BBB Device V2.4
I have created a successfully sealed flow chamber with this current device (V2.4), with the caveat that I sealed off the top chamber and was unable to test leaking from the top to the bottom.
Again, the dialysis chips from Dean’s work were used, as well as the same bulk microscope slide electrodes.
Improvements over V2.3:
– Fabricate Thin coverslip electrodes using URnano. The slides should be precisely cut to better match the gasket
I wasn’t able to finish these in time. These are the same as in V2.3. I have currently sputtered my own ITO on these thin coverslips, I just need to do lithography and etch
– Make the insterstitial, electrode gaskets slightly wider than the design to allow for better seating of the elements into the gaskets
Done. It’s precise enough to create a tight fit such that UV-ozone appears to seal it off.
– Keep layers clean using silicone gasket backing to make jigs, keep surfaces adhered if not being bonded at that point.
Done. It makes a big difference in terms of cleanliness. The backing plastic is too sticky though; I want the elements to slide off of the jig and bond to each other, not stay on the jig.
– Make fluidic vias through the film stack larger to allow for alignment error.
Done, increased from 1 to 2mm diameter circles. Makes a huge difference in terms of alignment.
– Determine whether PDMS is necessary to seal up the interstitial layer around the whole device, or just at the pertinent edges.
From my experience, it doesnt seem to be the case; no leaking was observed from the bottom layer to the top layer (more on that in a moment).
– The mousebite collapsed on one side, find a better way to keep the mousebite open. The good side was sufficient for access.
Reverting to a design without the mousebite seems to keep both sides open for access. I can minimize the contact error in the future by using a silver pen to create some conductive pathways for external contact (thanks Charles).
I fabricated all of the gasket film stacks using UV-ozone bonding and my jigs to align everything nicely. It worked pretty well. The only thing I forgot to do was attach PDMS wings using UV-Ozone to provide structural support for the capillaries. I had to use Mortar bonding because I had previously cured the stack, making it unable to be ozone bonded again.
I then inserted capillaries into the feet and did some DIC microscopy to see 5.6 micron nanobeads flowing in each of the channels, taking some pictures before I did so.
Tomogram of BBB V2.4 bottom up to membrane
Tomogram of BBB V2.4 top to bottom through pnc-Si filter
The tomograms clearly show some good flow underneath the membrane. When I tried to use the top channel, all the flow went into the bottom channel. When I used PDMS mortar bonding to attach the wings for the capillaries, it appears I sealed off the top channel. I did not see any leaking at all around the edge of the silicon into the top channel, so I believe UV-ozone bonding the wings onto the top electrode gasket will fix this problem.
Unfortunately, if I cannot get fluid into the top channel, I cannot perform any TEER with this particular device; airgaps in the channel will result in an open circuit. I will either need to remake this particular design, or plow ahead with V2.5.