Millisecond kinetics on a microfluidic chip using nanoliters of reagents

Abstract

This paper describes a microfluidic chip for performing kinetic measurements with better than millisecond resolution. Rapid kinetic measurements in microfluidic systems are complicated by two problems: mixing is slow and dispersion is large. These problems also complicate biochemical assays performed in microfluidic chips. We have recently shown (Song, H.; Tice, J. D.; Ismagilov, R. F. Angew. Chem., Int. Ed. 2003, 42, 768-772) how multiphase fluid flow in microchannels can be used to address both problems by transporting the reagents inside aqueous droplets (plugs) surrounded by an immiscible fluid. Here, this droplet-based microfluidic system was used to extract kinetic parameters of an enzymatic reaction. Rapid single-turnover kinetics of ribonuclease A (RNase A) was measured with better than millisecond resolution using sub-microliter volumes of solutions. To obtain the single-turnover rate constant (k = 1100 +/- 250 s(-1)), four new features for this microfluidics platform were demonstrated: (i) rapid on-chip dilution, (ii) multiple time range access, (iii) biocompatibility with RNase A, and (iv) explicit treatment of mixing for improving time resolution of the system. These features are discussed using kinetics of RNase A. From fluorescent images integrated for 2-4 s, each kinetic profile can be obtained using less than 150 nL of solutions of reagents because this system relies on chaotic advection inside moving droplets rather than on turbulence to achieve rapid mixing. Fabrication of these devices in PDMS is straightforward and no specialized equipment, except for a standard microscope with a CCD camera, is needed to run the experiments. This microfluidic platform could serve as an inexpensive and economical complement to stopped-flow methods for a broad range of time-resolved experiments and assays in chemistry and biochemistry.

Figure 1

Photograph of a PDMS microfluidic system next to a U.S. penny. The three adjacent Teflon needles were each filled with 2 μL of blue solution, sufficient to measure ~15 complete kinetic profiles on millisecond scale. The clear Teflon needle was filled with the fluorinated carrier fluid.

Figure 2

On-chip dilution was accomplished by varying the flow rates of the reagents. (a) A schematic of the microfluidic network. The blue rectangle outlines the field of view for images shown in parts c and d. (b) A graph quantifying this dilution method by measuring fluorescence of a solution of fluorescein diluted in plugs in the microchannel. Data are shown for 80 experiments in which fluorescein was flowed through one of the three inlets, where C_measured and C_theoretical [μM] are measured and expected fluorescein concentration. (c) and (d) Microphotographs illustrating this dilution method with streams of food dyes. Carrier fluid was flowed at 60 nL/s.

Figure 3

Figure 3. Design of microchannels for exponential kinetics. (a) A schematic of the microfluidic network. The blue rectangle outlines the field of view for the image shown in part b and • indicates time points, t_n [s]. The time interval Δt_n was defined as shown. Within the field of view, kinetic data were obtained at t_n for a unit length l [m] and flow velocity U [m/s]. (b) Left: a false-color fluorescence microphotograph (4 s exposure; sample consumption was 33 nL/s) within the field of view. The dashed white lines trace the walls of the microchannel. Right: an intensity profile across the region of the microphotograph indicated by the red arrow. Left axis shows time points t_n, and right axis shows time intervals Δt_n.

Figure 4

Measuring exponential kinetics of RNase A and Selwyn’s test. (a) Graph of experimental data (obtained from images such as that shown in Figure 3b) for 3.3 μM substrate at three RNase A concentrations (• 0.3 μM, ▪0.2 μM, ▴ 0.04 μM). (b) Graph of Selwyn’s test from experimental data in part a. Superimposable plots show absence of RNase A denaturation or product inhibition.

Figure 5

Measuring single-turnover kinetics of RNase A. (a) Left: a schematic of the microfluidic network. Right: a false-color fluorescence microphotograph (2 s exposure showing time-averaged fluorescence intensity of moving plugs and oil; sample consumption was 33 nL/s). The dashed white lines trace the walls of the microchannel. (b) Graph of reaction progress at a pH of 7.5. Shown are experimental kinetic data (left axis) for three substrate concentrations (• 5.8 μM, ▪ 3.3 μM, ▴ 0.8 μM) obtained from images such as that shown in part a with fits of the reaction progress (solid lines). Also shown is a mixing curve using the Fluo-4/Ca²⁺ system (right axis, ▽ in the same microfluidic device with fit (dashed line) of an explicit mixing function). (c) Graph of reaction progress at pH of 6.0. Shown are experimental kinetic data (left axis) for three substrate concentrations (• 5.8 μM, ▪ 3.3 μM, ♦ 1.6 μM) with fits of the reaction progress (solid lines). Also shown is the same mixing curve as in part b.