Development Of Microfluidic Platform For Manipulation And Detection Of Shewanella Oneidensis

In recent years,Shewanella oneidensis,which is a model organism for electrochemical activity bacteria,have gained attention,since it plays an important role in microbial fuel cell(MFC)applications,biofuel production,multiple metallic reduction and dye biodecolorization.These previous studies on interesting applications of metal-reducing organisms focused on large quantities of bacteria or biofilm.However,it is extremely difficult to conduct research on the respiratory system of bacteria in population-level experiments due to their increased complexity.Excluding the possible effects of biofilm and secreted materials in population-level experiments,research on S.oneidensis at the cell level,including studies on single bacterium or countable bacteria,can provide more sensitive and accurate information.Consequently,it is essential to analyze and measure S.oneidensis multi cells,without labels or secreted substances,because they play an important role in the environment and energy fields.For a rapid and efficient research of S.oneidensis at the cell level,a fine sample preparation is the first key element.It is desirable to sample one cell or countable multi cells to address potentially low analyte levels.However,the fine sample preparation of S.oneidensis is still difficult to achieve due to its sub-micron size,heterogeneous shape and motile behavior.Micro fluidic chip,which have found an array of important applications in areas such as analytical systems,biomedical devices,tools for chemistry and biochemistry,is considered as an appropriate solution due to their distinct advantages such as small sample consumption,fast analysis times,and parallel operation.Herein,the rapid and label-free microfluidic platform for trapping and measuring S.oneidensis at cell level were studied.The goal of this work was to establish a rapid and label-free microfluidic platform and realize trapping and measuring of countable submicron rod-shaped S.oneidensis.In view of microfluidic chip with the characteristics of miniaturization and integration,the miniaturized unit modules were firstly developed,including the manipulation module for sample preparation and the detection module for sample detection.With the help of unit modules,the microfluidic platform was developed to provide the rapid.effective and label-free trap and measure S.oneidensis at cell level.Three kinds of manipulation substrates were developed,which were microstructure arrays,sidewall microelectrode arrayss and non-contact microelectrodes.These manipulation modules were all rapid,high-throughput,label-free.Although S.oneidensis were trapped in microstructure arrays,the bacteria could still move away.The manipulation model of S.oneidensis based on dielectrophoresis was established.The development of three manipulation modules provides the foundation and experience for the development and integration of the manipulation module in the subsequent microfluidics.Two kinds of detection modules are proposed.Firstly,a rapid and readily accessible microfluidic fabrication method is presented to realize observation with high magnification microscopy.With the one-step molding process,the interconnections,the thin observation interface of polydimethylsiloxane(PDMS)membrane and microfluidic channels were integrated into an intact PDMS replica.Three kinds of PDMS replicas with different auxiliary beams were designed and optimized by leakage experiments and analytical software.The observation interfaces of a 170?m thickness PDMS membrane enlarges the application domain of microfluidic chips.The submicron particles and bacteria were successfully observed in microfluidic chips under ordinary microscope.In addition,the two-layer electrodes substrates are also prepared for the detection of S.oneidensis.We designed and fabricated the three-layer structures of the detection electrode-polymethyl methacrylate(PMMA)insulator-dielectrophoresis electrode.Finally,we propose a microfluidic platform integrated with a DEP technique,hole-array structures and fluorescence to trap,count and detect label-free S.oneidensis multi cells.To capture countable S.oneidensis multi cells,the proposed chip utilized a modified DEP method by changing and rearranging the electric field distribution.The rearrangement mechanisms were studied by numerical analysis.The correlation between holes and amount of trapped bacteria was also determined by designing a series of hole-arrays.Real time fluorescence imaging was used to monitor the trapping process.Additionally,Raman spectroscopy was used to characterize the potential of the chip for the analysis of trapped bacteria.Once the trapping process was accomplished,the bacteria were simultaneously detected by Raman spectroscopy.The microfluidic platform provided a rapid and effective bacteria trap and cell level measurements.The microfluidic chip was integrated with modified dielectrophoresis(DEP)trap technique and various sizes of hole arrays.The microfluidic platform provides a quantitative sample preparation and analysis method at the cell level that could be widely applied in the environmental and energy fields.