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 spectroscopy and quartz crystal microbalance technique. The formation of the serial assembly was first achieved by linearizing circular plasmid dsDNA using BamH I endonuclease enzyme. This was followed by a bisulfite-catalyzed transamination reaction in order to biotinylate the dsDNA. The reaction is single-strand specific, and it specifically targets unpaired cytosine bases generated during the enzyme cleavage. The biotinylated dsDNA was then used as a ligand at a gold electrode containing avidin. The process was monitored by ac impedance spectroscopy that was used to probe the changes in interfacial electron-transfer resistance upon binding and a microgravimetric quartz crystal microbalance that reflected in situ mass changes on the dsDNA-functionalized substrates. Our results demonstrated that this approach could be employed for the determination of small-molecular-weight organics such as cisplatin, daunomycin, bisphenol A, chlorinated phenols, and ethidium bromide. A detection limit in the magnitude of ca. 10 nM was achieved. This immobilization technique provides a generic approach for dsDNA-based sensor development and for the monitoring of DNA-analyte interactions.