Spelling suggestions: "subject:"singlemolecule"" "subject:"intermolecule""
211 |
Experimental realization of a feedback ratchet and a method for single-molecule binding studiesLopez, Benjamin J., 1982- 12 1900 (has links)
xii, 112 p. : ill. (some col.) / Biological molecular motors exist in an interesting regime of physics where momentum is unimportant and diffusive motion is large. While only exerting small forces, these motors still manage to achieve directed motion and do work. Brownian motors induce directed motion of diffusive particles and are used as models for biological and artificial molecular motors.
A flashing ratchet is a Brownian motor that rectifies thermal fluctuations of diffusive particles through the use of a time-dependent, periodic, and asymmetric potential. It has been predicted that a feedback-controlled flashing ratchet has a center of mass speed as much as one order of magnitude larger than the optimal periodically flashing ratchet. We have successfully implemented the first experimental feedback ratchet and observed the predicted order of magnitude increase in velocity. We experimentally compare two feedback algorithms for small particle numbers and find good agreement with Langevin dynamics simulations. We also find that existing algorithms can be improved to be more tolerant to feedback delay times. This experiment was implemented by a scanning line optical trap system.
In a bottom-up approach to understanding molecular motors, a synthetic protein-based molecular motor, the "tumbleweed", is being designed and constructed. This design uses three ligand dependent DNA repressor proteins to rectify diffusive motion of the construct along a DNA track. To predict the behavior of this artificial motor one needs to understand the binding and unbinding kinetics of the repressor proteins at a single-molecule level. An assay, similar to tethered particle motions assays, has been developed to measure the unbinding rates of these three DNA repressor proteins. In this assay the repressor is immobilized to a surface in a microchamber. Long DNA with the correct recognition sequence for one of the repressors is attached to a microsphere. As the DNA-microsphere construct diffuses through the microchamber it will sometimes bind to the repressor protein. Using brightfield microscopy and a CCD camera the diffusive motion of the microsphere can be characterized and bound and unbound states can be differentiated. This method is tested for feasibility and shown to have sufficient resolution to measure the unbinding rates of the repressor proteins. / Committee in charge: Dr. Raghu Parthasarathy, Chair;
Dr. Heiner Linke, Research Advisor;
Dr. Dan Steck;
Dr. John Toner;
Dr. Brad Nolan
|
212 |
Single-molecule fluorescence detection in molecular biology / Single-molecule fluorescence detection in molecular biologyFESSL, Tomáš January 2012 (has links)
SMFD techniques offer genuine detection possibilities which are often inaccessible using ensemble methods. This was demonstrated in three projects investigating translocation activity of CHD4 protein, analysis of MS2 phage capsid assembly and in-cell characterization of DNA structure. In other projects, binding interactions between two fluorescent probes and a short oligonucleotide were characterized and all optical depth of focus extended microscope configuration for imaging of individual molecules inside bacterial cells was developed and tested.
|
213 |
Role časových škál interakce systém-lázeň ve fotosyntetickém přenosu excitační energie / Role of system-bath interaction time-scales in photosynthetic excitation energy transferMalý, Pavel January 2018 (has links)
ROLE ASOVÉ 'KÁLY INTERAKCE SYSTÉM-LÁZE VE FOTOSYNTETICKÉM P ENOSU EXCITANÍ ENERGIE Tato práce se věnuje vlivu rychlého a pomalého molekulárno pohybu na přenos excitační energie ve fotosyntetick- ých světlosběrných komplexech. Vyvinuli jsme nový teoretický popis vnitromolekulárních vibračních mod· a zjistili jsme, že jejich resonance s energetickými rozdíly mezi fotosyntetickými pigmenty m·že vést ke zrychlení přenosu energie. Použitím jednomolekulární spektroskopie jsme pozorovali jak pomalé změny bílkovinné konformace mohou zcela změnit stav světlosběrného komplexu LHCII vyšších rostlin. Také jsme vyvinuli novou experimentální techniku, dvoupulzní ultrarychlou jednomolekulární spektroskopii. S její pomocí m·žeme pozorovat jak pomalý pohyb bílkoviny bakteriální antény LH2 ovlivňuje ultrarychlou relaxaci energie uvnitř komplexu. Konstrukcí jednotného modelu pro ultrarychlé objemové a jednomolekulární experimenty se nám podařilo zakomponovat rychlou a pomalou časovou škálu molekulárního pohybu do jednoho pohledu na fotosyntetický sběr světla.
|
214 |
Single-molecule diffusion measurements for material characterization in one-dimensional nanostructured polymer filmsTran-Ba, Khanh-Hoa January 1900 (has links)
Doctor of Philosophy / Department of Chemistry / Takashi Ito / This dissertation describes single-molecule tracking (SMT) measurements for the quantitative characterization of one-dimensional (1D) nanostructures in 200 nm-thick surfactant-templated mesoporous silica (STMS) and cylinder-forming polystyrene-poly(ethylene oxide) diblock copolymer (CF-PS-b-PEO) films with a μm-scale thickness. SMT is advantageous for the characterization of nanomaterials over conventional methods because it permits the simultaneous and quantitative assessment of the nanoscale and microscale morphologies, and mass-transport properties of the materials with a high nanometer-scale resolution under ambient conditions. It offers a unique means for the assessment and evaluation of the μm-scale nanostructure alignment in polymer films induced by vertical spin-coating (for STMS films), directional solution flow and solvent-vapor penetration (SVP) methods (both for CF-PS-b-PEO films), highly crucial for many potential technological applications using the materials. Through this work, we have identified suitable sample preparation conditions (e.g. solvent, temperature or solution flow rate) for obtaining highly-ordered mesoporous and microdomain structures over a long-range (> 5 μm). For the quantitative assessment of the 1D SMT data, orthogonal regression analysis was employed, providing assessment of the in-plane orientation and size of individual nanostructures with nanometer-scale precision. The analysis of the 1D trajectory data allowed the radius (ca. 11 nm) of cylindrical PEO microdomains to be estimated, yielding results consistent with the AFM results (ca. 14 nm). The distribution of the trajectory angles offered the estimation of the average orientation and order of the nanostructures in domains/grains for a μm-wide region of the polymer films, revealing the higher efficiency of SVP in the nanostructure alignment as compared to the spin coating and solution flow approaches. Systematic SMT measurements across the film depth and along lateral mm-scale distances afforded valuable insights into the shear- and solvent-evaporation-based alignment mechanisms induced by solution flow and SVP/spin coating approaches, respectively. Fluorescence recovery after photobleaching (FRAP) measurements in a SVP-aligned CF-PS-b-PEO film permitted the longer-range mass-transport properties to be probed, reflecting the effective continuity of the aligned cylindrical nanostructures over > 100 μm in length. In this dissertation, FRAP and more importantly SMT methods have provided a unique and useful means for the in-depth characterization of morphology and mass-transport characteristics in thin polymer films under ambient conditions, in confined spaces, and with a nanometer-scale resolution.
|
215 |
Single molecule tracking studies of flow-aligned mesoporous silica monoliths: pore order and pore wall permeabilityPark, Seok Chan January 1900 (has links)
Doctor of Philosophy / Department of Chemistry / Daniel A. Higgins / This dissertation describes single-molecule tracking (SMT) studies for the quantitative characterization of one-dimensional (1D) nanostructures in surfactant-templated mesoporous silica monoliths prepared within microfluidic channels. Single molecule diffusion of fluorescent probe molecules within the cylindrical mesopores reflects microscopic morphologies and mass-transport properties of the materials with high temporal and spatial resolution. The pore organization and materials order are initially investigated as a function of sol aging prior to loading into the microfluidic channels. Mesopores in these materials are templated by Cetyltrimethylammonium bromide (CTAB). Wide-field fluorescence videos depict 1D motion of the dyes within the individual mesopores. Orthogonal regression analysis of these motions provides a measure of the mesopore orientation. Channels filled prior to gelation of the sol produce monoliths incorporating large monodomains with highly aligned mesopores. In contrast, channels filled close to or after gelation yield monoliths with misaligned pores that are also more disordered. Two-dimensional (2D) small angle X-ray scattering (SAXS) experiments support the results obtained by SMT. These studies help to identify conditions under which highly aligned mesoporous monoliths can be obtained and also demonstrate the utility of SMT for characterization of mesopore order.
The non-ionic surfactant Pluronic F127 is also utilized as the structural-directing agent. The diffusive motions of PDI dyes that are uncharged, cationic and anionic are explored by SMT and fluorescence correlation spectroscopy (FCS). The SMT studies for the uncharged dye show development of 1D diffusion along the flow direction while charged dyes exhibit predominant isotropic diffusion, with each of these behaviors becoming more prevalent as a function of aging time after filling of the microfluidic channels. SMT studies from silica-free F127 gels suggest that partitioning plays a important role in governing the diffusion behavior of the PDI dyes within the surfactant-filled mesopores. FCS results exhibit similar mean diffusion coefficients for all three dyes that suggest these dyes diffuse through similar sample regions. These studies demonstrate that the silica pore walls in the mesoporous silica monoliths remain permeable after gelation and that partitioning of solute species to different regions within the pores plays an important role in restricting the dimensionality of their diffusive motion
|
216 |
Photophysical Properties and Applications of Fluorescent Probes in Studying DNA Conformation and DynamicsJanuary 2015 (has links)
abstract: Fluorescence spectroscopy is a popular technique that has been particularly useful in probing biological systems, especially with the invention of single molecule fluorescence. For example, Förster resonance energy transfer (FRET) is one tool that has been helpful in probing distances and conformational changes in biomolecules. In this work, important properties necessary in the quantification of FRET were investigated while FRET was also applied to gain insight into the dynamics of biological molecules. In particular, dynamics of damaged DNA was investigated. While damages in DNA are known to affect DNA structure, what remains unclear is how the presence of a lesion, or multiple lesions, affects the flexibility of DNA, especially in relation to damage recognition by repair enzymes. DNA conformational dynamics was probed by combining FRET and fluorescence anisotropy along with biochemical assays. The focus of this work was to investigate the relationship between dynamics and enzymatic repair. In addition, to properly quantify fluorescence and FRET data, photophysical phenomena of fluorophores, such as blinking, needs to be understood. The triplet formation of the single molecule dye TAMRA and the photoisomerization yield of two different modifications of the single molecule cyanine dye Cy3 were examined spectroscopically to aid in accurate data interpretation. The combination of the biophysical and physiochemical studies illustrates how fluorescence spectroscopy can be used to answer biological questions. / Dissertation/Thesis / Doctoral Dissertation Chemistry 2015
|
217 |
Charge Transport in Single MoleculesJanuary 2017 (has links)
abstract: Studying charge transport through single molecules is of great importance for unravelling charge transport mechanisms, investigating fundamentals of chemistry, and developing functional building blocks in molecular electronics.
First, a study of the thermoelectric effect in single DNA molecules is reported. By varying the molecular length and sequence, the charge transport in DNA was tuned to either a hopping- or tunneling-dominated regimes. In the hopping regime, the thermoelectric effect is small and insensitive to the molecular length. Meanwhile, in the tunneling regime, the thermoelectric effect is large and sensitive to the length. These findings indicate that by varying its sequence and length, the thermoelectric effect in DNA can be controlled. The experimental results are then described in terms of hopping and tunneling charge transport models.
Then, I showed that the electron transfer reaction of a single ferrocene molecule can be controlled with a mechanical force. I monitor the redox state of the molecule from its characteristic conductance, detect the switching events of the molecule from reduced to oxidized states with the force, and determine a negative shift of ~34 mV in the redox potential under force. The theoretical modeling is in good agreement with the observations, and reveals the role of the coupling between the electronic states and structure of the molecule.
Finally, conclusions and perspectives were discussed to point out the implications of the above works and future studies that can be performed based on the findings. / Dissertation/Thesis / Doctoral Dissertation Chemistry 2017
|
218 |
Characterization of single proteins using double nanohole optical tweezersHacohen, Noa 28 May 2018 (has links)
Proteomic studies at the single molecular level could provide better understanding of the protein’s behaviour and the effects of its interactions with other biomolecules. This could
have an impact on drug development methods, disease diagnosis, and targeted therapy.
Aperture assisted optical trapping is a proven technique for isolating single proteins in solution without the use of tethers or labels, and without denaturing them. Thus enabling studies of protein-protein interactions, protein-small molecule interactions, and protein-DNA interactions.
In this work, double nanohole (DNH) optical tweezers were used to analyze the protein composition of heterogeneous mixtures. The trapped proteins were grouped by molecular
mass based on two metrics: standard deviation of the trapping laser intensity fluctuations, and the time constant of the autocorrelation function of these fluctuations.
The quantitative analysis is demonstrated first for two separate standard-size proteins, then for a mixed solution of both. Finally, the approach is applied to real unprocessed egg white solution. The results correspond well with the known protein composition of egg white found in the literature. The DNH optical tweezers’ ability to distinguish proteins in unpurified heterogeneous mixtures, can progress this technique to the next level, allowing for single biomolecular studies of unprocessed physiological solutions like blood, urine, or saliva. / Graduate
|
219 |
Mechanism of the F1 ATPase Molecular Motor as Revealed by Single Molecule StudiesJanuary 2012 (has links)
abstract: The F1Fo ATP synthase is required for energy conversion in almost all living organisms. The F1 complex is a molecular motor that uses ATP hydrolysis to drive rotation of the γ–subunit. It has not been previously possible to resolve the speed and position of the γ–subunit of the F1–ATPase as it rotates during a power stroke. The single molecule experiments presented here measured light scattered from 45X91 nm gold nanorods attached to the γ–subunit that provide an unprecedented 5 μs resolution of rotational position as a function of time. The product of velocity and drag, which were both measured directly, resulted in an average torque of 63±8 pN nm for the Escherichia coli F1-ATPase that was determined to be independent of the load. The rotational velocity had an initial (I) acceleration phase 15° from the end of the catalytic dwell, a slow (S) acceleration phase during ATP binding/ADP release (15°–60°), and a fast (F) acceleration phase (60°–90°) containing an interim deceleration (ID) phase (75°–82°). High ADP concentrations decreased the velocity of the S phase proportional to 'ADP-release' dwells, and the F phase proportional to the free energy derived from the [ADP][Pi]/[ATP] chemical equilibrium. The decreased affinity for ITP increased ITP-binding dwells by 10%, but decreased velocity by 40% during the S phase. This is the first direct evidence that nucleotide binding contributes to F1–ATPase torque. Mutations that affect specific phases of rotation were identified, some in regions of F1 previously considered not to contribute to rotation. Mutations βD372V and γK9I increased the F phase velocity, and γK9I increased the depth of the ID phase. The conversion between S and F phases was specifically affected by γQ269L. While βT273D, βD305E, and αR283Q decreased the velocity of all phases, decreases in velocity due to βD302T, γR268L and γT82A were confined to the I and S phases. The correlations between the structural locations of these mutations and the phases of rotation they affect provide new insight into the molecular basis for F1–ATPase γ-subunit rotation. / Dissertation/Thesis / Ph.D. Molecular and Cellular Biology 2012
|
220 |
Espalhamento Raman intensificado pela superfície (SERS) no regime de detecção de uma molécula / Surface-enhanced Raman scattering at single-molecule detection regimeDiego Pereira dos Santos 18 February 2013 (has links)
Nesta tese foi estudado o espalhamento Raman intensificado pela superfície (SERS) em regime de detecção de uma molécula em eletrodo de prata ativado por ciclos de oxidação e redução. Neste regime, de baixas concentrações, são observadas intensas flutuações de intensidade SERS as quais foram controladas neste substrato pela aplicação de potencial ao eletrodo, o que foi associado a alterações na concentração de moléculas adsorvidas na superfície do eletrodo. Além da dependência com o potencial aplicado, foram estudadas através de simulações Monte Carlo, a contribuição nestas flutuações da constante de adsorção das moléculas, do número de \"hot spots\" (regiões de altas intensificações SERS) e do tipo de \"hot spot\" (em termos de eficiência para detecção de espectros de uma molécula). Através destas simulações foram verificadas flutuações de intensidade muito semelhantes às observadas experimentalmente. Além das flutuações de intensidade foram também observadas flutuações de intensidades relativas, como por exemplo, das relações de intensidades anti-Stokes/Stokes, as quais foram interpretadas segundo um modelo de ressonância, através do qual foi possível estimar as energias de ressonância nos \"hot spots\". Alguns dos resultados indicaram a contribuição de ressonâncias finas, as quais foram interpretadas como resultado de interferências entre ressonâncias de plasmon de superfície. Interferências como estas foram demonstradas através de simulações pelo método DDA (\"Discrete Dipole appoximation\") em modelos simples de \"hot spots\" formados por nanobastões de Au. / In this thesis it was studied surface-enhanced Raman scattering (SERS) at single-molecule detection on Ag electrode activated by oxidation and reduction cycles. At this low concentration limit it was observed strong SERS intensity fluctuations that were controlled by the applied potential to the electrode and this control was associated to changes in surface concentration of adsorbed molecules. Furthermore, it was studied through Monte Carlo simulations the influence of adsorption constant, number of \"hot spots\" (regions of high SERS enhancements) and type of \"hot pot\" (in terms of efficiency for single-molecule detection). With such simulations, it was verified fluctuations of SERS intensities very similar to experimental observations. Besides absolute intensity fluctuations, we also observed fluctuations of relative intensities as, for instance, the. anti-Stokes to Stokes intensity ratios. These fluctuations were interpreted according to a resonance model, which made possible the estimative of resonance energies at the SERS \"hot spots\". Some of these results indicated the existence of sharp resonances that were interpreted as a result of interferences among surface plasmon resonances, which were demonstrated through DDA (Discrete Dipole Approximation) simulations in simple models of \"hot spots\" formed by Au nanorods
|
Page generated in 0.0348 seconds