July 7, 2019  |  

High-throughput platform for real-time monitoring of biological processes by multicolor single-molecule fluorescence.

Authors: Chen, Jin and Dalal, Ravindra V and Petrov, Alexey N and Tsai, Albert and O'Leary, Seán E and Chapin, Karen and Cheng, Janice and Ewan, Mark and Hsiung, Pei-Lin and Lundquist, Paul and Turner, Stephen W and Hsu, David R and Puglisi, Joseph D

Zero-mode waveguides provide a powerful technology for studying single-molecule real-time dynamics of biological systems at physiological ligand concentrations. We customized a commercial zero-mode waveguide-based DNA sequencer for use as a versatile instrument for single-molecule fluorescence detection and showed that the system provides long fluorophore lifetimes with good signal to noise and low spectral cross-talk. We then used a ribosomal translation assay to show real-time fluidic delivery during data acquisition, showing it is possible to follow the conformation and composition of thousands of single biomolecules simultaneously through four spectral channels. This instrument allows high-throughput multiplexed dynamics of single-molecule biological processes over long timescales. The instrumentation presented here has broad applications to single-molecule studies of biological systems and is easily accessible to the biophysical community.

Journal: Proceedings of the National Academy of Sciences of the United States of America
DOI: 10.1073/pnas.1315735111
Year: 2014

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