Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, 8057, Zurich, Switzerland.
Department of Physics, University of Zurich, Winterthurerstrasse 190, 8057, Zurich, Switzerland.
Angew Chem Int Ed Engl. 2017 Jun 12;56(25):7126-7129. doi: 10.1002/anie.201702439. Epub 2017 May 16.
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.
为了能够使用共焦单分子光谱技术研究低亲和力生物分子复合物，我们开发了一种微流控装置，该装置可在毫秒级内将浓度样品稀释五个数量级，达到由扩散决定的物理极限。我们通过使用单分子双色和三色Förster 共振能量转移（FRET）研究低亲和力蛋白质复合物的离解动力学和结构特性，展示了该装置的功能。我们表明，该装置的多功能性使其适合研究解离常数从低纳摩尔到 10 μm 的复合物，从而涵盖了广泛的生物分子相互作用。该装置的设计和精确制造确保了简单但可靠的操作和结果的高度重现性。
