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. More recently, nanoaperture optical tweezers have been used to probe the low-frequency (in the single digit wavenumber range) Raman active modes of single nanoparticles and proteins. Here we review recent developments in the field of nanoaperture optical tweezers and how they have been applied to protein-antibody interactions, protein-small molecule interactions including single molecule binding kinetics, and protein-DNA interactions. In addition, recent works on the integration of nanoaperture optical tweezers at the tip of optical fiber and in microfluidic environments are presented.

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. Using fluorescence correlation spectroscopy and time-correlated photon counting, we measure fluorescence enhancement up to 100-fold, together with local density of optical states (LDOS) enhancement of 30-fold. The distinctive features of double nanoholes combining high local field enhancement, efficient background screening and relative nanofabrication simplicity offer new strategies for real time investigation of biochemical events with single molecule resolution at high concentrations.

The interaction of proteins with small molecules is fundamental to their function in living organisms and it is widely studied in drug development. Here we compare optical trapping dynamics of streptavidin and biotinylated streptavidin using a double nanohole optical trap in a metal film. Consistent and clearly distinct behavior is seen between the protein with and without the small molecule binding. The real-time dynamics at the single protein level are accessible with this technique, which also has advantages of not requiring tethering to a surface or the need for exogeneous markers.Protein-small molecule interactions (PSMIs) play an important role in biological functions. PSMIs are also of primary interest for the development of drugs, for example through inhibition of protein interactions. 1 While many works have studied PSMIs, only few approaches

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. Similar optical transmission behavior was not observed when streptavidin binding sites where blocked by mixing streptavidin with excess biotin. Furthermore, interaction of non-functionalized PS particles with streptavidin did not induce a change in the optical transmission through the DNH. These results are promising as the DNH trap can make an excellent single molecule resolution sensor which would enable studying biomolecular interactions and dynamics at a single particle/molecule level.

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. The binding kinetics of human serum albumin (HSA) to tolbutamide and to phenytoin are in quantitative agreement with previous measurements, and our single-molecule approach reveals a biexponential behavior characteristic of a multistep process. The DNH optical tweezer is an inexpensive platform for studying the real-time binding kinetics of protein-small molecule interactions in a label-free, free-solution environment, which will be of interest to future studies including drug discovery.