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.

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. In addition, we demonstrate co-trapping of bovine serum albumin (BSA) with anti-BSA by sequential delivery in a microfluidic channel. This co-trapping opens up exciting possibilities for the study of protein interactions at the single particle level.

We study the influence of fluid flow on the ability to trap optically a 20 nm polystyrene particle from a stationary microfluidic environment and then hold it against flow. Increased laser power is required to hold nanoparticles as the flow rate is increased, with an empirical linear dependence of 1 μl/(min×mW). This is promising for the delivery of additional nanoparticles to interact with a trapped nanoparticle; for example, to study protein-protein interactions, and for the ability to move the trapped particle in solution from one location to another.

Double nanohole (DNH) laser tweezers can optically trap and manipulate objects such as proteins, nanospheres, and other nanoparticles; however, precise fabrication of those DNHs has been expensive with low throughput. In this work, template stripping was used to pattern DNHs with gaps as small as 7 nm, in optically thick Au films. These DNHs were used to trap streptavidin as proof of operation. The structures were processed multiple times from the same template to demonstrate reusability. Template stripping is a promising method for high-throughput, reproducible, and cost efficient fabrication of DNH apertures for optical trapping.

Optical trapping is a technique for immobilizing and manipulating small objects in a gentle way using light, and it has been widely applied in trapping and manipulating small biological particles. Ashkin and co-workers first demonstrated optical tweezers using a single focused beam 1 . The single beam trap can be described accurately using the perturbative gradient force formulation in the case of small Rayleigh regime particles 1 . In the perturbative regime, the optical power required for trapping a particle scales as the inverse fourth power of the particle size. High optical powers can damage dielectric particles and cause heating. For instance, trapped latex spheres of 109 nm in diameter were destroyed by a 15 mW beam in 25 sec 1 , which has serious implications for biological matter 2,3 .A self-induced back-action (SIBA) optical trapping was proposed to trap 50 nm polystyrene spheres in the non-perturbative regime 4 . In a nonperturbative regime, even a small particle with little permittivity contrast to the background can influence significantly the ambient electromagnetic field and induce a large optical force. As a particle enters an illuminated aperture, light transmission increases dramatically because of dielectric loading. If the particle attempts to leave the aperture, decreased transmission causes a change in momentum outwards from the hole and, by Newton's Third Law, results in a force on the particle inwards into the hole, trapping the particle. The light transmission can be monitored; hence, the trap can become a sensor. The SIBA trapping technique can be further improved by using a double-nanohole structure.The double-nanohole structure has been shown to give a strong local field enhancement 5,6 . Between the two sharp tips of the double-nanohole, a small particle can cause a large change in optical transmission, thereby inducing a large optical force. As a result, smaller nanoparticles can be trapped, such as 12 nm silicate spheres 7 and 3.4 nm hydrodynamic radius bovine serum albumin proteins 8 . In this work, the experimental configuration used for nanoparticle trapping is outlined. First, we detail the assembly of the trapping setup which is based on a Thorlabs Optical Tweezer Kit. Next, we explain the nanofabrication procedure of the double-nanohole in a metal film, the fabrication of the microfluidic chamber and the sample preparation. Finally, we detail the data acquisition procedure and provide typical results for trapping 20 nm polystyrene nanospheres. Video LinkThe video component of this article can be found at https://www.jove.com/video/4424/ ProtocolThe principle of the SIBA trapping technique is illustrated in Figure 1. Figure 2 is a schematic of the experimental setup.