High-Resolution smFRET in a Microfluidic Gas Exchange Platform

Abstract

Single-molecule Forster Resonance Energy Transfer (smFRET) is a powerful technique providing new insights in the physics and chemistry of bio-molecules. The smFRET signal quality depends on the photon-flux that can be harvested from the fluorescent dyes. Mere use of high excitation intensities produces counter-productive results, mainly because oxygen-mediated photobleaching of the fluorophores rapidly destroys useful FRET signals. Here, we developed a microfluidic device that ensures a large decrease in the oxygen content of buffers in-situ, allowing us to substantially reduce photobleaching even at high laser powers and obtain dramatic enhancement of signal for several dye-pairs. The principle of the deoxygenation is simple: the microchannels carrying the samples are flanked by large channels ventilated by nitrogen; oxygen is removed by molecular diffusion through porous walls. The device combines this deoxygenation with on-chip mixing and generation of dual-component triplet quenchers (increasing the burst brightness), and also has laminar-flow mixing for kinetic studies.The increased photon flux obtained in the flow device leads to resolution improvements in two key dimensions: (i) it reduces considerably the time (by a factor of ∼ 10 from the typical 500μs) needed to collect high-quality FRET signal, thus providing higher time-resolution, and (ii) it allows the use of higher thresholds, which significantly reduces the width of the smFRET distributions and allows a better resolution of molecular subpopulations. In contrast with a popular enzymatic oxygen scavenger, the method can be used for de-oxygenation in denaturing conditions, hence enabling studies of protein folding. Overall, the platform combines multiple enabling features that can accommodate a range of equilibrium and kinetic and biochemical experiments, along with a simple construction and fabrication, and robust operation. Together, these features have the potential to make it useful for a broad variety of single-molecule fluorescence experiments.