Compact microfluidic structures for generating spatial and temporal gradients

Abstract

We present an improved microfluidic design for generating spatial and temporal gradients. The basic functional elements are bifurcated and trifurcated channels used to split flow between two and three channels, respectively. We use bifurcated channels on the exterior of the channel manifold and trifurcated channels in the interior with mixing tees to recombine flows. For N gradient-forming levels, the number of discrete steps in the gradient is 2(N) + 1, allowing a compact gradient-forming structure that is only 1.6 mm long and 0.5 mm wide. Control of the relative sample concentration at the inlets enables generation of gradients with varying slopes and offsets. The small total channel length allows faster switching (only 2.6 s) between gradients of different compositions than did previous designs, allowing complex temporal sequences and reducing total displacement volume and reagent use. The design permits opposing-gradient experiments and generation of complex nonlinear gradients. We fabricated and tested three channel designs with either three or four gradient-forming levels, 20- or 40-microm channel widths, 60- or 120-microm center-to-center channel spacings, and 9 or 17 output steps. These devices produced essentially identical high-quality linear gradients using both pressure-driven and electrokinetic flow.

Figure 1

Schematic of microfluidic device for generating controlled chemical gradients showing the inlets, gradient-forming region, and gradient chamber. Inlets 1 and 2 each have two input reservoirs, denoted a and b. See Table 1 for the parameters and Figure 2 for an enlarged view of the gradient-forming region.

Figure 2

Schematics of (a) gradient-forming region for device 3-20-60 (see Figure 3a), (b) bifurcated channel, (c) trifurcated channel, and (d) mixing tee. The gradient-forming region has three levels, L = 1–3. The channels have uniform cross section with lengths chosen to balance flow resistance.

Figure 3

(a) Transmitted-light image of the gradient-forming region for device 3-20-60 with three levels, 20-μm-wide channels, and 9 output channels with 60-μm center-to-center spacing. Fluorescence images of the gradient for pressure-driven flow with (b) 100% sample at inlet 1 and 0% sample at inlet 2 and (c) 0% sample at inlet 1 and 100% sample at inlet 2. The output channels have concentration steps of 12.5% from 100 to 0% in (b) and 0 to 100% in (c). The scale is the same in all images.

Figure 4

(a) Transmitted-light image of the gradient chamber for device 3-20-60. The chamber is 540 μm wide. Fluorescence images of the gradient for pressure-driven flow with (b) 100% sample at inlet 1 and 0% sample at inlet 2 and (c) 0% sample at inlet 1 and 100% sample at inlet 2. The inflow to the gradient chamber is in 12.5% increments from 100 to 0% in (b) and 0 to 100% in (c). The dashed line shows the position of the profiles in Figures 5, 6, and 8. The scale is the same in all images.

Figure 5

Gradient profiles (C/C₀) with varying (a) slopes and (b) offsets for device 3-20-60 with pressure-driven flow at l = 100 μm along the dashed line shown in Figure 4. Inputs from inlets 1 and 2 correspond to positions in the gradient chamber at 0 and 540 μm, respectively.

Figure 6

Transmitted-light images of the gradient-forming region for (a) device 3-40-120 and (b) device 4-20-60. Device 3-40-120 has three levels, 40-μm-wide channels, and 9 output channels with 120-μm center-to-center spacing, and device 4-20-60 has four levels, 20-μm-wide channels, and 17 output channels with 60-μm center-to-center spacing. The scale is the same in both images.

Figure 7

Gradient profiles for pressure-driven flow generated with devices 3-20-60, 3-40-120, and 4-20-60. See Table 1 for design parameters. The relative concentration of the sample (C/C₀) was measured at l = 100 μm for devices 3-20-60 and 4-20-60 and l = 400 μm for device 3-20-120. Inlet 1 was held at C/C₀ = 1.0, and inlet 2 at C/C₀ = 0. The 0-μm position marks the center of the chamber.

Figure 8

Gradient profiles at l = 100 μm along the dashed line shown in Figure 4 for device 3-20-60 with (a) pressure-driven and (b) electrokinetic flow. The gradients had a constant offset (C/C₀ = 0.5) and varying slopes. Inputs from inlets 1 and 2 correspond to positions in the gradient chamber at 0 and 540 μm, respectively.