Fluorometric Detection Of Ochratoxin A Based On Aptamer

Chapter1:This part briefly introduced the definition and properties of aptamer. The significance of ochratoxin A (OTA) detection and the commonly used methods for OTA detection were also introduced. Different aptamer based methods for ochratoxin A detection were reviewed. It was described that the research background, main research work, and innovation of the thesis.Chapter2:By using fluorescent aptamer probe, a fluorometric method for OTA detection was established based on fluorescence resonance energy transfer. OTA showed fluorescece emission at430nm with an excitation at375nm, when OTA bound to fluorescein labeled aptamer, the fluorescence intensity of fluoreophore was increased at518nm due to fluorescence resonance energy transfer between OTA and fluorophore. According to the changes of the fluorescence intensity at518nm, we can achieve the detection of OTA. The results showed that this method was simple, rapid, and of high specificity. The detection limit was1nmol/L.Chapter3:By using fluorescein labeled aptamer (FDNA) for ochratoxin A and quencher labeled complementary oligonucleotides (QDNA), a fluorometric method for OTA detection was developed. In the absence of OTA, the fluorescein-labeled aptamer bound to complementary ssDNA containing a quencher moiety of BHQ, the fluorescein and the quencher were close, resulting in fluorescence quenching. In the presence of OTA, FDNA bound to OTA with high affinity and specificity, and the FDNA did not bind with QDNA, so the fluorescence signal of FDNA was recovered. According to the changes of the fluorescence intensity, OTA could be quantitatively detected.Chapter4:By taking advantage of the intrinsic fluorescence of ochratoxin A, this part presented a fluorescence anisotropy (FA) approach for rapid analysis of the interaction between OTA and aptamer. The specific binding of OTA with the aptamer can induce increased FA of OTA as the result of the increase of molecular volume and the slowdown of molecular rotation of OTA. We investigated the effect of metal ions in the binding solution on the binding affinity of aptamer by the FA approach. FA analysis showed the interaction between OTA and aptamer was greatly enhanced by the simultaneous presence of Ca2+and Na+, while the binding affinity of aptamer decreased more than18fold when only Ca2+existed, and the binding was completely lost when Ca+was absent. Additionally, crucial bases of the aptamer for binding can be mapped through FA analysis and aptamer mutation. An aptamer-based FA approach for OTA detection was developed. In the solution containing fixed concentrations of aptamer and OTA analyte, the fluorescence anisotropy of OTA analyte decreased with the increasing of the concentration of OTA analyte, and OTA could be detected by measuring the change of FA.Chapter5:We explored a fluorescent strategy for sensing ochratoxin A by using single fluorophore labeled aptamer against OTA. When fluorophore labeled aptamer bound with OTA, the surrounding environment of the fluorophore changed, causing a change in fluorescence intensity. OTA can be detected by measuring the change in fluorescence intensity of fluorophore labeled aptamer. Different fluorescein labeling sites of aptamers were screened, including the internal thymine (T) bases,3’end and5’end of the aptamer, and the effect of the labeling on the aptamer affinity and the response of the fluorescein labeled aptamer to OTA were investigated. Some fluorescein labeled aptamers could show signal-on or signal-off response to OTA. With the fluorescent aptamer switch, simple, rapid and selective sensing ochratoxin A was achieved. OTA spiked in diluted red wine could be detected, showing the feasibility of the fluorescent aptamer for complex matrix.