A lateral flow immunoassay kit and method for detecting aflatoxin B1

Abstract

This invention discloses a lateral immunoassay kit and method for detecting aflatoxin B1, belonging to the field of lateral immunoassay technology. The kit includes a nanobody-biotin / streptavidin@quantum dot probe and a lateral immunoassay test strip. The lateral immunoassay test strip has a T-line and a C-line on its nitrocellulose membrane. The T-line is coated with AFB1-BSA antigen, and the C-line is coated with biotin-labeled bovine serum albumin. The directional coupling strategy of this invention overcomes the defect of reduced nanobody activity caused by random modification, greatly preserving the activity of the nanobody. Utilizing smartphone photography for grayscale reading, it features rapid, accurate, stable, and instrument-free on-site analysis, possessing broad application potential and significant market prospects.

Description

Technical Field

[0001] This invention belongs to the field of lateral flow immunoassay technology, and in particular relates to a lateral flow immunoassay kit and method for detecting aflatoxin B1. Background Technology

[0002] Currently, human and animal safety is facing a serious threat from mycotoxins, which are secondary metabolites produced by specific fungi growing on various crops. Aflatoxins are a class of carcinogenic mycotoxins, mainly produced by Aspergillus fungi. Among the more than 20 identified aflatoxins, aflatoxin B1 (AFB1) is the most toxic. Carcinogenicity, teratogenicity, acute toxicity, mutagenicity, and other biological activities are the main reasons for its harm to human and animal health. Numerous reports indicate that AFB1 can cause hepatocellular carcinoma (HCC) in humans and animals, directly or indirectly participating in cancer development, seriously endangering people's lives and property. Therefore, rapid quantitative analysis of AFB1 is crucial for ensuring food safety and protecting health.

[0003] Nanobodies (recombinant pure heavy chain antibody with variable domains, Nb) have become emerging reagents for analyzing environmental and food chemicals due to their high thermal stability, ease of genetic modification, and low-cost production. Many nanobody-based methods have been developed for detecting mycotoxins, such as AFB1, OTA, and DON. These methods are primarily based on traditional ELISA, which is time-consuming and involves complex procedures, hindering the application of nanobodies in field detection. Lateral flow immunoassay (LFIA) test strips are increasingly popular in field detection techniques for mycotoxins due to their ease of operation, low cost, ease of production, and reduced time consumption. Most LFIA test strips consist of monoclonal antibodies (mAbs) or polyclonal antibodies (pAbs) coupled with nanomaterials such as gold nanoparticles, quantum beads, and fluorescent microspheres. Antibodies are mainly randomly directed onto nanomaterials via electrostatic adsorption or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)-mediated carbodiimide method, which may degrade the performance of the immunoassay, resulting in unsatisfactory antibody performance. To overcome these bottlenecks, several methods have been developed to achieve directional coupling between antibodies and nanomaterials, such as protein A / G, brush-type ligand-coated quantum beads, and quantum dots with hydrazide groups, which help antibodies maintain their antigen capture efficiency. While many LFIA test strips have been reported, there are few reports on LFIA strips based on nanobodies, especially for mycotoxin detection. A possible reason is that the size of nanobodies is too small, and their activity is affected during the connection with nanomaterials. Therefore, developing a nanobodies-based lateral flow immunoassay test strip for the detection of mycotoxins, especially aflatoxin B1, is of great significance for mycotoxin detection. Summary of the Invention

[0004] The purpose of this invention is to provide a lateral flow immunoassay kit and method for detecting aflatoxin B1, thereby addressing the problems existing in the prior art. This invention utilizes a directional conjugation strategy based on biotin and streptavidin to overcome the deficiency of reduced nanobody activity caused by random modification, thus greatly preserving the activity of the nanobody. Using smartphone photography for grayscale readings, it features rapid, accurate, stable, and instrument-free on-site analysis, possessing broad application potential and significant market prospects.

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

[0006] This invention provides a lateral flow immunoassay kit for detecting aflatoxin B1, comprising: a nanobody-biotin / streptavidin@quantum dot probe and a lateral flow immunoassay test strip. The lateral flow immunoassay test strip has a T line and a C line on its nitrocellulose membrane. The T line is coated with AFB1-BSA antigen, and the C line is coated with biotin-labeled bovine serum albumin.

[0007] Furthermore, the nanobody-biotin / streptavidin@quantum dot probe is generated by combining nanobody-biotin and streptavidin@quantum dots through a directional coupling strategy of biotin and streptavidin; the amino acid sequence of the nanobody-biotin is shown in SEQ ID NO.4; the streptavidin@quantum dots are obtained by modifying COOH-QDs with streptavidin.

[0008] Furthermore, the preparation method of the streptavidin@quantum dots includes the following steps: adding COOH-QDs to borate buffer, then adding EDC and NHS, vertically incubating at 37°C for 60 minutes, adding streptavidin, vortexing at 25°C for 60 minutes, adding 2-mercaptoethanol and incubating for 30 minutes, centrifuging to collect the precipitate, and obtaining streptavidin@quantum dots.

[0009] Furthermore, the step of centrifuging to collect the precipitate also includes washing the precipitate with borate buffer solution twice.

[0010] Furthermore, the emission wavelength of the COOH-QDs is 585 nm.

[0011] Furthermore, the concentration of the AFB1-BSA antigen is 0.6 mg / mL. -1 .

[0012] The present invention also provides a method for detecting aflatoxin B1, comprising the following steps:

[0013] (1) The nanobody-biotin / streptavidin@quantum dot probe and aflatoxin B1 molecular solution were incubated with vibration for 10 minutes under light protection at room temperature. Then, the solution was added to the sample pad of the lateral flow immunoassay strip and reacted at room temperature for 15 minutes. After the fluorescence intensity of the T line and C line stabilized, the gray values ​​of the T line and C line were read.

[0014] (2) Establish a standard curve based on the grayscale results of the T-line and C-line;

[0015] (3) Using the same method as in step (1) and the standard curve obtained in step (2), the content of aflatoxin B1 in the sample to be tested is obtained.

[0016] Further, in step (1), the concentration of the aflatoxin B1 molecule solution is 1 × 10⁻⁶. 0-1×10 10 ng·mL -1 .

[0017] Furthermore, in step (1), after the fluorescence intensity of the T line and the C line stabilizes, a smartphone is used to take a picture at an excitation wavelength of 435 nm to record and read the grayscale results of the T line and the C line.

[0018] The present invention also provides the application of the kit in the detection of aflatoxin B1 in food.

[0019] The present invention discloses the following technical effects:

[0020] 1. This invention is the first to develop a nanobody LFIA test strip based on a biotin-streptavidin directional conjugation strategy. The directional conjugation strategy can overcome the defect of reduced nanobody activity caused by random modification and greatly preserve the activity of the nanobody. The assembly of the biotin-streptavidin system is highly flexible and can be developed into different types of test strips by using different streptavidin@nanomaterials. Compared with traditional mAb-based LFIA, the nanobody LFIA test strip of this invention exhibits higher stability and tolerance under different detection environments.

[0021] 2. This invention provides a rapid, instrument-free, on-site analysis LFIA (NQ&SC-LFIA) method for AFB1 detection using smartphone photography. Utilizing smartphone photography as a grayscale reading, it features rapid, accurate, stable, and instrument-free on-site analysis. After optimization, the limit of detection (LOD) for AFB1 is 0.106 ng·mL. -1 IC 50 It is 0.86 ng·mL -1 Furthermore, it does not cross-react with other structural analogs during detection, exhibiting high specificity; it also possesses long-term stability, making it highly practical, easy to store, and possessing market development value. It has broad application potential and a promising market outlook. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 This is a schematic diagram illustrating the detection principle of the method of the present invention;

[0024] Figure 2 SDS-PAGE of the Nb81C-Avi fusion protein;

[0025] Figure 3 The structure of the CDR region in the Nb81C nanobody was obtained using the online software Swiss-Model.

[0026] Figure 4 Ramachandran analysis for Nb81C;

[0027] Figure 5 Molecular docking of Nb81C with AFB1 (a) and analysis of the molecular docking results (b);

[0028] Figure 6 Sensitivity curve for Nb81C;

[0029] Figure 7 The binding activity of Nb81C and anti-AFB1 mAb was measured when incubated at 75°C for different times (a), PBS buffer at different pH values ​​(b), methanol at different concentrations (c), and NaCl at different concentrations (d) were used as diluents.

[0030] Figure 8 TEM results for QDs with green fluorescence (a), orange fluorescence (b), and red fluorescence (c);

[0031] Figure 9 Zeta potentials for individual COOH-QDs, SA, SA@QDs, Nb-Avi, and Nb-Avi / SA@QDs;

[0032] Figure 10 DLS results for individual COOH-QDs, SA@QDs, and Nb-Avi / SA@QDs;

[0033] Figure 11 The fluorescence intensities of SA@QDs and Nb-Avi / SA@QDs at excitation and emission wavelengths of 525 nm (a), 585 nm (b), and 605 nm (c) are given.

[0034] Figure 12 The results of optimizing the emission wavelength of 525nm quantum dots with different concentrations of complete antigen are shown, where a is a bar chart and b is a grayscale analysis chart.

[0035] Figure 13 The results of optimizing the emission wavelength of 585nm quantum dots with different concentrations of complete antigen are shown, where a is a bar chart and b is a grayscale analysis chart.

[0036] Figure 14 The results of optimizing the emission wavelength of 605nm quantum dots with different concentrations of complete antigen are shown, where a is a bar chart and b is a grayscale analysis graph.

[0037] Figure 15The results show the optimization of methanol concentration in the sample loading buffer for the test strips, where a is a bar chart and b is a grayscale analysis graph;

[0038] Figure 16 The results show the optimization of the pH value of the sample loading buffer for the test strips, where a is a bar chart and b is a grayscale analysis graph;

[0039] Figure 17 The results show the optimization of the Tween concentration in the sample loading buffer for the test strips, where a is a bar chart and b is a grayscale analysis graph;

[0040] Figure 18 The linear relationship between ΔG and the concentration of AFB1 in bovine serum albumin (BSA);

[0041] Figure 19 The fluorescence visual intensity triggered by different concentrations of AFB1 in bovine serum albumin (BSA) (a) and the fluorescence visual intensity triggered by different mycotoxins (b);

[0042] Figure 20 The grayscale results are the fluorescence visual intensities corresponding to different concentrations of AFB1 in bovine serum albumin (BSA).

[0043] Figure 21 The binding activity of nanobody-based and mAb-based test strips at different methanol concentrations (a), different NaCl concentrations (b), and different temperatures (c), as well as ΔG(d) after storage in a 50°C incubator for 7, 15, and 23 days, are calculated. Detailed Implementation

[0044] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0045] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0046] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0047] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0048] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0049] This invention is the first to develop a nanobody LFIA test strip based on a biotin-streptavidin directional conjugation strategy, which utilizes a smartphone camera to detect aflatoxin B1 (AFB1) on-site. Figure 1 As shown, AFB1-BSA antigen was sprayed onto the detection line (T), and biotin-labeled bovine serum albumin (BSA) was sprayed onto the control line (C) of the NC membrane to replace the conventional antibody and provide a stable signal on the C line. Nanobody-biotin / streptavidin@quantum dot (Nb-Avi / SA@QDs) probes were prepared and evaluated on LFIA test strips. The Nb-Avi / SA@QDs probes showed satisfactory fluorescence lines, indicating that the directional coupling strategy based on biotin tags and streptavidin@quantum dots commendably preserves the activity of the nanobody. Then, AFB1 detection was performed on the Nb-Avi / SA@QDs-based LFIA test strip (NQ&SC-LFIA) using a smartphone, showing satisfactory performance. The nanobody-based and mAb-based test strips were then compared under different environments, and the nanobody bands showed higher methanol tolerance, better ion concentration tolerance, and wider temperature adaptability.

[0050] Example 1: Preparation of anti-aflatoxin B1 nanobodies

[0051] 1. Preparation method

[0052] Based on the camel-derived anti-AFB1 immune nanobody library successfully constructed in the previous stage, high-sensitivity and high-stability anti-AFB1 nanobodies were screened out through phage panning and named Nb81C(Nb). The specific method is described in the patent application document with application number 202210890250.1.

[0053] Nanobody-Avi (Nb-Avi, nanobody-biotin, corresponding to Nb81C-Avi) is based on Nb, with the addition of an Avi-tagged protein, which enables it to be naturally modified with biotin in E. coli.

[0054] The Nb81C-Avi gene fragment was obtained by polymerase chain reaction (PCR). The amplification primers are shown in Table 1, the amplification system is shown in Table 2, and the amplification reaction conditions are: 98℃ for 30s; 98℃ for 10s, 55℃ for 30s, 72℃ for 30s, 36 cycles; 72℃ for 5min; 4℃ forever.

[0055] Table 1 Amplification Primers

[0056]

[0057] Table 2 Reaction System

[0058] reagents Volume (μL) Nb81C gene fragment 1 Nanobody-Avi-F 1 Nanobody-Avi-R 1 2×Q5High-FidelityMasterMix 25 ddH2O 22

[0059] The PCR amplified product Nb-Avi gene fragment was ligated into the plasmid vector pET-22b. The pET-22b vector, containing Nb and Nb-Avi gene fragments in the periplasm of the plasmid, was used to express Nb and Nb-Avi carrying histidine tags, respectively. Positive Nb-pET-22b and Nb-Avi-pET-22b vectors transformed with *E. coli* TransB(DE3) were induced with IPTG (0.1 mM) at 16°C for 12 hours to obtain Nb-81C and Nb-81C-Avi resistant to AFB1.

[0060] Nb and Nb-Avi were purified from the periplasmic extract using a nickel-affinity chromatography column (Ni-NTA). The expression and purification of Nb and Nb-Avi were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The SDS-PAGE results of the Nb-Avi fusion protein are shown below. Figure 2 As shown, the target protein is located at approximately 17 kDa and exhibits a single band, indicating the successful preparation of the Nb-Avi nanobody. The amino acid sequence of the target protein is shown in SEQ ID NO. 3-4.

[0061] Nb-81C amino acid sequence (SEQ ID NO.3):

[0062] VLAALLQGVQAQVQLVDSGGGSVQAGGSLRLSCVASGYTLSNYCMGWFRQVS GKEREGVAGIWTGGGSIWYADSVKGRFTISQDKDKKTLYLQMNSLKPEDTAVYYCA AEPWGGPSSSCPGRPDEYRYWGQGTQVTVSSLEHHHHHH.

[0063] Nb-81C-Avi amino acid sequence (SEQ ID NO.4):

[0064] VLAALLQGVQAQVQLVDSGGGSVQAGGSLRLSCVASGYTLSNYCMGWFRQVS GKEREGVAGIWTGGGSIWYADSVKGRFTISQDKDKKTLYLQMNSLKPEDTAVYYCA AEPWGGPSSSCPGRPDEYRYWGQGTQVTVSSGLNDIFEAQKIEWHELEHHHHHH, bold italics are Avi tags.

[0065] The amino acid sequence of Nb81C was submitted to the Swiss-Model homology model (https: / / swissmodel.expasy.org / ) for homology modeling and 3D structure construction. Figure 3 ), and optimized using the OPLS-AA / L force field from GROMACS. Ramachandran analysis of Nb81C ( Figure 4 The results showed that 92.8% of the amino acids were located in the core region, indicating that the Nb81C structure was rationally optimized (the main parameters of Nb81C were QMEAN 0.77±0.07 and GMQE 0.68). The AFB1 structure was downloaded from the Pubchem Small Molecule Bioactivity Database (https: / / pubchem.ncbi.nlm.nih.gov / ), and molecular docking between AFB1 and Nb81C was successfully completed using Autodock 4.2.6. Figure 5 (a). Molecular docking results showed that AFB1 was located in the nanobody active pocket composed of CDR2 and CDR3 regions. Interaction analysis showed Pi-PiT type interaction with TRP113; and conventional hydrogen bonds with GLY114 / GLY115 / SER117 / SER118. Figure 5 b) determines its high specificity and affinity.

[0066] 2. Bioactivity of nanobodies

[0067] Indirect competitive enzyme-linked immunosorbent assay (icELISA) is used to determine the sensitivity of Nb, such as... Figure 6 As shown, icELISA demonstrated the sensitivity of Nb81C with anti-VHHIgG-HRP as the secondary antibody, with a linear range from 0.286 ng / mL. -1 Up to 4.150 ng·mL -1 The LOD was 0.135 ng·mL. -1 IC 50 It is 0.969 ng·mL -1 .

[0068] To improve the stability of the immunoassay method, the binding activity and tolerance of Nb and anti-AFB1 mAbs under various factors were verified by indirect enzyme-linked immunosorbent assay (iELISA), including pH (2.0, 4.0, 6.0, 8.0, 10.0, 12.0); methanol content of PBS dilution (0%, 20%, 40%, 80%, 100%, v / v); NaCl concentration (0, 100, 200, 500, 1000 mM reaction solution); and thermal stability of Nb and mAbs (0, 5, 10, 20, 30, 40 min at 75 °C). Results are as follows: Figure 7 As shown.

[0069] Thermostability of an antibody reflects its ability to maintain activity under extreme temperature conditions or after repeated freeze-thaw cycles. Compared with anti-AFB1 mAbs, Nb81C exhibits higher thermostability. Figure 7 a) This is because Nb81C retains 71.4% binding activity after incubation at 75°C for 40 minutes, while mAb shows almost no activity within just 5 minutes at 75°C. Figure 7 As shown in b, Nb81C exhibits better pH tolerance than mAb. Nb81C reaches its peak binding activity at pH=8, while mAb reaches its peak binding activity at pH=6. However, nanobodies show higher activity under acidic or alkaline conditions. Figure 7 As shown in Figure c, the binding activity of mAb decreased significantly (95.9-26.8%) when the methanol concentration was above 20%, but the binding activity of Nb81C remained at 63.4% even at a methanol concentration as high as 100%. Immunoassays based on antibodies with high tolerance to organic solvents and acids / bases offer additional advantages for applications, such as saving sample pretreatment time and avoiding errors caused by multiple sample dilutions. Since food or feed analysis often involves high-salt conditions, the salt tolerance of nanobodies and mAbs was examined. Figure 7As shown in Figure d, when the NaCl concentration is less than 500 mM, Nb81C is almost unaffected (from 100% to 91.2%), while mAb binding activity decreases slightly (from 100% to 77.9%). However, when the NaCl concentration reaches 1000 mM, Nb81C exhibits slightly poorer tolerance. In summary, Nb81C shows greater potential in immunoassay applications.

[0070] Example 2: Preparation of test strips

[0071] A test line (T line) and a control line (C line) were set on a nitrocellulose membrane (NC membrane). A BioDotXYZ3050 plotter (Irvine, CA) was used to measure the flow rate at 1 μL / min. -1 The partition rate was determined by coating different concentrations of AFB1-BSA (0.3, 0.6, 0.9 mg·mL⁻¹) onto the T-line. -1 Apply Avi-BSA (1.0 mg·mL⁻¹) to line C. -1 Assemble the strips, with each end of the NC membrane partially overlapping the sample pad and absorbent pad on the plastic backing plate. Cut the strips to 3.5 mm width using a CTS300 (Shanghai, China) automatic paper cutter and store at 4°C with a desiccant.

[0072] Example 3: Preparation of SA@QDs and Characterization of Nb-Avi / SA@QDs Probes

[0073] Streptavidin (SA) was modified onto COOH-QDs using the EDC / NHS method. 0.16 nmol of COOH-QDs (20 μL; 8 μM) was diluted in 80 μL of borate buffer (0.05 M, pH 7.4), and then 77.6 μL of EDC (10.0 mg / mL) was added. -1 ) and 28.7 μL of NHS (10.0 mg·mL) -1 To enable the carboxyl group to be active on fluorescent QDs, the mixture was incubated in a vertical mixer at 37°C for 60 min, then 100 μL of 16 μMSA was added and vortexed for 60 min (25°C). To stop the reaction, 1.2 μL of 2-mercaptoethanol was added and incubated for 30 min. The mixture was then centrifuged at 12,000 rpm for 5 min at 4°C, and the precipitate was washed with 500 μL of borate buffer, repeated twice. The SA@QDs precipitate was resuspended in 200 μL of resuspension (0.05% NaN3 in borate buffer, v / v) and stored at 4°C before use.

[0074] Quantum dots of different sizes were characterized using transmission electron microscopy (TEM) to observe their morphology. Zeta potential was used to characterize COOH-QDs, SA / QDS, Nb-Avi, and Nb-Avi-SA / QDs to verify whether Nb was modified onto the quantum dot microspheres. Dynamic light scattering (DLS) was used to characterize COOH-QDs, SA / QDS, and Nb-Avi-SA / QDs to verify the particle size changes before and after modification. The characterization results show... Figure 8-11 In the middle, the TEM results of the three QDs are as follows Figure 8 As shown, Figure 8 In the figure, 'a' represents the green fluorescent QDs (Em525nm); Figure 8 b represents orange fluorescent QDs (Em585nm); Figure 8 c represents the red fluorescent QDs (Em 605 nm). In Zeta potential characterization, compared to COOH-QDs alone (-8.92 mV), SA@QDs, after modification with negatively charged SA (-17.57 mV), showed a significant charge change (-16.43 mV), confirming the successful preparation of SA@QDs. Figure 9 Furthermore, the above conclusions were also confirmed by DLS results, which showed a significant change in particle size before and after modification. Figure 10 On the other hand, the binding products of Nb-Avi and SA@QDs showed significant changes in zeta potential and DLS results compared to the two independent products. The effect of the modification on the excitation and emission wavelengths of the QDs was negligible. Figure 11 Therefore, Nb81C-based probes were prepared as Nb-Avi / SA@QDs.

[0075] Example 4: Optimization of NQ&SC-LFIA and experiments on the sensitivity and specificity of NQ&SC-LFIA

[0076] A competitive reaction was established using NQ&SC-LFIA. Nb-Avi / SA@QDs probes and AFB1 small molecule solution were incubated in a light-proof EP tube at room temperature with vibration for 10 minutes. The reaction solution from the EP tube was then added to the sample pad of the test strip and run at room temperature for 15 minutes. The Nb-Avi / SA@QDs probes reacted with AFB1 to form immune complexes. The higher the AFB1 concentration, the more immune complexes were formed, resulting in fewer probes being captured by the T-line, leading to lower fluorescence intensity. The remaining probes were then captured by the Avi-BSA in the C-line. After the fluorescence intensity of the T-line and C-line stabilized, images were taken using a smartphone at an excitation wavelength of 435 nm. A smartphone-based grayscale readout system was established, characterized by its speed, convenience, sensitivity, and instrument-free operation. Two signal readout methods were compared: qualitative analysis based on visual observation and quantitative analysis of grayscale values. Grayscale results (G) of the T-line and C-line were obtained using smartphone photography. T and G C The results of NQ&SC-LFIA show that G T0 and G C0 This indicates that the concentration of AFB1 is 0 ng / mL. -1 G T / C / G T0 / C0 The ratio (ΔG) was used for the optimization of NQ&SC-LFIA and the quantitative detection of AFB1. Results were qualitatively determined by visual inspection. For quantitative detection, images of the T and C lines were analyzed by grayscale comparison. A standard curve was established by plotting ΔG against the common logarithm of AFB1 concentration (10⁻⁵ - 2.5 - 1.25 - 0.625 - 0.15625 - 0.078125 - 0). The results were obtained using 5 ng / mL... -1 The specificity of NQ&SC-LFIA was investigated by testing four structural analogs and four functional analogs at a concentration of [specific value].

[0077] 1. Optimization of NQ & SC-LFIA

[0078] Optimization is key to improving the analytical performance of the test strip. In this test strip, the analytical performance of NQ&SC-LFIA was demonstrated by the AFB1-BSA concentrations (0.3, 0.6, 0.9 mg / mL) on the T line. -1 The data are represented by three different emission wavelengths (Em, 525nm, 585nm, 605nm), the methanol content of the AFB1 molecule dilution buffer (10%, 30%, 50%, 70%, v / v), and the loading buffer (pH = 2.0, 4.0, 6.0, 8.0, 10.0; Tween-20 concentration, 0.1%, 0.25%, 0.5%, 1.0%, 2.0%, v / v).

[0079] The amount and ratio of Nb-Avi have always been considered key factors for probe coupling. Purified Nb-Avi (20 μg·mL⁻¹) -1 The concentration of Nb-Avi was determined by BCA protein quantification, and the final Nb-Avi dose for each assay system was set at 10 ng. (2 ng / mL) -1 The effects of AFB1 molecules as a control on the concentrations of AFB1-BSA in the QDs and T-line were investigated. The concentrations of AFB1-BSA in the T-line were 0.3, 0.6, and 0.9 mg·mL⁻¹. -1 The emission wavelengths of the three QDs are 525nm ( Figure 12 ), 585nm Figure 13 ), 605nm Figure 14 ). At 0.6 mg·mL -1 At AFB1-BSA and 585nm QDs, ΔG was calculated to be 0.462, which is relatively low and stable, meaning that NQ&SC-LFIA exhibits the best sensitivity under these conditions. Figure 13 Therefore, further research was conducted based on this. Although ΔG (0.423) was at 0.3 mg·mL -1 The AFB1-BSA level was lowest, but the visual effect was poor based on qualitative tests conducted with the naked eye. Therefore, the optimized condition was 0.6 mg / mL. -1 AFB1-BSA and 585nm QDs.

[0080] The composition and concentration of the loading buffer are also key factors affecting ΔG. This invention also investigated the effect of methanol concentration in the AFB1 molecular solution. Figure 15 The results showed that when the methanol concentration in PBS was 30% (v / v), ΔG was at 2 ng·mL⁻¹. -1 The AFB1 molecular weight reached its lowest value under certain conditions and remained relatively stable. Stable analytical performance was also observed when the methanol concentration in PBS was 70% (v / v). Then, buffers with different pH values ​​(pH = 2.0, 4.0, 6.0, 8.0, 10.0) were used to increase the concentration of 1 ng / mL. -1 Analytical performance under AFB1 molecular conditions. As... Figure 16 As shown, ΔG decreased with increasing pH (2.0–6.0) until reaching pH 6.0, and then increased with increasing pH (6.0–10.0). Subsequently, at 1 ng / mL... -1 Under AFB1 molecular conditions, a series of Tween-20 buffer solutions (0.1%, 0.25%, 0.5%, 1.0%, and 2.0%, v / v) were also analyzed. Figure 17The results showed that at low concentrations of Tween-20, the visible fluorescence intensity and grayscale value of the T-line were high, but the sensitivity of NQ&SC-LFIA was not significantly improved; conversely, the optimal analytical performance was achieved at 1.0% Tween-20. In summary, by exploring and optimizing analytical conditions, a simple, easy-to-use, and highly sensitive NQ&SC-LFIA method for AFB1 was successfully developed.

[0081] 2. Sensitivity and specificity tests of NQ&SC-LFIA

[0082] Quantitative analysis results showed that NQ&SC-LFIA can sensitively analyze AFB1 under optimal conditions. Different concentrations of AFB1 were analyzed by visual qualitative analysis and quantitative grayscale contrast detection. The loading buffer was optimized as a mixture of 1% sucrose + 0.5% BSA + 1% PEG4000 + 1% Tween-20 in PB buffer (0.05M, pH=6). Figure 18 The relationship between ΔG and AFB1 concentration was shown. The method of this invention exhibits good linearity for the detection of AFB1, from 0.195 ng·mL⁻¹. -1 Up to 4.370 ng·mL -1 The LOD was 0.106 ng·mL. -1 IC 50 It is 0.86 ng·mL -1 1.25 ng·mL -1 Qualitative analysis can be performed using the naked eye. Figure 19 The grayscale result of 'a' is displayed in the image. Figure 20 In addition, specificity is also a key analytical performance of NQ&SC-LFIA, which is isolated from several mycotoxins (5 ng / mL). -1 Disorders include aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), aflatoxin G2 (AFG2), aflatoxin M1 (AFM1), fumonisin B1 (FB1), deoxynivalenol (DON), zearalenone (ZEN), and trichothecene-2 ​​(T2). Figure 19 Figure b shows that, compared to the fluorescence intensity triggered by other mycotoxins, the signal intensity generated by AFB1 is almost close to the background signal. Therefore, there is no cross-reactivity with functional analogues FB1, DON, ZEN, and T2, and the cross-reactivity with structural analogues of AFB1, AFB2, AFG1, AFG2, and AFM1, is very low.

[0083] Example 6 Stability Experiment

[0084] The stability of NQ&SC-LFIA was verified through accelerated aging experiments. This invention employs accelerated aging experiments based on the Arrhenius equation, an empirical formula expressing the dependence of the chemical reaction rate constant (k) on temperature (T). Eα represents the apparent activation energy (≈19.5 Kcal / mol), and R is the molar gas constant. The general trend is that as T increases, k also increases, allowing the calculation of the relationship between temperature and the number of aging days. Calculations show that 23 days at 50°C is equivalent to one year at room temperature (25°C). The prepared test strips were placed in a constant temperature incubator (50°C) for 7, 15, and 23 days respectively to verify their analytical performance. All experiments were performed five times. Test strips based on nanobodies and mAbs were compared under various conditions, including running buffer concentrations of methanol (10%, 30%, 50%, 70%, v / v), running buffer concentrations of NaCl (0, 100, 200, 500, 800 mM), and ambient test temperatures (4°C, 16°C, 32°C, 40°C).

[0085] The results are as follows Figure 21 As shown, Figure 21 As shown in a, the nanobody test strips exhibit high methanol tolerance; when the methanol concentration is above 30%, the mAb-based probes can hardly adhere to the T line of the test strip, but the Nb-based probes are almost unaffected. Figure 21 Figure b shows that the nanobody test strips exhibit better salt concentration tolerance between 100mM and 500mM. When the NaCl concentration exceeds 500mM, the binding activity of the Nb-based probe decreases sharply, showing lower activity than the mAb-based probe at 800mM, a phenomenon consistent with the iELISA results. Compared to mAb strips, the nanobody-based test strips show better temperature tolerance. From 4℃ to 40℃, the Nb-based probe is almost unaffected by the ambient temperature and still shows good binding activity at low temperatures, while mAb has almost no effect under these conditions. Figure 21 (c) This invention also found that nanobody-based probes can function normally at low temperatures, indicating that they can be used directly without pre-incubation to room temperature, which is essential for nanobody-based probes. These results suggest that nanobody-based strips have better application potential in complex sample matrices and less-than-ideal testing environments. Finally, to evaluate the stability of our test strips, they were stored in a 50°C incubator for 7, 15, and 23 days, respectively, with the following results: Figure 21As shown in d, even after being stored at 50°C for 23 days, the test strips still exhibit perfect performance. According to the Arrhenius equation, these test strips can be stored at room temperature for one year, which demonstrates the great potential of nanobody-based test strips in practical applications.

[0086] Example 7: Practical application of NQ&SC-LFIA in actual samples

[0087] To demonstrate the analytical performance of our method in real samples, quantification of AFB1 in oats was performed. First, matrix effects had to be eliminated because the composition of real sample extracts is complex, and biotin is present in many crop samples, which could affect the efficacy of NQ&SC-LFIA. Biotin is slightly soluble in water and ethanol but insoluble in other organic solvents, so pure methanol was considered for use.

[0088] Multiple concentrations of AFB1 solution were added to negative oat samples to obtain actual AFB1-added samples. First, 1g of oat samples containing different concentrations of AFB1 (20ng, 10ng, and 5ng) were dissolved in 4mL of methanol. The samples were thoroughly mixed for 15 minutes, and then centrifuged at 8000rpm for 10 minutes. Subsequently, the supernatant was collected, diluted 4-fold with PBS, and centrifuged at 12000rpm for 10 minutes. The extract was collected and used for the study. The final concentrations of AFB1 in the oat samples were 20, 10, and 5 μg·kg⁻¹, respectively. -1 The concentrations in the extracts were 1.25, 0.625, and 0.3125 ng·mL, respectively. -1 The extracts at three AFB1 concentrations were analyzed using NQ&SC-LFIA. Five replicate experiments were performed, and the recovery rate was calculated as: recovery rate = (measured AFB1 concentration / added AFB1 concentration) × 100. The results are shown in Table 3.

[0089] Table 3

[0090]

[0091] Table 3 shows that NQ&SC-LFIA has excellent performance in detecting AFB1 in diluted oat samples in practical applications, with recoveries ranging from 88.8% to 116.7%. This indicates that NQ&SC-LFIA, as a highly sensitive analytical method, has great potential for analyzing AFB1 in real samples.

[0092] In summary, we have successfully developed, for the first time, a smartphone-assisted lateral flow immunoassay strip based on nanobodies (NQ&SC-LFIA), achieving highly sensitive and convenient detection of AFB1. The Avi / SA coupling strategy overcomes the limitation of Nb activity being affected by random modifications of nanomaterials. Besides the advantages of LFIA's ease of operation and time-saving, data analysis can be performed via smartphone photography, enabling on-site detection without the need for instruments. Furthermore, compared to mAbs, the thermal stability and tolerance of Nb to complex analytical environments ensure the stability and anti-interference capabilities of our method. Real sample analysis has also validated our approach, with recoveries ranging from 88.8% to 116.7%, making it an effective method for on-site detection. Moreover, the Avi / SA coupling strategy shows broad application prospects in other nanobody applications. Therefore, this invention not only provides a stable and efficient on-site detection instrument for AFB1 monitoring but also promotes the development of Nb applications.

[0093] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A lateral flow immunoassay kit for detecting aflatoxin B1, characterized by, The application relates to a nanobody-biotin / streptavidin quantum dot probe and a lateral flow immunoassay test strip, wherein a T line and a C line are arranged on a nitrocellulose membrane of the lateral flow immunoassay test strip, the T line is coated with an AFB1-BSA antigen, and the C line is coated with a biotin-labeled bovine serum protein. The nanobody-biotin / streptavidin quantum dot probe is combined by nanobody-biotin and streptavidin quantum dots through a directional coupling strategy of biotin and streptavidin; the amino acid sequence of the nanobody-biotin is shown in SEQ ID NO. 4; and the streptavidin quantum dots are obtained by modifying streptavidin to COOH-QDs. The preparation method of the streptavidin quantum dots comprises the following steps: COOH-QDs are added into a boric acid buffer solution, EDC and NHS are added, vertical culture is carried out at 37 DEG C for 60 minutes, streptavidin is added, vortex is carried out at 25 DEG C for 60 minutes, 2-mercaptoethanol is added and incubated for 30 minutes, the precipitate is obtained by centrifugation, and the streptavidin quantum dots are obtained. The concentration of the AFB1-BSA antigen is 0.6 mg mL -1 .

2. The kit of claim 1, wherein After the centrifugation and the precipitation, the precipitate is washed with a boric acid buffer solution, and the washing is carried out twice.

3. The kit of claim 2, wherein The emission wavelength of the COOH-QDs is 585 nm.

4. The kit of claim 2, wherein The application further discloses a detection method of the nanobody-biotin / streptavidin quantum dot probe and the lateral flow immunoassay test strip.

5. A method of detecting aflatoxin B1, characterized by, (1) the nanobody-biotin / streptavidin quantum dot probe in claim 1 is vibrated and incubated with a yellow aspergillus toxin B1 molecular solution under room temperature and light-proof conditions for 10 minutes, then is added into the sample pad of the lateral flow immunoassay test strip in claim 1, and reaction is carried out at room temperature for 15 minutes; when the fluorescence intensity of the T line and the C line is stable, the gray scale results of the T line and the C line are read; (2) a standard curve is established according to the gray scale results of the T line and the C line; (3) the same method as in step (1) is adopted to obtain the content of the yellow aspergillus toxin B1 in the sample to be detected in combination with the standard curve obtained in step (2). In step (1), when the fluorescence intensity of the T line and the C line is stable, a smart phone is used to take a picture under an excitation wavelength of 435 nm, and the gray scale results of the T line and the C line are read.

6. The method of claim 5, wherein, In step (1), the concentration of the aflatoxin B1 molecule solution is 1 x 10 0 -1 x 10 10 ng·mL -1 .

7. The method of claim 5, wherein, 8. The kit according to any one of claims 1-4 for detecting the yellow aspergillus toxin B1 in food. ​

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