A Visible-Light-Powered Polymerization Method for the Immobilization of Enantioselective Organocatalysts into Microreactors

A versatile one-step photopolymerization approach for the immobilization of enantioselective organocatalysts is presented. Chiral organocatalyst-containing monoliths based on polystyrene divinylbenzene copolymer were generated inside channels of microfluidic chips. Exemplary performance tests were performed for the monolithic Hayashi–Jørgensen catalyst in continuous flow, which showed good results for the Michael addition of aldehydes to nitroalkenes in terms of stereoselectivity and catalyst stability with minimal consumption of reagents and solvents.     

Photochemical generation of highly destabilized vinyl cations: the effects of alpha- and beta-trifluoromethyl versus alpha- and beta-methyl substituents

The photochemical reactions in methanol of the vinylic halides 1-4, halostyrenes with a methyl or a trifluoromethyl substituent at the alpha- or beta-position, have been investigated quantitatively. Next to E/Z isomerization, the reactions are formation of vinyl radicals, leading to reductive dehalogenation products, and formation of vinyl cations, leading to elimination, nucleophilic substitution, and rearrangement products. The vinyl cations are parts of tight ion pairs with halide as the counterion. The elimination products are the result of beta-proton loss from the primarily generated alpha-CH(3) and alpha-CF(3) vinyl cations, or from the alpha-CH(3) vinyl cation formed from the beta-CH(3) vinyl cation via a 1,2-phenyl shift. The beta-CF(3) vinyl cation reacts with methanol yielding nucleophilic substitution products, no migration of the phenyl ring producing the alpha-CF(3) vinyl cation occurs. The alpha-CF(3) vinyl cation, which is the most destabilized vinyl cation generated thus far, gives a 1,2-fluorine shift in competition with proton loss. The experimentally derived order of stabilization of the vinyl cations photogenerated in this study, alpha-CF(3) < beta-CF(3) < beta-CH(3) < alpha-CH(3), is corroborated by quantum chemical calculations, provided the effect of solvent is taken into account.     

Label-free fluorescence detection in capillary and microchip electrophoresis

Herein, we summarize the current status of native fluorescence detection in microchannel electrophoresis, with a strong focus on chip-based systems. Fluorescence detection is a powerful technique with unsurpassed sensitivity down to the single-molecule level. Accordingly fluorescence detection is attractive in combination with miniaturised separation techniques. A drawback is, however, the need to derivatize most analytes prior to analysis. This can often be circumvented by utilising excitation light in the UV spectral range in order to excite intrinsic fluorescence. As sensitive absorbance detection is challenging in chip-based systems, deep-UV fluorescence detection is currently one of the most general optical detection techniques in microchip electrophoresis, which is especially attractive for the detection of unlabelled proteins. This review gives an overview of research on native fluorescence detection in capillary (CE) and microchip electrophoresis (MCE) between 1998 and 2008. It discusses material aspects of native fluorescence detection and the instrumentation used, with particular focus on the detector design. Newer developments, featured techniques, and their prospects in the future are also included. In the last section, applications in bioanalysis, drug determination, and environmental analysis are reviewed with regard to limits of detection.     

Multistep liquid-phase lithography for fast prototyping of microfluidic free-flow-electrophoresis chips

We present a fast and versatile method to produce functional micro free-flow electrophoresis chips. Microfluidic structures were generated between two glass slides applying multistep liquid-phase lithography, omitting troublesome bonding steps or cost-intensive master structures. Utilizing a novel spacer-less approach with the photodefinable polymer polyethyleneglycol dimethacrylate (PEG-DA), microfluidic devices with hydrophilic channels of only 25 μm in height were generated. The microfluidic chips feature ion-permeable segregation walls between the electrode channels and the separation bed and hydrophilic surfaces. The performance of the chip is demonstrated by free-flow electrophoretic separation of fluorescent xanthene dyes and fluorescently labeled amino acids.     

Rapid quantitative determination of ephedra alkaloids in tablet formulations and human urine by microchip electrophoresis

Microchip electrophoresis with fluorescence detection has been applied for fast separation and determination of ephedra alkaloids in pharmaceutical formulations and body fluids. A custom epifluorescence microscope setup was employed and the compounds were separated within 40?s, allowing the detection of less than 200?ng/L for both analytes. Quantitation of the two stimulants was performed via a derivatization step using FITC without any extraction or preconcentration steps. The effects of different microchip types and excitation light sources were investigated and the method was successfully applied for the analysis of these compounds in tablet formulations, yielding recovery rates from 100.2 to 101.1% and relative standard deviations from 1.5 to 3.4%. Analysis of ephedrines was also carried out with human urine samples at detection limits of 500-1000?ng/L and relative standard deviations from 2.2 to 3.3% using argon ion LIF detection.     

Expression of ionotropic glutamate receptors in the retina of the rdta transgenic mouse

Background

One mechanism that directs the action of the second messengers, cAMP and diacylglycerol, is the compartmentalization of protein kinase A (PKA) and protein kinase C (PKC). A-kinase anchoring proteins (AKAPs) can recruit both enzymes to specific subcellular locations via interactions with the various isoforms of each family of kinases. We found previously that a new class of AKAPs, dual-specific AKAPs, denoted D-AKAP1 and D-AKAP2, bind to RIα in addition to the RII subunits.

Results

Immunohistochemistry and confocal microscopy were used here to determine that D-AKAP1 colocalizes with RIα at the postsynaptic membrane of the vertebrate neuromuscular junction (NMJ) and the adjacent muscle, but not in the presynaptic region. The labeling pattern for RIα and D-AKAP1 overlapped with mitochondrial staining in the muscle fibers, consistent with our previous work showing D-AKAP1 association with mitochondria in cultured cells. The immunoreactivity of D-AKAP2 was distinct from that of D-AKAP1. We also report here that even though the PKA type II subunits (RIIα and RIIβ) are localized at the NMJ, their patterns are distinctive and differ from the other R and D-AKAP patterns examined. PKCβ appeared to colocalize with the AKAP, gravin, at the postsynaptic membrane.

Conclusions

The kinases and AKAPs investigated have distinct patterns of colocalization, which suggest a complex arrangement of signaling micro-environments. Because the labeling patterns for RIα and D-AKAP 1 are similar in the muscle fibers and at the postsynaptic membrane, it may be that this AKAP anchors RIα in these regions. Likewise, gravin may be an anchor of PKCβ at the NMJ.