Author: Nan Jing
Publisher:
ISBN:
Category :
Languages : en
Pages :
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The objective of this work is to develop a micro/nanofluidic-based single molecule detection (SMD) scheme, which would allow us to inspect individual protein or protein complex study protein-protein interactions and their dynamics. This is a collaboration work with MD Anderson Cancer Center and we applied this scheme to study functions of various proteins related to cancer progression in hope to shed new light on cancer research. State-of-the-art micro/nano-fabrication technology is used to provide fused silica micro/nano-fluidic channel devices as our detection platform. Standard contact photolithography, projection photolithography and advanced electron-beam lithography are used to fabricate micro/nano-fluidic channel with width ranging from 100nm to 2[micron]. The dimensions of these miniaturized biochips are designed to ensure single molecule resolution during detection and shrinking the detection volume leads to increase in signal-to-noise ratio, which is very critical for SMD. To minimize surface adsorption of protein, a fused silica channel surface coating procedure is also developed and significantly improved the detection efficiency. A fluorescent-labeled protein sample solution is filled in the fluidic channel by capillary force, and proteins are electro-kinetically driven through the fluidic channel with external voltage source. Commercial functionalized Quantum Dots (Qdots) are used as fluorescent labels due to its various advantages over conventional organic dyes for single molecule multi-color detection application. A fluorescence correlation spectrometer system, equipped with a 375nm diode laser, 60x water immersion objective with N.A. of 1.2 and two avalanche photodiodes (APD) is implemented to excite single molecules as well as collect emitted fluorescence signals. A two-dimensional photon burst analysis technique (photon counts vs. burst width) is developed to analyze individual single molecule events. We are able to identify target protein or protein complex directly from cell lysate based on fluorescence photon counts, as well as study the dynamics of protein-protein interactions. More importantly, with this technique we are also able to assess interactions between three proteins, which cannot be done with current ensemble measurement techniques. In summary, the technique described in this work has the advantages of high sensitivity, short processing time (2-3 minutes), very small sample consumption and high resolution quantitative analysis. It could potentially revolutionize the area of protein interaction research and provides us with more clues for the future of cancer diagnostics and treatments.
Author: Benjamin Ryan Cipriany
Publisher:
ISBN:
Category :
Languages : en
Pages : 306
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Microfluidics and nanofluidics have recently emerged as analytical tools for the study of biology. These devices have enabled the miniaturization of biological sample preparation and detection methods, toward consuming less sample volume and improving the sensitivity and speed of analysis. This thesis explores methods for rapid detection and sorting of individual biomolecules within a nanofluidic channel. In these devices, constructed using thin-film processing techniques, attoliter-scale volume confinement is formed to isolate individual, fluorophore-labeled biomolecules in solution for absolute quantification. These devices enable studies of the unique attributes of each molecule, often masked in ensemble-averaged measurements. Statistical sampling of many molecules is achieved by voltage-actuated, electrokinetic flow within the nanofluidic device to precisely control molecule analysis rate and achieve high throughput single molecule detection (SMD). This nanofluidic technology is applied to epigenetic analysis, enabling the study of epigenetic modifications at a single molecule level. Viable epigenetic analysis within a nanofluidic device is demonstrated using chromatin, DNA bound with histone proteins, which is shown to remain in its native state during nanofluidic confinement and electrokinetic flow under physiologically-relevant conditions. Detection of an epigenetic modification, DNA methylation, is also demonstrated to elucidate its potential for detecting multiple epigenetic marks on an individual molecule. Subsequently, an architecture for automated, high-speed sorting of individual molecules is developed. In this architecture, digital signal processing methods are implemented in a field programmable gate array to achieve real-time SMD. An electric circuit model is developed to actuate and switch electrokinetic flow of molecules, partitioning them into branches of a bifurcated nanofluidic device. An optical system for parallel SMD is realized to experimentally validate the actuation of molecule sorting in-situ. Combined, these components are utilized in automated, fluorescence-activated sorting of individual, methylated DNA molecules, which were then collected for further analysis. This device is reconfigurable and can be generalized for application to fluorescence-activated separations of other molecule types. Finally, a study of various methods for optofluidic integration is presented. The optical properties of fused-silica, silicon nitride, polydimethylsiloxane, hydrogen silisequioxane, and chemical vapor deposited oxides are investigated to consider their use in SMD applications requiring ultra-low autofluorescence and high confinement of the optical probe volume. Findings were then applied to form an optical waveguide as an fluorescence excitation source toward the dense integration of optical and nanofluidic components.
Author: Sung Min Hong
Publisher:
ISBN:
Category :
Languages : en
Pages :
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In the cancer research, it is important to understand protein dynamics which are involved in cell signaling. Therefore, particular protein detection and analysis of target protein behavior are indispensable for current basic cancer research. However, it usually performed by conventional biochemical approaches, which require long process time and a large amount of samples. We have been developed the new applications based on microfluidics and Raster image Correlation spectroscopy (RICS) techniques. A simple microfluidic 3D hydrodynamic flow focusing device has been developed for quantitative determinations of target protein concentrations. The analyte stream was pinched not only horizontally, but also vertically by two sheath streams by introducing step depth cross junction structure. As a result, a triangular cross-sectional flow profile was formed and the laser was focused on the top of the triangular shaped analyte stream. Through this approach, the target protein concentration was successfully determined in cell lysate samples. The RICS technique has been applied to characterize the dynamics of protein 53 (p53) in living cells before and after the treatment with DNA damaging agents. P53 tagged with Green Fluores-cent Protein (GFP) were incubated with and without DNA damaging agents, cisplatin or eptoposide. Then, the diffusion coefficient of GFP-p53 was determined by RICS and it was significantly reduced after the drug treatment while that of the one without drug treatment was not. It is suggested that the drugs induced the interaction of p53 with either other proteins or DNA. This result demonstrates that RICS is able to detect protein-protein or protein-DNA interactions in living cells and it may be useful for the drug screening. As another application of microfluidics, an integrated microfluidic platform was developed for generating collagen microspheres with encapsulation of viable cells. The platform integrated four automated functions on a microfluidic chip, (1) collagen solution cooling system, (2) cell-in-collagen microdroplet generation, (3) collagen microdroplet polymerization, and (4) incubation and extraction of the microspheres. This platform provided a high throughput and easy way to generate uniform dimensions of collagen microspheres encapsulating viable cells that were able to proliferate for more than 1 week.
Author:
Publisher: Elsevier
ISBN: 0128164646
Category : Science
Languages : en
Pages : 404
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Spectroscopy and Dynamics of Single Molecules: Methods and Applications reviews the most recent developments in spectroscopic methods and applications. Spectroscopic techniques are the chief experimental methods for testing theoretical models and research in this area plays an important role in stimulating new theoretical developments in physical chemistry. This book provides an authoritative insight into the latest advances in the field, highlighting new techniques, current applications, and potential future developments An ideal reference for chemists and physicists alike, Spectroscopy and Dynamics of Single Molecules: Methods and Applications is a useful guide for all those working in the research, design, or application of spectroscopic tools and techniques across a wide range of fields. - Includes the latest research on ultrafast vibrational and electronic dynamics, nonlinear spectroscopies, and single-molecule methods - Makes the content accessible to researchers in chemistry, biophysics, and chemical physics through a rigorous multi-disciplinary approach - Provides content edited by a world-renowned chemist with more than 30 years of experience in research and instruction
Author:
Publisher:
ISBN: 9789179053437
Category :
Languages : en
Pages :
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Author: Kumuditha Madushanka Weerakoon Ratnayake
Publisher:
ISBN:
Category :
Languages : en
Pages :
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Author: Melanie M. West
Publisher:
ISBN:
Category : Nanofluids
Languages : en
Pages : 107
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Single molecule detection is limited by the small scattering cross-section of molecules which leads to weak optical signals that can be obscured by background noise. The combination of plasmonics and nanofluidics in an integrated nano-device has the potential to provide the signal enhancement necessary for the detection of single molecules. The purpose of this investigation was to optimize the fabrication of an optofluidic device that integrates a nanochannel with a plasmonic bowtie antenna. The fluidic structure of the device was fabricated using UV-nanoimprint lithography, and the gold plasmonic antennas were fabricated using a shadow evaporation and lift-off process. The effect of electron beam lithography doses on the resolution of antenna-nanochannel configurations was studied to minimize antenna gap size while maintaining the integrity of the imprinted features. The smallest antenna gap size that was achieved was 46 nm. The antennas were characterized using dark field spectroscopy to find the resonance shift, which indicated the appropriate range for optical signal enhancement. The dark field scattering results showed antennas with a broad and well-defined resonance shift that ranged from 650 - 800 nm. The Raman scattering results showed the highest enhancement factor (EF = 2) for antennas with an "inverted configuration," which involved having the triangles of the antenna facing back-to-back rather than the more conventional tip-to-tip bowtie arrangement.
Author: Paolo Cadinu
Publisher:
ISBN:
Category :
Languages : en
Pages :
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Author: Baldassare Di Bartolo
Publisher: Springer
ISBN: 9400753136
Category : Science
Languages : en
Pages : 470
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This volume presents a considerable number of interrelated contributions dealing with the new scientific ability to shape and control matter and electromagnetic fields on a sub-wavelength scale. The topics range from the fundamental ones, such as photonic metamateriials, plasmonics and sub-wavelength resolution to the more applicative, such as detection of single molecules, tomography on a micro-chip, fluorescence spectroscopy of biological systems, coherent control of biomolecules, biosensing of single proteins, terahertz spectroscopy of nanoparticles, rare earth ion-doped nanoparticles, random lasing, and nanocoax array architecture. The various subjects bridge over the disciplines of physics, biology and chemistry, making this volume of interest to people working in these fields. The emphasis is on the principles behind each technique and on examining the full potential of each technique. The contributions that appear in this volume were presented at a NATO Advanced Study Institute that was held in Erice, Italy, 3-18 July, 2011. The pedagogical aspect of the Institute is reflected in the topics presented in this volume.
Author: M. Muthukumar
Publisher: CRC Press
ISBN: 1420075179
Category : Science
Languages : en
Pages : 362
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Book Description
Polymer translocation occurs in many biological and biotechnological phenomena where electrically charged polymer molecules move through narrow spaces in crowded environments. Unraveling the rich phenomenology of polymer translocation requires a grasp of modern concepts of polymer physics and polyelectrolyte behavior. Polymer Translocation discusse
