detection lines for detecting kanamycin and chloramphenicol.

[0069] The composite luminescent nanomaterial provided by this invention consists of molybdenum disulfide nanosheets and quantum dots. The nano-detection tag provided by this invention is a film-like colorimetric-fluorescence dual-enhanced tag, composed of a highly stable molybdenum disulfide substrate and a three-layer quantum dot shell containing thousands of small quantum dots. The molybdenum disulfide substrate provides a broad reaction interface and a strong colorimetric signal, while the three-layer quantum dot shell significantly enhances luminescence. Compared with existing immunochromatographic techniques, this invention utilizes the colorimetric properties of molybdenum disulfide itself combined with the fluorescence properties of the three-layer quantum dots to achieve colorimetric / fluorescence activity in immunochromatographic methods, which is superior to ordinary colorimetric nanotags and fluorescent quantum dots.

[0070] This invention prepares red (QD) by layer-by-layer (LbL) electrostatic self-assembly modifying three dense layers of CdSe / ZnS-MPA QDs or CdSe / ZnS-COOH QDs onto a monolayer molybdenum disulfide surface. 625 ) or green (QD) 525The MTQD white label is used for colorimetric and fluorescence dual-mode analysis of various agricultural pesticides, and exhibits good dispersibility, stability, and compatibility in immunochromatographic systems. The dual-mode immunochromatographic method proposed in this invention shows great potential as a convenient, low-cost, and accurate immunoassay tool for on-site monitoring of agricultural pesticides, particularly suitable for resource-constrained environments.

[0071] This invention provides a chromatographic immunoassay strip, comprising a nitrocellulose membrane, a sample pad, an absorbent pad, and a base plate. The nitrocellulose membrane is loaded with four detection lines and one control line. The four detection lines are respectively coated with KAN-BSA binding antigen, CAP-BSA binding antigen, CLO-BSA binding antigen, and CBZ-BSA binding antigen. The control line is modified with goat anti-mouse IgG.

[0072] In this invention, the sample pad is used for loading the sample solution. The absorbent pad is used to provide capillary force. The base plate is preferably made of PVC and serves as the backing card for the immunochromatographic test strip. The nitrocellulose membrane is loaded with four detection lines and one control line. The four detection lines are used to detect thiamethoxam, carbendazim, kanamycin, and chloramphenicol, respectively. In this invention, the four detection lines are respectively coated with KAN-BSA binding antigen, CAP-BSA binding antigen, CLO-BSA binding antigen, and CBZ-BSA binding antigen. The control line is preferably modified with goat anti-mouse IgG.

[0073] This invention provides a method for loading detection lines and control lines onto nitrocellulose membranes in immunochromatographic test strips, preferably comprising the following steps:

[0074] A nitrocellulose (NC) membrane was coated with KAN-BSA solution, CAP-BSA solution, CLO-BSA solution, CBZ-BSA solution, and goat anti-mouse IgG solution, respectively. The coated NC membrane was then dried, resulting in four detection lines and one control line on the nitrocellulose membrane. The preferred mass concentration of the KAN-BSA solution was 0.5–1 mg / mL, more preferably 0.8 mg / mL; the preferred mass concentration of the CAP-BSA solution was 0.5–1 mg / mL, more preferably 1 mg / mL; the preferred mass concentration of the CLO-BSA solution was 0.5–1 mg / mL, more preferably 0.8 mg / mL; and the preferred mass concentration of the CBZ-BSA solution was 0.5–1 mg / mL, more preferably 0.8 mg / mL. The preferred mass concentration of the goat anti-mouse IgG solution was 0.5–2 mg / mL, more preferably 1 mg / mL. The drying was performed in a constant temperature oven at a temperature preferably 37°C.

[0075] In this invention, the width of the immunochromatographic test strip is preferably 3.5 mm. The immunochromatographic test strip is preferably stored in a vacuum desiccator for use.

[0076] The competitive immunochromatographic method of the immunochromatographic test strip provided by this invention offers two detection modes: in colorimetric mode, rapid qualitative detection is achieved by visually identifying the colorimetric signal generated by the black molybdenum disulfide material on the T line; in fluorescence mode, precise quantitative analysis is achieved by observing the change in fluorescence intensity on the T line.

[0077] This invention provides a method for fluorescence immunochromatographic detection using an immunochromatographic test strip, comprising the following steps: loading a nano-detection tag and sample solution onto the sample pad of the immunochromatographic test strip using a running buffer, and bringing them into contact with the four detection lines on the immunochromatographic test strip for chromatographic reaction. After the chromatographic reaction, reading the colorimetric and fluorescence intensities on the four detection lines of the test strip. The running buffer is preferably PBST buffer. The mass content of the PBST buffer is preferably 10%. The chromatographic reaction time is 15 min.

[0078] This invention provides a qualitative detection method for thiamethoxam, carbendazim, kanamycin, and chloramphenicol, comprising the following steps:

[0079] The nano-detection tag described in the above technical solution is resuspended in the sample solution, and the immunochromatographic test strip described in the above technical solution is inserted. The colorimetric signals of the four detection lines on the immunochromatographic test strip are visually observed to obtain the qualitative detection results of thiamethoxam, carbendazim, kanamycin and chloramphenicol in the sample solution.

[0080] This invention provides a quantitative detection method for thiamethoxam, carbendazim, kanamycin, and chloramphenicol, comprising the following steps:

[0081] The nano-detection tag described in the above technical solution is resuspended in the sample solution and inserted into the immunochromatographic test strip described in the above technical solution. After being excited by ultraviolet light, the fluorescence intensity of the sample to be tested is obtained.

[0082] The fluorescence intensity of the sample solution is input into the standard linear curve or standard linear equation of the content of thiamethoxam, carbendazim, kanamycin and chloramphenicol versus fluorescence intensity to obtain the quantitative detection results of thiamethoxam, carbendazim, kanamycin and chloramphenicol in the sample solution; the standard linear curve or standard linear equation of the content of thiamethoxam, carbendazim, kanamycin and chloramphenicol versus fluorescence intensity is a standard linear curve or standard linear equation with the content of thiamethoxam, carbendazim, kanamycin and chloramphenicol as independent variables and the corresponding fluorescence intensity as dependent variables.

[0083] Compared with the prior art, the advantages of the present invention are:

[0084] (1) The three-dimensional molybdenum disulfide sheet composite nanomaterial proposed in this invention has a dual enhancement effect of colorimetry and fluorescence. The continuous coating of three layers of quantum dot shell on the surface of two-dimensional molybdenum disulfide sheet can effectively overcome the fluorescence quenching ability and internal filtration effect of molybdenum disulfide, which is superior to ordinary colorimetric nanotags and fluorescent quantum dots.

[0085] (2) The preparation method of the film composite nanomaterial proposed in this invention adopts the PEI-mediated layer-by-layer self-assembly method. Through the assembly and loading of multiple layers of PEI, film signal tags with different quantum dot layers can be prepared.

[0086] (3) The present invention proposes a molybdenum disulfide (MTQD) tag with a 3-layer quantum dot shell to support colorimetric-fluorescence dual-mode analysis, which greatly expands the application range of traditional colorimetric or fluorescence immunochromatographic analysis and improves sensitivity.

[0087] (4) The MTQD tags with red and green fluorescence intensity proposed in this invention are applied to multi-channel immunochromatography to simultaneously analyze two common pesticides (CLO and CBZ) and two common veterinary drugs (KAN and CAP). The detection method provided by this invention effectively reduces the mutual interference between multiple detection lines and improves the specificity of multi-target detection.

[0088] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention. Example

[0089] according to Figure 1 The preparation process shown is as follows:

[0090] (1) Preparation of MoS2@PEI nanosheets:

[0091] 10 mL of monolayer molybdenum disulfide (1 mg / mL) with sheet size of 200~1000 nm was mixed with 20 mL of deionized water, sonicated for 10 min, and the molybdenum disulfide nanosheets were separated by centrifugation. The nanosheets were then reacted with 20 mL of polyethyleneimine (PEI) aqueous solution (0.2 mg / mL) under strong sonication for 30 min. The nanosheets were rinsed twice with deionized water to remove excess PEI and then resuspended in 10 mL of deionized water.

[0092] (2) Preparation of MQD composite nanosheets:

[0093] 100 µL of CdSe / ZnS QDs (QD) were added to the prepared MoS2@PEI solution. 625 or QD 525(0.1M). The mixture was sonicated for 30 min to form monolayer QD-coated nanosheets (MQDs). The obtained MQDs were centrifuged and washed once with deionized water to remove excess quantum dots.

[0094] (3) Preparation of MDQD composite nanosheets:

[0095] The prepared MQDs were dispersed in 10 mL of deionized water and then reacted with 10 mL of PEI solution (0.2 mg / mL) under strong sonication for 30 min. The mixture was rinsed twice with deionized water to remove excess PEI and resuspended in 10 mL of deionized water. Then, 100 µL of CdSe / ZnS QDs (0.1 M) was added. The mixture was sonicated for 30 min to form nanosheets (MDQDs) coated with a double-layer QD shell.

[0096] (4) Preparation of MTQD composite nanosheets:

[0097] The prepared MDQDs were dispersed in 10 mL of deionized water, and then reacted with 10 mL of PEI solution (0.2 mg / mL) under strong sonication for 30 min. The mixture was rinsed twice with deionized water to remove excess PEI and resuspended in 10 mL of deionized water. Then, 100 µL of CdSe / ZnS QDs (0.1 M) was added. The mixture was sonicated for 30 min to form nanosheets (MTQDs) coated with a three-layer QD shell, which were then stored in 10 mL of ethanol for later use.

[0098] Figure 2 Transmission electron microscopy (TEM) images of the MoS2 nanosheets and MoS2@PEI nanosheets obtained in step (1) of this embodiment, the MQD composite nanosheets prepared in step (2), the MDQD composite nanosheets prepared in step (3), and the MTQD composite nanosheets prepared in step (4). Figure 2 a, b, and b in the image are TEM images of molybdenum disulfide nanosheets separated by centrifugation. Figure 2 In the image, d represents the TEM image of MoS2@PEI nanosheets. Figure 2 In the image, 'e' represents the TEM image of CdSe@ZnS. Figure 2 f in the image represents the TEM image of the MQDs. Figure 2 In the image, g represents the TEM image of MDQDs. Figure 2 In the image, h represents the TEM image of MTQDs. Figure 2 In the image, i represents a magnified TEM image of CdSe@ZnS. Figure 2 In the image, j is a magnified TEM image of a localized MQD. Figure 2 In the image, k is a magnified TEM image of a localized MDQD. Figure 2In the image, 'l' represents a magnified TEM image of a localized MTQD, derived from... Figure 2 This indicates that many small quantum dots are continuously assembled onto a two-dimensional nanostructure, and the density of quantum dots loaded on the molybdenum disulfide surface increases significantly with the increase of the number of PEI / QD layers. Example

[0099] KAN-BSA solution (0.8 mg / mL), CAP-BSA solution (1 mg / mL), CLO-BSA solution (0.8 mg / mL), CBZ-BSA solution (0.8 mg / mL), and goat anti-mouse IgG solution (1 mg / mL) were sprayed onto nitrocellulose membranes, respectively, and then dried in a constant temperature oven at 37°C to obtain nitrocellulose membranes with four independent detection lines (KAN-BSA, CAP-BSA, CLO-BSA, and CBZ-BSA) and one control line (goat anti-mouse IgG).

[0100] The dried nitrocellulose membrane is attached to a PVC backing card and assembled with the sample pad and absorbent pad. Finally, the fully assembled card is cut into individual strips to obtain immunochromatographic test strips and stored in a vacuum desiccator.

[0101] Optimization of antigen concentration in online spraying test cases

[0102] (1) CLO-BSA was diluted to 0.2, 0.4, 0.6, 0.8, and 1 mg / mL using PBS (10 mM, pH 7.4) buffer; CBZ-BSA was diluted to 0.2, 0.4, 0.6, 0.8, and 1 mg / mL; CAP-BSA was diluted to 0.6, 0.8, 1, 1.2, and 1.4 mg / mL; and KAN-BSA was diluted to 0.4, 0.6, 0.8, 1, and 1.2 mg / mL.

[0103] (2) Using a membrane coating instrument, four groups of different concentrations of binding antigens were sprayed onto nitrocellulose membranes to prepare immunochromatographic test strips;

[0104] (3) Resuspend the MTQD tag in the prepared running buffer, insert the immunochromatographic test strip, and after 15 min of immunoreaction, use a fluorescence reader (brand: Suzhou Hemai Precision Instruments Co., Ltd., model: FIC-S100) to record the fluorescence signal of the four detection lines under 365 nm excitation. By calculating and comparing the competitive inhibition rate of different spraying concentration groups, the most suitable spraying concentration for binding antigen is determined.

[0105] Figure 3To determine the optimal concentration of the online coated antigen, the immunochromatography based on the dual-signal molybdenum disulfide sheet-like composite fluorescent nanomaterials showed the highest competitive inhibition rate against the target when the concentrations of CLO-BSA, CBZ-BSA, KAN-BSA, and CAP-BSA (coated antigen) on the detection line reached 0.8 mg / mL, 0.8 mg / mL, 1 mg / mL, and 0.8 mg / mL, respectively.

[0106] Optimization of immune response time in test cases

[0107] (1) Resuspend the MTQD tag in the running buffer and insert the immunochromatographic test strip;

[0108] (2) After 5, 10, 15, 20 and 25 min of the immunoassay, the fluorescence signals of the four detection lines were recorded by a fluorescence reader (brand: Suzhou Hemai Precision Instruments Co., Ltd., model: FIC-S100) under 365 nm excitation. The optimal immunochromatographic detection time was determined by calculating the competitive inhibition rate.

[0109] Figure 4 The results show that a chromatography reaction time of 15 min is sufficient to achieve strong fluorescence intensity and the highest competitive inhibition in immunochromatography based on dual-signal molybdenum disulfide sheet-like composite fluorescent nanomaterials. Example

[0110] In evaluating the detection performance of the immunochromatographic assay, this invention used six groups of PBS samples containing different concentrations of CLO / CBZ / KAN / CAP (1: 10 / 10 / 10 / 10 ng / mL; 2: 0 / 0 / 0 / 0 ng / mL; 3: 10 / 0 / 0 / 0 ng / mL; 4: 0 / 0 / 10 / 0 ng / mL; 5: 0 / 10 / 0 / 0 ng / mL; 6: 0 / 0 / 0 / 10 ng / mL) to evaluate the selectivity of each T line of the multiplex MTQD-LFA assay. The MQD, MDQD, and MTQD tags were resuspended in the six groups of PBS samples and inserted into the detection lines in Example 2, achieving CLO-BSA, CBZ-BSA, KAN-BSA, and CAP-BSA (coating antigen) concentrations of 0.8 mg / mL, 0.8 mg / mL, 1 mg / mL, and 0.8 mg / mL, respectively. The performance of mg / mL test strips was compared with that of MQD, MDQD, and MTQD labels.

[0111] The detection principle and process are as follows Figure 5 As shown, the test results are as follows Figure 6 , Figure 7 and Figure 8It can be concluded that, compared with MQD and MDQD tags, MTQD with a three-layer quantum dot shell can produce a stable colorimetric signal and has higher sensitivity based on the red and green fluorescence intensity of the immunochromatographic system. SEM images of the T-line region in immunochromatography show a large number of membrane-like MTQD tags in the negative control test. Figure 8 (a) in the positive test, while no nanofilm was found in the positive test. Figure 8 b) indicates that the MTQD tag can function properly on the immunochromatographic system. Figure 8 The colorimetric image (I) and fluorescence image (II) in c show that only samples containing the target drug can suppress the colorimetric / fluorescence bands in the corresponding T region. Figure 8 Image (III) in c shows detailed red / green fluorescence intensities on the four T lines, clearly demonstrating that the method of the present invention has good selectivity and that there is no cross-reaction between the two fluorescence intensities on the CLO, CBZ, KAN and CAP lines. Example

[0112] In this embodiment, different concentrations of CLO, CBZ, CAP, and KAN (10 ng / mL to 0.004 ng / mL) were simultaneously added to PBS solution (10 mM, pH 7.4) to simulate a sample co-contaminated with multiple small molecule pollutants.

[0113] The MTQD tag was resuspended in the simulated sample and the immunochromatographic test strip obtained in Example 2 was inserted to perform an immunoassay.

[0114] Figure 9 This embodiment presents a flowchart of a dual-mode immunochromatographic assay for multi-pathway detection of CAP, KAN, CBZ, and CLO, as well as the analytical results of an immunochromatographic system based on dual-signal molybdenum disulfide sheet-like composite fluorescent nanomaterials for detecting CAP, KAN, CBZ, and CLO.

[0115] Figure 10 In the figure, 'a' represents the detection results of the immunochromatographic test strip based on MTQD in colorimetric mode. In MTQD-based immunochromatographic colorimetric mode, the vLOD values ​​for CLO, CBZ, KAN, and CAP are 0.12, 0.36, 0.12, and 0.36 ng / mL, respectively. Simultaneously, the cutoff value for the four target molecules is 1.1 ng / mL. Figure 10 In the figure, b represents the detection results of the immunochromatographic test strip based on MTQD in fluorescence mode. The vLOD values ​​of CLO, CBZ, KAN and CAP are 0.04, 0.12, 0.04 and 0.12 ng / mL, respectively. Figure 10 In the figure, c represents the heatmap result based on red / green fluorescence; Figure 10In the figure, d represents the detailed fluorescence intensity of the detection line region of the immunochromatographic band based on MTQD. The results show the changes in fluorescence intensity of the four detection line regions under 365 nm ultraviolet light excitation. Figure 10 In the figure, d represents the detailed fluorescence intensity results of four detection lines recorded simultaneously using an immunochromatographic reader (brand: Suzhou Hemai Precision Instruments Co., Ltd., model: FIC-S100).

[0116] Figure 11 In the figure, a~d are the fitted curves of CLO, CBZ, KAN and CAP plotted by fluorescence intensity. The calculated limits of detection (LOD) are 2.58, 4.88, 1.53 and 3.43 pg / mL, respectively.

[0117] Figure 12 The test strip image shows that the standard colloidal gold-based immunochromatographic method yielded vlods of 3.6, 3.6, 11, and 11 ng / mL for CLO, CBZ, KAN, and CAP, respectively. Figure 12 As shown in b, the ELISA detection limits for CBZ and CAP reached 0.32 and 0.21 ng / mL, respectively.

[0118] From these results, the present invention can conclude that the dual-mode immunochromatographic system of the present invention has higher sensitivity than ELISA, while effectively reducing the detection time from 3 hours (ELISA kit) to 15 minutes.

[0119] This invention further evaluated the reliability of dual-mode immunochromatography by testing real-world environmental samples (lakes and rivers) and food samples (orange juice and milk). Figure 13 , Figure 14 , Figure 15 and Figure 16 As shown, river water samples ( Figure 13 ), lake water samples ( Figure 14 ), orange juice sample ( Figure 15 ), milk samples ( Figure 16 The colorimetric images and measured fluorescence intensity results of the test strips were consistent with those of the PBS samples. Figure 13 , Figure 14 , Figure 15 and Figure 16 The results clearly demonstrate that MTQD-based dual-mode immunochromatography exhibits good stability, accuracy, and multiplex detection capability in the quantitative analysis of multiple drugs in real-world complex samples.

[0120] Test case

[0121] In Example 1 of this invention, molybdenum disulfide (MoS2) nanosheets and several layers of quantum dots (QDs) supported on the MoS2 nanosheets exhibit a co-enhanced colorimetric fluorescence signal. This co-enhancing in this invention involves both enhancement of the colorimetric signal and the fluorescence signal, an enhancement distinct from traditional colorimetric colloidal gold and fluorescent quantum dots. Because molybdenum disulfide possesses a large surface area and excellent colorimetric properties, and the loading of multiple quantum dots further enhances their fluorescence performance, a co-enhancing effect is achieved.

[0122] Figure 19 MTQD prepared in Example 1 525 ( Figure 19 a) and MTQD 625 ( Figure 19 b) Schematic diagram of the multilayer quantum dot shell structure of nanomaterials. Figure 19 c in the text stands for MTQD 525 Fluorescence spectrum of nanofilms Figure 19 d in the figure represents MTQD 625 Fluorescence spectrum of nanofilms. Figure 19 The illustrations are colorimetric / fluorescence images of QD, MoS2, MQD, MDQD, and MTQD solutions.

[0123] like Figure 19 As shown in a and b, in the MoS2@QDs structure, a large number of quantum dots are directly adsorbed onto the MoS2-PEI surface. The distance between the MoS2 sheet and the quantum dots is 1.5 nm (PEI layer), which is too narrow to suppress the quenching effect of MoS2. When a second layer of quantum dots is coated on the MQD surface, the distance between the MoS2 sheet and the outermost quantum dots can reach 15 nm (quantum dot diameter + two PEI layers), which can effectively overcome the IFE of the MoS2 sheet. Furthermore, with the increase of the number of quantum dot layers, the loading of quantum dots adsorbed on the surface of the MoS2 nanosheet increases exponentially, further enhancing the luminescence capability of the tag of this invention. Figure 19 The red and green fluorescence spectra of c and d in the data validated MTQD (MTQD 525 and MTQD 625 It has stronger fluorescence ability, more layers of QD and two or more PEI / QD shells, which can overcome the internal filtering effect (IFE) of MoS2 nanosheets.

[0124] This invention conducted five tests on mixed samples (containing CLO, CBZ, CAP, and KAN) at two different concentrations (1 / 0.1 ng / mL) to further determine the detection stability and accuracy of the proposed dual-mode MTQD-LFA method. Results are shown below. Figure 17When the target analyte concentration in the detection solution was the same, the immunochromatographic strips in the same group all showed stable and consistent colorimetric and fluorescence intensities. The relative standard deviations (RSDs) of fluorescence intensity in samples containing 1 / 1 / 1 / 1 ng / mL and 0.1 / 0.1 / 0.1 / 0.1 ng / mL CLO / CBZ / KAN / CAP were as low as 4.06% / 5.82% / 7.48% / 3.42% and 5.31% / 5.90% / 7.98% / 5.81%, respectively. Figure 17 The results show that the dual-mode MTQD-LFA method established in this invention has good stability.

[0125] For veterinary drug interference, streptomycin (STR), ofloxacin (OFLX), and gentamicin (GEN) are selected; for pesticide interference, imidacloprid (IMI) and chlorpyrifos (CPF) are selected. Figure 18 The results showed that when only four target analytes (CLO, CBZ, CAP, and KAN) were present simultaneously, the colorimetric and fluorescence intensities on the T-line of MTQD-LFA were completely suppressed. Therefore, even when a high concentration of interfering substances (100 ng / mL) was present in the detection solution, the colorimetric and fluorescence intensities on each T-line of MTQD-LFA showed no change compared to the blank control. This indicates that the dual-mode MTQD-LFA method designed in this invention has good specificity for detecting target chemical residues.

[0126] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. Other embodiments can be obtained based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A qualitative detection method for thiamethoxam, carbendazim, kanamycin, and chloramphenicol, characterized in that, Includes the following steps: The nano-detection tag is resuspended in the sample solution and inserted into the immunochromatographic test strip. The colorimetric signals of the four detection lines on the immunochromatographic test strip are visually observed to obtain the qualitative detection results of thiamethoxam, carbendazim, kanamycin and chloramphenicol in the sample solution. The nano-detection tag comprises a composite luminescent nanomaterial and a detection antibody modified on the surface of the composite luminescent nanomaterial. The composite luminescent nanomaterial comprises molybdenum disulfide nanosheets and a fluorescent layer loaded on the surface of the molybdenum disulfide nanosheets. The fluorescent layer consists of several fluorescent unit layers, each of which includes a cationic polymer self-assembled layer and a negatively charged quantum dot layer stacked together. The cationic polymer self-assembled layer and the negatively charged quantum dot layer are alternately arranged in the fluorescent layer. The cationic polymer self-assembled layer in the fluorescent layer is in contact with the surface of the molybdenum disulfide nanosheets. The fluorescent layer consists of three fluorescent unit layers. The molybdenum disulfide nanosheets are monolayer molybdenum disulfide nanosheets with a sheet size of 200-2000 nm. The cationic polymer self-assembled layer is a polyethyleneimine self-assembled layer with a weight-average molecular weight of 5000-80000. The negatively charged quantum dot layer is a carboxylated quantum dot self-assembled layer. The content of the detection antibody in the nano-detection tag is 5-10 mg / mL. The immunochromatographic test strip includes a nitrocellulose membrane, a sample pad, an absorbent pad, and a base plate. The nitrocellulose membrane is loaded with four detection lines and one control line. The four detection lines are respectively coated with KAN-BSA binding antigen, CAP-BSA binding antigen, CLO-BSA binding antigen, and CBZ-BSA binding antigen. The control line is modified with goat anti-mouse IgG.

2. The qualitative detection method according to claim 1, characterized in that, The preparation method of the composite nanoluminescent material includes the following steps: A plurality of fluorescent unit layers are prepared on the surface of the molybdenum disulfide nanosheets, and a fluorescent layer is obtained on the surface of the molybdenum disulfide nanosheets to obtain the composite nanoluminescent material; the preparation method of the single fluorescent unit layer includes: mixing a carrier aqueous dispersion and a cationic polymer aqueous solution for self-assembly to obtain a pre-coated carrier; mixing the aqueous dispersion of the pre-coated carrier and a negatively charged quantum dot aqueous dispersion for modification to obtain a single fluorescent unit layer; the carrier aqueous dispersion is an aqueous dispersion of molybdenum disulfide nanosheets or an aqueous dispersion of molybdenum disulfide nanosheets with a plurality of fluorescent unit layers loaded on the surface.

3. A quantitative detection method for thiamethoxam, carbendazim, kanamycin, and chloramphenicol, characterized in that, Includes the following steps: The nano-detection tag is resuspended in the sample solution, inserted into an immunochromatographic test strip, and excited by ultraviolet light to obtain the fluorescence intensity of the sample to be tested. The nano-detection tag comprises a composite nanoluminescent material and a detection antibody modified on the surface of the composite nanoluminescent material. The composite nanoluminescent material comprises molybdenum disulfide nanosheets and a fluorescent layer loaded on the surface of the molybdenum disulfide nanosheets. The fluorescent layer consists of several fluorescent unit layers, each of which includes a layer of cationic polymer self-assembled layer and a layer of negatively charged quantum dot layer stacked together. The cationic polymer self-assembled layer and the negatively charged quantum dot layer are alternately arranged in the fluorescent layer. The cationic polymer self-assembled layer in the fluorescent layer is in contact with the surface of the molybdenum disulfide nanosheets. The fluorescent layer consists of three fluorescent unit layers. The molybdenum disulfide nanosheets are single-layer molybdenum disulfide nanosheets with a sheet size of 200-2000 nm. The cationic polymer self-assembled layer is a polyethyleneimine self-assembled layer with a weight-average molecular weight of 5000-80000. The negatively charged quantum dot layer is a carboxylated quantum dot self-assembled layer. The content of the detection antibody in the nano-detection tag is 5-10 mg / mL. The immunochromatographic test strip includes a nitrocellulose membrane, a sample pad, an absorbent pad, and a base plate. The nitrocellulose membrane is loaded with four detection lines and one control line. The four detection lines are respectively coated with KAN-BSA binding antigen, CAP-BSA binding antigen, CLO-BSA binding antigen, and CBZ-BSA binding antigen. The control line is modified with goat anti-mouse IgG. The fluorescence intensity of the sample solution is input into the standard linear curve or standard linear equation of the content of thiamethoxam, carbendazim, kanamycin and chloramphenicol versus fluorescence intensity to obtain the quantitative detection results of thiamethoxam, carbendazim, kanamycin and chloramphenicol in the sample solution; the standard linear curve or standard linear equation of the content of thiamethoxam, carbendazim, kanamycin and chloramphenicol versus fluorescence intensity is a standard linear curve or standard linear equation with the content of thiamethoxam, carbendazim, kanamycin and chloramphenicol as independent variables and the corresponding fluorescence intensity as dependent variables.

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