期刊
ANGEWANDTE CHEMIE-INTERNATIONAL EDITION
卷 56, 期 25, 页码 7126-7129出版社
WILEY-V C H VERLAG GMBH
DOI: 10.1002/anie.201702439
关键词
FRET; intrinsically disordered proteins; microfluidics; protein-protein interactions; single-molecule studies
资金
- Swiss National Science Foundation [205321_149624, 31003A_152839]
- Postdoctoral Fellowship of the Forschungskredit of the University of Zurich [FK-16-049]
- Swiss National Science Foundation (SNF) [31003A_152839, 205321_149624] Funding Source: Swiss National Science Foundation (SNF)
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 Forster 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 mm, 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.
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