Versatile and high-throughput strategy for the quantification of proteins bound to nanoparticles

Biofunctionalized nanomaterials have been extensively studied as a tool for a wide range of applications in biomedical fields. Despite many existing strategies to conjugate proteins to colloidal particles, determining the grafting efficiency—that is, the amount of protein conjugated to the surface of a nanoparticle (NP)—remains challenging. Formulations for biomedical applications are subjected to strict constraints, and a lack of precise characterization can prevent otherwise promising formulations to be explored further. Here, we propose a simple approach to precisely measure the grafting efficiency of biological molecules on the surface of three types of widely used NPs: polymeric NPs, inorganic NPs, and metallic NPs. This approach relies on the simultaneous hydrolysis of the grafted protein and the NP degradation in acidic conditions, followed by a spectrophotometric quantification of primary amines in solution. This strategy can be applied to any type of protein and does not require any labeling agent. It can be performed in a high-throughput manner as a routine experiment and only requires a conventional oven and a microplate reader.