Rod-shaped plasmonic sensors

Plasmonic nanoparticles exhibit strong light scattering efficiency due to the oscillations of their conductive electrons (plasmon), which are excited by light. For rod-shaped nanoparticles, the resonance position is highly tunable by the aspect ratio (length/width) and the sensitivity to changes in the refractive index in the local environment depends on their diameter, hence, their volume. Therefore, rod-shaped nanoparticles are highly suitable as plasmonic sensors.rnWithin this thesis, I study the formation of gold nanorods and nanorods from a gold-copper alloy using a combination of small-angle X-ray scattering and optical extinction spectroscopy. The latter represents one of the first metal alloy nanoparticle synthesis protocols for producing rod-shaped single crystalline gold-copper (AuxCu(1-x)) alloyed nanoparticles. I find that both length and width independently follow an exponential growth behavior with different time-constants, which intrinsically leads to a switch between positive and negative aspect ratio growth during the course of the synthesis. In a parameter study, I find linear relations for the rate constants as a function of [HAuCl4]/[CTAB] ratio and [HAuCl4]/[seed] ratio. Furthermore, I find a correlation of final aspect ratio and ratio of rate constants for length and width growth rate for different [AgNO3]/[HAuCl4] ratios. I identify ascorbic acid as the yield limiting species in the reaction by the use of spectroscopic monitoring and TEM. Finally, I present the use of plasmonic nanorods that absorb light at 1064nm as contrast agents for photoacoustic imaging (BMBF project Polysound). rnIn the physics part, I present my automated dark-field microscope that is capable of collecting spectra in the range of 450nm to 1750 nm. I show the characteristics of that setup for the spectra acquisition in the UV-VIS range and how I use this information to simulate measurements. I show the major noise sources of the measurements and ways to reduce the noise and how the combination of setup charactersitics and simulations of sensitivity and sensing volume can be used to select appropriate gold rods for single unlabeled protein detection. Using my setup, I show how to estimate the size of gold nano-rods directly from the plasmon linewidth measured from optical single particle spectra. Then, I use this information to reduce the distribution (between particles) of the measured plasmonic sensitivity S by 30% by correcting for the systematic error introduced from the variation in particle size. I investigate the single particle scattering of bowtie structures — structures consisting of two (mostly) equilateral triangles pointing one tip at each other. I simulate the spectra of the structures considering the oblique illumination angle in my setup, which leads to additional plasmon modes in the spectra. The simulations agree well with the measurements form a qualitative point of view.rn

Reaction Control in Quiescent Systems of Free-Radical Retrograde-Precipitation Polymerization

Free-radical retrograde-precipitation polymerization, FRRPP in short, is a novel polymerization process discovered by Dr. Gerard Caneba in the late 1980s. The current study is aimed at gaining a better understanding of the reaction mechanism of the FRRPP and its thermodynamically-driven features that are predominant in controlling the chain reaction. A previously developed mathematical model to represent free radical polymerization kinetics was used to simulate a classic bulk polymerization system from the literature. Unlike other existing models, such a sparse-matrix-based representation allows one to explicitly accommodate the chain length dependent kinetic parameters. Extrapolating from the past results, mixing was experimentally shown to be exerting a significant influence on reaction control in FRRPP systems. Mixing alone drives the otherwise severely diffusion-controlled reaction propagation in phase-separated polymer domains. Therefore, in a quiescent system, in the absence of mixing, it is possible to retard the growth of phase-separated domains, thus producing isolated polymer nanoparticles (globules). Such a diffusion-controlled, self-limiting phenomenon of chain growth was also observed using time-resolved small angle x-ray scattering studies of reaction kinetics in quiescent systems of FRRPP. Combining the concept of self-limiting chain growth in quiescent FRRPP systems with spatioselective reaction initiation of lithography, microgel structures were synthesized in a single step, without the use of molds or additives. Hard x-rays from the bending magnet radiation of a synchrotron were used as an initiation source, instead of the more statistally-oriented chemical initiators. Such a spatially-defined reaction was shown to be self-limiting to the irradiated regions following a polymerization-induced self-assembly phenomenon. The pattern transfer aspects of this technique were, therefore, studied in the FRRP polymerization of N-isopropylacrylamide (NIPAm) and methacrylic acid (MAA), a thermoreversible and ionic hydrogel, respectively. Reaction temperature increases the contrast between the exposed and unexposed zones of the formed microgels, while the irradiation dose is directly proportional to the extent of phase separation. The response of Poly (NIPAm) microgels prepared from the technique described in this study was also characterized by small angle neutron scattering.

Molecular mechanism by which the Escherichia coli nucleoid occlusion factor, SlmA, keeps cytokinesis in check

Cell division or cytokinesis is one of the most fundamental processes in biology and is essential for the propagation of all living species. In Escherichia coli, cell division occurs by ingrowth of the membrane envelope at the cell center and is orchestrated by the FtsZ protein. FtsZ self-assembles into linear protofilaments in a GTP dependent manner to form a cytoskeletal scaffold called the Z-ring. The Z-ring provides the framework for the assembly of the division apparatus and determines the site of cytokinesis. The total amount of FtsZ molecules in a cell significantly exceeds the concentration required for Z-ring formation. Hence, Z-ring formation must be highly regulated, both temporally and spatially. In particular, the assembly of Z-rings at the cell poles and over chromosomal DNA must be prevented. These inhibitory roles are played by two key regulatory systems called the Min and nucleoid occlusion (NO) systems. In E. coli, Min proteins oscillate from pole to pole; the net result of this oscillatory process is the formation of a zone of FtsZ inhibition at the cell poles. However, the replicated nucleoid DNA near the midcell must also be protected from bisection by the Z-ring which is ensured by NO. A protein called SlmA was shown to be the effector of NO in E. coli. SlmA was identified in a screen designed to isolate mutations that were lethal in the absence of Min, hence the name SlmA (synthetic lethal with a defective Min system). Furthers SlmA was shown to bind DNA and localize to the nucleoid fraction of the cell. Additionally, light scattering experiments suggested that SlmA interacts with FtsZ-GTP and alters its polymerization properties. Here we describe studies that reveal the molecular mechanism by which SlmA mediates NO in E. coli. Specifically, we determined the crystal structure of SlmA, identified its DNA binding site specificity, and mapped its binding sites on the E. coli chromosome by chromatin immuno-precipitation experiments. We went on to determine the SlmA-FtsZ structure by small angle X-ray scattering and examined the effect of SlmA-DNA on FtsZ polymerization by electron microscopy. Our combined data show how SlmA is able to disrupt Z-ring formation through its interaction with FtsZ in a specific temporal and spatial manner and hence prevent nucleoid guillotining during cell division.

Molecular mechanism by which the nucleoid occlusion factor, SlmA, keeps cytokinesis in check.

In Escherichia coli, cytokinesis is orchestrated by FtsZ, which forms a Z-ring to drive septation. Spatial and temporal control of Z-ring formation is achieved by the Min and nucleoid occlusion (NO) systems. Unlike the well-studied Min system, less is known about the anti-DNA guillotining NO process. Here, we describe studies addressing the molecular mechanism of SlmA (synthetic lethal with a defective Min system)-mediated NO. SlmA contains a TetR-like DNA-binding fold, and chromatin immunoprecipitation analyses show that SlmA-binding sites are dispersed on the chromosome except the Ter region, which segregates immediately before septation. SlmA binds DNA and FtsZ simultaneously, and the SlmA-FtsZ structure reveals that two FtsZ molecules sandwich a SlmA dimer. In this complex, FtsZ can still bind GTP and form protofilaments, but the separated protofilaments are forced into an anti-parallel arrangement. This suggests that SlmA may alter FtsZ polymer assembly. Indeed, electron microscopy data, showing that SlmA-DNA disrupts the formation of normal FtsZ polymers and induces distinct spiral structures, supports this. Thus, the combined data reveal how SlmA derails Z-ring formation at the correct place and time to effect NO.

Refractive index changes in amorphous SiO2 (silica) by swift ion irradiation

The refractive index changes induced by swift ion-beam irradiation in silica have been measured either by spectroscopic ellipsometry or through the effective indices of the optical modes propagating through the irradiated structure. The optical response has been analyzed by considering an effective homogeneous medium to simulate the nanostructured irradiated system consisting of cylindrical tracks, associated to the ion impacts, embedded into a virgin material. The role of both, irradiation fluence and stopping power, has been investigated. Above a certain electronic stopping power threshold (∼2.5 keV/nm), every ion impact creates an axial region around the trajectory with a fixed refractive index (around n = 1.475) corresponding to a certain structural phase that is independent of stopping power. The results have been compared with previous data measured by means of infrared spectroscopy and small-angle X-ray scattering; possible mechanisms and theoretical models are discussed.

DNA packing in stable lipid complexes designed for gene transfer imitates DNA compaction in bacteriophage

The structure of complexes made from DNA and suitable lipids (lipoplex, Lx) was examined by cryo-electron microscopy (cryoEM). We observed a distinct concentric ring-like pattern with striated shells when using plasmid DNA. These spherical multilamellar particles have a mean diameter of 254 nm with repetitive spacing of 7.5 nm with striation of 5.3 nm width. Small angle x-ray scattering revealed repetitive ordering of 6.9 nm, suggesting a lamellar structure containing at least 12 layers. This concentric and lamellar structure with different packing regimes also was observed by cryoEM when using linear double-stranded DNA, single-stranded DNA, and oligodeoxynucleotides. DNA chains could be visualized in DNA/lipid complexes. Such specific supramolecular organization is the result of thermodynamic forces, which cause compaction to occur through concentric winding of DNA in a liquid crystalline phase. CryoEM examination of T4 phage DNA packed either in T4 capsides or in lipidic particles showed similar patterns. Small angle x-ray scattering suggested an hexagonal phase in Lx-T4 DNA. Our results indicate that both lamellar and hexagonal phases may coexist in the same Lx preparation or particle and that transition between both phases may depend on equilibrium influenced by type and length of the DNA used.

The thermodynamic origin of the stability of a thermophilic ribozyme

Understanding the mechanism of thermodynamic stability of an RNA structure has significant implications for the function and design of RNA. We investigated the equilibrium folding of a thermophilic ribozyme and its mesophilic homologue by using hydroxyl radical protection, small-angle x-ray scattering, and circular dichroism. Both RNAs require Mg2+ to fold to their native structures that are very similar. The stability is measured as a function of Mg2+ and urea concentrations at different temperatures. The enhanced stability of the thermophilic ribozyme primarily is derived from a tremendous increase in the amount of structure formed in the ultimate folding transition. This increase in structure formation and cooperativity arises because the penultimate and the ultimate folding transitions in the mesophilic ribozyme become linked into a single transition in the folding of the thermophilic ribozyme. Therefore, the starting point, or reference state, for the transition to the native, functional thermophilic ribozyme is significantly less structured. The shift in the reference state, and the resulting increase in folding cooperativity, is likely due to the stabilization of selected native interactions that only form in the ultimate transition. This mechanism of using a less structured intermediate and increased cooperativity to achieve higher functional stability for tertiary RNAs is fundamentally different from that commonly proposed to explain the increased stability of thermophilic proteins.

Estudo e desenvolvimento de lipossomas com potencial para aplicação em base cosmética

A acne vulgar é uma das doenças cutâneas mais comuns, apresentando como um de seus fatores fisiopatológicos primários a colonização pelo microrganismo Propionibacterium acnes. Atualmente, têm-se buscado terapias alternativas para o combate ao P. acnes, destacando-se alguns ácidos graxos, como o ácido laúrico (LA). O LA é uma molécula pouco solúvel em água, sendo possível sua incorporação em lipossomas. Os lipossomas apresentam capacidade de encapsulação/ liberação de ativos e impedem a desidratação da pele, tornando-se ingredientes inovadores na área de cosméticos. Foram preparados lipossomas de dipalmitoilfosfatidilcolina (DPPC) contendo diferentes concentrações de LA, que variaram de 0 a 50% da concentração total em mol, em quatro pHs: 9,0, 7,4, 5,0 e 3,0. Nestes pHs o estado de protonação do LA muda variando de 0 a -1. Os lipossomas foram extrusados por filtros com poros de 100 nm de diâmetro visando à obtenção de vesículas unilamelares grandes (LUV). As LUV foram caracterizadas quanto a sua estabilidade em condições de prateleira, temperatura de transição de fase da bicamada, encapsulamento no interior aquoso, liberação do LA, difusão das vesículas na pele e seus aspectos morfológicos foram caracterizados por espalhamento de raios-X a baixo ângulo (SAXS) e crio-microscopia eletrônica de transmissão. Estudos de estabilidade mostraram que independentemente da concentração de LA, as formulações são mais estáveis em pHs mais altos, quando LA está em sua maioria na forma de laurato. Os experimentos de DSC revelaram que em pHs 3,0 e 5,0 e concentrações maiores de LA, a interação deste ácido graxo com as bicamadas é favorecida, havendo um aumento da temperatura de transição de fase (Tm) e diminuição da cooperatividade. Análises de taxa de incorporação de sondas hidrofílicas confirmaram a presença de um compartimento aquoso interno para as vesículas de DPPC:LA. O LA conseguiu permear a pele no período avaliado e pouco LA foi liberado das vesículas em condições de temperatura ambiente. A morfologia das LUV se mostrou bem diferente da esperada e se observaram vesículas com mais de uma bicamada e outros formatos que não o esférico. Estes resultados podem auxiliar na otimização das condições para uma formulação que poderá ser usada no tratamento da acne, aumentando a eficácia do LA no sítio alvo.

Nanostructures, Semicrytalline Morphology, and Nanoscale Confinement Effect on the Crystallization Kinetics in Self-Organized Block Copolymer/Thermoset Blends

This work reports the first instance of self-organized thermoset blends containing diblock copolymers with a crystallizable thermoset-immiscible block. Nanostructured thermoset blends of bisphenol A-type epoxy resin (ER) and a low-molecular-weight (M-n = 1400) amphiphilic polyethylene-block-poly(ethylene oxide) (EEO) symmetric diblock copolymer were prepared using 4,4'-methylenedianiline (MDA) as curing agent and were characterized by transmission electron microscopy (TEM), atomic force microscopy (AFM), small-angle X-ray scattering (SAXS), and differential scanning calorimetry (DSC). All the MDA-cured ER/EEO blends do not show macroscopic phase separation but exhibit microstructures. The ER selectively mixes with the epoxy-miscible PEO block in the EEO diblock copolymer whereas the crystallizable PE blocks that are immiscible with ER form separate microdomains at nanoscales in the blends. The PE crystals with size on nanoscales are formed and restricted within the individual spherical micelles in the nanostructured ER/EEO blends with EEO content up to 30 wt %. The spherical micelles are highly aggregated in the blends containing 40 and 50 wt % EEO. The PE dentritic crystallites exist in the blend containing 50 wt % EEO whereas the blends with even higher EEO content are completely volume-filled with PE spherulites. The semicrystalline microphase-separated lamellae in the symmetric EEO diblock copolymer are swollen in the blend with decreasing EEO content, followed by a structural transition to aggregated spherical micellar phase morphology and, eventually, spherical micellar phase morphology at the lowest EEO contents. Three morphological regimes are identified, corresponding precisely to the three regimes of crystallization kinetics of the PE blocks. The nanoscale confinement effect on the crystallization kinetics in nanostructured thermoset blends is revealed for the first time. This new phenomenon is explained on the basis of homogeneous nucleation controlled crystallization within nanoscale confined environments in the block copolymer/thermoset blends.

Determination of domain sizes in blends of poly(ethylene) and poly(styrene) formed in the presence of supercritical carbon dioxide

Well-mixed blends of poly(ethylene) and poly(styrene) have been synthesized using supercritical carbon dioxide as a solvent. The morphology of the blends has been conclusively characterized using differential scanning calorimetry (DSC), small-angle X-ray scattering (SAXS), Raman microprobe microscopy, and C-13 solid-state cross-polarization magic angle spinning NMR (C-13 CPMAS NMR). DSC measurements demonstrate that poly(styrene) in the blends resides solely in the amorphous regions of the poly(ethylene) matrix; however, corroborative evidence from the SAXS experiments shows that poly(styrene) resides within the interlamellar spaces. The existence of nanometer-sized domains of poly(styrene) was shown within a blend of poly(styrene) and poly(ethylene) when formed in supercritical carbon dioxide using Raman microprobe microscopy and C-13 CPMAS NMR spectroscopy coupled with a spin diffusion model. This contrasts with blends formed at ambient pressure in the absence of solvent, in which domains of poly(styrene) in the micrometer size range are formed. This apparent improved miscibility of the two components was attributed to better penetration of the monomer prior to polymerization and increased swelling of the polymer substrate by the supercritical carbon dioxide solvent.

The cyclic antimicrobial peptide RTD-1 induces stabilized lipid-peptide domains more efficiently than its open-chain analogue

The effects of a mammalian cyclic antimicrobial peptide, rhesus theta defensin 1 (RTD-1) and its open chain analogue (oRTD-1), on the phase behaviour and structure of model membrane systems (dipalmitoyl phosphatidylcholine, DPPC and dipalmitoyl phosphatidylglycerol, DPPG) were studied. The increased selectivity of RTD-1 for anionic DPPG over zwitterionic DPPC was shown by differential scanning calorimetry. RTD-1, at a molar peptide-lipid ratio of 1:100, induced considerable changes in the phase behaviour of DPPG, but not of DPPC. The main transition temperature, T-m, Was unchanged, but additional phase transitions appeared above T-m. oRTD-1 induced similar effects. However, the effects were not observable below a peptide:lipid molar ratio of 1:50, which correlates with the weaker biological activity of oRTD-1. Small-and wide-angle X-ray scattering revealed for DPPG the appearance of additional structural features induced by RTP-1 above T-m, which were interpreted as correlated lamellar structures, with increased order of the fatty acyl side chains of the lipid. It is proposed that after initial electrostatic interaction of the cationic rim of the peptide with the anionic DPPG headgroups, leading to stabilized lipid-peptide clusters, the hydrophobic face of the peptide assists in its interaction with the fatty acyl side chains eventually leading to membrane disruption. (C) 2004 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Effect of pore packing defects in 2-D ordered mesoporous carbons on ionic transport

Ordered mesoporous materials show great importance in energy, environmental, and chemical engineering. The diffusion of guest species in mesoporous networks plays an important role in these applications, especially for energy storage, such as supercapacitors based on ordered mesoporous carbons ( OMCs). The ion diffusion behavior in two different 2-D hexagonal OMCs was investigated by using cyclic voltametry and electrochemical impedance spectroscopy. In addition, transmission electron microscopy, small-angle X-ray diffraction, and nitrogen cryosorption methods were used to study the pore structure variations of these two OMCs. It was found that, for the OMC with defective pore channels ( termed as pore packing defects), the gravimetric capacitance was greatly decayed when the voltage scan rate was increased. The experimental results suggest that, for the ion diffusion in 2-D hexagonal OMCs with similar mesopore size distribution, the pore packing defect is a dominant dynamic factor.

Phase behavior, crystallization, and nanostructures in thermoset blends of epoxy resin and amphiphilic star-shaped block copolymers

This article reports thermoset blends of bisphenol A-type epoxy resin (ER) and two amphiphilic four-arm star-shaped diblock copolymers based on hydrophilic poly(ethylene oxide) (PEO) and hydrophobic poly(propylene oxide) (PPO). 4,4'-Methylenedianiline (MDA) was used as a curing agent. The first star-shaped diblock copolymer with 70 wt% ethylene oxide (EO), denoted as (PPO-PEO)(4), consists of four PPO-PEO diblock arms with PPO blocks attached on an ethylenediamine core; the second one with 40 wt% EO, denoted as (PEO-PPO)(4), contains four PEO-PPO diblock arms with PEO blocks attached on an ethylenediamine core. The phase behavior, crystallization, and nanoscale structures were investigated by differential scanning calorimetry, transmission electron microscopy, and small-angle X-ray scattering. It was found that the MDA-cured ER/(PPO-PEO)(4) blends are not macroscopically phase-separated over the entire blend composition range. There exist, however, two microphases in the ER/(PPO-PEO)(4) blends. The PPO blocks form a separated microphase, whereas the ER and the PEO blocks, which are miscible, form another microphase. The ER/(PPO-PEO)(4) blends show composition-dependent nanostructures on the order of 10-30 nm. The 80/20 ER/(PPO-PEO)(4) blend displays spherical PPO micelles uniformly dispersed in a continuous ER-rich matrix. The 60/40 ER/(PPO-PEO)(4) blend displays a combined morphology of worm-like micelles and spherical micelles with characteristic of a bicontinuous microphase structure. Macroscopic phase separation took place in the MDA-cured ER/(PEO-PPO)(4) blends. The MDA-cured ER/(PEO-PPO)(4) blends with (PEO-PPO)(4) content up to 50 wt% exhibit phase-separated structures on the order of 0.5-1 mu m. This can be considered to be due to the different EO content and block sequence of the (PEO-PPO)(4) copolymer. (c) 2006 Wiley Periodicals, Inc.

Quantifying hydrogel response using laser light scattering

The pH and counter-ion response of a microphase separated poly(methyl methacrylate)-block-poly(2-(diethylamino)ethyl methacrylate)-block-poly(methyl methacrylate) hydrogel has been investigated using laser light scattering on an imprinted micron scale topography. A quartz diffraction grating was used to create a micron-sized periodic structure on the surface of a thin film of the polymer and the resulting diffraction pattern used to calculate the swelling ratio of the polymer film in situ. A potentiometric titration and a sequence of counter ion species, taken from the Hofmeister series, have been used to compare the results obtained using this novel technique against small angle X-ray scattering (nanoscopic) and gravimetric studies of bulk gel pieces (macroscopic). For the first time, the technique has been proven to be an inexpensive and effective analytical tool for measuring hydrogel response on the microscopic scale.

Reentrant condensation of proteins in solution induced by multivalent counterions

Negatively charged globular proteins in solution undergo a condensation upon adding trivalent counterions between two critical concentrations C* and C**, C*Small-angle x-ray scattering indicates a short-ranged attraction between counterion-bound proteins near C* and C**. Monte Carlo simulations (under these strong electrostatic coupling conditions) support an effective inversion of charge on surface side chains through binding of the multivalent counterions.