P.S: carboxylate surface was created from mercaptobenzoic acid

Is there still free edc/nhs roaming around in the solution when the second nanoparticle is added, or have you filtered it out?

Au@Citrate particles are realy not stable, which is caused by massive non specific binding to anything, even to negatively charged surfaces (should be repelled by elstat force) - this is my experience.

My questions to you:

How did you measure non-specific binding of Au@cit particles to the Au@EDC/NHS ones? Are they really binding to the other particles or only they agglomerate themselves (talking about Au@cit)?

Adam asked you very important question. Any charged molecule can enhance clustering.

Possible solution:

I don't reccomend PEG for passivation. Try to use some shorter easy molecule - some thioalkanes should be fine. But it will be more challenging to bind them to anything else afterwards.

I would suggest to read our work, but since any journal haven't accepted our manuscript yet, I can't :(

Thanks guys for kindly response,

To Adam: Yes I did rinse EDC/NHS activated sample with MES buffer and coupling buffer (PBS) with sonications.

To Rostislav: I'm happy to answer your questions:

After blocking PEG 5000Da around citrate-AuNP, they didnt aggregate in PBS buffer anymore. I put PEG-AuNP into EDC/NHS activated particles, within 20mins or less, I centrifuge PEG-AuNP-(EDC/NHS)-particle and they showed pink pellet, indicating non-specific binding of AuNP on my carboxylated particles. Meanwhile, PEG-AuNP itself didnt show any agglomerate after being centrifuged.

I think your suggestion is good for me, since I doubted myself a lot when coming to this situation. I'll try THPC stabilized AuNP instead. By the way, do you think THPC-AuNP is a good replacement for citrate-AuNP in terms of charge induced-clustering?

Many thanks guys for reading

I'll add a couple comments to the previous answers. First, the pink color you see only indicates the AuNPs are not aggregated to each other (thus quenching the SPR scattering/absorbance). They could co-exist in the pellet with your modified carboxylate particles as separate entities and not necessarily bound together. Second, as someone else has said, citrate AuNPs (and really AuNPs period) tend to form nonspecific interactions quite a bit. See Nanoscale, 2012, 4, 3208 on anti-TNF nonspecific binding to TNF functionalized AuNPs. PEG can block the surface, but if the  ligand is small enough it will find it's way to the surface. We have seen this in multiple systems where we studied competitive adsorption with PEG on AuNPs.

If your reactions are in aqueous solution, then alkane thiols will likely produce a less stable AuNP. The alkane is hydrophobic unless functionalized and both the alkane and the thio group will bind tightly to the gold surface providing little stability. If your goal is to react AuNPs with a thiol-functionalized particle, then citrate AuNPs are usually a good bet, because they are negatively charged (should not interact with a carboxylated surface very strongly) and the citrate is easily displaced during the thiol reaction. It's still not clear to me how you differentiated nonspecific from specific interactions in this case.

It is still not clear to me either. You dont know what is causing the pink color.

Did you try centrifuging them and load them in gel electrophoresis? this should provide you more info about what is going on in your solutions.

First of all, many thanks for your reading and valuable suggestions,

To Professor Hackley: thank you for your informative advice and reference, I've read it thoroughly and found some demerits in my nanohybrid design.

My answer to Professor Hackley and Rostislav about my so-called non-specific binding phenomenon:

Franky speaking, freshly-prepared citrate AuNP is well dispersed and reflective so it has its unique color depending on its size. In my case, my carboxylate particle is way too much bigger than AuNP (200nm vs 10nm, respectively) so I used 5000 rpm/1min centrifugation in order to centrifuge down carboxylate particle only, and I also included a solution with only AuNP to see if AuNP could possibly be centrifuged down at 5000 rpm. And I got my carboxylate particle pellet in pink, so I made my early conclusion.

Following your suggestions, I'll confirm the non specific binding using gel electrophoresis,

Again, thank you so much for your consideration given for my questions.

Best,

Hello again,

finally we published our results, you can read it here:

http://pubs.acs.org/doi/abs/10.1021/acsami.5b01191

Cheers

http://pubs.acs.org/doi/abs/10.1021/acsami.5b01191