Time-Resolved Ratiometric Detection of the Plasmonic Coupling Between Gold Nanoparticles: A Novel Technique for Single-Molecule Biophysics.

Abstract

Single-molecule biophysics enables interrogation of specific biomolecules, providing details of transient behaviors underpinning biomolecular interactions. Given the nanoscale distances and fast dynamics of biomolecular systems, techniques must be capable of high spatial and temporal resolution. Fluorescence-based techniques are the current gold standard to observe and measure dynamic interactions occurring on millisecond timescales, but suffer from the trade-off between fluorescence intensity and photostability. Gold nanoparticles, with extreme photostability and high signal strength, are ideally suited to overcome the limitations of fluorescence microscopy. Further, through the plasmonic coupling effects between gold nanoparticles, distance measurements with nanometer resolution are theoretically possible over distances nearly twice the nanoparticle diameter, far exceeding the detectable range of fluorescence-based techniques. Previous plasmonic coupling measurements have been limited by the use of white light excitation and detailed spectral characterization, both which limit the spatial and temporal resolution due to low efficiency in collecting and measuring the scattered light. This work aimed to advance the plasmonic coupling technique for high temporal resolution applications through the ratiometric detection of scattered light from two excitation wavelengths. Specifically, using a novel implementation of monochromatic laser excitation and total internal reflection-based darkfield microscopy, the scattered light from gold nanoparticles at two excitation wavelengths was collected and spatially separated onto a CCD array with a dichroic mirror. As a proof-of-principle to establish this ratiometric approach, the plasmonic coupling of surface-bound biotin-functionalized gold nanoparticles upon binding neutravidinconjugated gold nanoparticles from solution was measured. The first demonstrated detection of plasmonic coupling between two gold nanoparticles with >25 Hz temporal resolution is reported. At this time resolution, the signal-to-noise ratio is >100-fold above background, and the intensity ratio more than doubles upon binding by the second nanoparticle. Importantly, and in contrast to previous methods, this technique is fully extendable to faster timescales, limited largely by the choice of detector, making possible high spatial and temporal resolution measurements with long time durations and over a broad distance range. Moreover, the experimental technique may be applied to diverse systems involving plasmonic nanoparticles including high spatial and temporal resolution single particle tracking, highly sensitive biomolecular binding sensors, and plasmonic nanostructures.