Research On The Sensitive Detection Of Ochratoxin A Based On Fluorescence Signal Amplification Technology

At present,mycotoxins have been found in various cereals and coffees,and they have received widely attention because of their wide range of pollution,high toxicity,large yield and wide range of pollution,in recently years.The intake of mycotoxins is very harmful to animals and humans.Ochratoxin A is one of the most extensive and toxic toxins in mycotoxins.When human body ingest Ochratoxin A,it is very slow in metabolism and has a long half-life,which poses a great threat to human health.Therefore,the design of a simple and efficient aptamer sensor,while establishing a simple and feasible,rapid and accurate detection of ochratoxin A method for the detection of mycotoxins in food,to ensure food safety and human health is of great significance.In this paper,ochratoxin A was used as the detection target,and a new method based on aptamer sensor for fluorescence detection was established.The simple,sensitive and specific detection methods of ochratoxin A was realized.The main research contents and results can be summarized into the following two aspects:1.DNasel-assisted target recycling signal amplification strategy for ochratoxin A monitoring.In this method,a simple and sensitive aptamer/PVP-protected graphene oxide based assay for ochratoxin A(OTA)detection was developed.The polymer materials(poly(vinyl pyrrolidone),PVP)was used as coating materials to prevent the unspecific adsorption between target and graphene oxide.Meanwhile graphene oxide protect aptamer from DNase I cleavage.Aptamers can be detached from the surface of PVP-protected graphene oxide through specific target binding,contacting them with enzymatic cleavage and induces the target for a new cycle.Cycling of targets and PVP-protected graphene oxide contribute to significant signal amplification.As compared to the non-protccted graphene oxide/non-target recycling based biosensor,the new method reveals much lower detection limit(9.38 nM).Moreover,the application of this method in real sample has been verified using red wine samples spiked with a series of concentration of ochratoxin A.This aptasensor offers a great practical importance in food safety and can be widely extended for detection of other toxins by replacing the sequence of the recognition aptamer.2.Exonuclease-assisted DNA recycling signal amplification strategy for ochratoxin A sensitive detection.In this method,a sinple and homogeneous fluroscence aptasensor based on exonuclease-assisted recycling amplification for OTA detection was developed.The aptamer for OTA partially hybridized with complementary DNA(cDNA),which was released due to the recognition of aptaner to OTA.Then,cDNA immediately hybridized with specifically designed hairpin DNA,the short single-stranded DNA and cDNA was respectively released by hydrolysis of exonuclease Ⅲ on the double-stranded DNA.The cDNA induced next ring opening and digestion,the short single-stranded DNA captured the signal DNA absorbed on graphene oxide by DNA hybridization.The quenched fluorescence of the system can be recovered owing to the signal DNA divorced from graphene oxide.High sensitivity and good selectivity for OTA detection were observed.As compared to the non-recycling based biosensor,the new method reveals much lower detection limit(0.96 nM).Moreover,the application of this method in real sample has been verified using real samples spiked with a series of concentration of ochratoxin A.