Team:TU Eindhoven/Microfluidics/Results


iGEM Team TU Eindhoven 2014

iGEM Team TU Eindhoven 2014

Results Droplet Device

Before the droplets were formed, the injected fluids had to be stabilized and had to be controlled until both the oil and continuous phase appeared at the flow-focusing cross junction. If this is not done properly, air may be trapped inside the device which will lead to incorrect droplet formation. If the flow rate of the oil phase is too high in comparison with the continuous phase, the oil phase will enter the continuous inlet. This also occurs vice versa. It was necessary to test different flow rates of both the continuous and oil phase. Due to the viscosity of the fluids and the resistance of the walls of the channels, the injection speed was not exactly the same as the actual speed of the flows. The size of the droplets could be changed by alternating the flow rates of the oil and continuous phase. When the droplets were formed, it was possible to measure the size and the formation frequency of the droplets. Droplet formation can be seen in the video below.

Figure 1. Microscope image of polyacrylamide droplets. The sample
was put on a microscope glass with a cover glass on top.

Polyacrylamide Droplets

First tests were performed to form polyacrylamide droplets. The continuous phase consists of a solution of N,N’-methylenebisacrylamide (bisacrylamide), acrylamide monomers and ammonium persulfate. The oil phase contains N,N,N’,N’-tetramethylethylenediamine (TEMED) and a surfactant in HFE-7500. The surfactant prevents coalescence of the droplets and makes them more stable. TEMED will diffuse to the continuous phase inside the droplets because of its better solubility in water. It acts as a catalyst activating ammonium persulfate by forming free radicals. These free radicals in turn interact with the acrylamide monomers. The bisacrylamide also turns into free radicals. This mechanism continues until a network of polyacrylamide is formed with random cross-linkers of bisacrylamide in between.

Figure 2. Histogram of droplet size. Total Droplet count is 200.

A flow ratio oil:continuous of 13:1 µL/min was used to obtain droplets as seen in Figure 1. A histogram (Figure 2) of another test showed the mean size of the droplets was 18 µm.

After samples were collected, the polyacrylamide droplets were separated from the oil phase. This was done by first adding a 20% v/v solution of perfluorooctane (PFO) (Figure 3). This breaks and gets rid of the surfactant. The polymerized droplets float freely in the oil.

Figure 3. Polyacrylamide droplets after treatment with PFO (left). And on the right the sample has been spun.
Figure 4. Polyacrylamide droplets after completion of the protocol.

To separate the oil, the sample was spun to create a pellet of polymerized droplets. However, this caused to break the droplets completely as can be seen in Figure 3 on the right. Further treatment of the sample was unnecessary as the sample was already broken down. Upon completion of the protocol droplet separation much of the sample was lost (Figure 4).

To find out at which step the sample was damaged to the extent droplets were broken down, different photos were taken at different steps of the protocol to look at the effects of the chemicals (Figure 5).

As can be seen in the top left image (Figure 5) there are polymerized droplets visible in our sample. As stated before, adding PFO causes to break the surfactant and let the droplets freely float into the oil. Hereto a solution of span80 in hexane was added. Hexane is miscible with oil but immiscible with water. Span80 makes the droplets disperse evenly. However, after addition of Span80 the droplets appeared to coagulate as can be seen in Figure 5 at the bottom left. The bottom right shows addition of PFO after span80. As before, the droplets also started to coagulate in this case. Final steps of the protocol were not further analyzed at this point because it involved addition of TEBST buffer. This is used as a physiological buffer when droplet separation is performed on cell containing droplets. Also due to the fact that a new design was made where droplets could simply be analyzed in a droplet chamber this was of no further interest.

Figure 5. Photos at different steps of the protocol of droplet separation. On the top left: the original sample without addition of chemicals. Top right: addition of 20% PFO to the sample. Bottom left: addition of 20% PFO and Span80 to the sample. Bottom right: addition of Span80 to the sample.
iGEM Team TU Eindhoven 2014