EDC/NHS renders the Au nanoparticles unstable if they are coated with citrate ions. EDC/sulfo-NHS is supposed to be better at this, but I have never corroborated this. When adding the protein to the nanoparticles (considering that they survived the activation process), physical adsorption will most likely dominate over chemical linkage (moreover NHS is extremely susceptible to hydrolysis). I recommend running physical adsorption with streptavidin up to the point where the nanosystem is stable. Afterward, you can check blocking agents, again physically adsorbed. If the protein is expensive, I would first stabilize the nanoparticles using thiol-peg-cooh or thiol-peg-amine, I prefer the latter.

Omar,

I deeply appreciate your answer. I've decided to go with physical adsorption.

Any tips/advice or recommended protocols for AuNP-Protein immobilization?

I'm looking at a protocol from Bioconjugate Techniques (3rd Ed. 2013) where performs mixing at pH 6~7. I also read that AuNP@Streptavidin coating yields best at pH 9.0. And from my pre-understanding, proteins should be made to adsorb maximally at or near their isoelectric points (pI 5.2 for streptavidin).

Does pH play a critical role in the reaction? I'm all mixed up.

Thanks again.

Ref: https://www.semanticscholar.org/paper/Synthesis-of-the-Most-Effective-Streptavidin-with-Samokhvalov-Razo/81043c99a6bd5b156b1a93954db0336c5293e38d

pH is important for adsorption (at the isoelectric point adsorption will be favored) but also for stability of the nanosystem (away for the isoelectric point will impart electrostatic stability with the steric stability introduced by the polymer (protein)). Perform the adsorption at various pH values and with different protein concentration if possible.