Analytica Chimica Acta (v.561, #1-2)

Contents (v-vii).

The principal and the most important error sources in quantitative electron paramagnetic resonance (EPR) measurements arising from sample-associated factors are the influence of the variation of the sample material (dielectric constant), sample size and shape, sample tube wall thickness, and sample orientation and positioning within the microwave cavity on the EPR signal intensity. Variation in these parameters can cause significant and serious errors in the primary phase of quantitative EPR analysis (i.e., data acquisition). The primary aim of this review is to provide useful suggestions, recommendations and simple procedures to minimise the influence of such primary error sources in quantitative EPR measurements. According to the literature, as well as results obtained in our EPR laboratory, the following are recommendations for samples, which are compared in quantitative EPR studies: (i) the shape of all samples should be identical; (ii) the position of the sample/reference in the cavity should be identical; (iii) a special alignment procedure for precise sample positioning within the cavity should be adopted; (iv) a special/consistent procedure for sample packing for a powder material should be used; (v) the wall thickness of sample tubes should be identical; (vi) the shape and wall thickness of quartz Dewars, where used, should be identical; (vii) where possible a double TE104 cavity should be used in quantitative EPR spectroscopy; (viii) the dielectric properties of unknown and standard samples should be as close as possible; (ix) sample length less than double the cavity length should be used; (x) the optimised sample geometry for the X-band cavity is a 30 mm-length capillary with i.d. less then 1.5 mm; (xi) use of commercially distributed software for post-recording spectra manipulation is a basic necessity; and (xii) the sample and laboratory temperature should be kept constant during measurements. When the above recommendations and procedures were used in our quantitative EPR experiments (with a Bruker ER 200 D-SRC EPR spectrometer with a Bruker double TE104 rectangular cavity and air conditioning) the EPR signal intensity of a wide range of samples could be obtained with the experimental errors between 3 and 5%. However, if these special precautions were not employed the errors could be in excess of 20% or more. We believe that these tips are helpful in quantitative EPR practice.
Keywords: Review; Electron paramagnetic resonance (EPR) spectroscopy; Quantitative; Error sources; Reproducibility;

Investigation of the enzyme Bacillus agaradhaerens Cel 5a as an analytical tool in mass spectral characterisation of methylcelluloses by Arieh S. Cohen; Carina Nilsson; Herje Schagerlöf; Folke Tjerneld; Lo Gorton (16-24).
Two methylcelluloses (MC) were investigated by partial hydrolysis followed by direct infusion electrospray ionisation mass spectrometry (ESI-MS). Partial hydrolysis was achieved either enzymatically or by acid hydrolysis. Enzyme hydrolysis was performed using the endoglucanase Bacillus agaradhaerens Cel 5a. Three different hydrolysis buffer solutions were investigated in order to determine which was most compatible with the subsequent MS analysis. ESI-MS experiments showed that when using enzymatic hydrolysis as an analytical tool for characterisation of modified cellulose one has to be watchful for the investigated polymer to have a sufficient amount of available cleavage sites in order to ensure that the original ends of the polymer do not influence the results. MS2 experiments confirmed that the detected products originated from methylcellulose and provided structural information on the investigated products. MS data proved that B. agaradhaerens Cel 5a is able to cleave β1 → 4 glycosidic linkages adjacent to a permethylated glucosyl unit. MS2 data showed that methylation on the C-2 and C-6 hydroxyl groups of the glucosyl unit on the non-reducing side of the cleavage site hindered the enzyme.
Keywords: Methylcellulose; MC; Mass spectrometry; ESI-MS; Bacillus agaradhaerens Cel 5a; Cellulase; Modification pattern;

Application of probes having 2′-deoxyinosine for typing of single nucleotide polymorphisms (SNPs) using DNA microarray by Hideki Kinoshita; Shigemasa Ishiwata; Yasuhiko Tsuji; Makiko Dejima; Kazuyoshi Yano; Ichiro Takase; Isao Karube (25-31).
The principle of hybridizing oligonucleotides is employed with single nucleotide polymorphisms (SNPs) typing, but that typing frequently failed. We investigated the SNPs typing of multi-alleles on a chip. Two bases adjacent to the SNPs on the immobilized oligonucleotide probes were substituted for the 2′-deoxyinosine (dI) residues. The biotinylated probes containing the partial sequence of CYP2C9, CYP2C19, GLC1A and ApoE alleles were applied to the streptavidin-embedded plasma-polymerized (PP) DNA microarrays. For the probes containing dI, up to a 2–5-fold difference in the match to a mismatch oligonucleotide fluorescence intensity was found as the probes without dI were 1.5-fold or less. According to the substitution for dI, the hybridization accuracy was enhanced, and then several single-base mismatches were detected on a chip.
Keywords: Single nucleotide polymorphisms; Oligonucleotide array; Gene chips; 2′-Deoxyinosine;

Biomarkers play a potential role in the early detection and diagnosis of a disease. Our aim is to derivatize carbon nanotubes for exploration of the differences in human body fluids e.g. serum, through matrix assisted laser desorption ionisation/time of flight mass spectrometry (MALDI/TOF-MS) that can be related to disease and subsequently to be employed in the biomarker discovery process. This application we termed as the material enhanced laser desorption ionisation (MELDI). The versatility of this technology is meant to increase the amount of information from biological samples on the protein level, which will have a major impact to serve the cause of diagnostic markers.Serum peptides and proteins are immobilized on derivatized carbon nanotubes, which function as binding material. Protein-loaded suspension is placed on a stainless steel target or buckypaper on aluminum target for direct analysis with MALDI-MS. The elution method to wash the bound proteins from carbon nanotubes was employed to compare with the direct analysis procedure. Elution is carried out by MALDI matrix solution to get them out of the entangled nanotubes, which are difficult to desorb by laser due to the complex nanotube structures. The advantage of these optimized methods compared to the conventional screening methods is the improved sensitivity, selectivity and the short analysis time without prior albumin and immunoglobulin depletion. The comparison of similarly modified diamond and carbon nanotubes exhibit differences in their nature to bind the proteins out of serum due to the differences in their physical characteristics. Infrared (IR) spectroscopy provided hint for the presence of tertiary amine peak at the crucial chemical step of iminodiacetic acid addition to acid chloride functionality on carbon nanotubes. Atomic absorption spectroscopy (AAS) was utilized to quantitatively measure the copper capacity of these derivatized carbon nanotubes which is a direct measure of capacity of this material to bind the number of proteins from serum.
Keywords: Carbon nanotubes; MALDI; Protein profiling; Elution; IMAC; Biomarker;

Continuous monitoring of the organophosphate pesticide chlorpyrifos in real water samples has been performed using a portable surface plasmon resonance (SPR) immunosensor. The immunoassay was based on a binding inhibition test by using the covalent immobilization of an analyte derivative onto the gold-coated sensing surface. Sensor reusability was assured through the formation of alkanethiol self-assembled monolayers (SAMs). In this format, the biosensor surface remained stable over 190 measurements including regeneration cycles. The reproducibility and precision of SPR measurements were evaluated by performing independent immunoassays for three replicates of each sample. The batch-assay variability was also estimated using three different gold-coated sensor surfaces. The assay provided a highly sensitive detection of chlorpyrifos at nanogram per litre levels (55 ng L−1) in laboratory buffer. No significant matrix effects were found for ground, river and drinking water samples, where the limits of detection ranged from 45 to 64 ng L−1 and the recovery values were between 80 and 120%. The validation of chlorpyrifos immunoassay was performed using conventional chromatographic methods (GC–MS). The correlation between both techniques was proved by the good correlation coefficient (r 2  = 0.995) obtained. Chlorpyrifos immunoassays were developed using the β-SPR sensor system commercialised by the company SENSIA, S.L. (Spain). Rapid analysis of samples, without previous extraction or clean-up, was accomplished in only 20 min, including the regeneration cycle. The high sensitivity, low-time of response and portability of the β-SPR biosensor platform, would allow its application as a field analytical method for environmental waters.
Keywords: SPR; Immunosensor; Self-assembled monolayer; Chlorpyrifos; Environmental analysis;

A novel amperometric immunosensor for the detection of α-fetoprotein (AFP) based on the integration of microelectronic technology, mixed self-assembled monolayers (mixed SAMs), gold nanoparticles (nanogold) and enzyme amplification has been developed. Using microelectronic technology, an immunosensor was fabricated which has an “Au, Pt, Pt” three-microelectrode system and two microwells constructed by SU-8 photoresist on silicon wafer. Using mixed SAMs and nanogold, a mixed monolayer comprising cysteamine and 1,6-hexanedithiol was formed on the working electrode surface to assemble nanogold and further to immobilize AFP antibody for detecting AFP in human serum samples. The stepwise mixed SAMs and nanogold based immobilization procedure was characterized by means of cyclic voltammetry. The factors influencing the performance of the resulting immunosensor were studied in detail. After the addition of H2O2 and KI to the immunosensor incubated with AFP and further with horseradish peroxidase-labeled AFP antibody, the cathodic current varied linearly in concentration range of AFP from 15 to 350 ng/ml with a detection limit of 5 ng/ml. Moreover, the studied immunosensor has attractive advantages, such as miniaturization, compatibility with the complementary metal oxide semiconductor (CMOS) techniques, high specificity, good reproducibility and long-term stability, which make it potentially attractive for clinical immunoassays.
Keywords: Amperometric immunosensor; Microelectronic technology; Mixed self-assembled monolayers; Gold nanoparticles; α-Fetoprotein;

A reusable capacitive immunosensor for carcinoembryonic antigen (CEA) detection using thiourea modified gold electrode by Warakorn Limbut; Proespichaya Kanatharana; Bo Mattiasson; Punnee Asawatreratanakul; Panote Thavarungkul (55-61).
A capacitive immunosensor based on a self-assembled monolayer (SAM) of thiourea on gold electrode has been developed. Anti-carcinoembryonic antigen (anti-CEA) was immobilized on a self-assembled thiourea monolayer (SATUM) via covalent coupling. Under optimum conditions, the decrease in capacitive signal when carcinoembryonic antigen (CEA) standard was injected could be determined with a detection limit of 10 pg ml−1 and linearity in the range of 0.01–10 ng ml−1. The immobilized anti-CEA on SATUM gold electrode was stable and after regeneration good reproducibility of the signal could be obtained up to 45 times with an RSD lower than 3.4%. Good agreement was obtained when CEA concentrations of human serum samples determined by the flow injection capacitive immunosensor system were compared to those obtained using an enzyme linked fluorescent assay (ELFA) method (P  < 0.05).
Keywords: Capacitance; Immunosensor; Carcinoembryonic antigen; Thiourea; Self-assembled monolayer;

The uses of five antimicrobial growth promoters (AGPs) zinc bacitracin, spiramycin, tylosin, virginiamycin and olaquindox in animal feedingstuffs have been prohibited in the European Union (EU) since 1999. The development and validation (in-house) of an immunoassay-based multi-analyte screening procedure for simultaneously detecting five banned AGPs was achieved as part of an EC funded project (Feedstuffs-RADIUS). The resultant test has been adapted to an enzyme-linked immunosorbent assay (ELISA) kit format and its performance was assessed in a collaborative study. A total of 15 laboratories drawn from DG SANCO's Committee of Experts on Methods of Analysis of Feedingstuffs (CEMA) group representing government agencies and private industry in the EU participated in this collaborative study. Six incurred materials of three different feed types were analysed. The analysis of 231 reported results revealed an overall false non-compliant rate of 1.9% and a false compliant rate of 0% at concentrations of antibiotic in the region of 20% of their previous minimum inclusion rates (MIR). The developed procedure, when used in control laboratories, to monitor for the presence of the banned antibiotics, will have a major impact on the ability to enforce the European ban on these substances.
Keywords: Antimicrobial growth promoter (AGP); Feedingstuffs; Collaborative study; Immunoassay; Screening; Kit;

Application of an automated fluidic system using electrochemical bead-based immunoassay to detect the bacteriophage MS2 and ovalbumin by Hideki Kuramitz; Michael Dziewatkoski; Ben Barnett; H. Brian Halsall; William R. Heineman (69-77).
A fully-automated fluidic system for a bead-based immunoassay with electrochemical detection was developed. Assays for the bacteriophage MS2 and ovalbumin (OVA) were demonstrated using this system. Streptavidin-coated paramagnetic microbeads were used as a mobile solid phase. The immunoassay sandwich was made by attaching a biotinylated antibody to the streptavidin-coated beads, capturing antigen, and then exposing the antigen to an antibody conjugated with β-galactosidase. β-Galactosidase converts p-aminophenyl galactopyranoside (PAPG) to p-aminophenol (PAP), which is electrochemically oxidized to p-quinone imine (PQI). The behavior of paramagnetic microbeads in the fluidic system was investigated using beads with immobilized β-galactosidase at different concentrations of beads and flow rates for each procedure in the assay. Furthermore, the fully-automated MS2 and OVA assays were demonstrated using the fluidic system. The limits of detection for MS2 and OVA were 990 (1.6 × 1011 particles mL−1) and 470 ng mL−1, respectively.
Keywords: Electrochemical immunoassay; Paramagnetic microbeads; Fully-automated fluidic system; MS2; Ovalbumin;

Continuous flow configuration for total grease and surfactant determination in industrial degreasing baths by Eva Aguilera-Herrador; Rafael Lucena; Esther Trullols; Soledad Cárdenas; Miguel Valcárcel (78-82).
A simple and robust continuous system for the sequential determination of total grease and surfactant contents in industrial degreasing baths has been developed. The two families of compounds are extracted from the samples by liquid–liquid extraction and aliquots of 250 μl of the organic extract are automatically passed through a silica sorbent column for surfactants retention while the greases are directed to the evaporative light scattering detector for fraction monitoring. Complete separation of both families of compounds in the continuous flow system and right peak assignment was corroborated by ATR-FTIR analysis. The proposed method exhibits an adequate linear ranges and excellent precision, which makes it suitable for the determination of both parameters in these industrial samples. Finally, 15 real samples obtained from pre-cleaning and cleaning baths from a production line of one of these industries were analyzed by the proposed continuous flow analyzer and by a reference gravimetric method with satisfactory results.
Keywords: Industrial degreasing baths; Total grease; Total surfactant; Evaporative light scattering detector;

The feasibility of employing cloud point extraction (CPE) as a simple and effective alternative for recovery of alkaloids from tobacco samples followed by GC–MS analysis is demonstrated. An aqueous surfactant solution containing 5% Triton X114 was used for extraction of tobacco alkaloids. Then, the analytes were back-extracted with ultrasonic assistance from the surfactant-rich phase into dichloromethane. No other cleanup step preceded GC–MS analysis. Seven alkaloids in tobacco sample with different concentrations were analyzed simultaneously. Results show that the recovery of nicotine is 80.4% and the limit of detection (LOD) is 7.1 μg g−1. The relative standard deviations for the seven alkaloids are in the range of 2.77–9.97%. The results yielded by the proposed method were almost identical with those achieved by the more laborious industrial standard method, i.e., the continuous flow method.
Keywords: Cloud point extraction; GC–MS analysis; Alkaloids; Plant sample;

A headspace solvent microextraction–gas chromatography–mass spectrometry (HSME–GC–MS) method was described for the analysis of volatile compounds from Curcuma wenyujin Y.H. Chen et C. Ling. A comparison was made between HSME–GC–MS, solid-phase microextraction (SPME)–GC–MS and steam distillation (SD)–GC–MS methods. The number of the identified compounds was 66 for HSME–GC–MS, 71 for SPME–GC–MS and 63 for SD–GC–MS, respectively. HSME–GC–MS was found to share most of the compounds in common with the other two methods. Compared with the other two methods, HSME–GC–MS has a lower cost, more choices of extraction solvents and requires a smaller amount of sample and can directly utilizes the ground powder of traditional Chinese medicines (TCMs) for the analysis. In conclusion, HSME–GC–MS method is a simple, inexpensive and effective method for the analysis of volatile compounds in TCMs.
Keywords: Headspace solvent microextraction; Solid-phase microextraction; Steam distillation; Volatile compounds; Gas chromatography–mass spectrometry;

The gas chromatographic retention data of 90 saturated esters obtained isothermally at 150 °C on seven stationary phases (SE-30, OV-7, DC-710, OV-25, XE-60, OV-225 and Silar-5CP) are well-correlated with recently introduced Lu index and novel distance-based atom-type DAI topological indices using multiple linear regression (MLR) method. For the retention indices (RI) based on each stationary phase, the correlation coefficient r of the final models is larger than 0.994, and particularly, the decrease in the standard error is within the range of 53.0–64.8% as compared with the simple linear models with Lu index alone. The results indicate the efficiency of these indices in the structure–retention index correlations of complex compounds. The role of each of the molecular size and individual groups in the molecules is illustrated by analyzing the relative or fraction contributions of individual indices. The results also indicate that the molecular size makes a dominant contribution to retention behaviors, while some atomic type or groups have smaller influence dependent on the polarity of the column. The leave-one-out cross-validation demonstrates the final models to be statistically significant and reliable.
Keywords: Gas chromatographic retention indices; Lu index; DAI indices; Esters; QSRR;

A method was developed for the gas chromatographic (GC) determination of free fatty acids (FFA) in olive mill waste waters (OMW). The sample under examination was acidified to pH 2–3 to cause protein precipitation. Elimination of the colloidal fraction was obtained by addition of acetone. Polyvynilpirrolidone was added to eliminate the interference by phenolic compounds. The sample was then clarified by centrifugation and an aliquot was injected into the GC system. Both the C2–C8 FFA coming from microbial metabolism and the C16–C18 FFA coming from the oil originally present in the olives were quantified in a single run by employing nonanoic acid as the internal standard. Rather high amounts in particular of acetic acid were generally found, usually in the range from 1000 to 4000 mg/L of OMW.
Keywords: Gas chromatography; Free fatty acids; Olive mill waste waters;

Capillary-scale monolithic silica columns containing an entrapped transmembrane receptor were prepared by a sol–gel processing method and evaluated by frontal affinity chromatography with tandem mass spectrometric detection (FAC/MS/MS). Monolithic stationary phases were made by infusing micro-bore capillaries with a mixture containing the water soluble polymer polyethylene glycol (PEG), the biocompatible silica precursor diglycerylsilane (DGS) and membranes containing the nicotinic acetylcholine receptor (nAChR) from Torpedo californica. Through multivariate factorial analysis, the conditions used to form the stationary phase materials were analyzed to determine which factors were the most significant in controlling the silica pore diameter, silica gelation time, column reproducibility and the apparent activity of the immobilized receptor. Of the variables studied it was found that altering the concentration of PEG and the additive 3-(aminopropyl)triethoxysilane had the largest effect on the silica pore diameter and gelation times, as well as the apparent receptor activity. The information gained through factorial analysis was used to prepare an optimized “second-generation” material that was suitable for the reproducible preparation of nAChR-based bioaffinity columns. Using FAC/MS/MS, it was determined that the immobilized receptor-doped columns retained 100% of the receptor in active form, and that the binding constant for the nanomolar agonist epibatidine was very close to that obtained in buffered aqueous media. These results indicate that monolithic receptor columns should be useful for screening compound mixtures and identifying small molecules with nanomolar binding affinities.
Keywords: Acetylcholine receptor; Sol–gel; Affinity chromatography; Factor analysis; Mass spectrometry;

A microemulsion electrokinetic chromatography (MEEKC) method has been firstly developed and validated for the separation of aristolochic acids A, B, C and D, 7-hydroxy-aristolochic acid A and aristolic acid in herbal medicines. With the optimized experimental conditions (octane 0.81% (w/w), SDS 3.31% (w/w), butan-1-ol 6.61% (w/w) and 10 mM sodium tetraborate buffer 89.27% (w/w), with 15 kV as applied voltage, pH 9.2, 25 °C as capillary temperature), all of the six analytes were baseline-separated (any resolution was over 2) in peaks of good shapes within 16 min. Aristolochic acids A, B, C and aristolic acid were quantitative determined and acceptable precision (intra-day repeatability (n  = 6) and inter-day reproducibility (n  = 3) of the method in terms of migration times were within 0.98 and 2.75% R.S.D. in terms of migration times and within 5.09 and 5.93% R.S.D. in terms of peak areas, respectively) and linearity (r  > 0.995) and recovery (ranged between 97 and 104%) were obtained. Moreover, the method was successfully validated and applied to the determination of four analytes in herbal medicines.
Keywords: Microemulsion electrokinetic chromatography; Aristolochic acids; Chinese herbs nephropathy;

A simple and fast method for the simultaneous determination of the antioxidants propyl gallate (PG), octyl gallate (OG), lauryl gallate (LG), and nordihydroguaiaretic acid (NDGA) has been established by using microchip micellar electrokinetic chromatography with pulsed amperometric detection. Under the optimum conditions (30 mM borate buffer, pH 9.7, 30 mM sodium dodecyl sulfate, separation voltage of 1200 V and 5 s injection time) the analytes were baseline separated. Linear relationships were found between the concentration and peak current for all the selected antioxidants. The measured detection limits (S/N ≥ 3) of PG, OG, LG, and NDGA were 2.2, 1.4, 2.3, and 4.6 μM, respectively, which corresponds to 2–6 fmol of analyte. This approach has remarkable advantages with respect to other methodologies involving separations and electrochemical detection including minimal sample consumption, higher analysis speed, lower cost, and portability. Additionally, a highly reproducible signal (migration time and peak current) was obtained for a series of injections (n  = 30). In order to demonstrate the capabilities of the method, the determination of antioxidants in a commercial food sample is also presented.
Keywords: Microchip; Phenolic antioxidants; MEKC; Pulsed amperometric detection; Food analysis;

Polyhexamethylene biguanide hydrochloride (PHMB-HCl) and chlorhexidine digluconate (CHG) are cationic disinfectants widely used in aqueous personal products such as eye drops, lotions and creams. The antiseptics belong to the biguanide group, which are cationic substances well known for their effective action against microbial infection. In this work, capillary electrophoresis combined with contactless conductivity detection was used to determinate these substances in ophthalmic solutions. Statistical analysis of the results showed satisfactory precision ranging from 3 to 6% in peak area. The detection limits were determined to be 4 μg/mL for PHMB and 0.4 μg/mL for CHG. It is also demonstrated that the high sensitivity allows the method to be used for in vitro perfusion studies with rabbit cornea.
Keywords: Chlorhexidine digluconate; Polyhexamethylene biguanide hydrochloride; Capillary electrophoresis; Contactless conductivity detection;

Numerical simulation of Joule heating effect on sample band transport in capillary electrophoresis by G.Y. Tang; C. Yang; H.Q. Gong; J.C. Chai; Y.C. Lam (138-149).
It is known that the presence of Joule heating imposes limitations for the performance of capillary electrophoresis. This study reports modeling and numerical study of the Joule heating effects on the electrokinetic dispersion of a finite sample plug in capillary electrophoresis. Due to the sharp concentration gradient and large Peclet number for the finite sample plug transport, it is necessary to use a high-order numerical scheme to minimize the numerical pseudo-diffusion. This is demonstrated through the comparison of the numerical results between the widely used first-order Power-law scheme and the second-order Crank–Nicolson scheme in the presence of Joule heating effects and the comparison among the numerical results of these two numerical schemes and the analytical solutions in the absence of Joule heating effects. In addition, numerical simulations are carried out on the basis of the Crank–Nicolson scheme to study the Joule heating effect on the electrokinetic dispersion of a finite sample plug for various applied voltages, capillary radii, and sample electrophoretic mobilities. The results show that the Joule heating causes sample band to move faster and alters the sample shape in terms of lower sample peak and wider sample band.
Keywords: Electrokinetic dispersion of a finite sample plug; Joule heating effect; Capillary electrophoresis; Crank–Nicolson scheme; Power-law scheme;

Discrimination of DNA mismatches by direct force measurement for identification of tuna species by Tsuyoshi Tanaka; Tomohito Sasaki; Yosuke Amemiya; Haruko Takeyama; Seinen Chow; Tadashi Matsunaga (150-155).
The interaction between oligonucleotides and long DNA fragments was analyzed by force curve measurements using atomic force microscopy (AFM). DNA fragments (150-base or 406-base) from the mitochondrial ATPase and cytochrome oxidase subunit III genes that contained a mismatch of one to three bases among Tunnus species were immobilized on glass slides. The statistical distribution of disruption forces between oligonucleotide probes (21-mer or 29-mer) and single stranded DNA fragments (150-base or 406-base) were analyzed by 40 or 180 force curve measurements. Histograms plotting the frequencies of disruption forces showed a wide distribution with a highest peak. The highest mean values in disruption force were obtained when DNA fragments with perfectly match sequences were employed. These results demonstrated that the specific sequence differences between long DNA fragments can be measured using force-based detection. A single base mismatch yielded a statistically significant 10% decrease in disruption force, furthermore, 2-base and 3-base mismatches provided approximately 20 and 25–30% decreases, respectively. Our results indicated that force-based detection potentially can be applied toward many other mismatched DNA detection techniques besides species-specific identification of tuna.
Keywords: Atomic force microscopy; Force curve measurement; Disruption force; Long DNA fragment; Tuna species identification;

Logit linearization of analytical response curves in optical disposable sensors based on coextraction for monovalent anions by L.F. Capitán-Vallvey; E. Arroyo-Guerrero; M.D. Fernández-Ramos; L. Cuadros-Rodríguez (156-163).
The application of a decimal logistic transformation to the sigmoidal calibration curve of ion-selective bulk optodes for the determination of anions based on hydrophobic membranes containing neutral ionophore and chromoionophore is formally established and, consequently, a wide linear calibration function is obtained. The problems derived from the use of a sigmoidal curve in the calibration are therefore solved and the linear dynamic range is increased.The general equation resulting from the logistic transformation is discussed considering the stoichiometric factors for monovalent anions, and the linearization of the theoretical fit to experimental data was checked for two real cases. The strategy was applied to the determination of chloride and nitrate using disposable sensors for different types of waters (tap, well, stream, rain, snow and sea), validating the results against a reference procedure. This new linear calibration proposed for anion determination increases the linear dynamic range up to six orders of magnitude. The main advantage is that it is possible to directly quantify samples with very different analyte contents in a fast and simple way. The methods are easy to use and eliminate the need for prior treatment of the sample.
Keywords: Linearization; Anion determination; Disposable optical sensor; Ionophore–chromoionophore chemistry; Water analysis;

Development of Vitamin B12 based disposable sensor for dissolved oxygen by Meng Shan Lin; Hoang Jyh Leu; Chien Hung Lai (164-170).
We report herein a new type of Vitamin B12 based screen-printed sensor for the detection of dissolved oxygen (DO). The electrochemical behavior of Vitamin B12 modified electrode has been studied carefully, and the amperometric scheme for oxygen sensing has also been investigated. The experimental conditions were systematically optimized in acetate solution, at pH 3 and an applied potential of −0.2 V (versus Ag/AgCl with 3 M NaCl). Under the optimal conditions, the linearity of calibration is between 15 and 45 μmol L−1 (r  = 0.9991) with an estimated detection limit of 9.2 μmol L−1  (S/N = 3). The reproducibility, and response time are 1.37% (R.S.D., n  = 20), and 8.8 s. Further, the feasibility of using disposable strip for format DO was manifested by chronoamperometry. The results exhibited significant characteristics with a concentration range up to 0.5 mmol L−1 (r  = 0.9986) at an applied potential of −0.4 V (versus Ag/AgCl with 0.1 M NaCl). A correlation study between disposable sensor strip and a commercially available DO probe has also been studied with a correlation coefficient of 0.9996. Thus, this sensor strip provides rapid, sensitive, and compatible accurate characteristics.
Keywords: Amperometric; Dissolved oxygen; Vitamin B12; Screen-printed sensor; Chronoamperometry;

The preparation and the recognition property of molecularly imprinted polymer of podophyllotoxin by Xiufang Zhu; Qiue Cao; Nengbang Hou; Guosong Wang; Zhongtao Ding (171-177).
A selective and affinitive molecularly imprinting polymer (MIP) was prepared to podophyllotoxin (PPT) using a non-covalent imprinting approach. The imprinted conditions including the kind and relative amounts of functional and cross-linking monomers were carefully optimized. The binding characteristics of the imprinted polymer were evaluated in different solvent systems using equilibrium binding experiments. UV spectrum revealed cooperative hydrogen-bonding complex between PPT and acylamide (AM) might be formed at the ratio of 1:2 in the prepolymerized system, and produced two classes of binding sites in the resulting MIP with dissociation constants of 0.337 and 5.76 mmol l−1, and the affinity binding sites of 78.67 and 346.0 μmol g−1, respectively. Selective binding of the template molecule is demonstrated in comparison to the analogue 4′-demethylpipodophyllotoxin (DMEP). Finally, the optimized MIP was used as an adsorbent to extract PPT directly from the ethanol extraction of Chinese traditional Tibetan medicine, Sinopodophyllum emodi. The binding selectivity and recovery of the optimized imprinted polymer had been further estimated by HPCE and compared to its non-imprinted polymer. The obtained results implied a possibility to extract PPT directly from plants by a MIP technique shown in this paper.
Keywords: Molecular imprinting polymers; Podophyllotoxin; Sinopodophyllum emodi;

A sinomenine (SIN) molecularly imprinted polymer (MIP) with specific recognition ability for SIN was prepared by in situ molecularly imprinted technique. The polymer was then packed into a polypropylene column and used as a solid-phase extraction (SPE) sorbent. Utilizing this MIP-SPE as the separation medium, a crude extract of SIN from Sinomenium acutum Reht. et Wils was purified, and relatively pure SIN was obtained. At the same time, based on the specific molecular recognition ability of MIP, a determination method of SIN in blood was established with MIP as the separation medium. The linear range was 5–60 μg ml−1, and the linear correlation coefficient of the standard curve was 0.9975. The average recovery was 98%, with the relative standard deviation (R.S.D.) 7% (within-day) and 8% (between-day). These results reveal that the established method can be used for analysis of SIN in biological samples.
Keywords: Molecularly imprinted polymer; Sinomenine; In situ technique; Application;

Synthesis of hydrophilic macroporous chelating polymers and their versatility in the preconcentration of metals in seawater samples by Mamoru Haratake; Kazuyoshi Yasumoto; Masahiro Ono; Makoto Akashi; Morio Nakayama (183-190).
We systematically synthesized hydrophilic matrix polymers using glycidyl methacrylate (GMA) and ethylene glycol dimethacrylate (EGDMA) by the radical suspension copolymerization method. Methyl iso-butylketone added to the monomer phase as a porogen was effective in the formation of macroporous structures. Triethylenetetramine (TTA) was introduced on the matrix polymers as a chelating moiety (PGMA-EGDMA-TTA), and the adsorption and desorption characteristics of the obtained chelating polymers for several metals were investigated. A better candidate, PGMA-EGDMA-TTA polymer, was packed into a column and the applicability for the preconcentration of metals in seawater samples was tested. A PGMA-EGDMA-TTA possesses the preferable adsorptivity for metals at the seawater pH and a favorable desorption profile using an acid eluent. In addition, a PGMA-EGDMA-TTA-packed column was effective for the preconcentration of metals in the seawater samples.
Keywords: Suspension polymerization; Porogen; Macroporous structure; Metal; Preconcentration; Seawater;

Determination of DNA by solid substrate room temperature phosphorescence enhancing method based on the Morin·SiO2 luminescent nanoparticles–Pd system as a phosphorescence probe by Jia-Ming Liu; Tian-Long Yang; Fei Gao; Li-Xiang Hu; Hang-Xia He; Qin-Ying Liu; Zhen-Bo Liu; Xiao-Mei Huang; Guo-hui Zhu (191-197).
Sodium carbonate (Na2SiO3) as the precursor, was mixed with Morin organic dye to synthesize silicon dioxide luminescent nanoparticles containing Morin (Morin·SiO2) by sol–gel method. The particle sizes of SiO2·nH2O and Morin·SiO2 were both 50 nm, measured with TEM (transmission electron microscope). Morin·SiO2 modified by HS–CH2COOH could be dissolved by water. In the HMTA (hexamethylenetetramine)–HCl buffer solution, Pd2+ could coordinate with Morin in Morin·SiO2 to form complex Pd2+–Morin·SiO2, which could emit phosphorescence on polyamide membrane. And DNA (deoxyribonucleic acid) could cause a sharp enhancement of the room temperature phosphorescence (RTP) intensity of complex Pd2+–Morin·SiO2. Thus a new method of solid substrate room temperature phosphorescence (SS-RTP) enhancing for the determination of DNA was established based on the Morin·SiO2 luminescent nanoparticles–Pd system as a phosphorescence probe. The ΔIp is directly proportional to the content of DNA in the range of 4.00–1000.0 fg spot−1 (corresponding concentration: 0.010–2.50 ng ml−1). The regression equation of working curve was ΔIp = 21.13 + 0.2076mDNA (fg spot−1) (r  = 0.9990) and the detection limit was 0.61 fg spot−1 (corresponding concentration: 1.5 pg ml−1). This method had a wide linear range, high sensitivity, convenience, rapidity and only a little sample was needed. Samples containing 0.10 and 25.0 ng ml−1 DNA were measured repeatedly for 11 times and RSDs were 3.2 and 4.1% (n  = 11), respectively, which indicated that the method had a good repeatability. Disturbance of common ions, such as Mg2+, K+, and Ca2+, was small, and there was no disturbance in the presence of protein and RNA. This method has been applied to the determination of DNA in nectar successfully.
Keywords: Morin·SiO2 luminescent nanoparticles–Pd as phosphorescence probe; Solid substrate room temperature phosphorescence enhancing method; DNA;

A new solid substrate-room temperature phosphorescence (SS-RTP) quenching method for the determination of trace molybdenum has been established. The SS-RTP can be quenched by H2O2 oxidizing [Rhod.B]+·B[(C6H6)4] ion association complex, which is formed of Rhod.B reacting with NaB(C6H6)4 and [Rhod.B]+·B[(C6H6)4] ion association complex can emit strong and stable room temperature phosphorescence on filter paper substrate, while molybdenum can catalyze H2O2 to oxidize [Rhod.B]+·B[(C6H6)4] ion association complex to quench the SS-RTP sharply in HCl medium, heated at 90 °C for 10 min. Based on the facts above, a new method for the determination of trace molybdenum has been proposed. When using PVA–H3BO3–NaOH to modify the surface of filter, the results showed that ΔI p of the system enhanced 1.5 times than that of unmodified. The linear range of this method is 1.6–160 ag spot−1 (the sample volume: 0.40 μl spot−1), and the regression equation of working curve is ΔI p  = 18.23 + 0.8935m Mo(VI)  ag spot−1, n  = 6, r  = 0.9980. The detection limit is 0.34 ag spot−1 (corresponding concentration: 8.5 × 10−16  g ml−1). This method has been successfully applied to the determination of trace molybdenum in actual samples with special feature such as wide linear range, high sensitivity, good repeatability, high selectivity and so on. Meanwhile, the mechanism of catalysis reaction is also discussed.
Keywords: Molybdenum; [Rhod.B]+·B[(C6H6)4] ion association complex; Solid substrate-room temperature phosphorescence quenching method;

Trace amount of lecithin (PC) was determined in the buffer solution of pH 5.7, using tetracycline (TC)–europium ion (Eu3+) as a fluorescent probe. PC can remarkably enhance the fluorescence intensity of the TC–Eu3+ complex at λ  = 612 nm and the enhanced fluorescence intensity of Eu3+ is in proportion to the concentration of PC. Optimum conditions for the determination of PC were also investigated. The linear range and detection limit for the determination of PC are 4.0 × 10−7 to 1.4 × 10−5  mol/L and 3.9 × 10−8  mol/L. This method is simple, practical and relatively free of interference from coexisting substances and can be successfully applied to assess PC in serum samples. Moreover, the enhancement mechanism of the fluorescence intensity in the TC–Eu3+ system, the TC–Eu3+–PC system, and the TC–Eu3+–PC–sodium dodecyl benzene sulfonate (SDS) system is also discussed.
Keywords: Spectrofluorimetry; Lecithin; Tetracycline; Europium;

In the present work, experimental design was used for the fast optimization of three kinds of sample digestion procedures with the final aim of obtaining the COD value of wastewater samples. The digestion methods evaluated were “closed microwave-assisted” (CMWD), “open microwave-assisted” (OMWD) and “ultrasound-assisted” (USD). Classical digestion was used as reference method. The optimum values for the different variables studied in each method were: 90 psi pressure, 475 W power and 4 min irradiation time (CMWD); 150 °C temperature and 4 min irradiation time (OMWD); 90% of maximum nominal power (180 W), 0.9 s (s−1) cycles and 1 min irradiation time (USD). In all cases, interference concentration that produces a deviation of 10% in COD values is 13.4, 23.4, 21.1 and 2819 mg/L for S2−, Fe2+, NO2 and Cl, respectively. Under optimum conditions, the proposed digestion methods have been successfully applied, with the exception of pyridine, to several pure organic compounds and COD recoveries for 10 real wastewater samples were ranged between 88 and 104% of the values obtained with the classical (open reflux) method used as reference, with R.S.D. lower than 4% in most cases. Thus, the use of ultrasound energy for COD determination seems to be an interesting and promising alternative to conventional open reflux and microwave-assisted digestion methods used for the same purpose since the instrumentation is simpler, cheaper and safer and the digestion step faster than the ones used for the same purpose.
Keywords: COD determination; Ultrasound energy; Microwave radiation; Optimization; Experimental design;

The utility of attenuated total reflectance-mid-infrared spectroscopy (ATR-MIR) to monitor the concentrations of the key analytes ammonium, glucose, methyl oleate and biomass in a Streptomyces clavuligerus bioprocess for the synthesis of clavulanic acid was investigated. The cultivation medium was complex and the process fluid (matrix) underwent profound changes as culture growth proceeded, representing a considerable challenge from a spectroscopic viewpoint. Quantitative models were developed using the multivariate statistical technique, partial least square (PLS) for the key analytes over the entire time course of the bioprocess, which were validated externally using samples not incorporated in the original modelling exercise. The reasoning behind the choice of modelling strategy for each analyte is discussed, with particular focus on biomass modelling in order to generate clearer insight into the modelling process in this complex matrix. Despite the heterogeneous nature of the sample matrix, and the complexity of the spectral information arising, at-line models were developed giving low prediction error values for the analytes: ammonium 0.013 g/l; glucose 0.56 g/l; methyl oleate 0.38 g/l and biomass 0.39 g/l, respectively. These findings represent a considerable advance on previous studies using NIR in antibiotic processes, as all key analytes have been successfully modelled here, and the use of ATR-MIR in complex bioprocess fluids has been shown to be a viable method of near real-time monitoring.
Keywords: ATR-MIR; Streptomyces; PLS; Models;

In this work, simultaneous determination of two groups of elements consisting of Pb(II)–Cd(II) and Cu(II)–Pb(II)–Cd(II) using adsorptive cathodic stripping voltammetry are described. The method is based on accumulation of these metal ions on mercury electrode using xylenol orange as a suitable complexing agent. The potential was scanned to the negative direction and the differential pulse stripping voltammograms were recorded. The instrumental and chemical factors were optimized using artificial neural network. The optimized conditions were obtained in pH of 5.5, xylenol orange concentration of 4.0 μM, accumulation potential of −0.50 V, accumulation time of 30 s, scan rate of 10 mV/s and pulse height of 70 mV. The relationship between the peak current versus concentration was linear over the range of 5.0–150.0 ng ml−1 for cadmium and 5.0–150.0 ng ml−1 for lead. The limits of detection were 0.98 and 1.18 ng ml−1 for lead and cadmium ions, respectively. In simultaneous determination of Cu(II), Pb(II) and Cd(II) there are inter-metallic interactions, which result a non-linear relationship between the peak current and the ionic concentration for each of the element. Therefore, an artificial neural network was used as the multivariate calibration method. The ANN was constructed with three neurons as the output layer for the simultaneous determination of the three elements. The constructed model was able to predict the concentration of the elements in the ranges of 1.0–50.0, 5.0–200.0 and 10.0–200.0 ng ml−1, for Cu(II), Pb(II) and Cd(II), respectively.
Keywords: Adsorptive cathodic stripping voltammetry; Artificial neural network; Xylenol orange; Copper; Lead; Cadmium;

Author Index (233-235).