Research And Application Of Aflatoxin B₁ Detection Method Based On Nanomaterials And Aptamers

Mycotoxins have become an important bottleneck in the development of China’s grain,oil and food industry due to the existence of widespread contamination,high hazard,difficulty in prevention and control,low content and complex contamination of substrates and other characteristics.At present,most of the front-line grain warehouses into the rapid detection method of mycotoxin is immunochromatographic detection technology,the detection equipment and test strips used mainly rely on imports.However,imported immunochromatographic test strips are expensive,with large detection errors and long sample pre-processing time,which can no longer meet the needs of China’s grain in warehouse detection.In this project,Aflatoxin B₁（AFB₁）,a representative mycotoxin in food security,was used as the research target.Based on the traditional colloidal gold immunochromatography assay,a nano-composite material with a stable and strong colour signal was synthesized as the signal-amplifying element,and the aptamer was used as the recognition molecule instead of the antibody in the test strip for target recognition.Therefore,in this study,an aptamer colourimetric sensor and lateral flow test strip detection system with the simple experimental operation,fast signal response and visualization of results were prepared to achieve highly sensitive and rapid detection of mycotoxins,while expanding the detection range and reducing experimental cost.The specific studies are as follows:1.Colorimetric aptasensor was used to detect aflatoxin B₁ in maizeBased on the fact that gold nanoparticles（Au NPs）of different sizes and morphologies will exhibit different colours and interact with each other under the action of external environmental factors,thus causing changes in their sizes and solution colours as well as shifts in resonance peaks,this chapter established Au NPs-aptamer colourimetric method for the detection of AFB₁ in maize by electrostatic binding of Au NPs and aptamer as signal probes.If the solution contains AFB₁,the aptamer（Apt）will fall off from the surface of Au NPs so the solution exposed to sodium chloride will aggregate and produce changes in colour and absorbance.In this way,visual and rapid detection of aflatoxin B₁ can be achieved.By the optimal laboratory conditions,the method was found to detect AFB₁ at a minimum concentration of 0.5 ng/m L and the calibration curves had been shown to be well linear in the range of 0.5～50 ng/m L.The spiked samples were analyzed,and the calibration recovery of this method ranged from 90.3%to 94.8%with the relevant standard deviation（RSD）of 7.3%to 12.2%.2.Au NPs-based aptamer chromatography strip method for the detection of aflatoxin B₁in maizeBased on the experimental results obtained in the previous chapter,we can demonstrate that the sequence of aptamer used in this experiment has high specificity for aflatoxin B₁.In order to improve the accuracy and sensitivity of AFB₁ detection,based on the traditional Au NPs immunochromatography strip,the biotin-modified aptamer was used as the recognition molecule of the strips instead of the antibody,and the complementary chain of sulfhydryl modified was covalently bonded to Au NPs as the recognition probe,meanwhile,the streptavidin was used as the detection line of the strips to capture the conjugate.The colour of the strips was observed visually and scanned using a colloidal gold immunochromatography analyzer for the quantitative detection of AFB₁.By optimizing the conditions affecting the experimental results,the limit of detection of the aptamer test strip method was 0.5 ng/m L,a detection range of 0.5～200 ng/m L,a spiked rate of 97.3%～120%and a relative standard deviation of 2.3%～8.3%.3.An aptamer test strip based on the flower-like MoS₂@Au composite nanomaterial for the detection of aflatoxin B₁ in maizeIn order to further enhance the sensitivity of the assay and make the chromogenic effect of the test strip more obvious,based on the Au NPs-Apt lateral flow chromatographic strip in the previous chapter,a nanocomposite material Mo S₂@Au with stable and strong colour signal was synthesized to replace a single gold nanoparticle as the signal probe of the test strip.According to the large specific surface area of flower-like molybdenum disulfide,the colloidal gold attached to its surface can improve the utilization of Au NPs,while biotin-modified aptamer was used as recognition molecule instead of antibody in the test strip for target identification,so as to achieve rapid,accurate and quantitative detection purposes.Through the optimization of key factors,the assay limit of detection was 0.3ng/m L,the detection scope was from 0.2 to 200 ng/m L,and the method showed good linearity within the range of 1～50 ng/m L.The recovery of corn samples was 99.4%～109.4%,and the relative standard deviation was 3.2%～10.1%.