Tailoring reactive handles on the surface of nanoparticles for covalent conjugation of biomolecules

Surface modification of nanoparticles involves numerous types of active molecules such as DNA, antibodies, enzymes, or carbohydrates. These modifications usually require reactive handles like amines, carboxylic acids, azides, etc. on the nanoparticles. In this work, utilizing poly-benzyl methacrylate based nanoparticles as a model nanoparticle system, the number of functional groups was tuned with functional comonomers, amino ethyl methacrylate for the amino groups or methyl methacrylate for the carboxylic groups. Herein a systematic study is presented where the functional groups in the nanoparticles are differentiated between total, visible and accessible functional groups. The concentration of each type of functional group is compared using various methods. Polymers synthesized using free radical polymerization were analyzed using ¹H-NMR spectroscopy to obtain the total number of functional groups. Via a miniemulsion–solvent evaporation technique, these polymers were used to synthesize the nanoparticles. Zeta potential, pH value and particle charge detection measurements were used to determine the number of visible functional groups. The number of accessible functional groups was quantified by conjugating small dyes and fluorescence measurements were directly executed on the system under investigation, hence eliminating errors associated with indirect measurements and detecting very low concentrations (e.g. 80 nM). Lastly, human serum albumin was conjugated to investigate the effect of a bulky molecule on the accessibility of these reactive handles.