PEGylation of Coverslips and Chips
I’ve taken fluorescence measurements with the fluorescence microscope on PEGylated and unPEGylated glass coverslips and SiN chips.
Six coverslips and six chips were prepared. Samples were treated exactly the same, save for the fact that half the samples had PEG Silane in their treatment and half did not (though they were still bathed in toluene with HCl.) I then pipetted 33.3uL of 100x diluted 1mg/mL fluorescein conjugate BSA in PBS onto each sample, allowed them to incubate covered in tin foil for five minutes, and then rinsed each sample briefly in water before drying them on a hotplate. I then recorded fluorescence immediately after.
There was no significant difference in protein adsorption between PEGylated and unPEGylated samples. All 11 samples (one glass coverslip broke during treatment) displayed very comparable amounts of fluorescence, with only very small amounts of protein dotting their surfaces. I would not expect this to be problematic, given the small volumes that will be used in CytoVu assemblies.
I plan on testing the adsorption of PDMS that has been treated with n-Dodecyl B-D-Maltoside (DDM) versus untreated PDMS next week. Supposedly PDMS has much more dramatic protein adsorption issues, so the difference will likely be more noticeable. I’ll be making another post with the results of that experiment.
What was the starting concentration of BSA? Diluting it 100 fold does not sound like a good idea. Incubate at 1 mg/ml or higher for 1-2 hours.
You also need to quantify the fluorescence and compare to background negative control. Dean Johnson could help you with this.
The BSA came as a solid which I then mixed with filtered 1X PBS at 1mg/mL. When I was originally learning from Tejas to take fluorescence measurements he suggested that I dilute it 100 times, but it’s possible that for this specific application that’s unnecessary. I only incubated for five minutes because a few papers I found mentioned that they incubated proteins for fluorescence tests for that time, including the PDMS n-Dodecyl B-D-Maltoside paper. I can and will rerun the tests incubating at 1mg/mL for over an hour, however.
My understanding of the interpretation of results was that it wasn’t so much to quantify protein adsorption as it was to compare adsorption between treated and untreated samples. Jamie asked me to think of the untreated samples as displaying “100% fluorescence” and representing the treated samples fluorescence as a percentage of that. If you believe getting more exact quantifications of adsorption is important, I’ll follow up with Dean.
Thanks!