Zero-Mode Waveguides: A Powerful Tool for Single-Molecule Optical Studies

Single-molecule fluorescence studies of enzymes that incorporate fluorescently labeled substrate nucleotides typically operate at substrate concentrations well below their Km values. While this is inevitable in conventional fluorescence microscopy, the biological relevance of the insights gained into enzyme mechanism may be compromised. Zero-mode waveguides (ZMWs) provide an excellent solution to this problem by greatly reducing the observation volume. We report the nanofabrication of ZMWs, the surface treatment for controlled immobilization of biomolecules and the reduction of background noise. We also present the development of an assay to monitor in real time the incorporation of fluorescently-labeled nucleotides, which paves the way for the studies of nucleic acid polymerizing enzymes, e.g. DNA/RNA polymerase, reverse transcriptase, telomerase, etc.