| The specific function of biomacromolecules, such as proteins or nucleic acid highly depends on their three-dimensional structure. Folding kinetics investigation can reveal the mechanism of how the biomolecules transform from the free linear structure to the functional high-level structure, thus much attention has been paid to this scientific field recently. For example, Science journal listed the protein folding problem as one of the125significant unsolved problems in science area. The biomolecular folding events often occur in the time scale of millisecond or even microsecond, which means it is necessary to initiate the reaction before that time. Rapid mixing technique can initiate reactions in a short period of time and has been recognized as an attractive approach to analyze kinetics of fast reactions.Traditionally, experimental studies on protein folding kinetics are performed by stopped-flow, which has provided valuable information on mechanisms in protein folding. However, the milliseconds dead time and large sample consumption restrict its further application in analyzing faster reactions. Recently, instead of the stopped-flow, the concept of continuous-flow has been widely accepted in microfluidic mixer to analyze the kinetics of chemical and biological reactions due to its rapid mixing time. In this thesis, we developed three unique rapid micromixers for interrogating the folding (unfolding) kinetics of biomacromolecules under various conditions.(1) To mix solutions with low viscosity, a novel zigzag micromixer was designed with rapid mixing time, low sample consumption and simple structure. Numerical simulation and experimental evaluation confirmed that this mixer achieved complete mixing in16μs. We used this micromixer to investigate the kinetics process of the chemiluminescence reaction. Further, a revised zigzag micromixer was developed and the mixing time was decreased to be5.5μs, which was the fastest mixing time among those of the reported turbulent mixers. The folding kinetics of human telomere G-quadruplex under monovalent cations was tracked using this optimum mixer. For the first time, we found the experimental evidence of the collapsing process from the linear human telomere DNA to the hairpin structure.(2) To mix solutions with high viscosity, a co micromixer with advantages of simple structure and ease of fabrication was developed. The co micromixer was evaluated with numerical simulation and experimental experiments. The mixing time of this mixer was579.4μs for the solution with viscosity of33.6times of water, which represented a mixing speed about1000-fold faster than those reported previously. Further, the folding kinetics of human telomere G-quadruplex under molecular crowding conditions was investigated and it was discovered that there existed a folding event in the sub-millisecond time scale.(3) To characterizing the interaction kinetics of biomacromolecules with low sample consumption, a dual-hydrodynamic focusing laminar mixer was reported. Based on numerical simulation and experiments evaluation, it proved that the micromixer could mix solutions with low or high molecular weights and it has the unique advantages of wide observation time window (710μs-5.36s, four orders of magnitude) and ultra-low sample consumption (less than0.55μL/min for both samples).We further studied the interaction kinetics between G-quadruplex and single-stranded DNA binding protein (SSBP). The results indicated that the G-quadruplex was unfolded with the help of SSBP and the unfolding rate was slower under higher concentration of Na+solution.In summary, three unique rapid micromixers based on continuous-flow were reported for interrogations of the folding kinetics of biomacromolecules. Numerical simulations and experimental evaluations confirmed the mixing efficiency of these mixers. The applications to the chemical or biochemical reactions proved that these mixers were reliable and useful. They may be widely used in various researches of protein or nucleic acid folding kinetics. |
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