Photoactivated fluorescence from individual silver nanoclusters ranging in size from 2 8 atoms has been demonstrated at room temperature. The optical properties of such clusters are far superior to those of fluorescence dyes with absorption cross sections ~50 times stronger than those of even the best organic dyes. The strong oscillator strengths produced from such nanoclusters has been shown to yield comparable enhancement factors in the surface-enhanced Raman spectroscopy (SERS) process to those observed in the presence of a plasmon- supporting nanoparticle. Raman transitions are in fact so strong that antistokes scattering is also observable on a single molecule (SM) level marking the first true demonstration of SM-SERS to date. Capable of generating true scaffold specific Raman scattering on the single molecule level, the combination of fluorescence from the small nanoclusters and strong observed Raman signals in the absence of a nanoparticle strongly indicate a chemical or charge transfer SERS enhancement mechanism.

The Severe Acute Respiratory Syndrome (SARS) virus encodes several unusual RNA processing enzymes, including Nsp15, an endoribonuclease that preferentially cleaves 3? of uridylates through a Ribonuclease A-like mechanism. Crystal structures of Nsp15 confirmed that the Nsp15 active site is structurally similar to that of Ribonuclease A. These similarities and our molecular docking analysis lead us to hypothesize that previously characterized Ribonuclease A inhibitors will also inhibit the SARS-CoV Nsp15. Benzopurpurin B, C-467929, C-473872, N-36711, N-65828, N-103018 and Congo red were tested for effects on Nsp15 endoribonuclease activity. A real-time fluorescence assay revealed that the IC50 values for inhibiting Nsp15 were between 0.2 ?M and 40 ?M. Benzopurpurin B, C-473872, and Congo red are competitive inhibitors, according to kinetic studies and were demonstrated to bind SARS-CoV Nsp15 by a differential scanning fluorimetry assay. Benzopurpurin B also inhibited the Nsp15 orthologs from two other coronaviruses: mouse hepatitis virus (MHV) and infectious bronchitis virus. The three compounds reduced infectivity of MHV in L2 cells by 8 to 26 fold. The more effective drugs also caused a decrease in MHV RNA accumulation.

The objective of this work is to develop a micro/nanofluidic-based single molecule detection (SMD) scheme, which would allow us to inspect individual protein or protein complex study protein-protein interactions and their dynamics. This is a collaboration work with MD Anderson Cancer Center and we applied this scheme to study functions of various proteins related to cancer progression in hope to shed new light on cancer research. State-of-the-art micro/nano-fabrication technology is used to provide fused silica micro/nano-fluidic channel devices as our detection platform. Standard contact photolithography, projection photolithography and advanced electron-beam lithography are used to fabricate micro/nano-fluidic channel with width ranging from 100nm to 2?m. The dimensions of these miniaturized biochips are designed to ensure single molecule resolution during detection and shrinking the detection volume leads to increase in signal-to-noise ratio, which is very critical for SMD. To minimize surface adsorption of protein, a fused silica channel surface coating procedure is also developed and significantly improved the detection efficiency. A fluorescent-labeled protein sample solution is filled in the fluidic channel by capillary force, and proteins are electro-kinetically driven through the fluidic channel with external voltage source. Commercial functionalized Quantum Dots (Qdots) are used as fluorescent labels due to its various advantages over conventional organic dyes for single molecule multi-color detection application. A fluorescence correlation spectrometer system, equipped with a 375nm diode laser, 60x water immersion objective with N.A. of 1.2 and two avalanche photodiodes (APD) is implemented to excite single molecules as well as collect emitted fluorescence signals. A two-dimensional photon burst analysis technique (photon counts vs. burst width) is developed to analyze individual single molecule events. We are able to identify target protein or protein complex directly from cell lysate based on fluorescence photon counts, as well as study the dynamics of protein-protein interactions. More importantly, with this technique we are also able to assess interactions between three proteins, which cannot be done with current ensemble measurement techniques. In summary, the technique described in this work has the advantages of high sensitivity, short processing time (2-3 minutes), very small sample consumption and high resolution quantitative analysis. It could potentially revolutionize the area of protein interaction research and provides us with more clues for the future of cancer diagnostics and treatments.

This thesis aims to study external field induced alignment of semiconductor quantum dot by utilizing single molecule spectroscopy. Wurtzite structure semiconductors, such as CdSe, exhibit strong electric dipole moment along its c-axis. It is proposed that quantum dot can be aligned along the applied field with sufficient strength. Experiments with two kind of matrix: PMMA mixing with wax, and liquid crystal thin film, were performed for that quantum dots are able to rotate freely in the matrix. Experiments with PMMA matrix were also performed as its rigid matrix for comparison. Interdigitated structure electrodes was deposited on the cover glass for the electric field experiments. The topical transition (absorption and emission) of CdSe quantum dots has a bright plane perpendicular to its c-axis, and a dark axis along the c-axis. It thus used for characterizing the field alignment. For each observing quantum dot, we record the fluorescence intensity, anti-bunching, polarization anisotropy, and fluorescence lifetime information. In addition, we also analyze the fluorescence correlation spectroscopy to probe the small modulation signal from the fluctuating fluorescence intensity. However, the results indicate that we didn¡¦t observe the field induced change with the field up to 1E7(v/m).

Optical second harmonic generation (SHG) is a nonlinear process that converts two photons of frequency w to a single photon of frequency 2w. In the electricdipole approximation, this process requires a noncentrosymmetric medium. For thin films present at the interface between two centrosymmetric media¡Ðsuch as the interface between two liquids, the interface between a liquid and air, or for the surface of a variety of insulators and semiconductors¡Ðonly the molecules which participate in the asymmetry of the interface contribute to the SHG. Surface SGH from molecules which exhibit a large nonlinear optical response at a specific wavelength can be used to measure the relative surface coverage of molecules at the interface as a function of the external electrochemical parameters. The polarization dependence of the surface SHG can be used to estimate the average molecular orientation of chromophores at the liquid/liquid electrochemical interface. The potential dependence of the SHG from the interface provides important information on the position of the adsorbed chromophores with respect to the interface.

SERS

Understanding the radiolysis of impure water and resulting reactions is crucial to many fields. Reactions driven by low energy electrons (LEE) are of special interest, as high-energy radiation generates large quantities of these electrons, which then provide the energy for most subsequent reactions. Interfacially located reactions are also of particular interest, both as models for heterogeneously distributed reactions occurring during radiolysis, and in their own right, as radiation-driven reactions at interfaces are responsible for key processes such as corrosion and DNA damage. To study LEE-driven reactions at interfaces, thin-layered films of amorphous solid water (ASW) and methanol were grown under ultra-high vacuum conditions using molecular beam techniques. The films were exposed to a beam of low-energy (100eV or less) electrons, and studied using electron-stimulated desorption (ESD) and temperature programmed desorption (TPD). ESD studies indicated that methanol moves through a water film during deposition at 80 K but not at 50 K. This transport was not seen during thermal annealing, but radiation-induced mixing was observed at all temperatures. Major and minor LEE radiation products of pure methanol films were identified and found to be consistent with previous results. Products of LEE irradiated layered methanol/water films were determined for the first time using ESD and TPD spectra, and found to be limited to H₂, O, O₂, CH₂O, C₂H₆, CO, CO₂, CH₃OCH₃, and CH₃CH₂OH. The effect of adding methanol to an ASW film on the production in ASW of H₂ and O₂ was also examined. The interface created by the addition of CH₃OH to ASW was found to generate H₂ in previously non-reactive regions of the water film by increasing water-water and water-methanol reactions. Radiative mixing of CH₃OH and ASW enhanced this effect, presumably by increasing the region of disrupted H-bonding in the ASW. In contrast, the addition of CH₃OH at low coverages suppressed O₂ production in both unprocessed and preprocessed ASW layers. Modeling indicates that methanol scavenging of the O₂ precursor OH and of the reaction-driving electrons is responsible for this reduction in O₂ signal.

I present two applications of fluorescence spectroscopy in biophysics. The first is an instrument, the anti-Brownian electrokinetic (ABEL) trap, which is capable of trapping individual small molecules in aqueous solution at room temperature. The second is an investigation of the bending mechanics of double-stranded DNA using a novel DNA structure called a "molecular vise". Both projects take advantage of the sensitivity and specificity of fluorescence spectroscopy, and both benefit from the interplay of experimental work with theoretical and computational modeling. The ABEL trap uses fluorescence microscopy to track a freely diffusing particle, and applies real-time electrokinetic feedback forces to oppose observed motion. Small molecules are difficult to trap because they diffuse quickly and because their fluorescence emission is typically weak. I describe the experimental and algorithmic approaches that enabled small-molecule fluorophores to be trapped at room temperature. I additionally derive and discuss the theory of the molecules' behavior in the trap; this mathematical work informed the design of the trapping algorithm and additionally enabled trapped molecules to be distinguished on the basis of their diffusion coefficient and electrokinetic mobility. Molecular vises are DNA hairpins that use the free energy of hybridization to exert a compressive force on a sub-persistence length segment of double-stranded DNA. In response to the applied force, this "target strand" may either remain straight or bend, depending on its flexibility and length. Experimentally, the conformation can be monitored via Förster resonance energy transfer (FRET) between appended fluorophores. The experimental results quantitatively matched the predictions of the classic wormlike chain (WLC) model of DNA elasticity at low-to-moderate salt concentrations. Higher ionic strength induced an apparent softening of the DNA which was best accounted for by a high-curvature "kinked" state. The molecular vise is exquisitely sensitive to the sequence-dependent linear and nonlinear elastic properties of dsDNA and provides a platform for studying the effects of chemical modifications and small-molecule or protein binding on these properties.

Due to the fast increasing capabilities of modern computers it is now feasible to calculate spectra of small atom and molecules with the greater level of accuracy than high-resolution measurements. The mathematical algorithms developed and implemented on high performance supercomputers for the quantum mechanical calculations are directly derived from the first principles of quantum mechanics. The codes developed are primarily used to verify, refine, and predict the energies associated within a given system and given angular momentum state of interest. The Hamiltonian operator used to determine the total energy in the approach presented is called the internal Hamiltonian and is obtained by rigorously separating out the center-of-mass motion (or the elimination of translational motion) from the laboratory-frame Hamiltonian. The methods utilized in the articles presented in this dissertation do not include relativistic corrections and quantum electrodynamic effects, nor do these articles assume the Born-Oppenheimer (BO) approximation with the exception of one publication. There is one major review article included herein which describes the major differences between the non-BO method and the BO approximation using explicitly correlated Gaussian (ECG) basis functions. The physical systems studied in this dissertation are the atomic elements with Z < 7 (although the discussion is not limited to these) and diatomic molecules such as H₂⁺ and H₂ including nuclear isotopic substitution studies with deuterium and tritium, as well as electronic substitutions with the muon particle. Preliminary testing for triatomic molecular functionals using a model potential is also included in this dissertation. It has been concluded that using all-particle ECGs with including the addition of nonzero angular momentum functions to describe nonzero angular momentum states is sufficient in determining the energies of these states for both the atomic and molecular case.

During development, Dictyostelium discoideum cells assume muticellularity via their collective aggregation. Cell-cell adhesion is required for morphogenesis, cell differentiation, cell sorting and gene expression during development. TgrC1 is a heterophilic cell adhesion molecule which is indispendable for complete development. TgrC1 can be considered as the most important cell adhesion molecule for D. discoideum development because deletion of the tgrC1 gene completely arrests development at the loose aggregate stage and inhibits fruiting body formation. In order to investigate the biological role of TgrC1 during development, I have chosen to identify and charactize the extracellular heterophilic partner and the cytoplasmic binding partner(s) of TgrC1. Using different biochemical approaches, we identified TgrB1 as the heterophilic binding partner of TgrC1 and demonstrated that their association is mediated through IPT/TIG domains in the extracellular region of both proteins. Both tgrB1 and tgrC1 share the same transcriptional promoter and their spatiotemporal expression pattern is identical during development. We also examined the assembly of TgrC1-TgrB1 complexes via the split green fluorescence protein complementation assay and the fluorescence resonance energy transfer approach. Whereas TgrC1 is capable of forming cis-homodimers spontaneously, cis-homodimerization of TgrB1 depends on its trans-interaction with TgrC1. A model of the assembly process has been proposed. To investigate signalling events initiated by the interaction between TgrB1 and TgrC1, pull-down assays were employed and led to the identification of myosin heavy chain kinase C as the cytoplamic partner of TgrC1. Mutational analysis showed that the basic residues in the short cytoplasmic domain of TgrC1 are critical to the binding with MHCK-C. Disruption of the interation between MHCK-C and TgrC1 results in an alteration of cell motility at the aggregation stage and aberrant cell sorting in slugs. These studies have highlighted the role of TgrB1-TgrC1 complexes in the regulation of morphogenesis during Dictyostelium development.