Browsing by Subject "Single molecule spectroscopy"
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Item Fӧrster Resonance Energy Transfer (FRET) between phycobiliproteins and tandem conjugates(2012-08) Tian, Yu; Pappas, Dimitri; Thompson, Jonathan E.; Shi, HuazhongSingle molecule recrossing events was applied to study light tolerance and photobleaching of allophycocyanin (APC), which is one group of phycobiliprotein. Normalized recrossing ratio was defined as Nr/Nt, where Nr was the number of molecules that reenter the probe volume, and Nt was the total number of detected molecules. The ratio of Nr/Nt was regarded as a parameter for light tolerance of the molecules of interest. Normalized molecular recrossings as a function of excitation irradiation showed that the triplet-state formation and photobleaching occurred at high excitation power. Moreover, the similar method was employed to study energy transfer between allophycocyanins and a second conjugated organic dye. The saturation irradiance and triplet states for Streptavidin Allophycocyanin - AlexaFluor-680 (APC-680) was investigated using single molecule recrossings method. By comparing the saturation irradiance and normalized molecular recrossings Nr/Nt for APC and APC-680, it showed that the light tolerance of APC-680 was improved over APC. This improvement resulted from conjugating a second dye to APC, which provided another pathway for energy transfer. The alternative pathway decreased the possibility of photobleaching and triplet state conversion. Therefore, by linking a second dye to phycobiliproteins, the photostability and light tolerance of this system was improved. In order to ease the process of extracting single molecule fluorescence peaks from background noise, the power transform was applied to both simulated and experimental single molecule fluorescence time trace. Power transformations increased the contrast between fluorescence signal bursts and noise. The power transform simplified discerning single molecule signals from background. The results also showed that power transforms had the capability to distinguish the signals that could not be identified in original dataset. The power transform is a simple and reversible method to increase signal to noise ratio of single molecule time traces.Item Investigation of the photo-induced charge transfer in organic semiconductors via single molecule spectroscopy techniques(2009-12) Lee, Kwang Jik; Barbara, P. F. (Paul F.), 1953-Photo-induced charge transfer which occurs between molecules or different parts of a large molecule is the pivotal process related to performances of organic electronics. In particular, injection of charge carriers into conjugated polymers and dissociation of photo-generated excitons at the heterojunction between a donor and acceptor system are of great importance in determining the luminescence efficiency of organic light emitting diodes (OLEDs) and solar energy conversion efficiency of organic solar cells, respectively. However, the complex nature of organic semiconductors as well as complicated primary processes involved in the functioning of these devices have prevented us from understanding unique characteristics of these processes and thereby engineering better materials for higher performances. In this dissertation, two different types of photo-induced (or -related) charge transfer processes occurring in organic semiconductors were investigated by using single molecule spectroscopy (SMS) techniques to unravel the complexities of these processes. The carefully designed functioning capacitor-like model devices similar to OLEDs and photovoltaic cells were fabricated where isolated single nanoparticles were introduced as an active medium to mitigate the complexities of these materials. We observed that injection of positively charged carriers (holes) into poly[2-methoxy-5-(2'-ethyl-hexyloxy)-1,4-phenylene vinylene] (MEH-PPV) single nanoparticles from the carbazole hole transport layer does not occur in the absence of light. We denoted the observed hole injection in aid of light as the light-induced hole transfer mechanism (LIHT). It was revealed that the charging dynamics are highly consistent with a cooperative charging effect. In addition, the LIHT was proposed as the possible source for the formation of deep trapped hole in organic devices. Local exciton dissociation yields across a nanostructured domain between poly(9,9-dioctylfluorene-alt-benzothiadiazole) (F8BT) single nanoparticles and either poly(9,9- dioctylfluorene - co - bis-N,N- (4 -butylphenyl)-bis-N,N-phenyl-1,4-phenylene diamine) (PFB) or poly(9,9-dioctylfluorene-co-N-(4-butylphenyl)diphenylamine) (TFB) film in model photovoltaic devices was also investigated. A wide distribution of exciton dissociation yields was observed from each nanodomain due to the device geometry. The observed hysteresis in fluorescence voltage curve was ascribed to accumulated charges following charge separations. The dynamics of charge separation under the applied electric field was described in more detail.Item Studies on HIV-1 nucleocapsid chaperone role in protein/nucleic acid interactions by single molecule spectroscopy approaches(2009-12) Ma, Xiaojing, 1982-; Barbara, P. F. (Paul F.), 1953-; Korgel, Brian A.; Vanden Bout, David A.; Henkelman, Graeme A.; Russell, RickHIV-NC is a multifunctional protein which plays an important role in almost every step of the retroviral life cycle. NC is essential in catalyzing stand transfers of HIV-1 reverse transcription, including the annealing of the transactivation response element (TAR) of the viral genome to the complementary TAR DNA in minus-strong-stop DNA. In this dissertation, the research starts with focus on elucidating the reaction mechanism of NC-facilitated TAR DNA/RNA annealing using single molecule spectroscopy (SMS) approaches. The results indicate that nucleation of TAR DNA/RNA annealing occurs in an encounter complex form in which one or two DNA/RNA strands in the partially open “Y” form associated with multiple NC molecules. This encounter complex leads to annealing through the 3’/5’ termini, namely “zipper” pathway and the annealing through the hairpin loop region, namely “kissing” pathway. By employing target oligonucleotides for specific TAR regions, we directly probed kinetic reversibility and the chaperone role of NC. Concentration-dependence of NC chaperoned melting and annealing of TAR hairpins was investigated and the results further support the proposed reaction mechanism. Additionally, we used a single-stranded DNA (ssDNA) as model to study ssDNA conformational change upon NC binding. Here we present observation of NC binding to d(TG)n and d(T)n, including NC effect on flexibility and conformation of these oligonucleotides chains. Our results reveal that the rigidity of ssDNA chain is dramatically reduced through interaction with NC. Meanwhile the results of NC dissociation experiments indicate the interaction of NC/ssDNA is complex and heterogeneous. Finally, we used SMS in vitro to systematically compare and contrast the RNA/protein interactions for the zinc-finger-binding-motif protein (NC) and the arginine-rich-binding-motif (ARM) protein (Tat) encoded by HIV-1. Tat and NC use different RNA binding motifs to recognize and interact with RNA hairpin, giving rise to very different changes in the RNA secondary structure upon protein binding. Competition experiments show that the presence of Tat can effectively inhibit the NC binding-induced local melting of TAR RNA hairpins. These results indicate that Tat specifically binds and stabilizes the TAR RNA hairpin structure, which likely inhibits the local melting of the hairpin induced by NC.Item Understanding of conjugated polymer morphology formation and the structure-property relationships from the single chain level to the bulk level(2012-08) Adachi, Takuji; Vanden Bout, David A.Morphology is the origin of life and function. Defining and designing morphology, understanding the relationship between morphology and function, is an essential theme in a number of research areas. In conjugated polymer research, the major obstacles to achieving these goals are the heterogeneity and complexity of conjugated polymer films. In the study presented in this dissertation, various single molecule spectroscopy techniques were used as an approach to minimize the complexity of these problems. By using excitation polarization spectroscopy, it was discovered that single chains of poly[2-methoxy-5-(2'-ethylhexyloxy)-1,4-phenylenevinylene] (MEH-PPV) assume a highly ordered rod conformation despite the fact that the morphology of bulk films is known to be amorphous. The comparison of results from experiments and a coarse grained bead-on-a-chain simulation suggested that single chains have the ability to use a thermally induced defect to maximize [pi]-[pi] stacking and adopt a rod conformation as a stable conformation. Bias-induced centroid spectroscopy (BIC) on highly ordered single chains demonstrated that the energy transfer scale could be an order of magnitude larger than the value typically measured for bulk films. It was further demonstrated that such an extraordinary long energy transfer was not a unique property of single chains but was also observed in aggregates as long as the morphology was ordered. These studies were extended to another model compound poly(3-hexylthiophene) (P3HT) to generalize the mechanism of morphology formation and the structure-property relationship. For P3HT, it was shown that side-chains were a very important factor in determining single chain conformation, while the conformation of MEH-PPV was not affected by side-chains. By controlling the side-chains, both ordered and disordered P3HT chains were obtained. The comparison of results from experiments and an energy transfer model simulation quantified that energy transfer was at least twice as efficient in ordered chains as in disordered chains. In aggregates, the difference between the energy transfer efficiency of ordered and disordered morphology was even larger than that in the case of single chains. These results could suggest that there is a very fast energy transfer mechanism that occurs through interchain interactions when chains are packed in ordered fashion.