CN116840460A - Lateral flow immunoassay kit and method for detecting aflatoxin B1 - Google Patents

Lateral flow immunoassay kit and method for detecting aflatoxin B1 Download PDF

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CN116840460A
CN116840460A CN202310516218.1A CN202310516218A CN116840460A CN 116840460 A CN116840460 A CN 116840460A CN 202310516218 A CN202310516218 A CN 202310516218A CN 116840460 A CN116840460 A CN 116840460A
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line
streptavidin
biotin
afb1
lateral flow
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孙铁强
王�锋
王新阳
张予弦
虞立霞
姚站馨
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Environmental Medicine and Operational Medicine Institute of Military Medicine Institute of Academy of Military Sciences
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    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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    • G01N33/54386—Analytical elements
    • G01N33/54387—Immunochromatographic test strips
    • G01N33/54388—Immunochromatographic test strips based on lateral flow
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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    • G01N33/588—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with semiconductor nanocrystal label, e.g. quantum dots
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Abstract

The application discloses a lateral flow immunoassay kit and a lateral flow immunoassay method for detecting aflatoxin B1, and belongs to the technical field of lateral immunoassay. The kit comprises 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, AFB1-BSA antigen is coated on the T line, and biotin-marked bovine serum albumin is coated on the C line. The directional coupling strategy of the application can overcome the defect of reduced activity of the nano antibody caused by random modification, and greatly retains the activity of the nano antibody. The intelligent mobile phone photography is used as gray reading, and the intelligent mobile phone has the characteristics of rapidness, accuracy, stability, field analysis without instruments and the like, has wide popularization space and has great market prospect.

Description

Lateral flow immunoassay kit and method for detecting aflatoxin B1
Technical Field
The application belongs to the technical field of lateral flow immunoassay, and particularly relates to a lateral flow immunoassay kit and a lateral flow immunoassay method for detecting aflatoxin B1.
Background
Currently, human and animal safety is facing serious threats from mycotoxins, which are secondary metabolites produced by specific fungi grown on various crops. Aflatoxins are a kind of oncogenic mycotoxins mainly produced by aspergillus, and out of 20 or more established aflatoxins, aflatoxin B1 (AFB 1) is the most virulent one, and carcinogenicity, teratogenicity, acute toxicity, mutagenicity and other biological activities are main causes of harm to human and animal health, and a large number of reports indicate that AFB1 can cause human and animal hepatocellular carcinoma (HCC) to take part directly or indirectly in the occurrence of cancer, and seriously endanger life and property safety of people. Thus, rapid quantitative analysis of AFB1 is critical to ensure food safety and health protection.
Nanobodies (Nb), recombinant pure heavy chain antibody variable domains, are emerging reagents for analytical environments and food chemicals due to their high thermal stability, ease of genetic modification and low cost production. A number of nanobody-based methods have been developed for the detection of mycotoxins, such as AFB1, OTA, DON. These methods are mainly based on traditional ELISA, which would take a lot of time and complicated steps, preventing the application of nanobodies in field detection. Lateral Flow Immunoassay (LFIA) test strips are becoming 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) and are coupled to nanomaterials such as gold nanoparticles, quantum beads, fluorescent microspheres, etc. Antibodies are randomly oriented to some nanomaterials, primarily by electrostatic adsorption or 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) mediated carbodiimide methods, which may reduce the performance of the immunoassay, resulting in unsatisfactory performance of the antibody. 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, quantum dots with hydrazide groups, which help the antibody maintain its antigen capture efficiency. Although many LFIA test strips have been reported, few LFIA strips based on nanobodies have been reported, particularly in the detection of mycotoxins. The possible reason is that nanobodies are too small in size and their activity can be affected during the attachment to the nanomaterial. Therefore, development of a lateral flow immunoassay test strip based on nano antibodies for detecting mycotoxins, especially aflatoxin B1, has important significance for detecting mycotoxins.
Disclosure of Invention
The application aims to provide a lateral flow immunoassay kit and a lateral flow immunoassay method for detecting aflatoxin B1, which are used for solving the problems in the prior art. The application uses the directional coupling strategy based on biotin and streptavidin, can overcome the defect of reduced activity of nano antibody caused by random modification, and greatly retains the activity of nano antibody. The intelligent mobile phone photography is used as gray reading, and the intelligent mobile phone has the characteristics of rapidness, accuracy, stability, field analysis without instruments and the like, has wide popularization space and has great market prospect.
In order to achieve the above object, the present application provides the following solutions:
the application provides a lateral flow immunoassay kit for detecting aflatoxin B1, comprising: the kit comprises 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, AFB1-BSA antigen is coated on the T line, and biotin-labeled bovine serum albumin is coated on the C line.
Further, the nanobody-biotin/streptavidin@quantum dot probe is formed by combining nanobody-biotin and streptavidin@quantum dots through a directional coupling strategy of biotin and streptavidin; the amino acid sequence of the nano antibody-biotin is shown as SEQ ID NO. 4; the streptavidin@quantum dots are obtained by modifying streptavidin to COOH-QDs.
Further, the preparation method of the streptavidin@quantum dots comprises the following steps: adding COOH-QDs into boric acid buffer solution, adding EDC and NHS, vertically culturing at 37 ℃ for 60 minutes, adding streptavidin, swirling at 25 ℃ for 60 minutes, adding 2-mercaptoethanol, incubating for 30 minutes, centrifuging, and taking precipitate to obtain the streptavidin@quantum dot.
Further, the step of washing the precipitate with borate buffer solution is also included after the centrifugation to obtain the precipitate, and the washing is carried out for 2 times.
Further, the emission wavelength of the COOH-QDs is 585nm.
Further, the concentration of the AFB1-BSA antigen was 0.6mg.multidot.mL -1 。
The application also provides a method for detecting aflatoxin B1, which comprises the following steps:
(1) Vibrating and incubating a nanobody-biotin/streptavidin@quantum dot probe and an aflatoxin B1 molecular solution for 10 minutes at room temperature under a light-proof condition, then adding the solution into a sample pad of a lateral flow immunoassay test strip, reacting for 15 minutes at room temperature, and reading gray results of a T line and a C line after the fluorescence intensities of the T line and the C line are stable;
(2) Establishing a standard curve according to the gray scale results of the T line and the C line;
(3) And (3) combining the standard curve obtained in the step (2) by adopting the same method in the step (1) to obtain the content of aflatoxin B1 in the sample to be detected.
Further, in the step (1), the concentration of the aflatoxin B1 molecule solution is 1×10 0 -1×10 10 ng·mL -1 。
Further, in the step (1), after the fluorescence intensities of the T line and the C line are stable, the smart phone is used to record a photograph at an excitation wavelength of 435 nm, and the gray scale results of the T line and the C line are read.
The application also provides application of the kit in detecting aflatoxin B1 in food.
The application discloses the following technical effects:
1. the application develops a nano antibody LFIA test strip based on a biotin-streptavidin directional coupling strategy for the first time, and the directional coupling strategy can overcome the defect of reduced activity of the nano antibody caused by random modification, and greatly retains the activity of the nano antibody; the assembly of the biotin-streptavidin system is highly flexible, and can be developed into different types of test strips through different streptavidin@nano materials; compared with the traditional mAb-based LFIA, the nanobody LFIA test strip provided by the application has higher stability and tolerance under different detection environments.
2. The application provides a rapid instrumentalless field analysis LFIA (NQ) for AFB1 detection by utilizing smart phone photography&SC-LFIA). The intelligent mobile phone photography is used as gray reading, has the characteristics of rapidness, accuracy, stability, no instrument, field analysis and the like, and is optimized, wherein the detection Limit (LOD) of the AFB1 is 0.106 ng.mL -1 ，IC 50 Is 0.86 ng.mL -1 . Meanwhile, the kit does not cross react with other structural analogues during detection, and has high specificity; the product has the characteristics of long-term stability, strong practicability, easy preservation and market development value. Has wide popularization space and great market prospect.
Drawings
In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present application, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram of the detection principle of the method of the present application;
FIG. 2 is SDS-PAGE of Nb81C-Avi fusion proteins;
FIG. 3 shows the structure of the CDR regions of Nb81C nanobodies, obtained by the on-line software Swiss-Model;
FIG. 4 is a Ramachandran analysis of Nb 81C;
FIG. 5 shows molecular docking of Nb81C with AFB1 (a) and molecular docking result analysis (b);
FIG. 6 is a sensitivity curve of Nb 81C;
FIG. 7 shows the binding activity of Nb81C incubated with mAb against AFB1 at 75deg.C for various times (a), PBS buffer at various pH values (b), methanol at various concentrations (C) and NaCl at various concentrations (d) as dilution reagents;
FIG. 8 shows TEM results of QDs of green fluorescence (a), orange fluorescence (b) and red fluorescence (c);
FIG. 9 is Zeta potential of COOH-QDs, SA, SA@qds, nb-Avi, and Nb-Avi/SA@qds alone;
FIG. 10 is a DLS result for COOH-QDs, SA@QDs, and Nb-Avi/SA@QDs alone;
FIG. 11 is a graph showing fluorescence intensities of SA@QDs and Nb-Avi/SA@QDs at excitation and emission wavelengths of 525nm (a), 585nm (b) and 605nm (c);
FIG. 12 shows the results of optimizing complete antigens at different concentrations of quantum dots with emission wavelength of 525nm, wherein a is a histogram and b is a gray scale analysis chart;
FIG. 13 is a graph showing the results of optimizing complete antigens at different concentrations of quantum dots with emission wavelength of 585nm, wherein a is a histogram and b is a gray scale analysis graph;
FIG. 14 shows the results of optimizing complete antigens at different concentrations of quantum dots with emission wavelength of 605nm, wherein a is a histogram and b is a gray scale analysis chart;
FIG. 15 is a graph showing the results of optimizing the methanol concentration of the buffer solution for loading the test strip, wherein a is a histogram, and b is a gray scale analysis chart;
FIG. 16 is a graph showing the pH optimization result of the sample buffer of the test strip, wherein a is a histogram, and b is a gray scale analysis chart;
FIG. 17 is a graph showing the result of optimizing Tween concentration of a sample buffer solution of a test strip, wherein a is a histogram, and b is a gray scale analysis chart;
FIG. 18 is a linear relationship between ΔG and AFB1 concentration in Bovine Serum Albumin (BSA);
FIG. 19 shows fluorescence visual intensity (a) and fluorescence visual intensity (b) triggered by different mycotoxins at different concentrations of AFB1 in Bovine Serum Albumin (BSA);
FIG. 20 shows the gray scale results corresponding to the intensity of fluorescence emitted by AFB1 at various concentrations in Bovine Serum Albumin (BSA);
FIG. 21 shows binding activity of nanobody-based and mAb-based test strips at different methanol concentrations (a), different NaCl concentrations (b) and different temperatures (c) and ΔG (d) after 7 days, 15 days and 23 days, respectively, of storage in a 50℃incubator.
Detailed Description
Various exemplary embodiments of the application will now be described in detail, which should not be considered as limiting the application, but rather as more detailed descriptions of certain aspects, features and embodiments of the application.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the application. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the application described herein without departing from the scope or spirit of the application. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present application. The specification and examples of the present application are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.
The application develops a nanometer antibody LFIA test strip based on a biotin-streptavidin directional coupling strategy for the first time, and detects aflatoxin B1 (AFB 1) on a photographing site by using a smart phone, as shown in figure 1. AFB1-BSA antigen was sprayed on the detection line (T), and biotin-labeled Bovine Serum Albumin (BSA) was sprayed on the control line (C) of 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, nb-Avi/sa @ qds probes showed satisfactory fluorescent lines, indicating that a directional coupling strategy based on biotin tags and streptavidin @ quantum dots would be worth retaining nanobody activity. Then, an LFIA test strip (NQ & SC-LFIA) based on Nb-Avi/SA@QDs was photographed by a smart phone to detect AFB1 and show satisfactory performance. Then, the nanobody and mAb based strips were compared under different environments and found to show higher methanol tolerance, better ion concentration tolerance and wider temperature adaptability.
Example 1 preparation of anti-aflatoxin B1 nanobody
1. Preparation method
According to the camel-derived anti-AFB 1 immune nanobody library which is successfully constructed in the early stage, screening out the anti-AFB 1 nanobody with high sensitivity and high stability through phage panning, wherein the anti-AFB 1 nanobody is named as Nb81C (Nb), and the specific method is referred to the patent application document with the application number of 202210890250.1.
Nanobody-Avi (Nb-Avi, nanobody-biotin, corresponding to Nb 81C-Avi) is based on Nb, with Avi tag protein added to make it possible to naturally modify biotin in e.coli.
The Nb81C-Avi gene fragment is obtained by Polymerase Chain Reaction (PCR) amplification, and the amplification primers are as follows: see table 1, amplification system see table 2, amplification reaction conditions: 98 ℃ for 30s;98 ℃ for 10s,55 ℃ for 30s,72 ℃ for 30s,36 cycles; 72 ℃ for 5min;4℃forever.
TABLE 1 amplification primers
TABLE 2 reaction system
Reagent(s) Volume (mu L)
Nb81C gene fragment 1
Nanobody-Avi-F 1
Nanobody-Avi-R 1
2×Q5High-FidelityMasterMix 25
ddH2O 22
The PCR amplified product Nb-Avi gene fragment was ligated into plasmid vector pET-22b, and Nb-Avi carrying histidine tag (His-tag) were expressed by using pET-22b vector carrying Nb and Nb-Avi gene fragments in the plasmid periplasm, respectively. Positive Nb-pET-22b and Nb-Avi-pET-22b vectors transformed with E.coli TransB (DE 3) were induced with IPTG (0.1 mM) at 16℃for 12 hours to obtain Nb-81C and Nb-81C-Avi against AFB1.
Nb and Nb-Avi were purified from the periplasmic extract using a nickel-affinity 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 in FIG. 2, the target protein is located at about 17kDa, and the band is single, which indicates that the Nb-Avi nanobody is successfully prepared. The amino acid sequence of the target protein is shown as SEQ ID NO. 3-4.
Nb-81C amino acid sequence (SEQ ID NO. 3):
VLAALLQGVQAQVQLVDSGGGSVQAGGSLRLSCVASGYTLSNYCMGWFRQVS GKEREGVAGIWTGGGSIWYADSVKGRFTISQDKDKKTLYLQMNSLKPEDTAVYYCA AEPWGGPSSSCPGRPDEYRYWGQGTQVTVSSLEHHHHHH。
Nb-81C-Avi amino acid sequence (SEQ ID NO. 4):
VLAALLQGVQAQVQLVDSGGGSVQAGGSLRLSCVASGYTLSNYCMGWFRQVS GKEREGVAGIWTGGGSIWYADSVKGRFTISQDKDKKTLYLQMNSLKPEDTAVYYCA AEPWGGPSSSCPGRPDEYRYWGQGTQVTVSSGLNDIFEAQKIEWHELEHHHHHH, bold italics Avi tag.
The amino acid sequence of Nb81C was submitted to the Swiss-Model homology Model (https:// swissmodel. Expasy. Org /) for homology modeling and construction of three-dimensional structures (FIG. 3), and optimized using the OPLS-AA/L force field of GROMACS. Ramachandran analysis of Nb81C (fig. 4) showed 92.8% amino acids in the core region, indicating reasonable optimization of Nb81C structure (main parameters of Nb81C are QMEAN 0.77±0.07 and GMQE 0.68). The AFB1 structural formula was downloaded from the Pubchem organic small molecule bioactivity database (https:// Pubchem. Ncbi. Nl. Gov /), and molecular docking of AFB1 with Nb81C was successfully completed using Autodock4.2.6 (FIG. 5 a). Molecular docking results show that AFB1 is located in a nano antibody active pocket formed by CDR2 and CDR3 regions, and interaction analysis shows that the AFB1 is in Pi-PiT type with TRP 113; the conventional hydrogen bonding with GLY114/GLY115/SER117/SER118 (b of FIG. 5) determines its high specificity and affinity.
2. Biological Activity of nanobodies
Indirect competitive enzyme-linked immunosorbent assayExperiments (icELISA) were used to determine the sensitivity of Nb, and as shown in FIG. 6, icELISA showed sensitivity of Nb81C with anti-VHHIgG-HRP as secondary antibody, with linearity ranging from 0.286 ng.mL -1 To 4.150 ng-mL -1 LOD is 0.135 ng.mL -1 ，IC 50 Is 0.969 ng.mL -1 。
To improve the stability of the immunoassay, the binding activity and tolerability of Nb and mAb against AFB1 under a variety of factors, including pH (2.0,4.0,6.0,8.0, 10.0, 12.0), was verified by indirect enzyme-linked immunosorbent assay (iisa); methanol content (0%, 20%,40%,80%,100%, v/v) of PBS dilution; concentration of NaCl (0, 100, 200, 500, 1000mM reaction solution); thermal stability of Nb and mAb (0, 5, 10, 20, 30, 40 min at 75 ℃). The results are shown in FIG. 7.
The thermal stability of the antibody reflects its ability to remain active under extreme temperature conditions or after repeated freeze-thawing, nb81C exhibits higher thermal stability (fig. 7 a) than the mAb against AFB1, since Nb81C can retain 71.4% of the binding activity after 40 min incubation at 75 ℃ whereas mAb is almost completely inactive for only 5min at 75 ℃. As shown in fig. 7 b, nb81C is better tolerant to pH than mAb, nb81C reaches the highest peak of binding activity at ph=8, mAb reaches the highest peak of binding activity at ph=6, but nanobody shows higher activity under acidic or basic conditions. As shown in FIG. 7C, the binding activity of mAb was significantly reduced (95.9-26.8%) at methanol concentrations above 20%, but the binding activity of Nb81C was maintained at 63.4% at methanol concentrations 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 due to multiple sample dilutions. Since food or feed analysis is often accompanied by high salt conditions, salt tolerance of nanobodies and mabs was examined, nb81C was hardly affected (from 100 to 91.2%) when NaCl concentration was less than 500mM, mAb binding activity was slightly decreased (from 100 to 77.9%) but Nb81C tolerance was slightly worse when NaCl concentration reached 1000mM, as shown in fig. 7 d. In short, nb81C shows greater potential in immunoassay applications.
Example 2 preparation of test strips
A test line (T line) and a control line (C line) were provided on a nitrocellulose membrane (NC membrane) and were measured at 1. Mu.L.min using a BioDotXYZ3050 plotter (Irvine, calif.) -1 The distribution ratio of AFB1-BSA (0.3, 0.6, 0.9mg.mL) was coated on the T line at different concentrations -1 ) Avi-BSA (1.0 mg. Multidot. ML) was applied to line C -1 ). The strips were assembled and either end of the NC film overlapped partially with the sample pad and absorbent pad, respectively, on the plastic back plate. The strips were cut to 3.5 mm width using a CTS300 (shanghai, china) automatic paper cutter and stored at 4 ℃ with a desiccant.
Example 3sA@QDs preparation and characterization of Nb-Avi/SA@QDs probes
Modification of Streptavidin (SA) onto COOH-QDs by EDC/NHS method, 0.16nmol of COOH-QDs (20. Mu.L; 8. Mu.M) was diluted in 80. Mu.L of boric acid buffer (0.05M, pH 7.4), followed by addition of 77.6. Mu.L of EDC (10.0 mg. Mu.mL -1 ) And 28.7. Mu.L of NHS (10.0 mg.mL -1 ) So that the carboxyl groups are active on fluorescent QDs. The mixture was incubated on a constant temperature incubation vertical mixer (37 ℃ C., 60 minutes), 100. Mu.L of 16. Mu.MSA was added, and vortexed for 60 minutes (25 ℃ C.). To terminate the reaction, 1.2. Mu.L of 2-mercaptoethanol was added and incubated for 30 minutes. The pellet was then washed with 500. Mu.L of borate buffer and repeated twice by centrifugation at 12000rpm for 5 minutes at 4 ℃. The pellet of SA@QDs was resuspended in 200. Mu.L of a resuspension (0.05% NaN 3 V/v) in borate buffer, stored at 4℃prior to use.
Respectively characterizing quantum dots with different particle diameters by a Transmission Electron Microscope (TEM), and observing the morphology of the quantum dots; the Zeta potential is adopted to characterize COOH-QDs, SA/QDS, nb-Avi and Nb-Avi-SA/QDs, and whether Nb is modified on the quantum dot microsphere is verified; dynamic Light Scattering (DLS) was used to characterize COOH-QDs, SA/QDS, nb-Avi-SA/QDs, and the particle size change before and after modification was verified. The characterization results are shown in FIGS. 8-11, and TEM results for three QDs are shown in FIG. 8, where a in FIG. 8 is the green fluorescent QDs (Em 525 nm); FIG. 8 b is QDs (Em 585 nm) of orange fluorescence; FIG. 8 c shows QDs (Em 605 nm) of red fluorescence. In Zeta potential characterization, SA@qds showed a significant charge change (-16.43 mV) after modification of negatively charged SA (-17.57 mV) compared to COOH-QDs alone (-8.92 mV), confirming successful production of SA@qds (FIG. 9). Furthermore, the above conclusion was also confirmed by DLS results, which indicate a significant change in particle size before and after modification (fig. 10). On the other hand, the binding products of Nb-Avi and SA@QDs show a significant change in Zeta potential and DLS results compared to the two independent ones. The effect of the modification on the excitation and emission wavelengths of QDs is negligible (fig. 11). Thus, a probe based on Nb81C was prepared as Nb-Avi/SA@QDs.
Example 4 optimization of NQ & SC-LFIA and sensitivity and specificity experiments of NQ & SC-LFIA
NQ&SC-LFIA established a competitive reaction, and Nb-Avi/SA@QDs probe and AFB1 small molecule solution were incubated in a light-proof EP tube for 10 minutes with shaking at room temperature. The reaction solution in the EP tube was then added to the sample pad of the test paper and run at room temperature for 15 minutes. The higher the concentration of AFB1, the more immunocomplexes are formed, and therefore, less probes are captured by the T-line, and the lower the fluorescence intensity. The remaining probes were then captured by Avi-BSA on line C. After the fluorescence intensities of the T line and the C line are stable, the smart phone is used for photographing and recording at the excitation wavelength of 435 nanometers. A gray scale reading system based on a smart phone is established, and the system has the characteristics of rapidness, convenience, sensitivity and no instrument, and compares two signal readouts according to qualitative analysis of visual effect and quantitative analysis of gray scale value. Photographing by using a smart phone to obtain T line and C line gray scale results (G T And G C )，NQ&Results of SC-LFIA show G T0 And G C0 The concentration of AFB1 was 0ng mL -1 。G T/C /G T0/C0 Ratio (ΔG) of (A) for NQ&Optimization of SC-LFIA and quantitative detection of AFB1. The results can be detected qualitatively with the naked eye. For quantitative detection, photographs of T-line and C-line were analyzed by gray scale comparison. A standard curve was established by plotting the common logarithm of the ΔG and AFB1 concentration (10-5-2.5-1.25-0.625-0.15625-0.078125-0). By the method of the preparation method of the composite material in 5 ng.mL -1 4 structural analogues and 4 functional analogues were tested at the concentration of (2), NQ was studied&Specificity of SC-LFIASex.
Optimization of NQ & SC-LFIA
Optimization is the key to improving the analytical performance of the test strip. In the test strip, NQ&Analytical performance of SC-LFIA was determined by AFB1-BSA concentration on the T line (0.3, 0.6,0.9 mg.multidot.mL) -1 ) QDs (Em, 525nm,585nm,605 nm) at three different emission wavelengths, methanol content (10%, 30%,50%,70%, v/v) of AFB1 molecular dilution buffer, 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).
The amount and ratio of Nb-Avi has been considered as a key factor in the coupling with the probe, purified Nb-Avi (20. Mu.g. ML -1 ) The final Nb-Avi dose for each assay system was determined to be 10ng by BCA protein quantification. At 2 ng.mL -1 In the case of AFB1 molecules as controls, the effect of AFB1-BSA concentration in QDs and T lines was studied. The concentration of AFB1-BSA in the T line was 0.3,0.6, 0.9mg.multidot.mL -1 The method comprises the steps of carrying out a first treatment on the surface of the The emission wavelengths of the three QDs were 525nm (fig. 12), 585nm (fig. 13), and 605nm (fig. 14), respectively. At 0.6 mg.mL -1 With AFB1-BSA and 585nmQDs, ΔG was calculated to be 0.462, which reading was relatively low and stable, meaning NQ&SC-LFIA showed the best sensitivity under these conditions (FIG. 13), and therefore was further studied on this basis. Although ΔG (0.423) was 0.3 mg.multidot.mL -1 AFB1-BSA was the lowest but was visually inferior in qualitative test by naked eyes, so that the post-optimization condition was 0.6 mg.mL -1 AFB1-BSA and 585nmQDs.
The composition and concentration of the loading buffer is also a critical factor affecting Δg. The application also investigated the effect of methanol concentration in AFB1 molecular solutions. FIG. 15 shows that ΔG is at 2ng mL when the methanol concentration in PBS is 30% (v/v) -1 The AFB1 molecule reaches the minimum value under the condition and remains relatively stable. The stable analytical performance was still exhibited when the methanol concentration in PBS was 70% (v/v). Buffers of different pH values (pH= 2.0,4.0,6.0,8.0, 10.0) were then used to increase 1 ng.mL -1 Analytical performance under AFB1 molecular conditions. As shown in fig. 16, Δg decreases with pH (2.0-6.0) until pH reaches 6.0, and with pH (6.0-10).0) And (3) increasing. Then at 1 ng.mL -1 A series of concentrations of Tween-20 (0.1%, 0.25%,0.5%,1.0% and 2.0%, v/v) buffer were also analyzed under AFB1 molecular conditions, and FIG. 17 shows that at low concentrations of Tween-20, the visible fluorescence intensity and gray scale values of the T line are higher, but the NQ&The sensitivity of SC-LFIA is not obviously improved; in contrast, the best analytical performance was achieved at 1.0% Tween-20. In conclusion, the NQ of the AFB1 with simple and easy operation and high sensitivity is successfully developed by exploring and optimizing analysis conditions&SC-LFIA。
Sensitivity and specificity experiments of NQ & SC-LFIA
Quantitative detection results show that NQ&SC-LFIA is able to sensitively analyze AFB1 under optimal conditions. AFB1 was analyzed at different concentrations by visual qualitative analysis and quantitative gray scale contrast detection. The loading buffer was optimized as a mixture of 1% sucrose+0.5% bsa+1% peg4000+1% tween-20 in PB buffer (0.05 m, ph=6). FIG. 18 shows the relationship between ΔG and AFB1 concentration, the method of the present application has good linearity for AFB1 detection from 0.195 ng.mL -1 To 4.370 ng-mL -1 LOD is 0.106 ng.mL -1 ，IC 50 Is 0.86 ng.mL -1 ，1.25ng·mL -1 Qualitative analysis with the naked eye is possible (a of fig. 19, gray scale results are shown in fig. 20). In addition, the specificity is also NQ&A key analytical property of SC-LFIA, which is characterized by several mycotoxins (5 ng. ML -1 ) Jammers, including aflatoxin B2 (AFB 2). Aflatoxin G1 (AFG 1), aflatoxin G2 (AFG 2), aflatoxin M1 (AFM 1), fumonisin B1 (FB 1), deoxynivalenol (DON), zearalenone (ZEN) and trichothecene-2 (T2). FIG. 19 b shows that AFB1 produces a signal intensity nearly close to the background signal compared to the fluorescence visual intensity triggered by other mycotoxins. Therefore, there is no cross reaction with the functional analogues FB1, DON, ZEN, T2, and the structural analogues AFB2, AFG1, AFG2, AFM1 of AFB1 are very low.
Example 6 stability experiment
The stability of NQ & SC-LFIA was verified by accelerated aging experiments. The application adopts an accelerated aging experiment based on an Arrhenius equation to prove that the Arrhenius equation is an empirical formula, and expresses the dependence of a chemical reaction rate constant (k) on temperature (T). Eα represents the apparent activation energy (. Apprxeq.19.5 Kcal/mol), R is the molar gas constant. The general trend is that T increases and k increases, so that the relationship between temperature and days of aging can be calculated. Calculated, 23 days at 50℃corresponds to a period of one year at room temperature (25 ℃). The prepared test strips were placed in an incubator (50 ℃) for 7 days, 15 days and 23 days, respectively, and their analytical properties were verified. All experiments were performed 5 times. The nanobody and mAb-based strips were compared under various conditions, including running buffer concentration of methanol (10%, 30%,50%,70%, v/v), running buffer concentration of NaCl (0, 100, 200, 500, 800 mM), and test ambient temperature (4 ℃,16 ℃,32 ℃,40 ℃).
As a result, as shown in fig. 21, a in fig. 21 shows that the nanobody test strip shows very high methanol tolerance, and when the methanol concentration is higher than 30%, the mAb-based probe can hardly be immobilized on the T-line of the test strip, but the Nb-based probe is hardly affected. FIG. 21 b shows that nanobody test strips show better tolerance to salt concentration between 100mM and 500mM, and that binding activity of Nb-based probes decreases sharply when NaCl concentration is higher than 500mM, and that activity of mAb-based probes is lower than that of 800mM, which is consistent with the results of iELISA. Nanobody-based test strips showed better temperature tolerance compared to mAb strips, from 4 ℃ to 40 ℃, nb-based probes were hardly affected by the detection ambient temperature, and still showed good binding activity at low temperature, but mAb was hardly functional under this condition (c in fig. 21). The present application also found that nanobody-based probes can work normally in a low temperature environment, which means that nanobody-based probes can be used directly without pre-incubating to room temperature, which is essential for nanobody-based probes. The above results indicate that nanobody-based strips would have better application potential in cases where the sample matrix is complex and the test environmental conditions are not ideal. Finally, to evaluate the stability of our strips, after 7, 15 and 23 days of storage in an incubator at 50 ℃, the results are shown as d in fig. 21, the strips show perfect performance even when stored at 50 ℃ for 23 days, and these strips can be stored at room temperature for one year according to the Arrhenius equation, which indicates that nanobody-based strips have great potential in practical applications.
EXAMPLE 7 practical application of NQ & SC-LFIA in practical samples
To demonstrate the analytical performance of our method in real samples, quantitative detection of AFB1 in oat was performed. First, the matrix effect must be eliminated because the composition of the real sample extract is complex and biotin is present in many crop samples, which may affect the efficacy of NQ & SC-LFIA. Biotin is slightly soluble in water and ethanol and insoluble in other organic solvents, so pure methanol is considered.
Adding AFB1 solutions with various concentrations into the negative oat sample to obtain an actual sample added with the AFB1. First 1g of oat samples with different concentrations of AFB1 (20 ng, 10ng and 5 ng) were dissolved in 4mL of methanol, respectively, the samples were thoroughly mixed for 15 minutes and the mixture was centrifuged at 8000rpm for 10 minutes. Subsequently, the supernatant was collected, diluted 4-fold with PBS, centrifuged at 12000rpm for 10 minutes, and the extract was collected and used for the study, with final concentrations of AFB1 in oat samples of 20, 10, 5. Mu.g.kg, respectively -1 The concentration of the extract was 1.25, 0.625, 0.3125ng mL, respectively -1 . Extracts of three AFB1 concentrations were expressed as NQ&SC-LFIA was tested, 5 sets of replicates, and recovery was calculated, recovery = measured AFB1 concentration/added AFB1 concentration x 100. The results are shown in Table 3.
TABLE 3 Table 3
The data in Table 3 shows that NQ & SC-LFIA has good effect in detecting AFB1 in oat sample diluent, and the recovery rate is between 88.8% and 116.7%. This shows that NQ & SC-LFIA has great potential for analysis of AFB1 in actual samples as a highly sensitive assay.
In conclusion, the intelligent mobile phone auxiliary lateral flow immunoassay test strip (NQ & SC-LFIA) based on the nanobody is successfully developed for the first time, and high-sensitivity and convenience detection of AFB1 is realized. The Avi/SA coupling strategy solves the defect that the activity of Nb is affected by random modification of the nanomaterial. Besides the advantages of simple and convenient LFIA operation and time saving, the intelligent mobile phone can be used for photographing and data analysis, so that on-site detection is realized, and no instrument is needed. Meanwhile, compared with mAb, the stability and the anti-interference capability of our method are ensured due to the thermal stability of Nb and the tolerance to complex analysis environment. The true sample analysis is also verified, and the recovery rate is between 88.8 and 116.7 percent, so that the method is an effective method for realizing on-site detection. Meanwhile, the Avi/SA coupling strategy has wide application prospect in other nano-body applications. Therefore, the application not only provides a stable and efficient field detection instrument for AFB1 monitoring, but also plays a certain role in promoting the development of Nb application.
The above embodiments are only illustrative of the preferred embodiments of the present application and are not intended to limit the scope of the present application, and various modifications and improvements made by those skilled in the art to the technical solutions of the present application should fall within the protection scope defined by the claims of the present application without departing from the design spirit of the present application.

Claims (10)

1. A lateral flow immunoassay kit for the detection of aflatoxin B1 comprising: the kit comprises 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, AFB1-BSA antigen is coated on the T line, and biotin-labeled bovine serum albumin is coated on the C line.
2. The kit according to claim 1, wherein the nanobody-biotin/streptavidin @ quantum dot probe is generated by the directed coupling strategy of nanobody-biotin and streptavidin @ quantum dot binding by biotin and streptavidin; the amino acid sequence of the nano antibody-biotin is shown as SEQ ID NO. 4; the streptavidin@quantum dots are obtained by modifying streptavidin to COOH-QDs.
3. The kit according to claim 2, wherein the preparation method of the streptavidin @ quantum dot comprises the following steps: adding COOH-QDs into boric acid buffer solution, adding EDC and NHS, vertically culturing at 37 ℃ for 60 minutes, adding streptavidin, swirling at 25 ℃ for 60 minutes, adding 2-mercaptoethanol, incubating for 30 minutes, centrifuging, and taking precipitate to obtain the streptavidin@quantum dot.
4. The kit of claim 3, wherein the centrifugation step further comprises washing the pellet with borate buffer for 2 times.
5. The kit according to claim 3, wherein the emission wavelength of the COOH-QDs is 585nm.
6. The kit according to claim 1, wherein the concentration of the AFB1-BSA antigen is 0.6 mg/mL -1 。
7. A method for detecting aflatoxin B1, comprising the steps of:
(1) Incubating the nanobody-biotin/streptavidin @ quantum dot probe in claim 1 with aflatoxin B1 molecular solution for 10 minutes under the condition of room temperature light protection in a vibrating way, adding the solution into a sample pad of the lateral flow immunoassay test strip in claim 1, reacting for 15 minutes at room temperature, and reading gray results of T line and C line after the fluorescence intensity of the T line and the C line is stable;
(2) Establishing a standard curve according to the gray scale results of the T line and the C line;
(3) And (3) combining the standard curve obtained in the step (2) by adopting the same method in the step (1) to obtain the content of aflatoxin B1 in the sample to be detected.
8. The method according to claim 7, wherein in the step (1), the concentration of the aflatoxin B1 molecule solution is 1 x 10 0 -1×10 10 ng·mL -1 。
9. The method according to claim 7, wherein in the step (1), after the fluorescence intensities of the T line and the C line are stabilized, the smartphone is used to record a photograph at an excitation wavelength of 435 nm, and the gray scale results of the T line and the C line are read.
10. Use of a kit according to any one of claims 1-6 for detecting aflatoxin B1 in a foodstuff.
CN202310516218.1A 2023-05-09 2023-05-09 Lateral flow immunoassay kit and method for detecting aflatoxin B1 Pending CN116840460A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117147830A (en) * 2023-10-26 2023-12-01 德州国科医疗科技有限公司 Fluorescent staining solution for detecting specific fungus D-glucan

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117147830A (en) * 2023-10-26 2023-12-01 德州国科医疗科技有限公司 Fluorescent staining solution for detecting specific fungus D-glucan
CN117147830B (en) * 2023-10-26 2024-01-12 德州国科医疗科技有限公司 Fluorescent staining solution for detecting specific fungus D-glucan

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