Plasmonic Single-molecule Binding Detection Robert Riehn, Keith Weninger, Shuang Fang Lim, NC State University, Raleigh, NC Eukaryotic systems are characterized by multi-protein assemblies. These assemblies are frequently assembled in complex multi-step processes. The heterogeneity of assembly throughout a population demands single-molecule techniques for the study of formation kinetics. This proposal aims to sense single-molecule binding events between proteins and DNA without any labels. The sensor will be based on the transmission of light through a pair of zero-mode waveguides (ZMW), which are sub-wavelength apertures in a metal film. Without any biological molecules within either of the ZMW, the transmitted pattern will be symmetric. When a protein enters one of the ZMW, a phase shift is introduced, and the direction of the transmitted light through the ZMW pair will appear shifted. Directional shifts can be detected with very high precision. Because of the small mode volume, the strong coupling of plasmon and lumen of the ZMW, and the resulting low group velocity of light within the ZMW, even very small changes of the refractive index within one ZMW can be detected. This principle of detecting minute refractive index changes with a plasmon field with small group velocity mirrors that of a classical surface plasmon sensor. The assessment of the sensor is divided into 3 phases: 1) Dielectric particles doped with narrow-line 2-photon active ions will be use for basic characterization. Transmission signal will be referenced to two-photon emission. 2) Single DNA mismatch recognizing proteins (MutS) will be used to establish the detection limits of the transmission signal. Fluorescence resonance energy transfer (FRET) between capture DNA oligomer and MutS will verify specificity of signal. 3) Sequential recruitment of multiple MutS will be used to test whether enumeration of multiple binding events is possible. Time resolution will be established. By conclusion of the project we anticipate detecting protein binding events 1) with single molecule sensitivity, 2) without fluorescent labels, 3) at tens of microsecond time resolution.
This project aims a developing a novel analytical technique to detect the binding of proteins at the single-molecule level, in their native state without labeling, and with a time-resolution of tens of microseconds. If successful, it will provide a tool to studying assembly of protein complexes, which are pervasive in disease-relevant cellular processes.