Simultaneous generation of multiple aqueous droplets in a microfluidic device

Abstract

This paper describes a microfluidic platform for the on-demand generation of multiple aqueous droplets, with varying chemical contents or chemical concentrations, for use in droplet based experiments. This generation technique was developed as a complement to existing techniques of continuous-flow (streaming) and discrete-droplet generation by enabling the formation of multiple discrete droplets simultaneously. Here sets of droplets with varying chemical contents can be generated without running the risk of cross-contamination due to the isolated nature of each supply inlet. The use of pressure pulses to generate droplets in parallel is described, and the effect of droplet size is examined in the context of flow rates and surfactant concentrations. To illustrate this technique, an array of different dye-containing droplets was generated, as well as a set of droplets that displayed a concentration gradient of a fluorescent dye.

Figure 1

Schematic of microfluidic designs. (a) shows the design for generation of droplets with differing contents, while (b) shows the design for generating droplets with a gradient of chemical contents. Five syringes are intended for use with design (a), while two syringes are intended for design (b). For both designs, the aqueous-phase channels are 100 μm in width, while the oil phase channel is 750 μm wide. The system is fabricated to a height of 50 μm, except for the 7 μm wide and 100 μm long nozzles shown in the inset, which are 7 μm in height.

Figure 2

Effects of surfactant concentration on droplet formation. The concentration of Span 80 was increased from (a) 0.01%, (b) 0.05%, (c) 0.1%, (d) 0.2%, to (e) 1% in AS 4 silicone oil. A nine-outlet gradient-generator design was used. The oil-flow program used had a pulse magnitude of 350 μL min⁻¹. The scale bar is 100 μm.

Figure 3

Droplet formation using programmed pulsed oil flow. (a) Schematic of oil flow controlled using a programmable syringe pump. The pulse illustrated has the following steps: i) infuse (pump pushes the oil phase) for 1 second at 250 μL min⁻¹, ii) withdraw (pump pulls the oil phase) for 1 second at 250 μL min⁻¹, and iii) infuse for 5 seconds at 4 μL min⁻¹. (b) Schematic illustrating the corresponding movements of oil at the nozzle. (c) Sequence of images showing droplet formation using the oil-flow pulse in panel a. A nine-outlet gradient-generator was used, with the aqueous phase being 10 μM fluorescein in 1 mM phosphate; the oil phase was 0.1% Span 80 in AR 20. The white arrows point to the location of the water/oil interface. The scale bar for the panels is 50 μm.

Figure 4

Droplet formation as a function of applied pressure by way of oil-phase pulse magnitude. The applied pressure ranged from 190 kPa to 310 kPa. The corresponding pulse magnitudes are shown in the figure inset, with 190 kPa resulting from 250 μL min⁻¹ pulse and 310 kPa from 400 μL min⁻¹ pulse. A nine-outlet gradient-generator design was used. The aqueous phase was 10 μM fluorescein in 1 mM phosphate; the oil phase was 0.1% Span 80 in AR 20 silicone oil. The scale bar is 100 μm and applies to all panels.

Figure 5

(a) Simultaneous generation of multiple droplets containing different dye solutions, for which a five-outlet parallel-channel design was used. (b) Linescan of the normalized intensity across five channels seen in the inset, which consisted of a concentration gradient of fluorescein, made with a five-outlet gradient-generator design. The inset is a fluorescence image of five droplets formed using the gradient device, with 5 and 0 μM fluorescein in 1 mM phosphate and 0.125 w/w% PEG being introduced into the two inlets of the gradient-generation network. Oil phase was a 1:1 mix of 50 and 100 cSt silicone oil with 0.1% Span 80. Scale bar is 65 μm. The channel and droplet that contained 0 μM fluorescein was traced with dotted lines to aid visualization. (c) Plot of the average normalized intensity for each droplet. (d) Plots of normalized fluorescence intensities as a function of time for droplets containing different concentrations of esterase. The percent esterase refers to the concentration of each esterase solution with respect to the stock solution (0.33mg mL⁻¹ esterase dissolved in 1X PBS buffer at pH 7.4), and thus 75% esterase denotes a concentration that is 75% of the stock solution: ◆: 75% esterase; ●: 50% esterase; ■: 25% esterase; ▲: buffer. The buffer solution contained the same amount of substrate but with no esterase. The substrate was dissolved in both the aqueous solution and oil solution, since it has comparable solubility in both solutions. Oil phase was a 1:1 mix of 50 and 100 cSt silicone oil with 0.1% Span 80. The inset shows a schematic of the channel layout as well as an image of each type of droplet. The scale bar for the images represents 75 μm.