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Rapid microfluidic dilution for single-molecule spectroscopy of low-affinity biomolecular complexes


Zijlstra, Niels; Dingfelder, Fabian; Wunderlich, Bengt; Zosel, Franziska; Benke, Stephan; Nettels, Daniel; Schuler, Benjamin (2017). Rapid microfluidic dilution for single-molecule spectroscopy of low-affinity biomolecular complexes. Angewandte Chemie Internationale Edition, 56(25):7126-7129.

Abstract

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.

Abstract

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.

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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > Department of Biochemistry
07 Faculty of Science > Department of Biochemistry
Dewey Decimal Classification:570 Life sciences; biology
610 Medicine & health
Uncontrolled Keywords:General Chemistry, Catalysis
Language:English
Date:12 June 2017
Deposited On:07 Aug 2017 13:47
Last Modified:19 Aug 2018 09:45
Publisher:Wiley-VCH Verlag
ISSN:1433-7851
Additional Information:For accepted manuscripts: This is the peer reviewed version of the following article: N. Zijlstra, F. Dingfelder, B. Wunderlich, F. Zosel, S. Benke, D. Nettels, B. Schuler, Angew. Chem. Int. Ed. 2017, 56, 7126., which has been published in final form at https://doi.org/10.1002/anie.201702439. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving (http://olabout.wiley.com/WileyCDA/Section/id-820227.html#terms).
OA Status:Green
Publisher DOI:https://doi.org/10.1002/anie.201702439
PubMed ID:28510311

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