Assembling Functional Proteins on Gold-Glass Surfaces

Abstract

The generation of functional surfaces using biological molecules is of great interest for cell culture and diagnostic applications. Control of protein orientation is difficult but advantageous in many cases to retain activity. The results from simple adsorption methods can be extremely variable and often result in denatured or inactive proteins. The industrial sponsor of this work, Orla Protein Technologies, utilise gold-thiol chemistry and the self-assembling nature of a beta-barrel outer membrane protein (OMP) from E. coli to create reproducible protein arrays on gold surfaces. The E. coli OMP is a robust protein scaffold that can be engineered to display a wide variety of functional motifs or domains such as, cell adhesion proteins, single chain antibodies or antigens. Sputter coating is often used to achieve thin and even gold surfaces suitable for most uses but the method requires expensive equipment and specialist training and the layers are poorly transparent, limiting their use in cell biology and microscopy. This has led our group to develop a simple, flexible method for depositing high densities of gold nanoparticles on to glass substrates. These are cheap to produce using inexpensive equipment and all steps are carried out at room temperature without harsh solvents. Moving to a gold-glass platform enables the more flexible use of microscopy and spectroscopic techniques in cell culture and biosensing applications. Characterisation of the new surfaces by electron microscopy, fluorescence microscopy and neutron reflection reveals well-ordered fields of nanoparticles coated in a functional protein layer. Further analysis of functionalised nanoparticles in solution has been carried out using UV-Visible spectroscopy, dynamic light scattering, small angle neutron scattering and electron microscopy. This shows several proteins bound to the nanoparticle surface through a gold-thiol bond. Functional motifs are displayed away from the surface and retain the ability to bind specifically to antigens in the solution. These new surface topologies open new avenues for applications of self assembling protein layers.