Determining the Optimal Flow Rate
With a preliminary experiment together with a calculation of the channel volume, we determined the time it takes for the fresh medium to arrive at image bacteria. This depends, of course, on the flow rate with which the medium is sucked in from the reservoir. This information is crucial to fine tune the sucking pump for an optimized medium exchange. In the chemotaxis approach (Approach A, for which we designed the whole set-up), the bacteria are tethered to the glass surface at the bottom of the chip. There, the medium exchange has to be done in a fast manner to capture the full response of the bacteria to the concentration change, but still slow enough to not flush away the bacteria. The ideal flow rate turned out to be 10 μL/min.
Microfluidic chips have the advantage of a simple and straightforward fabrication procedure. The material used for this is Polydimethylsiloxane (PDMS). It is transparent, which makes it ideal for our imaging application. PDMS is liquid at room temperature, but after mixing it with a curing agent and heating it up, it will solidify. Therefore, liquid PDMS can be applied to a wafer, which acts as a negative template of the final structure of the chip. After the heating step, the solid PDMS is plasma-activated and applied onto a thin glass slide to cover the channels. To create an inlet and outlet opening for the reservoir and the tubing, small holes are punched into the chip. The reservoir can either be glued onto the chip or one can use a pipette tip and stick it into the hole. The tubes are connected by just pressing them into the hole. The simplicity of microfluidics is crucial, since it allows high modularity for our biosensor. Hence, once our imaging hardware is set up, the microfluidic chip containing the bacteria is the only part, which has to be exchanged for the adaptation of the biosensor to another ligand.