| We developed a system for amperometric detection of pathogen 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 catalyst. The analysis protocol can be conducted with solution volumes on the order of a few microliters and completed in 40 minutes. The detection system was capable of detecting single Escherichia coli cell without polymerase chain reaction with high specificity for E. coli vs. the bacteria Bordetella bronchiseptica. The kinetics of SAM formation onto electrode surface is investigated with surface plasmon resonance (SPR) and 80% of the protein deposition can be achieved within four seconds. The quality of the protein monolayer can be monitored based on topographical information from atomic force microscopy (AFM). |
