Single-cell protein analysis of a single mouse embryo by two-dimensional capillary electrophoresis

The characterization of protein expression from a single-cell mouse embryo using two-dimensional capillary electrophoresis (2D-CE) is described. These zygotes were obtained from Hsf1 gene knockout mice. Single zygotes were lysed off-column and proteins were fluorescently labeled using the fluorogenic dye 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ). After injection, analytes were separated first according to molecular weight using capillary sieving electrophoresis (CSE) and then by micellar electrokinetic capillary chromatography (MEKC) to obtain protein expression fingerprints. Analytes were detected in a sheath flow cuvette using laser-induced fluorescence. In a 1-h 2D-CE separation, over 100 components were resolved with a spot capacity of 380.     

Background electrolytes in 50% methanol/water for the determination of acidity constants of basic drugs by capillary zone electrophoresis

The acidic dissociation constants of several hydrophobic drugs, amiodarone and a series of antidepressants that show a secondary or tertiary amino group, were determined in a 50% methanol/water mixture by capillary zone electrophoresis. The electrophoretic behavior of buffers prepared from sodium acetate, tris(hydroxymethyl) aminomethane hydrochloride, sodium hydrogenphosphate, ammonium chloride, ethanolamine, butilammonium chloride, and sodium borate in the hydroalcoholic solution was tested. Thus, all of them follow the Ohm's law until about 25 kV and, therefore, they can be used without significant Joule heat dissipation at 20 kV. For the studied drugs, buffers prepared with phosphate or borate give effective mobility measurements lower than those from other buffers. The wide pKa range of the studied drugs provides a wide pH range where the protonated forms of the amino compounds coexist with hydrogenphosphate ions and where the neutral amines coexist with boric acid. The decrease of the experimental effective mobilities in these instances can be explained through the interactions between coexisting species. Therefore, phosphate and borate buffers should be avoided to determine the mobility of amines with aqueous pKa higher than 8, at least in solutions with high methanol content. Independent measurements of acidic dissociation constants of drugs validate this statement.     

Characterization of Ta and TaN diffusion barriers beneath Cu layers using picosecond ultrasonics

In computer chips, aluminum is being replaced with copper in order to produce smaller, faster and more efficient electronic devices. The usage of copper allows higher current densities and thus higher packaging densities than aluminum. However, copper leads to new challenges and problems. It has different mechanical properties and a tendency to migrate into the surrounding dielectric and/or semiconducting layers. These diffusion processes can be prevented by so called diffusion barriers. A diffusion barrier is a very thin layer consisting of tantalum and tantalum nitride or titanium and titanium nitride, deposited between the copper and the substrate. A pump-probe setup is used to determine the mechanical properties of the barrier layers and of the copper layer. This short-pulse-laser-acoustic method is contact-free and non-destructive. Mechanical waves are excited and detected thermoelastically using laser pulses of 70 fs duration. Thin film measurements of buried diffusion layers are provided and compared with scanning electron microscopy (SEM), transmission electron microscopy (TEM), and Rutherford Backscattering Spectroscopy measurements (RBS). Results of a thermo-elasto-mechanical simulation are presented and a short overview of the simulation procedure is given. Current limits of the presented method are discussed and future directions of the on-going research project are presented.     

Synthesis and properties of an anthraquinone-based redox switch for molecular electronics

[reaction: see text] The synthesis of a molecular wire bearing an anthraquinone core and thioacetyl end groups for gold electrode binding is described. A model anthraquinone system, substituted with tert-butylthio groups, can be reversibly switched electrochemically from cross conjugated (low conductance "off") to linear conjugated (high conductance "on") via two-electron reduction/oxidation reactions. This feature holds promise for the anthraquinone-based wires to be used as redox-controlled switches in molecular electronic devices.     

Intrinsic Photophysics of Light-harvesting Charge-tagged Chlorophyll a and b Pigments

Chlorophylls a and b (Chla/b) are responsible for light-harvesting by photosynthetic proteins in plants. They display broad absorption in the visible region with multiple bands, due to the asymmetry of the macrocycle and strong vibronic coupling. Their photophysics relies on the microenvironment, with regard to transition energies as well as quenching of triplet states. Here, we firmly establish the splitting of the Q and Soret bands into x- and y- polarized bands for the isolated molecules in vacuo, and resolve vibronic features. Storage-ring experiments reveal that dissociation of photoexcited charge-tagged complexes occurs over several milliseconds, but with two different time constants. A fast decay is ascribed to dissociation after internal conversion and a slow decay to the population of a triplet state that acts as a bottleneck. Support for the latter is provided by pump-probe experiments, where a second laser pulse probes the long-lived triplet state.     

Synthesis of a Counteranion‐Stabilized Bis(silylium) Ion

The preparation of a molecule with two alkyl‐tethered silylium‐ion sites from the corresponding bis(hydrosilanes) by two‐fold hydride abstraction is reported. The length of the conformationally flexible alkyl bridge is crucial as otherwise the hydride abstraction stops at the stage of a cyclic bissilylated hydronium ion. With an ethylene tether, the open form of the hydronium‐ion intermediate is energetically accessible and engages in another hydride abstraction. The resulting bis(silylium) ion has been NMR spectroscopically and structurally characterized. Related systems based on rigid naphthalen‐n,m‐diyl platforms can only be converted into the dications when the positively charged silylium‐ion units are remote from each other (1,8 versus 1,5 and 2,6).     

Development of a liquid chromatography/atmospheric pressure photo‐ionization high‐resolution mass spectrometry analytical method for the simultaneous determination of polybrominated diphenyl ethers and their metabolites: application to BDE‐47 metabolism in human hepatocytes

Polybrominated diphenyl ethers (PBDEs) are flame retardants widely used in electronic and domestic goods. These persistent pollutants are present in the environment and in humans, and their toxicological properties are of growing concern. PBDEs can be metabolised into compounds suspected to be responsible for their toxicity. These metabolites have been characterised quite well in rodents and fish, but available information in humans remains scarce. For their identification, an efficient method for the simultaneous analysis of PBDEs, hydroxylated PBDEs (OH‐PBDEs), and other PBDE metabolites in a single run was needed and has been developed in this work. Atmospheric pressure ionisation modes were compared, and Atmospheric Pressure Photo‐Ionization (APPI) was selected. After careful setting of APPI parameters such as dopant and operating temperature, the optimised method was based on APPI ionization coupled to High‐Resolution Mass Spectrometry operating in the full scan mode at a resolution of 60 000. This provided excellent sensitivity and specificity, allowing the discrimination of signals which could not be resolved on a triple quadrupole used as a reference. The full‐scan high‐resolution acquisition mode allowed monitoring of both parent PBDEs and their metabolites, including hydroxylated PBDEs, with detection limits ranging from 0.1 pg to 4.5 pg injected on‐column based on the investigated standard compounds. The method was applied to the study of BDE‐47 metabolism after incubation with human primary cultures of hepatocytes, and proved to be efficient not only for monitoring the parent compound and expected hydroxylated metabolites, but also for the identification of other non‐targeted metabolites. In addition to hydroxy‐BDE‐47, several conjugated metabolites could be located, and the formation of a dihydrodiol derivative was evidenced for the first time in the case of PBDEs in this work. Copyright © 2012 John Wiley & Sons, Ltd.