Click chemistry is a molecular synthesis strategy based on reliable, highly selective reactions with thermodynamic driving forces typically in excess of 20 kcal mol-1. The 1,3-dipolar cycloaddition of azides and alkynes developed by Rolf Huisgen saw dramatic rate acceleration using Cu(I) as a catalyst in 2002 reports by Barry Sharpless and Morten Meldal enabling its click chemistry eligibility. Since these seminal reports, the copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) has become the quintessential click reaction finding diverse utility. The popularity of the CuAAC has naturally led to interest in new catalyst systems with improved efficiency, robustness, and reusability with particular focus on nanomaterial catalysts, a common trend across the field of catalysis. The high surface area of nanomaterials lends to their efficacy as colloidal and heterogeneous nanocatalysts, but the latter boasts the added benefit of easy separation and recyclability. With any heterogeneous catalyst, a common question arises as to whether the active catalyst species is truly heterogeneous or rather homogeneous through metal ion leaching. Differentiating these processes is critical, as the latter would result in reduced efficiency, higher cost, and inevitable environmental and heath side effects. This thesis explores the CuAAC from an interdisciplary approach. First as a synthetic tool, applying CuAAC-formed triazoles as functional, modular building blocks in the synthesis of optical cation sensors by combining azide and alkyne modified components to create a series of sensors selective for different metal cations. Next, single-molecule spectroscopy techniques are employed to observe the CuNP-catalyzed CuAAC in real time. Combining bench-top techniques with single-molecule microscopy to monitor single-catalytically generated products proves to be an effective method to establish catalysis occurs directly at the surface of copper nanoparticles, ruling out catalysis by ions leached into solution. This methodology is extended to mapping the catalytic activity of a commercial heterogeneous catalyst by applying super-localization analysis of single-catalytic events. The approach detailed herein is a general one that can be applied to any catalytic system through the development of appropriate probes. This thesis demonstrates single-molecule microscopy as an accessible, effective, and unparalleled tool for exploring the catalytic activity of nanomaterials by monitoring single-catalytic events as they occur.

No description available.

Master of Science / Department of Chemistry / Daniel A. Higgins / Single molecule tracking (SMT) measurements have been applied to the study of molecular mobility in sol-gel-derived silica gradient films in this thesis. Such gradient films have broad potential applications in controlled adhesion and transport of cells, vesicles and polymers; separation of complex chemical mixtures and in the development of new catalysts. Silica films were prepared by “infusion-withdrawal dip-coating”. In this method, a suitable substrate is slowly withdrawn from a silica sol of time varying composition. The deposition reservoir is initially filled with a sol derived from one silica precursor (tetramethoxysilane). A second sol, prepared from a different precursor (methyltrimethoxysilane), is then infused into the deposition reservoir, as the mixed sol is withdrawn. Films thus prepared were initially characterized by bulk fluorescence spectroscopy, infrared (IR) microscopy, contact angle goniometry, spectroscopic ellipsometry and surface profilometry. The fluorescence, IR and contact angle data all demonstrate the presence of a gradient in the methyl content of the silica film. The primary objective of the work performed under this thesis was to investigate the diffusion of Nile Red molecules in and on these films, as a function of position along the gradient, by SMT methods. Histograms of the mean-square displacement of the molecules depict the presence of at least two distinct populations: one incorporating fixed (entrapped or adsorbed) molecules and the other clearly reflecting the presence of mobile molecules. The latter population was observed to vary along the gradient dimension and also changed as the films aged over the course of five days. Molecular mobility is attributed to the presence of liquid-like silica oligomers in the films. Spatial variations in the observed mobility are tentatively assigned to variations in oligomer viscosity along the gradient. Film viscosity also changes as the polymerization of the oligomers continues during film aging.

Doctor of Philosophy / Department of Chemistry / Daniel A. Higgins / The preparation of mesoporous/macroporous silica materials and polarity gradient thin film are introduced in this thesis. These porous silica materials and gradient materials have the potential applications as stationary phases for chemical separations, as materials for combinatorial catalysis and as absorbent/adsorbent layers for use in chemical or biological sensors. Single molecule spectroscopy is used to probe the chemical interaction between single dye molecule and porous silica matrix. Bulk fluorescence spectroscopy is used to investigate the properties of gradient film. In Chapter one, the applications of single molecule spectroscopic methods to sol-gel silica materials are reviewed, which covers a subset of the recent literature in this area and provided salient examples of the new information that can be obtained by single molecule studies. In Chapter two, both the sample preparation and experiment setup are covered. In Chapter three, the preparation of mesoporous silica film is presented. Single molecule spectroscopy is used to probe the mass transport and molecule-matrix interactions in mesoporous thin-film systems. Three different dyes of varying size, charge, and hydrophilicity are used. Silica films with/without surfactant or containing different kind surfactant are studied. The results provide new information on mass transport through the films, evidence of reversible surface adsorption, and quantitative information on variations in these phenomena with film hydration. In Chapter four, a new model describing how to explore the actual dye concentration in single molecule experiment with considering the molecule orientation is presented, which is verified to be correct by both experimental and simulated data. In Chapter five, the growth process of Methylsilsesquioxane (MSQ) particle is studied by single molecule spectroscopy, in which, the MSQ particle is treated as “native” dye molecule. In Chapter six, silica films incorporating polarity gradients are produced by using “infusion-withdrawal dip-coating” method. The gradient film is characterized by bulk fluorescence spectroscopy, water contact angle and FTIR. In Chapter seven, a brief conclusion is drawn and future directions are presented.

In this work organic molecules, namely derivatives of BODIPY and poly-para-phenyls are investigated on different metal surfaces by means of LT-STM. These molecule are important for the development of molecular electronics and spintronics. I show that aza-BODIPY molecules form a weak chemical bond with the Au(111) substrate and the molecular structure significantly changes upon adsorption. Due to the low corrugation of the Au(111) surface, diffusion of the molecule is observed for applied bias in excess of ±1 V. The temperature dependent formation of different molecular nanostructures formed by polyparaphenyls and Au adatoms is discussed. The diffusing Au adatoms act as coordination centers for the cyano groups present on one end of the molecules. The structure of the super molecular assemblies completely changes in a temperature range of only 60 K. Furthermore, I investigate in this work the hybridization of atomic orbitals within the molecular ligand. The Kondo resonance of a Co atom incorporated into an other aza-BODIPY derivative is investigated in detail on Ag(100). The hybridization of the atomic Co orbital with the organic ligands molecular orbitals is shown by spectroscopy measurements with submolecular resolution. The changing line shape of the Kondo resonance for the molecule-substrate system is discussed. This data is compared to measurements of Co incorporated in another molecular binding motive and on different metal samples to show the importance of the local environment for molecular materials. / In dieser Arbeit werden organische Moleküle, Derivate von BODIPY und poly-para-Phenyl, auf verschiedenen Metalloberflächen mittels Tief-Temperatur Rastertunnelmikroskopie (LT-STM) untersucht. Diese Moleküle sind wichtig für die Entwicklung von molekularer Elektronik und Spintronik. Ich zeige, dass aza-BODIPY-Moleküle eine schwache chemische Bindung mit dem Au(111)- Substrat eingehen und die molekulare Struktur bei der Adsorption deutlich verändert wird. Wegen der geringen Rauigkeit der Au(111)-Oberfläche wird bereits bei einer angelegten Spannungen über ±1 V die Diffusion der Moleküle beobachtet. Die temperaturabhängige Bildung verschiedener molekularer Nanostrukturen aus poly-para-Phenyl und frei beweglichen Goldatomen wird diskutiert. Die diffundierenden Goldatome agieren hierbei als Koordinationszentren für die Cyanogruppen am einen Ende der Moleküle. Die Struktur der supramolekularen Anordnungen verändert sich dabei in einem Temperaturbereich von nur 60 K vollkommen. Außerdem beschäftige ich mich in dieser Arbeit mit der Hybridisierung atomare Orbitale im molekularen Verbund. Die Kondo-Resonanz eine Co-Atoms, welches in einem anderen aza-BODIPY-Derivat gebunden ist, wird detailliert auf der Ag(100)-Oberfläche untersucht. Die Hybridisierung des atomaren Co-Orbitals mit den molekularen Orbitalen des organischen Liganden wird an Hand von Spektroskopiemessungen mit submolekularer Auflösung gezeigt. Die veränderte Form der Kondo-Resonanz für dieses Molekül-Substrat-System wird diskutiert. Diese Daten werden mit Messungen an Co-Atomen in anderen molekularen Bindungsschemen und auf anderen Substraten verglichen um dieWichtigkeit der lokalen Umgebung für molekulare Materialien zu verdeutlichen.

During the last decade high throughput DNA sequencing of single cells has evolved from an idea to one of the most high profile fields of research. Much of this development has been possible due to the dramatic reduction in costs for massively parallel sequencing. The four papers included in this thesis describe or evaluate technological advancements for high throughput DNA sequencing of single cells and single molecules. As the sequencing technologies improve, more samples are analyzed in parallel. In paper 1, an automated procedure for preparation of samples prior to massively parallel sequencing is presented. The method has been applied to several projects and further development by others has enabled even higher sample throughputs. Amplification of single cell genomes is a prerequisite for sequence analysis. Paper 2 evaluates four commercially available kits for whole genome amplification of single cells. The results show that coverage of the genome differs significantly among the protocols and as expected this has impact on the downstream analysis. In Paper 3, single cell genotyping by exome sequencing is used to confirm the presence of fat cells derived from donated bone marrow within the recipients’ fat tissue. Close to hundred single cells were exome sequenced and a subset was validated by whole genome sequencing. In the last paper, a new method for phasing (i.e. determining the physical connection of variant alleles) is presented. The method barcodes amplicons from single molecules in emulsion droplets. The barcodes can then be used to determine which variants were present on the same original DNA molecule. The method is applied to two variable regions in the bacterial 16S gene in a metagenomic sample. Thus, two of the papers (1 and 4) present development of new methods for increasing the throughput and information content of data from massively parallel sequencing. Paper 2 evaluates and compares currently available methods and in paper 3, a biological question is answered using some of these tools. / <p>QC 20160127</p>

This thesis is focused on understanding the interactions between molecules and surface-enhanced Raman scattering (SERS) substrates that are typically unresolved due to the diffraction limit of light. Towards this end, we have developed and tested several different sub-diffraction-limited imaging techniques in order to observe these interactions. First, we utilize an isotope-edited bianalyte approach combined with super-resolution imaging via Gaussian point-spread function fitting to elucidate the role of Raman reporter molecules on the location of the SERS emission centroids. By using low concentrations of two different analyte molecules, we find that the location of the SERS emission centroid depends on the number and positions of the molecules present on the SERS substrate. It is also known that SERS enhancement partially results from the molecule coupling its emission into the far-field through the plasmonic nanostructure. This results in a particle-dictated, dipole-like emission pattern, which cannot be accurately modeled as a Gaussian, so we tested the applicability of super-resolution imaging using a dipole-emission fitting model to this data. To test this model, we first fit gold nanorod (AuNR) luminescence images, as AuNR luminescence is primarily coupled out through the longitudinal dipole plasmon mode. This study showed that a three-dimensional dipole model is necessary to fit the AuNR emission, with the model providing accurate orientation and emission wavelength parameters for the nanostructure, as confirmed using correlated AFM and spectroscopy. The dipole fitting technique was next applied to single- and multiple-molecule SERS emission from silver nanoparticle dimers. We again found that a three-dimensional dipole PSF was necessary to accurately model the emission and orientation parameters of the dimer, but that at the single molecule level, the movement of the molecule causes increased uncertainty in the orientation parameters determined by the fit. Finally, we describe progress towards using a combined atomic force/optical microscope system in order to position a carbon nanotube analyte at known locations on the nanoparticle substrate. This would allow for the simultaneous mapping of nanoparticle topography and exact locations of plasmonic enhancement around the nanostructure, but consistently low signal-to-noise kept this technique from being viable. / text

Mechanics and biological function of nucleic acids are intimately coupled. The DNA double helix must be opened to allow base pairing of RNA during transcription; RNA must bend and fold in its many cellular functions. Presented in this dissertation are two investigations of mechanical deformations of nucleic acids, conducted with optical tweezers.In the introduction, the mechanical properties of DNA and RNA and their relevance to their cellular functions are introduced, to give the reader context for the results presented in the Chapters 2 and 3. This is followed by an introduction to the theory of semiflexible polymer elasticity. The optical tweezers instruments used in conducting these investigations are then presented, along with calibration procedures and a short introduction to optical trapping physics.Chapter 2 presents an investigation of the effect of downstream DNA tension on initiation by T7 RNA polymerase. A hidden Markov model is fit to force-dependent lifetimes obtained from optical tweezers experiments, allowing us to identify which steps in initiation are force-dependent and estimate rates and transition state distances. We find that 1-2 pN of tension is sufficient to turn o gene expression by causing transcription bubble collapse and destabilizing the bound state. Our force-dependence scheme and estimated transition distances provide independent supportfor the \scrunching" model of initiation.The effects of cation binding and screening on single-stranded helix formation in poly(A) RNA are presented in Chapter 3. Magnesium and calcium bind to poly(A), stabilize the helix, and change its mechanical properties. A new model of helix-coil transitions is presented and used to estimate energetics and mechanical properties.Chapter 4 presents the first fully objective algorithm for use in analyzing the noisy staircaselike data that is often produced by single-molecule fluorescence experiments. A test based on the SIC (BIC) statistic is used in conjunction with a progressive step-placement scheme to locate changepoints (steps) in noisy data. Its performance is compared to other step detection algorithms in use by biophysicists by repeating tests performed in a recent review.Experimental protocols and computer codes used in these investigations are presentedin detail in the appendices.

Interfacial charge transfer between metal oxides and organic semiconductors has been found to limit the efficiency of organic optoelectronic devices. Although a number of investigations of inorganic/organic systems exist, very few generally applicable rules for oxide/organic interfaces have been developed and many questions about these systems remain unanswered. Thus the studies presented in this dissertation were designed to improve the understanding of the fundamental interface physics of metal oxide/organic systems. Single molecule fluorescence microscopy was employed to determine the charge transfer mechanism while photoelectron spectroscopy was used to determine the energy level alignment of model systems. Additional computational studies allowed the examination of the properties of the charged organic molecules involved in charge transfer and modeling of the molecule-surface interaction. Calculations of the ground state properties and excited state transitions of the neutral and singly charged states of a modified perylene molecule were performed to provide insight into the orbitals of the initial and final states involved in the interfacial charge transfer process. The design and implementation of a novel UHV single molecule microscope is described. This microscope was used to observe the excited state charge transfer between a modified perylene molecule and Al₂O₃ (0001). The charge transfer mechanism was identified as involving activated trapping and detrapping of the defect derived states within the Al₂O₃ band gap, which resulted in the observation of strongly distributed kinetics for this system. The influence of defects and adsorbates on the electronic structure of ZnO and its interface with organic semiconductors was determined from photoelectron spectroscopy. Modified perylene molecules were found to have strong chemisorptive interactions with the ZnO surface involving charge transfer from defect derived ZnO states to the LUMO, while magnesium phthalocyanine molecules appear to have only weak physisorptive interactions with the ZnO surface. The interfacial investigations of the organic/oxide systems demonstrate the rich defect structure present in metal oxides. In both cases, defects were found to control the interfacial interactions between the metal oxide surface and the modified perylene molecules. Thus the manipulation of these defects states is of fundamental importance for optoelectronic device design.

The search for a satisfactory model for complexity, meant as an intermediate condition between total order and total disorder, is still subject of debate in the scientific community. In this dissertation the emergence of non-Poisson renewal processes in several complex systems is investigated. After reviewing the basics of renewal theory, another popular approach to complexity, called modulation, is introduced. I show how these two different approaches, given a suitable choice of the parameter involved, can generate the same macroscopic outcome, namely an inverse power law distribution density of events occurrence. To solve this ambiguity, a numerical instrument, based on the theoretical analysis of the aging properties of renewal systems, is introduced. The application of this method, called renewal aging experiment, allows us to distinguish if a time series has been generated by a renewal or a modulation process. This method of analysis is then applied to several physical systems, from blinking quantum dots, to the human brain activity, to seismic fluctuations. Theoretical conclusions about the underlying nature of the considered complex systems are drawn.