Development of a Plasmonic Nanoparticle-Based Assay to Observe Nanoscale Biological Dynamic Interactions

Abstract

To measure nanoscale distances relevant to quantifying bio-molecular dynamics, a widely used technique is fluorescence resonance energy transfer (FRET). While FRET is sensitive to distances between donor and acceptor fluorophores below 10nm, the technique suffers from a low signal-to-noise ratio, limited range of distance sensitivity, and rapid photobleaching of the fluorescent dyes. These unavoidable experimental shortcomings greatly hinder the robustness of the technique in measurements characterizing dynamics at the single-molecule level. A new approach that exploits the plasmon coupling of gold nanoparticles has been introduced, which has a high signal-to-noise ratio, distance ranges from sub-nanometer to hundreds of nanometers (depending on particle size), and superior photostability. Previous work has demonstrated the viability of plasmon coupling to report the distance between two gold nanoparticles conjugated to the ends of DNA. We have extended the technique such that the distance between the gold nanoparticles and the plasmon coupling spectral response are independently determined through the use of image analysis and spectrophotometry. In this manner, this technique can be extended to observe the dynamics of any protein interaction where gold nanoparticles may be conjugated. We have designed and implemented a custom spectrometer with high spectral resolution (0.015nm) and low integration times (millisecond), surpassing commercially available instruments, by using a sensitive CCD array and off-axis parabolic mirrors. By simultaneously analyzing the collected spectra of single gold nanoparticle pairs and images of their diffraction-limited spots using a 2-D Gaussian fitting algorithm, the spectra are correlated to the measured distance between the gold nanoparticles. As a proof-of-concept, we are now creating calibrations between the plasmon coupling spectra and the distance between dually-labeled DNA molecules. This experimental technique has the potential to study DNA-protein and protein-protein interactions with high spatial (sub-nanometer) and temporal (millisecond) resolution.