Transfection of DNA molecules into mammalian cells with electric pulsations, which is so-called electroporation, is a powerful and widely used method that can be directly applied to gene therapy. However, very little is known about the basic mechanisms of DNA transfer and cell response to the electric pulse. We developed a microelectroporation chip with poly(dimethylsiloxane) (PDMS) to investigate the mechanism of electroporation as a first step of DNA transfer and to introduce the benefits of miniaturization into the genetic manipulation. The microelectroporation chip has a microchannel with a height of 20 microm and a length of 2 cm. Owing to the transparency of PDMS, we could in situ observe the uptake process of propidium iodide (PI) into SK-OV-3 cells, which shows promise in visualization of gene delivery in living cells. We also noticed the geometric effect on the degree of electroporation in microchannels with diverse channel width. This experimental result shows that the geometry can be another parameter to be considered for the electroporation when it is performed in microchannels with an exponential decaying pulse generator. Cell culturing is possible within the microelectroporation chip, and we also successfully transfected SK-OV-3 cells with enhanced green fluorescent protein genes, which demonstrates the feasibility of the microelectroporation chip in genetic manipulation.

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A portable flow cytometer has been recognized as an important tool for many clinical applications such as HIV/AIDS screening in developing countries and regions with limited medical facilities and resources. Conventional flow cytometers typically require multiple detectors for simultaneous identification of multiple subsets of immune cell. To minimize the number of detectors toward portable flow cytometry or to analyze multi-parametric cellular information with minimum number of detectors in conventional flow cytometers, we propose a versatile multiplexed cell-counting method using functional silica nanoparticles (SiNPs). FITC-doped SiNPs, which are 100 times brighter than the FITC molecules itself, were used as new intensity-based fluorescent dye complexes to simultaneously measure two subsets of leukocytes using a single detector. CD45(+)CD4(+) cells tagged with these FITC-doped SiNPs were 50 times brighter than CD45(+)CD4(-) cells tagged only with FITC. To make the overall system compact, a disposable microchip flow cytometer that does not require sheath flow was developed. Combining these dye-doped SiNPs based detection schemes and the sheathless microchip flow cytometer scheme, we successfully identified and counted two subsets of leukocytes simultaneously (R(2) = 0.876). These approaches can be the building blocks for a truly portable and disposable flow cytometer for various clinical cytometry applications.