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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
1

An optical tweezers assay for the study of T7 RNA polymerase : observation of transcription initiation at the single-molecule level

Skinner, Gary Mark January 2001 (has links)
No description available.
2

Solution-processable carbon nanotube molecular junctions

McMorrow, Joseph January 2018 (has links)
Nanotechnology is the manipulation of matter at the supramolecular, molecular and atomic scale. As a result, nanotechnology is included in various fields of science including surface science, organic chemistry, molecular biology, semiconductor physics, microfabrication and molecular engineering. One of the ambitions for nanotechnology is to develop electrical devices where the active component is a single molecule or nanomoiety. In order to fabricate such devices, it is of paramount importance to develop strategies beyond the current top-down lithographic approaches typically employed in the semiconductor industry. In this regard, the ability to control the assembly of single-molecules and individual nanomoieties directly in solution can allow for the development of solution-processable approaches in nanotechnology, towards the fabrication of single-molecule devices. In this thesis, it will be discussed how molecular junctions with functional single molecules are fabricated in aqueous solutions employing single-walled carbon nanotubes as potential nanoelectrodes. Furthermore, it will be demonstrated how the assembly of molecular junctions can facilitate other functions and the construction of both nanostructures and microstructures. To begin, relevant work will be discussed that has been done in this field to date and outline clear ambitions of the study presented here. Subsequently, the key characterisation techniques that underpin all the results in this study will be described. In this work, it will be reported how metallic carbon nanotubes can act as nanoelectrodes in molecular junction assemblies and how conductive measurements of individual molecules are performed. Therefore, for the first time, the molecular junction conductance of a series of oligophenyls were successfully measured, which were formed via a solution-based assembly method. Measured molecular conductance values of the series of oligophenyls resulted in a β value of 0.5 Å−1. Furthermore, it will be described how the approach outlined previously can be extended to the synthesis of tri-amine molecular linkers as well as the formation of three-terminal junctions as the foundation of carbon nanotube-based single-molecule electronic devices. This research resulted in an increase in the formation of Y-shape molecular junctions by ~25%. Next, this report will outline the formation of molecular junctions in two-dimensional structures, which can allow for the development of electrical devices into networks. Utilising modified DNA sequences, "click" chemistry can lead to nanotube network with dimensions ranging into the micrometre scale. Building on this work, it will be further report on the change in physical properties when these two-dimensional superstructures are embedded into polymeric thin films. Finally, conclusions of the research will be drawn and it will be discussed how the findings obtained in this work can contribute to the development of novel single-molecule electronic devices.
3

Single-molecule fluorescence microscopy studies of fluorescent probes in thin films and on nanoparticle surfaces

Lu, Yin 30 March 2011
Single-molecule (SM) fluorescence spectroscopy has become a useful and important experimental approach for investigating the optical properties of chemical systems. In this thesis, four subprojects in the field of SM fluorescence spectroscopy are presented in which SM spectroscopy has provided invaluable experimental insight into the systems of interest.<p> In the first project, the photophysical properties of Calcium-Green 1 (CG-1), a calcium-ion indicator, were studied at both the ensemble and SM levels. CG-1 is non-fluorescent in the absence of Ca2+ and becomes strongly fluorescent when bound to Ca2+. In the ensemble measurements, the absorption and fluorescence spectra were collected under various Ca2+ concentrations. In addition, the fluorescence lifetime of CG-2 was also studied as a function of [Ca2+]. From SM measurements, the photobleaching time and fluorescence intensity distributions of CG-1 were studied both in the presence and in absence of Ca2+. The results were compared with those obtained for the dual-fluorophoric variant, Calcium-Green 2 (CG-2), whose photophysical properties have been investigated by previous researchers. The experimental results reveal that CG-1 can exist in two different forms: a highly-quenched form due to the occurrence of photoinduced electron transfer (PET) in the absence of Ca2+, and a strongly fluorescent form when bound to Ca2+.<p> The second project is a continuation of a previous study on CG-2. In the dual-chromophore CG-2 system, energy transfer between chromophores is controlled by the orientation and spatial separation between chromophores. Dual polarization fluorescence microscopy was used to determine the relative conformation of the two fluorophores in the emissive form of CG-2. Distributions of fluorescence polarization of individual CG-2 molecules were collected for both Ca2+-free and Ca2+-saturated conditions. The experimental polarization results were compared to those calculated from a simple geometric model based on randomly-orientated fluorescent dimers. The results show good agreement with previous calculations of the molecular conformation of CG-2. This indicates that the dual polarization imaging approach has significant potential as a general tool for characterizing chromophore orientation in coupled-fluorophore systems.<p> In the third project, Nile Red (NR), a solvatochromic lipid stain, was incorporated into phase separated Langmuir-Blodgett (LB) films composed of arachidic acid (AA) and perfluorotetradecanoic acid (PA). According to previous studies by atomic force microscopy (AFM), two types of separated domains are formed in the LB films: micron-sized hexagonal discontinuous domains that are exclusively comprised of AA, and the surrounding continuous domains which are enriched in PA. The photophysical properties of NR were characterized in the two physically and chemically distinct domains via bulk and SM fluorescence measurements. In addition to fluorescence microscopy, fluorescence confocal spectromicroscopy was also applied in the ensemble measurements to determine the spectral properties of NR in different sub-environments. Experimental results indicated that a small sub-population of dye molecules localize on the perfluorinated regions of the sample, but this sub-population is spectroscopically indistinguishable from that associated with the hydrogenated domains. Contrast in images was primarily due to preferential accumulation of the hydrophobic dye on the hydrophobic regions of the LB films.<p> In the final project, the fluorescence quenching behavior of a strongly fluorescent probe Alexa Fluor 514 (AF514) was investigated when it was covalently bound to gold metal protected clusters (AuMPC) with negligible plasmon bands. The fluorescence emission of the dye-AuMPCs system was characterized at different dye/Au MPC loading ratios with a combination of steady state and time-resolved ensemble spectroscopic measurements. It was found that the extent of fluorescence quenching in the system was small. After correction of inner filter effects, the results from bulk measurement demonstrate that the weak quenching is due to static quenching of the dye by the AuMPCs. SM measurements provided further support for the bulk measurements, with the fluorescence intensity of coupled AF514 molecules being comparable with that of unconjugated molecules. The photobleaching of the dye-AuMPC conjugates took place as a series of consecutive photobleaching events, without additional blinking dynamics within the time resolution of the experiment. These results suggest that the fluorophores on the AuMPCs are either entirely quenched, or remaining unquenched, as is consistent with the ensemble measurements.
4

Single-molecule fluorescence microscopy studies of fluorescent probes in thin films and on nanoparticle surfaces

Lu, Yin 30 March 2011 (has links)
Single-molecule (SM) fluorescence spectroscopy has become a useful and important experimental approach for investigating the optical properties of chemical systems. In this thesis, four subprojects in the field of SM fluorescence spectroscopy are presented in which SM spectroscopy has provided invaluable experimental insight into the systems of interest.<p> In the first project, the photophysical properties of Calcium-Green 1 (CG-1), a calcium-ion indicator, were studied at both the ensemble and SM levels. CG-1 is non-fluorescent in the absence of Ca2+ and becomes strongly fluorescent when bound to Ca2+. In the ensemble measurements, the absorption and fluorescence spectra were collected under various Ca2+ concentrations. In addition, the fluorescence lifetime of CG-2 was also studied as a function of [Ca2+]. From SM measurements, the photobleaching time and fluorescence intensity distributions of CG-1 were studied both in the presence and in absence of Ca2+. The results were compared with those obtained for the dual-fluorophoric variant, Calcium-Green 2 (CG-2), whose photophysical properties have been investigated by previous researchers. The experimental results reveal that CG-1 can exist in two different forms: a highly-quenched form due to the occurrence of photoinduced electron transfer (PET) in the absence of Ca2+, and a strongly fluorescent form when bound to Ca2+.<p> The second project is a continuation of a previous study on CG-2. In the dual-chromophore CG-2 system, energy transfer between chromophores is controlled by the orientation and spatial separation between chromophores. Dual polarization fluorescence microscopy was used to determine the relative conformation of the two fluorophores in the emissive form of CG-2. Distributions of fluorescence polarization of individual CG-2 molecules were collected for both Ca2+-free and Ca2+-saturated conditions. The experimental polarization results were compared to those calculated from a simple geometric model based on randomly-orientated fluorescent dimers. The results show good agreement with previous calculations of the molecular conformation of CG-2. This indicates that the dual polarization imaging approach has significant potential as a general tool for characterizing chromophore orientation in coupled-fluorophore systems.<p> In the third project, Nile Red (NR), a solvatochromic lipid stain, was incorporated into phase separated Langmuir-Blodgett (LB) films composed of arachidic acid (AA) and perfluorotetradecanoic acid (PA). According to previous studies by atomic force microscopy (AFM), two types of separated domains are formed in the LB films: micron-sized hexagonal discontinuous domains that are exclusively comprised of AA, and the surrounding continuous domains which are enriched in PA. The photophysical properties of NR were characterized in the two physically and chemically distinct domains via bulk and SM fluorescence measurements. In addition to fluorescence microscopy, fluorescence confocal spectromicroscopy was also applied in the ensemble measurements to determine the spectral properties of NR in different sub-environments. Experimental results indicated that a small sub-population of dye molecules localize on the perfluorinated regions of the sample, but this sub-population is spectroscopically indistinguishable from that associated with the hydrogenated domains. Contrast in images was primarily due to preferential accumulation of the hydrophobic dye on the hydrophobic regions of the LB films.<p> In the final project, the fluorescence quenching behavior of a strongly fluorescent probe Alexa Fluor 514 (AF514) was investigated when it was covalently bound to gold metal protected clusters (AuMPC) with negligible plasmon bands. The fluorescence emission of the dye-AuMPCs system was characterized at different dye/Au MPC loading ratios with a combination of steady state and time-resolved ensemble spectroscopic measurements. It was found that the extent of fluorescence quenching in the system was small. After correction of inner filter effects, the results from bulk measurement demonstrate that the weak quenching is due to static quenching of the dye by the AuMPCs. SM measurements provided further support for the bulk measurements, with the fluorescence intensity of coupled AF514 molecules being comparable with that of unconjugated molecules. The photobleaching of the dye-AuMPC conjugates took place as a series of consecutive photobleaching events, without additional blinking dynamics within the time resolution of the experiment. These results suggest that the fluorophores on the AuMPCs are either entirely quenched, or remaining unquenched, as is consistent with the ensemble measurements.
5

Transducing Signals and Pre-Concentrating Molecules for Enhanced Solid-State Nanopore Biosensing

Roelen, Zachary 03 January 2024 (has links)
Single-molecule biosensors offer distinct advantages over their ensemble-averaged counterparts by being able to extract information related to rare targets and specific molecular configurations within a sample. In particular, solid-state nanopores embody a promising single-molecule technique that is based on detecting target molecules by the amount of ionic current they block as they pass through a nanoscale aperture across a thin membrane. In this thesis, I present extensions of the basic nanopore system aimed at addressing some of its main limitations at present, namely: 1) the low rates at which nanopores capture molecules from a bulk volume, which restricts their ability to work with dilute (≲ nM) samples, and 2) the difficulty in using nanopores to distinguish small or closely related molecules by their direct current blockage signatures alone. I begin by describing the design and construction of a nanopore-based instrument that integrates an optical detection channel in parallel with ionic current sensing. A particular emphasis was placed on minimizing the electrical noise contributions of the added optical equipment on the original ionic current channel. Measuring the optical signals of translocating molecules together with their current blockages can improve the discrimination of two fluorescently labelled targets (or two configurations of a single target) that normally produce similar ionic current signatures. I next investigate the combination of nanopore sensing with target pre-concentration, specifically, by embedding a nanopore membrane within a fluidic cell that features an insulator-based dielectrophoretic (iDEP) trap. Applying large (≳ 100 V) AC voltages across the iDEP channels of the cells resulted in the accumulation of polarizable targets (dsDNA, polystyrene beads) at the locations of the membranes, thus pointing toward a convenient method for the detection of ultra-dilute target samples in future nanopore devices. Finally, I introduce improved protocols for the synthesis and nanopore signal analysis of dsDNA-based molecular carriers. In a molecular carrier scheme, in order to enhance the target specificity of the system, target molecules are not sensed directly by a nanopore but instead interact specifically with secondary molecules (“carriers”) to recognizably alter the carrier translocation signals. Here, I present proof-of-principle analyses of DNA carrier experiments that highlight the multiplexing capabilities of our carrier design, which are based on separating targets by their interactions with carriers of different lengths. Developments of the nanopore sensing platform such as those presented in this work, which leverage the intrinsic versatility of solid-state nanopores to be integrated within complex devices and to detect a wide range of target molecules, will play an important role in continuing to increase the precision of single-molecule measurements into the future and to expand their breadth of potential applications.
6

Développement des méthodes de molécule unique pour la détection simultanée des interactions protéine-ADN et leur application à l'étude du mécanisme de translocation de SpoIIIE. / Development of single-molecule methods for the simultaneous detection of protein-DNA interactions and their application to the study of the mechanism of DNA translocation by SpoIIIE

Thakur, Shreyasi 03 September 2012 (has links)
Le transfert d'ADN chez les bactéries est un processus essentiel dans la ségrégation des chromosomes lors de la progression du cycle cellulaire et de nombreuses protéines sont impliquées dans ce processus. Parmi elles, on trouve la famille des protéines SpoIIIE / FtsK, et différentes fonctions leur ont été attribuées. SpoIIIE a été identifiée comme étant essentielle a la sporulation chez Bacillus subtilis. Au cours de la sporulation, un septum de division asymétrique se développe à proximité d'un pôle de la cellule et divise la cellule en deux compartiments : la préspore et la cellule mère. SpoIIIE est responsable de la translocation directionnelle de l'ADN de la cellule mère à la préspore. Ce transport implique l'interaction de SpoIIIE avec des séquences spécifiques qui sont distribuées de forme asymétrique le long des chromosomes (séquences de reconnaissance SpoIIIE ou SRS). Dans cette thèse, je développe des méthodes de molécule unique pour aborder les différents aspects des mécanismes de translocation de SpoIIIE. Cette thèse est divisée en trois sections : (1) les développements et de l'optimisation méthodologiques, (2) la caractérisation de sytox comme un nouveau colorant intercalant l ‘ADN pour les expériences en molécules uniques, et (3) l'utilisation de ces méthodes de molécules uniques pour tester les modèles de translocation d'ADN par SpoIIIE. Pour commencer, j'ai développé deux méthodes de détection et manipulation par molécules uniques: 1) le premier permet la visualisation simultanée de l'ADN et les protéines fluorescentes par microscopie TIRF et à épifluorescence, et (2) l'utilisation de pinces magnétiques dans une configuration transversale qui, couplée à la détection par fluorescence, permet la détection simultanée de l’ extension de l'ADN et la visualisation de la localisation des protéines. Au cours de la thèse, j'ai construit ces configurations optiques, les ai caractérisé, et optimisé. Deuxièmement, j’ai étudié le mécanisme de liaison et des propriétés de fluorescence de sytox, un nouveau colorant d’ADN. Plus précisément, j'ai déterminé que: (1) sytox se lie à l'ADN rapidement dans un processus en deux étapes séquentielles qui implique des interactions électrostatiques; (2) la dynamique rapide de liaison et de dissociation de sytox conduit à un taux extrêmement faible de photoblanchiment , (3) la dégradation de l'ADN par sytox est quatre fois inférieure à celle observée pour d'autres bis-intercalants, tels que YOYO-1, et 4) sytox est un intercalant d'ADN qui augmente la longueur de l'ADN de 43%, et n'affecte pas ses propriétés mécaniques (mesurée par la longueur de persistance). Enfin, pour observer l'interaction entre SpoIIIE et SRS, la protéine SpoIIIE a été chimiquement étiquetée et caractérisée. Substrats d'ADN contenant la séquence SRS ont été préparés et adaptés aux méthodes de molécule unique développées pendant ma thèse. L’observation directe des interactions SpoIIIE-SRS par ces méthodes ont permis de réfuter un des modèles existant. / DNA contains the genetic information of cells. Several cellular processes, including chromosome segregation during cell division and sporulation, and plasmid conjugation require the transport of double-stranded DNA (dsDNA) within and between bacterial cells. SpoIIIE/FtsK/Tra are a family of ring-shaped, membrane-anchored, ATP-fueled, directional motors required to segregate DNA across membranes during sporulation, cell division and conjugation. In particular, SpoIIIE is responsible for packaging the chromosome inside the prespore during the process of sporulation in Bacillus subtilis. This transport is directional and requires that SpoIIIE recognizes highly-skewed octameric sequences (SpoIIIE Recognition Sequences, or SRS) sparsely distributed along the whole chromosome. In this thesis, I developed different single-molecule methods to investigate the molecular mechanism by which SpoIIIE-SRS interactions lead to directional DNA transport. This thesis is divided in three sections: methodological developments and optimization, characterization of sytox as a new intercalating dye for single molecule experiments, and the use of single molecule methods to test the models for directional DNA translocation by SpoIIIE. First, I developed two single molecule methods that involved 1) the simultaneous visualization of DNA and protein by using intercalating dyes and direct protein labels to detect the localization of SpoIIIE on DNA using TIRF and epi-fluorescence microscopy; and (2) the use of a transverse magnetic tweezers setup coupled to fluorescence detection to simultaneously detect DNA extension and visualize protein localization. I built these optical setups, characterized them, and optimized several parameters. Secondly, we investigated the binding mechanism and fluorescence properties of sytox, a new bright, low photo-damage, multi-color DNA labeling agent. Specifically, I determined that: (1) sytox binds DNA rapidly in a two-step sequential process that involves electrostatic interactions; (2) the fast dynamics of binding and unbinding of sytox leads to an extremely low photobleaching rate; (3) DNA degradation by sytox is four-fold lower than that observed for other bis-intercalators, such as YOYO-1; and 4) sytox is a DNA intercalator that increases the DNA length upon binding by 43 %, while not affecting its mechanical properties (measured by the persistence length). Finally, to observe SpoIIIE-SRS interactions, SpoIIIE was chemically labeled and characterized. DNA substrates containing SRS sequence were prepared suitable for the different single molecule approaches undertaken and also characterized. Observation of SpoIIIE-SRS interactions allowed us to conclude that: (1) in the absence of SRS, SpoIIIE can bind DNA non-specifically (2) this first binding event does not require threading through the DNA end or assembly of monomers but rather the binding of a hexamer from an open to a closed conformation, (3) in the presence of ATP, SpoIIIE translocates on DNA and is predominantly located in DNA ends, and (4) can often condense DNA by looping, reconstituting the activity observed in magnetic tweezers assays, (5) when SRS sequences are present, SpoIIIE is redistributed from non-specific sites by a diffusional or 3D looping mechanism and locates SRS sequences where it remains bound with a higher affinity than to non-specific sequences.
7

Silver nanocluster single molecule optoelectronics and its applications

Lee, Tae-Hee 30 January 2004 (has links)
Charge transport dynamics through molecular scale materials is of common interest to both scientific and engineering disciplines. Putting molecules on nanoscale break junctions is the most straightforward setup to study charge transport dynamics through single molecules. Electromigration process can provide a simple and easy method of forming metallic oxide nanogap junctions. By using silver oxide thin films to form such nanogap junctions, silver nanoclusters (Ag2~Ag8) are also formed in-situ within the junctions. Formed silver nanoclusters strongly and stably electroluminesce under DC, AC, and customized pulse train excitation. By detecting extremely sensitive feedback, i.e. photons, two interesting behaviors of single molecule charge transport dynamics were revealed: 1) asymmetric charge transport and 2) discrete energy level tunneling. The discrete energy level tunneling of field emitted electrons yields negative differential resistance (NDR). Combined with photoconductivity and optical reduction of silver oxide to form silver nanoclusters, junction-asymmetry and NDR can be very useful in both electronic and optoelectronic applications such as on-demand electronics fabrication, single photon sources, and nanoscale photon detectors.
8

Extending and combining single-molecule fluorescence methods to study site-specific recombination

Pinkney, Justin N. M. January 2012 (has links)
Förster resonance energy transfer (FRET) has become an important tool for studying biochemical reactions at the single-molecule level, despite its increasing maturity there is an on-going effort to improve and expand the technique. This thesis presents methods for extending conventional two-colour single-molecule FRET measurements; by expanding the range and applicability of single-molecule fluorescence methods a greater variety of biological reactions can be studied, in greater detail than previously possible. To circumvent the complexities of multi-colour FRET measurements and extend the range of observable distances I developed and characterised a new single-molecule fluorescence method termed tethered fluorophore motion (TFM). TFM is based on the existing technique of tethered particle motion (TPM) which relies on Brownian motion of a particle, attached to a surface by DNA, to probe the effective length of the DNA tether. TFM takes this concept and applies it at the single-fluorophore level, allowing simultaneous measurement of other fluorescence observables such as FRET and protein induced fluorescence enhancement (PIFE). Having developed TFM I combined it with FRET to study site-specific recombinase proteins at the single-molecule level, in greater detail than possible by either technique alone. Studying the model tyrosine recombinase Cre, I extend and clarify previous ensemble observations regarding the order of DNA strand exchange, as well as uncovering a previously unobserved complex conformation and molecular heterogeneity. Finally, I used TFM-FRET to study the more complex XerCD recombination system and its interaction with the DNA translocase FtsK. I made observations, for the first time, of synaptic complex formation and of recombination at the single-molecule level, and these suggest intriguing and unexpected intermediates in the recombination reaction. I also combine TFM with PIFE to investigate the mechanism of DNA looping by FtsK. The introduction of TFM, and its combination with other fluorescence techniques, allows observation of complex protein-DNA interactions from a variety of perspectives and will help expand the repertoire and applicability of single-molecule biophysical experiments.
9

Synthesis and characterisation of lanthanide complexes as possible single-molecule magnets

King, Sara January 2016 (has links)
A range of lanthanide compounds incorporating soft bridging ligands or alkoxide ligands have been synthesised and their magnetic properties investigated. These two classes of compound have shown promise as single molecule magnets but have not been widely studied; this thesis aims to expand on this area of research. Softer bridging ligands are found to slightly increase superexchange interactions between metal centres compared to harder bridging ligands. The introduction to this thesis covers the basic properties of the lanthanides, paying special attention to their chemistry with soft donor ligands and alkoxide ligands. Also included is an introduction to the field of single-molecule magnetism and the role of lanthanide complexes in the study of this behaviour. In Chapter 2, four complexes are reported: the phosphine adducts [Cp'3Ln(H2PMes)] and the phosphide-bridged trimers [(Cp'2)Ln(μ-PHMes)]3 (Ln = Er, Gd). Their structures and magnetic properties are characterised. In Chapter 3, the novel dodecametallic thiolate-bridged lanthanide macrocycles [(Cp'2Ln)3({μ-SCH2}3CMe)]4 (Ln = Dy, Y, Gd) are reported and characterised by X-ray crystallography, NMR spectroscopy and magnetometry. [(Cp'2Dy)3({μ-SCH2}3CMe)]4 is shown to be a single-molecule magnet with Ueff = 69 cm-1. In Chapter 4, the novel thiolate-bridged lanthanide dimers [Cp'2Ln(μ-SCH2{C4H7S2})]2 (Ln = Dy, Y, Gd) are reported, showing sulfur-sulfur bonding leading to ring cyclisation of the bridging ligand [MeC(CH2S)3]3-. These complexes are characterised by X-ray crystallography, NMR spectroscopy and magnetometry. Extra NMR spectroscopic studies were performed to investigate the mechanism of ring closure on the bridging ligand. [Cp'2Dy(μ-SCH2{C4H7S2})]2 is shown to be a single-molecule magnet with Ueff = 87 cm-1. In Chapter 5, four new lanthanide siloxide clusters incorporating alkali metals are reported: the trigonal bipyramidal [Dy2K3(OSiMe3)9]; the octahedral [Dy2K4(OSiMe3)10]; the bi-capped cuboid [Y4K6O6(OSiMe3)12]6-; and the [Dy3K8O3(OSiMe3)12]- 'burger' cluster. All clusters are structurally characterised by X-ray crystallography and [Dy2K4(OSiMe3)10] is magnetically characterised. The synthetic rationalisation for formation of these diverse structures is investigated.
10

Monte Carlo Simulations of Single-Molecule Fluorescence Detection Experiments

Robinson, William Neil 01 August 2011 (has links)
Several Monte Carlo simulations of single-molecule fluorescence systems are developed to help evaluate and improve ongoing experiments. In the first simulation, trapping of a single molecule in a nanochannel is studied. Molecules move along the nanochannel by diffusion and electrokinetic flow. Single-molecule fluorescence signals excited by two spatially offset laser beams are detected and the direction of the flow is adjusted to try to equalize the signals and center the molecule between the beams. An algorithm is evaluated for trapping individual molecules in succession by rapidly reloading the trap after a molecule photobleaches or escapes. This is shown to be effective for trapping fast-diffusing single-chromophore molecules in succession within a micron-sized confocal region while accommodating the limited electrokinetic speed and the finite latency of feedback imposed by experimental hardware. In the second simulation, trapping of a molecule in a two-dimensional fluidic device consisting of sub-micron-separated glass plates is studied. Two different illumination schemes for sensing the molecule's position are compared: (i) a single continuous laser spot circularly scanned at 40 KHz or 240 KHz in the plane of the device; and (ii) four pulsed laser spots arranged in a square and temporally alternated at 304 MHz In either case, the times of detected photons are used by algorithms to control the electrokinetic flow in two dimensions to compensate diffusion and achieve single-molecule trapping. However each scheme is found to have limitations, as circular scanning produces a modulation in the fluorescence signal and in the autocorrelation function, whereas the four-pulse scheme becomes ineffective if the fluorescence lifetime of the molecule is greater than the time between laser pulses, The third simulation investigates appropriate conditions for detection of single molecules flowing through an array of fluidic channels for an application to high-throughput screening for pharmaceutical drug discovery. For parallelized single-molecule detection, illumination is provided by a continuous laser focused to a line intersecting all channels and fluorescence is imaged to a single row of pixels of an electron-multiplying CCD with sufficient gain for single-photon detection. The simulation separately models each channel to determine laser, flow, and camera operating conditions suitable for efficient detection.

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