Determination of the Adsorption Degree of Protein on Gold Nanoparticles by Gel Electrophoresis

Introduction Proteins can be immobilised on the surface of nanoparticles (NPs) by two main methods: covalent bonding and adsorption. Regardless the method, the immobilisation process allow for the design of multimodal... [ view full abstract ]

Introduction

Proteins can be immobilised on the surface of nanoparticles (NPs) by two main methods: covalent bonding and adsorption. Regardless the method, the immobilisation process allow for the design of multimodal structures that have the synergistic effect of being a protein-particle hybrid system. Although, there are several methods available to investigate NP-protein interactions, quantification of the amount of protein on the single NP surface is still challenging. In this work a simple, reproducible and highly sensitive analytical method to determine the amount of protein adsorbed on a single NP by gel electrophoresis will be presented.

Methods

Gold nanoparticles (AuNPs) synthesized via chemical reduction method were modified with proteins (catalase) by incubation. Dynamic Light Scattering was used to monitor the hydrodynamic size of AuNPs and the agglomeration state of colloids before and after the immobilisation of catalase. Morphological studies of the AuNPs before and after modification were performed by Scanning Transmission Electron Microscopy. Determination of catalase adsorption was carried out with different electrophoresis protocols to find the optimal conditions for quantification of the surface coverage of AuNPs by catalase (native-PAGE, non-reducing SDS-PAGE, b-mercaptoethanol-PAGE, and reducing SDS-PAGE).

Results

The obtained results confirmed that the modification process of AuNPs with catalase did not disturb the stability of NPs. AuNPs were colloidally stable and did not form any aggregates after modification. The hydrodynamic size of protein-modified AuNPs increased compared to non-modified which confirmed the successful modification process. The determination of the surface coverage of AuNPs with catalase was performed based on the native-PAGE for which the most sensitive conditions were found.

Discussion

The presented method allowed for the identification and quantification of the amount of CAT adsorbed on the surface of NPs in a colloidal state. The obtained results prove that the method is effective and versatile and can be successfully used for the identification and quantification of proteins adsorbed on the surface of different types of colloidal NPs.