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Master Research Outline

1. Development of a multiple electric field microfluidic chip.
    three electric field strength direct current electric fields and a EF-null region is created in a PMMA microfluidic chip. By using this new chip, experimental throughput on electrotaxis study can be accelerated.
    Engineering innovation: 
(a)Ability to create isolated structures for fluidic definition in polymeric tape construct for fluid channel.
(b)Complete bubble free microfluidic environment in PMMA microfluidic chip that now allows the cells to be cultured on chip for weeks to even months.
    Several new design based on the approach is completed and awaits for experimental confirmation.
    Publication: on Biomicrofluidics in 2012.
2. Numerical simulation of multiphysics in microfluidic environment using commercial software:
 CFD-ACE+(initial use), COMSOL Multiphysics(now primary), ANSYS(trial), flow-3D(trial)
3. A large electric field stimulation chamber for sample preparation.
   background: conventional study of electrotaxis was possible at a feasible low height microfluidic channel. This was most commonly down by flanking two coverslips in a 10cm dish. Although these convensional devices are suitable for tracking cell migration in dcEF, the cellular throughput in these devices are quite low and to acquire enough of sample for classical molecular biology and protein chemistry experimental especially, multiple devices may be required to collect enough of cells.
   It is intuitive to scale up the device to increase the experimental throughput. However, problems such as electric field a-uniformity and difficult to apply the electric field to desired strength arises.
   Engineering innovation:A large device with appropriate current rectifying chamber and salt bridge arrangement was designed. The new device can easily provide 10^7 cells in a single experiment.
   Also the new system allows rapid release of the device right after the experiment, thus allowing the cells be easily accessed for further processing.
   We have used this device for investigation of 
   (a) important cellular signaling node of phosphorylation. Publication on PLoSONE in 2013.
        Use of this new device with high cell culture area and uniform electric field stimulation. We can collected a large amount of crude protein extract of cell stimulate under dcEF that elucidate significant electrotaxis. We are challeging the most accepted electrotaxis hypothesis that the membrane receptors undergo in situ electrophoresis (electromigration of membrane receptors on the membrane towards the electrodes). The asymmetrical distribution of membrane receptors on cells is the reason why cells would acquire directional cue under uniform ligand environment. Asymmetrical distribution of EGFR on CL1-5 was observed when under dcEF stimulation, but the direction is contraindicatory to the electrotaxis. (cathodal EGFR polarization but the migration is anodal)
        Through western blotting, we have confirmed that CL1-5 have high EGFR expression but there was not EGFR phosphorylation or at least not conventional EGF stimulated phosphorylation residue when under dcEF stimulation.
         This is quite interesting because it is the first time of the report that EGFR is a known signaling pathway of electrotaxis in NIH3T3 fibroblasts and in breast cancer cells. CL1-5 is a lung adenocarcinoma that also express high level of EGFR but EGFR does not seem to participate in the electrotaxis of CL1-5. This finding is now under further thinking into the fundamental mechanism to electrotaxis.
        Also, through Western blot results, we have identified that the cells origin from the same patient tissue, CL1-0 and CL1-5 cells demonstrate different electrotaxis effect also have completely different cellular signaling under direct current electric field stimulation.
   (b) global phosphoproteome profiling
        We use a large electric field stimulation chamber to collect crude cell protein extracts and stabilize them for further downstream phosphoproteome profiling through HPLC/MS/MS. We've collect the experimental data and pending for detailed analysis.
   (c) surface glycome analysis.
4. Investigation into composite PMMA/PDMS microfluidic device.

Paul, Hsieh-Fu Tsai,
Jul 28, 2014, 12:26 AM