Involvement of the LSPR spectral overlap for energy transfer between a dye and Au nanoparticle.

Nanometal surface energy transfer (NSET) is a molecular ruler technique that has been utilized to optically probe long distances in biomolecular structures. We investigate the useful spectral range of donor dyes and the importance of overlap between the localized surface plasmon resonance (LSPR) and the donor photoluminescence (520-780 nm) and provide a comprehensive study of the R(0) values for the NSET processes from dyes to 2 nm Au NP (gold nanoparticle). The distance-dependent quenching results provide experimental evidence that the efficiency curve slope, R(0) value, and distance of quenching is best modeled as a surface-mediated NSET process analogous to the predictions of Persson-Lang and Chance-Prock-Silbey (CPS). The results show that the LSPR plays a very important role in the observed quenching of excited-state donors at the surface of the nanometal, and the correlation to the NSET model allows a compilation of the necessary biophysical constants for application within the toolbox of biophysics.

Tracking spatial disorder in an optical ruler by time-resolved NSET.

For biomolecular applications, potential interactions between newly developed dye molecules and the biomolecule of interest can dramatically influence the accuracy of optical ruler techniques. By utilizing nanometal surface energy transfer (NSET), an optical technique is developed that allows the nature of interactions between dyes and a biomolecule, namely DNA, to be directly assessed. To demonstrate the method, interactions between well-known molecular dyes based on carboxyfluorescein (FAM, noninteracting) and Cy5 (known intercalator) with DNA is probed. The results demonstrate that FAM exhibits no interactions with the DNA backbone and is adequately represented as a solvent exposed dye, while the commonly used near-IR dye Cy5 exhibits two discrete interactions that depend on the site of appendage and the length of the linker arm. The exact population and nature of Cy5 interaction with the DNA indicates a 37% ratio of intercalation for the internal C(6), a 42% ratio for an internal C(3) spacer length, and no evidence of interaction for terminal labeling. The results allow quantitative assignment of the site occupation of donors to be analyzed providing a powerful set of information for use of dyes in FRET based optical ruler technologies without the need of single molecule methods or the assumption of an averaged site occupation for the donor.