Gold nanoparticles (AuNPs) have garnered wide interest over the last decades in applications such as drug delivery, biosensing, electrochemical devices, and immunoassay development due to the unique optical properties and easily manipulatable surface chemistry. Protein-NP bioconjugates have gained particular interest in physiological applications, however a complete understanding of the protein-NP interaction to maximize bioconjugate stability and function is highly desired. In this work we explore chemical modification of a protein to precisely control the number of protein thiols and evaluate the impact of thiols on bioconjugation. Specifically, solvent exposed lysine residues on the model protein α-chymotrypsin (ChT) were reacted with 2-iminothiolane (Traut’s) to introduce additional thiol groups that facilitate adsorption to gold nanoparticles. We hypothesize that multi-point adsorption through several thiol-Au bonds will yield robust, stable bioconjugates that retain enzymatic structure/function and resist aggregation. Dynamic light scattering and UV-vis spectroscopy confirms the formation of conjugates while salt induced aggregation kinetics demonstrate the increased stability due to chemical modification. Further, fluorescence spectroscopy was used to investigate the secondary structure post conjugation and it was found that native ChT loses its secondary structure upon interaction with the gold surface. Ongoing efforts are focused on quantifying the activity of the immobilized ChT. The results of this study provide additional evidence for the importance and role of free thiols in the formation of stable and enzymatically active bioconjugates.

Protein modification as a facile approach to retain activity upon conjugation to gold nanoparticles

ACS Spring 2022

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