This thesis presents optofluidic nanostructures for analyte transport, concentration and sensing. This work was part of a larger collaborative project between the BC Cancer Agency and the departments of Chemistry, Electrical and Mechanical Engineering at the University of Victoria. In this work, arrays of nanoholes are used as optofluidic platforms for sensing, combining the characteristics of these nanostructures for both fluidic transport and plasmonic (optical) sensing. Two different modes are considered: flow-over mode, where the sample solution containing the analyte flows on top of the nanohole arrays, and a novel flow-through mode, where the nanoholes are used as nanochannels, enabling solution transport and analyte sieving. Flow-through nanohole array operation and sensing is first demonstrated, offering a six-fold improvement in sensor response compared to established flow-over sensing formats. Through a subsequent theoretical scaling analysis and computational analyses, the benefits of the flow-through nanohole sensing format are further quantified. A first analysis is dedicated to study the enhancement offered by the flow-through operation mode using a mass transport approach. A second analysis offers an ample study of benefits and limitations of the flow-through nanostructure operation using the combination of mass transport and binding kinetic parameters for different analytes with characteristics of clinical relevance. The mass transport analysis indicates much higher analyte collection efficiency (~ 99%) offered by the flow-through mode, compared to the flow-over platform (~ 2%). The analysis including both mass transport and binding kinetics demonstrate up to 20-fold improvement in response time for typical biomarkers. This thesis also presents the use of the flow-through optofluidic platform as an active analyte concentrator. In combination with a pressure bias, an electric field is used to concentrate electrically charged analyte for subsequent sensing. Fluorescein enrichment of 180-fold in 60 s was achieved, and 100-fold enrichment and simultaneous surface plasmon resonance (SPR) sensing of a protein (bovine serum albumin, BSA) was demonstrated. These experiments represent the first active utilization of a nanohole metallic layer as an electrode, and the first demonstration of a photonic nanostructure achieving both concentration and sensing of analytes. Towards the integration of optofluidic nanostructures into microfluidic environments for portable lab-on-chip diagnostic systems, this dissertation also includes the development of two nanohole array based sensing systems with simple flow-over operation. The first system consisted of a hand-held device with a dual-wavelength light source to increase the spectral diversity. The second system consisted of nanohole arrays integrated with a microfluidic concentration gradient generator for the detection and quantification of ovarian cancer antibody and antigen. Additionally, this dissertation includes a novel technique to actuate liquids in microchannels through ground-directed electric discharges. Experiments demonstrate average fluid velocities on the order of 5cm/s and applicability of the technique in serpentine channels, for on-demand fluid routing, to initiate a mixing process, and through an on-chip integrated microelectrode. / Graduate

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

Since Ebbesen's seminal work in 1998 observing extraordinary optical transmission (EOT) through nanohole arrays, much research has focused on the role of surface plasmon polaritons (SPPs) in EOT. While the energy and momentum conditions have become clear, no consensus has been reached on the role of incident light polarization. This study presents a simple model that captures Bloch-SPP excitation, including the role of polarization, in general periodic plasmonic structures. Our model predicts that under certain conditions polarization conversion should occur in EOT light transmitted through the nanohole array. We experimentally measure polarization conversion in EOT and compare the experimentally obtained results to the predictions of our model. Using numerical simulations, we tie the far field experimental results to the near field underlying physics described by our model. In using polarization conversion to provide evidence supporting our model, we also establish a novel approach to achieving polarization conversion based on SPPs instead of hole shape or other techniques in literature, and present reasons why this approach to achieving polarization conversion may be better suited for applications in biomedical sensing and optical elements. / Master of Science / In 1998, Ebbesen et al¹ observed that when light is shown on a metal nanofilm perforated with nanoholes more light appears on the other side of the metal film than was incident on the nanoholes. The unexpectedly high transmission of light through the nanohole array was termed extraordinary optical transmission (EOT), and quickly found applications in diverse fields such as biomedical sensing^13,14, energy harvesting^12,31, and nonlinear optics^12–14,24 . As the importance of EOT in applications became clear, interest developed in understanding the fundamental physics involved. Over the next 20 years, researchers showed that the incident light (made up of electromagnetic fields) excites conduction electrons on the surface of the metal film¹¹ . Specifically, the light and the electrons couple to form quasiparticles known as surface plasmon polaritons (SPP) which propagate along the surfaces of the metal film. The SPPs on the back surface of the metal film then radiate free space transmitted light, which is observed as EOT. However, much of the physics involved how SPPs mediate EOT has remained unclear. The first focus of this work is theoretical, presenting a microscopic model for SPP mediated EOT. In contrast to many groups which aim to characterize SPPs from their far field properties, our model focuses on the near field microscopic physics and presents the far field properties as a consequence of the near field physics. Since the near field cannot be probed iv experimentally, we use numerical simulations to both verify our model’s predictions in the near field and predict the properties that should be measured in the far field. The second focus of this work is more applications driven. We notice that our model predicts that under certain conditions SPPs should cause a phenomenon known as polarization conversion to occur, which is when the polarization of the transmitted light is different from the polarization of the incident light. We experimentally measure the predicted polarization conversion, thereby providing substantial experimental evidence in support of our theoretical model. Our novel approach to achieving polarization conversion based on the behavior of SPPs differs substantially from the approaches in literature (usually based on hole shape²⁴). We present the reasons why our SPP-based approach to achieving polarization conversion is more robust to fabrication imperfections than the conventional approaches, and describe how our approach could affect various applications.

Proteomic studies at the single molecular level could provide better understanding of the protein’s behaviour and the effects of its interactions with other biomolecules. This could have an impact on drug development methods, disease diagnosis, and targeted therapy. Aperture assisted optical trapping is a proven technique for isolating single proteins in solution without the use of tethers or labels, and without denaturing them. Thus enabling studies of protein-protein interactions, protein-small molecule interactions, and protein-DNA interactions. In this work, double nanohole (DNH) optical tweezers were used to analyze the protein composition of heterogeneous mixtures. The trapped proteins were grouped by molecular mass based on two metrics: standard deviation of the trapping laser intensity fluctuations, and the time constant of the autocorrelation function of these fluctuations. The quantitative analysis is demonstrated first for two separate standard-size proteins, then for a mixed solution of both. Finally, the approach is applied to real unprocessed egg white solution. The results correspond well with the known protein composition of egg white found in the literature. The DNH optical tweezers’ ability to distinguish proteins in unpurified heterogeneous mixtures, can progress this technique to the next level, allowing for single biomolecular studies of unprocessed physiological solutions like blood, urine, or saliva. / Graduate

Different types of plasmonic nanostructures are proposed and examined experimentally and theoretically, with a view towards sensing applications. First, a self-assembly approach was developed to create arrays of well-ordered glass-supported gold nanoparticles (AuNPs) with controllable particle size and inter-particle spacing. Then, a periodic array of gold nano-disks (AuNDs) supported by a Bragg reflector was proposed and examined in a search for Fano resonances in its optical response. Arrays of heptamer-arranged nanoholes (HNH) in a thin gold film were also proposed and explored theoretically and experimentally, revealing a very rich spectrum of resonances, several exhibiting a Fano lineshape. A commercial implementation of the vectorial finite element method (FEM) was used to model our plasmonic structures. Taking advantage of the periodic nature of the structures, a unit cell containing a single element was modelled. The transmittance, reflectance or absorbance spectra were computed, and the associated electromagnetic fields were obtained by solving the vector wave equations for the electromagnetic field vectors throughout the structures, subject to the applicable boundary conditions, and the applied source fields. The sensing performance of the structures, based on the bulk sensitivity, surface sensitivity and figure of merit (FOM) was calculated. First, a novel bottom-up fabrication approach was applied (by our collaborators) to form a periodic array of AuNPs with controllable size over large areas on SiO2 substrates. In this method, self-assembly of block copolymer micelles loaded with metal precursors was combined with a seeding growth route to create ordered AuNPs of desired size. It was shown that this new fabrication method offers a new approach to tune the AuNP size and edge-to-edge inter-particle spacing while preserving the AuNP ordering. The optical characteristics of the AuNP arrays, such as their size, interparticle spacing, localized surface plasmon resonance (LSPR) wavelength, and bulk sensitivity, were examined, numerically and experimentally. This proposed novel fabrication method is applicable for low-cost mass-production of large-area arrays of high-quality AuNPs on a substrate for sensing applications. Then, we proposed and examined the formation of Fano resonances in a plasmonic-dielectric system consisting of uncoupled gold nano-disk (AuND) arrays on a quarter-wave dielectric stack. The mechanism behind the creation of Fano resonances was explained based on the coherent interference between the reflection of the Bragg stack and the LSPPs of the AuNDs. Fano parameters were obtained by fitting the computational data to the Fano formula. The bulk sensitivities and figure of merit of the Fano resonances were calculated. This plasmonic structure supports Fano resonances with a linewidth around 9 nm which is much narrower than the individual AuND LSPP bandwidth ( 80 nm) and the Bragg stack bandwidth ( 100 nm). Supporting Fano resonances with such a narrow linewidth, the structure has a great potential to be used for sensing applications. Also, this metallic-dielectric nanostructure requires no near-field coupling between AuNDs to generate the Fano resonances. So, the AuNDs can be located far enough from each other to simplify the potential fabrication process. The optical properties of HNH arrays on an SiO2 substrate were investigated, numerically and experimentally. Helium focused ion beam (HeFIB) milling was applied (by Dr. Choloong Hahn) to fabricate well-ordered and well-defined arrays of HNHs. Transmittance spectra of the structures were obtained as the optical response, which exhibits several Fano resonances. Then, the mechanism behind the formation of the Fano resonances was explained, and the sensing performance of the structure was inspected by measuring the bulk sensitivities. This array of nanohole cluster is exciting because it supports propagating SPPs and LSPPs, and also Wood’s anomaly waves, which makes the optical response very rich in excitations and spectral features. Also, as a periodic array of sub-wavelength metallic nanoholes, the system produces extraordinary optical transmission - highly enhanced transmission through (otherwise) opaque metallic films at specific wavelengths, facilitating measurement acquisition in transmission.

No description available.

The main objective of this thesis is to present a simple and robust hands-on technology for the fabrication of a microfluidic chip in a laboratory. The purpose of this new technology is to replace the existing PDMS based microfluidic chip used for optical trapping of diverse single nano particles. It also lists the different fabrication methods attempted and the successful integration of this chip to the optical trap system which is used to study binding at the single molecular level. Microfluidics is a quickly growing field which deals with manipulating the fluids in channels whose dimensions are few tens of micrometers. Its potential has a major impact on fields like chemical analysis and synthesis techniques, biological analysis and separation techniques, and optics and information technology. One of the main application of these microfluidic chips is in optofluidics, which is the emerging field of integrated photonics with fluidics. This provides freedom to both fields and permits the realization of optical and fluidic property. It requires small volumes of fluids and connections and eventually performs better than conventional methods of robotic fluid handling. Here, the microfluidic chip is targeted for optical trapping with double nano-hole aperture to trap a single protein. The double nanoholes integrated with this microfluidic chip show that stable trapping can be achieved below flow rates of few μL/min. This has provided many possibilities of co-trapping of proteins and study their interactions. / Graduate

Recent advances in nanofabrication techniques have opened up new avenues and numerous possible applications in both nanoscale electrochemistry and analytical nanoscience by enabling the fabrication of reproducible nanoelectrodes with different new geometries. Nanoelectrodes exhibit advantages including enhanced mass transport, higher current densities, improved signal-to-noise ratios, and lower ohmic drop. In this dissertation, the use of nanoelectrodes in the electrochemical response properties investigations or in the spectroelectrochemical studies is the unifying factor among all the chapters. First (in Chapter 4), we presented a direct comparison between the electrochemical characteristics of two finite nanoelectrodes arrays with different geometries: 6 × 6 recessed nanodiscs and nanorings microarrays. Using computational methods, it was demonstrated that the electrode geometry’s parameters have a drastic influence on the mass transport properties of the nanoelectrodes. The results presented here are the first combination of experimental and numerical studies that elucidate the transport on nanoring electrode arrays. The comparison of the electrochemical behavior between nanostructures using full 3D simulations is also unique. Second, we have provided a comprehensive numerical study on the redox cycling performance properties of a 6 × 6 recessed nanorings-ring electrode array configuration. The simulation results were in good agreement with the experimental data. After validating the model against experiments, a comprehensive computational investigation revealed avenues to optimize the performance of the structure in terms of geometric parameters and scan rates. The second half of this dissertation is comprised of the spectroelectrochemical studies. The combination of surface plasmon resonance with electrochemistry presents new paths to investigateredox reaction events at the electrode surface since it brings an additional dimension to the classical electrochemical approaches. Third, we have reported a novel active plasmonic device based on a new switching mechanism for the nanohole electrodes array to bridge between photonics and electronics at nanoscales. The inner surfaces of the nanohole electrodes in the array were coated with an electroconductive polymer, polypyrrole, (PPy). Then, it was shown that light transmitted through the PPy- modified nanohole electrodes can be easily tuned and controled by applying an external potential. We were also able to switch on and off the transmitted light intensity through the modified nanohole arrays by potential steps, demonstrating the potential of this platform to be incorporated into optoelectronic devices. Finally, we have fabricated larger area plasmonic periodic nanopillar 3D electrodes using a rapid, high-throughput, and cost-effective approach: the laser interference lithography. Then, the electrochemical behavior of these electrodes was investigated both experimentally and computationally. The properties were ‘compared with a flat electrode with an equivalent geometric area. Afterward, we have successfully probed the changes in the concentration of a reversible redox pair near the electrode surface induced by various applied potentials, in an in-situ EC-SPR experiment. / Graduate

Extracellular vesicles (EVs) are lipid bilayer encapsulated nanoparticles which have emerged as an excellent source of biomarkers for multiple diseases, including cancer. However, they are highly heterogeneous in their molecular compositions which remains a major challenge hindering the utilization of their biomarker potential. A single-EV analysis is essential to both discovery and detect EVs that carry disease-specific signature. In this work, we designed plasmonic nanohole array for capturing single EVs and perform fluorescence detection of their membrane proteins by exploiting plasmonic amplification of the fluorescence signal. The design of the array was optimized using COMSOL Multiphysics-based simulation. Nanohole arrays with three different periodicities were fabricated on aluminum thin film on glass substrate. The substrates were then functionalized with three different methods for investigation of antibody-free capturing techniques, which are electrostatic interaction, hydrophobic interaction, and size-selective capturing. After surface functionalization with each of the techniques, genetically engineered EVs expressing mNeonGreen (mNG) were incubated and their capture efficiency were compared. The presence of single-EVs within plasmonic nanoholes was verified through both fluorescence analysis and atomic force microscopy (AFM). Fluorescence intensities of mNG-EVs recorded with the plasmonic chip with different periodicities showed intensity variations in agreement with the simulation results. Furthermore, the EVs were immunostained with R-phycoerythrin (R-PE) conjugated CD-9 to demonstrate the possibility of general and multimarker fluorescence detection. In a separate experiment, DOPC liposomes were synthesized and their deformability was analyzed by using AFM. The nanohole array provides a basis for a future platform of EV analyses, promising to capture the signature arising from low expressing proteins. / Extracellulära vesiklar (EV) är lipadmembranförsedda nanopartiklar som har dykt upp som en utmärkt källa till biomarkörer för flera sjukdomar, däribland cancer. De är dock mycket heterogena i sina molekylära sammansättningar, vilket skapar en stor utmaning och hindrar utnyttjandet av deras potential som biomarkörer. EV-analys på enpartikelnivå är nödvändig både för att upptäcka och detektera vesiklar som har en sjukdomsspecifik signatur. I detta arbete designade vi en plasmonisk uppsättning av nanohål för att fånga enstaka EVs och utföra fluorescensdetektion av deras membranproteiner genom att utnyttja plasmonisk amplifiering av fluorescenssignaler. Designen av uppsättningen optimerades med hjälp av COMSOL Multiphysics-baserad simulering. Nanohålsuppsättningar med tre olika periodiciteter tillverkades på tunn aluminiumfilm på glassubstrat. Substraten funktionaliserades sedan enligt tre olika metoder för undersökning av antikroppsfria bindningsmetoder. De tre metoderna är elektrostatisk interaktion, hydrofob interaktion och storleksselektiv bindning. Efter ytfunktionalisering med var och en av teknikerna inkuberades vesiklar genetiskt modifierade att uttrycka mNeonGreen (mNG) och deras bindningseffektivitet jämfördes. Närvaron av individuella EVs i plasmoniska nanohål bekräftades genom både fluorescensmikroskopi och atomkraftsmikroskopi (AFM). Fluorescensintensiteter för mNG-EVs registrerades med plasmonchipet med olika periodiciteter och visade intensitetsvariationer i överensstämmelse med simuleringsresultaten. Dessutom immunfärgades vesiklarna med R-fykoerytrin (R-PE) konjugerad CD-9 för att påvisa möjligheten till allmän och multimarkör fluorescensdetektion. I ett separat experiment syntetiserades DOPC-liposomer och deras deformerbarhet analyserades med AFM. Nanohåluppsättningen lägger grund för en framtida plattform för EV-analys, som lovar att fånga signaturen som uppstår från låguttryckande proteiner.