This dissertation presents novel techniques applied to double nanohole (DNH) optical tweezer with the idea of characterizing and developing capabilities of nanoaperture trap, for single molecule and nanoparticle analysis. In addition, an alternative approach for fabrication of double nanoholes using template stripping is presented. The strength of the DNH tweezer was characterized quantitatively in terms of trap stiffness using two techniques: autocorrelation of Brownian-induced intensity fluctuations and trapping transient. These experimental techniques have, for the first time, been applied to an aperture based trap used for trapping Rayleigh particles in the range of few nanometres. These techniques can be used for calibration and comparison of the aperture based traps among themselves and with other nano-optical tweezers. A statistical technique based on the parameters, time-to-trap and the transient jump due to optical trapping was used for sensing the concentration, size and refractive index of the nanoparticles. The time-to-trap showed a linear dependence with particle size and a -2/3 power dependence with particle concentration, which is in agreement with the diffusion theory based on simple microfluidic considerations. The transient jump in the trapping signal at the trapping instant scales empirically as the Clausius–Mossotti factor for different refractive index particles. The ability of the DNH tweezer to hold small Rayleigh particles with high efficiency and also the increased sensitivity of the transmission signal to the trapped particle during detection makes it favourable for studying the dynamics and interactions of biomolecules. In this direction, the unzipping of the hairpin DNA and its interaction with the tumour suppressor p53 transcription protein, which suppresses the unzipping, were detected using double nanohole optical tweezer. The energy associated with the suppression of unzipping was found to be close to the binding energy of p53-DNA complex. The mutant p53 inability to supress the unzipping of the DNA was also confirmed, showing the ability of the DNH tweezer to distinguish between the mutant p53 and the wild-type. An extraordinary acoustic Raman (EAR) technique was used to study the vibrational modes of ssDNA molecule. The resonant vibrational modes were found to be in the sub 100 GHz range and could be tuned based on the base sequence and length of the DNA strand. The vibrational modes were verified using 1-D lattice vibration theory. Finally, an alternative approach of template stripping for fast and cheaper fabrication of DNH is presented. The template strip process can be used reliably for mass production of gold slide containing DNH’s and also results in cost reduction by 70 % for a single gold slide. Also, we have successfully used this approach to transfer DNH structure to the tip of the cleaved fiber, which would make the DNH tweezer module more compact and scalable. This would open up opportunities for many other applications for single molecule and nanoparticle analysis such as transfer of molecules in-situ to other biomolecular solution for studying their interactions and many others. / Graduate

This thesis presents a route towards a single photon source based on erbium-doped nanocrystals, fabricated with methods that use double nanohole optical tweezers. Single photon sources are an exciting quantum technology and erbium is good candidate as it emits in the low-loss fiber optic C-band, but it is a weak emitter. Double nanohole apertures can be designed with plasmonic resonances to enhance the local electric field. In this thesis, double nanohole optical tweezers are used to isolate and enhance the emission of erbium-doped nanocrystals, with the tuned geometry showing a factor of 50 additional enhancement over rectangular apertures. With the enhanced emission, nanocrystals with discrete levels of erbium emitters are detected and isolated in real-time, based on their level of emission. This real-time process demonstrates a major improvement over typical post-processing approaches. A novel method to anchor nanocrystals in a double nanohole using a photochemical thiol reaction was investigated which yielded 40% of nanoparticles anchoring within 2 μm of the DNH, with 5% inside. This is useful as otherwise the trapping laser must be maintained to keep the nanocrystal in the trap. Another challenge is coupling to an optical fiber, for which a method to combine trapping and coupling was explored. Colloidal pattern transfer is presented as a low-cost fabrication method for nanoaperture optical fiber tweezers, with fiber-based trapping demonstrated using 40 nm polystyrene nanospheres and hexagonal boron nitride. The preliminary results from these methods show great potential, and with further refinement they may lead towards a method to fabricate a low-cost fiber-coupled single photon source based on erbium-doped nanocrystals. / Graduate

Optical trapping is a method which uses focused laser light to manipulate small objects. This optical manipulation can be scaled below the diffraction limit by using interactions between light and apertures in a metal film to localize electric fields. This method can trap objects as small as several nanometers. The ability to determine the properties of a trapped nanoparticle is among the most pressing issues to the utilization of this method to a broader range of research and industrial applications. Presented here are two methods which demonstrate the ability to determine the properties of a trapped nanoparticle. The first method incorporates Raman spectroscopy into a trapping setup to obtain single particle identification. Raman spectroscopy provides a way to uniquely identify an object based on the light it scatters. Because Raman scattering is an intrinsically weak process, it has been difficult to obtain single particle sensitivity. Using localized electric fields at the trapping aperture, the Raman integrated trapping setup greatly enhances the optical interaction with the trapped particle enabling the required sensitivity. In this work, the trapping and identification of 20 nm titania and polystyrene nanoparticles is demonstrated. The second method uses an aperture assisted optical trap to detect the response of a magnetite nanoparticle to a varying applied magnetic field. This information is then used to determine the magnetic susceptibility, remanence, refractive index, and size distribution of the trapped particle. / Graduate / 0544 / 0752 / stevenjones3.14@gmail.com

Non-contact manipulation technologies present a useful and powerful means of handling particles or cells. Such techniques are of interest in regenerative medicine applications, and in particular the scalability of these techniques is an area of active research. Optical trapping is a precise and dextrous method of manipulating particles with the forces exerted by a laser beam, while acoustic trapping is a scalable technique capable of exerting a force on particles through standing wave resonance. These complimentary modalities can be utilised in a hybrid system to give a resultant technique that borrows from the strengths of each individual method. In this thesis, methods of force balancing, using optics and acoustics, are explored, both independently and in combination with each other. A technique for 3D acoustic trapping in glass capillaries is shown, utilising the two pairs of opposing channel walls and the air-water interfaces of two air bubbles as acoustic reflectors. Standing waves set up between these surfaces show discrete acoustic trapping sites for varying lengths of fluid cavity. A method of optical radiation force balancing is observed in a 3D potential energy landscape, using similar principles as seen in particle trapping with counter-propagating beams. Tuning of the radiation force balance in this system allows particles to, instead of being pinned to the surface by the radiation force from the optical pattern, become localised at discrete planes of trapping sites throughout the fluid volume. A hybrid force balance separation method using the optical and acoustic forces is devised using a single laser beam as the primary deflection mechanism with acoustic trapping providing both localisation and a force balance with the optics. Separation of different sized particles is observed, with larger scale optical deflection mechanisms and their resultant thermal effects demonstrated.

This thesis presents the optical trapping of various nanometric particles (both biological and non-biological) and methods that can be used to extract information about the trapped particle from the signal transmitted through a nanoaperture trap. These methods are used to detect the excitation of vibrational modes in trapped particles due to the presence of a beat signal between two tunable trapping lasers and the molecular weight of the particle by examining the transmitted signal. Optical trapping has long been used to trap ever smaller particles in gentle non-destructive ways. In its infancy, only the optical trapping of micron sized particles was feasible. Due to various limitations, changes to the optical trapping scheme were needed to push its limits into the nanometric regime. Nanoaperture assisted optical trapping has allowed for the optical trapping of particles as small as 5 nm in diameter. By making use of specially chosen nanoapertures in gold films higher trapping strengths with lower incident laser powers have become possible. While this is an accomplishment in and of itself there are several issues associated with working with such small systems. Most notably, the ability to observe such systems is very limited. Traditional optical trapping of micron sized particles could make easy use of optical inspection, however in the nanometric regime this is not possible. It has since become a focus of the trapping community to find sophisticated ways to use the limited data available to probe these systems and their trapped targets. Once a particle is trapped the only information available about the particle is contained in the signal transmitted through the nanoaperture. The first main area of research in this thesis covers using this information to extract the molecular weight of the trapped particles for identification. In the same vein, Raman has been a tool widely used in the past to identify and probe systems of large ensembles of particles. While this is incredibly effective in some situations, it is not effective at the single particle limit. To form an analog that can be used within an optical trapping setup a new method of exciting Raman active vibrational modes with twin trapping lasers is presented. The low wavenumber vibrational spectra are presented for several different particles as well as a wide array of globular proteins. / Graduate

No description available.

Nanoaperture optical tweezers are emerging as useful 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 platform for single molecule studies without the need to use tethers or labeling. This thesis gives a general description of conventional optical tweezers and how they are limited in terms of their capability to trapping biological molecules. It also looks at nanoaperture-based optical tweezers which have been suggested to overcome the limitations of conventional optical tweezers. The thesis then focuses on the double nanohole optical tweezer as a tool for trapping biological molecules and studying their behaviour and interactions with other molecules. The double nanohole aperture trap integrated with microfluidic channels has been used to detect single protein binding. In that experiment a double-syringe pump was used to deliver biotin-coated polystyrene particles to the double nanohole trapping site. Once stable trapping of biotin-coated polystyrene particle was achieved, the double-syringe pump was used to flow in streptavidin solution to the trapping site and binding was detected by measuring the transmission through the double nanohole aperture. In addition, the double nanohole optical tweezer has been used to observe the real-time dynamic variations in protein-small molecule interaction (PSMI) with the primary focus on the effect of single and multiple binding events on the dynamics of the protein in the trap. Time traces of the bare form of the streptavidin showed slower timescale dynamics as compared to the biotinylated forms of the protein. Furthermore, the double nanohole aperture tweezer has been used to study the real-time binding kinetics of PSMIs and to determine their disassociation constants. The interaction of blood protein human serum albumin (HSA) with tolbutamide and phenytoin was considered in that study. The dissociation constants of the interaction of HSA with tolbutamide and phenytoin obtained using our technique were in good agreement with the values reported in the literature. These results would open up new windows for studying 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. / Graduate

Arthur Ashkin's Nobel Prize-winning single-beam gradient force optical tweezers have revolutionized research in many fields of science. The invention has enabled various atomic and single molecular studies, proving to be an essential tool for observing and understanding nature at the nanoscale. This thesis showcases the uniqueness of single-beam gradient force traps and the advances necessary to overcome the limitations inherent in conventional techniques of optical trapping. With decreasing particle sizes, the power required for a stable trap increases and could potentially damage a particle. This is a significant limitation for studying biomolecules using conventional optical traps. Plasmonic nanoaperture optical trapping using double-nanohole apertures is introduced as a solution to overcoming these limitations. Achievements in double-nanohole optical trapping made possible by the pioneering work of Gordon et. al are highlighted as well. This thesis focuses on the advances in nanoaperture fabrication methods and improvements to experimental techniques adopted in single molecular optical trapping studies. The technique of colloidal lithography is discussed as a cost-effective high-throughput alternative method for nanofabrication. The limitation in using this technique for producing double-nanohole apertures with feature sizes essential for optical trapping is analyzed. Improvements to enable tuning of aperture diameter and cusp separation is one of the main achievements of the work detailed in this thesis. Furthermore, the thesis explains the modified fabrication process tailor-made for designing double-nanohole apertures optimized for optical trapping. Transmission characterization of various apertures fabricated using colloidal lithography is carried out experimentally and estimated by computational electrodynamics simulations using the finite-difference time-domain (FDTD) method. Optical trapping with double-nanohole apertures fabricated using colloidal lithography is demonstrated with distinct results revealing trapping of a single polystyrene molecule, a rubisco enzyme and a bovine serum albumin (BSA) protein. / Graduate

There are a growing variety of research fields requiring non-contact micro- manipulation. An increasing number of these fields are turning to optical tweezers as a solution, owing to their high spatial and temporal resolution. Optical tweezers have the ability to quantitively exert and measure forces on the piconewton scale, a convenient force scale for soft biological materials, and are hugely versatile due to the wide assortment of beam shaping techniques that can be employed. The work in this thesis can be broadly divided into two main themes: that quantifying the optical trapping forces in shaped beams; and bringing control and simplification of complex systems to non-expert users who may utilise optical tweezers as part of interdisciplinary collaborations. Static beam shaping is used to generate a conically refracted optical trap and the trapping properties are characterised. It is shown that trapping in the lower Raman spot gives full, 3D gradient trapping, while the upper Raman spot allows for particle guiding due to its levitation properties. Particles in the Lloyd/Poggendorff rings experience a lower trap stiffness than particles in the lower Raman spot but benefit from rotational control. Dynamic beam shaping techniques are exploited for the simplification of complex systems through the development and testing of the HoloHands program. This software allows a holographic optical tweezers experiment to be controlled by gestures that are detected by a Microsoft Kinect. Multiple particle manipulation is demonstrated, as well as a calibration of the tweezers system. Application of trapping forces is demonstrated through an examination of integrin – ligand bond strength. Both wild type effector T cells and those with a kindlin-3 binding site mutation similar to that found in neutrophils from Leukocyte Adhesion Deficiency sufferers are investigated. Through the use of back focal plane interferometry, a bond rupture force of (17.9 ± 0.6) pN at a force loading rate of (30 ± 4) pN/s, was measured for single integrins expressed on wild type cells. As expected, a significant drop in rupture force of bonds was found for mutated cells, with a measured rupture force of (10.1 ± 0.9) pN at the same pulling rate. Therefore, kindlin-3 binding to the cytoplasmic tail of the β2-tail directly affects bond strength of single integrin-ligand bonds. An experimental system for studying these cells under more physiologically relevant conditions is also presented. Additionally, a low-cost optical micromanipulation system that makes use of simple microfabricated components coupled to a smartphone camera for imaging is proposed and demonstrated. Through the layering of hanging droplets of polydimethylsiloxane (PDMS) on microscope coverslips, lenses capable of optical trapping are created. Combination of PDMS with Sudan II dye led to the fabrication of long pass filters. An extension of this low-cost system into the life sciences is proposed through the adaptive use of bubble wrap, which allows for the culturing of cells in a chamber compatible with optical trapping.

This thesis presents optical trapping of various single nanoparticles, and the method for integrating the optical trap system into a microfluidic channel to examine the trapping stiffness and to study binding at the single molecule level. Optical trapping is the capability to immobilize, move, and manipulate small objects in a gentle way. Conventional trapping methods are able to trap dielectric particles with size greater than 100 nm. Optical trapping using nanostructures has overcome this limitation so that it has been of interest to trap nanoparticles for bio-analytical studies. In particular, aperture optical trapping allows for trapping at low powers, and easy detection of the trapping events by noting abrupt jumps in the transmission intensity of the trapping beam through the aperture. Improved trapping efficiency has been achieved by changing the aperture shape from a circle; for example, to a rectangle, double nanohole (DNH), or coaxial aperture. The DNH has the advantage of a well-defined trapping region between the two cusps where the nanoholes overlap, which typically allows only single particle trapping due to steric hindrance. Trapping of 21 nm encapsulated quantum dot has been achieved which shows optical trapping can be used in technologies that seek to place a quantum dot at a specific location in a plasmonic or nanophotonic structure. The DNH has been used to trap and unfold a single protein. The high signal-to-noise ratio of 33 in monitoring single protein trapping and unfolding shows a tremendous potential for using the double nanohole as a sensor for protein binding events at a single molecule level. The DNH integrated in a microfluidic chip with flow to show that stable trapping can be achieved under reasonable flow rates of a few µL/min. With such stable trapping under flow, it is possible to envision co-trapping of proteins to study their interactions. Co-trapping is achieved for the case where we flow in a protein (bovine serum albumin – BSA) and co-trap its antibody (anti-BSA). / Graduate / 0544 / 0752 / oskuie@uvic.ca