Visual Biochemistry: modular microfluidics enables kinetic insight from time-resolved cryo-EM

Abstract

Mechanistic understanding of biochemical reactions requires structural and kinetic characterization of the underlying chemical processes. However, no single experimental technique can provide this information in a broadly applicable manner and thus structural studies of static macromolecules are often complemented by biophysical analysis. Moreover, the common strategy of utilizing mutants or crosslinking probes to stabilize otherwise shortlived reaction intermediates is prone to trapping off-pathway artefacts and precludes determining the order of molecular events. To overcome these limitations and allow visualisation of biochemical processes at near-atomic spatial resolution and millisecond time scales, we developed a time-resolved sample preparation method for cryo-electron microscopy (trEM). We integrated a modular microfluidic device, featuring a 3D-mixing unit and a delay line of variable length, with a gas-assisted nozzle and motorised plunge-freeze set-up that enables automated, fast, and blot-free sample vitrification. This sample preparation not only preserves high-resolution structural detail but also substantially improves protein distribution across the vitreous ice. We validated the method by examining the formation of RecA filaments on single-stranded DNA. We could reliably visualise reaction intermediates of early filament growth across three orders of magnitude on sub-second timescales. Quantification of the trEM data allowed us to characterize the kinetics of RecA filament growth. The trEM method reported here is versatile, easy to reproduce and thus readily adaptable to a broad spectrum of fundamental questions in biology.

Mixing and jetting analysis using continuous flow microfluidic sample delivery devices

“…Therefore, by increasing the ow rate, uniform mixing can be achieved faster and at a point which is closer to the inlet (lower t(mix)) due to the increase in the amount of chaotic mixing. This agrees with the recent results reported by Maeots et al 42 demonstrating that higher mixing efficiencies can be achieved at higher liquid ow rates as shown by using Fig. 4, and the results of the mixing experiments, we can see that a cylindrical liquid jet with uniform mixing of the two solutions is achieved at ow rates higher than 100 mL min À1 and at medium gas ow rates between 126 and 162 mg min À1 .…”

A millisecond passive micromixer with low flow rate, low sample consumption and easy fabrication

“…With this design, the mixing will be improved by transverse Dean flows developed in curved channels due to the action of the centrifugal force. The principle is the following: in this circularly shaped channel, the flow will be bent in 3D, and the centrifugal force will pull the inner stream to the outer wall radially, forcing the fluid originally located close to the outer wall to move laterally inwards through the top and bottom of the channel based on the conserve mass principle 62 , 63 . The generated transverse flow causes an exponential growth of the interfacial area, which significantly improves the mixing.…”