Nanopores are apertures of nanometric dimensions in an insulating matrix. They are routinely used to sense and measure properties of single molecules such as DNA. This sensing technique relies on the process of translocation, whereby a molecule in aqueous solution moves through the pore under an applied electric field. The presence of the molecule modulates the ionic current through the pore, from which information can be obtained regarding the molecule's properties. Whereas the electrical properties of the nanopore are relatively well known, much less work has been done regarding their fluidic properties. In this thesis I investigate the effects of fluid flow within the nanopore system. In particular, the charged nature of the DNA and pore walls results in electrically-driven flows called electroosmosis. Using a setup which combines the nanopore with an optical trap to measure forces with piconewton sensitivity, we elucidate the electroosmotic coupling between multiple DNA molecules inside the confined environment of the pore. Outside the pore, these flows produce a nanofluidic jet, since the pore behaves like a small electroosmotic pump. We show that this jet is well-described by the low Reynolds number limit of the classical Landau-Squire solution of the Navier-Stokes equations. The properties of this jet vary in a complex way with changing conditions: the jet reverses direction as a function of salt concentration, and exhibits asymmetry with respect to voltage reversal. Using a combination of simulations and analytic modelling, we are able to account for all of these effects. The result of this work is a more complete understanding of the fluidic properties of the nanopore. These effects govern the translocation process, and thus have consequences for better control of single molecule sensing. Additionally, the phenomena we have uncovered could potentially be harnessed in novel microfluidic applications, whose technological implications range from lab-on-a-chip devices to personalised medicine.

The material that is covered in this thesis concerns the calibration and application of a set of optical tweezers to be used for one- and two-point micro-rheology experiments on hard sphere colloidal suspensions. The colloidal suspensions that were used in this study were all quasi-monodisperse density- and refractive index-matched PMMA particles that had a radii, a = 0:90 ± 0:05μm or a = 0:86 ± 0:07 for one-point microrheology experiments and radii a = 0:90 ± 0:05μm or a = 0:133 ± 0:010μm for the two-point micro-rheology experiments. By collecting the forward scattered light from a single optically trapped particle the particle's displacements in time were used to determine passive microviscosity, η(Passive) μ , for colloidal suspension in the range of 0:10 < Ø < 0:57 and comparison with literature data has been made and agreement found. Actively dragging an optically trapped particle through suspensions with volume fractions of the same range has yielded the active microviscosities, η(Active) μ , for both high and low shear regimes, displaying shear thinning behaviour. Comparison to literature data has been made and agreement found as well. Collecting the forward scattered light from two optically trapped particles has been used to determine the cross-correlated motion of the two particles in bare solvent and in suspensions with volume fraction Ø = 0:02. The friction coefficients ξ1;1 and ξ1;2 were extracted from the cross-correlated motion of the particles and agreement was found with theoretical predictions for bare solvent only. The suspensions with volume fraction Ø = 0:02 were found to have a friction coefficient ξ1;1 that was greater than what theory predicted with the suspension with bath particles a = 0:90 ± 0:05μm had the greater magnitude. The magnitude ξ1;2 was found to decrease for the suspension with bath particles of radius a = 0:133 ± 0:010μm and to increase for the suspension with bath particles a = 0:90 ± 0:05μm.

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

Nanopore-based resistive pulse sensing represents an important class of single-molecule measurements. It provides information about many molecules of interest (i.e. DNA, proteins, peptides, clusters, polymers, etc.) without the need for labeling. Two experiments that are especially well suited for studying with nanopore sensors are DNA sequencing and DNA-protein force measurements. This thesis will describe progress that has been made in both areas. DNA sequencing has become an active area of research for stochastic single-molecule sensing, with many researchers striving for the ultimate goal of single-molecule de novo DNA sequencing. One intriguing method towards that goal involves the use of a DNA exonuclease or polymerase enzyme, which when attached close to the mouth of a pore, leads to cleavage of individual DNA nucleotide bases for loading into the pore for sensing. Though this method seems promising, the end goal has been elusive because the nucleotide motion is dominated by diffusion over the relevant length scales. This limits the likelihood of the cleaved nucleotide entering the pore to be characterized. The first part of this thesis will describe a method for addressing this problem, where it is shown that increasing the nucleotide capture probability can be achieved by lowering the bulk diffusion coefficient relative to the pore diffusion coefficient. The second part of this thesis will describe the design and implementation of a new type of sensor that combines a biological nanopore experimental apparatus with optical tweezers. The goal of this apparatus is to develop a means to independently measure the force on a charged molecule inside of the pore. The setup will be thoroughly described, and preliminary results showing that it is possible to optically trap a micron sized bead within a few microns of an isolated biological nanopore while simultaneously making current measurements through that pore will be presented. This will enable force measurements on DNA molecules tethered to the bead, which opens the door for the study of molecular force interactions between DNA and biological nanopores, DNA-bound protein interactions that cause diseased states, and controlled translocation of DNA through biological nanopores.

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 dissertation describes the development of a device capable of suspending a microscale object in a controlled flow. The uPIVOT is a system integrating two laser-based techniques: micron particle image velocimetry (uPIV) and optical tweezers (OT). The OT allows the suspension and manipulation of micron-sized objects such as microspheres or biological cells. uPIV provides imaging of the suspended object and velocity measurements from which fluid induced stresses can be determined. Using this device, we measured fluid velocities around an optically suspended polystyrene microsphere (an experimental first) and studied the interaction between two particles suspended in a uniform flow. The results were consistent with theoretical low Reynolds number, Newtonian flow predictions. Additionally, we analyzed a single cell's mechanical response to a controlled and measurable multiaxial external force (fluid flow) without the cell being physically attached to a surface. The cell's mechanical response was monitored by observing its morphology and measuring its deformation. The results show significant deformations of optically suspended cells at substantially smaller stresses than previously reported and demonstrate the opportunity to optically distinguish a cell by its trapping efficiency. These initial applications of the uPIVOT demonstrate the potential of this unique device as a research tool for novel studies in the fields of fluid/particle(s) interactions, non-Newtonian fluid mechanics, and single cell biomechanics.

In the late 19th and early 20th centuries famous scientists like Boltzmann, Loschmidt, Maxwell and Einstein tried, unsuccessfully, to find the link between the time-reversible equations of motion of individual molecules and irreversible thermodynamics. The solution to this puzzle was found in 1993, and the link is now known as the Fluctuation Theorem (FT). In the decade that followed theory and computer simulation tested the FT and, in 2002, an experiment indirectly demonstrated the FT.¶ This thesis describes original experiments that demonstrate the FT directly using Optical Tweezers. A related expression, known as the Kawasaki Identity, is also experimentally demonstrated. These experimental results provide a rigorous demonstration that irreversible dynamics can be obtained from a system with time-reversible dynamics.

Riboswitches are gene regulatory elements found in messenger RNA that function by changing structure upon the binding of a ligand to an aptamer domain. Single adenine-binding pbuE riboswitch aptamer RNAs were unfolded and refolded co-transcriptionally using optical tweezers for single molecule force spectroscopy. The kinetic and energetic properties of distinct folding intermediates were characterised with and without the binding of adenine. These observed intermediates were related to structural elements of the aptamer, which were found to fold sequentially, in a transcriptionally independent manner. The mechanical switch underlying the regulatory action of the riboswitch was observed directly (adenine stabilisation of the weakest helix), and the energy landscape for the folding was reconstructed. The construction of a dual-beam optical trap with separate detection and trapping laser beams manipulated and focused into a rigid, modified inverted microscope is also described. This instrument aims to achieve ngstrm-level resolution through careful design to reduce noise.

This study proposes a novel microfluidic system based on a computer controlled digital image processing (DIP) technique and optical tweezers for automatic cell/microparticle recognition, counting and sorting in a continuous flow environment. In the proposed system, the cells/microparticles are focused electrokinetically into a narrow sample stream and are then driven through the region of interest (ROI), where they are recognized and traced in real time using a proprietary DIP system. Synchronized control signals generated by the DIP system are then used to actuate a focused IR laser beam to displace the target cells from the main sample stream into a neighboring sheath flow, which carries them to a downstream collection channel where they are automatically counted. The proposed approach makes possible the continuous sorting and counting of cell samples without the need for any moving parts or embedded transducers. The experimental results show that the proposed system is capable of sorting 5 £gm or 10 £gm PS bead from a mixture of 5 £gm and 10 £gm samples in the flow speed 300 £gm/sec. The proposed system provides a simple, low-cost, high-performance solution for cell manipulation in microfluidic devices.