Protein surface labeling reactivity of N-hydroxysuccinimide esters conjugated to Fe3O4@SiO2 magnetic nanoparticles

The N-hydroxysuccinimide (NHS) ester moiety is one of the most widely used amine reactive groups for covalent conjugation of proteins/peptides to other functional targets. In this study, a cleave-analyze approach was developed to quantify NHS ester groups conjugated to silica-coated iron oxide magnetic nanoparticles (Fe3O4@SiO2 MNPs). The fluorophore dansylcadaverine was attached to Fe3O4@SiO2 magnetic nanoparticles (MNPs) via reaction with NHS ester groups, and then released from the MNPs by cleavage of the disulfide bond in the linker between the fluorophore and the MNPs moiety. The fluorophore released from Fe3O4@SiO2 MNPs was fluorometrically measured, and the amount of fluorophore should be equivalent to the quantity of the NHS ester groups on the surface of Fe3O4@SiO2 MNPs that participated in the fluorophore conjugation reaction. Another sensitive and semiquantitative fluorescence microscopic test was also developed to confirm the presence of NHS ester groups on the surface of Fe3O4@SiO2 MNPs. Surface-conjugated NHS ester group measurements were primarily performed on Fe3O4@SiO2 MNPs of 100-150 nm in diameter and also on 20-nm nanoparticles of the same type but prepared by a different method. The efficiency of labeling native proteins by NHS ester-coated Fe3O4@SiO2 MNPs was explored in terms of maximizing the number of MNPs conjugated per BSA molecule or maximizing the number of BSA molecules conjugated per each nanoparticle. Maintaining the amount of fresh NHS ester moieties in the labeling reaction system was essential especially when maximizing the number of MNPs conjugated per protein molecule. The methodology demonstrated in this study can serve as a guide in labeling the exposed portions of proteins by bulky multivalent labeling reagents.