Nanoscale volume confinement and fluorescence enhancement with double nanohole aperture.

Diffraction ultimately limits the fluorescence collected from a single molecule, and sets an upper limit to the maximum concentration to isolate a single molecule in the detection volume. To overcome these limitations, we introduce here the use of a double nanohole structure with 25 nm gap, and report enhanced detection of single fluorescent molecules in concentrated solutions exceeding 20 micromolar. The nanometer gap concentrates the light into an apex volume down to 70 zeptoliter (10(-21) L), 7000-fold below the diffraction-limited confocal volume.

Label-free free-solution nanoaperture optical tweezers for single molecule protein studies.

Nanoaperture optical tweezers are emerging as useful label-free, free-solution tools for the detection and identification of biological molecules and their interactions at the single molecule level. Nanoaperture optical tweezers provide a low-cost, scalable, straight-forward, high-speed and highly sensitive (SNR ∼ 33) platform to observe real-time dynamics and to quantify binding kinetics of protein-small molecule interactions without the need to use tethers or labeling. Such nanoaperture-based optical tweezers, which are 1000 times more efficient than conventional optical tweezers, have been used to trap and isolate single DNA molecules and to study proteins like p53, which has been claimed to be in mutant form for 75% of human cancers.

A label-free untethered approach to single-molecule protein binding kinetics.

Single molecule approaches provide rich real-time dynamics of molecular interactions that are not accessible to ensemble measurements. Previous single molecule studies have relied on labeling and tethering, which alters the natural state of the protein. Here we use the double-nanohole (DNH) optical tweezer approach to measure protein binding kinetics at the single molecule level in a label-free, free-solution (untethered) way.

Observing single protein binding by optical transmission through a double nanohole aperture in a metal film.

We experimentally demonstrate protein binding at the single particle level. A double nanohole (DNH) optical trap was used to hold onto a 20 nm biotin-coated polystyrene (PS) particle which subsequently is bound to streptavidin. Biotin-streptavidin binding has been detected by an increase in the optical transmission through the DNH.

Double nanohole optical trapping: dynamics and protein-antibody co-trapping.

A double nanohole in a metal film can optically trap nanoparticles such as polystyrene/silica spheres, encapsulated quantum dots and up-converting nanoparticles. Here we study the dynamics of trapped particles, showing a skewed distribution and low roll-off frequency that are indicative of Kramers-hopping at the nanoscale. Numerical simulations of trapped particles show a double-well potential normally found in Kramers-hopping systems, as well as providing quantitative agreement with the overall trapping potential.