Team:ETH Zurich/Process/Microfluidics

We figured out relatively early that we need some kind of channel to establish the acetaldehyde or xylene gradient required for SmoColi (see Circuit Design). However, there were several different possible channel designs, and the final design evolved through an iterative series of design steps and design validations. The first designs were validated based on vast simulations, the final design furthermore by biological experiments in the lab (see Systems Validation).

Initial Channel Designs

Microfluidic channel with flow and recycling of the medium

We came up with two different possible microfluidic channel designs, both involving a flow of toxic molecule in medium through the channel:

• Variant 1: Plate with cells-in-agarose-filled cubic "pixels" and a microfluidic channel above. This channel design consists of two parts: A PDMS plate with regular cubic "pixels" that is filled with cells in agarose by letting the liquid cell-agarose solution flow over the pixels by gravity. The cell-agarose liquid is then cooled down and the solidified surplus cell-agarose gel scraped off. The second part is a larger PDMS channel, which is several pixels wide and several pixels high. It is then laid on top of the pixel structure and clamped tightly in order to create a seal. Although this setup is rather complex, it has the advantage of having immobilized cells and thus being more robust, i.e. we could vary flow speeds or even put aerosol in the channel without the cells being washed out. Additionally, cell density can be varied very easily in this design as the cells can be diluted before being immobilized in agarose.

Experimental setup for SmoColi.

Evaluation of the model.

• Variant 2: Microfluidic channel with cells sitting in pockets in the channel. This channel design only consists of one part: A PDMS channel that is fixed onto a glass carrier. The PDMS channel contains pockets which hold the cells given a constant flow from the direction of the "open end" of the pockets. This channel design does not have the advantages of the above one, i.e. it is not as robust and cell density cannot be reliably varied. However, manufacturing it is a standard process and thus is easier.

Experimental setup for SmoColi.

A problem with both of these designs is that for the AHL-based RFP alarm to work, recycling of the flow back into the channel would be required. Since the tubing and pumps would have very high volumes compared to the channel volume, the AHL signal would be diluted to the point where no detection is possible anymore. Also, several pumps would be required to accomplish this, further complicating the process design and making it more error-prone.

Improved microfluidic channel without flow

Experimental setup for SmoColi.

Modeling showed that diffusion and degradation of acetaldehyde/ xylene is enough to create a concentration gradient in the tube. Without a flow there is no need for a moving liquid and thus no need for any of the complex designs above, as one can simply fill the whole channel with cell-agarose liquid. We can then wait until the cell-agarose liquid solidifies to a gel and then connect one side of the channel to a reservoir with the toxic molecule while sealing the other. Likewise we do not need recycling because AHL can diffuse through the whole channel and does not have to diffuse against a flow.

Modeling the system thus had a profound effect on the process design, leading to an extensive reduction of complexity and error-proneness. Additionally, the AHL recycling idea would not have worked in the initial design, and was "saved" by the new channel design we developed by modeling our system.

Photo of the channel in action. The channel (the long thin tube at the right) is physically attached to a reservoir filled with the sample medium containing acetaldehyde or xylene (the Eppendorf tube at the lower left). In the case of acetaldehyde, the whole setup is packed in an impermeable plastic bag to significantly reduce the vaporization of acetaldehyde (not shown).

After the experiment, the agarose gel containing the cells is removed from the tubing. The bald interior of the channel is placed on a petri dish (see picture) and analyzed under a fluorescence microscope.

Further improving the channel material

With the tube live imaging is not possible, that´s why we develop our channel design further. To image the channel at the top and bottom of our channel a glas slide is needed. The channel itself we tried to build up by using a plastic mask and casting the boundaries with different materials. We tried Two-component glue, Silicon and molten parafilm. Two-component glue turned out to be toxic for our cells. Silicon and molten parafilm in contracts seen to be adequate for our channel.