Analytica Chimica Acta (v.422, #2)

Fiber optic detection of in situ lux reporter gene activity in porous media: system design and performance by Irfan Yolcubal; Joseph J. Piatt; Shelley A. Pierce; Mark L. Brusseau; Raina M. Maier (121-130).
A luminescence detection system is described that couples a genetically engineered bioluminescent reporter organism and fiber optic technology for monitoring in situ reporter gene activity in porous media under dynamic conditions. The reporter bacterium used was Pseudomonas putida RB1353, which carries plasmids NAH7 and pUTK9 that encode genes for salicylate degradation (nah) and luminescence (lux) that are regulated by the same promoter. The system can be used to examine the relationship between microbial activity and the resultant impact on biodegradation and transport of salicylate in porous media. Several batch and column experiments were conducted to analyze the performance of the fiber optic detection system. Batch studies showed that the fiber optic response in liquid cultures was linearly correlated to a calibrated system response using a liquid scintillation counter in the single photon counting mode. In column studies, it was demonstrated that decreases in salicylate and dissolved oxygen concentrations associated with biodegradation were correlated with an increase in luminescence response. The maximum luminescence given off by the reporter organism was linearly related to salicylate concentration. These results indicate that the fiber optic system can be used to monitor microbial activity under dynamic conditions. This system allows rapid, real-time, and non-destructive measurements of luminescence from a specific lux reporter microbial population in porous media.
Keywords: Bioluminescence; Reporter organism; Biosensor; Biodegradation;

To overcome the quenching effect of oxygen in fluorescence spectrometry, an in-line flow degassing device (FDD) was successfully applied to the removal of oxygen from sample solutions. The FDD consisted of a 3 m long coil of capillary silicone rubber tubing, installed in a glass flask maintained under reduced pressure of N2 (2.400 Pa). To avoid recontamination of the emerging flow with O2, the connection of the silicone tube with the fluorimetric cell is made with flexible silica capillary tube. Solutions can be injected with a syringe or feed with a pump. Aqueous solution of ruthenium(II) tris-bipyridil chloride, subject to quenching by oxygen, served as a luminescent probe. The performance was checked against independent amperometric measurements of O2 with a Clark cell, that correlated well with the O2 obtained from the fluorescence data using the Stern–Volmer relationship. At a flow rate of 1.0 ml min−1, the concentration of O2 (measured amperometrically) dropped from 8.24 mg l−1 to 15 μg l−1 for a residence time of 34 s in the FDD, rendering the oxygen quenching effect on [Ru(bipy)3]2+ undetectable. The FDD compares favorably with direct inert gas purging of the solution in efficiency and speed. Moreover, it is compatible with flow injection analysis. A frequency of 35 determinations per hour was attained at 1 ml min−1, with good repeatability of the peaks (R.S.D.=0.8%, n=25 injections of 200 μl of 1×10−4  M [Ru(bipy)3]2+).
Keywords: Fluorimetry; Flow analysis; FIA; Quenching by oxygen; In-line oxygen removal; Oxygen sensor;

A novel assay for the voltammetric detection of DNA sequences related to the hepatitis B virus (HBV), using MB as the hybridization indicator was performed. The voltammetric signals of MB have been investigated at bare CPE, double stranded DNA (dsDNA)-modified CPE and single stranded DNA (ssDNA)-modified CPE by means of differential pulse voltammetry and cyclic voltammetry and the increased peak currents were observed, in respect to the order of electrodes. The extent of hybridization between the 21-mer oligonucleotides of complementary sequences was determined by the enhancement of the voltammetric signal of MB. The response of the hybridization of the probe with the single-base mismatch oligonucleotide at CPE was detected by MB. In this case, the unbound guanine bases increased the voltammetric signal obtained with the hybrid-modified CPE. Control experiments with the noncomplementary oligonucleotides were performed to assess whether the DNA biosensor responds selectively, via hybridization, to the target.Numerous factors, affecting the probe immobilization, target hybridization and indicator binding reactions are optimized to maximize the sensitivity and reduce the assay time. The new indicator MB holds great promise for rapid screening of HBV infection.
Keywords: DNA biosensors; Hepatitis B virus (HBV); Methylene blue (MB); DNA; Hybridization;

In this paper, a determinating method of nucleic acids at nanogram levels by a resonance light scattering (RLS) technique with a common spectrofluorometer has been reported. The characteristics of RLS spectra of acridine red (AR) with nucleic acids, the effective factors and the optimum conditions have been studied. In the pH range 6.40–7.10, nucleic acids and surfactant CTMAB can jointly enhance the intensity of resonance light scattering of AR and the interaction of AR with nucleic acids results in three characteristic peaks of RLS at 410, 470 and 555 nm. Mechanistic studies shows that when the molar ratio of nucleic acids to AR (m) is smaller than 5:1, the assembly of AR on the molecular surface of nucleic acids occurs and the enhancement of RLS can be observed. The binding numbers resulting from the Scatchard plot are increased in the presence of CTMAB. When m is higher than 5:1, AR intercalates into the base pairs of DNA. The enhanced intensity of RLS is in proportion to the concentration of nucleic acids in the range 5.0×10−8 to 8.0×10−6  g ml−1 for yeast RNA, 5.0×10−9 to 5.0×10−6  g ml−1 for fish sperm DNA and 5.0×10−8 to 1.2×10−6  g ml−1 for calf thymus DNA. The limits of detection are 1.28, 0.095 and 8.53 ng ml−1, respectively. Synthetic samples were determined satisfactorily. Especially, the detection limit of fish sperm DNA has reached 10−11  g ml−1. It shows that this method has high sensitivity.
Keywords: Acridine red; Nucleic acids; Resonance light scattering (RLS);

The paper describes the use of the palladium(II) complex with 2-(2-thiazolylazo)-5-dimethylamino-benzoic acid (TAMB) for the spectrophotometric determination of nucleic acids with the detection limit at ng levels. At pH 5.9, Pd(II), TAMB and nucleic acids interact rapidly at room temperature to form a supermolecular complex, leading to absorbance decreases at 621 and 675 nm of the palladium(II)–TAMB complex. With 675 nm as the measurement wavelength, the calibration linearity for calf thymus DNA (ct DNA), fish sperm DNA (fs DNA), and yeast RNA extend up to 3.5, 3.5 and 2.0 μg ml−1, respectively, with corresponding 3σ detection of 22, 27 and 25 ng ml−1. Relative standard deviations for the synthetic samples are within 2.8% (n=5). DNA extracted from pig liver is determined with a calibration graph for ct DNA, the result being very close to obtained by ethidium bromide fluorimetry. Compared to other colorimetric nucleic acids assays, the outstanding aspects of the present method are its sensitivity, simplicity and practicality. By the Rosenthanl and Meehan graphical procedures, the binding number and association constant of ct DNA with the complex are estimated, and the binding reaction is considered to be an intercalation.
Keywords: Nucleic acids; Spectrophotometry; 2-(2-Thiazolylazo)-5-dimethylaminobenzoic acid; Palladium(II); Binding reaction;

The development and performance of a competitive indirect immunoassay for the quantitation of polychlorinated biphenyl compounds (PCBs) in insulating oils is described. Reagent preparation and the assay characterisation, optimisation and validation steps are described. The dynamic range of the assay for Aroclors 1254 and 1260 in methanol was 30–1000 ng ml−1 with 50% signal inhibition values of 217 and 212 ng ml−1, respectively. Impending legislation in the UK is likely to decree that oils containing >50 μg ml−1 PCBs be considered contaminated. Assay sensitivity increased with the degree of PCB chlorination. The assay of structurally related compounds of environmental concern yielded cross-reactivity values of <0.6%. The immunoassay proved reliable for the analysis of diluted transformer oils containing >35 μg ml−1 PCB (neat) but over-estimated PCB levels in diluted oils containing <10 μg ml−1 of neat analyte. The oils required pre-treatment using either solid-phase extraction or washing with KOH–ethanol/sulphuric acid to remove matrix interferents. The analytical performance of the assay was compared against a commercially available semi-quantitative immunoassay kit for PCBs in soil and water.
Keywords: Polychlorinated biphenyl compounds; Insulating oils; Enzyme immunoassay; Legislation; Organic phase; Extraction;

CO2 laser photoacoustic spectroscopy was used for quantitative analysis of a multicomponent gaseous mixture of pollutants with similar chemical structures (toluene, ortho-, para- and meta-xylenes) in trace concentrations (50–700 ppm). The agreement with the results of gravimetric measurements was better than 5% in most cases. The following detection limits were found for this method in the given arrangement: 3.57±0.04 ppm for toluene, 1.82±0.02 ppm for o-xylene, 1.00±0.01 ppm for p-xylene and 2.86±0.04 ppm for m-xylene. Determination of the concentration using the classical least squares method was compared with a method based on the principle of calculation of the lowest component entropy. While the least squares method yielded the most selective analysis when four lines were used for four-components mixture, introduction of the component entropy yielded an increase in the selectivity level by measurement of six lines.
Keywords: Photoacoustic spectroscopy; Pollutants; Multicomponent analysis;

A UV spectroscopic method for monitoring aromatic hydrocarbons dissolved in water by F. Vogt; M. Tacke; M. Jakusch; B. Mizaikoff (187-198).
An enhanced UV spectrometric method is applied to trace measurements of aromatic hydrocarbons dissolved in water. This approach gains selectivity and sensitivity by the use of optically generated first and second derivatives of transmission spectra. The augmented spectroscopic technique is combined with chemometric algorithms like principal component regression or partial least squares which are used for calibration of the spectrometer and quantitative evaluation of spectra. Laboratory measurements were performed on mixtures containing up to five substances, i.e. benzene, toluene, ethylbenzene, the three xylene isomers, chlorobenzene, and gasoline. Due to the difficulty of preparing precisely defined calibration and test samples of these very volatile compounds, a novel mixing device was developed and is presented. From these first investigations it can be estimated that the detection limits are down to ca. 10 μg analyte per liter of water by using a 10 cm absorption pathlength and a few minutes measurement time.
Keywords: UV derivative spectrometry; Water analysis; Aromatic hydrocarbons; Environmental monitoring; Automated calibration system; Sequential injection analysis;

Tantalum (0–20 μg) can be determined spectrophotometrically at 482 nm after extraction as tetrabutylammonium hexathiocyanatotantalate(V) into ethyl acetate. The effects of diverse ions, reagent conditions and masking agents are reported. The system has been applied to the determination of tantalum in tin slag samples.
Keywords: Spectrophotometry; Tantalum; Thiocyanate; Liquid–liquid extraction; Tetrabutylammonium; Tin slag;

234U belongs to the 238U natural radioactive decay series, and at equilibrium, the abundance ratio, 234U/238U, corresponds to the ratio of their half-lives, i.e. 54.8×10−6. In natural waters, deviations from this ratio reflect radioactive and geochemical processes and can be correlated with the flow path of the water. Alpha spectrometry or thermal ionization mass spectrometry require tedious procedures for separation and preconcentration of the uranium prior to analysis. Inductively coupled plasma quadrupole mass spectrometry can yield reliable results for the 234U/238U ratio without any treatment of the sample (except acidification). Here, we show that combining a flow-injection sample introduction system with an ultrasonic nebulizer, significantly increases the sensitivity and enhances the ability to cope with a relatively high salt content in non-saline water samples. The relative standard deviation attained is 5% at 50 pg l−1 234U. The method is extremely fast (1 min per measurement), pre-treatment is not required and 1 ml of the sample is needed for triplicate measurements. Therefore, it may be used as a fast screening method.
Keywords: 234U/238U ratio; Surface water; ICPMS; Flow injection; Ultrasonic nebulizer;

Highly sensitive beryllium detection with microwave plasma source atomic emission spectrometry by Yongxuan Su; Zhe Jin; Yixiang Duan; Martin Koby; Vahid Majidi; Jose A Olivares; Stephen P Abeln (209-216).
A highly sensitive technique for beryllium determination using microwave induced plasma atomic emission spectrometry (MIP-AES) is explored in this work based on a self-assembled instrumental system. The analytical performance of this system for beryllium determination was examined using argon as working gas and an ultrasonic nebulization–desolvation system for solution sample introduction. Experimental operating parameters, such as working gas flow rate, microwave power, and observation height were optimized during the system characterization. Influence of matrix elements on beryllium determination was studied. The matrix elements, such as Cu and Ni, were found to have little influence on Be(I) 265.06 nm spectral line even at a concentration of 50 μg ml−1, which is 1000 times higher than that of beryllium. Short-term stability of the MIP-AES system was evaluated and a relative standard deviation of 1.8% is achieved. A calibration range of five orders of magnitude for beryllium determination could be obtained with its multiple spectral lines. The detection limits of Be(II) 313.04/313.11, and Be(I) 234.86 nm were calculated based on 3σ using 1% HNO3 blank solution and shown to be in parts per trillion level.
Keywords: Beryllium; Atomic emission spectrometry; Microwave plasma; Elemental analysis;

It has been shown that by using pyrolysis gas chromatography/mass spectrometry it is possible to discriminate between members within a series of modified alkyd resins which were indistinguishable by Fourier transform infrared spectroscopy.
Keywords: Pyrolysis gas chromatography (PyGC); Fourier transform infrared (FT-IR) spectrometer; Alkyd paint resins;

Retention properties of mesoporous silica-based materials by M.C Bruzzoniti; E Mentasti; C Sarzanini; B Onida; B Bonelli; E Garrone (231-238).
The partition and retention properties have been studied of mesoporous silicas prepared using a surfactant-based supramolecular templating approach towards environmental pollutants (trichloroacetic acid and haloderivatives such as chloroform, 1,1,1-trichloroethane, trichloroethylene and tetrachloroethylene). Gas-chromatography and suppressed anion-exchange chromatography have been used to evaluate the amount of retained analytes. The effect of the composition (presence and absence of the surfactant templating agent and of aluminum) and the influence of synthesis procedure of mesoporous silicates on the sorption performances have been considered. Calcined materials do not show affinity for ionic analytes. The same materials show good affinity for non-ionic analytes: different retention properties towards the tested molecules are observed, interpreted in terms of van der Waals and hydrogen bonding interactions between silica surface and analytes. As-prepared materials show excellent retention performances towards TCA, which depend on the conditions used for the synthesis.All this provides the basis for potential applications of these materials as preconcentrators and sorptive chromatographic substrates for selective separation and/or enrichment (removal) of specific classes of analytes.
Keywords: Mesoporous materials; Volatile hydrocarbons; Trichloroacetic acid; Sorption; Chromatography;

Index (239-240).