Microfluidic mixer designed for performing single-molecule kinetics with confocal detection on timescales from milliseconds to minutes

Abstract

Microfluidic mixing in combination with single-molecule spectroscopy allows the investigation of complex biomolecular processes under non-equilibrium conditions. Here we present a protocol for building, installing and operating microfluidic mixing devices optimized for this purpose. The mixer is fabricated by replica molding with polydimethylsiloxane (PDMS), which allows the production of large numbers of devices at a low cost using a single microfabricated silicon mold. The design is based on hydrodynamic focusing combined with diffusive mixing and allows single-molecule kinetics to be recorded over five orders of magnitude in time, from 1 ms to ∼100 s. Owing to microfabricated particle filters incorporated in the inlet channels, the devices provide stable flow for many hours to days without channel blockage, which allows reliable collection of high-quality data. Modular design enables rapid exchange of samples and mixing devices, which are mounted in a specifically designed holder for use with a confocal microscopy detection system. Integrated Peltier elements provide temperature control from 4 to 37 °C. The protocol includes the fabrication of a silicon master, production of the microfluidic devices, instrumentation setup and data acquisition. Once a silicon master is available, devices can be produced and experiments started within ∼1 d of preparation. We demonstrate the performance of the system with single-molecule Förster resonance energy transfer (FRET) measurements of kinetics of protein folding and conformational changes. The dead time of 1 ms, as predicted from finite element calculations, was confirmed by the measurements.

Integrated view of internal friction in unfolded proteins from single-molecule FRET, contact quenching, theory, and simulations

“…This process results in an approximately exponential decay in the fluorescence correlation curve, where the corresponding , it is thus necessary to quantify both c q and τ q , ideally under conditions where the protein is completely unfolded, so that f u does not affect the amplitude. Here, we used a microfluidic mixing device that allows us to dilute the protein rapidly from fully denaturing (3 M GdmCl) to native conditions (0.3 M GdmCl) or intermediate GdmCl concentrations with millisecond dead time (51), enabling the observation of unfolded-state dynamics for all conditions before the protein folds. Measurements of the FRETlabeled protein in the microfluidic device confirmed that the protein was predominantly unfolded at times less than 5 ms after dilution of the denaturant (SI Appendix and SI Appendix, Fig.…”

Single-molecule spectroscopy reveals chaperone-mediated expansion of substrate protein

“…Dual-focus fluorescence correlation spectroscopy measurements were performed at 22°C on a MicroTime 200 confocal microscope equipped with a differential interference contrast prism as described previously (29). For rapid mixing experiments, microfluidic mixers were fabricated and used as described previously (23). Molecular dynamics simulations of DnaKrhodanese complexes were performed with CafeMol 2.0 (43) based on a Cα representation of the rhodanese chain and a structure-based model of DnaK bound to the binding sites predicted by LIMBO (44).…”

“…1C, colored histograms). As a reference state in the absence of chaperones and denaturant, we thus transiently populate denatured rhodanese in a microfluidic mixing device by rapid dilution of guanidinium chloride (GdmCl) (19,(21)(22)(23). In contrast to native rhodanese, all denatured rhodanese variants exhibit a similar and high transfer efficiency of ∼0.8, which suggests the presence of a compact denatured state ensemble under these conditions (24).…”

“…To quantify the dynamics underlying this nonequilibrium process, we use a combination of manual and microfluidic mixing experiments, which allows us to monitor the process from milliseconds to hours. For measurements in the microfluidic device (19,21,23), preformed rhodanese-DnaJ complexes and ATP entering from the two side channels are rapidly mixed with DnaK in the main channel (Fig. 3A).…”

Soluble Oligomers of the Pore-forming Toxin Cytolysin A from Escherichia coli Are Off-pathway Products of Pore Assembly

“…The measurements were recorded on a modified MicroTime 200 instrument (PicoQuant) using a setup for pulsed interleaved excitation (PIE (26)) with two excitation pulses for the donor dye alternating with a single acceptor pulse. The initial phase of protomer formation was measured in a microfluidic mixer (27), and the later phase as well as the pore formation kinetics was constructed from repeated manual mixing measurements (Table 1). All measurements were done at a nominal concentration of labeled ClyACys of 300 pM in PBS with 0.001% (w/v) Tween 20 and 0.1% (w/v) DDM at 22°C.…”