Summary of diffusion through a pnc-Si array in a 384 well plate

During the past few weeks, I have been experimenting with different array setups for diffusion. I have been working with BSA, but I will move to APOBEC3G protein once the arrays Barrett designed are finished.

All of the pnc-Si arrays used were treated with UV-ozone. Treating with UV-ozone cleans the surface and helps prevents the pipetted drop from jumping onto the wafer surface. This makes it easier to load the top wells and to prevent mixing of samples on the wafer surface.

I. Test for flow, which would mean that there is enough fluid in the wells of the plate to wet the backsides of the membranes

Test 1: 28µl of DI water in the plate; 2µl of fluorescein sodium in pnc-Si wells (3µl spread out too much on the chip)

After 15 minutes, I lifted the array and there was no fluorescein in the wells.

Test 2: 30µl of DI water in the plate; 2µl of fluorescein sodium in pnc-Si wells

After 15 minutes, I lifted the array and the fluorescein had visibly diffused into the wells.

Conclusion: 30µl of DI water is needed in the 384 well plates to initiate diffusion.

II. Test for cross-contamination between the wells of the 384 well plate when an array is overlaid

Test 1: Alternate 30µl of fluorescein and DI water in the wells of the plate. (Note: the fluorescein has more of a meniscus than the DI water.) Place array over the wells.

After 1 minute, I lifted the array and there was mixing between all of the wells.

Test 2: Because the fluorescein had more of a meniscus, I used 28µl of fluorescein  and 30µl of DI water in the same configuration.

After 1 minute, I lifted the array and there was no mixing between the wells. Fluorescein and DI water residue could be seen on the backside of their respective membranes when the array was lifted, so the backsides were assumed to have been wetted.

Conclusion: The type of fluid affects the amount of fluid that can be put in the wells before cross-contamination occurs.

III. Standard BSA curve: Bradford assay in a 384 well plate

Setup: 30µl 1:4 diluted Bradford dye reagent in wells + 1µl of standard BSA solution

Absorption is taken at 595nm in the TECAN.

Conclusion: The problem with this setup is that the Bradford dye is not certified to be RNase free, which would nullify experiments with APOBEC3G.

IV. Standard BSA curve: mixed in a 384 well plate, Bradford assay in a 96 well plate

–> With this set-up, we don’t have to worry about whether or not the Bradford dye is RNase free because the assay will be done after diffusion has already been completed. If RNase is introduced to the diffusion set-up, the APOBEC3G protein will break up, and all of the pieces will diffuse through the membrane. Since the Bradford assay just measures the amount of protein present in solution, it doesn’t matter if the APOBEC3G protein breaks up during the assay process.

Setup: 30µl PBS + 1µl of standard BSA solution.

10µl is taken from well and placed in a 96 well plate. 200µl of 1:4 diluted Bradford dye reagent is added.

Absorption is taken at 595nm in the TECAN.

V. Diffusion of BSA diffusion through a pnc-Si array in a 384 well plate

Experiment:

• Pipette 30µl of 1xPBS in wells of 384 well plate
• Place array over wells
• Add 1µl of 10mg/ml BSA to top left and bottom right membranes. Add 1xPBS to remaining two membranes.
• Add 30µl of 1xPBS to surrounding four wells, to help prevent evaporation.
• Place lid on plate and use parafilm to seal the setup.

After two hours, I checked on the setup. The top left membrane was broken and the bottom left membrane looked almost completely dry on top.

The setup is estimated to have dried out after three hours. Without the parafilm, the drops dried up after about 15 minutes.

Note: Only 1µl was added to the top of the membranes in this experiment in order to help prevent mixing between the wells. A 2µl drop seems to be more likely to jump onto the surface of the pnc-Si wafer and to spread out enough for the wells to mix.

Results from diffusion: