Hao Huang

Zwitterionic polymers exhibit excellent nonfouling performance due to their strong surface hydrations. However, salt molecules may severely reduce the surface hydrations of typical zwitterionic polymers, making the application of these polymers in real biological and marine environments challenging. Recently, a new zwitterionic polymer brush based on the protein stabilizer trimethylamine N-oxide (TMAO) was developed as an outstanding nonfouling material. Using surface-sensitive sum frequency generation (SFG) vibrational spectroscopy, we investigated the surface hydration of TMAO polymer brushes (pTMAO) and the effects of salts and proteins on such surface hydration. It was discovered that exposure to highly concentrated salt solutions such as seawater only moderately reduced surface hydration. This superior resistance to salt effects compared to other zwitterionic polymers is due to the shorter distance between the positively and negatively charged groups, thus a smaller dipole in pTMAO and strong hydration around TMAO zwitterion. This results in strong bonding interactions between the O– in pTMAO and water, and weaker interaction between O– and metal cations due to the strong repulsion from the N+ and hydration water. Computer simulations at quantum and atomistic scales were performed to support SFG analyses. In addition to the salt effect, it was discovered that exposure to proteins in seawater exerted minimal influence on the pTMAO surface hydration, indicating complete exclusion of protein attachment. The excellent nonfouling performance of pTMAO originates from its extremely strong surface hydration that exhibits effective resistance to disruptions induced by salts and proteins.

Graphene-based biosensors have attracted considerable attention due to their advantages of label-free detection and high sensitivity. Many such biosensors utilize noncovalent van der Waals force to attach proteins onto graphene surface while preserving graphene's high conductivity. Maintaining the protein structure without denaturation/substantial conformational change and controlling proper protein orientation on the graphene surface are critical for biosensing applications of these biosensors fabricated with proteins on graphene. Based on the knowledge we obtained from our previous experimental study and computer modeling of amino acid residual level interactions between graphene and peptides, here we systemically redesigned an important protein for better conformational stability and desirable orientation on graphene. In this paper, immunoglobulin G (IgG) antibody-binding domain of protein G (protein GB1) was studied to demonstrate how we can preserve the protein native structure and control the protein orientation on graphene surface by redesigning protein mutants. Various experimental tools including sum frequency generation vibrational spectroscopy, attenuated total refection-Fourier transform infrared spectroscopy, fluorescence spectroscopy, and circular dichroism spectroscopy were used to study the protein GB1 structure on graphene, supplemented by molecular dynamics simulations. By carefully designing the protein GB1 mutant, we can avoid strong unfavorable interactions between protein and graphene to preserve protein conformation and to enable the protein to adopt a preferred orientation. The methodology developed in this study is general and can be applied to study different proteins on graphene and beyond. With the knowledge obtained from this research, one could apply this method to optimize protein function on surfaces (e.g., to enhance biosensor sensitivity).