Visualized Study Of The Cycle Reaction System With Total Internal Reflection Fluorescence Microscopy

In this thesis, several nucleic molecular machines were established based on cycling DNA reactions, and total internal reflection fluorescence microscope (TIRFM) and confocal laser scanning microscope (CLSM) were employed to study the operation of these molecular machines. It was shown that the visualization studies of nucleic acid molecular machines could be carried out at single-molecular level with the two imaging techniques.Chapter1introduced the technique backgrouds. Biological fluorescence and the application of fluorescence tagging techniques were introduced. The CLSM technique and TIRFM technique were introduced together with their applications in single-molecule detection and cell biology. Progress on DNA molecular machines were reviewed. The main subject of the thesis was introduced:the operation of DNA molecular machines, and its performance in living cells, were supposed to be investigated by single-molecular visualization techniques.In chapter two, a cycling DNA reaction network was studied on the surface of the ITO electrodes which were coated with sonicated poly(o-phenyldiamine) thin films; home-made quantum dots were used as fluorescence tags to visualize the molecular machine. CLSM was employed to visualize the release of the fluorescence tagged DNA signal strands in the cycling reaction network.The reaction network was initial izeed by aptameric recognition of the target, lysozyme, and was operated in3reaction phases, providing cascading signal amplifications:a main cycle including strand-displacement polymerization and target-displacement polymerization, a downstream cycleof strand-displacement polymerization, and a series of DNA nicking-polymerization cycles. The reaction network resulted in the repetitive releasing of the short signal strands tagged with quantum dots, which caused the fluorescence dots to scatter and translocate in the visualization process. The releasing quantity of the quantum dot-tagged signal strands increased along with the increasing of the inputted Iysozyme concentration, thus the operation of the cycling molecular machine could be verified.In Chapter3, the signal amplification in an artificial DNA molecular machine was directly visualized via TIRFM with the help of triggered hybridization chain reaction as a reporting device for visualization. The molecular machie consisted of two parts, the reaction part and the reporting part. The reaction part was triggered by Iysozyme (the target), and perform signal amplification in two progressive reaction phases, producing "triggering DNA strands" in amplified concentration; and the "trigger strand" was in turn fed-forward to the reporting part, in which it triggered hybridization chain reaction (HCR) to produce a long thread-like DNA complex with periodic repeated double-stranded short segments, which would be intercalated with fluorescence tags. The fluorescence-tagged HCR products could then be visualized via TIRF.In chapter4, a DNA molecular machine based on cycling DNA polymerization and coupled with the interaction between DNA and graphene was studied by confocal laser scanning microscopy; the molecular machine initiated by the aptermeric recognition; the interaction between the molecular machine and graphene in cancer cells were investigated. It was shown that, coupled with graphene, this molecular machine could exhibit the existence and concentration of Iysozyme, thus could be used to detect Iysozyme in cancer cells.