Conjugation of carbon coated-iron nanoparticles with biomolecules for NMR-based assay

In this work, we developed and optimized conjugates of carbon-coated iron nanoparticles (Fe@C) with streptavidin and monoclonal antibodies. The conjugation procedure included two stages. First, amino groups were grafted onto the carbon shell to facilitate noncovalent sorption of bovine serum albumin (BSA). Further, the covalent attachment of proteins to the BSA layer via glutaraldehyde coupling was performed. It was established and confirmed that the synthesis procedure is reproducible and allows preparation of stable conjugates. The resulting nanoparticles are clusters of Fe@C particles coated by proteins. The size of the clusters is in the range of 100-190 nm and can be controlled via the tuning of conjugation conditions, including pH, BSA-to-Fe@C ratio, etc. Conjugates of Fe@C with streptavidin and monoclonal antibodies (sizes of approximately 140-150 nm) were synthesized. Proton T₂ relaxometry was used to detect these conjugates with very high sensitivity due to the magnetic markers, Fe@C. The relaxivity (r₂) of different conjugates varied within the range of 290-450 1/s*mM. Conjugate applicability for relaxometry-based assay was confirmed by direct detection of streptococcal protein G and biotinylated BSA in a dot immunoassay.