Study On The Detection Method Of Aflatoxin B₁ And Zearalenone Optical Aptamer

Aflatoxin B₁（Aflatoxin B₁,AFB₁）and Zearalenone（Zearalenone,ZEA）have strong carcinogenic,teratogenic,mutagenic and immune damage,etc.,which can pollute agricultural products,food and feed or indirectly contaminate animal products（Milk,meat,eggs）in the food chain,which seriously affects food safety and poses a threat to human health.Therefore,improving the supervision of the contamination level and content of AFB₁and ZEA in food,and establishing an accurate and effective detection method for AFB₁ and ZEA is of great significance to human health.This project combines the characteristics of visualization of colorimetric and etching methods,as well as the advantages of simple operation and fast signal response of fluorescence methods,with the strong specific nucleic acid aptamers（Apt）and have established three aptamer optical sensors for the detection of mycotoxins AFB₁ and ZEA by using the enzyme-like activity and fluorescence quenching characteristics of gold nanoparticles（Au NPs）,as well as the etching effect of gold nanorods（Au NRs）The specific research contents are as follows:1.The glucose oxidase-like activity of gold nanoparticles（Gold nanoparticles,Au NPs,Au）was utilized as a material to catalyze the production of hydrogen peroxide（H₂O₂）from glucose（Glu）,and nucleic acid aptamers was used as specific recognition elements The competitive combination of Apt and AFB₁ with Au NPs can controls the enzyme-like catalytic activity of Au NPs,which exhibits different shades of visualization effects under the action of the chromogenic agent ABTS and was used for the detection of AFB₁.Studies have shown that metal nanomaterials has better stability and lower cost than the HRP enzyme.The experimental parameters were optimized,and 1/4 Au had the best catalytic activity,0.2M Glu was the best concentration,the reaction time for catalyzing Glu was 30 min,the reaction temperature was room temperature,the best p H was 7,and the best organic solvent system is 10%methanol,the best reaction time is 10 min,the best aptamer concentration is0.1μM.The linear range of AFB₁ obtained under the best conditions was 1～70 ng/m L,and the detection（LOD）is 0.8 ng/m L.The spiked samples were tested and the recovery rate was88.1%～109.26%.2 In order to improve the sensitivity and response speed of visual detection,horseradish peroxidase（HRP）was used to etch blue-green Au NRs,showing rich color changes of blue,blue,purple,pink,no,and yellow.Established a detection method for AFB₁ multicolor sensor based on aptamer combined with gold nanorod etching.According to experimental optimization,when the concentration of HRP was 0.02μg/m L,the reaction time of enzyme activity was 5 min,and the reaction was carried out at room temperature,the optimal ratio volume of Au NRs:CTAB was 1:4,and the concentration of aptamer was 0.5μM,the concentration of AFB₁ in the range of 0.01～0.5 ng/m L hads a good linear correlation with the change of UV-visible absorption spectrum,and the limit of detection（LOD）was 0.008ng/m L.This colorimetric sensor hads good selectivity to AFB₁.The recovery rate of AFB₁in the actual samples were 81%-119%,and the relative standard deviation（RSD）was less than 10%.3.In order to further improve the sensitivity of the sensor signal and the diversification of detection targets.Carboxyfluorescein（FAM）labeled AFB₁ aptamer（FAM/Apt）and rhodamine（ROX）labeled ZEA aptamer（ROX/Apt）are used as fluorescent probes,and Au NPS-FAM/Apt-ROX/Apt dual-color fluorescence signal augmented aptamer sensor for simultaneous detection of AFB₁ and ZEA.was constructed.Optimal working conditions are obtained after optimization:FAM-Apt-1 and ROX-Apt-2 were the best aptamer chains;the incubation time of Apt and Au NPs was 20 min,and the reaction volume was 40μL;the incubation time of Apt and toxin was 10 min,The reaction volume was 50μL;PBS（p H=8.5）buffer was the working solution,and room temperature was the working environment.The standard curve was established under the above optimal conditions,and the linear range of AFB₁ was 0.01-50 ng/m L;the linearity of ZEA The range was 0.01-200ng/m L.The detection limits of AFB₁ and ZEA were 8 pg/m L and 6 pg/m L,respectively.The sensor has good specific selectivity for AFB₁ and ZEA detection.The actual samples were spiked and tested,the recovery rate of AFB₁ was 81%～119%,and the recovery rate of ZEA was 86%～104.6%.