I had successfully self-assemble protein nanoparticles into 100 nm size range and require crosslinking for indicidual particles. My protein is 40 kDa and contains only 1 lysine but lots of acidic residues. I am thinking that crosslinking the particle using EDC/NHS might be more appropriate, but thought that the amount of crosslink might not be sufficient.

Initially I tried using ethylenediamine in hopes of crosslinking the carboxylic acids, but secondary side reactions of protein-protein crosslinking is unevitable. This is confirmed by the size increase to >1 um since the ethylenediamine spacer arm is happily sticking out wanting to bind to nearby particles.

Also as I am required to functionalise the surface with another amine ligand, equimolar ratio of ligand and ethylenediamine was added for equal competition with the activated carboxylic acid.

Did anyone manage to overcome this challenge? I activated the carboxylic acid with EDC/NHS for 15 minutes with MES pH 7.2 before throwing in ethylenediamine and reacting for 3 hours. The excessive long time might have contributed to the size increase. Unfortunately I cannot change the pH as the nanoparticle size and polydispersity will be greatly affected.