PDMS Coating Issues

I’ve performed what might be the last test of the n-Dodecyl β-D-Maltoside (“DDM”) coating. This time I washed for three minutes each sample in HBS just like in the reference, used precisely the same concentration (and type, for that matter) of protein that was used in the reference, and tested a positive control, a negative control, a sham sample, an untreated sample, a once-DDM-treated sample, and a twice-DDM-treated sample. The results are unimpressive:

Both DDM’d samples actually display more fluorescence than both the Sham and Untreated samples, certainly due to measurement error. In any event, the difference in protein adsorption between a DDM treated sample and an untreated sample simply doesn’t appear to be noticeable when tested in this manner. I cannot say why the reference got such spectacular results, but they’re certainly not showing up here.

Jim and I discussed the possibility that maybe this is a non-issue. It is known that PDMS tends to adsorb some protein, but it’s conceivable that in the CytoVu format the rather low wetted PDMS surface area will not present an issue. Thus, I’ll be moving on to testing just how much protein is lost to the system when a diffusion test is performed — a bit backwards, but if we knew what we were doing it wouldn’t be called research.

For that, I’ll be diffusing Cytochrome C from the top chamber of a CytoVu assembly into the bottom chamber, which will initially contain only DI water. Right before I pipette the Cytochrome C solution into the top chamber, I’ll record the amount of it in solution using the TECAN, and after the system reaches equilibrium (I’ll have to run some tests to determine when exactly that is,) I’ll take a sample of the solution in the bottom chamber and test for Cytochrome C there. By performing this test with various starting concentrations of Cytochrome C in the top chamber, I should be able to determine just how much protein is adsorbed to the system as a function of its starting concentration, and we can make a judgement from there on whether or not we need to worry about preventing that loss.

Assuming we do decide to worry about it, it’s likely that the best option for attempting to reduce it will be good ol’ PEGylation of the glass coverslip, and possibly of the chips. The wetted areas of the glass and chip are much larger than the wetted area of the PDMS gaskets, after all, and it’s also possible that we’ll move away from PDMS entirely, in favor of Teflon or perhaps acrylic.

I’ll post again when I know more!

UPDATE: I’m told we might move away from glass coverslips all together in favor of a less adsorptive substance. Between that and the replacement of PDMS, this problem might solve itself.