| Food safety problems caused by mycotoxins have been widely concerned all over the world.Ochratoxin A(OTA)is a secondary metabolite produced by some strains of Aspergillus and Penicillium.It is the most polluted in nuts,cereal agricultural products and feed.A large number of studies have shown that OTA has strong nephrotoxicity,hepatotoxicity and neurotoxicity,and can be teratogenic or carcinogenic to humans and mammals.At present,the conventional detection methods of OTA have the problems of long detection time,high cost of instruments and equipment,cumbersome pretreatment steps and high requirements for the professional quality of operators.Therefore,the development of rapid,accurate,effective and economical safety analysis methods is of great significance to ensure food safety and human health.Aptamers have the advantages of high stability,strong specificity,high affinity,small variation between batches,easy chemical synthesis and modification,low production cost and so on.In recent years,biosensors developed using aptamers as recognition elements have attracted the favor and attention of scientific researchers.Gold nanoparticles(AuNPs)is an important nano enzyme material with excellent catalytic properties.It can simulate the activities of peroxidase,glucose oxidase and catalase and catalyze the reaction of corresponding substrates.AuNPs have attracted extensive attention in the field of catalytic detection because of their non-toxic,high catalytic activity and good stability.Based on the specific recognition,high affinity and conformational transformation of aptamers and the excellent peroxidase properties of AuNPs,a visual colorimetric sensor based on aptamer and gold nanoenzyme was constructed to realize the quantitative detection of OTA in food.The main contents are as follows:Part Ⅰ:Study on the mechanism of constructing OTA visual sensor based on label-free aptamer and gold nanoenzyme.AuNPs with particle size of 13 nm were synthesized in this chapter.Firstly,to dye fluorescence test showed that the aptamer and OTA could bind to each other.UV-Vis absorption spectrum and Zeta potential measurement showed that AuNPs did not have nonspecific binding(electrostatic adsorption and coordination,etc.)with OTA,which determined the feasibility of constructing OTA visual colorimetric sensor based on aptamer and AuNPs.Secondly,the mechanism of aptamer enhancing AuNPs enzyme activity was explored.Zeta potential measurement showed that the adsorption of aptamer on the surface of AuNPs increased the negative charge density of AuNPs,which was conducive to the adsorption of substrate TMB.The fluorescence test of terephthalic acid probe showed that the aptamer promoted the generation of ·OH,and XPS test showed that the aptamer promoted the release of Au3+,which were beneficial to promote the oxidation of TMB.Therefore,the aptamer is shown to enhance the peroxidase activity of AuNPs.When OTA does not exist,the aptamer is adsorbed on AuNPs,the peroxidase activity of AuNPs is enhanced,and the color of TMB oxidation product is darker;When OTA exists,the aptamer binds to OTA,the peroxidase activity of AuNPs decreases,and the color of TMB oxidation products becomes lighter,so as to realize the colorimetric detection of OTA.Part Ⅱ:Research and development of OTA visualization kit based on label-free aptamer and gold nanoenzyme.Based on the theory of the previous chapter,a colorimetric sensor for visual detection of OTA based on label-free aptamers and gold nanoenzymes was constructed and a kit was developed.The system optimization,performance evaluation and actual sample detection in food matrix were carried out.The optimal experimental parameters of the sensor are as follows:the concentration of AuNPs is about 6 nM,the concentration of aptamer is 500 nM,the buffer is acetic acid sodium acetate(10 mM,pH 3.8),the construction time of sensing system is 10 min,the concentrations of substrate TMB and H2O2 are 2 mM and 1 M respectively,the substrate reaction temperature is 37℃ and the substrate reaction time is 15 min.The detection range in buffer is 10～600 nM,which has a good linear relationship,and the detection limit is 6.20 nM.When other common coexisting mycotoxins were added,no obvious interference effect was found,and the specificity was good.It was applied to the detection of OTA in food samples such as oats,corn,soybeans,rice and glutinous rice.The detection sensitivity was 7.43 nM～10.57 nM,the recovery was 91.42%～109.35%,and the coefficient of variation was 1.13%～8.98%.In addition,the reagent quality is stable after 120 days of storage,and the standard curve and sensitivity can keep basically unchanged. |
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