Zero-Mode Waveguides for Single-Molecule Analysis at High Concentrations

Levene, M. J.
Korlach, J.
Turner, S. W.
Foquet, M.
Craighead, H. G.
Webb, W. W.

¹Applied and Engineering Physics, ²Graduate Program in Biochemistry, Molecular, and Cell Biology, Cornell University, Clark Hall, Ithaca, NY 14853, USA.

*Present address: Nanofluidics Incorporated, 17 Sheraton Drive, Ithaca, NY 14850, USA.

†To whom correspondence should be addressed. E-mail: [email protected]

Supporting Online Material: www.sciencemag.org/cgi/content/full/299/5607/682/DC1; Materials and Methods; SOM Text; Figs. S1 and S2; Movies S1 and S2

23 October 2002; accepted 12 December 2002

Science 299(5607):p 682-686, January 31, 2003.

Optical approaches for observing the dynamics of single molecules have required pico- to nanomolar concentrations of fluorophore in order to isolate individual molecules. However, many biologically relevant processes occur at micromolar ligand concentrations, necessitating a reduction in the conventional observation volume by three orders of magnitude. We show that arrays of zero-mode waveguides consisting of subwavelength holes in a metal film provide a simple and highly parallel means for studying single-molecule dynamics at micromolar concentrations with microsecond temporal resolution. We present observations of DNA polymerase activity as an example of the effectiveness of zero-mode waveguides for performing single-molecule experiments at high concentrations.