Silane treatment: If the PDMS is hard to be peeled off from the wafer and/or removes the photoresist, this is likely a result of ineffective silane treatment. This is possily a result of either silane that has been left open and exposed to the ambient for an extended period of time, or an insufficiently strong vacuum. Test the vacuum desiccator to insure that it is pulling a strong vacuum, and if the silane is believed to be significantly oxidized, purchase new silane.
Bonding issue: If the bonds between layers in the microfluidic device do not appear sufficiently strong, either causing leakage or is non-conforming, there could be several culprits. If the layers thermo-bonded are delaminating, it is possible that one or all of the layers are overcured. Repeat the fabrication process but bake the thin, spin-coated layer for a few minutes less. If the bond between layers that are plasma activated is weak, there could be several causes. If the plasma chamber is not as clean, the plasma activation process may not work as properly. Clean according to manufacturer’s recommendations and test by bonding blank pieces of PDMS to each other or to glass. Properly bonded PDMS should be very difficult to remove. Additionally, evaluate the protocol and be sure that the bonding surfaces are clean and are not touched by gloves or tweezers. You may need to vary the plasma treatment time to find an optimal condition.
Alignment: Proper alignment is critical. If the device is offset slightly the partial closure valves will operate slightly differently and change the hydrodynamic resistance. To facilitate good alignment, we use a ring-LED light source connected to a stereomicroscope, and perform bonding at high magnification. Your Cleanroom may have equipment (e.g. aligners) that may facilitate these processes.
Leakage: Leakage typically results from one of two sources: bonding issues, or a poor seal between the device and connected tubing/pins. If the cause seems to be bonding, see the bonding troubleshooting entry. If the problem appears to be a result of a bad seal between the connected pins and the device, it could be as a result of either incorrectly sized pins or a tearing around the holes in the device. The silicone material of the device is used to form a strong seal around pins and tubing connected to it. When the holes in the device are punched, however, it is possible to create small tears around the hole that prevent a good seal from forming. If this is the case, make sure to take care when punching holes, and possibly sharpen the pins used for punching holes in the device.
Cooling: If animals do not appear to be immobilized, the temperature on chip may not be sufficiently low. To find the optimal temperature for the coolant, measure the coolant temperature before arriving on chip and optimize the power input to the Peltier. An optimal starting point is ~3 °C, but the exact condition also depends on the room temperature and the flow rates of the coolant and the animal suspension.
Device clogging: When operating the device, occasionally multiple animals or a piece of debris may clog the channels. Increase the pressure that drives the flow to push the animals and debris out of the channel. Some animals may be lost in that process, so depending on whether more false positives or false negatives can be tolerated, open either the sorted sample outlet or the waste outlet.