Combining Microfluidics, Electrophysiology, and Fluorescence Detection to Study Drug Transport Across Biomembranes

Abstract

Nanoparticles have been conjugated to proteins to create unique imaging agents, multifunctional particles, and drug delivery vehicles. However, the biggest barrier for the success of these applications is understanding the interface of biomolecules with nanoparticles. Often conjugation of proteins and DNA with nanoparticles results in protein denaturation and non-specific adsorption, which are due to the many non-covalent interactions at the inorganic-biological interface. While development of new biological applications of nanoparticles has garnered a great deal of attention, the protein-nanoparticle interface has remained poorly characterized. As a result, insufficient understanding of the interface has limited the capabilities of nano-bio hybrids. We present work in which we study the interface between inorganic nanoparticles of Au and CoFe2O4 and the protein cytochrome c, which is covalently linked to the nanoparticle. We devise a method to site-specifically label the protein, minimizing non-specific adsorption. We study the effect of nanoparticle ligand, nanoparticle material, and protein labeling site on the structure of the protein. Biophysical techniques such as quantitative gel electrophoresis, circular dichroism, and optical spectroscopy are used to characterize the structure of the protein in the conjugate. These experiments allow us to understand the chemical interactions involved in non-specific adsorption, and come up with general design rules for optimal conjugation. We determine that nanoparticle labeling generally destabilizes the motif containing the labeling site, and that when the nanoparticle is labeled on certain motifs, protein denaturation is not recoverable.