Ratiometric Electrochemical Sensor Based On TID For Ochratoxin A Detection In Liquor And Wine

Ochratoxin A(OTA),a toxic secondary metabolite generated by Aspergillus and Penicillium.is widely presented in agricultural、wine and condiments.According to data from the Food and Agriculture Organization of the United Nations(FAO),OTA contaminated 25%of the world’s agricultural products.OTA can be accumulated in humans and animals by the food chain,causing nephrotoxicity,liver toxicity,teratogenicity,immunotoxicity,and carcinogenicity.OTA has been listed as a 2B human carcinogen(probably carcinogenic)by the International Agency for Research on Cancer(IARC).Currently,OTA is mainly detected by large instruments with the requirements of expensive instruments,professional operators,cumbersome process and long detection cycle.In addition,enzyme-linked immunosorbent assay(ELISA)is also one of the commonly used detection methods,but the poor stability of antibody and the high false positive rate limit its further development.Therefore,it is urgent to develop a simple,fast,low-cost,high-sensitivity and specific detection method to solve the food safety problems caused by OTA.Electrochemical methods have the advantages of high sensitivity,short timeconsuming,which attract much attention in the field of mycotoxin detection.Aptamer is a type of mononucleotides obtained by artificial synthesis in vitro with high affinity and specificity for targets,which possessing good application prospects in the field of detection.In this study,a ratiometric electrochemical aptasensor was constructed to detect OTA based on the principle of target-induced aptamer dissociation and dual signal molecules of ferrocene(Fc)and methylene blue(MB).Cyclic voltammetry and electrochemical impedance spectroscopy were used to characterize the modified states of electrode surface,and altering cyclic voltammetry was used to verified the feasibility of the developed aptasensor.In this study,the reactant concentration and reaction conditions of the ratiometric electrochemical aptasensor were optimized,and the effects of different hybridization methods and hybridization buffers on the detection performance of the aptasensor were compared.The optimal reaction system contained:the concentration of Fc-cDNA immobilized on the electrode surface(10 μM),hybridization time(120 min),OTA ncubation time(60 min),hybridization method(immersion method),and the hybridization buffer(Tris-HCl buffer).In the optimal conditions,ACV was used to test the sensitivity,specificity,reproducibility and stability.The sensitivity test results showed that the detection limit of the ratiometric electrochemical aptasensor was 0.349 pg/mL(LOD=3 S/m),the linear detection range was 1 pg/mL～1 ng/mL,and the detection time was 1 h.The specificity detection of the proposed aptasensor was performed using five non-specific substances.Five non-specific substances and their mixtures and the blank control group showed negative results,OTA and the mixtures(OTA and non-specific substances)presented positive results.Therefore,the proposed aptasensor has good specificity.By comparing the reproducibility of the electrochemical aptasensors based on single-signal(MB and Fc)and ratiometric electrochemical aptasensor,the following result was obtained:the variances of ratiometric electrochemical aptasensor and single-signal electrochemical aptasensors were 0.01,0.048(Fc)and 0.031(MB),respectively.It can be seen that the reproducibility of ratiometric electrochemical aptasensor was significantly better than the single-signal electrochemical aptasensors.Stability test results showed that the best use period of the electrode was 5 days.According to standard addition method,the OTA standard solutions with different concentrations were added to the liquor and wine samples as the actual samples,and then the samples were detected by the proposed ratiometric electrochemical aptasensor,the results showed that the recovery rate was between 98.6%and 107.0%,indicating that the proposed aptasensor can be used in actual detection.In summary,a new ratiometric electrochemical aptasensor was constructed successfully in this study and used to detect OTA in the liquor and wine samples.The aptasensor integrated three major advantages of fast and sensitive electrochemical analysis technology,highly specific target-induced aptamer dissociation strategy and reproducible ratiometric detection technology,which was conducive to rapid and efficient on-site detection.It meet the requirements of the food industry for the mycotoxins detection and has great development prospects.