Active plasmonic devices have recently emerged as plasmonic/nanophotonic technologies with tunable optical characteristics. However, electro-optic effects in plasmonic metals are weaker than their photonic counterpart since plasmonic materials such as Au and Ag have extremely high electron densities, resulting in the effective screening of the externally applied electrical fields. In this thesis, to overcome these limitations, I introduce electric field tunable loading of plasmonic nanostructures and demonstrate wireless electric-field sensors for electrophysiology and flat optic modulators.

First, I introduce an ultrasensitive and extremely bright nanoscale electric-field probe overcoming the low photon count limitations of existing optical field reporters. I demonstrate that electrochromic loading of plasmonic nanoantenna allows us to realize optical field probes with 10 − 100 million times larger cross sections than fluorescence molecules and ∼ 3000- fold enhanced sensitivities than conventional plasmonic nanoantennas. Using our nanoprobes, I realize optical detection of electric-field dynamics from diffraction limited spots and high- speed recordings with sub-millisecond temporal response times (∼ 191 μs). Furthermore, I demonstrate label-free optical recording of electrogenic activity of cardiomyocyte cells with low-intensity light. Our nanoprobes offering high spatiotemporal resolution measurement ca- pability opens the door to label-free electrophysiological studies with photons.

Finally, I introduce a subwavelength-thick (< 250 nm) nano-electrochromic flat optic modulator that utilizes the electrochromic modulation mechanism in combination with highly dispersive Fano resonances in extraordinary light transmission (EOT) effect. We demonstrate electro-optic switching capability that can simultaneously deliver remarkably high modulation depth (∼ 17.6 dB) and high speed ( ∼ 500 μs, ∼ 2 kHz) switching capability beyond the video rates. The field-effect flat optic modulator shown here paves the way to the advancement of technologies based on electrochromic soft materials.

Abstract

Plasmonic Zero Waveguide Modes and Radiative Decay Engineering of Quantum

Emitters

by

David Cota Gonzalez

This paper documents an investigation of plasmonic behavior and the application of plasmonic phenomena with the intended purpose of fabricating and manufacturing a novel zero-mode waveguide (ZMW). The conventional ZMW can focus incident light to tiny volumes on the order of zeptoliters (zL) in magnitude. This ability to focus light beyond the diffraction limit is promising for single molecule detection (SMD) particularly when investigating a sample of µM concentrations or beyond.

While the prospect of having the excitation light intensity focused to zL volumes is desirable for SMD, the observer still requires that the intensity of light emissions from the molecule under investigation be sufficiently larger than the noise from the excitation light source, any fluorophores beyond the observation volume and other light emissions which may disrupt the sample study. The solutions under investigation may be susceptible to quenching; thus, the waveguide must be designed so as to apply a maximum intensity into a volume of minimal size all while detecting the enhanced emissions without damaging the sample.

A conventional ZMW consisting of a 50nm diameter cylindrical etch of a 100nm thick Aluminum (Al) metal film lying atop a glass substrate produces zL-effective observation volumes.[16] We proceeded to design and simulate ZMW structures consisting of two or more metallic columns standing in a rectangular etch of a heterogeneous stack — Gold and Aluminum — atop a glass substrate. The waveguide takes advantage of plasmonic devices’ capacity to both confine light and also enhance the fluorescence signal emanating from the confined volume.

The integration of surface acoustic wave (SAW) technology with Lab-on-a-Chip (LOC) devices has enabled powerful acoustic mechanisms for cell sorting, cell deflection, and other types of manipulation of biological specimen. Their control can be further enhanced and specialized with the integration of phononic crystals, which are materials designed to have phononic bandgaps to control the propagation of acoustic waves through a substrate. While many methods of designing phononic crystals require fabrication techniques that alter the surface topology with pillar or hole arrays, our electropoling technique creates a completely monolithic substrate. Electropoling involves using a strong electric field to reverse the domains of a piezoelectric substrate in a periodic pattern to generate a phononic bandgap. The technique is both simple and allows for compatibility with microfluidic components. By integrating phononic crystal substrates into a LOC device, we can enable a new degree of control to SAW particle manipulation. Here we demonstrate the use of phononic crystals to reflect SAWs in order to generate standing SAW fields. Particle focusing experiments are performed to observe the standing wave pattern. This is a novel demonstration of utilizing reflecting phononic energy towards a functional device, as well as integration of phononic crystal technology into a microfluidic system device.

Cancer is a very serious disease; it is the second leading cause of death worldwide. Most cancers can be effectively treated if discovered early, when the tumor is in its early stage. Current clinical cancer diagnose methods lack the necessary timelines and sensitivity, which needs 20-40 years to diagnose the cancer after first parental tumor cell genesis. However, according to the present method, it still needs more than ten years for the cancer to be diagnosed. In addition, most clinical blood biomarkers lack the requisite sensitivity and specificity to detect cancer at early stages. Therefore, there is a pressing need for discovering new biomarkers that possess higher sensitivity and specificity for diagnosing and prognosing aggressive cancers. We found that extracellular vesicle (EV) –exosomes contain many types of known biomarker proteins DNA and miRNA. The specific exosomes secreted by tumor cells normally contain the biomarkers that can be used to predict the existence of cancer. The biogenesis of exosomes is regulated by the machinery of endosomal sorting complexes required for lipid ceramide or transport (ESCRT). Since exosomes possess large enough density and size that can be isolated and purified from blood using ultracentrifugation or magnetic separation methods. After re-suspending the isolated exosomes in a pure solution, the concentration of the specific biomarkers in the exosomes can be significantly increased compared to the traditional blood based biomarker method. Moreover, the volume of re-suspension solution is controllable, which can adjust the concentration of the specific biomarkers accordingly. Therefore, exosomes based method is superior over the blood based biomarker method and other traditional cancer detection methods.

However, it is still unclear how early the exosomes based detection can diagnose cancer. In order to demonstrate the feasibility of exosomes based cancer diagnosis method, we developed a mathematical model to simulate the kinetics of cancer specific exosomes in relation to the growth of tumors that begin with a single parental cancer cell. To exemplify a more realistic scenario, we primed the model on pancreatic tumor growth using Glypican-1(GPC1) circulating exosomes (crExo). The simulation parameters are obtained from published literature.

As the result, after first parental tumor cell genesis, it requires 940 days for tumor cells to secrete 105 numbers of the GPC1 specific exosomes. Followed by isolation and purification of the secreted exosomes, the isolated and purified exosomes can be re-suspended in any desirable volume of solution, which can greatly increase the concentrations of all the biological contents including the biomarkers enclosed by the exosomes compared to the traditional blood based biomarker. Hence, the required number of secreted exosomes is significantly reduced in order to reach the same equipment detection limit. In comparision, exosome based method can diagnose the cancer in 2.58 years compared to about 10 years diagosis timeframe using traditional blood based biomarker.

My developed mathematical model is capable of predicting the cancer detection time and helping the researchers simulate the growth and size for any tumor cell type. The model presented here can be extended to virtually any solid cancer and associated biomarkers.

Recently, the technology integrating acoustic with microfluidics, also called acoustofluidics, has drawn significant attention based on its unique advantages through the combinations of solids and fluids, and the interplay of mechanics and electronics. Despite its promise to address many challenges in biomedical research, it's still in its infancy. Much work needs to be done to fully explore the potential of acoustofluidics as an everyday tool for real-world applications. To achieve this goal, many challenging problems related to physics, engineering, and biomedical applications of acoustofluidics need to be solved. In this dissertation, I systematically investigated the pivotal components in SAW-based acoustofluidics and developed novel acoustofluidic devices with innovative configurations. The main works involved in this dissertation are as follows:First, we explore a fully integrated chip-scale artificial graphene-like piezo-optomechanical structure and investigate the characteristics of surface phonon transport in it. We exploit the surface phonon polariton (SPhP) coupling between electromagnetic (EM) waves and atomic-level vibrations in the piezo-optomechanical metamaterial to create a SAW bandgap. We monolithically integrate the metamaterial with a lithium niobate substrate through a non-destructive and ad-hoc electric field poling technique. By merging the metamaterial with an acoustofluidic system, we present the first experimental demonstration of both phase and amplitude coherent scattering of SAW by monitoring the response of micrometer-sized non-organic particles to the resulting SAW field established in the microfluidic channel. Then, we present an integrated phononic crystal acoustofluidic device (IPAD) that incorporates phononic crystal into a SAW-based acoustofluidic system to achieve a multi-functional acoustofluidic platform. To perform band structure tuning, we systematically investigate the band gap properties of 2D SPhP phononic crystals and analyzed the band gap variation with different filling ratios. With the assistance of the frequency domain engineering technique, we integrate multiple SAW schemes to perform multi-operations by merging standing SAW-induced particle alignment and traveling SAW-induced off-center particle translocation. We experimentally demonstrate the ability of the IPAD platform to perform particle separation of different sizes with a better separation efficiency and higher throughput than one single SAW scheme without compromising the device’s compactness. In the third part (Chapter 4), we develop a Two-Stage Acoustic Focusing Compressibility Cytometry (AF-CC) technique that merges standing surface acoustic wave (SSAW)-based cell focusing and SSAW-based off-center cell translocation within a single microfluidic channel in a cascaded manner. By three-dimensional (3D) accurately tight-focusing cells into a single line, we can achieve real-time quantification of whole-cell compressibility by monitoring the trajectories of suspended cells. A one-to-one mapping of cell compressibility to the cell transit path length over its trajectory is established, allowing us to realize in-situ and high-throughput (~ 10 cells/second) quantitative analysis of single-cell. We demonstrate that AF-CC technique is sensitive enough to precisely distinguish the compressibility of three distinct murine hematopoietic cell types and their inhibitor-treated counterparts with cytoskeleton-perturbing molecules. In the final part of the thesis (Chapter 5), we present a label-, contact-, flow perturbation-free acoustic drifting effect (ADE) technology significantly boosts the advective transport of suspended colloids and biological cells across the channel in a remarkably short distance. Our ADE platform uses a simple and fabrication-friendly configuration by integrating a straight microfluidic channel with a pair of metallic interdigitated transducers (IDTs) lithographically deposited on a LiNbO3 substrate. By exploiting the well-engineered acoustic field, this ADE platform allows us to selectively separate target particles/cells and achieve immunoaffinity-based isolation. Our platform is proven to efficiently translate the target particles to the functionalized bottom of the microfluidic channel by the exerted downward acoustic radiation force, where the immobilized capture agents can bind with the target particles. Finally, We also demonstrate the selective capture of rare cells (T lymphocyte cells) with a 90% separation efficiency at a 1 mL/hr flow rate, comparable to current rare cell isolation technologies.

Living cells undergoing cellular differentiation, chronological aging, and malignant progression could exhibit dramatically different biophysical characteristics. Accurate quantification of the intrinsic mechanical properties of cells is of both practical and fundamental significance in biological research and clinical diagnostics. Here, we introduce Non-invaSIve acousTo-mechanophEnotyping (ON-SITE) for high-throughput (~ 5 × 104 cells/hr) on-the-fly quantification of single-cell mechanics in continuous flow without any physical contact and labeling. ON-SITE integrates two sequential modes of acousto-dynamic operation in a staged fashion, enabling three-dimensional focusing of cells into a tight line and subsequent touchless steering of them orthogonal to laminar flow, tracing out a parabola-like trajectory. This unique dual functionality offers real-time probing and evaluation of cell mechanical properties with excellent spatiotemporal resolution by tracking cell axial transit path lengths over its entire trajectory. We identified multiple critical biophysical factors (size, density, compressibility) that contribute to the dynamic motion of cells by analytical modeling and experimental approaches. We demonstrate highly efficient, reliable, and precise quantification of the cell elastic modulus of three distinct wild-type hematopoietic progenitors and their conditionally reprogrammed counterparts upon treatment with cytoskeleton-perturbing molecules. Remarkably, the subtle microscopic differences (~ 1 – 12 %) in cell elastic modulus are unraveled on an ultrafast timescale (~ 0.3 s) using acoustic waves that are generated at a clinically safe power (~ 2 W/cm2) and frequency (~ 13 MHz) level.

Exosomes have emerged as novel biomarkers for disease diagnostics and prognosis. Exosomes are present in bodily fluids and closely resemble the contents of their parental cells; thus, they have a huge potential to serve as a liquid biopsy tool in the diagnosis of multiple diseases. In particular, tumor exosomes have the potential as biomarkers for the early detection of cancer since their contents reflect the genomic and metabolic abnormalities in their parental cells. With the development of techniques for high-throughput purification and isolation of exosomes and exosome content analysis, exosomal proteins is rapidly becoming an important tool for the early diagnosis of cancer.

Exosomes are extracellular vesicles with a diameter of 30 - 150 nm. These nanovesicles are produced by almost all types of mammalian cells and cancer cells through fusion of an intermediate endocytic compartment, namely, multivesicular bodies, with the plasma membrane.

In the introduction part of this thesis, I introduce the concept of the exosome, its definition, biogenesis and characteristics, and composition. Then I discuss the use of exosomes for cancer prognosis, and α-synuclein circulating exosomes as an example to explain how exosomal proteins work and its high specificity to Parkinson Disease. Finally, I discuss the different methods of isolating and collecting specific types of exosomes.

One unanswered question is at what stage of disease and cancer development can the exosome-based method be useful for diagnosis. To test the practicability of exosome-based methods in cancer and diseases diagnosis, I will establish a mathematical model and analytically calculate the concentrations of cancer-specific exosomes based on tumor growth. Then I will predict how early the exosome-based method can detect cancer and other diseases considering the detection limits of current diagnostic technologies. In addition, I will introduce and discuss the parameters required to inspect and verify the feasibility of my mathematical model. This thesis is focused on two specific exosomes: α-synuclein circulating exosomes for Parkinson disease and HSP70 proteins for liver cancer.

After the exosomes are extracted and purified, they can be resuspended into a small volume solution. Therefore, exosomal samples yield higher concentrations of biomarkers in a resuspension solution than those in blood. In the following, we show that it takes about 2.05 years for a parental cell to expand to a cell population that can secrete a baseline value of the α-synuclein circulating exosomes in Parkinson disease (PD) patients, and 0.73 years for a parental tumor cell to expand to a cell population that can secrete the baseline number of HSP70 proteins circulating exosomes in liver cancer patients. In conclusion, the mathematical model I established can help us predict how exosomal protein can be used to detect cancer and PD.

We are in a thrilling period defined by the rapid integration of many research disciplines and groundbreaking technical advancements fundamentally transforming our diagnostic and clinical procedures. The fusion of micro-nanotechnology and biotechnology is driving our technical capabilities to unprecedented levels. Despite notable advancements in sensitivity and multiplexing capability over the past few decades, there remains ample opportunity for innovation to enhance performance as a practical instrument for real-world applications. In order to accomplish this objective, numerous complex issues pertaining to the engineering, plasmonics, electrochemistry, and biomedical applications of bead-based assays must be addressed. To achieve this, I present a novel method in assay development, plasmonic and electrochemical sensing techniques, and automation in this dissertation. The first chapter introduces reagents used in the diagnostic assays, and current diagnostic methods such as ELISA, PCR, and bead-based assays. We then discuss three examples of widely used detection techniques. These techniques are plasmonic sensing, electrochemical sensing, and fluorescence sensing.The second chapter introduces a novel sandwich bioassay called Biomarker-to-surrogate conversion (B2S). We develop this assay to overcome the one challenge that has been neglected over the past decades: the need for solutions in the current diagnostic world when it comes to amplifying signals for protein detection. We convert target proteins (antigens) into surrogate beads that are much heavier and larger than antigens. That enables us to detect them much more reliably at attomolar-level concentrations. This remarkable noble assay enables us to have background free surrogate particles as final product of the assay. Hence, it opens us many doors to develop and execute different detection setups. The third chapter aims to develop a novel signal collection method to generate signal from surrogate products of B2S described in Chapter 2. In this chapter, we overcome the mass transport problem that has been prevented most research from going below pg/mL detection ranges, by introducing a flow-through setup for processing the final buffer obtained through our B2S assay. We introduce plasmonic nanohole arrays (NHA) and their implementation in surrogate particle sensing. We employ Extraordinary Optical Transmission (EOT) with the manufactured NHA chips, combining nanofluidics and nanoplasmonics into a single unit. We measure the peak shift in the spectrum of the transmitted light due to the resonance wavelength, and hence detect the target molecule in our sample. This setup also enables us power-free and naked-eye detection of the samples, by utilizing merely sun light, at femtomolar levels. The fourth chapter is about automating the novel assay, B2S, described in Chapter 2, with the novel electrochemical sensing technique we have developed and explained in this chapter. By using a low-cost and open-source liquid handling platform called OT-2, we are able to automate the B2S assay procedure which significantly reduces the workload on the scientist. Also, we designed a printed circuit board (PCB) to control multiple SPEs, allowing us to do electrophoretic deposition simultaneously at eight different chips. Then, by combining this PCB with a commercial potentiostat, we can extract the readout data of that automated and high-throughput diagnostic setup. The finalized setup is automated from start to finish and can process 8 samples at a time, with the possibility of increasing the number.

Abstract

A Surrogate Based Characterization Technique

For Antibody Coupling Efficiency

by

Mustafa Mutlu

The immunoprecipitation protocol is of key importance to antigen purification, to downstream protein analysis, and to clinical research. The success of an immunoprecipitation experiment depends on many variables of which antibody immobilization is the most important. Antibodies must be coupled with specific solid surfaces, such as agarose beads or superparamagnetic beads, during immunoprecipitation. Although antibody coupling is crucial for the protocol, there is not an easy-to-apply, low cost, or fast method to maximize and characterize the immuno-binding capacity. In this work we propose a characterization method which gives a clear result of the coupling efficiency, which does not require complicated experimental steps or expensive laboratory equipment. By forming a two-particle complex that sandwiches an antibody, the total amount of antibody coupled to the given number of magnetic beads can be readily translated into the number of surrogate beads that are attached to the magnetic beads through the antibodies. This method doesn’t require additional biological reagents. The surrogate bead alone suffices to quantify immobilization efficiency. This method requires only fluorescence imaging, which can be done with any portable fluorescence platform. It has multiplexing capability, provides high-throughput and high-yield, is cost-effective and time-efficient. Researchers can easily track the shelf-life, quality, degradation, and deterioration of the immobilized beads. This research has characterized a couple of variables, as well as tested automating the protocol. Our design also allows other researchers to explore more variables and automate their protocols.

We are living in an exciting era of a fast-paced confluence of wide-ranging research fields and technological breakthroughs that are profoundly altering our biomedical practices. In particular, the marriage of micro-nanotechnology and biotechnology is propelling our technical capabilities to new horizons. Despite tremendous progress made over the past 20 years, many challenges still exist for a broad range of biomedical applications spanning from few nanometers to tens of microns.

In this thesis, I aim to address three important biomedical problems at three different length scales. I first present an innovative and low-cost acousto-microfluidic approach that overcomes the diffusion limitations imposed by laminar flow profile in microfluidic channels at a challenging flow regime corresponding to extremely low Reynolds (Re~10-4) and high Péclet (Pe~106) numbers. Using this novel approach, we achieve immunoaffinity based isolation of cells using a simple rectangular channel without resorting to chaotic mixing processes or magnetic forces. Our acousto-microfluidic method uses a combination of pure and quasi-standing surface acoustic waves that are generated at a safe power (~ 1-10 W/cm2) and frequency (~10 MHz) regime comparable to those used in ultrasound imaging (e.g. fetal imaging). By exploiting the local shear forces in the vicinity of the bottom channel boundary (defined capture surface), we achieve selective separation of desired particles/cells using pre-functionalized cell specific biomolecules. We demonstrate selective enrichment of low-abundance T lymphocyte cells with a separation efficiency of about 80% at a high flow rate of 1.2 mL/hr, a separation efficiency that is comparable to most advanced rare cell isolation technologies. In the second part of my thesis (Chapter 2), I introduce a novel nanophotonic approach for enhanced inactivation of multidrug-resistant bacterial pathogens using visible light at 405 nm. To achieve this, I introduce precisely engineered aluminum nanoantenna arrays using a space mapping algorithm enabling inverse design of nanostructures from the desired spectral response. The nanoantenna arrays are optimized using radiative decay engineering principles to maximize the near-field enhancements and photothermal heating at 405 nm, a wavelength which bacteria is extremely sensitive to light and used as an emerging light-based disinfection technology alternative to ultraviolent (UV) light. In our experiments with low illumination intensities (2.5 mW/mm2), we demonstrate that our aluminum nanoantenna array (ANA) enables rapid eradication (~20 min) of multidrug-resistant Vibrio Cholerae biofilms with more than 500-fold inactivation efficiency (~99.995%) with respect to the 405 nm light alone. In the final part of my thesis (Chapter 3), I introduce a subwavelength thick (<200 nm) Optofluidic PlasmonIC (OPtIC) microlens that effortlessly achieves objective-free focusing and self-alignment of opposing optical scattering and fluidic drag forces for selective separation of exosome size bioparticles at low flow rates (< 5 m/s). Our optofluidic microlens provides a self-collimating mechanism for particle trajectories with a spatial dispersion that is inherently minimized by the optical gradient and radial fluidic drag forces working together to align the particles along the optical axis. I demonstrate that this facile platform facilitates complete separation of small size bioparticles (i.e., exosomes) from a heterogenous mixture through negative depletion and provides a robust selective separation capability for same size nanoparticles based on their differences in chemical composition. Unlike existing optical chromatography techniques that require complicated instrumentation (lasers, objectives and precise alignment stages), our OPtIC microlenses with a foot-print of 4 μm × 4 μm open up the possibility of multiplexed sorting of nanoparticles on a chip using low-cost broadband light sources (LEDs).