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Detection and identification of nucleic acid engineered fluorescent labels in submicrometre fluidic channels

Samuel M Stavis1,3, Joshua B Edel1,3, Yougen Li2, Kevan T Samiee1, Dan Luo2 and Harold G Craighead1,4

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Nucleic acid engineers have created nanoscale fluorescent labels that are uniquely identifiable by the number of conjugated fluorophores, and with binding characteristics that permit recognition of individual specific biomolecules. The viability of this technology for use in multi-analyte homogeneous assays depends on the ability to optically detect individual labels, and distinguish the fluorescence emission of each label. We describe the use of fluidic channels with submicrometre dimensions to rapidly detect individual labels in solution. Labels with small differences in fluorophore composition were differentiated with varying degrees of accuracy. Labels were synthesized at the molecular level from dendrimer-like DNA, with the identity encoded into the number of Alexa Fluor 488 and BODIPY 630/650 fluorophores conjugated with the structure. To explore the decoding resolution limit, labels with a single fluorophore of each colour were detected, and were found to be distinguishable as a group, but not individually, from labels with one additional red fluorophore. Labels with one green and three red fluorophores were individually distinguishable with greater than 80% accuracy from labels with one red and three green fluorophores. Photon counting histograms were analysed to differentiate the various labels, and fluorescence correlation spectroscopy was used to measure their mobilities. Fluidic channels were fabricated in fused silica with a 500 nm square cross section, resulting in a focal volume of approximately 500 al. Because the entire channel width was illuminated, every fluorescent molecule in solution passing through the channel was uniformly excited and analyzed. Flow control enabled a balance of rapid data acquisition and efficient fluorescence collection with these nanoscale systems.


PACS

87.85.Lf Tissue engineering

87.85.Qr Nanotechnologies-design

87.15.B- Structure of biomolecules

85.85.+j Micro- and nano-electromechanical systems (MEMS/NEMS) and devices

Subjects

Electronics and devices

Biological physics

Nanoscale science and low-D systems

Dates

Issue 7 (July 2005)

Received 7 January 2005, in final form 1 March 2005

Published 15 April 2005



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