To prevent aggregation of 500 nm carboxyl-modified polystyrene microparticles during EDC/NHS activation, use fresh reagents, optimize buffer conditions, and consider adding a small amount of surfactant. Additionally, gentle resuspension techniques and short intervals of sonication are employed to break up aggregates before proceeding to the coupling step.

Thanks Abdelhak Maghchiche

Do you have any suggestions for buffer components? We have tried using sodium chloride and 0.05% Tween 20 in the buffers. We still see aggregation only after the activation step. We have been resuspending using vortex and sonication, do you suggest any other method of resuspension?

Thanks for your help!

The original nanoparticles must be stabilized electrostatically through the carboxylic acid groups. The active intermediate generated after adding EDC/NHS "deletes" the charge of the carboxylic groups rendering the nanosystem unstable. This will promote particle aggregation (not agglomeration) and there is no going back (even when using ultrasonic probes) from there. Adding sodium chloride does not help as it contributes to further destabilize the nanoparticles. Adding tween-20 could help (as it will become adsorbed to the nanoparticles and impart steric stabilization) but you need to determine a concentration where the system remains stable but the carboxylic groups as still accessible for activation. This concentration might not exist. It has been reported that EDC/sufo-NHS is better as the active intermediate is charged. From my experience, carbodiimide chemistry sucks and hydrolysis is faster than the intended reaction.