Zero-mode waveguides for single-molecule analysis at high concentrations.
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.
Levene
M J
MJ
Applied and Engineering Physics, Graduate Program in Biochemistry, Molecular, and Cell Biology, Cornell University, Clark Hall, Ithaca, NY 14853, USA.
Korlach
J
J
Turner
S W
SW
Foquet
M
M
Craighead
H G
HG
Webb
W W
WW
D001671:Biochemistry / Q000295:instrumentation / Q000379:methods*; D003198:Computer Simulation; D003374:Coumarins; D004247:DNA / Q000096:biosynthesis; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D003845:Deoxycytosine Nucleotides / Q000378:metabolism*; D004058:Diffusion; D004800:Enzymes, Immobilized / Q000378:metabolism; D020342:Finite Element Analysis; D005453:Fluorescence; D005456:Fluorescent Dyes; D008024:Ligands; D008027:Light; D036103:Nanotechnology / Q000295:instrumentation; D055095:Optics and Photonics; D013050:Spectrometry, Fluorescence / Q000295:instrumentation / Q000379:methods*
D003374:Coumarins; D003845:Deoxycytosine Nucleotides; D004800:Enzymes, Immobilized; D005456:Fluorescent Dyes; D008024:Ligands; C024107:2'-deoxycytidine 5'-triphosphate; D004247:DNA; C030123:coumarin; C034970:bacteriophage T7 induced DNA polymerase; D004259:DNA-Directed DNA Polymerase