Biosensors based on the binding-induced folding of proteins and polypeptides.

摘要

While classic immunochemical detection assays such as ELISA and Western blot tests exhibit excellent sensitivity and selectivity, and have been generalized to the detection of an enormous range of molecular analytes, their use outside of a laboratory setting has proven difficult due to the numerous reagents, specialized equipment and personnel training required to perform such tests. The development of platforms that can detect a similarly wide array of biomolecular targets quantitatively, in real time, at the point of care (POC), remains a major research goal.; Thus, the primary focus of my thesis work has been to develop general signal transduction mechanisms in support of protein- and peptide-based biosensors for POC testing applications. The approach I have taken is based on the optical detection of binding-induced folding. In order to convert protein and polypeptide-based binding events into measurable signals, I have developed a platform, termed peptide beacons (PB), and several different PB biosensors were synthesized using both well established and completely novel conjugation techniques. Specifically, the synthesis of these PB biosensors entailed the attachment of fluorescent-based reporting groups to biological recognition elements capable of binding to the biomolecular targets of interest. As such, in the presence of the target, the PBs exhibit unique fluorescent signals capable of detection using standard, inexpensive desktop fluorimeters. The PBs also detected the presence of their respective targets at low nanomolar, typically close to or below the solution phase dissociation constants of the recognition polypeptide. I also determined the extent to which the PBs could detect their respective targets in more complex media, such as blood and saliva. By coupling exogenous fluorescent molecules to biological recognition elements, I have developed a series of different detection architectures that convert protein and polypeptide-based binding events into measurable signals, and in doing so, helped expand the potential targets for POC testing.