Enzyme-modulated cleavage of dsDNA for studying interfacial biomolecular interactions

This work describes the chemistry and methodology for constructing multilay ers of bis-biotinylated dsDNA on metal substrates after enzyme cleavage and demonstrates its use for amplified microgravimetric and impedimetric analy ses of anticancer drug, cisplatin. Specific chemical modification of dsDNA prior to immobilization was achieved via a bisulfite-catalyzed transaminati on of cytosine after endonuclease cleavage of plasmid DNA. The specificity of the reaction of cytosine residues at ss- versus dsDNA loci after endonuc lease cleavage was characterized using circular dichroism, mass spectrometr y, and absorption spectrophotometry. The biotinylated dsDNA consisting of 2 961 base pairs was then used as a ligand at avidin-modified gold electrodes . Ac impedance spectroscopy and quartz crystal microbalance measurements cl early showed that the response to cisplatin increased linearly with target concentrations. The impedance spectroscopy resulted in a detection limit of 1 nM and a surface density of 4.8 x 10(13) molecules/0.1 cm(2). The immobi lization of dsDNA on surfaces is a significant improvement over existing ap proaches in that it enables the attachment of long pieces of unmodified dou ble-stranded DNA via a simple biotinylation step, The immobilization techni que provides a generic approach for dsDNA-based sensor development and for monitoring DNA-analyte interactions.