太古嗜鹽菌Halobacterium salinarum紫膜 (purple membrane, PM) 內具有獨特光敏感蛋白，被稱為細菌視紫質 (bacteriorhodopsin, BR)。由於BR受光後會使PM膜兩側產生質子梯度差而誘導光電流產生，且此光電流與入射光強度成正向關係，因此再利用菌體本身可散射光特性，本實驗室先前已分別開發以抗體及核酸適體為辨識分子的PM膜微生物感測器。本研究首先以連續注流式即時檢測系統延續探討抗體及aptamer被固定化於PM膜上及微生物被吸附於辨識分子上之穩定性，發現兩種辨識分子在10 μL/min低流速時均具有最佳穩定性；反之150 μL/min高流速時皆會被脫附於晶片表面，其次，分別被抗體及核酸適體辨識分子捕捉於PM膜晶片上之Escherichia coli K-12及Lactobacillus acidophilus兩種微生物，在100 μL/min 較高流速下也會完全脫離固定於PM晶片上之辨識分子，因此可藉由調高流速而使辨識分子-PM晶片再生利用。此外，在10 μL/min 低流速流場中辨識分子-PM晶片可連續檢測不同濃度之微生物體，研究中發現抗體-PM晶片測菌時所需平衡時間為30分鐘；核酸適體-PM晶片則只要5分鐘，都比原先靜態吸附檢測菌時需要反應兩小時更為迅速。再者，本研究也發現空白PM晶片在流場下所能承受之最大流速為2 mL/min，因此可再次藉由粉體及菌體之光遮蔽效應直接以空白PM晶片進行檢測。於250 μL /min高流速下，發現空白PM晶片能檢測到體積平均粒徑為45 μm Al2O3粉末之最低濃度為0.2 ppb；E. coli K-12能被檢測到的最低濃度為102 CFU/mL。最後，我們以E. coli核酸適體取代現有固定化在PM膜上之抗體辨識分子，先以模擬軟體探討其二級結構並選出最適化固定化與檢測條件，再分別對E. coli、L. acidophilus及Bacillus subtilis進行檢測，結果發現能檢測到E. coli之最低濃度為1 CFU/10 mL；然而對於106 CFU/mL L. acidophilus及Bacillus，此E. coli感測晶片仍分別有45 %及29 %因非特異性吸附所引起的光電流下降，因此未來需調整檢測條件，以提升其專一性。

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.

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