5 years ago

Rapid Microfluidic Dilution for Single-Molecule Spectroscopy of Low-Affinity Biomolecular Complexes

Rapid Microfluidic Dilution for Single-Molecule Spectroscopy of Low-Affinity Biomolecular Complexes
Stephan Benke, Fabian Dingfelder, Niels Zijlstra, Bengt Wunderlich, Franziska Zosel, Daniel Nettels, Benjamin Schuler
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 μm, 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. Rapid microfluidic dilution: A novel microfluidic device for single-molecule spectroscopy capable of diluting a concentrated sample by almost five orders of magnitude in milliseconds has been developed. This device can be used to transiently populate and study the structural properties of low-affinity complexes and to quantify the dynamics of the dissociation process over a wide range of timescales.

Publisher URL: http://onlinelibrary.wiley.com/resolve/doi

DOI: 10.1002/anie.201702439

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