Red fluorescence signal label and preparation method thereof, color-changing fluorescence immunochromatography kit and application

Abstract

The invention discloses a red fluorescence signal label, a kit prepared from the red fluorescence signal label and application of the red fluorescence signal label. The signal label comprises a graphene oxide carrier, a fluorescent layer arranged on the graphene oxide carrier and a detection antibody modified on the fluorescent layer, the detection antibody is one of an anti-ICP monoclonal antibody, an anti-FB1 monoclonal antibody and an anti-CLE monoclonal antibody; the fluorescent layer comprises one or more quantum dot layers; each quantum dot layer comprises a PEI self-assembly layer and a red quantum dot layer in the direction far away from the graphene oxide carrier; the red quantum dot layer is formed by self-assembling red fluorescent quantum dots or coating the red fluorescent quantum dots with silicon dioxide after the red fluorescent quantum dots are self-assembled. The signal label is combined with green fluorescent microspheres embedded in a test line, and a wider fluorescence gradient can be generated according to the change of the concentration of target molecules, so that the sensitivity, the stability and the quantitative range of immunochromatographic analysis (ICA) are remarkably improved.

Description

Technical Field

[0001] The present invention relates to the technical field of immunological detection, and in particular to a red fluorescent signal label and a preparation method thereof, a color-changing fluorescent immunochromatography kit and applications thereof. Background Art

[0002] Small agricultural molecule pollutants, such as drug residues (including antibiotics and veterinary stimulants), pesticides, mycotoxins, and heavy metals, are ubiquitous in water and soil pollution and remain persistently difficult to degrade, posing significant risks to the ecological environment. Furthermore, these small molecules can bioaccumulate through the food chain, posing serious risks to human health and potentially leading to acute poisoning and a range of chronic health problems, including liver and kidney toxicity, neurological damage, and increased cancer risk. To protect consumer health, many countries and international organizations have established strict restrictions on hazardous substances. For example, the European Commission has set a maximum residue limit (MRL) of 50 ng / mL for imidacloprid (ICP) in tea and honey, while the Food and Agriculture Organization of the United Nations (FAO) has set a maximum residue limit (MRL) of 0.05 ng / mL for chlorpyrifos (CLE) in milk. Therefore, regular monitoring of these residues is crucial to ensuring food safety and improving overall quality. While large-scale analytical instruments such as high-performance liquid chromatography and liquid chromatography-mass spectrometry can accurately detect small molecules, effectively identifying and quantifying a wide range of small molecule pollutants under field conditions remains a challenge.

[0003] Immunochromatographic assays (ICAs) have gained prominence in point-of-care (POCT) testing due to their low cost, ease of use, instrument independence, and rapid detection capabilities. Competitive assay results rely on signal labeling competition between haptens and target molecules, resulting in reduced or absent test line (t-line) color for positive samples. However, traditional competitive ICA methods have three significant drawbacks. ① They rely on single-color labels (such as colorimetric or fluorescent nanospheres) to provide a visible color change, resulting in low sensitivity and poor quantitative signal readout. ② The accuracy of the "signal-off" strategy in competitive assays is limited by an inherently low signal-to-noise ratio (SNR), which results in insufficient color responsiveness and sensitivity for detecting weakly positive samples. ③ The drastic intermediate color changes between "yes" and "no" test results result in a narrow detection range, which can easily lead to misinterpretations. Therefore, it is imperative to address these challenges in competitive ICA and promote its widespread application in the detection of small molecule pollutants.

[0004] In recent years, biosensors using dual-emission fluorescent materials have shown considerable advantages in detection and analysis, achieving precise quantitative results by monitoring the emission ratio of two fluorescent signals. This method effectively reduces errors caused by external factors such as fluctuations in excitation light intensity and uneven sample concentration, thereby improving the stability and accuracy of the detection system. Dual-emission fluorescence has been successfully applied to the rapid detection of targets such as proteins, viruses, and mycotoxins, significantly improving the sensitivity and detection range of the ICA method. However, existing dual-emission fluorescence ICA still cannot meet the requirements for on-site screening of small molecule pollutants due to limitations in the stability of its fluorescent labeling, the method of reading the fluorescent signal, and the sensitivity of the detection results.

[0005] A Chinese patent application document with publication number CN117007793A discloses a competitive fluorescent immunochromatographic test strip, fluorescent probe, and application for rapid joint detection of imidacloprid and carbendazim. The test strip includes a PVC base, a sample pad, a nitrocellulose membrane, and an absorbent pad. The T line on the nitrocellulose membrane is coated with a pesticide hapten, and the C line is coated with a goat anti-mouse antibody. The fluorescent probe is a silicon core multilayer quantum dot shell nanosphere-pesticide monoclonal antibody immune complex. The test strip has good stability, excellent dispersibility, high luminescence performance, and immune properties. After mixing with the test solution, it flows sideways to the T line and binds to the coated antigen. The T line exhibits red fluorescence under ultraviolet light, and the rest binds to the coated antibody on the C line. Using a commercial fluorescence reader, quantitative detection of imidacloprid and carbendazim residues can be achieved, but its detection sensitivity and range are still not ideal. Summary of the Invention

[0006] The technical problem to be solved by the present invention is how to improve the sensitivity and stability of the immunochromatographic method for detecting small molecule pollutants.

[0007] The present invention solves the above technical problems through the following technical means:

[0008] A red fluorescent signal label comprises a graphene oxide carrier, a fluorescent layer disposed on the graphene oxide carrier, and a detection antibody modified on the fluorescent layer; the detection antibody is one of an anti-imidacloprid monoclonal antibody, an anti-fumonisin B1 monoclonal antibody, and an anti-clenbuterol monoclonal antibody; the fluorescent layer comprises one or more quantum dot layers; each quantum dot layer comprises a PEI self-assembled layer and a red quantum dot layer in a direction away from the graphene oxide carrier; the red quantum dot layer is formed by self-assembly of red fluorescent quantum dots or by self-assembly of red fluorescent quantum dots and then coating them with silicon dioxide.

[0009] Preferably, the fluorescent layer includes a quantum dot layer, and the red quantum dot layer is formed by self-assembly of red fluorescent quantum dots and then coating with silica; or, the fluorescent layer includes multiple quantum dot layers; the red quantum dot layer of the outermost quantum dot layer away from the graphene oxide carrier is formed by self-assembly of red fluorescent quantum dots and then coating with silica, and the red quantum dot layers of the remaining quantum dot layers are formed by self-assembly of red fluorescent quantum dots.

[0010] Preferably, the fluorescent layer includes three quantum dot layers; along the direction away from graphene oxide, there are the first, second and third quantum dot layers, the red quantum dot layers of the first quantum dot layer and the second quantum dot layer are self-assembled by red fluorescent quantum dots, and the red quantum dot layer of the third quantum dot layer is self-assembled by red fluorescent quantum dots and then coated with silica.

[0011] Preferably, the fluorescent layer further comprises BSA to block non-specific binding sites.

[0012] The present invention also provides a method for preparing the red fluorescent signal label, comprising the following steps:

[0013] S1. Graphene oxide and PEI solution are mixed and ultrasonicated, and PEI self-assembles on the graphene oxide to form a PEI layer. The product is mixed with a red fluorescent quantum dot solution and ultrasonicated, and the quantum dots self-assemble on the PEI layer.

[0014] S2. The product in S1 is evenly mixed with ethanol, mixed with ammonia water, TEOS, and TEPSA, and sonicated; the product is resuspended in MES buffer, mixed with EDC and NHS, and sonicated. After centrifugation, it is redispersed in PBS buffer, the detection antibody is added for incubation, and then BSA is added and shaken to obtain the red fluorescent signal label.

[0015] Preferably, in S1, the step of repeating S1 is further included, using a product obtained by self-assembly of quantum dots on the PEI layer instead of graphene oxide; preferably, the number of repetitions is 1-2 times.

[0016] Preferably, in the method for preparing the red fluorescent signal label, the ultrasonication time is 15-80 min.

[0017] Preferably, the usage ratio of ammonia water, TEOS, and TEPSA is 3 mL:0.2 mL:100 μL.

[0018] During the preparation process, strongly negatively charged GO nanosheets rapidly self-assemble with the cationic polymer polyethyleneimine (PEI) on their surface. Under ultrasound, a large number of red QD625 particles rapidly assemble onto the GO-PEI surface through electrostatic adsorption, forming a GQD structure. Through repeated PEI self-assembly and QD adsorption, GTQDs are formed. Under alkaline conditions, a smooth SiO2 shell is directly coated on the GTQD surface by uniformly mixing with ammonia, tetraethoxysilane (TEOS), and [(3-triethoxysilyl)propyl] succinic anhydride (TEPSA).

[0019] Preferably, the graphene oxide carrier is a single-layer graphene oxide (GO) with a size of 400-800 nm; through a PEI-mediated self-assembly strategy, three layers of red fluorescent QDs625 are ultrasonically adsorbed on the surface of the graphene oxide carrier; then, a SiO2 shell on the surface of the outermost quantum dots is synthesized by ammonia, TEOS and TEPSA to obtain a fluorescent layer; the thickness of the formed SiO2 shell is about 20 nm.

[0020] The present invention also provides a fluorescent immunochromatography kit, comprising one or more red fluorescent signal labels and a fluorescent immunochromatography test strip;

[0021] The fluorescent immunochromatographic test strip includes a sample pad, an absorption pad, a bottom plate and a nitrocellulose membrane; the sample pad, the nitrocellulose membrane and the absorption pad are sequentially arranged on the bottom plate; one or more detection lines and quality control lines are sequentially provided on the nitrocellulose membrane along the chromatography direction; the quality control line is coated with goat anti-mouse IgG; the detection line is coated with one of the coating antigens ICP-BSA, the coating antigen CLE-BSA and the coating antigen FB1-BSA and a green signal label; the green signal label includes silica sphere quantum dot nanoparticles and BSA modified on the surface of the silica sphere quantum dot nanoparticles; the silica sphere quantum dot nanoparticles include a SiO2 nanoparticle core and a double-layer quantum dot shell wrapped on the surface of the SiO2 nanoparticle core; each layer of the quantum dot shell includes a polyethyleneimine layer and green fluorescent quantum dots distributed on the surface of the polyethyleneimine layer in a direction away from the SiO2 nanoparticle core.

[0022] Preferably, the detection antibodies of different types of red fluorescent signal labels are different; the number of types of red fluorescent signal labels is consistent with the number of test lines and the type of analyte; and the detection antibodies on the red fluorescent signal labels correspond to the coating antigens coated on the test lines.

[0023] Preferably, the green fluorescent quantum dots and the red fluorescent quantum dots are both CdSe / ZnS QDs.

[0024] Preferably, the fluorescent immunochromatography kit further comprises a buffer solution.

[0025] Preferably, the fluorescent immunochromatography kit comprises three red fluorescent signal labels, wherein one red fluorescent signal label comprises a graphene oxide carrier, a fluorescent layer disposed on the graphene oxide carrier, and an anti-imidacloprid monoclonal antibody modified on the fluorescent layer; one red fluorescent signal label comprises a graphene oxide carrier, a fluorescent layer disposed on the graphene oxide carrier, and an anti-fumonisin B1 monoclonal antibody modified on the fluorescent layer; and another red fluorescent signal label comprises a graphene oxide carrier, a fluorescent layer disposed on the graphene oxide carrier, and an anti-clenbuterol monoclonal antibody modified on the fluorescent layer.

[0026] Preferably, the fluorescent immunochromatographic test strip has three detection lines on the nitrocellulose membrane; one detection line is coated with the coating antigen ICP-BSA and a green signal label, one detection line is coated with the coating antigen CLE-BSA and a green signal label, and the other detection line is coated with the coating antigen FB1-BSA and a green signal label.

[0027] Preferably, the same PEI-mediated self-assembly technology as that used in the preparation of the red fluorescent signal tag is used to ultrasonically adsorb two layers of green QD525 on the SiO2 surface to prepare a SiDQD nanocomposite material that provides a stable green fluorescent signal on the ICA test line.

[0028] Preferably, the method for preparing the green signal label comprises the following steps:

[0029] S1, mixing SiO2 nanoparticles with polyethyleneimine solution and then ultrasonicating to obtain SiO2-PEI nanospheres;

[0030] S2, mixing SiO2-PEI nanospheres, water, and green fluorescent quantum dots and then ultrasonicating to obtain SiQDs;

[0031] S3, mixing the SiQDs obtained in S2 with the polyethyleneimine solution and then sonicating;

[0032] S4, mixing the product obtained in S3, water, and green fluorescent quantum dots and then ultrasonicating to obtain SiDQDs;

[0033] S5. Disperse the SiDQD product obtained in S4 into MES buffer, add EDC and NHS, obtain carboxyl-activated SiDQD after sonication, resuspend in PBST solution, then add BSA, and incubate with vibration to obtain the green signal label.

[0034] Preferably, in the method for preparing the green signal label, the ultrasonication time is 15-40 minutes.

[0035] Preferably, the size of the SiO2 nanoparticles used is 200 nm.

[0036] Preferably, the SiO2 nanoparticles are prepared by mixing ammonia water, water, ethanol and TEOS and then stirring them, and two layers of QDs525 are adsorbed on the SiO2 surface by PEI self-assembly and ultrasonic adsorption to obtain silicon sphere quantum dot nanoparticles.

[0037] Preferably, in the preparation process of silicon sphere quantum dot nanoparticles, the usage ratio of ammonia water, water, ethanol and TEOS is 8 mL:12 mL:200 mL:8 mL, and the mixing and stirring time is two hours.

[0038] Preferably, the nitrocellulose membrane model is CN140.

[0039] Preferably, the sample pad is a cellulose material.

[0040] Preferably, the absorbent pad is a cellulosic material.

[0041] The present invention also provides an application of the fluorescent immunochromatographic kit in detecting one or more of imidacloprid, fumonisin B1 and clenbuterol.

[0042] Preferably, the fluorescent immunochromatographic kit simultaneously detects three small molecule pollutants: ICP, FB1, and CLE.

[0043] The present invention also proposes a method for detecting one or more of imidacloprid, fumonisin B1, and clenbuterol using the fluorescent immunochromatography kit, comprising the following steps: uniformly mixing a detection target, a buffer solution, and a red fluorescent signal label, or uniformly mixing the detection target and a red fluorescent signal label to obtain a test liquid, adding the test liquid to the sample pad of an immunochromatographic test strip for reaction; and performing qualitative analysis and / or quantitative analysis after ultraviolet irradiation after the reaction is completed.

[0044] Preferably, the fluorescence signal is observed under ultraviolet light for qualitative analysis and / or read using a fluorescence reader for accurate quantitative analysis.

[0045] Preferably, in the method for detecting one or more of ICP, FB1, and CLE, the reaction time is 15 minutes.

[0046] Preferably, the wavelength of the ultraviolet light is 365 nm.

[0047] Preferably, the concentration gradient of the detection target in the test solution is 100-0.001 ng / mL.

[0048] In the present invention, a red 2D thin film label (GTQD@Si) is introduced into the multi-fluorescence ICA, and a dual signal amplification effect is achieved through a "large reaction interface and high quantum dot (QD) loading". At the same time, green fluorescent nanospheres (SiDQD-BSA) are embedded in the test line (T line) area as indicator labels to provide green background fluorescence. A multi-channel fluorescence gradient ICA (FGICA) platform based on the interaction between dual-emission labels is constructed, which can conveniently, ultra-sensitively, over a large range, and simultaneously detect fumonisin B1 (FB1), imidacloprid (ICP) and clenbuterol (CLE) pollutants in complex sample matrices.

[0049] The present invention establishes a universal fluorescence gradient immunochromatographic assay (FGICA) that utilizes dual signal superposition to achieve ultrasensitive, wide-range, and simultaneous quantitative detection of multiple small molecules. A red fluorescent nanofilm (GTQD@Si) was synthesized by continuously self-assembling multilayer quantum dots and SiO2 shells on a graphene oxide surface. This nanofilm exhibits high stability in complex environments and provides superior fluorescence and a larger sensing-reaction interface. Combining GTQD@Si with green fluorescent microspheres embedded in the test line generates a wide fluorescence gradient based on changes in the concentration of the target molecule, significantly improving the sensitivity, stability, and quantitative range of the immunochromatographic assay (ICA). By directly reading the ratio of red and green image signals, FGICA can simultaneously and highly sensitively and quantitatively detect three different types of small molecule pollutants, including fumonisin B1, imidacloprid, and clenbuterol, within 15 minutes, extending the detection range by 2-3 orders of magnitude compared to traditional methods. Furthermore, comprehensive testing of FGICA on a variety of real samples has verified its strong practicality and demonstrated its great potential for on-site detection of small molecules.

[0050] Compared with the prior art, the present invention has the following beneficial effects:

[0051] ① The use of a film-like tag (GTQD@Si) protected by a SiO2 shell enhances the sensitivity and stability of ICA in complex systems, enabling accurate and simultaneous detection of multiple targets.

[0052] ② The strong red fluorescence is superimposed on the green fluorescence to form a fluorescence gradient detection range from orange-red to orange and then to green, which significantly broadens the detection range of the competitive ICA method.

[0053] ③ By directly reading the red (R) and green (G) values in the fluorescence image, accurate analysis of the fluorescence signals of multiple detection areas on the FGICA strip is achieved, thereby improving the accuracy of multi-target detection.

[0054] The FGICA technique of this invention can rapidly and sensitively detect FB1, CLE, and ICP within 15 minutes, with a detection limit as low as pg / mL and a quantitative range spanning five orders of magnitude. Furthermore, the FGICA method demonstrated excellent accuracy and stability in the detection of real-world food samples, environmental samples, and clinical urine samples, demonstrating the broad potential of the proposed detection technique for real-time environmental and biological pollutant analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 This is a schematic diagram of the preparation principle of the FGICA material according to Example 1 of the present invention;

[0056] Figure 2 This is a schematic diagram of the FGICA principle for simultaneously detecting three target small molecule pollutants in Example 1 of the present invention;

[0057] Figure 3 The morphological characterization of the red fluorescent GTQD@Si nanofilm and the green fluorescent SiDQD nanospheres in Example 2 of the present invention;

[0058] Figure 4 Characterization of the physical / chemical properties of the GTQD@Si nanofilm in Example 3 of the present invention;

[0059] Figure 5 Schematic diagram of the detection results of GQD@Si-FGICA, GDQD@Si-FGICA and GTQD@Si-FGICA in Example 4 of the present invention, as well as the sensitivity comparison of GQD@Si-FGICA, GDQD@Si-FGICA and GTQD@Si-FGICA bands at different ICP concentrations and SEM images in the T-line area;

[0060] Figure 6 The cross-validation and sensitivity test results of GTQD@Si-FGICA in Example 5 of the present invention are shown;

[0061] Figure 7 This is to verify the detection performance of GTQD@Si-FGICA in Example 6 of the present invention when applied to real samples;

[0062] Figure 8 This is the repeatability diagram of GTQD@Si-FGICA in Example 6 of the present invention. DETAILED DESCRIPTION

[0063] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0064] Unless otherwise specified, the test materials and reagents used in the following examples can be obtained from commercial sources.

[0065] If no specific techniques or conditions are specified in the examples, they can be carried out according to the techniques or conditions described in the literature in the field or according to the product instructions.

[0066] The pesticide standard solutions used in the examples, including imidacloprid (ICP), fumonisin B1 (FB1), and clenbuterol (CLE), were obtained from Shanghai Macklin Co., Ltd., China. Anti-ICP monoclonal antibody (Ab8017), anti-FB1 monoclonal antibody (Ab3033), anti-CLE monoclonal antibody (QY-C0109), coating antigens ICP-BSA (Ag8017), CLE-BSA (QYL-040101), and FB1-BSA (Ag3033) were purchased from Jiangnan University. Goat anti-mouse IgG was provided by Shanghai Sangon Biotechnology Co., Ltd.

[0067] Example 1

[0068] Figure 1 and Figure 2 This is a schematic diagram of the fluorescent gradient immunochromatography (FGICA) method shown in Example 1 of the present invention. The immunochromatographic test strip used includes a base plate, a sample pad, an absorption pad, and a nitrocellulose membrane. The nitrocellulose membrane is attached to the base plate, and the sample pad and the absorption pad are attached to both ends of the base plate, overlapping the nitrocellulose membrane. The nitrocellulose membrane is provided with three test lines and a quality control line. The quality control line is close to the absorption pad, and the test line is close to the sample pad. The three test lines (T1, T2, and T3) of the nitrocellulose membrane are sprayed with SiDQD-ICP-BSA (i.e., a solution containing SiDQD-BSA and ICP-BSA), SiDQD-FB1-BSA (i.e., a solution containing SiDQD-BSA and FB1-BSA), and SiDQD-CLE-BSA (i.e., a solution containing SiDQD-BSA and CLE-BSA), respectively. The quality control line (C) is sprayed with goat anti-mouse antibody IgG. The schematic diagram is shown in FIG. Figure 2 shown.

[0069] A method for preparing an immunochromatographic test strip for detecting small molecule pollutants ICP, FB1, and CLE comprises the following steps:

[0070] (1) Preparation of GTQD@Si-COOH and SiDQD as fluorescent signal labels, the process is shown in the figure Figure 1 As shown;

[0071] GTQD@Si-COOH: 5 ml of 2 mg / mL dispersed monolayer graphene oxide (GO) (400-800 nm) was mixed with 5 ml of 2 mg / mL PEI solution and sonicated for 30 minutes to form a GO-PEI with a positive surface layer. The GO-PEI was centrifuged at 14,000 rpm for 8 minutes and washed twice to remove excess PEI. Subsequently, 100 μL of a 0.1 M solution of red fluorescent quantum dots (QD625) (i.e., CdSe / ZnS QDs with an excitation wavelength of 625 nm) was added and sonicated for 30 minutes to achieve uniform distribution. The GQD product was recovered by centrifugation at 7,000 rpm for 8 minutes. The GQD product was repeated with the above process of forming a positive layer and loading QD625 to load a second layer of QD625 to obtain GDQDs. The GDQDs were then repeated with the above process of forming a positive layer and loading QD625 to load a third layer of QD625 to prepare GTQDs. The resulting GTQDs were sonicated with 50 mL of ethanol solution to ensure complete mixing. The suspension was then mixed with 3 mL of ammonia, 0.2 mL of TEOS, and 100 μL of TEPSA and sonicated for 80 minutes. After the reaction, the GTQD@Si-COOH solution was washed twice with deionized water and resuspended in 10 mL of anhydrous ethanol for later use.

[0072] SiDQDs: 8 mL of ammonia and 12 mL of deionized water were added to 200 mL of anhydrous ethanol. After thorough mixing, 8 mL of TEOS was added and the mixture was stirred for 2 hours. The mixture was then washed thoroughly with anhydrous ethanol by centrifugation to prepare SiO2. The resulting SiO2 was added to 40 mL of a 2 mg / mL PEI solution and sonicated for 30 minutes to form SiO2-PEI. SiO2-PEI nanospheres were obtained by centrifugation (6000 rpm, 6 minutes) and washed twice to remove excess PEI. The SiO2-PEI nanospheres were dispersed in 30 mL of deionized water and 100 μL of a 0.1 M solution of green fluorescent quantum dots (QD525) (i.e., CdSe / ZnS QDs with an excitation wavelength of 525 nm) was added. After sonication for 40 minutes, the mixture was centrifuged (5500 rpm, 6 minutes) to obtain SiQDs. The above steps of adsorbing PEI and QD525 were repeated to coat the nanospheres with a second layer of QD525 to form SiDQDs. Green SiDQDs were obtained by centrifugation (5000 rpm, 6 min) and dispersed in 10 ml of anhydrous ethanol for later use.

[0073] (2) Preparation of GTQD@Si-Ab and SiDQD-BSA

[0074] GTQD@Si-Ab: The GTQD@Si-COOH obtained in step (1) was resuspended in 10 ml of anhydrous ethanol and centrifuged at 5500 rpm for 8 min. The suspension was then resuspended in 0.5 mL of 0.1 M MES buffer (pH 5.8). 5 μL of EDC (10 mM) and 10 μL of NHS (10 mM) were added and sonicated for 15 min. After centrifugation, the suspension was redispersed in 500 μL of PBS buffer. Detection antibodies (8 μg each of anti-ICP monoclonal antibody, anti-FB1 monoclonal antibody, and anti-CLE monoclonal antibody) were then introduced and incubated for 2 h. 100 μL of bovine serum albumin (BSA) (0.1 g / mL) was added and the suspension was shaken for 1 h. The GTQD@Si-Ab was washed by centrifugation and resuspended in 200 μL of PBS buffer to obtain a GTQD@Si-Ab solution.

[0075] SiDQD-BSA: SiDQD-BSA was synthesized using the EDC / NHS carbodiimide chemistry method. After centrifuging 1 ml of the SiDQD solution prepared in step (1) at 5000 rpm for 6 min, the sample was redispersed in 0.5 mL of MES buffer (0.1 M, pH 5.5). Carboxyl activation was initiated by adding 5 μL of freshly prepared EDC (100 mM) and 10 μL of NHS (100 mM), followed by sonication for 15 min. The carboxyl-activated SiDQDs were collected by centrifugation and resuspended in 200 μL of PBST solution. 100 μL of BSA (10% w / v) was then added, incubated with shaking for 2 h, and centrifuged to obtain SiDQD-BSA, which was resuspended in 0.5 mL of PBS solution (0.01 M, pH = 7.4) to obtain the SiDQD-BSA solution for later use.

[0076] Then, the hapten ICP-BSA of ICP, the hapten FB1-BSA of FB1, and the hapten CLE-BSA of CLE were mixed with the SiDQD-BSA solution obtained above to make the concentrations of ICP-BSA, FB1-BSA, and CLE-BSA 0.06, 0.6, and 0.4 mg / mL, respectively, to obtain mixed solutions, which were sequentially recorded as SiDQD-ICP-BSA, SiDQD-FB1-BSA, and SiDQD-CLE-BSA, and were used for spraying on the T1, T2, and T3 lines, respectively. Figure 2 As shown in b.

[0077] (3) Construction of immunochromatographic test strips

[0078] This test uses competitive immunochromatography to detect small molecule contaminants. Competitive immunochromatography is based on the specific binding of antigens and antibodies, detecting target molecules through a competitive reaction. The target antigen and the labeled antigen in the sample compete for binding to the immobilized antibody. The more target antigen there is, the less antibody there is for the labeled antigen to bind, and the weaker the signal, indicating a higher concentration of the target antigen.

[0079] The test paper of the present invention adopts a four-component design, including a basic plastic backing card (base plate), a sample receiving pad (sample pad), a nitrocellulose (NC) membrane with three integrated detection lines (T1 / T2 / T3) and a quality control line (C line), and an absorption pad that allows capillary flow. The nitrocellulose membrane is attached to the base plate, and the sample pad and absorption pad are attached to both ends of the base plate, overlapping the nitrocellulose membrane. The quality control line is close to the absorption pad, and the detection line is close to the sample pad. The construction principle diagram is shown in FIG. Figure 2 As shown in the figure, during the preparation process, SiDQD-ICP-BSA, SiDQD-FB1-BSA, and SiDQD-CLE-BSA were precisely sprayed onto the detection lines T1 / T2 / T3 of the NC membrane, respectively. Goat anti-mouse IgG (0.5 mg / mL) was sprayed onto line C using an automatic sprayer (Biodot xyz5050) to produce the antibody-loaded NC membrane. The antibody-loaded NC membrane was dried in a drying oven and then attached to a base plate. The sample pad and absorbent pad were assembled on the plastic base plate, with the sample pad overlapping one end of the NC membrane and the absorbent pad overlapping the other end. Finally, the card was cut into 3 mm wide strips and dried in a dark place until ready for use.

[0080] like Figure 2 As shown in Figure 2 (b), during the test, the target solution is mixed with the analyte, a GTQD@Si-Ab solution, and a buffer solution (PBS buffer containing 1% Tween and 0.1% fetal bovine serum (FBS) by volume). The volume ratio of the GTQD@Si-Ab solution to the buffer solution is 1:100. The target solution is added dropwise to the sample pad of the test strip. Following the chromatography effect, the solution flows through the entire nitrocellulose membrane and finally reaches the absorption pad. During this process, when the target analyte is absent, the GTQD@Si-Ab antibody binds to the BSA on the corresponding T line, forming a color-changing fluorescent signal. Conversely, when the target analyte is present, the T line retains its original green fluorescent signal.

[0081] (4) Fluorescence signal reading

[0082] After irradiation with 365nm ultraviolet laser light, the results can be read qualitatively and semi-quantitatively by naked eyes. The fluorescent signal can be accurately and quantitatively read using a portable fluorescence reader, such as Figure 2 As shown in c, the schematic diagram of negative and positive test results on the test line is as follows Figure 2 As shown in d.

[0083] Example 2

[0084] Figure 3 This is the morphological characterization of the red fluorescent GTQD@Si-COOH nanofilm and green fluorescent SiDQD nanospheres prepared in Example 1 of the present invention.

[0085] Figure 3 ae are TEM images of the initial carrier GO, intermediate products GQD, GDQD, GTQD and final product GTQD@Si-COOH, respectively.

[0086] Interpretation of results: GO is a semi-transparent nanosheet with a thickness of 1-2 nm ( Figure 3 a), driven by ultrasound, a large number of red QD625 quickly self-assembled onto the GO-PEI surface through electrostatic adsorption, forming a GO@QD structure with densely arranged quantum dots on the surface, namely GQD ( Figure 3 b). By repeating the PEI self-assembly and QD adsorption process, the second and third layers of dense QDs can be easily loaded onto the GO surface, thus forming a composite nanomembrane with a multilayer QD shell ( Figure 3 cd).

[0087] Figure 3 fj are high-resolution TEM (HRTEM) images of GQD (f, g), GDQD (h), GTQD (i) and GTQD@Si-COOH (j).

[0088] Interpretation of results: By HRTEM, coherent lattice fringes with uniform spacing (~0.348 nm) were observed on the surface of the adsorbed quantum dots, indicating that the main component of the quantum dots is CdSe ( Figure 3 f). Figure 3 hi shows that with the continuous coating of multiple QD shell layers, the density and number of QDs on the nanosheet surface increase significantly, Figure 3 The thickness of the SiO2 shell formed is about 20 nm.

[0089] Figure 3 km is the HAADF and EDS element scanning diagram of a single GTQD@Si-COOH.

[0090] Interpretation of results: These clearly present the distribution of Si, O, Cd, Se, Zn and S elements on the surface of C element in the composite nanofilm, proving the multilayer structure and elemental composition of the composite film-like fluorescent label.

[0091] Figure 3 nq is the TEM images of the prepared SiO2, SiQD, SiDQD and surface-loaded QD525. Figure 3 rs is SiQD, Figure 3tu is a locally enlarged TEM image of SiDQD.

[0092] Interpretation of the results: The adsorbed QD525 completely covered the entire SiO2 surface with the continuous coating of PEI and QD525. HRTEM observations revealed uniformly spaced coherent lattice fringes (~0.349 nm) on the surface of the adsorbed QDs, indicating that the primary component of the QDs is CdSe.

[0093] Example 3

[0094] Figure 4 This is the physical / chemical property characterization of the GTQD@Si-COOH nanofilm of Example 1 of the present invention. Figure 4 a is a schematic diagram of the structure of the film-like GTQD@Si-COOH, in which the inner layer of two-dimensional GO provides a large reaction interface, the three-layer PEI / QD625 produces a high signal loading capacity, and the outermost SiO2 shell provides better stability and dispersion ability in complex samples.

[0095] Figure 4 bh are wide scan and high-resolution XPS spectra of GTQD@Si-COOH.

[0096] Interpretation of results: Obvious signal peaks of Si, Cd, Se, Zn, O and C elements were observed, and no signals of other impurity elements appeared, confirming that the outer layer of the nanofilm is composed of quantum dots and SiO2.

[0097] The fluorescence of SiO2, SiQD, SiDQD, GO, GQD, GDQD, GTQD and GTQD@Si-COOH as well as the mixture of SiDQD and GTQD@Si-COOH was tested using a microplate reader. Figure 4 il is the fluorescence spectrum data of red fluorescent GTQD@Si-COOH and green fluorescent SiDQD and their mixture at different stages.

[0098] Interpretation of the results: Continuous adsorption of two layers of QD525 on the SiO2 surface, which has no fluorescence signal itself, can produce strong green fluorescence. The fluorescence intensity of SiDQD is 1.43 times that of SiQD nanocomposites ( Figure 4 i). Similarly, continuous adsorption of multiple layers of QD625 (1-3 layers) on the GO surface can produce a gradually enhanced red fluorescence signal of the membrane marker, where the fluorescence intensity of GTQD reaches 1.26 times and 2.04 times that of GQD and GDQD, respectively ( Figure 4 j). The mixture of red (R) fluorescent material GTQD@Si-COOH and green (G) fluorescent material SiDQD produces orange fluorescence under ultraviolet excitation ( Figure 4k). Compared with GTQD, GTQD@Si-COOH (i.e., GTQD@Si) exhibits better stability ( Figure 4 l).

[0099] Example 4

[0100] GQDs and GDQDs were prepared according to the steps in Example 1. The resulting GQDs and GDQDs were each sonicated with 50 mL of ethanol solution to ensure complete mixing. The suspension was then mixed with 3 mL of ammonia, 0.2 mL of TEOS, and 100 μL of TESA and sonicated for 80 minutes. After the reaction, the suspensions were washed twice with deionized water to obtain GQD@Si and GDQD@Si, respectively. Each suspension was resuspended in 10 mL of anhydrous ethanol, centrifuged at 5500 rpm for 8 minutes, and resuspended in 0.5 mL of 0.1 M MES buffer (pH 5.8). 5 μL of 10 mM EDC and 10 μL of 10 mM NHS were then added, and sonicated for 15 minutes. After centrifugation, the suspensions were redispersed in 500 μL of PBS buffer. Detection antibodies (anti-ICP monoclonal antibody, 8 μg) were then introduced and incubated for 2 hours. 100 μL of 0.1 g / mL bovine serum albumin (BSA) was added and shaken for 1 hour. GQD@Si-Ab and GDQD@Si-Ab were obtained by centrifugation and washing, and then resuspended in 200 μl PBS buffer.

[0101] Using GQD@Si-Ab, GDQD@Si-Ab, and GTQD@Si-Ab as signal labels, test strips containing only one detection line (sprayed with SiDQD-ICP-BSA) were constructed according to the method in Example 1 and then tested. They were recorded as GQD@Si-FGICA, GDQD@Si-FGICA, and GTQD@Si-FGICA, respectively. Figure 5 Schematic diagram of the constructed test strip and the detection results of GQD@Si-FGICA, GDQD@Si-FGICA and GTQD@Si-FGICA (Figure a), the detection images and the measured fluorescence signals (Figures bc), the comparison of the detection performance of the three FGICAs (Figure de), and the SEM images of the NC film under positive and negative conditions of the test strip prepared in Example 1 (Figure f), where the concentration in Figure b is the concentration of ICP contained in the test solution;

[0102] Interpretation of the results: When combined with green fluorescent tags (SiDQDs), all three types of red fluorescent nanofilms produced a fluorescence gradient effect on the T line; however, the fluorescence response intensity of the GTQD@Si tag on the FGICA strip was significantly higher than that of the GQD@Si and GDQD@Si tags ( Figure 5ac). In addition, according to the s-curve fitted by the T-line R / G value on the FGICA test paper, the detection limits (LODs) of GQD@Si-FGICA, GDQD@Si-FGICA and GTQD@Si-FGICA for ICP were 0.068 ng / mL, 0.012 ng / mL and 0.00102 ng / mL, respectively ( Figure 5 d). GTQD@Si exhibits a wider range of fluorescence color changes on the FGICA strips, thereby improving the sensitivity of the proposed fluorescence gradient biosensor and expanding its detection range ( Figure 5 e). The lines of the FGICA strips were observed using a scanning electron microscope. SiDQD-BSA particles can be observed in the internal structure of the T line of the positive sample test paper ( Figure 5 fI). This result also verifies that SiDQD-BSA can be firmly bound to the NC membrane of the T line and will not be washed away by the sample solution. In the internal structure of the T line of the negative sample, GTQD@Si nanofilm and spherical SiDQD-BSA labels can be observed simultaneously within the T line structure, indicating that the fluorescence gradient of the T line comes from the interaction between these two labels ( Figure 5 fII). These SEM characterization results confirm that the combination of red and green tags can work stably and sensitively in the FGICA system.

[0103] Example 5

[0104] Figure 6 This is a cross-validation of the three detection targets of Example 1 of the present invention and a sensitivity comparison of FGICA and ELISA.

[0105] Figure 6 Figure a shows a cross-validation of three targets, showing photographs of test strips (test strip iv) with different CLE / FB1 / ICP concentrations and the corresponding R / G ratios of the three T lines on the FGICA test strip (sample CLE / FB1 / ICP concentrations are as follows: i: 0 / 0 / 0 ng / mL; ii: 0 / 0 / 100 ng / mL; iii: 0 / 100 / 0 ng / mL; iv: 100 / 0 / 0 ng / mL; v: 100 / 100 / 100 ng / mL). From these results, we can clearly observe that only in the presence of the target small molecule is the red fluorescence signal of the corresponding T line on the test strip suppressed, resulting in a rapid decrease in the R / G ratio. Notably, T lines with uniform red and green fluorescence only appear when testing the corresponding negative and positive samples, further demonstrating the absence of fluorescence interference between the three T lines on the test strip.

[0106] Interpretation of the results: Only in the presence of the target small molecule will the red fluorescence signal on the corresponding T line on the test strip be suppressed, resulting in a rapid decrease in the R / G ratio, indicating that there is no fluorescence interference between the three T lines on the test strip. Therefore, the established FGICA has good selectivity for multiplex detection of different targets.

[0107] According to the method of the target solution in Example 1, the three targets were serially diluted with buffer, and then the red signal tag GTQD@Si-Ab was added for quantitative reading. The target concentration is as follows: Figure 6 As shown in b, Figure 6 bf is the sensitivity detection of CLE, FB1 and ICP.

[0108] Interpretation of results: As the concentrations of the three target analytes in the solution increased, the fluorescence colors of the three T lines on the FGICA strip gradually changed from orange-red to orange and finally to green, showing a clear fluorescence gradient process. The precise ratio of red and green fluorescence on the three T lines (R / G value) was quickly and accurately read using the fluorescence reading software. The results showed that the R / G value, the ratio of red fluorescence to green fluorescence of the T1 / T2 / T3 lines, decreased with the increase in the concentration of the target analytes (ICP / FB1 / CLE), indicating that the R / G value had a good negative correlation with the small molecule concentration. The calibration curve of the competitive ICA of the three target small molecule pollutants was constructed using the s-type function of the target concentration and the obtained R / G value, which had a good quantitative relationship within the detection range ( Figure 6 bf).

[0109] Figure 6 gi is ELISA detection of CLE, FB1 and ICP

[0110] Interpretation of results: The LODs for CLE, FB1, and ICP detected by ELISA were 0.184, 0.119, and 0.195 ng / mL, respectively, which are 57.5, 117.82, and 191.18 times higher than those of our method. Due to the cumbersome ELISA procedure and long detection time (2-3 hours), our proposed FGICA offers greater user-friendliness and rapid on-site detection capabilities.

[0111] Example 6

[0112] Example 6 of the present invention is Figure 7 and Figure 8 Repeatability verification of urine samples using real samples and GTQD@Si-FGICA

[0113] Figure 7ad are four real sample verifications. Specifically, the analyte sample was diluted with real samples instead of the buffer of the target solution in Example 1, and then the red signal label GTQD@Si-Ab was added and the data was read;

[0114] Results: FGICA maintained a stable fluorescence signal across four defined concentration gradients (1, 0.1, 0.01, and 0 ng / mL) in various complex samples, with changes in red and green fluorescence exhibiting a good concentration-dependent pattern. The average recoveries of FGICA for small molecule targets ranged from 90.58% to 107.90%, with coefficients of variation (CV) below 9.68%, demonstrating its reliability in food and environmental testing.

[0115] Figure 8 ac are the urine sample repeatability verification of GTQD@Si-FGICA.

[0116] Interpretation of results: We collected 5 urine samples instead of the buffer solution of the target solution and prepared small molecule targets (ICP / FB1 / CLE) of different concentrations (0.01-1 ng / mL) for immunochromatographic detection. The FGICA method can accurately and simultaneously detect the three target small molecules in real urine samples and achieve precise quantification through the ratio of red and green fluorescence. The R / G values measured in 5 independent experiments remained stable within each concentration group, and the CV was all below 5.2%. In Figures ac, the concentrations of the small molecule targets were 1 ng / mL, 0.1 ng / mL, and 0.01 ng / mL, respectively.

[0117] Based on the interaction between the red and green fluorescent markers on the test line (T line), the fluorescence on the T line changes significantly from orange-red (negative) to orange (weakly positive) and then to green (positive). A fluorescence gradient ICA platform for the simultaneous quantitative detection of three small molecule pollutants was developed. The SiO2 shell-protected GTQD nanofilm (GTQD@Si) was used as a high-brightness red fluorescent label, and BSA-modified green fluorescent microspheres (SiDQD-BSA) were used as fluorescent indicators in the nitrocellulose membrane detection area. In the GTQD@Si fluorescent label, multiple layers of QD625 (1-3 layers) were continuously adsorbed on the graphene oxide surface, which could produce a gradually enhanced film-like labeled red fluorescent signal. The fluorescence intensity of GTQD reached 1.26 times and 2.04 times that of GQD and GDQD, respectively. In addition, the fluorescence performance of GTQD@Si reached 95.2% of that of GTQD, indicating that the SiO2 shell has little effect on the luminescence performance of the QD-based material. Continuously adsorbing two layers of QD525 on a SiO2 surface produces strong green fluorescence, with the resulting SiDQDs exhibiting a fluorescence intensity 1.43 times greater than that of SiQD nanocomposites. This further enhances the intensity of the red / green fluorescence signal. The green-fluorescent SiDQDs were used in a test line, and GTQD@Si antibodies were prepared via an amidation reaction, enabling the preparation of target immunochromatographic test strips.

[0118] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A red fluorescent signal tag, characterized in that: The invention comprises a graphene oxide carrier, a fluorescent layer arranged on the graphene oxide carrier, and a detection antibody modified on the fluorescent layer; the detection antibody is one of an anti-imidacloprid monoclonal antibody, an anti-fumonisin B1 monoclonal antibody, and an anti-clenbuterol monoclonal antibody; the fluorescent layer comprises one or more quantum dot layers; each quantum dot layer comprises a PEI self-assembled layer and a red quantum dot layer in a direction away from the graphene oxide carrier; the red quantum dot layer is formed by self-assembly of red fluorescent quantum dots or by self-assembly of red fluorescent quantum dots and then coating them with silicon dioxide.

2. The red fluorescent signal tag according to claim 1, characterized in that: The fluorescent layer includes a quantum dot layer, and the red quantum dot layer is formed by self-assembly of red fluorescent quantum dots and then coating with silicon dioxide; or, the fluorescent layer includes multiple quantum dot layers; the red quantum dot layer of the outermost quantum dot layer away from the graphene oxide carrier is formed by self-assembly of red fluorescent quantum dots and then coating with silicon dioxide, and the red quantum dot layers of the remaining quantum dot layers are formed by self-assembly of red fluorescent quantum dots.

3. A method for preparing a red fluorescent signal tag according to claim 1 or 2, characterized in that: The following steps are involved: S1. Graphene oxide and PEI solution are mixed and ultrasonicated, and PEI self-assembles on the graphene oxide to form a PEI layer. The product is mixed with a red fluorescent quantum dot solution and ultrasonicated, and the quantum dots self-assemble on the PEI layer. S2. The product in S1 is evenly mixed with ethanol, mixed with ammonia water, TEOS, and TEPSA, and sonicated; the product is resuspended in MES buffer, mixed with EDC and NHS, and sonicated. After centrifugation, it is redispersed in PBS buffer, the detection antibody is added for incubation, and then BSA is added and shaken to obtain the red fluorescent signal label.

4. The method for preparing a red fluorescent signal label according to claim 3, wherein: In S1, the step of S1 is further comprised of replacing graphene oxide with a product obtained by self-assembly of quantum dots on the PEI layer and repeating the steps of S1.

5. A fluorescent immunochromatography kit, characterized in that: Comprising one or more red fluorescent signal labels and fluorescent immunochromatographic test strips as described in claim 1 or 2 or prepared as claimed in claim 3 or 4; The fluorescent immunochromatographic test strip includes a sample pad, an absorption pad, a bottom plate and a nitrocellulose membrane; the sample pad, the nitrocellulose membrane and the absorption pad are sequentially arranged on the bottom plate; one or more detection lines and quality control lines are sequentially provided on the nitrocellulose membrane along the chromatography direction; the quality control line is coated with goat anti-mouse IgG; the detection line is coated with one of the coating antigens ICP-BSA, the coating antigen CLE-BSA and the coating antigen FB1-BSA and a green signal label; the green signal label includes silica sphere quantum dot nanoparticles and BSA modified on the surface of the silica sphere quantum dot nanoparticles; the silica sphere quantum dot nanoparticles include a SiO2 nanoparticle core and a double-layer quantum dot shell wrapped on the surface of the SiO2 nanoparticle core; each layer of the quantum dot shell includes a polyethyleneimine layer and green fluorescent quantum dots distributed on the surface of the polyethyleneimine layer in a direction away from the SiO2 nanoparticle core.

6. The fluorescent immunochromatography kit according to claim 5, wherein: Different types of red fluorescent signal labels have different detection antibodies; the number of types of red fluorescent signal labels is consistent with the number of test lines and the type of analyte; and the detection antibody on the red fluorescent signal label corresponds to the coating antigen coated on the test line.

7. The fluorescent immunochromatography kit according to claim 5 or 6, characterized in that: The green fluorescent quantum dots and the red fluorescent quantum dots are both CdSe / ZnS QDs.

8. The fluorescent immunochromatography kit according to claim 5 or 6, characterized in that: Buffer is also included.

9. Use of the fluorescent immunochromatographic kit according to any one of claims 5 to 8 in detecting one or more of imidacloprid, fumonisin B1, and clenbuterol.

10. A method for detecting one or more of imidacloprid, fumonisin B1, and clenbuterol using the fluorescent immunochromatographic kit according to any one of claims 5 to 8, characterized in that: The following steps are involved: The detection target, buffer solution and red fluorescent signal label are evenly mixed, or the detection target and red fluorescent signal label are evenly mixed to obtain a test solution, which is added to the sample pad of the immunochromatographic test strip for reaction; after the reaction is completed, qualitative analysis and / or quantitative analysis is performed after ultraviolet light irradiation.

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