Colorimetric detection of immunoglobulin G by use of functionalized gold nanoparticles on polyethylenimine film

A method based on use of functionalized gold nanoparticles on polyethylenimine film has been developed for colorimetric detection of immunoglobulin G (IgG). The immunogold nanoparticles were immobilized on quartz slides by recognition between antibody and antigen, with the antigen chemically adsorbed on the polyethylenimine film. By measurement of the UV–visible spectra of the immobilized immunogold, detection of h-IgG was achieved. The detection limit for h-IgG by use of this method can be as low as 0.01 μg mL⁻¹. This method is quite promising for numerous applications in immunoassay. Figure     

Spatially resolved element analysis of historical violin varnishes by use of μPIXE

External μPIXE has been used for characterisation of small samples of varnish from historical violins, and pieces of varnished wood from historical and modern stringed instruments. To obtain spatially resolved information about the distribution of elements across the varnish layers single-spot analysis, line-scans, and area-mapping were performed. Local resolution of approximately 20 μm was obtained from the 3 MeV, 1 nA proton micro-probe. Results from simultaneous multi-element determination of Na, Mg, Al, Si, P, S, K, Ca, Ti, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, As, Br, Rb, Sr, Ag, Cd, Sn, Ba, and Pb in historical varnishes are presented. Semi-quantitative evaluation of line-scans recorded on diverse historical varnishes is reported. The applied method is discussed in detail and the results obtained are critically reviewed and compared with those in the literature.      

Determination of glucosinolates in traditional Chinese herbs by high-performance liquid chromatography and electrospray ionization mass spectrometry

A reversed-phase HPLC method has been developed for determination of twelve intact glucosinolates—glucoiberin, glucocheirolin, progoitrin, sinigrin, epiprogoitrin, glucoraphenin, sinalbin, gluconapin, glucosibarin, glucotropaeolin, glucoerucin, and gluconasturtiin—in ten traditional Chinese plants. The samples were extracted with methanol and the extracts were cleaned on an activated Florisil column. A mobile phase gradient prepared from methanol and 30 mmol L⁻¹ ammonium acetate at pH 5.0 enabled baseline separation of the glucosinolates. Glucosinolate detection was confirmed by quadrupole time-of-flight tandem mass spectrometric analysis in negative-ionization mode. Detection limits ranged from 0.06 to 0.36 μg g⁻¹ when 5 g of dried plant was analyzed. Recoveries of the glucosinolates were better than 85% and precision (relative standard derivation, n = 3) ranged from 5.3 to 14.6%. Analysis of the glucosinolates provided scientific evidence enabling differentiation of three pairs of easily confused plants. Figure Glucosinolates Analysis for the Differentiation of Easily-Confusing Herbs     

Determining sulfamonomethoxine and its acetyl/hydroxyl metabolites in chicken plasma under organic solvent-free conditions

A quantitative technique is described for a sample preparation followed by high performance liquid chromatography method for the simultaneous determination of sulfamonomethoxine and its metabolites, N ⁴-acetyl SMM and 2,6-dihydroxy SMM, in chicken plasma. The average recoveries, analytical total time, and limits of quantitation were ≥80% (relative standard deviations (SD) ≤6%), <30 min sample-1 (12 samples in 2 h), and ≤0.09 μg ml⁻¹, respectively. The procedure, performed under 100% aqueous conditions, uses no organic solvents and toxic reagents at all and is, therefore, harmless to the environment and humans.      

Analysis of glycans in glycoproteins by diffusion-ordered nuclear magnetic resonance spectroscopy

Enzymatically cleaved glycans from sub-milligram quantities of erythropoietin (EPO) and ovalbumin have been analyzed, without further purification, by two-dimensional diffusion-ordered nuclear magnetic resonance spectroscopy. At NMR sample concentrations below 50 μmol L⁻¹ the major components of the oligosaccharide fractions could be distinguished by their anomeric proton chemical shift and their size-dependent diffusion coefficients. Figure ¹H NMR diffusion decay curves of anomeric protons in the EPO glycan fraction     

Determination of the titanium content of human transferrin by inductively-coupled plasma-atomic-emission spectroscopy

We report a simple method that combines dialysis, as a purification method, with the multielement capability of ICP to determine the titanium-to-transferrin mole ratio at physiological pH, under buffer conditions. The method, by means of which titanium and transferrin are determined simultaneously, enabled us to assess the binding capacities of different titanocene complexes. Figure Titanocene dichloride     

31P NMR study on the autophosphorylation of insulin receptors in the plasma membrane

A nonradioactive ³¹P nuclear magnetic resonance (NMR) spectroscopy protocol has been developed and used to investigate in vitro autophosphorylation of insulin receptors. Optimum experimental conditions have been explored, and the effects of Mn²⁺ and phosphocreatine (PCr) on the determination of the phosphorylation reaction have been assayed. The method was used to monitor the time courses of the phosphorylation reaction in solution. The results from this NMR study were in agreement with observations of insulin receptor phosphorylation made by using Western blotting.      

Surface modification of amine-functionalised graphite for preparation of cobalt hexacyanoferrate (CoHCF)-modified electrode: an amperometric sensor for determination of butylated hydroxyanisole (BHA)

A cobalt hexacyanoferrate (CoHCF)-modified graphite paraffin wax composite electrode was prepared by a new approach. An amine-functionalised graphite powder was used for the fabrication of the electrode. A functionalised graphite paraffin wax composite electrode was prepared and the surface of the electrode was modified with a thin film of CoHCF. Various parameters that influence the electrochemical behaviour of the modified electrode were studied by varying the background electrolytes, scan rates and pH. The modified electrode showed good electrocatalytic activity towards the oxidation of butylated hydroxyanisole (BHA) under optimal conditions and showed a linear response over the range from 7.9 × 10⁻⁷ to 1.9 × 10⁻⁴ M of BHA with a correlation coefficient of 0.9988. The limit of detection was 1.9 × 10⁻⁷ M. Electrocatalytic oxidation of BHA was effective at the modified electrode at a significantly reduced potential and at a broader pH range. The utility of the modified electrode as an amperometric sensor for the determination of BHA in flow systems was evaluated by carrying out hydrodynamic and chronoamperometric experiments. The modified electrode showed very good stability and a longer shelf life. The modified electrode was applied for the determination of BHA in spiked samples of chewing gum and edible sunflower oil. The advantage of this method is the ease of electrode fabrication, good stability, longer shelf life, low cost and its diverse application for BHA determination. Figure Cyclic Voltammogram of () CoHCF modified electrode, () in presence of 1.9 x 10⁻⁵ M of BHA and () bare electrode, () in the presence of 1.9 x 10⁻⁵ M of BHA in 1.0 M NaCl, pH 7.0     

A new ICP–TOFMS. Measurement and readout of mass spectra with 30 μs time resolution, applied to in-torch LA–ICP–MS

In-torch LA–ICP–MS was implemented into an in-house-built ICP–TOFMS system. The fast data acquisition capabilities of the new configuration allowed simultaneous multi-element measurement and readout of in-torch LA–ICP–MS signals with 30 μs time resolution. The measurements confirmed previously observed fine structures of in-torch generated signals and provided new insights in the dynamic processes in the plasma on a microsecond time scale. The new setup is described in detail and first figures of merit are given. Figure Time dependent multi element signal after laser ablation in the torch of an ICP-TOFMS instrument     

Fluoroquinolones in soil—risks and challenges

Fluoroquinolones (FQs) are among the most important antibacterial agents used in human and veterinary medicine. Because of the growing practice of adding manure and sewage sludge to agricultural fields these drugs end up in soils, where they can accumulate and have adverse effects on organisms. This paper presents an overview of recent developments in the determination of FQs in solid environmental matrices and describes the risks and challenges (persistence, fate, effects, and remediation) which result from their presence in soil. Figure Pathways into the environment for FQs     

Urea nitrate, an exceptionally easy-to-make improvised explosive: studies towards trace characterization

Urea nitrate is a powerful improvised explosive, frequently used by terrorists in the Israeli arena. It was also used in the first World Trade Center bombing in New York in February 1993. It is difficult to identify urea nitrate in post-explosion debris, since only a very small fraction survives the blast. Also, in the presence of water, it readily decomposes to its original components, urea and nitric acid. It is suspected that post-blast debris of urea nitrate can be confused with ammonium nitrate, the main solid product of urea nitrate thermal decomposition. In a comprehensive study towards identification of urea nitrate in post-blast traces, a spectrophotometric technique for quantitative determination of urea nitrate was developed, and conditions were found for extraction and separation of un-exploded traces of urea nitrate with minimal decomposition. Nevertheless, out of 28 samples collected from a series of three controlled firings of urea nitrate charges, only one gave the typical adduct ion by liquid chromatography/mass spectrometry analysis. We found that urea nitrate can be extracted from solid mixtures to organic solvents by using Crown ethers as “host compounds.” The adducts thus formed are solid, crystalline compounds that can be characterized by microanalysis and spectroscopic techniques. Figure Adduct formation between urea nitrate and 18-crown-6     

In vitro and in vivo metabolisms of K-48

Metabolic pathways of the oxime K-48 have been elucidated by means of in vitro and in vivo experiments. K-48 exposure to rat liver microsomal fraction resulted in the formation of a hydroxylated derivative, in addition to a small molecular fragment. The in vivo metabolism in rats was investigated after intramuscular administration of 50 μmol oxime. K-48 was present in the rat serum in unchanged form. Similarly, the analysis of rat cerebrospinal fluid indicated the sole occurrence of unchanged K-48. In contrast, unchanged K-48 was not found in the rat urine, where only the metabolite generated by epoxidation on the alkyl chain connecting the two pyridinium rings was present. The presence of unchanged K-48 in the serum and cerebrospinal fluid facilitates quantitative determination using HPLC separation and ultraviolet absorbance detection. Figure Suggested metabolic pathways of K-48     

Amperometric biosensor for the determination of creatine

An amperometric biosensor for the determination of creatine was developed. The carbon rod electrode surface was coated with sarcosine oxidase (SOX) and creatine amidinohydrolase by cross-linking under glutaraldehyde vapour. The SOX from Arthrobacter sp. 1–1 N was purified and previously used for creation of a creatine biosensor. The natural SOX electron acceptor, oxygen, was replaced by an redox mediating system, which allowed amperometric detection of an analytical signal at +400-mV potential. The response time of the biosensor was less than 1 min. The biosensor showed a linear dependence of the signal vs. creatine concentration at physiological creatine concentration levels. The optimal pH in 0.1 M tris(hydroxymethyl)aminomethane (Tris)–HCl buffer was found to be at pH 8.0. The half-life of the biosensor was 8 days in 0.1 M Tris–HCl buffer (pH 8.0) at 20 °C. Principal scheme of consecutively followed catalytic reactions used to design a biosensor for the determination of creatine     

Evaluation of CD detection in an HPLC system for analysis of DHEA and related steroids

The biological importance of dehydroepiandrosterone (DHEA) is reflected by the fact that DHEA is a crucial precursor of the biosynthesis of the steroidal sex hormones. Simultaneous separation of DHEA, dehydroepiandrosterone sulfate (DHEA-S), pregnenolone, androstenedione and testosterone has been accomplished by reversed-phase ion-pair high-performance liquid chromatography (RP-IP-HPLC) based on isocratic elution applying circular dichroism (CD) detection at 295 nm. Addition of tetrabutylammonium hydrogensulfate to the mobile phase increases the retention of DHEA-S on the C8-silica column by an apparent ion-pairing mechanism without affecting the retention of the other (non-ionic) steroids. CD spectroscopy provides highly selective detection of compounds possessing optically active absorption bands and the separation is even more selective in the higher wavelength range applied. The linearity of the steroid concentration (c, mg mL⁻¹) versus peak area was tested in the concentration range of 0.5–2 mg mL⁻¹ (injected quantities were 10–40 μg). The relative standard deviation (RSD) values for DHEA and DHEA-S indicated a good intra-assay and inter-assay precision of the method.      

Fluorometry of hydrogen peroxide using oxidative decomposition of folic acid

Hydrogen peroxide (H₂O₂) is one of the most important reactive oxygen species. In the present study, a fluorometry method for detecting H₂O₂ utilizing folic acid was evaluated. Folic acid was decomposed by H₂O₂ in the presence of Cu(II) into pterine-6-carboxylic acid, leading to strong fluorescence enhancement. In the absence of the metal ion, superoxide and H₂O₂ could not decompose folic acid. Also, H₂O₂ plus sodium hypochlorite (a source of singlet oxygen) could not induce fluorescence enhancement. These results demonstrate that H₂O₂ can be selectively detected using folic acid plus Cu(II). The limit of detection (LOD; at S/N=3) for H₂O₂ is 0.5 μM. This method based on the fluorescence enhancement of folic acid was applied in order to determine small amounts of H₂O₂ generated through the autooxidation of semicarbazide (generation rate: ∼0.01 μM min⁻¹), a carcinogenic compound.      

Chemiluminescence assay for quinones based on generation of reactive oxygen species through the redox cycle of quinone

A sensitive and selective chemiluminescence assay for the determination of quinones was developed. The method was based on generation of reactive oxygen species through the redox reaction between quinone and dithiothreitol as reductant, and then the generated reactive oxygen was detected by luminol chemiluminescence. The chemiluminescence was intense, long-lived, and proportional to quinone concentration. It is concluded that superoxide anion was involved in the proposed chemiluminescence reaction because the chemiluminescence intensity was decreased only in the presence of superoxide dismutase. Among the tested quinones, the chemiluminescence was observed from 9,10-phenanthrenequinone, 1,2-naphthoquinone, and 1,4-naphthoquinone, whereas it was not observed from 9,10-anthraquinone and 1,4-benzoquinone. The chemiluminescence property was greatly different according to the structure of quinones. The chemiluminescence was also observed for biologically important quinones such as ubiquinone. Therefore, a simple and rapid assay for ubiquinone in pharmaceutical preparation was developed based on the proposed chemiluminescence reaction. The detection limit (blank + 3SD) of ubiquinone was 0.05 μM (9 ng/assay) with an analysis time of 30 s per sample. The developed assay allowed the direct determination of ubiquinone in pharmaceutical preparation without any purification procedure. Figure Chemiluminescence generated through the redox cycle of quinone     

Application of anodized aluminum in fluorescence detection of biological species

Thin nanoporous alumina obtained by anodization of aluminum films offers promising advantages for application in fluorescence-based biological sensors including convenient preparation, increased density of binding sites, and improved collection efficiency of fluorescence. These advantages are illustrated in the detection of streptavidin using biotin covalently bound to the surface of alumina nanopores. Fluorescence intensity enhancement as high as 7 times is observed in nanopores in comparison to flat glass surface.      

Analysis of biosensors by chemically specific optical techniques. Chemiluminescence-imaging and infrared spectroscopic mapping ellipsometry

The standard methods currently used to read out microarrays are fluorescent and chemiluminesent imaging techniques. These methods require labeling of a component with a marker and, usually, only the concentration of the marker molecule is detected. A label-free imaging method that also enables quantitative spectroscopic analysis of the composition and component interaction would be of great advantage. In this article it is shown for the first time that IR mapping ellipsometry enables label-free imaging of a biochip before and after incubation with peptide solution. The measurements prove that IR ellipsometry is a sensitive tool for laterally resolved identification of the different materials and determination of the composition of a biochip. The lateral resolution required was achieved by using radiation from an infrared synchrotron beamline.      

Improved online coupling of planar chromatography with electrospray mass spectrometry: extraction of zones from glass plates

A plunger-based extraction device for HPTLC/MS coupling, which was originally designed for extraction on TLC aluminum foils, was enhanced. The threefold modifications enabled extraction of analytes from glass-backed HPTLC/TLC plates after separation. A buffering of the plunger reduced the occurrence of leakage. The involvement of a torque screwdriver for the fixation resulted in a reproducible contact pressure and avoided breaking the glass plates. The employment of this device was also extended to plates with a layer thickness of 100 μm by reducing the height of the plunger’s cutting edge. Repeatability of the extraction from glass-backed plates, linearity of the signal obtained, and detection capability were shown to be comparable to the original device, which was only usable with aluminum foils. The influence of the elution solvent on the intensity of the MS signal was demonstrated to be a compromise between high elution power of the solvent and good solubility of the analyte in the elution solvent. The extraction device was employed for plates from different lots and for plates with different stationary phases thereby proving its general applicability in planar chromatography.