A simple three-dimensional-focusing, continuous-flow mixer for the study of fast protein dynamics

Abstract

We present a simple, yet flexible microfluidic mixer with a demonstrated mixing time as short as 80 μs that is widely accessible because it is made of commercially available parts. To simplify the study of fast protein dynamics, we have developed an inexpensive continuous-flow microfluidic mixer, requiring no specialized equipment or techniques. The mixer uses three-dimensional, hydrodynamic focusing of a protein sample stream by a surrounding sheath solution to achieve rapid diffusional mixing between the sample and sheath. Mixing initiates the reaction of interest. Reactions can be spatially observed by fluorescence or absorbance spectroscopy. We characterized the pixel-to-time calibration and diffusional mixing experimentally. We achieved a mixing time as short as 80 μs. We studied the kinetics of horse apomyoglobin (apoMb) unfolding from the intermediate (I) state to its completely unfolded (U) state, induced by a pH jump from the initial pH of 4.5 in the sample stream to a final pH of 2.0 in the sheath solution. The reaction time was probed using the fluorescence of 1-anilinonaphthalene-8-sulfonate (1,8-ANS) bound to the folded protein. We observed unfolding of apoMb within 760 μs, without populating additional intermediate states under these conditions. We also studied the reaction kinetics of the conversion of pyruvate to lactate catalyzed by lactate dehydrogenase using the intrinsic tryptophan emission of the enzyme. We observe sub-millisecond kinetics that we attribute to Michaelis complex formation and loop domain closure. These results demonstrate the utility of the three-dimensional focusing mixer for biophysical studies of protein dynamics.

Figure 1

(A) Mixer design. The components include: (1) MicroCross that holds 360 µm outer diameter tubing and has a 150-µm through-hole (2) 1/32” PEEK tubing that has a 250 µm inner diameter and carries sheath flow from HPLC pumps (3) Fused silica capillary for sample flow: outer diameter = 90µm; inner diameter = 20µm (4) MicroTight tubing sleeve (5) Fused silica capillary for sheath flow: outer diameter = 350 µm; inner diameter = 200 µm. All parts are connected via fitting nuts and MicroFerrules with 0.025” diameters. (B) Overlay of bright field and fluorescence images of capillary flow system. The sample flows from left to right through the inner capillary, and the sheath solution flows from left to right through the outer capillary, with dimensions as indicated. The tapered capillary tip and 3D focusing by the sheath solution create a narrow sample jet that is 2–5 µm in diameter. A laminar flow of the sample is established and rapid mixing occurs via diffusion. The sample appears as a bright stream due to fluorescence of Eu beads. The sample flow rate is 0.025 µL/min, and the sheath flow rate is 100 µL/min. (C) Cutaway of MicroCross assembly showing how solutions are kept separate (not to scale). Ferrules (not shown) hold each tube or capillary in place. The sheath solution can only flow out through the outer capillary, whereas the inner capillary outlet is outside of the cross and therefore the mixing region is outside of the cross, in the vicinity of the microscope objective.

Figure 2

(A) Single-point laser excitation of Eu nanospheres flowing through the mixer. The top part of the image shows the excitation laser position and profile. The laser is focused to a narrow line perpendicular to the axis of sample flow. The bottom part of the image shows the nanosphere fluorescence as a white streak downstream of the line-focused laser. The nanospheres exit the inner capillary and are excited at the position of the focused laser, then as they move away from the excitation point, the intensity of their fluorescence decays at a known rate. (B) Mixer time resolution at various sheath flow rates. Experimental data (black circles) were fit to a single exponential function (red line): τ = 1.1 + 3.5e ^−3.5v. Also shown is the theoretical time resolution (blue dashes) based on Equation 2.

Figure 3

Fluorescence transients from the quenching of fluorecein fluorescence by mixing with potassium iodide at different sheath flow rates. Inset shows the observed mixing times as determined by exponential fits. Inset data were fit to a single exponential function: τ_mix = 82 + 530e ^−3.0·v.

Figure 4

Fluorescence transients from the rapid change in pH of apoMb from 4.5 to 2.0. Inset shows apoMb unfolding times as determined by exponential fits. The unfolding time decreases according to an exponential function: τ_unf = 760 + 80000e ^−8.8·v. The baseline indicates a flow rate independent unfolding time (I ↔ U) of 760 ± 8 µs.

Figure 5

Transient tryptophan fluorescence profile obtained from the rapid mixing of 200 µM pig heart lactate dehydrogenase/350 µM NADH with 1000 mM pyruvate. The observed increase in fluorescence intensity is due to a combination of enzyme conformational change and enzymatic turnover.