Ratiometric Electrochemical Aptasensor Based On CHA For The Detection Of Ochratoxin A In Food

Ochratoxin A(OTA)is a toxic secondary metabolite generated by Aspergillus and Penicillium.OTA is widespread in various cereal crops and animal foods,mainly contaminating grain(wheat,maize,barley,oats and so on)and its products.OTA has serious toxicity for the kidneys,liver and brain of humans and animals.Thus,realizing the accurate and sensitive detection for OTA is vital for the health and safety of human.In this study,a ratiometric electrochemical aptasensor based on CHA was fabricated for convenient,accurate and ultrasensitive detection of OTA in foods.The main research contents and achievements are as follows:Firstly,the construction of the ratiometric electrochemical aptasensor based on CHA.The ratiometric electrochemical aptasensor was fabricated by designing the DNA probe(tDNA,Fc-H1,MB-H2)and preparing the sensing interface of electrode,and its feasibility was verified.The conditions of electrochemistry and the reaction system were optimized to ascertain the suitable detection conditions.The optimal results are as follows:The concentration of Fc-H1 was 2.0 μmol/L,the assembly time for Fc-H1 was 4 h,the hybridization time of aptamer and tDNA was 60 min,the concentration of aptamer-tDNA(A-T)was 0.5 μmol/L,the concentration of MB-H2 was 2.0 μmol/L,the reaction time of CHA was 120 min.Secondly,the performance evaluation of ratiometric electrochemical aptasensor based on CHA,including sensitivity,specificity,stability and repeatability.The sensitivity of the ratiometric electrochemical aptasensor was assessed by detecting OTA with various concentrations.The results showed that there was a good linear relationship between IMB/IFc and LogCOTA in the range of 100 fg/mL～50 ng/mL.The linear regression equation was IMB/IFc=0.43751LogCoTA+2.2214.The linear correlation coefficient(R2)was 0.992 and the limit of detection(LOD)was 81 fg/mL.For the specificity,only the mixed group containing OTA was consistent with the OTA positive group,while the other results including the non-target toxins and their mixed groups were consistent with the blank group,indicating that the ratiometric electrochemical aptasensor had good specificity.The stability of the ratiometric electrochemical aptasensor was tested by storing at 4℃ for 0,3,6,9,12,15,and 18 days.The results manifested that the aptasensor remained 85%of its initial response on the 15th day,which was acceptable.For the repeatability,OTA detection was performed on dual signal and single signal electrochemical aptasensors,and their relative standard deviation(RSD)was 3.179%and 8.211%,respectively,indicating that the developted ratiometric electrochemical aptasensor had good repeatability.Thirdly,the practical application evaluation of the ratiometric electrochemical aptasensor based on CHA.In this study,the most common foods(wheat flour,oats,maize,and barley)were selected to detect OTA,and the results were compared with the high performance liquid chromatography-mass spectrometry(HPLC-MS).The results displayed that the recovery rate of the ratiometric electrochemical aptasensor was 93%～106%,which was consistent with the results of HPLC-MS,indicating that the developted aptasensor had high reliability and practical application value.In conclusion,a ratiometric electrochemical aptasensor based on CHA was constructed for OTA detection in food.The developted aptasensor has the advantages of accuracy,specificity,sensitivity and convenience,providing a newly feasible platform for OTA detection in food.Furthermore,The aptasensor could be widely used to detect various targets by changing the target aptamer,which provided a new idea for the detection of other mycotoxins and pathogenic microorganisms in food,having practical significance for food safety and quality control.