Matching Items (51)
Description
After a decade of efforts, accurate and affordable DNA sequencing continues to remain an important goal in current research landscape. This thesis starts with a brief overview of the recent updates in the field of DNA sequencing technologies followed by description of the nanofluidics route to single molecule DNA detection. Chapter 2 presents discusses carbon nanotube(CNT) based nanofluidics. The fabrication and DNA sensing measurements of CNT forest membrane devices are presented. Chapter 3 gives the background for functionalization and recognition aspects of reader molecules. Chapter 4 marks the transition to solid state nanopore nanofluidics. The fabrication of Imidazole functionalized nanopores is discussed. The Single Molecule detection results of DNA from Palladium nanopore devices are presented next. Combining chemical recognition to nanopore technology, it has been possible to prolong the duration of single molecule events from the order of a few micro seconds to upto a few milliseconds. Overall, the work presented in this thesis promises longer single molecule detection time in a nanofludic set up and paves way for novel nanopore- tunnel junction devices that combine recognition chemistry, tunneling device and nanopore approach.
ContributorsKrishnakumar, Padmini (Author) / Lindsay, Stuart (Thesis advisor) / He, Jin (Committee member) / Vaiana, Sara (Committee member) / Schmidt, Kevin (Committee member) / Arizona State University (Publisher)
Created2013
Description
One dimensional (1D) and quasi-one dimensional quantum wires have been a subject of both theoretical and experimental interest since 1990s and before. Phenomena such as the "0.7 structure" in the conductance leave many open questions. In this dissertation, I study the properties and the internal electron states of semiconductor quantum wires with the path integral Monte Carlo (PIMC) method. PIMC is a tool for simulating many-body quantum systems at finite temperature. Its ability to calculate thermodynamic properties and various correlation functions makes it an ideal tool in bridging experiments with theories. A general study of the features interpreted by the Luttinger liquid theory and observed in experiments is first presented, showing the need for new PIMC calculations in this field. I calculate the DC conductance at finite temperature for both noninteracting and interacting electrons. The quantized conductance is identified in PIMC simulations without making the same approximation in the Luttinger model. The low electron density regime is subject to strong interactions, since the kinetic energy decreases faster than the Coulomb interaction at low density. An electron state called the Wigner crystal has been proposed in this regime for quasi-1D wires. By using PIMC, I observe the zig-zag structure of the Wigner crystal. The quantum fluctuations suppress the long range correla- tions, making the order short-ranged. Spin correlations are calculated and used to evaluate the spin coupling strength in a zig-zag state. I also find that as the density increases, electrons undergo a structural phase transition to a dimer state, in which two electrons of opposite spins are coupled across the two rows of the zig-zag. A phase diagram is sketched for a range of densities and transverse confinements. The quantum point contact (QPC) is a typical realization of quantum wires. I study the QPC by explicitly simulating a system of electrons in and around a Timp potential (Timp, 1992). Localization of a single electron in the middle of the channel is observed at 5 K, as the split gate voltage increases. The DC conductance is calculated, which shows the effect of the Coulomb interaction. At 1 K and low electron density, a state similar to the Wigner crystal is found inside the channel.
ContributorsLiu, Jianheng, 1982- (Author) / Shumway, John B (Thesis advisor) / Schmidt, Kevin E (Committee member) / Chen, Tingyong (Committee member) / Yu, Hongbin (Committee member) / Ros, Robert (Committee member) / Arizona State University (Publisher)
Created2012
Description
This work demonstrated a novel microfluidic device based on direct current (DC) insulator based dielectrophoresis (iDEP) for trapping individual mammalian cells in a microfluidic device. The novel device is also applicable for selective trapping of weakly metastatic mammalian breast cancer cells (MCF-7) from mixtures with mammalian Peripheral Blood Mononuclear Cells (PBMC) and highly metastatic mammalian breast cancer cells, MDA-MB-231. The advantage of this approach is the ease of integration of iDEP structures in microfliudic channels using soft lithography, the use of DC electric fields, the addressability of the single cell traps for downstream analysis and the straightforward multiplexing for single cell trapping. These microfluidic devices are targeted for capturing of single cells based on their DEP behavior. The numerical simulations point out the trapping regions in which single cell DEP trapping occurs. This work also demonstrates the cell conductivity values of different cell types, calculated using the single-shell model. Low conductivity buffers are used for trapping experiments. These low conductivity buffers help reduce the Joule heating. Viability of the cells in the buffer system was studied in detail with a population size of approximately 100 cells for each study. The work also demonstrates the development of the parallelized single cell trap device with optimized traps. This device is also capable of being coupled detection of target protein using MALDI-MS.
ContributorsBhattacharya, Sanchari (Author) / Ros, Alexandra (Committee member) / Ros, Robert (Committee member) / Buttry, Daniel (Committee member) / Arizona State University (Publisher)
Created2013
Interactions driving the collapse of islet amyloid polypeptide: implications for amyloid aggregation
Description
Human islet amyloid polypeptide (hIAPP), also known as amylin, is a 37-residue intrinsically disordered hormone involved in glucose regulation and gastric emptying. The aggregation of hIAPP into amyloid fibrils is believed to play a causal role in type 2 diabetes. To date, not much is known about the monomeric state of hIAPP or how it undergoes an irreversible transformation from disordered peptide to insoluble aggregate. IAPP contains a highly conserved disulfide bond that restricts hIAPP(1-8) into a short ring-like structure: N_loop. Removal or chemical reduction of N_loop not only prevents cell response upon binding to the CGRP receptor, but also alters the mass per length distribution of hIAPP fibers and the kinetics of fibril formation. The mechanism by which N_loop affects hIAPP aggregation is not yet understood, but is important for rationalizing kinetics and developing potential inhibitors. By measuring end-to-end contact formation rates, Vaiana et al. showed that N_loop induces collapsed states in IAPP monomers, implying attractive interactions between N_loop and other regions of the disordered polypeptide chain . We show that in addition to being involved in intra-protein interactions, the N_loop is involved in inter-protein interactions, which lead to the formation of extremely long and stable β-turn fibers. These non-amyloid fibers are present in the 10 μM concentration range, under the same solution conditions in which hIAPP forms amyloid fibers. We discuss the effect of peptide cyclization on both intra- and inter-protein interactions, and its possible implications for aggregation. Our findings indicate a potential role of N_loop-N_loop interactions in hIAPP aggregation, which has not previously been explored. Though our findings suggest that N_loop plays an important role in the pathway of amyloid formation, other naturally occurring IAPP variants that contain this structural feature are incapable of forming amyloids. For example, hIAPP readily forms amyloid fibrils in vitro, whereas the rat variant (rIAPP), differing by six amino acids, does not. In addition to being highly soluble, rIAPP is an effective inhibitor of hIAPP fibril formation . Both of these properties have been attributed to rIAPP's three proline residues: A25P, S28P and S29P. Single proline mutants of hIAPP have also been shown to kinetically inhibit hIAPP fibril formation. Because of their intrinsic dihedral angle preferences, prolines are expected to affect conformational ensembles of intrinsically disordered proteins. The specific effect of proline substitutions on IAPP structure and dynamics has not yet been explored, as the detection of such properties is experimentally challenging due to the low molecular weight, fast reconfiguration times, and very low solubility of IAPP peptides. High-resolution techniques able to measure tertiary contact formations are needed to address this issue. We employ a nanosecond laser spectroscopy technique to measure end-to-end contact formation rates in IAPP mutants. We explore the proline substitutions in IAPP and quantify their effects in terms of intrinsic chain stiffness. We find that the three proline mutations found in rIAPP increase chain stiffness. Interestingly, we also find that residue R18 plays an important role in rIAPP's unique chain stiffness and, together with the proline residues, is a determinant for its non-amyloidogenic properties. We discuss the implications of our findings on the role of prolines in IDPs.
ContributorsCope, Stephanie M (Author) / Vaiana, Sara M (Thesis advisor) / Ghirlanda, Giovanna (Committee member) / Ros, Robert (Committee member) / Lindsay, Stuart M (Committee member) / Ozkan, Sefika B (Committee member) / Arizona State University (Publisher)
Created2013
Description
Single molecule identification is one essential application area of nanotechnology. The application areas including DNA sequencing, peptide sequencing, early disease detection and other industrial applications such as quantitative and quantitative analysis of impurities, etc. The recognition tunneling technique we have developed shows that after functionalization of the probe and substrate of a conventional Scanning Tunneling Microscope with recognition molecules ("tethered molecule-pair" configuration), analyte molecules trapped in the gap that is formed by probe and substrate will bond with the reagent molecules. The stochastic bond formation/breakage fluctuations give insight into the nature of the intermolecular bonding at a single molecule-pair level. The distinct time domain and frequency domain features of tunneling signals were extracted from raw signals of analytes such as amino acids and their enantiomers. The Support Vector Machine (a machine-learning method) was used to do classification and predication based on the signal features generated by analytes, giving over 90% accuracy of separation of up to seven analytes. This opens up a new interface between chemistry and electronics with immediate implications for rapid Peptide/DNA sequencing and molecule identification at single molecule level.
ContributorsZhao, Yanan, 1986- (Author) / Lindsay, Stuart (Thesis advisor) / Nemanich, Robert (Committee member) / Qing, Quan (Committee member) / Ros, Robert (Committee member) / Zhang, Peiming (Committee member) / Arizona State University (Publisher)
Created2014
Description
Biophysical techniques have been increasingly applied toward answering biological questions with more precision. Here, three different biological systems were studied with the goal of understanding their dynamic differences, either conformational dynamics within the system or oligomerization dynamics between monomers. With Cy3 on the 5' end of DNA, the effects of changing the terminal base pair were explored using temperature-dependent quantum yields. It was discovered, in combination with simulations, that a terminal thymine base has the weakest stacking interactions with the Cy3 dye compared to the other three bases. With ME1 heterodimers, the goal was to see if engineering a salt bridge at the dimerization interface could allow for control over dimerization in a pH-dependent manner. This was performed experimentally by measuring FRET between monomers containing either a Dap or an Asp mutation and comparing FRET efficiency at different pHs. It was demonstrated that the heterodimeric salt bridge would only form in a pH range near neutrality. Finally, with DNA processivity clamps, one aim was to compare the equilibrium dissociation constants, kinetic rate constants, and lifetimes of the closed rings for beta clamp and PCNA. This was done using a variety of biophysical techniques but with three as the main focus: fluorescence correlation spectroscopy, single-molecule experiments, and time-correlated single photon counting measurements. The stability of beta clamp was found to be three orders of magnitude higher when measuring solution stability but only one order of magnitude higher when measuring intrinsic stability, which is a result of salt bridge interactions in the interface of beta clamp. Ongoing work built upon the findings from this project by attempting to disrupt interface stability of different beta clamp mutants by adding salt or changing the pH of the solution. Lingering questions about the dynamics of different areas of the clamps has led to another project for which we have developed a control to demystify some unexpected similarities between beta clamp mutants. With that project, we show that single-labeled and double-labeled samples have similar autocorrelation decays in florescence correlation spectroscopy, allowing us to rule out the dyes themselves as causing fluctuations in the 10-100 microsecond timescale.
ContributorsBinder, Jennifer (Author) / Levitus, Marcia (Thesis advisor) / Wachter, Rebekka (Committee member) / Ros, Robert (Committee member) / Arizona State University (Publisher)
Created2015
Description
Calcitonin Gene-Related Peptide (CGRP) is an intrinsically disordered protein
that has no regular secondary structure, but plays an important role in vasodilation and pain transmission in migraine. Little is known about the structure and dynamics of the monomeric state of CGRP or how CGRP is able to function in the cell, despite the lack of regular secondary structure. This work focuses characterizing the non-local structural and dynamical properties of the CGRP monomer in solution, and understanding how these are affected by the sequence and the solution environment. The unbound, free state of CGRP is measured using a nanosecond laser-pump spectrophotometer, which allows measuring the end-to-end distance (a non-local structural property) and the rate of end-to-end contact formation (intra-chain diffusional dynamics). The data presented in this work show that electrostatic interactions strongly modulate the structure of CGRP, and that peptide-solvent interactions are sequence and charge dependent and can have a significant effect on the internal dynamics of the peptide. In the last few years migraine research has shifted focus to disrupting the CGRP-receptor pathway through the design of pharmacological drugs that bind to either CGRP or its receptor, inhibiting receptor activation and therefore preventing or reducing the frequency of migraine attacks. Understanding what types of intra- and inter-chain interactions dominate in CGRP can help better design drugs that disrupt the binding of CGRP to its receptor.
that has no regular secondary structure, but plays an important role in vasodilation and pain transmission in migraine. Little is known about the structure and dynamics of the monomeric state of CGRP or how CGRP is able to function in the cell, despite the lack of regular secondary structure. This work focuses characterizing the non-local structural and dynamical properties of the CGRP monomer in solution, and understanding how these are affected by the sequence and the solution environment. The unbound, free state of CGRP is measured using a nanosecond laser-pump spectrophotometer, which allows measuring the end-to-end distance (a non-local structural property) and the rate of end-to-end contact formation (intra-chain diffusional dynamics). The data presented in this work show that electrostatic interactions strongly modulate the structure of CGRP, and that peptide-solvent interactions are sequence and charge dependent and can have a significant effect on the internal dynamics of the peptide. In the last few years migraine research has shifted focus to disrupting the CGRP-receptor pathway through the design of pharmacological drugs that bind to either CGRP or its receptor, inhibiting receptor activation and therefore preventing or reducing the frequency of migraine attacks. Understanding what types of intra- and inter-chain interactions dominate in CGRP can help better design drugs that disrupt the binding of CGRP to its receptor.
ContributorsSizemore, Sara (Author) / Vaiana, Sara (Thesis advisor) / Ghirlanda, Giovanna (Committee member) / Ros, Robert (Committee member) / Lindsay, Stuart (Committee member) / Ozkan, Sefika (Committee member) / Arizona State University (Publisher)
Created2015
Description
Fluorescence spectroscopy is a popular technique that has been particularly useful in probing biological systems, especially with the invention of single molecule fluorescence. For example, Förster resonance energy transfer (FRET) is one tool that has been helpful in probing distances and conformational changes in biomolecules. In this work, important properties necessary in the quantification of FRET were investigated while FRET was also applied to gain insight into the dynamics of biological molecules. In particular, dynamics of damaged DNA was investigated. While damages in DNA are known to affect DNA structure, what remains unclear is how the presence of a lesion, or multiple lesions, affects the flexibility of DNA, especially in relation to damage recognition by repair enzymes. DNA conformational dynamics was probed by combining FRET and fluorescence anisotropy along with biochemical assays. The focus of this work was to investigate the relationship between dynamics and enzymatic repair. In addition, to properly quantify fluorescence and FRET data, photophysical phenomena of fluorophores, such as blinking, needs to be understood. The triplet formation of the single molecule dye TAMRA and the photoisomerization yield of two different modifications of the single molecule cyanine dye Cy3 were examined spectroscopically to aid in accurate data interpretation. The combination of the biophysical and physiochemical studies illustrates how fluorescence spectroscopy can be used to answer biological questions.
ContributorsShepherd Stennett, Elana Maria (Author) / Levitus, Marcia (Thesis advisor) / Ros, Robert (Committee member) / Liu, Yan (Committee member) / Arizona State University (Publisher)
Created2015
Description
An animal's ability to produce protein-based silk materials has evolved independently in many different arthropod lineages, satisfying various ecological necessities. However, regardless of their wide range of uses and their potential industrial and biomedical applications, advanced knowledge on the molecular structure of silk biopolymers is largely limited to those produced by spiders (order Araneae) and silkworms (order Lepidoptera). This thesis provides an in-depth molecular-level characterization of silk fibers produced by two vastly different insects: the caddisfly larvae (order Trichoptera) and the webspinner (order Embioptera).
The molecular structure of caddisfly larval silk from the species Hesperophylax consimilis was characterized using solid-state nuclear magnetic resonance (ss-NMR) and Wide Angle X-ray Diffraction (WAXD) techniques. This insect, which typically dwells in freshwater riverbeds and streams, uses silk fibers as a strong and sticky nanoadhesive material to construct cocoons and cases out available debris. Conformation-sensitive 13C chemical shifts and 31P chemical shift anisotropy (CSA) information strongly support a unique protein motif in which phosphorylated serine- rich repeats (pSX)4 complex with di- and trivalent cations to form rigid nanocrystalline β-sheets. Additionally, it is illustrated through 31P NMR and WAXD data that these nanocrystalline structures can be reversibly formed, and depend entirely on the presence of the stabilizing cations.
Nanofiber silks produced by webspinners (order Embioptera) were also studied herein. This work addresses discrepancies in the literature regarding fiber diameters and tensile properties, revealing that the nanofibers are about 100 nm in diameter, and are stronger (around 500 MPa mean ultimate stress) than previous works suggested. Fourier-transform Infrared Spectroscopy (FT-IR), NMR and WAXD results find that approximately 70% of the highly repetitive glycine- and serine-rich protein core is composed of β-sheet nanocrystalline structures. In addition, FT-IR and Gas-chromatography mass spectroscopy (GC-MS) data revealed a hydrophobic surface coating rich in long-chain lipids. The effect of this surface coating was studied with contact angle techniques; it is shown that the silk sheets are extremely hydrophobic, yet due to the microstructural and nanostructural details of the silk surface, are surprisingly adhesive to water.
The molecular structure of caddisfly larval silk from the species Hesperophylax consimilis was characterized using solid-state nuclear magnetic resonance (ss-NMR) and Wide Angle X-ray Diffraction (WAXD) techniques. This insect, which typically dwells in freshwater riverbeds and streams, uses silk fibers as a strong and sticky nanoadhesive material to construct cocoons and cases out available debris. Conformation-sensitive 13C chemical shifts and 31P chemical shift anisotropy (CSA) information strongly support a unique protein motif in which phosphorylated serine- rich repeats (pSX)4 complex with di- and trivalent cations to form rigid nanocrystalline β-sheets. Additionally, it is illustrated through 31P NMR and WAXD data that these nanocrystalline structures can be reversibly formed, and depend entirely on the presence of the stabilizing cations.
Nanofiber silks produced by webspinners (order Embioptera) were also studied herein. This work addresses discrepancies in the literature regarding fiber diameters and tensile properties, revealing that the nanofibers are about 100 nm in diameter, and are stronger (around 500 MPa mean ultimate stress) than previous works suggested. Fourier-transform Infrared Spectroscopy (FT-IR), NMR and WAXD results find that approximately 70% of the highly repetitive glycine- and serine-rich protein core is composed of β-sheet nanocrystalline structures. In addition, FT-IR and Gas-chromatography mass spectroscopy (GC-MS) data revealed a hydrophobic surface coating rich in long-chain lipids. The effect of this surface coating was studied with contact angle techniques; it is shown that the silk sheets are extremely hydrophobic, yet due to the microstructural and nanostructural details of the silk surface, are surprisingly adhesive to water.
ContributorsAddison, John Bennett (Author) / Yarger, Jeffery L (Thesis advisor) / Holland, Gregory P (Thesis advisor) / Wang, Xu (Committee member) / Ros, Robert (Committee member) / Arizona State University (Publisher)
Created2014
Description
This thesis describes several experiments based on carbon nanotube nanofludic devices and field-effect transistors. The first experiment detected ion and molecule translocation through one single-walled carbon nanotube (SWCNT) that spans a barrier between two fluid reservoirs. The electrical ionic current is measured. Translocation of small single stranded DNA oligomers is marked by large transient increases in current through the tube and confirmed by a PCR (polymerase chain reaction) analysis. Carbon nanotubes simplify the construction of nanopores, permit new types of electrical measurement, and open new avenues for control of DNA translocation. The second experiment constructed devices in which the interior of a single-walled carbon nanotube field-effect transistor (CNT-FET) acts as a nanofluidic channel that connects two fluid reservoirs, permitting measurement of the electronic properties of the SWCNT as it is wetted by an analyte. Wetting of the inside of the SWCNT by water turns the transistor on, while wetting of the outside has little effect. This finding may provide a new method to investigate water behavior at nanoscale. This also opens a new avenue for building sensors in which the SWCNT functions as an electronic detector. This thesis also presents some experiments that related to nanofabrication, such as construction of FET with tin sulfide (SnS) quantum ribbon. This work demonstrates the application of solution processed IV-VI semiconductor nanostructures in nanoscale devices.
ContributorsCao, Zhai (Author) / Lindsay, Stuart (Thesis advisor) / Vaiana, Sara (Committee member) / Ros, Robert (Committee member) / Marzke, Robert (Committee member) / Shumway, John (Committee member) / Arizona State University (Publisher)
Created2011