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Analytical and Bioanalytical Chemistry (v.376, #3)
Analysis of single mammalian cells with capillary electrophoresis
by Lori A. Woods; Andrew G. Ewing (pp. 281-283).
Genotyping of single nucleotide polymorphisms by liquid chromatography–electrospray ionization mass spectrometry
by Herbert Oberacher; Christian G. Huber (pp. 292-294).
Solid-state NMR spectroscopy and its application in analytical chemistry
by D. Reichert (pp. 308-310).
Process analytical chemistry—future trends in industry
by Stephan Kueppers; Markus Haider (pp. 313-315).
Biosensor for the specific detection of a single viable B. anthracis spore by Harriet A. Hartley; Antje J. Baeumner (pp. 319-327).
A simple membrane strip-based biosensor for the detection of viable B. anthracis spores was developed and combined with a spore germination procedure as well as a nucleic acid amplification reaction to identify as little as one viable B. anthracis spore in less than 12 h. The biosensor is based on identification of a unique mRNA sequence from the anthrax toxin activator (atxA) gene encoded on the toxin plasmid, pXO1. Preliminary work relied on plasmid vectors in both E. coli and B. thuringiensis expressing the atxA gene. Once the principle was firmly established, the vaccine strain of B. anthracis was used. After inducing germination and outgrowth of spores of B. anthracis (Sterne strain), RNA was extracted from lysed cells, amplified using nucleic acid sequence-based amplification (NASBA), and rapidly identified by the biosensor. While the biosensor assay requires only 15-min assay time, the overall process takes12 h for the detection of as little as one viable B. anthracis spore, and is shortened significantly, if larger amounts of spores are present. The biosensor is based on an oligonucleotide sandwich-hybridization assay format. It uses a membrane flow-through system with an immobilized oligonucleotide probe that hybridizes with the target sequence. Signal amplification is provided when the target sequence hybridizes to a second oligonucleotide probe that has been coupled to dye-encapsulating liposomes. The dye in the liposomes then provides a signal that can be read visually or quantified with a hand-held reflectometer. The biosensor can detect as little as 1.5 fmol of target mRNA. Specificity analysis revealed no crossreactivity with closely related species such as B. cereus, B. megaterium, B. subtilis, B. thuringiensis etc.
Keywords: Biosensor B. anthracis RNA Detection Spore Viable Biosecurity
Ionophore-based ion-selective potentiometric and optical sensors by R. Daniel Johnson; Leonidas G. Bachas (pp. 328-341).
This paper describes ion-selective electrodes (ISEs) and optodes with a focus on the fundamental mechanisms, response characteristics, and recognition elements (ionophores) described to date for these sensors. The topics covered review the most frequently encountered ideas about ionophore-based sensors, including the theoretical and experimental parameters that influence response, typical ionophore structures, useful membrane compositions, and applications. Since these sensors are applicable in a number of fields, such as clinical, environmental, and process monitoring, as well as more novel approaches such as microfluidic-based systems and micro/nanoprobes, this article provides descriptions that are oriented toward a more general audience. Furthermore, a large portion of this review describes the ionophore component itself in hopes of inspiring ideas in readers of how novel molecular architectures with new ionic targets or improved selectivity can be developed.
Keywords: Ion-selective electrodes Optodes Ionophores Sensors Potentiometry
Optical sensor systems for bioprocess monitoring by Roland Ulber; Jan-Gerd Frerichs; Sascha Beutel (pp. 342-348).
Bioreactors are closed systems in which microorganisms can be cultivated under defined, controllable conditions that can be optimized with regard to viability, reproducibility, and product-oriented productivity. To drive the biochemical reaction network of the biological system through the desired reaction optimally, the complex interactions of the overall system must be understood and controlled. Optical sensors which encompass all analytical methods based on interactions of light with matter are efficient tools to obtain this information. Optical sensors generally offer the advantages of noninvasive, nondestructive, continuous, and simultaneous multianalyte monitoring. However, at this time, no general optical detection system has been developed. Since modern bioprocesses are extremely complex and differ from process to process (e.g., fungal antibiotic production versus mammalian cell cultivation), appropriate analytical systems must be set up from different basic modules, designed to meet the special demands of each particular process. In this minireview, some new applications in bioprocess monitoring of the following optical sensing principles will be discussed: UV spectroscopy, IR spectroscopy, Raman spectroscopy, fluorescence spectroscopy, pulsed terahertz spectroscopy (PTS), optical biosensors, in situ microscope, surface plasmon resonance (SPR), and reflectometric interference spectroscopy (RIF).
Keywords: UV spectroscopy, IR spectroscopy Raman spectroscopy Fluorescence spectroscopy Pulsed terahertz spectroscopy (PTS) Optical biosensors, Affinity sensors (SPR and RIF)
Covalent immobilization of proteases and nucleases to poly(methylmethacrylate) by Wendy D. Dominick; Beniam T. Berhane; Justin S. Mecomber; Patrick A. Limbach (pp. 349-354).
The increased popularity of microfabricated devices formed from plastics such as poly(methylmethacrylate) (PMMA) will benefit from approaches adding (bio)chemical functionality to such surfaces. Here, various proteases and nucleases have been covalently immobilized to PMMA surfaces and shown to retain their enzymatic activity as monitored by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Immobilized enzymes yield structural information at a level equivalent to or exceeding that obtained from conventional homogeneous solution-based approaches. Such an approach could be used to expand the functionality of polymer-based microfabricated devices for biological mass spectrometry.
Keywords: Proteins Nucleic acids Oligonucleotides Peptides Proteomics Microchips
Determination of residual pesticides in olive oil by GC–MS and HPLC–MS after extraction by size-exclusion chromatography by Sami Barrek; Olivier Paisse; Marie-Florence Grenier-Loustalot (pp. 355-359).
This work describes the development of a method for analyzing pesticide residues in olive oil by GC–MS and HPLC–MS. Pesticides were separated from the oily matrix by size-exclusion chromatography. After extraction, 20 pesticides were separated and analyzed by GC–MS and 11 others HPLC–MS in electrospray mode. The development of this method enabled us to identify and quantify the pesticides of interest.
Keywords: Olive oil Pesticides SEC HPLC–MS GC–MS
In-house validation of an improved sample extraction and clean-up method for GC determination of isomers of nervonic acid in meat products by Marie-Elisabeth Agazzi; Andrea Bau; Robertino Barcarolo; Ernst Luecker; Josefa Barrero-Moreno; Elke Anklam (pp. 360-365).
An improved extraction and clean-up method for determination of brain-specific fatty acids, in particular lignoceric acid (C24:0) and the cis / trans isomers of nervonic acid (15 c-t C24:1), in meat products has been developed. The method is based on isolation of the polar lipids of interest from the bulk lipids by solid-phase extraction. The fatty acids, derivatised to their fatty acid methyl esters, are quantified by GC in a DB5 column. Fresh meat samples were extracted by using a mixture of n -butanol:hexane (1:9) as solvent. The extract was loaded in a silica gel cartridge column previously equilibrated with hexane. The first fraction containing the major part of the fat was eluted with hexane while acetone and methanol allowed the elution of fatty acids bound to polar moieties such as nervonic and lignoceric acids. This second fraction containing the analyte was methylated and injected into the GC for quantification after addition octacosane (C28) as internal standard.
Keywords: Central nervous system Fatty acids Food analysis Gas chromatography Solid-phase extraction (SPE)
A new approach to phosphoserine and phosphothreonine analysis in peptides and proteins: chemical modification, enrichment via solid-phase reversible binding, and analysis by mass spectrometry by Florian Thaler; Barbara Valsasina; Rosario Baldi; Jin Xie; Albert Stewart; Antonella Isacchi; Henryk M. Kalisz; Luisa Rusconi (pp. 366-373).
β-Elimination of the phosphate group on phosphoserine and phosphothreonine residues and addition of an alkyldithiol is a useful tool for analysis of the phosphorylation states of proteins and peptides. We have explored the influence of several conditions on the efficiency of this PO4 3− elimination reaction upon addition of propanedithiol. In addition to the described influence of different bases, the solvent composition was also found to have a major effect on the yield of the reaction. In particular, an increase in the percentage of DMSO enhances the conversion rate, whereas a higher amount of protic polar solvents, such as water or isopropanol, induces the opposite effect. We have also developed a protocol for enrichment of the modified peptides, which is based on solid-phase covalent capture/release with a dithiopyridino-resin. The procedure for β-elimination and isolation of phosphorylated peptides by solid-phase capture/release was developed with commercially available α-casein. Enriched peptide fragments were characterized by MALDI–TOF mass spectrometric analysis before and after alkylation with iodoacetamide, which allowed rapid confirmation of the purposely introduced thiol moiety. Sensitivity studies, carried out in order to determine the detection limit, demonstrated that samples could be detected even in the low picomolar range by mass spectrometry. The developed solid-phase enrichment procedure based on reversible covalent binding of the modified peptides is more effective and significantly simpler than methods based on the interaction between biotin and avidin, which require additional steps such as tagging the modified peptides and work-up of the samples prior to the affinity capture step.
Keywords: Protein phosphorylation Mass spectrometry Protein chemistry Beta-elimination of phosphate Dithiopyridino-resin MALDI
Analysis by partial reflection spectrometry of protonated tetraphenylporphyrin adsorbed at a liquid–liquid interface by Yoshio Moriya; Takeshi Hasegawa; Kotaro Hayashi; Machiko Maruyama; Shinichi Nakata; Nobuaki Ogawa (pp. 374-378).
Visible reflection spectra of diprotonated meso-tetraphenylporphyrin adsorbates spontaneously formed at a dodecane–aqueous sulfuric acid interface have been measured using a home-made device comprising a prism-cell and variable-angle optics. The tilt angle of the pyrrole ring plane was estimated to be 47° from the interface normal by use of an experimentally evaluated molecular density (1.20×10−10 mol cm−2) of the diprotonated molecule in a monolayer form at the liquid–liquid interface. Positive and negative bands have been observed in the p-polarized partial internal reflection (p-PIR) spectra, whose band locations correspond to those in p-polarized external reflection (p-ER) spectra. Nevertheless, the bands in the p-PIR exhibited reversed sign to those of p-ER spectra. These suggest that the surface selection rule of the p-PIR spectrometry has a reversal rule of p-ER and p-PIR can also be used for the analysis of molecular orientation.
Keywords: Liquid–liquid interface Partial reflection Adsorbate Orientation Protonated porphyrin
Capillary electrophoretic separation of dsDNA under nonuniform electric fields by Yang-Wei Lin; Chih-Ching Huang; Huan-Tsung Chang (pp. 379-383).
Improved sensitivity for the analysis of DNA by capillary electrophoresis has been achieved, based on simultaneous increases in optical path length and injection volume. To increase the optical path length, bubble cells with diameters ranging from 150 to 450 μm have been fabricated and tested. In terms of resolution and sensitivity, a bubble cell of 300 μm diameter is appropriate when using 75-μm capillaries. To allow greater injection volumes, we performed on-line concentration of DNA in the presence of electroosmotic flow (EOF) using 2.0% poly(ethylene oxide) (PEO). With a 300-μm bubble cell, a 170-fold improvement in the sensitivity for the 89-bp fragment has been accomplished when injecting about 0.33 μL DNA. In the presence of the bubble cell, the resolution for the large fragments improves while that for the small ones (<124 base pair) decreases. The effect of bubble cells was further investigated by conducting DNA separation in the absence of EOF, showing that improvements in resolution are mainly due to increased migration differences when DNA migrated at low electric field strengths in the bubble region. We have suggested that such an effect is more profound using shorter capillaries, leading to complete separation of φX 174 RF DNA-Hae III digest in 2 min.
Keywords: Bubble cell DNA separation Stacking Capillary electrophoresis Laser-induced fluorescence
Uncertainty evaluation of mass values determined by electronic balances in analytical chemistry: a new method to correct for air buoyancy by S. Wunderli; G. Fortunato; A. Reichmuth; Ph. Richard (pp. 384-391).
A new method to correct for the largest systematic influence in mass determination—air buoyancy—is outlined. A full description of the most relevant influence parameters is given and the combined measurement uncertainty is evaluated according to the ISO–GUM approach [1]. A new correction method for air buoyancy using an artefact is presented. This method has the advantage that only a mass artefact is used to correct for air buoyancy. The classical approach demands the determination of the air density and therefore suitable equipment to measure at least the air temperature, the air pressure and the relative air humidity within the demanded uncertainties (i.e. three independent measurement tasks have to be performed simultaneously). The calculated uncertainty is lower for the classical method. However a field laboratory may not always be in possession of fully traceable measurement systems for these room climatic parameters.A comparison of three approaches applied to the calculation of the combined uncertainty of mass values is presented. Namely the classical determination of air buoyancy, the artefact method, and the neglecting of this systematic effect as proposed in the new EURACHEM/CITAC guide [2]. The artefact method is suitable for high-precision measurement in analytical chemistry and especially for the production of certified reference materials, reference values and analytical chemical reference materials. The method could also be used either for volume determination of solids or for air density measurement by an independent method.
Keywords: Mass Uncertainty Weighing Air buoyancy Balance Metrology Reference materials
A coumarin derivative covalently immobilized on sensing membrane as a fluorescent carrier for nitrofurazone by Chen-Xu Jiao; Cheng-Gang Niu; Li-Xin Chen; Guo-Li Shen; Ru-Qin Yu (pp. 392-398).
A coumarin derivative 4-methyl-8-methylacrylamide-2H, 5H-pyrano [3, 2-C] benzpyran 2, 5-dione (MMPBD) has been synthesized as a fluorescent carrier for preparing an optical chemical sensor. The carrier is immobilized on a quartz glass plate surface treated with a silanizing agent to prevent the leakage of the dye. This MMPBD sensor can be utilized for a nitrofurazone (NF) assay based on fluorescence quenching. The sensor shows good repeatability, a long lifetime and a fast response of less then 50 s. NF can be determined in the range between 1.0×10-6–1.0×10-3 mol L-1 with a detection limit of 8.0×10-7 mol L-1 at pH 7.0.
Keywords: Optical sensor Coumarin derivative Fluorescence quenching Covalent immobilization Nitrofurazone
Silica accumulation in Triticum aestivum L. and Dactylis glomerata L. by Dagmar Dietrich; Simone Hinke; Wolfgang Baumann; Rüdiger Fehlhaber; Ernst Bäucker; Gebhardt Rühle; Otto Wienhaus; Günter Marx (pp. 399-404).
The silica accumulation in orchard grass (Dactylis glomerata L.) and wheat (Triticum aestivum L.) has been studied in plant samples grown under defined conditions in a pot trial. The plant habit and the quantity of biomineralised silica within the selected Gramineae depend to a remarkable extent on the soil. The plants grew with different soil pH values and silica additives. By means of atomic absorption spectrometry, the silicon enrichment in different plant parts was determined. In dried plant parts the silica bodies can be well distinguished by variable pressure scanning electron microscopy in the back scattering mode. They are located in silica cells below the epidermis and in epidermal appendices (bristles, prickle hairs). The silica bodies showed a defined morphology, structure and composition which was elucidated by the combined performance of scanning electron microscopy in combination with X-ray spectroscopy, solid-state nuclear resonance, X-ray diffraction and Raman spectroscopy. The composition was near to stoichiometric SiO2 (41 weight% silicon, 56 weight % oxygen), and the SiO4/2 tetrahedra were arranged preferentially in three-dimensional networks; a smaller proportion was in chains and layers. The silica bodies with an overall amorphous structure contained crystalline precipitates, which could be indexed by α-quartz.
Keywords: Biomineralisation Silica bodies Solid-state analytical methods
Complexation as the most important factor in the fate and transport of heavy metals in the Dnieper water bodies by Peter N. Linnik (pp. 405-412).
The results of long-term investigations of the concentrations of dissolved forms of some heavy metals (Mn, Cu, Zn, Pb, Cr, Cd) and their species in the water of the Dnieper reservoirs and the Dnieper-Bug estuary are considered. Chemiluminescent methods, anodic stripping voltammetry, membrane filtration, ion-exchange, and gel-permeation chromatography were used for study of the speciation of the metals. It has been found that binding of heavy metals into complexes with dissolved organic matter (DOM) is the dominant factor of their stabilization in solution. The molecular weight distribution of organic metal complexes and their chemical nature, as well as the potential complexing ability of DOM were investigated. Humic substances, particularly fulvic acids, play a major role in the complexation. These ligands bind from 45 to 80% of metals in the form of organic complexes. Metal complex compounds of relatively low molecular weight (<5 kDa) predominated in the organic complexes.
Keywords: Heavy metals Speciation Dissolved organic matter Humic substances Complexation Complexing ability Reservoirs
Unbiased spectrophotometric method for estimating phenol or o-cresol in unknown water samples by L. A. Tortajada-Genaro; P. Campíns-Falcó; F. Bosch-Reig (pp. 413-421).
The generalised H-point standard addition method (GHPSAM) was applied to quantify phenol and o-cresol in waters and compared with other analytical approaches. The method is based on spectroscopic and kinetic measurement of the formation of derivatives with p-aminophenol and KIO4 in presence of NaOH. First, pseudo-first order kinetic behaviour of the reaction products was demonstrated. The unbiased formation rate constants of phenol and o-cresol derivatives were calculated. The analytical figures of merit were determined using the GHPSAM as calibration method. Detection limits achieved were 0.2 μg L−1 of phenol and 0.2 μg L−1 of o-cresol using a preconcentration factor of 10. Styrene–divinyl benzene cartridges were used in the preconcentration step. Repeatability values were 3.7% for phenol and 2.0% for o-cresol; reproducibility values were 6.9% and 3.5%, respectively. Accurate and precise results were obtained when the method was applied to real samples of natural water.
Keywords: Solid-phase extraction UV–visible spectrophotometry Derivatization with p-aminophenol and KIO4 GHPSAM Phenol and o-cresol