Detection Of Small Molecules By Fluorescence Anisotropy Method Based On Fluorophore Labeled Aptamer

Chapter 1:The properties and selection of aptamers were briefly introduced. The definition of fluorescence anisotropy (FA) and its application in the field of bioanalytical chemistry were introduced subsequently. It was described that the research background, the main research work and innovation of the thesis.Chapter 2:By taking advantages of aptamers in high affinity, good stability, easy generation and labeling and merits of fluorescence anisotropy in simplicity and robustness, a noncompetitive FA analysis method based on tetramethylrhodamine (TMR)-labeled aptamer for detection of Ochratoxin A (OTA) was established. The intramolecular interaction between TMR and aptamer bases greatly affected the local ratation and FA of TMR. The conformation of aptamer changed upon OTA binding, leading to the change of intramolecular interaction, and FA of TMR changed. TMR was labeled on different sites (terminals and T bases) of aptamer to OTA. According to the study of FA responses of the TMR-labeled aptamers to OTA, we found the aptamer with a TMR labeled on the 10th T base exhibited a significant FA reduction. The FA method based on T10-TMR-O36 showed high sensitivity and good selectivity for OTA analysis with a detection limit of 3 nM and a detection range from 3 nM to 3μM. The tested compounds with similar structures of OTA did not cause interference.Chapter 3:A simple and efficient FA analysis method for direct detection of adenosine was developed by using TMR-labeled aptamers against adenosine. TMR was successfully labeled on the sites of 5’ teminal,3’ terminal and the A, C and T bases of the aptamers. The different FA responses of TMR-labeled aptamers to adenosine were comprehensive studied. The intramolecular interaction between TMR and the adjacent G bases altered upon adenosine binding, which affected local ratation and FA of TMR. Aptamer with TMR labeled on 16th C base (C16-TMR-Ad25) showed remarkable FA decrease FA response to adenosine. And the detection limit of adenosine was 2 μM. Moreover, the FA analysis method could be used for the detection of adenosine in complex matrix.Chapter 4:The detection princple in this paper was similar to the previous two chapters, and a sensitive method for adenosine triphosphate detection was presented. Aptamer for adenosine also could bind with ATP. The C16-TMR-Ad25 and A23-TMR-Ad25 aptamers with high FA responses were selected, these two aptamer probes both allowed for the quantitative detection of ATP with FA-decreasing or FA-increasing responses, respectively. The FA analyses also allowed to detect ATP in human urine and human serum sample.