Well you are disrupting the original particle's electrostatic stabilization. After attaching protein, you introduce hydrophobic patches that will look to aggregate the nanoparticles-protein system. If the protein is uncharged, this process will be favored. Blocking the remaining carboxylic groups with tris is also not helping towards electrostatic stabilization. Not sure why you add tween 85 though. You need to switch to steric stabilization

Your polystyrene nanoparticles(NP) are carboxylated which would make them neutral at low pH because there's excess hydronium ions in solution. When you react it with a protein through EDC conjugation, the carboxyl covalently attaches to the amines on your protein. Depending on the availability of carboxyl groups on your NP and whether or not you optimized your conjugation process with respect to the concentrations of the NP, the protein or the EDC as well as the pH of the reaction, there could be available carboxyl on the NPs and some free unconjugated amines on the proteins that are already attached to the NPs. And depending upon the pH and electrolyte levels at which you store your NP - protein conjugates, the free carboxyl from one NP- protein conjugates could be attracted by a free amine from another NP-protein conjugate. When multiple such interactions occur, you get your aggregated NP-protein conjugates. How to avoid it? Optimize your reactans and reaction conditions that I mentioned above and then react the available excess carboxyl on the NP with a neutral molecule to prevent interactions with free amines from the conjugated proteins.

Dear Researcher,

After taking the surface charge of you nano-complex, you can determine the cause of aggregation. So use the DLS spectroscopy to determine the surface charge of nano-system firstly; then, you can apply suitable chemistry in order to overcome aggregation by using suitable blocker that maintain the surface charge of nancarrier in desirable value and prevent from aggregation.