PEG Monolayer and Non-Specific Protein Adhesion

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mascott
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PEG Monolayer and Non-Specific Protein Adhesion

I have been having issues with non-specific protein adhesion to my PEG-monolayer that I have formed.

First things first, The surface was first cleaned using Nanostrip (stabilized piranha), and sonicated in DI for 1hr. The surface was then dried under a nitrogen stream. I then used a 2% solution of PEG-Silane (MW ~700from Gelest Inc.) in toluene, and allowed to react under nitrogen for 16 hours.

I find that the monolayer works well at resisting some protein adhesion. For example, I can prevent streptavidin from adhering to the surface. But sticker proteins, such as laminin or PDL (Poly-lysine) are non-specifically adhering to the background. I have seen very little literature on this matter of different proteins behaving differently on PEG-monolayers.

I also tried a glutaraldehyde crosslinking method of PEG-NH2 to an APTES monolayer, and a nucleophilic substitution method using PEG-NHS onto an APTES monolayer. None of these worked to prevent the adhesion either.

I cannot use a BSA blocking method, as my aim is to avoid cell adhesion, and my cell cultures are sticking to BSA passivated surfaces.

Has anyone experienced this before?

Mr. Gunn
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mascott wrote:I have been

mascott wrote:

I have been having issues with non-specific protein adhesion to my PEG-monolayer that I have formed. First things first, The surface was first cleaned using Nanostrip (stabilized piranha), and sonicated in DI for 1hr. The surface was then dried under a nitrogen stream. I then used a 2% solution of PEG-Silane (MW ~700from Gelest Inc.) in toluene, and allowed to react under nitrogen for 16 hours. I find that the monolayer works well at resisting some protein adhesion. For example, I can prevent streptavidin from adhering to the surface. But sticker proteins, such as laminin or PDL (Poly-lysine) are non-specifically adhering to the background. I have seen very little literature on this matter of different proteins behaving differently on PEG-monolayers. I also tried a glutaraldehyde crosslinking method of PEG-NH2 to an APTES monolayer, and a nucleophilic substitution method using PEG-NHS onto an APTES monolayer. None of these worked to prevent the adhesion either. I cannot use a BSA blocking method, as my aim is to avoid cell adhesion, and my cell cultures are sticking to BSA passivated surfaces. Has anyone experienced this before?

I'm not working with cells, but I've done something similar. I used APTMS(basically the same as APTES) and then I used PEG5-4FB-NHS(from Solulink, used at 350 uM in 150 mM NaCL, 100 mM PBS, pH 7.2 overnight) on the surface, and then 0.02% tween-20 in the running buffer. Under these conditions, my polyclonal IgG-HRP @ 0.2 mg/mL didn't stick. I don't know if that amount of tween is compatible with your cells, but you could probably go still lower.  Another thing you should try is the silanization at 70 C for one hour, with continuous gentle agitation. I've gotten the most uniform monolayers that way, so at least that removes the roughness that cells like to stick to, and I imagine with a 16 hour reaction you're getting a fairly rough multilayer surface, and not a monolayer at all.
See Pasternack et al. for more info on this method.