Gau J J, Lan E H, Dunn B, Ho C M, Woo J C
Department of Biomedical Engineering, University of California at Los Angeles, 7523 Boelter Hall, 405 Hilgard Avenue, Los Angeles, CA 90095-1595, USA.
Biosens Bioelectron. 2001 Dec;16(9-12):745-55. doi: 10.1016/s0956-5663(01)00216-0.
We developed a system for amperometric detection of Escherichia coli (E. coli) based on the integration of microelectromechanical systems (MEMS), self-assembled monolayers (SAMS), DNA hybridization, and enzyme amplification. Using MEMS technology, a detector array was fabricated which has multiple electrodes deposited on a Si wafer and was fully reusable. Using SAMs, a monolayer of the protein streptavidin was immobilized on the working electrode (Au) surface to capture rRNA from E. coli. Three different approaches can be used to immobilize streptavidin onto Au, direct adsorption of the protein on bare Au, binding the protein to a biotinylated thiol SAM on Au, and binding the protein to a biotinylated disulfide monolayer on Au. The biotinylated thiol approach yielded the best results. High specificity for E. coli was achieved using ssDNA-rRNA hybridization and high sensitivity was achieved using enzymatic amplification with peroxidase as the enzyme. The analysis protocol can be conducted with solution volumes on the order of a few microliters and completed in 40 min. The detection system was capable of detecting 1000 E. coli cells without polymerase chain reaction with high specificity for E. coli vs. the bacteria Bordetella bronchiseptica.
我们基于微机电系统（MEMS）、自组装单分子层（SAMS）、DNA杂交和酶扩增技术，开发了一种用于安培检测大肠杆菌（E. coli）的系统。利用MEMS技术制造了一种探测器阵列，该阵列在硅片上沉积有多个电极，并且可以完全重复使用。通过自组装单分子层，将一层链霉亲和素蛋白固定在工作电极（金电极）表面，以捕获大肠杆菌的rRNA。有三种不同的方法可用于将链霉亲和素固定到金电极上：将蛋白质直接吸附在裸露的金电极上、将蛋白质与金电极上生物素化的硫醇自组装单分子层结合，以及将蛋白质与金电极上生物素化的二硫化物单分子层结合。生物素化硫醇方法产生了最佳结果。利用单链DNA - rRNA杂交实现了对大肠杆菌的高特异性，利用以过氧化物酶为酶的酶扩增实现了高灵敏度。分析方案可以在几微升量级的溶液体积下进行，并在40分钟内完成。该检测系统能够在不进行聚合酶链反应的情况下检测1000个大肠杆菌细胞，对大肠杆菌相对于支气管败血波氏杆菌具有高特异性。
