Development Of New Electrochemical Detection Methods For Ochratoxin A

This research mainly focused on the development of simple, fast, sensitive detection methods for Ochratoxin A (OTA). On one hand, aptamer was applied to achieve rapid detection. On the other hand, suitable signal amplification techniques were adopted to improve the sensitivity, including nanoparticles and isothermal amplification. The concrete research is as follows:Firstly, DNA modified with redox reporter was immobilized on the surface of electrode. The competitive recognition and binding of OTA onto the surface of electrode induced the release of cDNA from the electrode and decreased the redox peak currents. According to the change of signal, the rapid electrochemical aptasensor for OTA detection was successfully developed.Secondly, rolling circle amplification (RCA) was used to amplify the detection signal and improve the sensitivity. A probe containing aptamer of OTA was used as the primer of RCA. Meanwhile, a template that could hybridize with primer was designed. When the primer and template were mixed, the RCA could be triggered under optimized conditions. Therefore, the length of primer could be amplified with repeated sequence, which acted as the carrier of methylene blue and was immobilized on the surface of electrode by hybridization. In principle, with the addition of OTA in sensing system, it will compete with the primer to combine with the padlock, inhibiting the process of RCA. Consequently, the redox signal of MB could be changed. Under optimized conditions, ultrasensitive detection of OTA was achieved with the limit of detection of 0.065ppt.Thirdly, functional probe combined with gold nanoparticles was applied to get rapid and ultrasensitive detection of OTA. Functional probe concludes recognition sequence, signal sequence and spacer sequence. In the presence of OTA, it will bind with the functional probe on the electrode, which induces the conformational change of the probe and facilitates the formation of redox reporter in the presence of hemin. Evidently, the sensing of OTA could be easily achieved and a relationship could be constructed between the redox signal variations and OTA concentration. Following, when OTA induces the conformational change of the probe, sensor could produce redox reporter with the addition of hemin and increase the sensing signal by applying gold nanoparticles with large surface area. Under optimized conditions, the ultrasensitive electrochemical aptasensor was achieved with the limit of detection of lppt.