Nanopores offer the potential to study a wide range of protein-related phenomena that includes unfolding kinetics, differences in unfolding pathways, protein structure stability and free energy profiles of DNA-protein and RNA-protein binding. In addition to providing a tool for fundamental protein characterization, nanopore measurement systems can also be used to study polymer confinement at the single molecule limit. We are developing new nanopore measurements that rapidly monitor the thermodynamic and kinetic limitations of these biosensors in order to interrogate their detailed molecular operation and provide a foundation for highly efficient biosensors that can be use in fundamental biophysics and biomedical applications.
We are developing new tools to investigate the energy landscape of single-molecule sensors. Our goal is to apply a wide-ranging array of technologies to probe the critical physicochemical properties of nanopore biosensors. These properties include the free energy of confinement for polymers as they partition into and interact with a nanopore (Fig. 1a), and the various roles of entropy and enthalpy governing their operation. Our measurements provide the fine detail needed to understand and manipulate the local environment in order to optimize molecular detection and characterization. We investigate the molecular details that regulate the capture efficiency (i.e., detection limit), retention time (i.e., signal-to-noise, and chemical selectivity), and transport. To achieve such these goals, we are developing interfaces that combine proteinaceous ion channels and local surface plasmon resonance (LSPR) objects such as gold nanoparticles (Fig. 1b) which enables precise temperature control with 100 MHz bandwidth or better at nanometer scales. We are using these measurements to study the dynamical properties of biomolecules, the application of LSPR particles as nanoscopic heating elements for precise control of temperature gradients on the nanosecond time scale, and ionic transport in confined environments. Together these tools provide a benchmark for optimization of single-molecule nanopore sensors.