Enzyme-modulated cleavage of dsDNA for supramolecular design of biosensors

Supramolecular docking and immobilization of biotinylated dsDNA onto a self -assembled monolayer of avidin have been measured using impedance spectrosc opy and quartz crystal microbalance technique. The formation of the serial assembly was first achieved by linearizing circular plasmid dsDNA using Bam H I endonuclease enzyme. This was followed by a bisulfite-catalyzed transam ination reaction in order to biotinylate the dsDNA. The reaction is single- strand specific, and it specifically targets impaired cytosine bases genera ted during the enzyme cleavage. The biotinylated dsDNA was then used as a l igand at a gold electrode containing avidin. The process was monitored by a c impedance spectroscopy that was used to probe the changes in interfacial electron-transfer resistance upon binding and a microgravimetric quartz cry stal microbalance that reflected in situ mass changes on the dsDNA-function alized substrates. Our results demonstrated that this approach could be emp loyed for the determination of small-molecular-weight organics such as cisp latin, daunomycin, bisphenol A, chlorinated phenols, and ethidium. bromide. A detection limit in the magnitude of ca. 10 nM was achieved. This immobil ization technique provides a generic approach for dsDNA-based sensor develo pment and for the monitoring of DNA-analyte interactions.