| Microfluidic devices feature numerous technical advantages,such as miniaturization,portability,multi-functional integration,high accuracy and reliability.Thus,they have been extensively used to monitor biological processes including cell proliferation,migration,differentiation,senescence and apoptosis at single-cell level,as well as to characterize and extract physiological properties of single cells.As a eukaryotic model organism,budding yeast,Saccharomyces cerevisiae(S.cerevisiae),has benefits of fast generation,short cell cycle,ease of maintenance and manipulation,and a fullysequenced genome.Microfluidic single-cell analysis using budding yeast as a model organism has been rapidly developed in recent years,thereby promising applications in the fields of drug development,disease mechanism investigation and aging researches.In this thesis,for the replicative aging studies of single budding yeast,microfluidic devices and systems for flexible manipulation,reliable culturing and wide-band electrical impedance measurement of single yeast cells,have been developed.The main contents are described as follows:(1)A microfluidic device and system integrated with multiplexing microelectrodes are developed to achieve precise manipulation,reliable culturing and wide-band electrical impedance measurement of single yeast cells.A microfluidic network consisting of main and side channels is designed for hydrodynamic immobilization of budding yeast by applying the suction pressure.By using multiplexing microelectrode pairs,the selective release based on negative dielectrophoresis and wideband electrical impedance measurement of budding yeast are achieved.In the electrical-impedancespectroscopy-(EIS)integrated system,real-time electrical impedance monitoring of yeast budding process is implemented to accurately identify dissection events of yeast daughter cells.(2)A microfluidic single yeast in-situ impedance array is designed and its sensing characteristics are investigated by using finite-element modeling and simulation.An addressable microelectrode array consisting of orthogonal row and column electrodes,and capture-culturing-dissection microstructures are proposed and modeled using finite element methods.In a single sensing unit,the linear relationship between impedance amplitudes at 1 MHz and cell volume is established to characterize cell growth and daughter dissections of budding yeast.In a 5×5 sensing array,effects of non-target cells on impedance signals and on the accurate identification of target daughter dissections are analyzed.Finally,an optimization scheme for the row and column spacing of the microelectrode array is proposed in terms of accuracy and throughput.(3)An electrode-array-integrated microfluidic device and system for real-time and high-throughput monitoring of yeast replicative aging are developed to accurately determine the yeast replicative lifespan.Yeast capture-culturing-dissection microstructures are designed and optimized.Combined with the microelectrode array,the microfluidic device is fabricated and implemented into an EISintegrated system.72-h long-term culturing and time-lapse microscopic imaging of budding yeast cells are performed to analyze multiple physiological properties and parameters including dissection consistency,replicative lifespan,yeast generation duration and terminal morphology.The innovative aspects in the work are described as follows:(1)Multiplexing microelectrodes for onchip integration of dielectrophoresis and electrical impedance spectroscopy;(2)Design concept of double-layer addressable microelectrode array for improving throughput of yeast in-situ impedance measurement;(3)Accurate identification of yeast daughter dissection events via time-lapse electrical impedance monitoring,and the development of EIS-integrated microfluidic device and system for real-time monitoring of yeast replicative aging. |
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