LASER INDUCED LYSIS OF ESCHERICHIA COLI CELLS AND BACILLUS SUBTILIS SPORES

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The principle of laser induced cell/spore lysis was explored in this project. E. coli K12 and B. subtilis spores were chosen as the target analytes. The lasers available for testing were of wavelength 980nm and 1480nm. As a proof of concept, a 2uL droplet of E. coli culture in nutrient broth was place on a hydrophobic surface. The tip of the laser fiber was placed directly above the droplet, and the droplet was exposed to 100mW (1480nm) or 200mW (980nm) for 4 minutes. An alternative setup of the droplet testing was also designed to prevent evaporation. The droplet was placed in a well made in 2mm thick PDMS and covered with a glass slide. The laser fiber was placed above the glass slide. The results showed that there was moderated lysis using both lasers. The percentage lysis was greater using 1480nm laser (21%) than the 980nm laser (14%). This was expected since the main cause of cell lysis is due to a sudden increase in temperature. The aqueous nature of cells thus makes the lysis highly dependent on the water absorption coefficient, which is 50 times higher at 1480 nm than at 980 nm. The alternative setup showed slightly less lysis (9% for 980nm). The difference to the droplet experiment results indicates that evaporation occurred during the droplet test which resulted in a smaller testing volume and thus in higher exposure to the laser. The next step was to test the laser in microfluidic channels in order to determine whether it would be applicable for a micro-bioanalytical system. A 25 parallel-channel design with channel widths of 50?m and depths of 100?m were fabricated on a silicon wafer and subsequently realized in poly(dimethylsiloxane) using soft-lithography. In addition to E. coli cells, the effectiveness of lysis of B. subtilis spores was also explored. Variable flow rates of 5?L/min, 2?L/min, 1?L/min and 0.5 ?L/min were implemented. The results showed a greater percentage of lysis of E. coli cells compared to the droplet method (31% for 5uL/min, 41% for 2uL/min, and 43% for 1uL/min). However, no lysis was observed for B. subtilis spores under any flow rate.

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2005-08-04T05:08:07Z
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cell lysis; laser; microfluidics
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dissertation or thesis
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