N. Zijlstra: Rapid microfluidic dilution device for probing low-affinity biomolecular complexes with single-molecule spectroscopy

Single-molecule spectroscopy has developed into a powerful strategy for investigating biomolecular complexes on nanometer length scales, especially in combination with Förster resonance energy transfer (FRET). However, a major challenge in studying biomolecular complexes by single-molecule spectroscopy is that their affinity is often low, resulting in rapid dissociation at the exceedingly low concentrations required for single-molecule detection. Here, we demonstrate a solution to this problem with a microfluidic device capable of diluting a concentrated sample almost five orders of magnitude within ~5 ms, which can be easily combined with confocal single-molecule detection. During this talk, I will first introduce the basic concepts of microfluidics and the design considerations for microfluidic devices. Second, I will show two applications of the rapid dilution device, where we used single-molecule two-color and threecolor single-molecule FRET to study a low-affinity protein complex. I will show that we can transiently populate and study the structural properties of these complexes and quantify the dynamics of the dissociation process over a wide range of timescales.