Bioconjugation Strategies for Microtoroidal Optical Resonators

The development of label-free biosensors with high sensitivity and specificity is of significant interest for medical diagnostics and environmental monitoring, where rapid and real-time detection of antigens, bacteria, viruses, etc., is necessary. Optical resonant devices, which have very high sensitivity resulting from their low optical loss, are uniquely suited to sensing applications. However, previous research efforts in this area have focused on the development of the sensor itself. While device sensitivity is an important feature of a sensor, specificity is an equally, if not more, important performance parameter. Therefore, it is crucial to develop a covalent surface functionalization process, which also maintains the device’s sensing capabilities or optical qualities. Here, we demonstrate a facile method to impart specificity to optical microcavities, without adversely impacting their optical performance. In this approach, we selectively functionalize the surface of the silica microtoroids with biotin, using amine-terminated silane coupling agents as linkers. The surface chemistry of these devices is demonstrated using X-ray photoelectron spectroscopy, and fluorescent and optical microscopy. The quality factors of the surface functionalized devices are also characterized to determine the impact of the chemistry methods on the device sensitivity. The resulting devices show uniform surface coverage, with no microstructural damage. This work represents one of the first examples of non-physisorption-based bioconjugation of microtoroidal optical resonators

Design and development of novel large scale applications in micro/nanophotonics and nanobiotechnology

Ankara : The Materials Science and Nanotechnology Program and the Graduate School of Engineering and Science of Bilkent University, 2014.Thesis (Ph. D.) -- Bilkent University, 2014.Includes bibliographical references leaves 91-101.Developments in micro/nanophotonics and nanobiotechnology creates new opportunities regarding development of devices with unprecedented capabilities, which could improve human civilization substantially. On the other hand, a certain level of maturity in transforming these possibilities into reality still requires considerable efforts. One of the main problems of these novel technologies is that their practical know-how is so scarce that they could only be utilized within strictly determined laboratory conditions, and by highly sophisticated scientists. This thesis focuses on large scale applications at the intersection of microphotonics and nanobiotechnology, and also in nanophotonics. On microphotonics side, optical microresonators with toroidal shape were successfully fabricated and optically integrated. Having an extremely high sensitivity towards perturbations in their environments, these microcavities could be used as biological sensors; however, they are also very sensitive for nonspecific interactions. Thus, a novel surface chemistry enabling bioconjugation of molecular probes without compromising their sensitivity and enhancing their selectivity was developed, based on methylphosphonate containing silane modification of the microtoroid surface. After this functionalization, microtoroids were used in biodetection in complex media. Also, a macroscopic photodetection device composed on intrinsically aligned semiconducting selenium nanowires were demonstrated. This device could be considered as a novel and efficient demonstration of nanowire integration to the macroscopic world. Together with the research on biosensors, these are important large scale applications of emergent science of our age.Özgür, ErolPh.D

A Whispering Gallery Mode Microlaser Biosensor

A biological sensor, commonly referred to simply as a biosensor, is a transducing device that allows quantitative information about specific interactions, analytes or other biological parameters to be monitored and recorded. The development of biosensors that are low-cost, reliable and simple to use stand to facilitate fundamental breakthroughs and revolutionize current medial diagnostic methods. Notably, there remains an unmet need for developing in-vivo biosensors, allowing insights to be directly gained from the precise location of biological interactions within the human body. Over the last two decades, whispering gallery modes (WGM) within microresonators have emerged as a promising technology for developing highly sensitive and selective biosensors, among many other applications. However, significant work remains to allow WGM sensors to make the transition from primarily being used within purely research environments to real-world applications. Specifically, one of the key limiting factors is the requirement of an external phase-matched coupling scheme (such as a tapered or angle polished optical fiber, prism or waveguide) to excite the WGMs, despite these devices displaying tremendous sensing performance. One way to lift this dependency on complex interrogation schemes is introduce a gain medium, such as a fluorescent dye or coating the resonator with quantum dots for example, thereby rendering it active and allowing remote excitation and collection of the WGM spectrum. Using active WGM resonators has allows the creation of novel sensing opportunities such as tagging, tracking and monitoring forces from insides living cells. Applications like these could not have been realized using external phase-matched coupling schemes. The biosensing platform presented here is based on combining WGM within active microspherical resonators with microstructured optical fibers (MOF). The MOF enables both the excitation and collection method for the WGM spectrum while simultaneously providing a robust and easy to manipulate dip sensing architecture that has the potential to address the unmet need for real time labelfree in-vivo sensing by combining with a catheter. The platform is investigated fundamentally as well as experimentally, beginning with the development of an analytical model that is able to generate the WGM spectrum of active microspherical resonators. This provides the opportunity to pinpoint the optimal choice of resonator to be used for undertaking refractive index based biosensing. Specifically by being able to extract the quality (Q) factor, a measure of the resonance linewidth, and refractive index sensitivity from the WGM spectrum, the optimal combination of resonator parameters (diameter and resonator refractive index) can be identified for optimizing the resonators sensing performance. Further, the availability, biocompatibility and cost, as well as fabrication requirements can be also considered when selecting the ideal resonator. Next, the inherently lower Q-factors observed in active resonators compared to their passive counterparts (i.e. resonators without a gain medium) is examined using a combination of theoretical, experimental and imaging methods. Through this examination process, the inherent asphericity of the resonator is identified as being the limiting factor on the Q-factor of active resonators, with its effect most notably being observed for measurements made in the far field. Experimentally, the first demonstration of this platform operating as a biosensor is presented by monitoring the well-documented specific interaction of Biotin/neutravidin in pure solutions. Including identifying ways to improve sensing performance and lower the detection limit, such as operating the resonator above its lasing threshold. Although, it is noted that in its current form, this platform is best suited for the monitoring of protein, preferably occurring in higher concentrations, until further improvements to the sensing performance can be implemented. However, the robust design coupled with its ability to provide access to previously difficult to obtain locations provides an insight into its potential future application capabilities. Finally, the extension of the platform to operating in complex samples, namely undiluted human serum, is outlined. By self-referencing the platform, through the addition of a second, almost identical resonator (only varying in its surface functionalization) into one of the remaining vacant holes on the tip of the fiber, the effects of non-specific binding as well as changes in local environmental conditions (i.e. temperature fluctuations), can be eliminated.Thesis (Ph.D.) -- University of Adelaide, School of Physical Sciences, 201

Expanding the toolbox for precision medicine with silicon photonic microring resonators and microfluidic technologies

The goal of precision medicine is to use molecular profiles of disease to identify a targeted treatment that results in the best available patient outcomes. Although the concept of individualizing treatment is not new to medicine, genomic technologies and therapies targeted to genetic drivers of disease have inspired an era of precision medicine. Obtaining molecular profiles of disease requires analyzing biological samples that present daunting analytical challenges with thousands of potentially interfering analytes, often at concentrations much higher than the analytes of interest. Many analytes harbor fragile chemical modifications, such as phosphorylation of signaling proteins, and therefore, require careful design of protocols for sample handling to preserve the relevant biological information. Classifying patients into subgroups that will benefit from tailored therapies demands moving beyond single biomarker diagnostics to multiplexed detection methods. The analytical toolbox for studying the molecular basis of human disease has grown tremendously in recent decades and has been motivated in part by the human genome project, with the most dramatic changes seen in sequencing technologies. The next frontier for molecular diagnostics is the development of diagnostic tools for non-genomic molecular profiles of disease, such as non-coding RNAs and proteins. This thesis details efforts to improve multiplexed protein detection for precision medicine diagnostics. Most of the following work uses microring resonator arrays as the detection platform, a versatile silicon photonic biosensing technology. Chapter 1 reviews the applications of optical resonators in analytical chemistry. Microring resonators arrays are a class of whispering gallery mode resonators featured throughout the review. A conventional focus of optical resonator development has been on designing label-free sensors for biomolecule detection. However, much of the recent work pushing limits of detection for the microring resonator platform have used immunoaffinity labels and enzymatic enhancement to perform detection in clinically relevant samples. Initially, we had anticipated needing to fractionate interfering species out of these biological samples to perform multiplex measurements on the fractions of interest using the microring resonator platform. We found solutions that avoided separations prior to sample analysis, but the microring resonators offered an interesting property uncommon among chromatography detectors: a universal detector with an enormous dynamic range. Chapter 2 details interfacing the microring resonator platform with liquid chromatography. Optical resonators are surface sensitive and most commonly used to observe binding events on a modified sensor, but they can also serve as bulk refractive index detectors. In comparison to commercial refractive index detectors, the microring resonator platform is compatible with solvent gradient chromatography because of the large dynamic range. Commonly studied small molecule pharmaceuticals were used for proof-of-concept experiments, and ongoing work seeks to extend the platform to polymer analysis, an analyte class that lacks chromogenic signatures. The next two chapters detail my contributions to protein detection on the microring resonator platform and can be summarized as the implementation of protein and phosphoprotein detection in whole cell lysates and tissue homogenates. Protein detection in cell lysate was achieved by modifying the signaling amplification schemes developed by previous lab members and altering the chemical strategy for covalent modification of proteins to the sensor surface. Chapters 3 and 4 describe the specifics of this strategy. These projects were designed in part as proof-of-principle studies to demonstrate the application of microring resonators to novel samples and biomolecules. Signaling pathways are often dysregulated in tumors, resulting in uncontrolled growth and proliferation, and these signaling pathways are driven by phosphorylation cascades. In Chapter 3, a multiplex protein and phosphoprotein panel was used to monitor the levels across multiple signaling pathways in glioblastoma, the most common and aggressive brain cancer in adults. Chapter 4 builds on the phosphoprotein panel developed in Chapter 3 to dynamically monitor signaling networks of patient derived xenografts in response to targeted therapeutics. The phosphoprotein levels in these samples indicated pathway signatures unique to treatment time and mutational status of the sample. This approach could potentially be used to provide actionable information to clinicians by determining tumor susceptibility to treatment based off its signaling state. The phosphoprotein panels described in Chapters 3 and 4 center around the PI3K/Akt/mTOR signaling network. However, these panels lack the membrane proteins that initiate the signaling cascade. To include membrane protein analysis, I developed a microfluidic platform for Nanodisc assembly and purification, referred to as the μNAP platform and detail in Chapter 5. The μNAP platform capitalizes on sample preservation and small volume processing inherent to miniaturization and microfluidics and achieves Nanodisc assembly by combining a reagent mixing chamber and packed detergent removal bed onto a microfluidic device. The platform also includes an affinity chromatography module for rapid purification on the microfluidic scale. Cytochrome P450 3A4 was used to demonstrate the capabilities of the μNAP platform. Chapter 6 details the future directions for each of the described projects. The next steps for phosphoprotein detection with microring resonator arrays is to use the targeted panel to reconstruct the aberrant signaling networks from tumor biopsy samples. Network reconstruction has been performed using global profiling methods, such as next generation sequencing and proteomics with mass spectrometry, but reconstructing key signaling networks from minimal network data could provide a less cumbersome approach to obtain actionable information with higher throughput. The future work for the μNAP platform will include generating Nanodisc libraries from valuable samples, such as tumor biopsies. Nanodisc libraries have been shown to accurately represent the membrane protein composition of the sample, and these libraries formed from patient samples could be used for functional screening of membrane proteins in response to targeted therapeutics. Finally, interfacing the microring resonator platform with on-chip electrophoresis could prove to be a remarkably useful combination. The microring resonator platform would allow for on-chip multiplexed detection of biomolecules combined with the separation efficiency of electrophoresis. The interface would substantially reduce reagent consumption and shrink the footprint of the sensor platform by eliminating the need for external pumping. By applying the previously developed electrophoretic methods for sample stacking along with the improved mass transfer properties of non-laminar flow, on-chip electrophoresis combined with microring resonator arrays could represent a significant analytical advancement

An Optical Microsensor Utilizing Genetically Programmed Bioreceptor Layers for Selective Sensing

Protein engineering is a rich technology that holds the potential to revolutionize sensors through the creation of highly selective peptides that encode unique recognition affinities. Their robust integration with sensor platforms is very challenging. The goal of this research project is to combine expertise in micro-electro-mechanical systems (MEMS) and biological/protein engineering to develop a selective sensor platform. The key enabling technology in this work is the use of biological molecules, the Tobacco mosaic virus (TMV) and its derivative, Virus-Like-Particle (VLP), as nanoreceptor layers, in conjunction with a highly sensitive microfabricated optical disk resonator. This work will present a novel method for the integration of biological molecules assembly on MEMS devices for chemical and biological sensing applications. Particularly in this research, TMV1Cys-TNT and TMV1Cys-VLP-FLAG bioreceptor layers have been genetically engineered to bind to an ultra-low vapor pressure explosive, Trinitrotoluene (TNT), and to a widely used FLAG antibody, respectively. TNT vapor was introduce to TMV1Cys-TNT coated resonator and induced a 12 Hz resonant frequency shift, corresponding to a mass increase of 76.9 ng, a 300% larger shift compared to resonators without receptor layer coating. Subsequently, a microfabricated optical disk resonator decorated with TMV1Cys-VLP-FLAG was used to conduct enzyme-linked immunosorbent assay and label-free immunoassays on-a-chip and demonstrated a resonant wavelength shift of 5.95 nm and 0.79 nm, respectively. The significance of these developments lies in demonstrating the capability to use genetically programmable viruses and VLPs as platforms for the display and integration of receptor peptides within microsystems. The work outlined here constitutes an interdisciplinary investigation on the integration capabilities of the bio-nanostructure materials with traditional microfabrication architectures. While previous works have focused on individual components of the system, this work addresses multi-component integration, including biological molecule surface assembly and fabrication utilizing both top-down and bottom-up approaches. Integrating biologically programmable material into traditional MEMS transducers enhances selectivity, sensitivity, and simplifies fabrication and testing methodologies. This research provides a new avenue for enhancing sensor platforms through the integration of biological species as the key to remedying challenges faced by conventional systems that utilize a wide range of polymers or metals for nonspecific bindings

Surface engineering of poly(methylmethacrylate): Effects on fluorescence immunoassay

The authors present surface engineering modifications through chemistry of poly(methylmethacrylate) (PMMA) that have dramatic effects on the result of surface-bound fluorescence immunoassays, both for specific and nonspecific signals. The authors deduce the most important effect to be clustering of antibodies on the surface leading to significant self-quenching. Secondary effects are attributable to the formation of sparse multilayers of antibody. The authors compare PMMA as an antibody support surface with ultraviolet-ozone oxidized PMMA and also to substrates that were, after the oxidation, surface modified by a four-unit poly(ethyleneglycol) carboxylic acid (PEG4), a branched tricarboxylic acid, and a series of carboxylic acid-terminated dendrimers, from generation 1.5 to 5.5. Fluorescence immunoassay and neutron reflectometry were used to compare the apparent antibody surface loading, antigen binding and nonspecific binding on these various surfaces using anti-human IgG as a model antibody, chemically coupled to the surface by amide formation. Simple physical adsorption of the antibody on PMMA resulted in a thick antibody multilayer with small antigen binding capacity. On the carboxylated surfaces, with chemical coupling, a simple monolayer was formed. The authors deduce that antibody clustering was driven by conformational inflexibility and high carboxylate density. The PEG4-modified surface was the most conformationally flexible. The dendrimer-modified interfaces showed a collapse and densification. In fluorescence immunoassay, the optimal combination of high specific and low nonspecific fluorescence signal was found for the G3.5 dendrimer

Surface engineering of poly(methylmethacrylate): Effects on fluorescence immunoassay

The authors present surface engineering modifications through chemistry of poly(methylmethacrylate) (PMMA) that have dramatic effects on the result of surface-bound fluorescence immunoassays, both for specific and nonspecific signals. The authors deduce the most important effect to be clustering of antibodies on the surface leading to significant self-quenching. Secondary effects are attributable to the formation of sparse multilayers of antibody. The authors compare PMMA as an antibody support surface with ultraviolet-ozone oxidized PMMA and also to substrates that were, after the oxidation, surface modified by a four-unit poly(ethyleneglycol) carboxylic acid (PEG4), a branched tricarboxylic acid, and a series of carboxylic acid-terminated dendrimers, from generation 1.5 to 5.5. Fluorescence immunoassay and neutron reflectometry were used to compare the apparent antibody surface loading, antigen binding and nonspecific binding on these various surfaces using anti-human IgG as a model antibody, chemically coupled to the surface by amide formation. Simple physical adsorption of the antibody on PMMA resulted in a thick antibody multilayer with small antigen binding capacity. On the carboxylated surfaces, with chemical coupling, a simple monolayer was formed. The authors deduce that antibody clustering was driven by conformational inflexibility and high carboxylate density. The PEG4-modified surface was the most conformationally flexible. The dendrimer-modified interfaces showed a collapse and densification. In fluorescence immunoassay, the optimal combination of high specific and low nonspecific fluorescence signal was found for the G3.5 dendrimer

Glassy Materials Based Microdevices

Microtechnology has changed our world since the last century, when silicon microelectronics revolutionized sensor, control and communication areas, with applications extending from domotics to automotive, and from security to biomedicine. The present century, however, is also seeing an accelerating pace of innovation in glassy materials; as an example, glass-ceramics, which successfully combine the properties of an amorphous matrix with those of micro- or nano-crystals, offer a very high flexibility of design to chemists, physicists and engineers, who can conceive and implement advanced microdevices. In a very similar way, the synthesis of glassy polymers in a very wide range of chemical structures offers unprecedented potential of applications. The contemporary availability of microfabrication technologies, such as direct laser writing or 3D printing, which add to the most common processes (deposition, lithography and etching), facilitates the development of novel or advanced microdevices based on glassy materials. Biochemical and biomedical sensors, especially with the lab-on-a-chip target, are one of the most evident proofs of the success of this material platform. Other applications have also emerged in environment, food, and chemical industries. The present Special Issue of Micromachines aims at reviewing the current state-of-the-art and presenting perspectives of further development. Contributions related to the technologies, glassy materials, design and fabrication processes, characterization, and, eventually, applications are welcome

Surface plasmon resonance sensing: an optical fibre based SPR platform with scattered light interrogation

This thesis describes the development, fabrication and optimisation of a Surface Plasmon Resonance (SPR) sensing architecture based on optical fibres. Motivated by biosensing applications, SPR was chosen as a simple and sensitive label-free technique that allows real time quantitative measurements of biomolecular interactions. Unlike conventional fibre SPR probes, this platform utilises a novel interrogation mechanism based on the analysis of scattered radiation facilitated by a rough plasmonic coating. A theoretical study is performed in order to determine the optimal parameters of the sensing configuration, i. e. the metal coating and fibre material. This analysis revealed a trade-off between the sensitivity of these devices, and their resolution. Optical fibres with cores made of lower refractive index materials were found to increase the sensitivity of the sensor, but broaden the SPR spectral signature. This broadening of the linewidth results in an unwanted increase in the sensor resolution, which leads to an undesirable increase in the detection limit. Therefore, experiments were performed to investigate the trade off between the sensitivity and resolution of the sensor to optimise both performance characteristics. The experimental demonstration and characterisation of a scattering SPR platform based on lead silicate fibres is described. The plasmonic coating with required surface roughness was fabricated using chemical electroless plating. In order to increase the refractive index sensitivity, a fibre SPR sensor with a lower refractive index core made of fused silica was produced. Due to the different surface properties of the silica glass and the lead silicate glass, surface modification with stannous chloride was required to fabricate suitable plasmonic coatings on the fused silica fibres. Characterisation of the new fused silica SPR sensors showed that the sensitivity of the sensing probe was improved, however, the spectral linewidth of the SPR signature was broadened, in agreement with the theoretical modelling. Nevertheless, analysis of the capability of the silica fibre based SPR sensors demonstrated potential for this platform in biological studies. To improve the resolution without affecting the sensitivity of a sensor, smaller core fibres can be used. However, using conventional small core fibres or fibre tapers is challenging due to their fragility and the requirement for fibre post processing to access the core. To overcome these difficulties, an SPR sensor based on a silica microstructured optical fibre with a core exposed along the entire fibre length was fabricated. Exposed Core Fibres (ECFs) have small cores that are supported by thin struts inside of a larger support structure, providing mechanical robustness to the fibre. The ECF SPR sensing platform doubled the improvement in the spectral linewidth when compared to the large core fused silica fibre sensor, without compromising sensitivity. Finally, the demonstration of Metal Enhanced Fluorescence (MEF) phenomena is presented. The effect of rough metallic coatings on the enhancement of fluorescence emission is investigated in planar glass substrates, showing significant improvement in emission when compared to smooth metal films. An optical fibre based MEF platform was demonstrated to illustrate the potential of rough metal coatings on a fibre for surface enhanced optical phenomena. This work is the first systematic study of a scattering based SPR sensing platform. This architecture addresses existing practical limitations associated with current SPR technologies, including but not limited to bulk design and affordability. Additionally, performance enhancement of the sensing probes is achieved through the use of alternative fibre material and geometry. The demonstrated performance improvements are not class-leading compared to commercial biosensing devices, however, the performance is in agreement with the theoretical analysis which provides a pathway for further improvement. This demonstrated that the scattering based SPR fibre platform is a practical new approach that offers the advantages of high sensitivity and signal to noise ratio, and low resolution, with the capability to improve the detection limit of SPR devices. Most importantly, this novel SPR interrogation approach allows the incorporation of two different sensing techniques, SPR and fluorescence, in the same fibre device, which opens pathways for novel biosensing applications combining the two phenomena.Thesis (Ph.D.)--University of Adelaide, School of Physical Sciences, 2017