Single-Molecule Magnets (SMMs) are discrete molecules that exhibit slow relaxation of magnetization. Unlike conventional magnets that rely on the long range magnetic ordering in the form of domains, these molecules act as magnets independently, that is without the influence of neighbouring molecules. SMMs have intrigued physicists and chemists alike for over twenty years with their potential future applications in data storage quantum computing, and with this communal interest there has been significant collaboration between the two fields of research. SMMs have brought forth an opportunity for coordination chemists to muster their creativity and synthetic expertise in the rational design and development of these magnetic materials. From these new and fascinating compounds, both experimental and theoretical physicists have sought to develop and refine our understanding of the aspects of these molecular magnets in order to improve their performance at higher temperatures. In this work, new topologies for lanthanide complexes are explored using a novel Schiff base ligand. The magnetic properties of dinuclear, tetranuclear and octanuclear lanthanide complexes are discussed and correlated to their structural properties. The rational design of tetrazine-based Schiff base ligands for magnetic studies is also discussed in hopes of developing high performance SMMs.

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Nanopores have emerged as a simple but effective tool to investigate the behavior of polymers in solution. They have shown great potential to simplify expensive and time consuming procedures like DNA sequencing, protein detection, and disease biomarker detection. With the development of in situ fabrication of solid-state nanopores by controlled breakdown (CBD) of a dielectric material, nanomanufacturing of nanopore-based technologies became feasible. However, there are still a lot of challenges to overcome for these applications to become reality. One of the major problems with solid-state nanopores is the rapid passage time of analytes going through the pore, complicating detection and reliable identification of molecules. In this thesis molecular structures are proposed that increase passage times due to increased interactions between analyte and pore wall, and at the same time increase signal amplitude due to increased blockage of the pore. These structures are short, branched DNA molecules that were assembled with built-in modifications and matching sequences to assume their structure. Nanopore experiments reveal that these structurally defined DNA produce higher detection rates than their linear DNA counterparts, making them better candidates for labels in single-molecule sensing experiments.

Master of Science / Department of Chemistry / Takashi Ito / Solvent swelling of block copolymer microdomains plays an essential role in the improvement of microdomain alignment by solvent vapor annealing and in chemical separations using block copolymer monoliths. Here, investigation of the effects of solvent swelling on the molecular permeability and dimensions of cylindrical microdomains in polystyrene-block-poly(ethylene oxide) (PS-b-PEO) films is done by using single molecule tracking. These films are prepared by sandwiching benzene (with/without methanol) or THF (with/without methanol) solutions containing 5 nM sulforhodamine B (SRB) between two glass substrates. The PEO microdomains are aligned in the solution flow direction during the film preparation. The diffusional motions of individual SRB molecules are measured at different drying times to assess the microdomain radius and permeability. These parameters, on average, gradually decrease with an increase in drying time; however the trend differs slightly from one solvent system to another. A sharp decrease of microdomain radius is observed for benzene, benzene-methanol, THF and THF-methanol swollen films at initial drying condition (for example 2 days). In contrast, microdomain permeability does not decrease sharply; instead a gradual decreasing trend is seen for all solvent systems. In addition, mixing of a small amount of methanol (14% in PEO microdomains) either with benzene or with THF does not produce noticeable difference in the swelling of PEO microdoamins. Importantly, both benzene and THF offer similar microdomain swelling behavior at the same drying temperature, which is evident from the microdomain radius values, however THF shows comparatively larger microdomain permeability and better correlation between permeability and microdomain radius compared with benzene.

This thesis describes the synthesis, characterization and magnetic investigation of homometallic lanthanide complexes based on two different o-vanillin-derived Schiff base ligands: H2ovph and H2ovgrd. The studies were performed using single crystal X-ray diffractometry, Powder XRD and SQUID magnetometry. Chapter 2 focuses on dinuclear systems 1-8 coordinated to the ligand H2ovph and presents their structural and magnetic properties, mainly with respect to their intramolecular interactions. Chapter 3 describes two hexanuclear systems, 9 (DyIII) and 10 (GdIII), with trigonal prism-assembled core structures. A structural comparison to other similar complexes in the literature is performed. A series of dinuclear complexes, 11-15, based on the ligand H2ovgrd are described in Chapter 4, focusing on the synthetic strategy, crystal structures and magnetism. The presence of the lanthanide contraction is evident in this system of complexes and is consistent with the intrinsic lanthanide contraction property.

This thesis will describe the magnetic behavior found in a scaling array of magnetic nanomaterials that have been uniquely designed, synthesized and characterised in order to better understand their properties with regards to potential future applications. Within Chapter 1 will be a detailed, yet accessible, introduction to nanomagnetism and the fundamental principles and practical techniques essential to the study of this unique mélange of physics and chemistry. This chapter will be designed to give the reader the necessary tools to understand key literature concepts found in Chapter 2, as well as the work presented in the following chapters. Chapter 2 will provide an overview of relevant literature in the field of magnetic nanomaterials, including: nanoparticles, single-molecule magnets, single-chain magnets and metal-organic frameworks. Chapter 3 will describe work performed on nanoparticles doped with lanthanide ions in order to explore their resulting size, shape, crystallinity and magnetic properties. The relevance of the chosen particles (NaYF4) pertains to their proposed use in a variety of applications due to their known luminescent properties, which we sought to hybridize with interesting magnetic properties, thus creating multimodal imaging capabilities. Doping with a variety of desired ratios of lanthanide ions (GdIII, TbIII, DyIII, ErIII and YbIII) was successful, producing crystalline nanoparticles with tunable size and shape. Magnetic measurements displayed a clear absence of superparamagnetic behavior, indicating that these materials have the potential to be well-suited to applications in biomedicine as multimodal imaging probes and MRI contrast agents. Chapter 4 will build on the previously explored doped nanomaterials through creating a hybrid nanomaterial by tethering lanthanide-based magnetic molecules to the surface of nanoparticles. This is performed through the synthetic design of a SMM with two anisotropic DyIII ions, which was synthesized and designed to bear terminal S-groups in order to promote the binding of the magnetic molecule to capping agent free gold nanoparticles. Upon confirmation of the successful surface attachment of the molecules, magnetic measurements displayed that the magnetic molecules maintained their static properties, however, their dynamic properties were altered. This system was the first example of this type of novel approach to the study of magnetic molecules on surfaces for data storage, spintronics, and quantum computing applications. Chapter 5 will expand on the previous study of ordering arrays of magnetic molecules on the surface of nanoparticles by tethering them into 1D chain networks. We successfully synthesized chain networks with YIII, EuIII, GdIII, TbIII and DyIII lanthanide ions. Magnetic characterisation revealed slow relaxation of the magnetization with no significant interactions between magnetic ions, thus these are discrete magnetic molecules in 1D. Rather surprisingly, the isotropic GdIII analogue displayed field induced slow relaxation of the magnetisation, necessitating the use of ab initio calculations in order to shed light on the potential causes of this unexpected behavior. Overall, through the formation and study of these structures, we observed a new potential method of SMM assembly for the study of ordered arrays of molecular magnets. Chapter 6 will focus on ordering of discrete magnetic systems in 3D. With this in mind, we successfully isolated the first Co8 cuboctahedron MOF. Magnetic measurements displayed that each SBU was well-isolated, with significant antiferromagnetic coupling between CoII ions, leading to an S = 0 ground state. These interactions were then modelled using density functional theory. This type of study promotes the future development of novel high-nuclearity MOF structures with interesting and tuneable magnetic properties, as well as the potential for assembly of discrete molecular magnetic units in 3D using MOFs. Chapter 7 utilizes the principles of Chapter 3, wherein magnetic ions are doped into a diamagnetic material; in this case, MOF-5. We sought to isolate one CoII ion in each SBU, and build upon this by adding additional magnetic ions and probing their interactions. Through magnetic measurements we observed a scaling magnetic moment with CoII content, and with higher dopant percentages we began to observe magnetic interactions occurring within the SBUs. Interestingly, we also observed a change in coordination environment with higher dopant percentages, likely as a result of the previously suggested capability of one ZnII ion within the MOF-5 SBU to become hexacoordinate, allowing CoII doping up to a maximum of 25%. Consequently, this study points to the cause of the structural instability that plagues MOF-5 in the presence of air and moisture. We probed this system further in Chapter 8 using FeIII as a dopant ion, and were able to obtain the first crystallographic evidence of the coordination change of ZnII in MOF-5. Furthermore, the structure obtained with FeIII was the first example of metal ion addition within a MOF that bound two interpenetrated frameworks together. This new MOF was found to have the potential to be a more practical material for gas storage and separation, and/or for catalysis. Thus, this study was informative in regards to the inherent instability of the parent framework, as well as a new method of metal addition to a known MOF structure. Chapter 9 will conclude the work with a discussion of what was performed in, and learned from, each thesis section, as well as provide an outlook and perspective on the novel work that may be derived from these projects going forward.

The miniaturization afforded by the integration of microfluidic technologies within lab-on-a-chip devices has greatly enhanced analytical capabilities in several key applications. Microfluidics has been utilized in a wide range of areas including sample preparation and analysis, DNA microarrays, cell detection, as well as environmental monitoring. The use of microfluidics in these applications offer many unique advantages: reduction in the required sample size, reduction in analysis time, lowered cost through batch fabrication, potentially higher throughput and the vision of having such devices used in portable systems. Nanopore sensors are a relatively new technology capable of detection and analysis with single-molecule sensitivity, and show promise in many applications related to the diagnosis and treatment of many diseases. Recently, some research groups demonstrated the integration of nanopores within microfluidic devices to increase analytical throughput. This thesis describes a methodology for integrating nanopore sensors within microfluidic devices with the aim of enhancing the analytical capabilities required to analyze biomolecular samples. In this work, the first generation of an integrated nanopore-microfluidic device contained multiple independently addressable microfluidic channels to fabricate an array of nanopore sensors using controlled breakdown (CBD). Next, for the second generation, we added pneumatic microvalves to manipulate electrical and fluidic access through connected microfluidic channels. As a proof-of-concept, single molecules (single- and double-stranded DNA, proteins) were successfully detected in the devices. It is also demonstrated that inclusion of the microfluidic via (microvia) limited the exposed area of the embedded silicon nitride membrane to the solution. This helped in localizing nanopore formation by confining the electric field to specific regions of the insulating membrane while significantly reducing high frequency noise in the ionic current signal through the reduction of chip capacitance. The devices highlighted in this thesis were designed and fabricated using soft lithography techniques which are available in most biotechnology laboratories. The core of this thesis is based on two scientific articles (Chapters 3 and 4), which are published in peer-reviewed scientific journals. These chapters are preceded by an introductory chapter and another chapter detailing the experimental setup and the methods used during the course of this study.

DNA replication is one of the most fundamental processes in all living organisms. Its semi-discontinuous nature dictates that the lagging strand is synthesized in short fragments called Okazaki fragments. In eukaryotes, each Okazaki fragment is initiated by an ~ 30-40 nucleotide-long RNA-DNA hybrid primer that is synthesized by Pol α-primase complex. To ensure genomic stability, the RNA primer has to be excised, any misincorporations by Pol α have to be corrected for and finally the resulting nick has to be sealed generating a contiguous strand. This feat is accomplished by a highly coordinated and regulated process called Okazaki fragment maturation. At the center of this process are 5’ nucleases, which are structure-specific nucleases that catalyze the incision of phosphodiester bonds one nucleotide into the 5’ end of ssDNA/dsDNA junctions. Previous structural and biochemical studies have shed some light on the mechanism of FEN1 substrate recognition, its catalysis and regulation. However, many gaps in our understanding of this remarkable nuclease still persist. Moreover, the choice between the short- and long-flap pathways is still elusive. Finally, the mechanism of the coordination among the different enzymatic activities of the polymerase, the nuclease and the ligase during Okazaki fragment maturation is still debatable. In this work, we set out to study FEN1 substrate recognition, catalysis and regulation using single molecule techniques. We show that FEN1 employs a sophisticated substrate recognition mechanism through which it actively distorts the DNA to ~100˚ bent angle. It also displays a remarkable selectivity towards its cognate substrate and avoids off-target substrate by a lock-down mechanism that commits the enzyme for catalysis on cognate substrates while promoting the dissociation of non-cognate substrates. We further characterized FEN1 reaction from substrate binding/bending to product handoff and built a comprehensive kinetic scheme that shows FEN1 releasing its product in two steps. Finally, we uncovered an unprecedented role of FEN1 in the choice between short- and long-flap pathways.

In the circular Escherichia coli chromosome, two replisomes are assembled at the unique origin of replication and drive DNA synthesis in opposite directions until they meet in the terminus region across from the origin. Despite the difference in rates of the two replisomes, their arrival at the terminus is synchronized through a highly specialized system consisting of the terminator protein (Tus) bound to the termination sites (Ter). This synchronicity is mediated by the polarity of the Tus−Ter complex that stops replisomes from one direction (non-permissive face) but not the other (permissive face). Two oppositely oriented clusters of five Tus–Ters that each block one of the two replisomes create a “replication fork trap” for the first arriving replisome while waiting for the late arriving one. Despite extensive biochemical and structural studies, the molecular mechanism behind Tus−Ter polar arrest activity remained controversial. Moreover, none of the previous work provided answers for the long-standing discrepancy between the ability of Tus−Ter to permanently stop replisomes in vitro and its low efficiency in vivo. Here, I spearheaded a collaborative project that combined single-molecule DNA replication assays, X-ray crystallography and binding studies to provide a true molecular-level understanding of the underlying mechanism of Tus−Ter polar arrest activity. We showed that efficiency of Tus−Ter is determined by a head-to-head kinetic competition between rate of strand separation by the replisome and rate of rearrangement of Tus−Ter interactions during the melting of the first 6 base pairs of Ter. This rearrangement maintains Tus’s strong grip on the DNA and stops the advancing replisome from breaking into Tus−Ter central interactions, but only transiently. We further showed how this kinetic competition functions within the context of two mechanisms to impose permanent fork stoppage. The rate-dependent fork arrest activity of Tus−Ter explains its low efficiency in vivo and why contradictory in vitro results from previous studies have led to controversial elucidations of the mechanism. It also provides the first example where the intrinsic heterogeneity in rate of individual replisomes could have different biological outcomes in its communication with double-stranded DNA-binding protein barriers.

Numerous short flapped DNA structures are created during the semi-discontinuous replication. These toxic intermediates are quickly resolved to produce a fully intact duplex of replicated DNA. Structure specific nuclease are key to resolving these structures, and show a high degree of specificity for their cognate substrate structures while being essentially insensitive to nucleotide sequence. Herein I demonstrate through confocal based single molecule experiments that the 5’ structure specific nuclease Flap Endonuclease 1 (FEN1) achieves its substrate specificity by coupling the bending of DNA substrate with structuring of the active site in a way that non-cognate structures binding is significantly destabilized and enzymatic features are incapable of structuring in the absence of particular substrate features, in particular a single nucleotide 3’ flap the FEN1 induces in nearly all DNA substrates. Debate remained over whether DNA was bound via a conformational capture or induced fit mechanism, and so I proceed to investigate the dynamics of the DNA itself in solution. Conclusions about conformational capture or induced fit remain elusive, however I did determine that DNA structures are rigidified by charge repulsion, an effect lessened by the salt concentration, which functions to shield the negative charge of DNA from itself. Additionally unstacking of the DNA in nicked structures incurs a significant free energy penalty, which FEN1 overcomes by its hydrophobic wedge motif, lending credence to an induced fit mechanism.