Polymeric microfluidic continuous flow mixer combined with hyperspectral FT-IR imaging for studying rapid...

Early reaction intermediates in protein folding, such as those resulting in β-amyloid formation due to transient misfolding, emerge within a few hundred microseconds. Here, we report a method to obtain sub-millisecond temporal resolution and molecular structural information of protein (mis-)folding events by using a microfluidic continuous-flow mixer (MCFM) in combination with Fourier transform infrared (FT-IR) imaging. The MCFMs are made out of cyclic olefin copolymer (COC) films, because this approach allows for rapid prototyping of different mixer designs. Furthermore, COC offers high IR transparency between 1500 and 2500 cm^-1, thus maximizing the signal to noise ratio of the IR data obtained from a sample of interest. By combining narrow and wide channel widths in MCFM design, the platform provides fast mixing (460 μs) to induce protein (mis-)folding, and it maximizes the residence time in the observing area, so a wide range of reaction timescales can be captured in a single image. We validated the platform for its ability to induce and observe sub-millisecond processes by studying two systems: (i) the mixing of H₂O and D₂O and (ii) the mixing induced deprotonation of carboxylic acid. First, we observed excellent agreement between simulated and experimental data of the on-chip mixing of H₂O and D₂O, which verifies the distance-reaction time relationships based on simulation. Second, deprotonation of carboxylic acid by on-chip mixing with sodium hydroxide solution validates the ability of the platform to induce rapid pH jump that is needed for some biomolecular reactions. Finally, we studied the methanol-induced partial-unfolding of ubiquitin to show that our platform can be used to study biomolecular events 'on-pathway' using FT-IR imaging. We successfully extracted kinetic and structural details of the conformational changes along the channel. Our results are in agreement with prior studies that required more elaborate stopped flow approaches to acquire data for different time points. In summary, the reported method uses an easy-to-fabricate microfluidic mixer platform integrated with hyperspectral FT-IR imaging for rapid acquisition of structural details and kinetic parameters of biomolecular reactions. This approach does not need stopped flow or molecular imaging probes, as required respectively for alternative FT-IR spectroscopy and fluorescence approaches.