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Nanoengineered glycan sensors may help realize the long-held goal of accurate and rapid glycoprotein profiling without labeling or glycan liberation steps. Current methods of profiling oligosaccharides displayed on protein surfaces, such as liquid chromatography, mass spectrometry, capillary electrophoresis, and microarray methods, are limited by sample pretreatment and quantitative accuracy. Microarrayed platforms can be improved with methods that better estimate kinetic parameters rather than simply reporting relative binding information. These quantitative glycan sensors are enabled by an emerging class of nanoengineered materials that differ in their mode of signal transduction from traditional methods. Platforms that respond to mass changes include a quartz crystal microbalance and cantilever sensors. Electronic response can be detected from electrochemical, field effect transistor, and pore impedance sensors. Optical methods include fluorescent frontal affinity chromatography, surface plasmon resonance methods, and fluorescent single walled carbon nanotubes-(SWNT). Advantages of carbon nanotube sensors include their sensitivity and ability to multiplex. The focus of this work has been to develop carbon nanotube-based sensors for glycans and proteins. Before detailing the development of these new sensors, the thesis will begin with a very brief primer on glycobiology, its connection to medicine, and the advantages and limitations of existing tools for glycan analysis. In the second chapter we model the use of quantitative nanosensors in a weak affinity dynamic microarray (WADM) to simulate practical uses of these sensors in bioprocessing and clinical diagnostics. There is significant interest in developing new detection platforms for characterizing glycosylated proteins, despite the lack of easily synthesized model glycans or high affinity receptors for this analytical problem. In the third chapter we experimentally demonstrate 'proof of concept' of carbon nanotubebased glycan sensors. This is done with a sensor array employing recombinant lectins as glycan recognition sites tethered via Histidine tags to Ni2l complexes that act as fluorescent quenchers for SWNT embedded in a chitosan hydrogel spot to measure binding kinetics of model glycans. We examine as model glycans both free and streptavidin-tethered biotinylated monosaccharides. Two higher-affined glycan-lectin pairs are explored: fucose (Fuc) to PA-IIL and N-acetylglucosamine (GlcNAc) to GafD. The dissociation constants (KD) for these pairs as free glycans (106 and 19 [mu]M respectively) and streptavidin-tethered (142 and 50 [mu]M respectively) were found. The absolute detection limit for the first-generation platform was found to be 2 pg of glycosylated protein or 100 ng of free glycan to 20 pg of lectin. Glycan detection (GlcNAc-streptavidin at 10 [mu]M) is demonstrated at the single nanotube level as well by monitoring the fluorescence from individual SWNT sensors tethered to GafD lectin. Over a population of 1000 nanotubes, 289 of the SWNT sensors had signals strong enough to yield kinetic information (KD of 250 ± 10 [mu]M). We are also able to identify the locations of "strong-transducers" on the basis of dissociation constant (4 sensors with KD 10 [Mu]) or overall signal modulation (8 sensors with 5% quench response). We report the key finding that the brightest SWNT are not the best transducers of glycan binding. SWNT ranging in intensity between 50 and 75% of the maximum show the greatest response. The ability to pinpoint strong-binding, single sensors is promising to build a nanoarray of glycan-lectin transducers as a high throughput method to profile glycans without protein labeling or glycan liberation pretreatment steps. In the fourth chapter we move from detection of model glycoproteins (streptavidin with biotinylated glycans) to a more applied problem: detection of antibodies and their glycosylation. We do this with a second generation array of SWNT nanosensors in an array format. It is widely recognized that an array of addressable sensors can be multiplexed for the label-free detection of a library of analytes. However, such arrays have useful properties that emerge from the ensemble, even when monofunctionalized. As examples, we show that an array of nanosensors can estimate the mean and variance of the observed dissociation constant (KD), using three different examples of binding IgG with Protein-A as the recognition site, including polyclonal human IgG (KD [mu] = 19 [mu]M, [sigma]2 = 1000 [mu]M2 ). murine IgG (KD = 4.3 [mu]M, 2= 3 [mu]M 2), and human IgG from CHO cells (KD [mu] = 2.5 nM, [sigma]F2 = 0.01 RM2). Second, we show that an array of nanosensors can uniquely monitor weakly-affined analyte interactions via the increased number of observed interactions. One application involves monitoring the metabolically-induced hypermannosylation of human IgG from CHO using PSA-lectin conjugated sensor arrays where temporal glycosylation patterns are measured and compared. Finally, the array of sensors can also spatially map the local production of an analyte from cellular biosynthesis. As an example we rank productivity of IgG-producing HEK colonies cultured directly on the array of nanosensors itself. One great limitation to these practical applications, common to other new sensor developments, are the constraints of large, bulky, and capital-intensive excitation sources, optics, and detectors. In the fifth chapter we detail the design of a lightweight, field-portable detection platform for SWNT based sensors using stock parts with a total cost below $3000. The portable detector is demonstrated with antibody detection in our lab and onsite at a commercial facility 3700 miles away with complex production samples. Along the course of developing these sensors, there was a need to analyze noisy data sets from signal nanotubes (Chapter 3) to determine distinct binding states. NoRSE was developed to analyze highfrequency data sets collected from multi-state, dynamic experiments, such as molecular adsorption and desorption onto carbon nanotubes. As technology improves sampling frequency, these stochastic data sets become increasingly large with faster dynamic events. More efficient algorithms are needed to accurately locate the unique states in each time trace. NoRSE adapts and optimizes a previously published noise reduction algorithm (Chung et al., 1991) and uses a custom peak flagging routine to rapidly identify unique event states. The algorithm is explained using experimental data from our lab and its fitting accuracy and efficiency are then shown with a generalized model of stochastic data sets. The algorithm is compared to another recently published state finding algorithm and is found to be 27 times faster and more accurate over 55% of the generalized experimental space. This work is detailed in Chapter 6. Future uses of these sensors include in vivo reporters of protein biomarkers. In Chapter 7, three-dimensional tracking of single walled carbon nanotubes (SWNT) with an orbital tracking microscope is demonstrated for this purpose. We determine the viscosity regime (above 250 cP) at which the rotational diffusion coefficient can be used for length estimation. We also demonstrate SWNT tracking within live HeLa cells and use these findings to spatially map corral volumes (0.27-1.32 Im 3), determine an active transport velocity (455 nm/s), and calculate local viscosities (54-179 cP) within the cell. With respect to the future use of SWNTs as sensors in living cells, we conclude that the sensor must change the fluorescence signal by at least 4-13% to allow separation of the sensor signal from fluctuations due to rotation of the SWNT when measuring with a time resolution of 32 ms. In the final chapter we draw conclusions from the development of this carbon nanotube-based sensor for glycan analysis and show the start of future work with arrays of SWNT sensors for glycoprofiling.
Optical biosensors based on fluorescent single-walled carbon nanotubes (SWNT) are a promising alternative to conventional biosensors due to the exceptional photophysical properties of SWNT. Such sensors can enable highly-sensitive, selective, and real-time detection of biological analytes. However, important questions regarding sensor fabrication and reproducibility must be addressed for these sensors to be of practical value. Herein we describe the use of highly-purified, single-chirality SWNT which are functionalized for antibody detection, and demonstrate that reproducibility is drastically improved with these SWNT. Further, we observe a concentration dependence of the effective equilibrium dissociation constant, KD,eff, which is in good agreement with previous reports, yet has eluded mechanistic description due to complexities associated with multivalent interactions. We show that a bivalent binding mechanism is able to describe this concentration dependence of KD,eff which varies from 100 pM to 1 uM for IgG concentrations from 1 ng/ml to 100 ug/ml, respectively. The mechanism is shown to describe the unusual concentration-dependent scaling demonstrated by other sensor platforms in the literature, and a comparison is made between resulting parameters. The platform is then extended to the detection of human growth hormone (hGH) using SWNT functionalized with a native hGH receptor (hGH-R), with potential use as a real-time and label-free measurement of protein activity. Native hGH is detected in the micromolar range, and an invariant equilibrium dissociation constant of 9 uM is revealed upon fitting the calibration curve to a single-site adsorption model. Selective detection of native hGH over thermally denatured hGH is shown at a concentration which is 1% of a clinical dose. Lastly, a multichannel detector was built to demonstrate real-time characterization of multiple protein properties. This work could find broad impact in biomanufacturing as real-time analysis of complex biologics is a long-standing goal in this field.
Semiconducting single-walled carbon nanotubes (SWCNTs) are attractive transducers for biosensor applications due to their unique photostability, single molecule sensitivity, and ease of multiplexing. Sensors can be rendered selective via several detection modalities including the use of natural recognition elements (e.g., proteins) as well as the formation of synthetic molecular recognition sites from adsorbed heteropolymers. However, to date, deployment of SWCNT-based biosensors has been limited. The aim of this thesis was to study the design and development of SWCNT-based optical sensors for analytes relevant to the food and pharmaceutical industries including neurotransmitters, proteins, and metal ions. The research described in this thesis spans several levels of nanosensor development including: i) the fundamental study of SWCNT-polymer interactions and their dependence on solution properties; ii) sensor development using existing detection modalities and the use of mathematical modeling to guide sensor design and interpret data; and iii) the invention of a new sensor form factor enabling long-term sensor stability and point-of-use measurements. Our fundamental work on SWCNT-polymer interactions investigates the influence of polymer structure, SWCNT structure, and solution properties on molecular recognition, using single-stranded DNA as a model polymer system. We find that specific ssDNA sequences are able to form distinct corona phases across SWCNT chiralities, resulting in varying response characteristics to a panel of biomolecule probe analytes. In addition, we find that ssDNA-SWCNT fluorescence and wrapping structure is significantly influenced by the solution ionic strength, pH, and dissolved oxygen in a sequence-dependent manner. We are able to model this phenomenon and demonstrate the implications of solution conditions on molecular recognition, modulating the recognition of riboflavin. These results provide insight into the unique molecular interactions between DNA and the SWCNT surface, and have implications for molecular sensing, assembly, and nanoparticle separations. In addition to our experimental work, we used mathematical modeling to guide sensor design for biopharmaceutical characterization. A mathematical formulation for glycoprotein characterization was developed as well as a dynamic kinetic model to describe the data output by a label-free array of non-selective glycan sensors. We use the formulated model to guide microarray design by answering questions regarding the number and type of sensors needed to quantitatively characterize a glycoprotein mixture. As a second example, we report the design of a novel, diffusion-based assay for the characterization of protein aggregation. Specifically, we design hydrogel-encapsulated SWCNT sensors with a tunable hydrogel layer to influence the diffusion of immunoglobulin G protein species of variable size, and we develop a combined model that describes both the diffusion of analyte and analyte-sensor binding. By measuring the sensor response to a series of well-characterized protein standards that have undergone varying levels of UV stress, we demonstrate the ability to detect protein aggregates at a concentration as low as one percent on a molar basis. Finally, we report the development of a new form factor for optical nanosensor deployment involving the immobilization of SWCNT sensors onto paper substrates. We find that SWCNT optical sensors can be immobilized onto many different paper materials without influencing sensor performance. Moreover, we pattern hydrophobic barriers onto the paper substrates to create 1-dimensional sensor arrays, or barcodes, that are used for rapid, multiplexed characterization of several metal ions including Pb(II), Cd(II) and Hg(II). In addition to providing a new form factor for conducting point-of-use sensor measurements, these findings have the potential to significantly enhance the functionality of SWCNT-based optical sensors by interfacing them with existing paper diagnostic technologies including the manipulation of fluid flow, chemical reaction, and separation.
Novel Nanomaterials for Biomedical, Environmental, and Energy Applications is a comprehensive study on the cutting-edge progress in the synthesis and characterization of novel nanomaterials and their subsequent advances and uses in biomedical, environmental and energy applications. Covering novel concepts and key points of interest, this book explores the frontier applications of nanomaterials. Chapters discuss the overall progress of novel nanomaterial applications in the biomedical, environmental and energy fields, introduce the synthesis, characterization, properties and applications of novel nanomaterials, discuss biomedical applications, and cover the electrocatalytical and photothermal effects of novel nanomaterials for efficient energy applications. The book will be invaluable to academic researchers and biomedical clinicians working with nanomaterials. Offers comprehensive details on novel and emerging nanomaterials Presents a comprehensive view of new and emerging tactics for the synthesis of efficient nanomaterials Describes and monitors the functions of applications of new and emerging nanomaterials in the biomedical, environmental and energy fields
This book will cover the full scope of nanobiosensing, which combines the newest research results in the cross-disciplines of chemistry, biology, and materials science with biosensing and bioanalysis to develop novel detection principles, sensing mechanisms, and device engineering methods. It not only covers the important types of nanomaterials for biosensing applications, including carbon nanotubes, carbon nanofiber, quantum dots, fullerenes, fluorescent and biological molecules, etc., but also illustrates a wide range of sensing principles, including electrochemical detection, fluorescence, chemiluminesence, antibody-antigen interactions, and magnetic detection. The book details novel developments in the methodology and devices of biosensing and bioanalysis combined with nanoscience and nanotechnology, as well as their applications in biomedicine and environmental monitoring. Furthermore, the reported works on the application and biofunction of nanoparticles have attracted extensive attention and interest, thus they are of particular interest to readers. The reader will obtain a rich survey of nanobiosensing technology, including the principles and application of biosensing, the design and biofunctionalization of bionanomaterials, as well as the methodology to develop biosensing devices and bioanalytical systems.
This work investigated single-walled carbon nanotube (SWNT)/polymer-protein A complexes for optically reporting antibody concentration via a change in near infrared fluorescent emission after antibody binding. SWNT have potential as biosensors because of extraordinary sensitivity, lack of photobleaching, and optical activity in a near-infrared window. A SWNT sensor could provide label-free measurements of antibody concentration in a continuous fashion, which may aid selection of production strains. Protein A itself, dextran, poly vinyl alcohol, DNA sequences, and chitosan were used as polymers for wrapping SWNT. Nonspecific binding to solution-phase constructs was found to be a major problem with these approaches. Chitosan hydrogels encapsulating SWNT also show nonspecific responses.
This book presents recent research on cancer detection methods based on nanobiosensors, which offer ultrasensitive point-of-care diagnosis. Several methods for diagnosing cancer have been discovered and many more are currently being developed. Conventional clinical approaches to detecting cancers are based on a biopsy followed by histopathology, or on the use of biomarkers (protein levels or nucleic acid content). Biopsy is the most widely used technique; however, it is an invasive technique and is not always applicable. Furthermore, biomarker-based detection cannot be relied on when the biomarkers are present in an extremely low concentration in the body fluids and in malignant tissues. Thus, in recent years highly sensitive and robust new cancer diagnosis techniques have been developed for clinical application, and may offer an alternative strategy for cancer diagnosis. As such, this book gathers the latest point-of-care cancer diagnostic methods and protocols based on biomedical sensors, microfluidics, and integrated systems engineering. It also discusses recent developments and diagnostics tests that can be conducted outside the laboratory in remote areas. These technologies include electrochemical sensors, paper-based microfluidics, and other kit-based diagnostic methods that can be adapted to bring cancer detection and diagnostics to more remote settings around the globe. Overall, the book provides students, researchers, and clinicians alike a comprehensive overview of interdisciplinary approaches to cancer diagnosis.
Bacterial Proteins—Advances in Research and Application: 2012 Edition is a ScholarlyEditions™ eBook that delivers timely, authoritative, and comprehensive information about Bacterial Proteins. The editors have built Bacterial Proteins—Advances in Research and Application: 2012 Edition on the vast information databases of ScholarlyNews.™ You can expect the information about Bacterial Proteins in this eBook to be deeper than what you can access anywhere else, as well as consistently reliable, authoritative, informed, and relevant. The content of Bacterial Proteins—Advances in Research and Application: 2012 Edition has been produced by the world’s leading scientists, engineers, analysts, research institutions, and companies. All of the content is from peer-reviewed sources, and all of it is written, assembled, and edited by the editors at ScholarlyEditions™ and available exclusively from us. You now have a source you can cite with authority, confidence, and credibility. More information is available at http://www.ScholarlyEditions.com/.
The text covers fiber optic sensors for biosensing and photo-detection, graphene and CNT-based sensors for glucose, cholesterol, and dopamine detection, and implantable sensors for detecting physiological, bio-electrical, biochemical, and metabolic changes in a comprehensive manner. It further presents a chapter on sensors for military and aerospace applications. It will be useful for senior undergraduate, graduate students, and academic researchers in the fields of electrical engineering, electronics, and communication engineering. The book Discusses implantable sensors for detecting physiological, bio-electrical, biochemical, and metabolic changes Covers applications of sensors in diverse fields including healthcare, industrial flow, consumer electronics, and military Includes experimental studies such as the detection of biomolecules using SPR sensors and electrochemical sensors for biomolecule detection Presents artificial neural networks (ANN) based industrial flow sensor modeling Highlights case studies on surface plasmon resonance sensors, MEMS-based fluidic sensors, and MEMS-based electrochemical gas sensors The text presents case studies on surface plasmon resonance sensors, MEMS-based fluidic sensors, and MEMS-based electrochemical gas sensors in a single volume. The text will be useful for senior undergraduate, graduate students, and academic researchers in the fields of electrical engineering, electronics, and communication engineering.
During the global COVID-19 pandemic, the needs and benefits of fast and specific analytical tools became apparent to everyone. In particular, advances in nanotechnology promise novel healthcare diagnostics, like the identification of bacterial pathogens. However, up to now, optical nanosensors for pathogen detection rarely exist, but could pave the way for fast, label-free in situ detection of infections in the future. One class of nanomaterials with extraordinary photophysical properties are semiconducting single-walled carbon nanotubes (SWCNTs) that can serve as building blocks for such o...