Zero-Mode Waveguides for Real-Time Observation of Single Nucleotide Incorporation

Single-molecule fluorescence studies on the incorporation of fluorescently labeled nucleotides by DNA polymerizing enzymes typically operate at nucleotide concentrations well below their Km values. While this is inevitable given the femtoliter observation volumes accessed via conventional fluorescence microscopy, the biological relevance of the insights gained into enzymatic kinetics may be compromised. Zero-mode waveguides (ZMWs), sub-wavelength holes in a thin metal film, provide an excellent solution to this problem by greatly reducing the observation volume [M. J. Levene, et al., 2003, Science 299, 682-686].We have successfully designed and fabricated ZMWs of about 100 nm in width. In addition we have developed a surface treatment protocol based on PEG functionalization to make the ZMWs biocompatible, and to facilitate the controlled tethering of biomolecules [A. Crut, et al., Nanotechnology, in press (2008)]. In these structures, we have observed the real-time observation of single nucleotide incorporation at biologically-relevant concentrations in ZMWs, The latest scientific results will be presented.