Optical studies of pulsed-laser fragmentation of biliary calculi

The fragmentation of gallstones and kidney stones using pulsed visible laser radiation has recently been demonstrated; however, the fragmentation mechanism is not well understood. The temporal and spectral characteristics of the bright flash of light accompanying fragmentation of gallstones were studied using 0.8 and 360-s-long, 690-nm-wavelength, dye-laser pulses. Time-resolved visible emission spectra show a broad continuum upon which line spectra are superimposed. The continuum emission is due to free-free and free-bound electron transitions indicative of a plasma and the line spectra are due to neutral and ionized calcium. Initiation of this plasma is fluence rather than intensity dependent. A model is proposed in which laser energy is coupled to the plasma, which then impulsively expands, generating intense acoustic transients which fracture the stone.     

Optical biosensor for pharmaceuticals, antibiotics, hormones, endocrine disrupting chemicals and pesticides in water: Assay optimization process for estrone as example

Certain contaminants at trace concentrations in surface waters can have dramatic effects on the hormonal system of organisms in the aquatic environment. Therefore, immunoanalytical methods at a very low limit of detection (LOD) and a low limit of quantification (LOQ) are becoming more and more important for environmental analysis and especially for monitoring drinking water quality. Environmental monitoring of antibiotics, hormones, endocrine disrupting chemicals, and pesticides in real water samples (e.g. surface, ground or drinking water) with difficult matrices places high demands on chemical analysis. Biosensors have suitable characteristics such as efficiency in allowing very fast, sensitive, and cost-effective detection. Here we describe an assay optimization process with a fully automated immunoassay for estrone which resulted in a LOD below 0.20 ng L⁻¹ and a LOQ below 1.40 ng L⁻¹. In contrast to common analytical methods such as GC-MS or HPLC-MS, the biosensor used requires no sample pre-treatment and pre-concentration. The very low validation parameters for estrone are the result of the continuous optimization of the immunoassay. The basis of our sensitive assay is the antibody with a high affinity constant towards estrone. During the optimization process, we reduced the amount of antibody per sample and improved the chip surface modification. Finally, this proceeding led to a calibration routine with an amount of antibody of only 3.0 ng per sample (sample volume: 1.0 mL). The reduction of the amount of antibody per sample results in better validation parameters (LOD, LOQ, and IC₅₀), but this reduction leads to the current device-related limitation of the River Analyser (RIANA).For some endocrine disrupting compounds, no effect levels (NOELs) in the lower nanogram per liter range are reported. This defines the challenge, which analytical methods have to compete with and our RIANA instrument with its improved sensitivity for the detection of a single hormone in the lower nanogram per liter range is a powerful tool in aquatic analytics in addition to the common analytical methods.     

GnRH binding RNA and DNA Spiegelmers: a novel approach toward GnRH antagonism

Mirror-image oligonucleotide ligands (Spiegelmers) that bind to the pharmacologically relevant target gonadotropin-releasing hormone I (GnRH) with high affinity and high specificity have been identified using the Spiegelmer technology. GnRH is a decapeptide that plays an important role in mammalian reproduction and sexual maturation and is associated with several benign and malignant diseases. First, aptamers that bind to D-GnRH with dissociation constants of 50-100 nM were isolated out of RNA and DNA libraries. The respective enantiomers of the DNA and RNA aptamers were synthesized, and their binding to L-GnRH was shown. These Spiegelmers bind to L-GnRH with similar affinity to that of the corresponding aptamers that bind to D-GnRH. We further demonstrated dose-dependent inhibition of GnRH-induced Ca(2+) release in Chinese hamster ovary cells that were stably transfected with the human GnRH receptor.     

Substituent-dependent reactivity in the photodimerization of N-substituted dibenz[b,f]azepines

The photoprocesses of a series of N-substituted dibenz[b,f]azepines (iminostilbenes) were studied by absorption and emission spectroscopy, by laser flash photolysis, and by preparative irradiation with NMR analysis. In solutions, 2pi+2pi photodimers of N-cyano and N-acyl dibenzazepines are formed via the triplet state upon acetone- or benzophenone-sensitized energy transfer. T-T absorption spectra were measured and absorption coefficients were determined. The triplet energy transfer is equally efficient for N-alkyl dibenzazepines, which do not dimerize. Excited states of npi* character in the latter cases are discussed to rationalize the different reactivities. In spite of negligible intersystem crossing of 21 dibenzazepine derivatives, photodimers of N-acyl and N-cyano dibenzazepines are formed upon direct excitation in concentrated solutions (0.01-0.1 mol dm(-3)) as well as in the solid state. A selective anti-configuration of the photodimers was found throughout.     

Thiol- and disulfide-modified oligonucleotide monolayer structures on polycrystalline and single-crystal Au(111) surfaces

We provide a comprehensive study of single- (ss) and double-strand (ds) oligonucleotides with either 25 or 10 bases or base pairs (bp) immobilized on polycrystalline and single-crystal Au(111) surfaces. The study is based on X-ray photoelectron spectroscopy, cyclic and differential pulse voltammetry, interfacial capacitance data, and electrochemical scanning tunnelling microscopy (in situ STM). The sequences used were the 25-bp sequence from the BRCA1 gene (25-mer), while the 10-bp oligonucleotides contained solely linear adenine and thymine sequences. The oligonucleotides were modified by the dimethoxytrityl group (DMT) via a disulfide group [DMT-S-S-ss25-mer and DMT-S-S-ds(AT)₁₀], a pure disulfide group (A₁₀-S-S-T₁₀), or a thiol group [HS-ss25-mer and HS-ds-(AT)₁₀], all via a hexamethylene linker. The overall pattern suggests strategies for controlled adsorption of DNA-based molecules and recognition of complementary strands or other molecules.     

Proton binding to proteins: pK(a) calculations with explicit and implicit solvent models

Ionizable residues play important roles in protein structure and activity, and proton binding is a valuable reporter of electrostatic interactions in these systems. We use molecular dynamics free energy simulations (MDFE) to compute proton pKa shifts, relative to a model compound in solution, for three aspartate side chains in two proteins. Simulations with explicit solvent and with an implicit, dielectric continuum solvent are reported. The implicit solvent simulations use the generalized Born (GB) model, which provides an approximate, analytical solution to Poisson's equation. With explicit solvent, the direction of the pKa shifts is correct in all three cases with one force field (AMBER) and in two out of three cases with another (CHARMM). For two aspartates, the dielectric response to ionization is found to be linear, even though the separate protein and solvent responses can be nonlinear. For thioredoxin Asp26, nonlinearity arises from the presence of two substates that correspond to the two possible orientations of the protonated carboxylate. For this side chain, which is partly buried and has a large pKa upshift, very long simulations are needed to correctly sample several slow degrees of freedom that reorganize in response to the ionization. Thus, nearby Lys57 rotates to form a salt bridge and becomes buried, while three waters intercalate along the opposite edge of Asp26. Such strong and anisotropic reorganization is very difficult to predict with Poisson-Boltzmann methods that only consider electrostatic interactions and employ a single protein structure. In contrast, MDFE with a GB dielectric continuum solvent, used for the first time for pKa calculations, can describe protein reorganization accurately and gives encouraging agreement with experiment and with the explicit solvent simulations.     

The Reactivity of Bis(para‐methoxyphenyl)telluroxide towards Triflic Acid and Diphenylphosphinic Acid. Theoretical Considerations of the Protonation and Hydration Process of Diorganotelluroxanes

The reaction of (p‐MeOC₆H₄)₂TeO with two equivalents of HO₃SCF₃ and HO₂PPh₂ provided the tetraorganoditelluroxanes (F₃CSO₃)(p‐MeOC₆H₄)₂TeOTe(p‐MeOC₆H₄)₂(O₃SCF₃) ( 1 ) and (Ph₂PO₂)(p‐MeOC₆H₄)₂TeOTe(p‐MeOC₆H₄)₂(O₂PPh₂)·2 Ph₂PO₂H ( 2 ) in good yields. Compounds 1 and 2 were characterized by solution and solid‐state ³¹P and ¹²⁵Te NMR spectroscopy, IR spectroscopy, electrospray mass spectrometry, conductivity measurements and single crystal X‐ray diffraction. In solution, compound 1 undergoes an electrolytic dissociation and reversibly reacts with traces of water to give the mononuclear cation [(p‐MeOC₆H₄)₂TeOH]⁺ and triflate anions. Theoretical aspects of the protonation and hydration of model telluroxanes R₂TeO (R = H, Me, Ph) were investigated by preliminary DFT calculations and compared to the corresponding selenoxanes R₂SeO. The tellurium dihydroxides R₂Te(OH)₂ seem to be more stable than the hydrogen‐bonded complexes R₂TeO·H₂O.     

Electron-impact and chemical ionization detection of nicotine and cotinine by gas chromatography-mass spectrometry in rat plasma and brain.

Nicotine and its metabolite, cotinine, were measured in rat plasma and brain by gas chromatography-mass spectrometry. Both agents were extracted from plasma and brain, separated on a capillary column, and quantified by single-ion monitoring. The major fragment ions of nicotine and cotinine at m/z 84 and m/z 98, respectively, were monitored by electron-impact ionization detection and the protonated molecular ions at m/z 163 and m/z 177, respectively, were monitored by chemical ionization detection. Both compounds were quantified using deuterium-labeled nicotine and cotinine, respectively, as internal standards.     

Specific determination of 20 primary aromatic amines in aqueous food simulants by liquid chromatography-electrospray ionization-tandem mass spectrometry

A multi-analyte method without any pre-treatment steps using reversed-phase liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) was developed and applied for the determination of 20 primary aromatic amines (PAA) associated with polyurethane (PUR) products or azo-colours. The method was validated in-house for water and 3% acetic acid food simulants using spiked migrates from plastic laminates. Detection limits ranged from 0.27 to 3 microg amine/L food simulants, and RSD values of within-laboratory reproducibility at the 2 microg PAA/L level ranged from 3.9 to 19%. PAA migration from plastic laminates and black nylon cooking utensils were determined with the method, and high levels of 4,4'-methylenedianiline and aniline were found in migrates from about half of the tested cooking utensils. The method fulfils present legislative demands in the EU for screening and verification of PAA migration from food contact materials.