Analytica Chimica Acta (v.487, #1)

Foreword by A.J. Baeumner; Richard A. Durst (1).

Spectrographic graphite electrodes were modified through adsorption with laccase from Trametes versicolor. The laccase-modified graphite electrode was used as the working electrode in an amperometric flow-through cell for monitoring phenolic compounds in a single line flow injection system. The experimental conditions for bioelectrochemical determination of catechol were studied and optimized. The relative standard deviation of the biosensor for catechol (10 μM, n=12) was 1.0% and the reproducibility for six laccase-modified graphite electrodes, prepared and used different days was about 11%. The optimal conditions for the biosensor operation were: 0.1 M citrate buffer solution ( at pH 5.0), flow rate of 0.51 ml min−1 and a working potential of −50 mV versus Ag|AgCl. At these conditions the responses of the biosensor for various phenolic compounds were recorded and the sensor characteristics were calculated and compared with those known for biosensors based on laccase from Coriolus hirsutus, cellobiose dehydrogenase (CDH) from Phanerochaete chrysosporium and horseradish peroxidase (HRP).
Keywords: Biosensors; Flow injection; Amperometry; Laccase; Phenols;

Optimization and validation of an enzyme immunoassay for the insect growth regulator fenoxycarb by András Székács; Hong T.M Le; Ferenc Szurdoki; Bruce D Hammock (15-29).
A competitive enzyme-linked immunosorbent assay (ELISA) was developed for the quantitative detection of the insect growth regulator fenoxycarb. Polyclonal rabbit antisera, raised against protein conjugates of four haptenic derivatives of fenoxycarb, were utilized in immobilized antigen-based, competitive immunoassays. With ELISA systems that were both hapten- and carrier-heterologous, most antiserum titers fell in the range of 1:1000–1:30,000. Assay conditions, including concentrations of antisera and coating antigens, were optimized. The effect of pH, organic solvents, and various blocking agents was also investigated. A hapten-homologous and two hapten-heterologous indirect ELISAs allowed fenoxycarb determination in the range of 0.1–85 ng ml−1 with apparent IC50 values of 1.2–2.8 ng ml−1. Cross-reactivities with a number of compounds (e.g. pesticides of related structure, hapten synthesis intermediates, fenoxycarb metabolite, photodegradation products) were determined, and the assay proved highly selective for fenoxycarb. In particular, no significant interference was found with selected pyrethroid and juvenile hormone analog insecticides, phenoxyacetic acid herbicides, and photodegradation products of fenoxycarb. Using spiked water samples, assay performance was validated by SPME/GC-MS.
Keywords: Fenoxycarb; ELISA; Immunoassay optimization; Characterization and validation; GC-MS; SPE; SPME;

Development of a non-labeled immunosensor for the herbicide trifluralin via optical waveguide lightmode spectroscopic detection by András Székács; Nikoletta Trummer; Nóra Adányi; Mária Váradi; István Szendrő (31-42).
A highly sensitive immunosensor using optical waveguide lightmode spectroscopy (OWLS) was developed for the detection of the herbicide trifluralin. OWLS as an in situ and label free method of detection, based on the measurement of the diffraction of a linearly polarized laser beam (He–Ne laser, 632.8 nm) on a diffraction grating in a thin waveguide layer (SiO2–TiO2), offered means to produce immunosensors utilizing immobilized antibodies raised against trifluralin allowing a non-competitive biosensor, or immobilized trifluralin conjugate allowing a competitive biosensor for this analyte. Immobilization of molecules sensitizing the sensor was undertaken on amino silanized waveguide surfaces in a two-step procedure using glutaraldehyde. Within the immobilized antibody (Ab) based immunosensor the signal measured was proportional to the trifluralin content in the samples, but the method allowed detection of trifluralin only above 100 ng ml−1 due to the small molecular size of the antigen (Ag). In the immobilized antigen based immunosensor, a trifluralin–bovine serum albumin (BSA) conjugate was covalently linked to the waveguide surface. During measurements the standard solutions and samples were mixed in 1:1 ratio with antiserum, containing constant amounts of antibodies. The amount of free antibodies bound to the surface was inversely proportional to the trifluralin content of the solutions measured. The immobilized antigen based method allowed detection of trifluralin in the concentration range of 2×10−7 to 3×10−5  ng ml−1. Results of trifluralin determinations were compared to those obtained in parallel enzyme-linked immunosorbent assay (ELISA) tests and in gas chromatorgraphic-mass spectrometric (GC-MS) analyses, and indicated an increase of six orders of magnitude in the limit of detection (LOD).
Keywords: Immunosensor; OWLS; Immunoassay; ELISA; GC-MS; SPME; Trifluralin; Detection in surface water;

A commercially available enzyme-linked immunosorbent assay (ELISA) kit, the PAH RIS c ® soil test, was evaluated with regard to cross-reactivity. Phenanthrene in methanol was used as reference substance. Anthracene, naphthalene and fluorene were chosen as representatives of the 16 US-EPA priority-pollutant polycyclic aromatic hydrocarbons (PAHs). In addition, a number of polycyclic aromatic compounds (PACs), including methyl-, phenyl-, and carbonyl-PAHs, as well as NSO-heterocyclic PACs, found at former industrial sites, were chosen for elucidation of structure/cross-reactivity relationships. The study emphasizes the importance of a priori knowledge of sample composition for accurate interpretation of test results.
Keywords: Cross-reactivity; PAH; Immunoassay; ELISA; SARs;

Bacterial Lux-Fluoro test for biological assessment of pollutants in water samples from urban and rural origin by Christa Baumstark-Khan; Riaz A. Khan; Petra Rettberg; Gerda Horneck (51-60).
During the 20th century, population growth and urbanization, together with changes in production and consumption, have placed unprecedented demands on the quality of water. The ongoing extraordinary economic growth, industrialization, and urbanization of many developing countries results in widespread water pollution from agricultural, industrial, and domestic sources. In consequence, people consume contaminated drinking water, thereby increasing the risk of exposure not only to infectious and parasitic disease but also to a growing volume of genotoxic and cytotoxic chemicals. In light of these trends, new, rapid and low-cost approaches are urgently needed to assess the quality of water supplies. Because of their simplicity and sensitivity, bacterial tests play an important role in the detection and screening of genotoxins or cytotoxins in water. Thus, the bacterial Lux-Fluoro test, which is a combination of two bioassays that simultaneously measure the genotoxicity (SOS-Lux test) and the cytotoxicity (LAC-Fluoro test), was used to identify polluted water from samples of rural and urban sources, collected from 10 different locations in the Punjab rivers’ basin. We identified at least three samples from rural origin having a high cytotoxic potential. The highest toxicity was found for the sample obtained from a draining canal collecting runoff water from the fields. The two other highly contaminated samples identified were taken from two ponds of different villages. The water samples obtained from the Ravi river and from the water tap in a suburb of the megacity Lahore showed no sign of genotoxicity or cytotoxicity. Seven control samples with differing genotoxic and cytotoxic potency were shown for comparison.
Keywords: Bioluminescence; Biosensor; Cytotoxicity; Genotoxicity; Green fluorescent protein; SOS-response; Environmental samples; Punjab rivers’ basin; Asia;

Whole cell immobilised biosensors for toxicity assessment of a wastewater treatment plant treating phenolics-containing waste by Jim C Philp; Séverine Balmand; Eva Hajto; Mark J Bailey; Siouxsie Wiles; Andrew S Whiteley; Andrew K Lilley; Janos Hajto; Sandra A Dunbar (61-74).
Wastewater treatment plants dealing with industrial wastes are often susceptible to overload of toxic influent that can partially or completely destroy treatment for extended periods. An obvious candidate for monitoring toxicity in such wastewater systems is bioluminescent bacteria. However, the natural bioluminescent bacteria can be particularly sensitive to some industrial wastes and therefore their response to normal operational conditions does not reflect the status of the microbial community responsible for treatment. Moreover, the salt dependence of the marine bioluminescent bacteria, and the temperature sensitivity of some strains, further complicate their use. Here we describe the construction of whole cell genetically modified bioluminescent biosensors and their immobilisation for use in monitoring the toxicity of a complex industrial wastewater containing phenolic materials. A hand-held luminometer was designed for laboratory or field use, and the immobilisation system designed with several things in mind: the geometry of the instrument; the need for containment of GM bacteria; the maximisation of the bioavailability of the wastewater to the biosensor. The performance of a candidate GM sensor was compared with Vibrio fischeri in liquid culture and after immobilisation in thin films of poly(vinyl alcohol) (PVA) cryogels. The biosensors were tested against pure phenol and 3-chlorophenol as a reference toxic chemical known to be much more toxic to bacteria than phenol. The biosensors were then tested with the phenolics-containing industrial wastewater. The immobilisation system proved to operate predictably with pure toxicants, and was able to discriminate toxicity of various zones within the wastewater treatment plant.
Keywords: Wastewater treatment plant; Phenolics; Immobilisation; PVA hydrogel;

In this work, a combination of complementary metal-oxide semiconductor (CMOS) microchip system with capillary array electrophoresis (CAE) is demonstrated as a system for optimizing conditions for enzymatic reaction. Dimethylacridinone (DDAO)-phosphate substrate and alkaline phosphatase conjugate were selected for the enzymatic reaction, which was applicable to the enzyme-linked immunosorbent assay (ELISA) technique. Laser-induced fluorometry with a miniature semiconductor laser was used to detect the enzymatic products. The speed of the enzymatic reaction between the DDAO-phosphate and the alkaline phosphatase conjugate was investigated as a function of reaction time. The microchip-CAE detection system could determine the pH condition and the concentration of enzyme that are suitable for rapid and low-cost analysis. This result shows the feasibility of using the microchip-CAE system for application to miniaturized screening systems.
Keywords: Complementary metal-oxide semiconductor microchip; Enzymatic reaction; Capillary array electrophoresis;

Electrochemical ELISA for the screening of DDT related compounds: analysis in waste waters by F Valentini; D Compagnone; G Giraudi; G Palleschi (83-90).
An electrochemical enzyme-linked immunosorbent assay (ELISA) for the detection of 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (p,p′-DTT), 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (p,p′-DDE), 1,1-dichloro-2,2-bis-(4-chlorophenyl)ethane (p,p′-DDD) and o,p-DDT was developed. Optimization of the ELISA competition conditions, led to similar response for the p,p′-isomers. The activity of the label enzyme (horseradish peroxidase) was measured electrochemically using 3,3′,5,5′-tetramethylbenzidine as substrate. The use of purified antiserum for p,p′-DDT resulted in a sensitive assay with a detection limit of 40 pg ml−1 and R.S.D. ranging from 1 to 3% intra-day and 3 to 6% inter-day. No matrix effect for waste water samples of different origin has been evidenced. The ELISA was used to detect DDTs in 20 samples after extraction in diethylether. This method appears suitable for routine screening of DDTs without sample pre-treatment other than dilution in PBS or after organic solvent extraction if high sensitivity is required.
Keywords: Electrochemistry; ELISA; Screening immunoassay; Flow injection analysis (FIA);

Enzyme-based determination of cholesterol using the quartz crystal acoustic wave sensor by S.P Martin; D.J Lamb; J.M Lynch; S.M Reddy (91-100).
We have used the AT-cut quartz crystal sensor to measure in real-time the total cholesterol concentration in buffer and serum, using the trienzyme system of cholesterol esterase (ChE), cholesterol oxidase (ChOx) and horseradish peroxidase (HRP). The hydrogen peroxide produced from the ChE–ChOx reaction oxidises diaminobenzidine (DAB), in the presence of HRP. The response of the sensor to cholesterol is optimal in the presence of 0.1% (v/v) Triton X-100 at 0.2 U/ml ChOx, and 1 U/ml ChE. A response is obtained in less than 25 min. Using the optimal concentrations of the reagents, the linear range for free cholesterol and low density lipoprotein (LDL) cholesterol determination was between 50 and 300 μM, and 25 and 400 μM, respectively. It was found that the concentration of high density lipoprotein (HDL) cholesterol could not be determined because it solubilised the oxidised DAB, leading to poor adsorption at the crystal surface. We obtained a response to the use of cholesterol in serum at 300 μM, demonstrating that this biosensor could be used for cholesterol determination in clinical samples.
Keywords: Cholesterol; Quartz crystal acoustic wave sensor; Cholesterol oxidase; Diaminobenzidine;

Characterization of a fluorescent sol–gel encapsulated erythrosin B dissolved oxygen sensor by R.T Bailey; F.R Cruickshank; G Deans; R.N Gillanders; M.C Tedford (101-108).
A thin film dissolved oxygen sensor was fabricated by trapping erythrosin B in a sol–gel matrix. No phosphorescence was observed when the sensor was immersed in water. Very weak phosphorescence was observed when erythrosin B was dissolved in aqueous ethanol and acetone solutions. However, fluorescence at 590 nm, which was efficiently quenched by dissolved oxygen, was observed from the doped sol–gel sensor in water. A singlet oxygen feedback mechanism was proposed. Good sensitivity and stability were observed for this detector.
Keywords: Erythrosin B; Dissolved oxygen; Sensor; Sol–gel; Fluorescence;

Simultaneous determination of methylene blue and new methylene blue by slab optical waveguide spectroscopy and artificial neural networks by Li-Xian Sun; A.Mahipal Reddy; Naoki Matsuda; Akiko Takatsu; Kenji Kato; Tatsuhiro Okada (109-116).
A slab optical waveguide (SOWG) has been used for study of adsorption of both methylene blue (MB) and new methylene blue (NMB) in liquid–solid interface. Adsorption characteristics of MB and NMB on both bare SOWG and silanized SOWG by octadecyltrichlorosilane (ODS) were compared. The simultaneous determinations of both MB and NMB were explored by flow injection SOWG spectrophotometric analysis and artificial neural networks (ANNs) for the first time. Concentrations of MB and NMB were estimated simultaneously with the ANNs. Results obtained with SOWG were compared with those got by conventional UV-visible spectrophotometry.
Keywords: Slab optical waveguide; Flow injection analysis; Methylene blue; New methylene blue; Artificial neural networks; Simultaneous multicomponent determination;

We report on the elemental results from sequential extractions of BCSS-1 (marine sediment), MESS-1 (estuarine sediment), MAG-1 (marine mud), SCo-1 (Cody shale), and NIST-1c (argillaceous limestone) to encourage future comparisons of sequential extraction results within the marine geochemical and paleoceanographic communities. We measured Si, Al, Ti, Fe, Mn, Ca, P, and Ba in sequential de-ionized water (loosely-bound), MgCl2 (exchangeable), acetic acid (carbonate), hydroxylammonium hydrochloride (oxide), H2O2 (organic), Na2CO3 (opal), and residual (lithogenic) leaches. The protocol and selected elements were tailored to be most relevant to paleoceanographic geochemical studies instead of to environmental studies. Our results show that the sequential extraction procedure faithfully yields elemental distributions that are consistent with individual SRM lithologies. Our results also show that the procedure is typically reproducible within approximately 15%. However, in almost all cases, the procedure suffers from a systematic under-recovery of material when compared with the certified, bulk chemical analysis, and the under-recovery appears to be related to the lithology of the SRM. Similar under-recovery appears to be typical of sequential extraction procedures as reported by other previous studies. While this is problematic in assessing closure, it does not diminish the potential of inter-lab comparisons and first-order accuracy comparisons. We found that the elemental totals for the sequential extractions of MAG-1 compared best with the certified, bulk totals, and we recommend using this SRM to facilitate future accuracy assessments and inter-lab comparisons.
Keywords: Sequential extraction; Standard reference materials; Major and trace elements; Reproducibility; Marine sediment;