Hi,
I have gold nanopaticles affixed to a glass surface and am trying to find an optimal protocol for coating with PEG molecules, in particular, CTPEG12 and MTPEG4 ((http://www.piercenet.com/product/carboxy-peg-thiol-compounds) at various mixing ratios.
We've noticed some interesting behaviors. First, at low mM concentrations, these PEG molecules will precipitate out of our solution (buffer is either DMSO and PBS or pure DMSO) over time; this can compromise the experiments. At lower concentrations/incubation times, we don't seem to get the coating that we want (not as resistant to non-specific binding).
Does anyone have a recommended functionalization protocol?
Thanks
I have modified gold particles in solution with similar type of PEG thiols. I have usually worked with just 1-10uM of the PEG, and 1-2 hour incubation (although my LSPR measurements show that at these concentrations in less than 5 minutes all the possible PEG that can adsorb do adsorb. I have also done mixed monolayers with ratio of 1:1 or 1:5 (short PEG in excess).
After the modification at low concentrations, how did you see that they were not very resistant to non-specific binding? Can you be sure it is the gold and not the glass around that was binding non-specifically?
yours,
Ron.
Hi Ron,
Thanks for your reply. I believe our reaction takes longer because the AuNPs are on a relatively small surface in the solution, so it's diffusion limited. Likewise, the response does have about a 12 hour time constant according to our data; whereas free solution AuNPs are free to interact.
It's not clear if we are seeing any binding to glass as you point out; however, in fibers that were sucessfully functionalized overnight, I can add a protein, get a very small signal, then rinse the solution and the signal fades. For the shorter PEG assembly time, the signal does not elute. Therefore, I believe it is a real effect, either on the AUNPs or the glass.
We seem to have more luck by using DMSO; the combo of PBS and DMSO seemed to cause salt precipitation over long time intervals.
Thanks. We have quantitative estimates for the number of AuNPs on the fiber, but remember the process is diffusion limited since they are fixed to a relatively small surface in the solution; there are only about 10^6-10^7 particles on the fiber.
Ron,
We are using silica optical fibers. They are coated in TMDSE, a silanization agent, then in gold. Therefore, the glass surface is very sticky, and this could explain the buildup of what seems to be salt or crust on the fibers.
Also, we just ran a very careful, new run with some modifications overnight. While there was some visible, white buildup on the fiber, the solution precipitation seemed reduced and the buildup washed off in water; maybe it's just a salt crystalization or something?
Do you guys think the DMSO could make for a poor self-assembled monolayer of the PEG coating?
Thanks for your insights.
Although I dissove my PEGs in DMSO for the stock solution, I always have diluted the stock 100-1000x in buffer, so I get a very small percentage of DMSA in my working concentrations. If you are only working with PEGs and not proteins, you don't have to use PBS, and 10mM of Tris, Posphate or HEPES buffer should work fine. Maybe that will solve your salt percipitation problems. You can also try to passivate excess amines on the surface with NHS-PEG. Alternatively, you can add PEG without a functional group (usually 5k or 10k MW, and 0.1% final concentration) to your working (when you flow proteins on it) and washing buffer. This usually helps reduce non-specific adsoprtion of the proteins. But because it works by non-specific adsorption of the PEG, you need to 'blank' your reading with a solution with PEG without your proteins, and then measure any differences when you introduce your protein solution, with the same amount of PEG.
Ron.
Thanks guys. Do you think the buffer conditions such as salinity, pH, might affect the monolayer formation?
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