Studies Of Novel Optical Sensing Technology Based On Conformational Switch Of G-quadruplex

Biosensors have provided novel strategies and technical support for the research and development of life sciences, which have large potential applications in the life sciences, medicine and environmental monitoring. Taking advantages of easily designing and implementing biosensors, this master thesis concerns on the research hotspot in biomolecules, the enzyme activity detection and common heavy metal ions, focusing on how to improve the sensitivity, reduce the cost simultaneously and so on. Based on the conformational switch of G-quadruplex, several label-free optical detection methods have been developed for the detection of thrombin, Adenosine monophosphate (AMP), heavy metal ions and biothiol. Compared with the traditional methods, the proposed detection methods are convenient, cost-effective and sensitive. In addition, the practicability of these developed strategies were also verified. The detailed contents are described as follows.In chapter 2, we developed a novel label-free biosensor for Ag+ and biothiol detection based on G-quadruplex conformational switching that can be sensitively probed by the fluorescence of ThT. It has been reported that thioflavin T(ThT), a commercially available water-soluble fluorescent dye, which is weakly fluorescent in the free state, but exhibits obvious enhancement in its fluorescence upon interacting with the quadruplex DNA structures. In the presence of the Ag+, the G-quadruplex structure is coordinated by Ag+, inducing the release of ThT with a concomitant decrease of the fluorescence signal. The decreased fluorescence signal gives a measure for the quantification of Ag+. However, the presence of biothiol competes with guanine for Ag+because of the stronger interaction of biothiol with Ag+. This interaction releases Ag+from guanine, restoring guanine for the formation of the G-quadruplex structure and re-activating the strong fluorescence of ThT for the quantification of biothiol. Compared with the traditional methods detection for Ag+and biothiol, this approach owns the merits over conventional assays in its label-free design, extreme operation simplicity, low cost and so on. Therefore, it is expected that the developed approach has a potential of becoming a universal platform for the detection of a wide range of analytes.In chapter 3, we report a novel label-free aptameric biosensor based on a self-assembled aptamer/GO architecture for sensitive detection of biomolecules. We designed an aptamer probe containing G-rich sequence, which was well adsorbed on the surface of graphene oxid and obtained a self-assembled aptamer/GO architecture. The presence of the target would specifically bind to the aptamer, which induces the release of the aptamer sequence away from the GO surface. Then, ThT can bind to the G-rich oligonucleotide region of the probe to form G-quadruplex conformation with a substantially enhanced fluorescence signal. Based on the principle of specific binding between target and aptamer, we apply the label-free aptamer/GO architecture to detecting adenosine monophosphate(AMP)and human a-thrombin. Simultaneously avoiding the defects of high-cost and strong background signal caused by mark, this method provided a sensitive, selective and robust platform for label-free quantitative detection of biomolecules.