Novel Detection Method for DNA Hybridization Based on Molecular Spotlighting

Tina H. Huang, Michael J. Tarlov
Process Sensing Group
Process Measurement Division

A new platform for monitoring hybridization of thiol-derivatized DNA probes (SH-DNA) immobilized on gold surfaces is currently being developed. The gold surface is modified with mixed monolayers of the SH-DNA probes and 6-mercaptohexanol. In fluorescence-based detection scheme, the fluorophore is typically attached to the "target" DNA. In our investigation, the "probe" DNA is tagged with the fluorophore instead. The detection principle is based on the dramatic structural changes between single- (ss-) and double-stranded (ds-) DNAs. Prior to hybridization, the fluorophore is expected to be in close proximity to the gold surface, resulting in the quenching of the fluorescence signal. Upon hybridization, the dsDNA adopts a rod-like structure that extends the fluorophore away from the gold surface. With the fluorphores located further from the gold surface, quenching is reduced and an increase in fluorescence intensity is observed. Parameters that affect fluorescence signal are examined and discussed.