Real-time monitoring the stability of antibodies and aptamers immobilized on purple menbrane-based photoelectric biochips and development Escherichia coli detection

• Main Authors: Min-Che Li, 黎閔哲
• Other Authors: Hsiu-Mei Chen
• Format: Others
• Language: zh-TW
• Published: 2018
• Online Access: http://ndltd.ncl.edu.tw/handle/maxug7

碩士 === 國立臺灣科技大學 === 化學工程系 === 106 === Halobacterium salinarum purple membranes (PM) contain a unique light-sensitive protein called bacteriorhodopsin (BR). When BR is illuminated, a proton gradient is formed across PM, which drives photocurrent production. The generated photocurrent is linearly correlated with the illumination intensity; thus both antibody-PM and aptamer-PM composite sensors have previously been developed in this laboratory to detect microorganisms based on the fact that bacteria scatter light. In this study, a real-time flow-injection analysis system was employed to investigate the stability of the immobilized antibodies and aptamers as well as the captured bacteria on PM-coated chips in a shear flow. Both recognition molecules exhibited the best stability at 10 μL/min, while their dissociation from the chip surface was observed at 150 μL/min. In addition, Escherichia coli K-12 and Lactobacillus acidophilus cells captured by the antibody-PM and aptamer-PM sensor chips, respectively, were completely detached from their respective immobilized recognition molecules at 100 μL/min, suggesting the feasibility of regenerating either sensor chip simply by raising flow rates. Moreover, microorganisms at different concentrations were readily detected at 10 μL/min with only 5-min and 30-min equilibrium time observed upon each cell injection for the aptamer-PM and antibody-PM chips, respectively, which were significantly shorter than what was observed (2 h) in a static detection. The maximal flow rate for the bare PM chip to sustain its full photoelectric activity was 2 mL/min, so we subsequently employed the bare PM chip to directly detect microparticles and bacteria based on their light scattering effects. At 250 μL /min, the bare PM chips directly detected Al2O3 powders with a volume-based particle size of 45 μm with a detection limit of 0.2 ppb, and a limit of 102 CFU/mL for E. coli K-12 was obtained. Finally, an E. coli aptamer-PM sensor chip was prepared by first simulating the secondary structure of the E. coli aptamer for its optimal immobilization and detection conditions. The chip readily detected E. coli K-12 with a limit of 1 CFU/10 mL. However, the chip also exhibited 45 % and 29 % photocurrent reductions on the detection of 106 CFU/mL L. acidophilus and Bacillus subtilis, respectively, indicating nonspecific adsorption of both cells. More investigations of the detection condition will be conducted to improve the chip selectivity in the future.