Study On OTA Fluorescence Detection Method Based On G-quadruplex/dye Complex

Ochratoxin A(OTA)is the most toxic,most widely distributed,highest toxin production,the heaviest pollution to agricultural products,and the most closely related to human health in Aspergillus Ochra.Traditional detection methods mostly use instrumental analysis and immunoassay.Instrumental analysis has high detection sensitivity and high accuracy,but it requires expensive instruments,skilled operator,and complex pre-processing and detection processes.It is cumbersome and time-consuming,not suitable for on-site detection and immediate detection.The immunoassay method shows high sensitivity,without requirement of expensive instruments,simple and easy operation,and high-throughput detection,but this method is susceptible to the instability of antigen and antibody and poorly reproducible and prone to false positives.The photonic crystal microspheres developed in this paper use the secondary structure of nucleic acid aptamers-G-tetramers and fluorescent dye molecules to form a fluorescent system to detect OTA,which can achieve low cost,easy operation,and realize purpose of fast and accurate detection of OTA.First,the microfluidic platform designed in the laboratory is used to prepare silica photonic crystal microspheres with different particle sizes by changing the output speed of water and oil.Then different chemical groups are modified on the surface of the microspheres,and the groups with the lowest background signal and the best fluorescence enhancement effect are selected to combine the aptamer.Subsequently,the aptamer is modified on the surface of the solid phase carrier.At last,the subsequent experiments are carried out.A parallel G-quadruplex/Th T complex fluorescence system method is established to detect OTA.First,circular dichromatic spectroscopy is used to verify the formation of parallel G-quadruplex.Then the key factors of the final experimental conditions are optimized.The standard curve of OTA according to the above conditions was obtained:Y=0.09064 lg X+0.23417,R~2=0.9906,the detection of the linear range is 0.1-100ng/m L.Then the structure of OTA analogues and other toxins OTA are used to verify the specificity of the aptamer.The results show that the OTA-aptamer structure of OTA have no obvious cross reaction among analogues and other toxins.This method is further verified by recovery rates for the three kinds of grain.The recovery rate is good.This method and ELISA method are applied to detect OTA in real samples at the same time.Comparing with the results of the two methods,it can be seen that the determining coefficient R~2 of the two methods reaches to 0.8208,which verified the effectiveness of this method.An antiparallel G-quadruplex/CV complex fluorescence system method is established to detect OTA.First,we prepare the suitable buffer condition to form the parallel G-quadruplex structure,and then circular dichroic spectroscopy is used to valid the formation of antiparallel G-quadruplex.After that,the key conditions are optimized.According to the optimized conditions,the standard curve for OTA is prepared:Y=3.36894 lg X+25.11219,R~2=0.9950.The detection linear range is 0.1-100 ng/m L,and the limit of detection is 0.06 ng/m L.Then the specificity of OTA-aptamer is verified by using structural analogs of OTA and other toxins.The results showed that OTA-Aptamer had no obvious cross reaction to structural analogs and other toxins of OTA.The recovery rates in the spiked cereal samples are detected.The standard ELISA method is used to verify the developed method and the results of them are agreement with.This new method and ELISA method are applied to detect OTA in real samples at the same time.The results show that the determining coefficient R~2of the two methods reached to 0.8623,which verified the effectiveness of this method.Compared with the results of the two methods,it can be seen that the CV method has faster detection speed and higher accuracy,and can realize naked eye detection;Th T method requires less reagents and lower detection cost;Compared with the national standard ELISA method,the detection method has wider linear scope,shorter detection time,lower cost,and the detection high-throughput,which is more suitable for on-site rapid detection.