| Molecular display technologies, in particular phage display, have been the hallmark for dissecting molecular reognition events. Large phage-displayed, combinatorial polypeptide libraries are extensively employed to elucidate molecular recognition principles, protein folding and stability, and structure-function relationships. The research described herein aims to explore the use of phage display for engineering protein thermostability and as a bioaffinity scaffold for the development of biosensors. First, a combination of computationally designed mutations and phage display selections were applied to the enzyme 5-epi-aristolochene synthase (TEAS) to create the first example of a thermostable terpene synthase. It is evident from these studies that conformational dynamics plays a key role in the activity of the terpene synthase. Second, vast phage-displayed peptide libraries can be used for the universal detection of analytes in real-time. A reagent-free biosensor composed of a covalent virus layer (CVL) attached to the surface of a gold electrode was evaluated and determined to be a robust, selective, and sensitive means for carrying out mass-based biodetection. Further characterization of the CVL used electrochemical impedance spectroscopy (EIS) to establish conditions of frequency and phase that would permit selective and sensitive detection of analytes. Lastly, the use of genetic and chemically modified phage has led to the development of a "library times library" (LxL) approach, which uses the entire surface of the virus for recognition and analyte binding. This LxL-based virus electrode was used to increase the selectivity and affinity for prostate specific membrane antigen (PSMA), and detect the cancer marker in real-time. |
