Analytica Chimica Acta (v.427, #2)

Preface by D Barceló (147).

Rapid detection of Escherichia coli in water by a culture-based amperometric method by Fidel Pérez; Ingun Tryland; Marco Mascini; Liv Fiksdal (149-154).
An amperometric culture-based method was developed for rapid detection of viable Escherichia coli in water. The bacteria were recovered by filtration and incubated in a selective medium, lauryl sulphate broth (LSB) supplemented with the substrate 4-aminophenyl-β-d-galactopyranoside (4-APGal) at 44.5°C. The electrochemically active molecule 4-aminophenol (4-AP) was produced after hydrolysis of 4-APGal by the enzyme β-galactosidase. 4-AP was measured by amperometry and was detected at a due concentration of E. coli. The time necessary for reaching that concentration was inversely related to the initial E. coli concentration of the sample. Environmental samples and suspensions of E coli IT1 were assayed. 4-AP was detected after 7.3 and 2.0 h in samples containing initial concentrations of E. coli IT1 of 4.5 and 4.5×106  cfu ml−1, respectively. For environmental samples with initial E. coli concentrations of 1.0 and 2.0×103  cfu ml−1, 4-AP were detected after 10 and 6.6 h, respectively.
Keywords: Escherichia coli; Amperometry; Rapid detection; Water;

Electrochemical DNA biosensor for environmental monitoring by Giacomo Chiti; Giovanna Marrazza; Marco Mascini (155-164).
A disposable electrochemical DNA biosensor for the determination of toxic aromatic amines has been developed. The device relies on the intercalative or electrostatic collection of aromatic amines onto an immobilized dsDNA or ssDNA layer (obtained from several sources), followed by a chronopotentiometric analysis. The anodic signal of the guanine bases of DNA coated screen printed electrodes (SPEs) is strongly influenced by structural or conformational modifications of the DNA layer accrued from DNA–analyte association. So the variation in the oxidative signal of guanine is taken as an index of the molecular recognition.When the analyte is electroactive, its oxidation peak gives an additional information; in fact interesting correlation have been found between the amount of analyte trapped on the electrode and the guanine peak variation. Submicromolar detection limits have been obtained for molecules with more than two aromatic rings after a 2 min accumulation.The biosensor has also been tested on wastewater real samples; the comparison with results of classical genotoxicity tests has confirmed the applicability of the method for real samples.
Keywords: DNA biosensor; Aromatic amines; Electrochemical detection; Environmental monitoring; Electrochemical DNA biosensor; Genotoxic detection;

A solid-phase fluoroimmunoassay combined with an optical transducer chemically modified with an analyte derivative coupled to a FIA system was used [A. Brecht, A. Klotz, C. Barzen, G. Gauglitz, R. Harris, G. Quigley, J. Wilkinson, P. Sztajnbok, R. Abuknesha, J. Gascón, A. Oubiña, D. Barceló, Anal. Chim. Acta 362 (1998) 69]. Excitation and collection of fluorescence from fluorescently labelled anti-paraquat antibodies locally bound at the planar interface allows the measurement of the fluorescent signal which is indirectly related to the paraquat concentration of the sample. Matrix effects on the immunosensor response were observed, thus leading to the following detection limits, 0.01 and 0.06 μg l−1, when analyzing paraquat in MilliQ and in river water, respectively. The validation of the biosensor was carried out analyzing paraquat samples by capillary zone electrophoresis with ultraviolet detection (CZE-UV) at 214 nm. Preconcentration of the samples prior to their injection in the capillary electrophoresis were performed using the automated solid-phase extraction system (ASPEC-XL). Paraquat samples were adjusted at pH 9 and were percolated through a silica cartridge, subsequent elution was carried out using a mixture of hydrochloric acid and methanol and afterwards samples were evaporated and injected.This optical immunosensor, also called RIver ANAlyzer (RIANA) with an optical immunosensor can be applied for fast monitoring of paraquat in river water samples being the total analysis time of 15 min.
Keywords: Optical immunosensor; Paraquat residues; Capillary zone electrophoresis-ultraviolet detection;

Amperometric separation-free immunosensor for real-time environmental monitoring by Anthony J Killard; Laura Micheli; Kathleen Grennan; Milan Franek; Vladimir Kolar; Danila Moscone; Ilaria Palchetti; Malcolm R Smyth (173-180).
Immunoanalytical techniques have found widespread use due to the characteristics of specificity and wide applicability for many analytes, from large polymer antigens, to simple haptens, and even single atoms. Electrochemical sensors offer benefits of technical simplicity, speed and convenience via direct transduction to electronic equipment. Together, these two systems offer the possibility of a convenient, ubiquitous assay technique with high selectivity. However, they are still not widely used, mainly due to the complexity of the associated immunoassay methodologies. A separation-free immunoanalytical technique is described here, which has allowed for the analysis of atrazine in real time and in both quasi-equilibrium and stirred batch configurations. It illustrated that determinations as low as 0.13 μM (28 ppb) could be made using equilibrium incubation with an analytical range of 0.1–10 μM. Measurements could be made between 1 and 10 mM within several minutes using a real-time, stirred batch method. This system offers the potential for fast, simple, cost-effective biosensors for the analysis of many substances of environmental, biomedical and pharmaceutical concern.
Keywords: Immunosensor; Atrazine; Polyaniline; Real-time;

Wastewater toxicity screening of non-ionic surfactants by Toxalert® and Microtox® bioluminescence inhibition assays by Marinel.la Farré; Marı́a-Jesús Garcı́a; Lluis Tirapu; Antoni Ginebreda; Damià Barceló (181-189).
The toxic response of commonly used non-ionic surfactants with different bioluminescence inhibition assays (ToxAlert®100 and Microtox®) were established. The 50% effective concentration (EC50) values were determined for every standard substance using each assay, together with toxicity units (TU). A sigmoidal curve was fitted and the (EC50) was calculated.Chemical analysis and bioassays were used in conjunction to provide a determination of aquatic toxicity in wastewaters. This methodology was applied to real samples of influent and effluent wastewater treatment works in Spain and Portugal and also for untreated textile wastewater effluents.The protocol used in this paper involved different steps. First, the aquatic toxicity evaluation for every effluent was carried out using ToxAlert®100. For every one, a sigmoidal curve was fitted, the (EC50) and the toxicity impact index (TII50) were calculated. Second, the identification and quantification of polar organic cytotoxic substances and their contribution to the total toxicity was obtained, using sequential solid-phase extraction (SSPE) followed by liquid chromatography-mass spectrometry (LC-MS).The use of toxicity results (in terms of bioluminescence inhibition) like a screening parameter, is proposed. This procedure excludes the chromatographic analysis if the sample has less than the 20% inhibition. Overall, this procedure presented here helps to reduce the large number of samples and sub-samples that need to be processed in wastewater monitoring.
Keywords: Toxicity; Surfactants; Bioluminescence; Wastewater;

Biomonitoring of cytotoxic and genotoxic effects in mammals which allows fast and reliable quantification of these effects requires the use of an appropriate reporter in a mammalian cell system. Enhanced green fluorescent protein (EGFP) as an in vivo reporter is extremely useful for examining constitutive gene expression and the time scale of induction of a promoter in question by a variety of pollutants.For measurement of cytotoxic effects on mammalian cells, the EGFP gene under control of the CMV promoter (pEGFP-N1) was stably transfected into Chinese hamster ovary (CHO) cells and EGFP expressing clones were selected and expanded using the aminoglycoside antibiotic G418. Growth was determined by cell counts and measurements of fluorescence intensities in a microplate reader. Increase of fluorescence is delayed compared to the increase of cell numbers. After UVC-irradiation CHO-pEGFP-N1 cells show a dose-dependent prolonged lag phase and a lower maximal fluorescence.For measuring genotoxic effects, the EGFP gene has to be under control of a DNA damage inducible promoter. The resistance of EGFP to intracellular protease digestion may be useful for measuring long-lasting effects on gene expression. Kinetic measurements concerning the up and down regulation of gene expression can be performed with the destabilised d2EGFP, which has a half-life of 3 h in CHO cells. Due to decreased accumulation of the reporter protein the d2EGFP expressing cells emit lower absolute fluorescence. In consequence, the measurement of d2EGFP expression regulated by constitutive or inducible promoters should be achieved by FACS-analysis.
Keywords: Bioassays; Reporter genes; Green fluorescent protein; Mammalian cells; Transfection; Cytotoxicity; Genotoxicity;

Determination of phenolic compounds using recombinant tyrosinase from Streptomyces antibioticus by Katrin Streffer; Erik Vijgenboom; Armand W.J.W Tepper; Alexander Makower; Frieder W Scheller; Gerard W Canters; Ulla Wollenberger (201-210).
Properties of Streptomyces antibioticus tyrosinase and the implementation of the enzyme in a biosensor for the detection of phenolic compounds were investigated. The tyrosinase from S. antibioticus is a monomer and has a molecular weight of 30.6 kD. The specific activity is about 5 U/mg with catechol as substrate and 1225 U/mg with l-dopa as substrate. The activity of tyrosinase upon long-term storage is best maintained in buffer at temperatures of −80 or +4°C. Storage at −18°C, with or without glycerol, resulted in quick enzyme inactivation.For the construction of the sensor bi-enzymatic substrate recycling was exploited. Quinoprotein glucose dehydrogenase (GDH) and tyrosinase were immobilised in polyvinyl alcohol and coupled to a Clark-type oxygen electrode that allowed for monitoring of the oxygen consumption during catechol conversion. This design of the sensor facilitates the determination of phenolic compounds in the nanomolar range. The lower limit of detection for l-dopa, dopamine, and adrenalin was 5 nM.
Keywords: Biosensor; Tyrosinase; Glucose dehydrogenase; Phenolic compounds; Substituted catechols;

Development of competitive enzyme-linked immunosorbent assays (ELISAs) based on monoclonal antibodies for chloronicotinoid insecticides imidacloprid and acetamiprid by Shigeyuki Wanatabe; Shigekazu Ito; Yoshio Kamata; Naiki Omoda; Tetsuo Yamazaki; Hiroshi Munakata; Takashi Kaneko; Yojiro Yuasa (211-219).
Enzyme-linked immunosorbent assays (ELISAs) based on monoclonal antibodies (MoAbs) for chloronicotinoid insecticides imidacloprid and acetamiprid were developed. The MoAbs for either imidacloprid or acetamiprid were obtained utilizing mice with 3-[5-(2-nitroiminoimidazolidin-1-ylmethyl)-2-pyridylthio]propionic acid or 3-[5-(N-(1-cyanoiminoethyl)-N-methylaminomethyl)-2-pyridylthio] propionic acid haptens conjugated to bovine serum albumin, respectively. Both MoAbs (33C3-1-1 for imidacloprid and 45E10-1-1 for acetamiprid) characterized by an indirect competitive ELISA showed high sensitivity and low cross-reaction to related compounds. Direct competitive ELISAs (DC-ELISAs) with 33C3-1-1 for imidacloprid or with 45E10-1-1 for acetamiprid were tested on spiked vegetable and fruit samples without clean-up procedures. The mean recovery of the either assay measured by DC-ELISA ranged from 80 to 120% for imidacloprid or from 80 to 100% for acetamiprid, respectively. Both recovery tests for imidacloprid and acetamiprid showed good performance by DC-ELISA at the permitted level (0.5 ppm for imidacloprid and 1 ppm for acetamiprid) in vegetable and fruit. The result demonstrates that these assays could be easier to handle and as well as lesser expensive than usual high-performance assays such as gas chromatography (GC), high performance liquid chromatography (HPLC) and GC-mass spectrometry (GC-MS).
Keywords: Immunoassay; DC-ELISA; Monoclonal antibody; Imidacloprid; Acetamiprid;

Chemometric methods applied to the differentiation of Fourier-transform Raman spectra of ivories by Rachel H Brody; Howell G.M Edwards; A.Mark Pollard (223-232).
Fourier-transform (FT) Raman spectroscopy and chemometrics are used as a non-destructive means of discriminating dentine from six mammalian species. The spectra are divided into eleven wavenumber regions and the area in each determined. Normalisation was achieved by dividing through by the values for one of the regions. Principal components and discriminant analysis of these values allows for classification of the six different species. The dentine spectra can also be differentiated from those of bone and cementum. The discrimination is based upon the ratio of the organic and inorganic components within the samples.
Keywords: FT Raman spectroscopy; Ivory; Chemometrics; Principal component analysis; Discriminant analysis; Forensic science;

A stepwise procedure is proposed that regard computer-supported structure elucidation relying on infrared spectra as leading to hypotheses that have to be verified in the subsequent steps. As test set compounds were selected by a preliminary counter-propagation neural network prediction. As target substructure the phenolic group was investigated, however, structures were also included not containing that group due to false positive predictions of the neural network. For its verification library search procedures, including the partial cross correlation approach, with maximum common substructure analysis were applied. In the majority of instances, erroneous propositions of the neural network could be corrected.
Keywords: Infrared spectroscopy; Computer-supported structure elucidation; Spectrum–structure-correlation; Partial cross correlation approach; Maximum common substructure; Neural networks;

A spectrophotometric method has been developed for the determination of vitamin C in a variety of samples. The procedure is based on the reduction of iron(III) to iron(II) and complexation of iron(II) with 4-(2-pyridylazo)resorcinol, followed by its extraction into n-butanol. The absorbance is measured at 710 nm. Beer’s law is observed up to 5.5 μg ml−1 ascorbic acid. Interference effects of various substances including sugars, vitamins, amino acids, inorganic cations and anions and some organic substances have been studied.
Keywords: Vitamin C determination; 4-(2-Pyridylazo)resorcinol; Potassium citrate; Extraction; n-Butanol;

Fourth derivative UV-spectrophotometric and high-performance liquid chromatographic (HPLC) methods for simultaneous determination of fosinopril and hydrochlorothiazide in tablets have been developed. Standard solutions were measured at zero crossing wavelengths of 217.7 and 227.9 nm for fosinopril and hydrochlorothiazide, respectively, by fourth derivative spectrophotometric method. Calibration curves were constructed by plotting d4 A/dλ 4 values at selected wavelengths against concentrations. HPLC analyses were carried out on C-18 column with gradient elution by using 10 mM H3PO4 and CH3CN as mobile phase. Benazepril was used as internal standard and the substances were detected at 215 nm. Commercially available tablets containing 20 mg fosinopril and 12.5 mg hydrochlorothiazide were analysed by fourth derivative spectrophotometric and HPLC methods. The results were compared statistically at 95% confidence level with each other. There was no significant difference between the mean percentage recoveries and precision of the two methods.
Keywords: Fosinopril; Hydrochlorothiazide; UV-derivative spectrophotometry; High-performance liquid chromatography;

Use of capillary electrophoresis in the analysis of aerosol and bulk/dry deposition collected on a daily basis by W.F.C Tam; P.A Tanner; P.T.R Law; K Bächmann; S Pötzsch (259-269).
Simple protocol is described for the collection of bulk and of dry deposition on a daily basis, and for size-selected aerosol sampling. The use of capillary electrophoresis (CE) as the instrumental method of analysis provides the sample throughput, accuracy and precision required for routine analyses of ionic components from these samples in the μeq. dm−3–neq. dm−3 ranges, with very low sample consumption. The scheme presented enables the simultaneous analyses of inorganic anions and organic acids, as well as of major cations. Representative results are presented for samples collected in Hong Kong.
Keywords: Capillary electrophoresis; Aerosol; Bulk/dry deposition;

Phosphate biosensor based on polyelectrolyte-stabilized pyruvate oxidase by Vasilis G Gavalas; Nikolas A Chaniotakis (271-277).
In this work the development of a pyruvate oxidase-based phosphate biosensor is illustrated. The use of polyelectrolyte stabilized recombinant pyruvate oxidase in conjunction with a porous conductive carbon results in the development of a simple, reproducible and stable phosphate biosensor. The polyelectrolyte diethylaminoethyl-dextran or DNA was used as the enzyme stabilizer, and the resulting enzyme–polyelectrolyte complexes were physically adsorbed into the transducer, a highly porous and conductive carbon electrode, for the construction of the biosensor. The optimized biosensor exhibits high operational (67% remaining activity after 220 h) and storage (49% remaining activity after 24 weeks) stability, and very good sensor-to-sensor reproducibility. The optimized phosphate biosensor was used for the measurement of the phosphate ion activity in serum.
Keywords: Recombinant pyruvate oxidase; Phosphate; Biosensor; Polyelectrolyte; Porous carbon electrode; Serum analysis;

In the present work, a sequential injection system with spectrophotometric detection was developed for the determination of free and total sulphur dioxide in wines. It was based on the formation of a coloured product from the reaction among SO2, formaldehyde and pararosaniline. A gas diffusion unit (GDU) was incorporated into the manifold to prevent the wine matrix interference in the spectrophotometric measurement. An acid solution was added to the sample prior to its passage through the donor channel of the GDU to promote gaseous SO2 formation. For the free SO2 determination, the sample was directly aspirated into the holding coil; for the total SO2 determination, the sample was processed after previous in-line hydrolysis of bound SO2 with an alkali solution.Two second-order calibration curves were established, defining two concentration ranges: 2–40 mg l−1 for the free SO2 determination and 25–250 mg l−1 for the total SO2 determination. Relative standard deviations (n=10) were lower than 1.2% for the determination of free SO2 and lower than 2.3% for the determination of total SO2. The sample frequency was about 16 h−1.This methodology was applied to the determination of free and total sulphur dioxide in 10 table wines and the results were statistically comparable with those furnished by the recommended procedure.
Keywords: Sequential injection; Sulphur dioxide; Wine; Gas diffusion; Spectrophotometry;

A novel polyacrylacylaminourea chelating fiber is synthesized simply and rapidly from nitrilon (an acrylonitrile-based synthetic fiber) and used for the preconcentration and separation of trace In(III), Bi(III), Cr(III), V(V) and Ti(IV) ions from solution samples. The analyzed ions can be quantitatively concentrated by the fiber up to a flow rate of 10.0 ml min−1 at pH 3, and can also be desorbed with 10 ml of 2 M HCl+2% thiourea from the fiber column with recoveries of 97–99%. The chelating fiber is reused for eight times, the recoveries of these ions are still over 92%, and 100–1000 times of excess of Fe(III), Al(III), Ca(II), Mg(II), Ni(II), Mn(II), Cu(II), Zn(II), and Cd(II) cause no interference in the determination of these ions by inductively-coupled plasma atomic emission spectrometry. The capacities of the fiber for the analytes are in the 0.23–1.17 mmol g−1 range. The results show the relative standard deviation for the determination of 50.0 ng ml−1 In(III) and Bi(III), 5.0 ng ml−1 Cr(III), V(V) and Ti(IV) are in the 0.5–3.2% range. The recoveries of a standard added in real solution samples are between 96 and 100%, and the concentration of each ion in mineral sample detected by the method is in good agreement with the certified value.
Keywords: Concentration; Separation; Polyacrylacylaminourea chelating fiber; Indium; Bismuth; Chromium; Vanadium; Titanium;

Extraction behaviour of metal ions in aqueous polyethylene glycol–sodium sulphate two-phase systems in the presence of iodide and thiocyanate ions by Masami Shibukawa; Noriko Nakayama; Tadashige Hayashi; Daigo Shibuya; Yukihiro Endo; Satoshi Kawamura (293-300).
The distribution behaviour of Mn(II), Fe(III), Co(II), Ni(II), Cu(II), Zn(II), Cd(II) and Li(I) was studied in an aqueous two-phase system formed from a polyethylene glycol and sodium sulphate in the presence of thiocyanate or iodide ion. From the distribution ratios determined as a function of the iodide or thiocyanate concentration, the extracted species were assumed. In the SCN system Cu(II), Zn(II) and Co(II) distribute into the PEG-rich phase predominantly as Cu(SCN)4 2−, Zn(SCN)4 2− and Co(SCN)4 2− and Fe(III) as Fe(SCN)4 , respectively. On the other hand, Cd(II) is extracted as Cd(SCN)2 in the SCN system, while as CdI4 2− in the I system. The extractabilities of the metal ions depend not only on the stabilities of the metal thiocyanate or iodide complexes but also on those of their sulphate complexes. A selective separation of Cd(II) from the other metal ions studied was accomplished in the aqueous two-phase system by using iodide ion as an extracting reagent for Cd(II) and an ammonium buffer as a masking reagent for Cu(II).
Keywords: Aqueous two-phase system; Extraction; Polyethylene glycol; Metal ions; Thiocyanate; Iodide;

Index (301-303).