Catechol determination in compost bioremediation using a laccase sensor and artificial neural networks

An electrochemical biosensor based on the immobilization of laccase on magnetic core-shell (Fe₃O₄–SiO₂) nanoparticles was combined with artificial neural networks (ANNs) for the determination of catechol concentration in compost bioremediation of municipal solid waste. The immobilization matrix provided a good microenvironment for retaining laccase bioactivity, and the combination with ANNs offered a good chemometric tool for data analysis in respect to the dynamic, nonlinear, and uncertain characteristics of the complex composting system. Catechol concentrations in compost samples were determined by using both the laccase sensor and HPLC for calibration. The detection range varied from 7.5 × 10^–7 to 4.4 × 10^–4 M, and the amperometric response current reached 95% of the steady-state current within about 70 s. The performance of the ANN model was compared with the linear regression model in respect to simulation accuracy, adaptability to uncertainty, etc. All the results showed that the combination of amperometric enzyme sensor and artificial neural networks was a rapid, sensitive, and robust method in the quantitative study of the composting system. Figure Structure of the magnetic carbon paste electrode used in the electrochemical biosensor     

Elucidation of n-butyl benzyl phthalate biodegradation using high-performance liquid chromatography and gas chromatography–mass spectrometry

n-Butyl benzyl phthalate (BBP) is an endocrine-disrupting chemical. A bacterium species capable of using BBP as the sole source of carbon and energy was isolated from mangrove sediment. Effects of BBP concentration, pH, temperature, and salinity on BBP biodegradation were studied. The optimum pH, temperature, and salinity for the BBP biodegradation were 7.0, 37°C, and 15 g L⁻¹, respectively. BBP was completely degraded within 6 days under optimum conditions, and the biodegradation of BBP could be fitted to a first-order kinetic model. The major metabolites of BBP biodegradation were identified as mono-butyl phthalate, mono-benzyl phthalate, phthalic acid, and benzoic acid by using high-performance liquid chromatography and gas chromatography–mass spectrometry. A preliminary metabolic pathway was proposed for the biodegradation of BBP.      

Multivariate classification and modeling in surface water pollution estimation

The present study deals with the application of self-organizing maps (SOM) and multiway principal-components analysis to classify, model, and interpret a large monitoring data set for surface water quality. The chemometric methods applied made it possible to reveal specific quality patterns of the chemical and biological parameters used to monitor the water quality (relation between water temperature, turbidity, hardness, colibacteria), seasonal impacts during the long period of observation and the relative independence on the spatial location of the sampling sites (water supply sources for the City of Trieste). Figure The schematic procedure for surface water pollution estimation supported by neural network-based classification and multivariate factor analysis     

In vitro assessment of pediococci- and lactobacilli-induced cholesterol-lowering effect using digitally enhanced high-performance thin-layer chromatography and confocal microscopy

The cholesterol-lowering properties of 12 lactic acid bacteria (LAB) in the absence or presence of 0.3% bile salts were assessed and compared quantitatively and qualitatively in vitro. A new, more sensitive and cost-effective high-performance thin-layer chromatography method combined with digital image evaluation of derivatised chromatographic plates was developed and validated to quantify cholesterol in LAB culture media. The performance of the method was compared with that of the o-phthalaldehyde method. For qualitative assessment, assimilated fluorescently tagged cholesterol was visualised by confocal microscopy. All LAB strains exhibited a cholesterol-lowering effect of various degrees (19–59% in the absence and 14–69% in the presence of bile salts). Lactobacillus plantarum LAB12 and Pentosaceus pentosaceus LAB6 were the two best strains of lactobacilli and pediococci. They lowered cholesterol levels by 59% and 54%, respectively, in the absence and by 69% and 58%, respectively, in the presence of bile salts. Confocal microscopy showed that cholesterol was localised at the outermost cell membranes of LAB12 and LAB6. The present findings warrant in-depth in vivo study.

(A) 3D plots based on scan at 525 nm of (B) derivatized HPTLC plate of separated cholesterol and (C) confocal microscopic image showing the localisation of NBD-cholesterol assimilated by LAB

     

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     

A sequential injection electronic tongue employing the transient response from potentiometric sensors for anion multidetermination

Intelligent and automatic systems based on arrays of non-specific-response chemical sensors were recently developed in our laboratory. For multidetermination applications, the normal choice is an array of potentiometric sensors to generate the signal, and an artificial neural network (ANN) correctly trained to obtain the calibration model. As a great amount of information is required for the proper modelling, we proposed its automated generation by using the sequential injection analysis (SIA) technique. First signals used were steady-state: the equilibrium signal after a step-change in concentration. We have now adapted our procedures to record the transient response corresponding to a sample step. The novelty in this approach is therefore the use of the dynamic components of the signal in order to better discriminate or differentiate a sample. In the developed electronic tongue systems, detection is carried out by using a sensor array formed by five potentiometric sensors based on PVC membranes. For the developed application we employed two different chloride-selective sensors, two nitrate-selective sensors and one generic response sensor. As the amount of raw data (fivefold recordings corresponding to the five sensors) is excessive for an ANN, some feature extraction step prior to the modelling was needed. In order to attain substantial data reduction and noise filtering, the data obtained were fitted with orthonormal Legendre polynomials. In this case, a third-degree Legendre polynomial was shown to be sufficient to fit the data. The coefficients of these polynomials were the input information fed into the ANN used to model the concentrations of the determined species (Cl⁻, and ). Best results were obtained by using a backpropagation neural network trained with the Bayesian regularisation algorithm; the net had a single hidden layer containing three neurons with the tansig transfer function. The results obtained from the time-dependent response were compared with those obtained from steady-state conditions, showing the former superior performance. Finally, the method was applied for determining anions in synthetic samples and real water samples, where a satisfactory comparison was also achieved.      

A synchrotron FTIR microspectroscopy investigation of fungal hyphae grown under optimal and stressed conditions

Synchrotron FTIR can provide high spatial resolution (<10 μm pixel size) in situ biochemical analyses of intact biotissues, an area of increasing importance in the post-genomic era, as gene functions and gene networks are coming under direct scrutiny. With this technique, we can simultaneously assess multiple aspects of cell biochemistry and cytoplasmic composition. In this paper, we report the first results of our synchrotron FTIR examination of hyphae of three important fungal model systems, each with sequenced genomes and a wealth of research: Aspergillus, Neurospora, and Rhizopus. We have analyzed the FTIR maps of Aspergillus nidulans cells containing the hypA1 allele, a well-characterized single-gene temperature-sensitive morphogenetic mutation. The hypA1 cells resemble wildtype at 28 °C but have growth defects at 42 °C. We have also investigated Neurospora and Rhizopus cultures grown in media with optimal or elevated pH. Significant differences between the spectra of the three fungi are likely related to differences in composition and structure. In addition, high spatial resolution synchrotron FTIR spectroscopy provides an outstanding method for monitoring subtle subcellular changes that accompany environmental stress. Figure Photomicrographs and FTIR spectra acquired along Rhizopus hyphae grown at pH 6.5 (a) and pH 8.5 (b). Scale bars 50 μm     

Detection of bacteria by surface-enhanced Raman spectroscopy

The detection and identification of dilute bacterial samples by surface-enhanced Raman spectroscopy has been explored by mixing aqueous suspensions of bacteria with a suspension of nanocolloidal silver particles. An estimate of the detection limit of E. coli was obtained by varying the concentration of bacteria. By correcting the Raman spectra for the broad librational OH band of water, reproducible spectra were obtained for E. coli concentrations as low as approximately 10³ cfu/mL. To aid in the assignment of Raman bands, spectra for E. coli in D₂O are also reported. Figure Light scattering apparatus used to detect bacteria     

Raman spectroscopic study of bacterial endospores

Endospores and endospore-forming bacteria were studied by Raman spectroscopy. Raman spectra were recorded from Bacillus licheniformis LMG 7634 at different steps during growth and spore formation, and from spore suspensions obtained from diverse Bacillus and Paenibacillus strains cultured in different conditions (growth media, temperature, peroxide treatment). Raman bands of calcium dipicolinate and amino acids such as phenylalanine and tyrosine are more intense in the spectra of sporulating bacteria compared with those of bacteria from earlier phases of growth. Raman spectroscopy can thus be used to detect sporulation of cells by a characteristic band at 1,018 cm^–1 from calcium dipicolinate. The increase in amino acids could possibly be explained by the formation of small acid-soluble proteins that saturate the endospore DNA. Large variations in Raman spectra of endospore suspensions of different strains or different culturing conditions were observed. Next to calcium dipicolinate, tyrosine and phenylalanine, band differences at 527 and 638 cm^–1 were observed in the spectra of some of the B. sporothermodurans spore suspensions. These bands were assigned to the incorporation of cysteine residues in spore coat proteins. In conclusion, Raman spectroscopy is a fast technique to provide useful information about several spore components. Figure A difference spectrum between Raman spectra of B. licheniformis LMG 7634 cultured for 6 days and 1 day, together with the reference Raman spectrum of calcium dipicolinate     

Simultaneous quantification of cilostazol and its primary metabolite 3,4-dehydrocilostazol in human plasma by rapid liquid chromatography/tandem mass spectrometry

A simple, rapid, sensitive and selective liquid chromatography/electrospray tandem mass spectrometry method was developed and validated for the simultaneous quantification of cilostazol and its primary metabolite 3,4-dehydrocilostazol in human plasma using mosapride as an internal standard. The method involves a simple one-step liquid-liquid extraction with a diethyl ether and dichloromethane mixture (7:3). The analytes were chromatographed using an isocratic mobile phase on a reversed-phase C₁₈ column and analyzed by mass spectrometry in the multiple reaction monitoring mode using the respective [M+H]⁺ ions, m/z 370/288 for cilostazol, m/z 368/286 for 3,4-dehydrocilostazol and m/z 422/198 for the internal standard. The assay exhibited a linear dynamic range of 5–2,000 ng/mL for cilostazol and 5–400 ng/mL for 3,4-dehydrocilostazol in human plasma. The lower limit of quantitation was 5 ng/mL for both cilostazol and its metabolite. Acceptable precision and accuracy were obtained for concentrations over the standard curve ranges. A run time of 2.5 min for each sample made it possible to analyze more than 400 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetics, bioavailability or bioequivalence studies.      

Headspace mass spectrometry methodology: application to oil spill identification in soils

In the present work we report the results obtained with a methodology based on direct coupling of a headspace generator to a mass spectrometer for the identification of different types of petroleum crudes in polluted soils. With no prior treatment, the samples are subjected to the headspace generation process and the volatiles generated are introduced directly into the mass spectrometer, thereby obtaining a fingerprint of volatiles in the sample analysed. The mass spectrum corresponding to the mass/charge ratios (m/z) contains the information related to the composition of the headspace and is used as the analytical signal for the characterization of the samples. The signals obtained for the different samples were treated by chemometric techniques to obtain the desired information. The main advantage of the proposed methodology is that no prior chromatographic separation and no sample manipulation are required. The method is rapid, simple and, in view of the results, highly promising for the implementation of a new approach for oil spill identification in soils. Figure PCA score plots illustrate clear discrimination of types of crude oil in polluted soil samples (e.g. results are shown for vertisol)     

Pattern recognition analysis for <Superscript>1</Superscript>H NMR spectra of plasma from hemodialysis patients

¹H NMR spectroscopic and pattern recognition-based methods (NMR-PR) were applied to the metabolic profiling studies on hemodialysis (HD). Plasma samples were collected from 37 patients before and after HD and measured by 600 MHz NMR spectroscopy. Each spectrum was data-processed and subjected to principal component analysis for pattern recognition. Spectral patterns of plasma between pre- and post-dialyses were clearly discriminated, together with significant fluctuations in the levels of creatinine, trimethylamine-N-oxide, glucose, lactate, and acetate, which were quantitated. We have first observed the significant elevation of lactate levels in post-dialysis plasma. The present study has demonstrated the high feasibility of NMR-PR method for monitoring the dialysis condition and comprehensive profiling of the change of low-molecular-weight metabolites in HD. Figure PCA for 1H NMR spectra of plasma from HD patients     

Amplification of noble gas ion lines in the afterglow of a pulsed hollow cathode discharge and possible benefit for analytical glow discharge mass spectrometry

A high-current pulsed hollow cathode discharge was used to study the role of atomic and ionic metastables involved in ionization plasma processes. We observed the enhancement of the spectral emission lines of noble gas ions in the afterglow. A study of the processes that involve atomic and ionic metastables is of great interest since it should lead to a better understanding of and enhanced control over the ionization mechanisms crucial to analytical glow discharge mass spectrometry (GDMS) analysis. Figure Time profile of Ti, Ti⁺, and Ne⁺ spectral lines     

Intensified biochip system using chemiluminescence for the detection of Bacillus globigii spores

This paper reports the first intensified biochip system for chemiluminescence detection and the feasibility of using this system for the analysis of biological warfare agents is demonstrated. An enzyme-linked immunosorbent assay targeting Bacillus globigii spores, a surrogate species for Bacillus anthracis, using a chemiluminescent alkaline phosphatase substrate is combined with a compact intensified biochip detection system. The enzymatic amplification was found to be an attractive method for detection of low spore concentrations when combined with the intensified biochip device. This system was capable of detecting approximately 1 × 10⁵ Bacillus globigii spores. Moreover, the chemiluminescence method, combined with the self-contained biochip design, allows for a simple, compact system that does not require laser excitation and is readily adaptable to field use. Figure Schematic diagram of the miniature biochip detection system     

Further research on the photo-SPME of triclosan

In this study the photoinduced degradation of triclosan has been investigated by photo-solid-phase microextraction (photo-SPME). In photo-SPME, photodegradation is carried out on the SPME fibre containing the target compound. Triclosan was extracted from aqueous solutions by use of polydimethylsiloxane SPME fibres and these were subsequently exposed to UV irradiation (power 8 W, wavelength 254 nm) for different times (from 2 to 60 min). The photodegradation kinetics of triclosan were investigated, the photoproducts generated were tentatively identified, and the photochemical behaviour of these products was studied by use of this on-fibre approach followed by gas chromatographic–mass spectrometric analysis. Eight photoproducts were tentatively identified, including chlorinated phenols, chlorohydroxydiphenyl ethers, 2,8-dichlorodibenzo-p-dioxin, and a possible dichlorodibenzodioxin isomer or dichlorohydroxydibenzofuran. The main photodegradation mechanisms were postulated and photodegradation pathways proposed. The effect of pH on triclosan degradation and on triclosan-to-dioxin conversion was also investigated. Triclosan degradation occurred, and generation of 2,8-dichlorodibenzo-p-dioxin was confirmed, throughout the pH range studied (from 3 to 9).      

Use of the diffusive gradients in thin films technique to evaluate (bio)available trace metal concentrations in river water

Concentrations of Cd, Cu, Cr, Pb, Ni and Zn were monitored in the Svitava River (the Czech Republic) during April and September 2005. Total concentrations and total dissolved concentrations were obtained through regular water sampling, and the diffusive gradients in thin films technique (DGT) were used to gain information on the kinetically labile metal concentrations. Each measured concentration was compared with the corresponding average (bio)available concentration calculated from the mass of metal accumulated by the moss species Fontinalis antipyretica. The concentrations of Cd, Pb, Cr and Zn measured using DGT corresponded well with those obtained after the deployment of Fontinalis antipyretica moss bags in the Svitava River, but the concentrations of Cu and Ni did not. The calculated (bio)available Cu concentration correlated well with the total dissolved concentration of Cu, whereas no correlation was found to exist between the concentrations of Ni. Scheme of the Svitava River monitoring station, including the DGT sampling units and Fontinalis antipyretica moss bags Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Nuclear magnetic resonance of mass-limited samples using small RF coils

Figure Schematic diagram of a typical arrangement used for hyphenating chemical microseparations (e.g. capillary HPLC, CE, or CEC) with microcoil NMR detection     

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.      

Laser ablation inductively coupled plasma mass spectrometry for direct analysis of the spatial distribution of trace elements in metallurgical-grade silicon

The spatial distribution and concentration of impurities in metallurgical-grade silicon (MG-Si) samples (97–99% w/w Si) were investigated by use of laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). The spatial resolution (120 μm) and low limits of detection (mg kg⁻¹) for quality assurance of such materials were studied in detail. The volume-dependent precision and accuracy of non-matrix-matched calibration for quantification of minor elements, using NIST SRM 610 (silicate standard), indicates that LA-ICP-MS is well suited to rapid process control of such materials. Quantitative results from LA-ICP-MS were compared with previously reported literature data obtained by use of ICP-OES and rf-GD-OES. In particular, the distribution of element impurities and their relationship to their different segregation coefficients in silicon is demonstrated. Dedicated to Professor Klaus G. Heumann