Aptamer Biosensing Technology For Detection Of Ochratoxin A

Ochratoxin is a class of mycotoxins produced by the genus Aspergillus and several Penicillium species,including seven compounds,Ochratoxin A?OTA?,Ochratoxin B?OTB?and Ochratoxin C.Of these ochratoxin compounds,OTA is the most widely distributed toxic and harmful.Study has shown that OTA has toxicity of liver and kidney,and has effects of carcinogenic,teratogenic,mutagenic and immunosuppressive.OTA is classified as CLASS 2B carcinogens by the United Nations Organization for Research on Cancer.At present,the OTA test in our country is mainly based on traditional instrumentation,which includes chromatography,atomic absorption spectrometry,capillary electrophoresis,and supercritical fluid chromatography.These traditional detection methods are often cumbersome.The detection cycle is long,and the sample preparation process is cumbersome and time-consuming,requiring professional operators,and to a certain extent,limiting their practical application in OTA detection.Therefore,the establishment of a simple,sensitive and selective method for OTA detection in actual samples such as food and related products is of great significance for the effective control of OTA and food safety and human health.In this dissertation,OTA was detected by the combination of specific aptamers and fluorescent dyes,and then the fluorescence quenching mechanism of fluorescence resonance energy transfer between gold nanoparticles?AuNPs?and fluorescent dyes.This study mainly consists of two parts:The detection platform for signal amplification and AuNPs fluorescence quenching was established based on Exo III.When an OTA is present,the aptamer recognizes and binds to OTA,leading to the formation of duplex DNA between the complimentary DNA?cDNA?stranding and the signal probe.Exo III is digested from the flat end of the 3'signal probe,releasing the fluorescent group and the the cDNA at the same time.After joining the AuNPs,the fluorophore can not be adsorbed and quelled.The released cDNA is hybridizes with other signal probes to initiate a new cleavage reaction.An OTA molecule can trigger the cleavage of a large quantity of signal probes through cyclic hybridization–hydrolysis process,thus significantly amplifying fluorescence.The aptamer sensor designed in this study has high selectivity to OTA with a detection limit of 4.82 nmol/L.The feasibility was verified by adding standard recovery about this study and the applicability of the aptamers,demonstrating that the detection method is not interfered by the sample matrix.The study can be effectively used to detect mycotoxins about aptamer sensor of the present invention.In the system,aptamers are adsorbed on the surface of the gold nanoparticles when OTA are absented the fluorescently labeled.The fluorescence signal that was emited by the OTA aptamer fluorescein-labeled is quenched by the fluorescence resonance energy transfer effect of gold nanoparticles.In the system,the fluorescent signal are retained owing to the fluorescently labeled aptamer binds to the OTA and forms a folded structure,which can resist the adsorption of gold nanoparticles when the OTA are present.The detection limit is 5 nmol/L and the linear detection range is 10¹000 nmol/L with this sensing platform.The detection method can be specific to OTA without interference from other analogues,such as ochratoxin B,ochratoxin C,aflatoxin B₁,zearalenone toxin,N-acetyl-L-phenylalanine,and warfarin.