G-quadruplexes represent a versatile sensing platform for the construction of label-free molecular detection assays due to their diverse structures that can be selectively recognized by G-quadruplex-specific luminescent probes. In this thesis, we have explored the applications of the label-free G-quadruplex-based luminescent detection platforms for the detection of biomolecules and metal ions. Chapter 1 provides an overview of the principles and recent developments of the field of luminescent oligonucleotide-based probes, and highlighting in particular the use of the “label-free” strategy for the construction of simple and inexpensive sensing platforms. Chapter 2 introduces the basic experiments performed during the course of this thesis, including UV/Vis absorption spectroscopy, luminescence spectroscopy, nuclear magnetic resonance, mass spectrometry circular dichroism spectroscopy and G-quadruplex fluorescent intercalator displacement assay. Chapter 3 describes a G-quadruplex-based switch-on luminescence assay for the detection of gene deletion using iridium(III) complex 1 as a G-quadruplex-selective probe. Our method is based on the formation of a split G-quadruplex upon hybridization of two critically designed quadruplex-forming sequences with the mutant DNA sequence, resulting in a “switch-on” luminescence response. Chapter 4 describes a label-free, oligonucleotide-based, switch-on luminescence detection method for T4 polynucleotide kinase activity using a G-quadruplex-selective luminescent iridium(III) complex 2. The application of the assay for screening potential T4 PNK inhibitors is also demonstrated. To our knowledge, this is the first metal-based assay for PNK activity that has been reported in the literature. Chapter 5 describes a label-free oligonucleotide-based luminescence switch-on assay for the selective detection of sub-nanomolar Pb2+ ions in aqueous solution and real water samples. Iridium(III) complex 1 was employed as a G-quadruplex-specific luminescent probe and a guanine-rich DNA sequence (PS2.M, 5.-GTG3TAG3CG3T2G2-3.) was employed as recognition unit for Pb2+ ions. The assay could detect Pb2+ ions in aqueous media with a limit of detection of 600 pM, and also exhibited good selectivity for Pb2+ ions over other heavy metal ions. Furthermore, the application of the assay for the detection of Pb2+ ions in spiked river water samples was demonstrated. Chapter 6 describes a label-free G-quadruplex-based luminescent switch-on assay for the selective detection of micromolar histidine in aqueous solution. Iridium(III) complex 8 was employed as a G-quadruplex-specific luminescent probe while a guanine-rich oligonucleotide (Pu27, 5.-TG4AG3TG4AG3TG4A2G2-3.)/cupric ion (Cu2+) ensemble was employed as a recognition unit for histidine. The assay could detect down to 1 µM of histidine in aqueous media, and also exhibited good selectivity for histidine over other amino acids with the use of the cysteine-masking agent N-ethylmaleimide. Furthermore, the application of the assay for the detection of histidine in diluted urine samples was demonstrated. Chapter 7 summarizes the work that was conducted in this thesis, and the future outlook of G-quadruplex-based sensing is presented.

The development of facile and reliable methods to image and detect important biomolecules has drawn considerable attention owing to their potential applications in clinical, bioanalytical and forensic analysis. One-photon microscopy (OPM) has traditionally been used in cell biology research. However, probes based on OPM are associated with shortcomings including photobleaching, cell damage, and intracellular autofluorescence interference. Many researchers are seeking better tools to overcome these obstacles. Two-photon microscopy (TPM) is a convenient and powerful tool to explore the intracellular environment and provides the opportunity to overcome the abovementioned obstacles. Probes based on TPM have become important for bioimaging and sensing because of their low photodamage, reduced fluorescence interference, and better tissue penetration depth. With the development of fluorescence molecules in recent decades, a wide range of organic fluorescence probes based on TPM has been rapidly developed and used in biomedicine and bioimaging. Cyanine dye, one of the classic synthetic dyes, continues to be used in many fields, especially in bio-related applications, owing to its ability to interact with biomolecules through non-covalent and electrostatic bonds. Based on cyanine models, we designed a series of structural modifications of cyanine fluorophores used as two-photon (TP) probes to detect and image the intracellular environment in which new cyanine compounds, namely SLSO3, SLCOOH-Pr, F-SLOH, SLOH, Me-SLM, SLE, SAM, SAOH, SLG, F-SPG, SLOH-Pr, SLAD, F-SLAD, Me-SLG, SLNA, SLAD-Pr, SLCOOH, SLAce, SLM, SPC, SIOH, PSIOH, DMA-SLOH, DBA-SLOH, DPA-SLM, GBPM, HBBM, HBLM, SBM, SIBM, SIM, PLOH, and PTM, was successfully synthesized. All of these newly designed compounds were characterized with 1H NMR, 13C NMR, and HRMS and found to show good agreement with the desired structures. To our surprise, some of the novel cyanine molecules were also able to detect and image amyloid-β (Aβ) peptide species and showed excellent biological properties including neuroprotective effects against the cytotoxicity induced by different forms of Aβ species, blood-brain barrier permeability, and high in vivo stability. The photophysical and biological properties of these newly synthesized compounds included optical properties such as UV-vis absorption, emission, fluorescence quantum yield in different solvents, dissociation constant determined by fluorescence titration, and circular dichroism spectroscopy, cytotoxicity assay, neuroprotection, and inhibition of Aβ aggregation were investigated.

It has long been recognized that our understanding of how RNA adapts its complex three-dimensional structure and undergoes conformational fluctuations has played a central role in our understanding of the biological functions of RNA. Our current understanding of the vast and diverse set of RNA conformational dynamics is the culmination of several decades of biophysical research applying several ensemble and single-molecule techniques. In this journey, each of the biophysical techniques have provided a unique perspective into the dynamic processes of RNA and revealed information about distinct RNA dynamics occurring over a broad range of timescales. In recent years, a new, promising single-molecule biophysical technique called single-molecule field effect transistors (smFETs) has been developed. Because smFETs do not rely on fluorophore reporters of conformation or mechanical (un)folding forces, they provide a unique approach that enables single-molecule studies of RNA conformational dynamics observed at microsecond temporal resolution for a long period of time. The broad range of timescales opens immediate prospects for smFETs to provide a unique perspective into understanding RNA conformational dynamics that are presently inaccessible in other single-molecule approaches. The primary focus of this thesis is to understand how RNA stem-loops undergo folding and unfolding. Stem-loops are one of the most common secondary structural motifs in RNA and act as a fundamental building block for complex RNA structures. Despite their fundamental importance, a complete unifying picture of the folding mechanism of RNA stem-loops has been difficult to achieve, primarily due to the rugged nature of their folding energy landscapes. In Chapter 2, experimental methods that were developed to enable smFET studies of RNA conformational dynamics are described. This includes the development of a high-throughput fabrication process that generates high signal to noise ratio (SNR) smFET devices and the development and validation of nucleic acid tethering strategies that enables controlled tethering of biomolecules onto smFET devices. Utilizing these methods, Chapter 3 establishes smFET as a general single-molecule approach to characterize the folding dynamics of RNA stem-loops. Finally, Chapter 4 explores the use of smFETs to investigate the molecular mechanism in which a model RNA stem-loop undergoes folding and unfolding. Collectively, this thesis demonstrates how smFETs can be applied to uniquely capture and describe the folding energy landscapes of RNA and reveal new insights to how RNAs fold and unfold.

As science and medicine advance, it becomes ever more important to be able to control and analyze smaller and smaller bioparticles all the way down to single molecules. In this dissertation several studies aimed at improving our ability to manipulate and monitor single biomolecules will be discussed. First, we will discuss a study on developing a way to map dielectrophoresis with nanoscale resolution using a novel atomic force microscopy technique. Dielectrophoresis can be applied on nanoparticles through micron-scale electrodes to separate and control said particles. Therefore, this new method of mapping this force will greatly improve our ability to manipulate single biomolecules through dielectrophoresis. The next two studies discussed will be aimed at using carbon nanotube nanocircuits to monitor single protein kinetics in real time. Drug development and delivery methods rely on the precise understanding of protein interactions, thus creating the need for information on single protein dynamics that our techniques provides. The proteins studied in these sections are MMP1 and HDAC8, both of which are known targets of anti-cancer drugs. Finally, we developed a new strategy for diagnosing pancreatic cancer. Our strategy involves using graphene nanotransistors to detect exosomes released from the pancreatic tumor. The ability to reliably diagnose pancreatic cancer before it reaches metastasis would greatly improve the life expectancy of patients who develop this condition. We were able to test our technique on samples from a number of patients and were successfully able to distinguish patients with pancreatic cancer from noncancerous patients.

Toxicity of nano-scaled Al2O3, SiO2, TiO2 and ZnO to bacteria (Bacillus subtilis, Escherichia coli and Pseudomonas fluorescens) was examined and compared to that of their respective bulk (micro-scaled) counterparts. All nanoparticles (NPs) but TiO2 showed higher toxicity than their bulk counterparts. Toxicity of released metal ions was differentiated from that of the oxide particles. ZnO was the most toxic among the three NPs, causing 100% mortality to the three tested bacteria. TEM images showed attachment of NPs to the bacteria, suggesting that the toxicity was affected by bacterial attachment. The effects of oxide NPs on bacteria cells and bacterial surface biomolecules were studied by FTIR spectroscopy to provide a better understanding of their cytotoxicity. Lipopolysaccharide (LPS) and lipoteichoic acid could bind to oxide NPs through hydrogen bonding and ligand exchange, but the cytotoxicity of NPs seemed largely related to the function-involved or structural changes to proteins and phospholipids. The three NPs decreased the intensity ratio of β-sheets/α-helices, indicating protein structure change, which may affect cell physiological activities. The phosphodiester bond of L-α- Phosphatidyl-ethanolamine (PE) was broken by ZnO NPs, forming phosphate monoesters and resulting in the highly disordered alkyl chain. Such damage to phospholipid molecular structure may lead to membrane rupture and cell leaking, which is consistent with the fact that ZnO is the most toxic of the three NPs. LPS and PE are amphiphilic biomolecules that are major constituents of the outer membrane of Gram-negative bacteria. Their micelles and vesicles were studied as model cell membranes to evaluate NP effects on membrane construction. The adsorption of polysaccharides on Al2O3 and TiO2 NPs dispersed LPS vesicles and micelles. LPS coated Al2O3 NPs, while it caused the aggregation of TiO2 NPs according to atom force microscopy images. Desorption from the two NPs was slow due to the firm hydrogen bonding. For PE, Al2O3 NPs induced large multilamillar vesicles, while ZnO NP converted vesicles to tiny aggregates due to molecular structure breakup. PE stability in solution was disturbed by adding NPs, but its stability was enhanced by increasing pH. The electrostatic force was the determining factor for the vesicle stability.

No description available.

<p> This thesis focuses on the use of time-resolved fluorescence anisotropy (TRF A) for the analysis of peptide-silica and protein-silica interactions. Previous studies from our group have shown that strong ionic binding of the cationic probe rhodamine 6G (R6G) to the anionic surface of silica particles in water provides a convenient labeling procedure to study both particle growth kinetics and surface modification by time-resolved fluorescence anisotropy (TRF A). The decays for R6G dispersed in diluted Ludox silica sols usually fit to a sum of picosecond and nanosecond decay components, along with a significant residual anisotropy component. The first objective of my work was to assess the nature of the R6G:silica interaction to determine the origin of the nanosecond decay component, and ultimately validate the model used to fit the TRFA data and gain further insight into the physical meaning of the anisotropy decay parameters. Our results show the origin of the nanosecond decay component ( ¢2) is due to the presence of a subpopulation of small nanoparticles in the Ludox sol. </p> <p> With the correct physical model in place, we have been able use TRFA ofR6G in aqueous Ludox to monitor peptide adsorption onto the silica particles in situ. Steady-state anisotropy and TRF A of R6G in Ludox sols were measured to characterize the extent of the ionic binding of the probe to silica particles in the presence of varying levels of tripeptides of varying charge, including Lys-Trp-Lys (KWK), N-acetylated Lys-Trp-Lys (Ac-KWK), Glu-Trp-Glu (EWE) and N-acetylated Glu-Trp-Glu (Ac-EWE). R6G showed significant decreases in anisotropy in the presence of cationic peptides, consistent with the addition of cationic peptides blocking the adsorption of the dye to the silica surface. The study shows that the competitive binding method can be used to assess the binding of various biologically relevant compounds onto silica surfaces, and demonstrates the potential of TRF A for probing peptide: silica and protein: silica interactions. </p> <p> We have also extended the application of TRF A to monitor protein adsorption onto plain and modified silica particles using a recently reported cationic long-lifetime quinolinium dye, CG437, which strongly binds to anionic silica particles through electrostatic interactions. In this case, alterations in the rotational correlation time of Ludox particles resulting from increases in the diameter of the rotating body upon binding of protein to the silica surface were monitored. The study shows that TRFA analysis of long-lived cationic probes such as CG437 can provide an effective method to investigate interactions between proteins and modified silica surfaces, extending the utility of the TRF A method. </p> / Thesis / Master of Science (MSc)

No / To fully harness the enormous potential offered by interfaces between graphitic nanostructures and biomolecules, detailed connections between adsorbed conformations and adsorption behaviour are needed. To elucidate these links, a key approach, in partnership with experimental techniques, is molecular simulation. For this, a force-field (FF) that can appropriately capture the relevant physics and chemistry of these complex bio-interfaces, while allowing extensive conformational sampling, and also supporting inter-operability with known biological FFs, is a pivotal requirement. Here, we present and apply such a force-field, GRAPPA, designed to work with the CHARMM FF. GRAPPA is an efficiently implemented polarisable force-field, informed by extensive plane-wave DFT calculations using the revPBE-vdW-DF functional. GRAPPA adequately recovers the spatial and orientational structuring of the aqueous interface of graphene and carbon nanotubes, compared with more sophisticated approaches. We apply GRAPPA to determine the free energy of adsorption for a range of amino acids, identifying Trp, Tyr and Arg to have the strongest binding affinity and Asp to be a weak binder. The GRAPPA FF can be readily incorporated into mainstream simulation packages, and will enable large-scale polarisable biointerfacial simulations at graphitic interfaces, that will aid the development of biomolecule-mediated, solution-based graphene processing and self-assembly strategies. / Veski

Les études et les résultats présentés dans ce manuscrit ont pour but de développer une meilleure compréhension des principes à la base de l'auto-organisation dans les systèmes biologiques. La théorie topologique des transitions de phase est l'un des approches possibles pour fournir une généralisation de la description des transitions de phase dans les systèmes petits ou mésoscopiques. Cette théorie a été rigoureusement enracinée dans deux théorèmes: un contre exemple à l'un de ces théorèmes a été récemment découvert. La première partie de ce manuscrit est donc consacré à mieux comprendre ce «contre-exemple » pour verifier si et comment la théorie peut être sauvé.Dans la deuxieme parte de ce manuscrit les résultats des recherches théoriques, numériques et expérimentales sur la condensation à la Fr "ohlich sont reportés. Ceci est une condition préalable à l'activation des oscillations dipolaires géantes qui entraînent des interactions électrodynamiques à long portée entre les molécules coresonnantes. Dans cette thèse, on montre que les interactions à longue portée affectent sensiblement les propriétés de diffusion des molécules en solution. Une empreinte des interactions à long portée pourrait être un phénomène de «transition» en ce qui concerne le coefficient de diffusion en fonction d'un paramètre de contrôle proportionnel à l’intensité d'interaction. Simulations analogues ont été réalisées afin de valider une approche expérimentale visant à trouver une telle «empreinte» dans les systèmes avec interactions à longue portée. / The studies and results reported in this manuscript are aimed to develop a deeper understanding of the principles at the basis of self-organization in biological system.The Topological Theory of phase transitions is one of the possible approaches to provide a generalization of description of phase transitions in small or mesoscopic systems. This theory has been rigorously rooted in two theorems: a counterexample to one of these theorems has been recently found. The first part of this manuscript is devoted to investigation of the "counterexample" to understand if and how the theory can be saved. In the second part of this manuscript the results of theoretical, numerical and experimental investigations on Fr"ohlich-like condensation for normal modes of biomolecules are reported. This is a prerequisite for the activation of giant dipole oscillations in biomolecules which entail long-range electrodynamic interactions between coresonant molecules. In this thesis is shown that long-range interactions markedly affect the self-diffusion properties of molecules in solution. A fingerprint of long-range interactions could be a "transitional" phenomenon concerning the self-diffusion coefficient as a function of a control parameter proportional to interaction strength. Analogous simulations have been performed to validate an experimental approach aimed at finding such "fingerprint" in systems with built-in long-range interactions.

L’étude des organismes vivants, la biologie, s’étend sur de nombreux domaines et notamment s’applique à comprendre le fonctionnement des êtres vivants. Les organismes les plus complexes comme les êtres Humains possèdent plusieurs niveaux d’organisation : ils sont constitués successivement d’organes, de tissus, de cellules, de biomolécules. On trouve plusieurs types de biomolécules dont les protéines, qui sont comme des minuscules outils qui permettent aux cellules de vivre et d’interagir avec leur environnement. Pour cela, les protéines doivent entrer en contact les unes avec les autres de manière très précise et déterminée. Cette thèse teste l’existence de forces électrodynamiques de longue portée qui leur permettraient d’interagir de manière rapide et guidée, via l’étude de l’absorption ou l’émission de ce type d’onde par des protéines, puis la diffusion de ces protéines en solution pour observer leur comportement. / The study of living organisms, biology, extends over many fields and in particular, applies to understanding the functioning of living beings. The most complex organisms, such as human beings, have several levels of organization: they are made up successively of organs, tissues, cells, and biomolecules. There are several types of biomolecules including proteins, which are like tiny tools that allow cells to live and interact with their environment. To do this, proteins must come into contact with each other in a very precise and determined way. This thesis tests the existence of long-range electrodynamic forces which would allow them to interact in a rapid and guided way, by studying the absorption or emission of this type of wave by proteins, then the diffusion of these proteins in solution to observe their behavior.