Single-molecule fluorescence detection in microfluidic channels—the Holy Grail in μTAS?

Both single-molecule detection (SMD) methods and miniaturization technologies have developed very rapidly over the last ten years. By merging these two techniques, it may be possible to achieve the optimal requirements for the analysis and manipulation of samples on a single molecule scale. While miniaturized structures and channels provide the interface required to handle small particles and molecules, SMD permits the discovery, localization, counting and identification of compounds. Widespread applications, across various bioscience/analytical science fields, such as DNA-analysis, cytometry and drug screening, are envisaged. In this review, the unique benefits of single fluorescent molecule detection in microfluidic channels are presented. Recent and possible future applications are discussed.Dedicated to the memory of Wilhelm Fresenius     

1-Alkyl-4-dialkylaminopyridinium Halides as Phase-Transfer Catalysts in Dichlorocarbene Reactions

 A series of 1-alkyl-4-dialkylaminopyridinium halides derived from 4-dimethylamino- and 4-morpholinopyridines were synthesized and tested as phase-transfer catalysts in three typical reactions of dichlorocarbene: dehydration of benzamide, N-formylation of diphenylamine, and dichlorocyclopropanation of styrene. The catalytic performance of the above compounds was found comparable or higher than that of conventional quaternary ammonium catalysts. The influence of catalyst structure on the reactivity was evaluated.     

A fluorimetric assay for cortisol

A simple, rapid and sensitive fluorimetric assay for the quantitative determination of cortisol is reported. The assay is based on the formation of a fluorescent dye when cortisol is incubated with a mixture of sulfuric acid and acetic acid. The fluorescence spectrum recorded for the resulting dye shows a maximum extinction at 475 nm and a maximum emission at 525 nm. The solvent 2-methyl-4-pentanone was used for extraction and was found to act as a fluorescence amplifier. A limit of detection of 2.7 μM was achieved, making it possible to forego solvent evaporation. The assay suffers minor interference from 11-deoxycortisol which exhibits low fluorescence at λ _ex: 460 nm; λ _em: 505 nm. Typical standard deviations were below 4%. We validated the assay using a biotransformation with recombinant Schizosaccharomyces pombe which regioselectively hydroxylates 11-deoxycortisol to cortisol. The method described herein is suitable for preliminary screening of microorganisms capable of steroid hydroxylation.     

The preparation of 7-substituted norbornadiene-2,3-dicarboxylic anhydrides and an experimental and theoretical study of their reactivity

Four new substituted methano-bridged or heteroatom-bridged norbornadienomaleic anhydrides have been prepared and converted to sesquinorbornadiene anhydrides by reaction with cyclic 1,3-dienes. The versatility of parity reversal, in conjunction with N-substituent steric effects, has been used to produce all three possible stereoisomers of the N,O-sesquinorbornadiene anhydrides in separate, stereoselective cycloadditions. The anhydrides have been synthesized by cyclization of their diacids (in situ production) or by flash vacuum pyrolysis of their furan adducts (yielding crystalline products); further fragmentation occurs at these or higher temperatures to produce five-membered carbocyclic or heterocyclic anhydrides. Activation energies have been evaluated for the fragmentation and cycloaddition processes using DFT calculations (B3LYP/6-31G∗) and these calculations correctly predict, which reaction can be intercepted at the norbornadienomaleic anhydride stage and preferred stereochemistry of cycloadducts.     

New intra-intermolecular criss-cross cycloaddition of unsymmetrical allenylazines with alkynes leading to three fused five-membered heterocycles

This report describes a new type of intra-intermolecular criss-cross cycloadditions. Thermal reactions of unsymmetrical allenylazines in the presence of alkynes led to three fused five-membered heterocycles in some cases. In the case of unsymmetrical substituted alkynes, a regioselectivity was observed. The molecular structures of all products are discussed. One X-ray crystal structure is also reported.     

Hydrogenation and double bond isomerization of hexenes over EUROPT-1

Hexenes give large amounts of hexane and isomeric hexenes over EUROPT-1. A constant hydrocarbon coverage can be assumed in the steady-state period of the reaction; the direction of reactions of these surface species depends on the hydrogen excess and also on the structure of starting hydrocarbon.

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EUROPT-1 , . , . , .

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Based on the diploma work of Mr. M. Räth and Ms. B. Brose.     

Optimization of a MALDI TOF-TOF mass spectrometer for intact protein analysis

A MALDI TOF-TOF instrument was optimized and evaluated for intact protein analysis by tandem mass spectrometry. Ion source voltages and delay times were adjusted to affect an up to a 10-fold improvement in fragment ion yield compared to data obtained using default settings employed in peptide analysis. For large peptides (3-4.5 kDa), up to 90% of all possible b- and y-fragment ions were observed, which provides sufficient information for de novo sequencing and unambiguous protein identification. Product ion signals associated with preferential cleavages C-terminal to aspartic acid and glutamic acid residues and N-terminal to proline residues became dominant with increased protein molecular weight. Matrix effects were also evaluated and, among the eight matrices examined, alpha-cyano-4-hydroxycinnamic acid (CHCA) was found to produce the best intact protein tandem mass spectra for proteins up to 12 kDa. Optimized performance yielded detection limits of 50-125 fmol for proteins of 4 and 12 kDa, respectively. This improved performance has yielded an instrument with potential to be a useful tool in proteomic investigations via analysis of intact proteins.     

Speciation analysis of tellurium by solid-phase extraction in the presence of ammonium pyrrolidine dithiocarbamate and inductively coupled plasma mass spectrometry

Under acidic conditions tellurium(IV) formed a complex with ammonium pyrrolidine dithiocarbamate (APDC). The tellurium(IV) complex was completely retained on a non-polar Isolute silica-based octadecyl (C(18)) sorbent-containing solid-phase extraction (SPE) cartridge, while the uncomplexed Te(VI) passed through the cartridge and remained as a free species in the solution. Only partial Te(IV) was retained on the SPE cartridge for samples without addition of APDC. On the basis of different retention behaviours of the complexed Te(IV) and uncomplexed Te(VI), a simple and highly sensitive method is proposed for the determination of total tellurium and Te(VI) by SPE separation and inductively coupled plasma mass spectrometry (ICP-MS) detection. The Te(IV) concentration was calculated as the difference between total tellurium and Te(VI) concentrations. The detection limit (3 sigma) is 3 ng L(-1) tellurium. Factors affecting the separation and detection of tellurium species were investigated. Coexisting ions did not show significant interferences with the Te(IV)-APDC complex retention and the subsequent ICP-MS detection of Te. The method has been successfully applied to the tellurium speciation analysis in waters with spiked recoveries for Te(IV) and Te(VI) of 86.0-108% and 87.1-97.4%, respectively.     

New polythiophenes bearing electron-acceptor phthalocyanine chromophores

Two new donor-acceptor copolymers comprising a polythiophene backbone, and bearing phthalocyanine chromophores on the side chains have been prepared. Preliminary photophysical characterization of these materials by FTIR photoinduced absorption indicates that electron transfer from the polythiophene to the phthalocyanine units takes place.     

Complete-active-space self-consistent-field/Amber parameterization of the Lys296–retinal–Glu113 rhodopsin chromophore-counterion system

A special hybrid quantum mechanics/molecular mechanics forcefield is defined, parameterized and validated for studying the photoisomerization path of the retinal chromophore in the rhodopsin protein. It couples a multireference ab initio Hamiltonian (CASSCF and second-order multireference many-body perturbation theory using a CASSCF reference) to describe the chromophore while the rest of the protein is approximated with the Amber forcefield. The frontier has been carefully parameterized in order to reproduce full quantum mechanics torsional energy profiles, for both the ground state and the first excited state. It is also shown that replacing the chromophore counterion with point charges is a valid approximation. This result is interpreted in terms of a cancellation effect for which a possible explanation is given.