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Rapid Microfluidic Dilution for Single‐Molecule Spectroscopy of Low‐Affinity Biomolecular Complexes
Author(s) -
Zijlstra Niels,
Dingfelder Fabian,
Wunderlich Bengt,
Zosel Franziska,
Benke Stephan,
Nettels Daniel,
Schuler Benjamin
Publication year - 2017
Publication title -
angewandte chemie international edition
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.831
H-Index - 550
eISSN - 1521-3773
pISSN - 1433-7851
DOI - 10.1002/anie.201702439
Subject(s) - förster resonance energy transfer , microfluidics , molecule , dissociation (chemistry) , chemistry , nanotechnology , spectroscopy , fabrication , reproducibility , dilution , kinetics , chemical physics , analytical chemistry (journal) , materials science , fluorescence , chromatography , organic chemistry , medicine , physics , alternative medicine , pathology , quantum mechanics , thermodynamics
To enable the investigation of low‐affinity biomolecular complexes with confocal single‐molecule spectroscopy, we have developed a microfluidic device that allows a concentrated sample to be diluted by up to five orders of magnitude within milliseconds, at the physical limit dictated by diffusion. We demonstrate the capabilities of the device by studying the dissociation kinetics and structural properties of low‐affinity protein complexes using single‐molecule two‐color and three‐color Förster resonance energy transfer (FRET). We show that the versatility of the device makes it suitable for studying complexes with dissociation constants from low nanomolar up to 10 μ m , thus covering a wide range of biomolecular interactions. The design and precise fabrication of the devices ensure simple yet reliable operation and high reproducibility of the results.