Detection Of Ochratoxin A By Enzyme-assisted Aptamer Biosensor

Ochratoxin,Whose basic structure is isocoumarin benzoate,is a secondary metabolite produced by some species of Aspergillus and Penicillium,and contains seven structural analogues.Ochratoxin A(OTA)is the most widely distributed and highly toxic in nature.OTA is a potentially toxic substance that can contaminate foods such as wheat,corn,coffee,spices,beer,grapes and derivatives,animal-derived products as well as feed.Ochratoxin A is a strong nephrotoxin with hepatotoxicity,teratogenicity,immunosuppressive and carcinogenicity and has been classified as a possible human carcinogen(2B)by the International Agency for Research on Cancer(IARC).In view of the potential threat to human health caused by Ochratoxin A,it is very important to develop a sensitive and rapid quantitative analysis method.Biosensor is a device that uses biometric elements to contact transducers directly to provide quantitative analysis information.It consists of two most important components:a biological component that responds to the target material and a signal generation module that detects the generation of results.Due to its good selectivity,high sensitivity,fast analysis speed,low cost,and on-line continuous monitoring in complex systems,especially its highly automated,miniaturized and integrated features,it has developed vigorously and rapidly in recent decades and has been widely used in the field of food detection.Traditional methods to detect OTA mainly include high performance liquid chromatography(HPLC),fluorescence,mass spectrometry and ELISA.Although these methods have high sensitivity and can provide qualitative and quantitative information,they limit the detection of OTA to a large extent due to the long analysis time and high cost.Therefore,it is particularly essential that we develop a faster,reliable,low-cost and sensitive method to detect OTA.Two fluorescent aptamer biosensor have been established to detect OTA:the first one:A aptamer biosensor was established to detect OTA quantificationally based on single walled carbon nanohorns(SWCNHs)and Exo III-aided signal amplification.The second:Based on the activity of Exo III endonuclease and the binding of SYBR Gold to intact DNA strand,a labeling-free fluorescent aptamer biosensor was established.The specific contents of the study included the following two aspects:A fluorescent aptamer biosensor based on Exo III signal amplification and SWCNHs as fluorescence quenching agent was established for the detection of OTA.We designed a hairpin probe(HP)and a signal probe(SP)labeled with carboxyfluorescein(FAM).The HP contained the OTA-specific aptamer sequence and was partially complementary to the SP.After addition of OTA,the aptamer binded OTA and thus exposed a single-stranded sequence that could hybridize with the SP to form a double-stranded structure through base pairing.Exo III digested the SP from double-strand to liberate the free fluorophore.The damaged SPs no longer were adsorbed by the SWCNHs and fluorescence could not be quenched.The method had a detection range that was linear from 10 nM to 1000 nM(with a correlation coefficient of 0.997)and the linear equation was Y=-0.5658+0.65063X.The limit of detection(LOD)was 4.2 nM(about 1.6 ng/mL)(S/N=3).A fluorescent aptamer biosensor based on exonuclease III(Exo III)and fluorescent dye SYBR Gold was established for detecting of OTA.This experiment consisted of aptamers specifically bound to OTA,single-stranded DNA complementary to aptamers,Exo III,fluorescent dye SYBR Gold and OTA.In the presence of OTA,the aptamer specificlly binded with OTA to form G-quadruplex structure,which released single-stranded DNA.When Exo III was added,it could only digest double-stranded DNA and had no effect on single-stranded DNA.Thus,when SYBR Gold was added to the system,the fluorescent probe binded to the intact DNA strand to generate fluorescence signal.In the absence of OTA,the aptamer combined with the complementary strand.When addition of the exonuclease III(Exo III),Exo III hydrolyzed the DNA strand along the 3?protruding terminus of the double-stranded DNA.Due to the DNA strand was destroyed by the hydrolysis of Exo III,after adding to SYBR Gold,it could not bind to DNA hydrolysate,there was only a weak fluorescence signal.Therefore,the quantitative detection of OTA can be realized according to the intensity of fluorescence signal.In our study,there was a good linear relationship between the relative fluorescence intensity and the logarithm of OTA concentration in the range of 8-1000 ng/mL,and the linear equation was y=0.07451 X-0.122(R~2=0.991).The detection limit was 4.7 ng·mL~-11 and the detection platform had good specificity and selectivity to OTA.In order to prove its practicability,we applied it to detect OTA in red wine.The results showed that the established detection system was suitable for the detection of actual samples.