To activate COOH groups on the surface of nanoparticles, I use EDC/NHS treatment and let the sample to incubate with EDC/NHS for about 2 hours. Then to remove the excess of EDC and NHS, I use ultrafiltration and washing with water for three times. Although before the ultrafiltration the solution which contains the nanoparticles and EDC/NHS (in water) is transparent and no aggregation is observed(even if it is kept for a long time), during ultrafiltration most of the nanoparticles would stick on the membrane surface and lead to aggregation of the sample.

I use 4500 rpm for centrifugation, I keep the ultrafiltration tube in water before using and even I pre-wash the tubes before adding my samples. I've noticed that diluting the solution before the ultrafiltration might be a little bit helpful but still a lot of sample would be wasted because of the irreversible aggregation.

I have also repeated the process in organic solvents such as DMSO and the same thing happened.

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