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Eluted metals from Enshu (a Chinese) and Stratford (an Australian) coal during pretreatments with methanol/water and dilute acetic acid solutions were characterized and quantitatively analyzed by solid-state 23Na NMR, cation chromatography, X-ray fluorescence (XRF), and Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP-AES) measurements. To achieve the requirements of solvent extracted coal (namely Hyper-Coal: HPC) for its direct utilization to gas turbine system, alkali and alkali-earth metals such as Na, K, and Ca should be removed from coals. A quantitative analytical method of alkali metals with ICP-AES in combination with flow injection system is established for the evaluation of remaining alkali metal contents, especially for the quantitative analysis of Na concentration in the original coal and its HPC. Microwave irradiation acidic digestion pretreatment under controlled high pressure and temperature is very essential to the prompt and quantitative analyses of all the metals in coals and their derived HPC. Combination of ICP-AES with XRF and/or cation chromatography is also effective for the respective analyses of the residue and the extract or filtrate. Solid-state 23Na NMR spectra of the coals before and after solvent soaking pretreatment or acetic acid treatment revealed that ion-exchangeable Na species might be rather selectively eluted by these treatments. It is also suggested that the remaining Na species could not be removed by dilute acetic acid solutions. [Copyright &y& Elsevier]

For determination of methylmercury (MeHg) and ethylmercury (EtHg) in seawater and industrial wastewater, a simple and robust analytical method was developed based on phenylation and solvent extraction followed by GC-MS measurement. Alkylmercury compounds were directly phenylated with sodium tetraphenylborate in water and extracted into toluene. The method detection limits obtained for MeHg and EtHg in pure water were 53.3 and 33.5 ng Hg L -1 , respectively, which are almost 10 times lower than the environmental quality standards for water pollution in Japan (EQSJ): 0.5 μg Hg L -1 . The recoveries of alkylmercury compounds from seawater and four kinds of industrial wastewater except for EtHg from treated wastewater of an optic lens factory were satisfactory (>90%) at 1- or 4-fold concentrations of the EQSJ. Contrarily, the low recovery of EtHg from the treated wastewater (75.4 ± 4.7%) was found to be caused by the rapid decomposition of EtHg into inorganic mercury.

We have developed a method for the determination of Cr(VI) and Cr(III) in industrial wastewater by liquid chromatography-inductively coupled plasma mass spectrometry (LC-ICP-MS) combined with a chelating pretreatment with 2,6-pyridinedicarboxylic acid (PDCA). The PDCA unified the chemical forms of the Cr(III) species in water samples by the formation of a stable Cr(III)-PDCA complex, which was then separated by a LC column. The chromatographic mobile phase at neutral pH and the column of a mixed-bed of anion and cation exchangers successfully separated not only the chromium species without any redox conversion, but also chloride, which interfered with ICP-MS detection. The method detection limits measured at m/z 53 were 0.66 μg of Cr L -1 for Cr(III) and 0.74 μg L -1 for Cr(VI) with a sample injection volume of 20 μL under a no gas mode. The recoveries of spiked Cr(VI) at 50 and 500 g L -1 into the fifteen kinds of industrial wastewater samples were satisfactory (>90%). The proposed method for the determination of Cr(VI) was also validated by comparing with a colorimetric method using 1,5-diphenylcarbazide prescribed by the ISO 11083 and the JIS K0102.

Inorganic arsenic (InAs) is a ubiquitous metalloid that has been shown to exert multiple adverse health outcomes. Urinary InAs and its metabolite concentration has been used as a biomarker of arsenic (As) exposure in some epidemiological studies, however, quantitative relationship between daily InAs exposure and urinary InAs metabolites concentration has not been well characterized. We collected a set of 24-h duplicated diet and spot urine sample of the next morning of diet sampling from 20 male and 19 female subjects in Japan from August 2011 to October 2012. Concentrations of As species in duplicated diet and urine samples were determined by using liquid chromatography-ICP mass spectrometry with a hydride generation system. Sum of the concentrations of urinary InAs and methylarsonic acid (MMA) was used as a measure of InAs exposure. Daily dietary InAs exposure was estimated to be 0.087 µg kg -1 day -1 (Geometric mean, GM), and GM of urinary InAs+MMA concentrations was 3.5 ng mL -1 . Analysis of covariance did not find gender-difference in regression coefficients as significant (P > 0.05). Regression equation Log 10 [urinary InAs+MMA concentration] = 0.570× Log 10 [dietary InAs exposure level per body weight] + 1.15 was obtained for whole data set. This equation would be valuable in converting urinary InAs concentration to daily InAs exposure, which will be important information in risk assessment.

As a result of the growing potential industrial and medical applications of multi-walled carbon nanotubes (MWCNTs), people working in or residing near facilities that manufacture them may be exposed to airborne MWCNTs in the future. Because of concerns regarding their toxicity, quantitative data on the long-term clearance of pristine MWCNTs from the lungs are required. We administered pristine MWCNTs well dispersed in 0.5 mg ml(-1) Triton-X solution to rats at doses of 0.20 or 0.55 mg via intratracheal instillation and investigated clearance over a 12-month observation period. The pristine MWCNTs pulmonary burden was determined 1, 3, 7, 28, 91, 175 and 364 days after instillation using a method involving combustive oxidation and infrared analysis, combined with acid digestion and heat pretreatment. As 0.15- and 0.38-mg MWCNTs were detected 1 day after administration of 0.20 and 0.55 mg MWCNTs, respectively, approximately 30% of administrated MWCNTs may have been cleared by bronchial ciliary motion within 24 h of administration. After that, the pulmonary MWCNT burden did not decrease significantly over time for up to 364 days after instillation, suggesting that MWCNTs were not readily cleared from the lung. Transmission electron microscopy (TEM) showed that alveolar macrophages internalized the MWCNTs and retained in the lung for at least 364 days after instillation. MWCNTs were not detected in the liver or brain within the 364-day study period (<0.04 mg per liver, < 0.006 mg per brain)., (Copyright © 2015 The Authors. Journal of Applied Toxicology Published by John Wiley & Sons Ltd.)

Rapid, simple, and low-cost screening procedures are necessary for the detection of harmful compounds in the effluent that flows out of point sources such as industrial outfall. The present study investigated the effects on a novel sensor of harmful compounds such as KCN, phenol, and herbicides such as 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), 2-chloro-4-ethylamino-6-isopropylamino-1,3,5-triazine (atrazine), and 2-N-tert-butyl-4-N-ethyl-6-methylsulfanyl-1,3,5-triazine-2,4-diamine (terbutryn). The sensor employed an electrode system that incorporated the photocurrent of intra-cytoplasmic membranes (so-called chromatophores) prepared from photosynthetic bacteria and linked using carbon paste electrodes. The amperometric curve (photocurrent-time curve) of photo-induced electron transfer from chromatophores of the purple photosynthetic bacterium Rhodobacter sphaeroides to the electrode via an exogenous electron acceptor was composed of two characteristic phases: an abrupt increase in current immediately after illumination (I₀), and constant current over time (Ic). Compared with other redox compounds, 2,5-dichloro-1,4-benzoquinone (DCBQ) was the most useful exogenous electron acceptor in this system. Photo-reduction of DCBQ exhibited Michaelis-Menten-like kinetics, and reduction rates were dependent on the amount of DCBQ and the photon flux intensity. The Ic decreased in the presence of KCN at concentrations over 0.05 μM (=μmol·dm(-3)). The I₀ decreased following the addition of phenol at concentrations over 20 μM. The Ic was affected by terbutryn at concentrations over 10 μM. In contrast, DCMU and atrazine had no effect on either I₀ or Ic. The utility of this electrode system for the detection of harmful compounds is discussed.

The present protocol introduces a single-chain probe carrying a functional peptide in the N-terminal domain of the androgen receptor (AR NTD) for illuminating androgenicity of ligands. In the single-chain probe, a functional peptide in the AR NTD was genetically fused to the ligand-binding domain of AR (AR LBD) via a flexible linker, and then sandwiched between the N- and C-terminal fragments of split-firefly luciferase (FLuc) dissected at D415. This single-chain probe exerts (1) a high signal-to-background ratio and (2) sensitive discrimination between agonists and antagonists, where the dimerization of AR LBD is not involved. The present protocol guides a fundamental methodology on how to discriminate weak protein-protein (peptide) binding, and provides a new insight into the intramolecular folding inside monomeric AR.

The present protocol demonstrates a new strategy for imaging ligand-triggered protein phosphorylation using circularly permutated luciferases (cpLuc): (1) a luciferase is first fragmented into two segments for creating new N- and C-terminal ends in the hydrophilic region, (2) the original N- and C-terminal ends are circularly permutated and linked via a GS linker, whereas the new ends made by fragmentation are correspondingly linked with two proteins of interest. When the new ends of the cpLuc are linked with the ligand-binding domain of estrogen receptor (ER LBD) and Src homology two domain of Src (SH2), the estrogen can trigger phosphorylation of the ER LBD and consequent intramolecular ER LBD-SH2 binding. This interaction triggers an approximation of the adjacent fragments of split-cpLuc recovering the enzyme activity. This probe design greatly improves signal-to-noise (S/N) ratios upon tracing weak protein-protein interactions (PPIs) in mammalian cells.

The concentrations of arsenic (As) species in 19 food composites prepared from 159 food items purchased in Shizuoka city, Japan, were determined (1) to estimate total daily intake of inorganic As (InAs) and some organic As species and (2) to determine food contributing to total daily InAs intake. As analysis included extraction of As species with a synthetic gastric juice (0.07 mol/L HCl + 0.01 % pepsin) from food composite and high-performance liquid chromatography-high efficiency photo-oxidation-hydride generation-inductively coupled plasma mass spectrometry. InAs was detected in 9 of 19 food composites at a concentration of 0.423-450 ng As/g fresh-weight. Daily intake of InAs from cereals was greatest (13 μg/person/day) followed by algae (5.7 μg/person/day), and the intake from the two categories constituted 90 % of the total daily InAs intake of adults (21 μg/person/day on a bioaccessible-fraction basis and 24 μg/person/day on a content basis). Analysis of individual food items showed that rice and hijiki contributed virtually 100 % of InAs from cereals and algae, respectively. The present survey indicated that InAs from rice and hijiki consumption contributed to total daily InAs intake and consequently to significant cancer risk of the general Japanese population. Daily intake of some organic forms of As and their contributing food categories was also estimated.

The present study demonstrates the creation of artificial luciferases (ALuc) for bioassays, inspired by a sequence alignment of copepod luciferases. Extraction of the consensus amino acids from the alignment enabled us to generate a series of ALucs with unique optical properties and sequential identities that are clearly different from those of any existing copepod luciferase. For example, some ALucs exhibited heat stability, dramatically prolonged optical intensities, broad full width at half-maximum, and strong optical intensities. The practical suitability of the luciferases as an optical readout was examined in diverse bioassays, including mammalian two-hybrid assays, live cell imaging, single-chain probes, bioluminescent capsules, and bioluminescent antibodies. We further determine the physical properties of ALucs through bioinformatic analysis and finally discuss detailed issues on the unique properties of ALucs. The present study shows how to create the artificial enzymes with excellent optical properties for bioassays and encourages researchers to fabricate their own unique artificial enzymes with designed properties and functionalities.

It is known that the distribution of the charge-states as well as the evaporation field shift to higher values as the specimen temperature is decreased at a constant rate of evaporation. This study has explored the effect of Mg or Ag addition on the evaporation field of Al in terms of the charge state distribution of the field evaporated Al ions. The fractional abundance of Al(2+) ions with respect to the total Al ions in Al-Mg alloy is lower than that in pure Al, whereas it shows higher level in the Al-Ag alloy at lower temperatures. The temperature dependence of the fractional abundance of Al(2+) ions has been also confirmed, suggesting that Al atoms in the Al-Mg alloy need lower evaporation field, while higher field is necessary to evaporate Al atoms in the Al-Ag alloy, compared with pure Al. This tendency is in agreement with that of the evaporation fields estimated theoretically by means of measurements of the work function and calculations of the binding energy of the pure Al, Al-Mg and Al-Ag alloys., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The reagent-free mineralization of dissolved organic matter (DOM) in river water was achieved within 1 min using a lamp-pass-through photoreactor containing a narrow reaction tube (2 mm i.d.) passing through a 40 W mercury lamp. The structure efficiently irradiated the sample solution in the tube with vacuum ultraviolet (VUV; 185 nm) light from the lamp, which rapidly decomposed the DOM with hydroxyl radicals generated efficiently from the water and oxygen that are naturally present in the solution. The photoreactor was also applicable to oxidizing reagent-free online toatal organic carbon (TOC) analysis of DOM in river-water samples using a non-dispersive infrared radiation detector after acidification of the sample using 20 mmol L(-1) phosphoric acid. The detection limit for phthalate at the injection of 390 μL was 6.2 μg of carbon L(-1). The repeatability, as expressed by the relative standard deviation, was 2.5% for thrice-repeated analyses of a river sample with 1.85 mg of carbon L(-1).

Multi-walled carbon nanotubes (MWCNTs), dispersed in suspensions consisting mainly of individual tubes, were used for intratracheal instillation and inhalation studies. Rats intratracheally received a dose of 0.2 mg, or 1 mg of MWCNTs and were sacrificed from 3 days to 6 months. MWCNTs induced a pulmonary inflammation, as evidenced by a transient neutrophil response in the low-dose groups, and presence of small granulomatous lesion and persistent neutrophil infiltration in the high-dose groups. In the inhalation study, rats were exposed to 0.37 mg/m(3) aerosols of well-dispersed MWCNTs (>70% of MWCNTs were individual fibers) for 4 weeks, and were sacrificed at 3 days, 1 month, and 3 months after the end of exposure. The inhalation exposures delivered less amounts of MWCNTs into the lungs, and therefore less pulmonary inflammation responses was observed, as compared to intratracheal instillation. The results of our study show that well-dispersed MWCNT can produce pulmonary lesions, including inflammation.

The daily dietary intake of inorganic arsenic (InAs) and some of organic arsenic (OrAs) species of Japanese subjects were estimated by determining the concentrations of As species in two different sets of total diet sample: duplicated diet samples collected from 25 subjects in Japan and a certified reference material with total diet matrix (NIES CRM No. 27 Typical Japanese Diet, TJD). The concentration of InAs and OrAs in diet samples were determined by LC-ICP-MS using a photo-oxidation and hydride generation system. The median intake of InAs for the 25 subjects was 3.8 μg day(-1) (2.0-57 μg day(-1)) and intake of 27 μg day(-1) was estimated from TJD. The median intake of MMA, DMA and TMAsO were <0.18, 1.1 and <0.053 μg day(-1) for the 25 subjects and that of MMA, DMA, AB and TMAsO was estimated to be 3.9, 11, 140 and 5.9 μg day(-1), respectively, based on TJD analysis. On the basis of InAs intakes estimated and the oral slope factor of the US EPA and Health Canada, excess cancer risk was estimated to exceed acceptable level. Cancer risk posed by the dietary InAs of the general Japanese may not be negligible., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

By exposure to appropriate UV intensities, rapid and quantitative oxidation/reduction of inorganic selenite, selenate and several organoselenium compounds representative of those of biochemical/metabolic interest, including selenomethionine, selenobetaine, L-selenocystine, selenomethylselenocysteine, γ-glutamyl-seleno-methylselenocysteine and selenocystamine, is achieved. In the presence of acetic acid, quantitative conversion to volatile SeH(2) and SeCO occurs using a flow-through system comprising a highly efficient 40 W UV lamp for oxidation in tandem with a lower power 8 W UV photocatalytic reactor utilizing a thin-film coating of titania. The volatile reduced species are detected by atomic absorption spectrometry using a heated quartz tube atomizer. Direct photochemical conversion of selenite, selenomethionine, L-selenocystine, γ-glutamyl-Se-methylselenocysteine and selenocystamine occurs in the presence of 5% acetic acid, following exposure to an 8 W UV field, to yield volatile detectable species, whereas selenobetaine and selenate are unresponsive unless the latter is first subjected to oxidation by exposure to a highly efficient 40 W UV lamp and the selenate reduced in the presence of titania.

In this study, a rational synthesis of superluminescent variants from marine luciferases with prolonged bioluminescence has been demonstrated. A putative active site of a model marine luciferase, Gaussia princeps Luciferase (GLuc), was assigned and modified by a site-directed mutagenesis. The potent variants were found to generate up to 10 times stronger bioluminescence, emitting red shifts of up to 33 nm with natural coelenterazine than native GLuc, rendering an efficient optical signature in bioassays. The advantageous properties were demonstrated with mammalian two-hybrid assays, single-chain probes, and metastases of murine B16 melanoma in BALB/c nude mice. The unique ideas for engineering GLuc are proved to be valid even for other marine luciferases.

A capillary-based pitch-variable array spotter, designed to dispense solutions in nanoliter volumes with high precision and density, was used to immobilize gene probes on a microelectrode array chip. Small volumes of the probe solutions were dispensed onto the microelectrodes, without any physical contact between the capillary ends and the electrode surfaces, to fabricate an electrochemical gene sensor array chip. The fabricated gene sensor array chip showed sequence-selective responses, expressed on a pseudocolor scale, to a target DNA sample. It was demonstrated that this dispensing technique provides integrated sensor array chips by dispensing small volumes of solutions of synthesized functional gene probes onto microelectrode array chips, a process not possible with conventional dispensing techniques.

The present study demonstrates a single-molecular bioluminescent probe carrying functional peptides in the N-terminal domain of the androgen receptor (AR NTD) with an improved sensorial property to androgens. The N-terminal peptides in AR were genetically fused to the ligand binding domain of AR (AR LBD) with a flexible linker, and then sandwiched between the N- and C-terminal fragments of split-firefly luciferase (FLuc) dissected at D415. We found that the proline-rich region in AR NTD efficiently interacts with AR LBD and exerts (i) an enhanced signal-to-background ratio and (ii) discrimination between agonists and antagonists with (iii) a 100-times improved sensitivity to androgens, upon comparison with previous references. A deletion mutation to the proline-rich region in AR revealed that this region is critical for the transcriptional activities. The quantum yields of these single-chain probes were estimated to be 37.8 +/- 0.6%. This monomeric AR LBD-peptide binding is necessary, and sufficient for discriminating an agonist and an antagonist, where the dimerization of AR LBD is not involved. The present study guides a fundamental methodology on how to discriminate weak protein-peptide binding, and provides a new insight into the contribution of functional peptides in AR NTD to the initial activation of monomeric AR.

This study demonstrates a unique, nontranscriptional assay system based on molecular tension of a luciferase artificially appended by protein-protein binding. We hypothesized that an artificially appended molecular tension to a full-length luciferase may diversify the enzymatic activity through a modification of the active site. For the basic probe design, a full-length luciferase was sandwiched between two component proteins of interest. The length of flexible linkers between the components was minimized to exert an efficient molecular tension to the sandwiched luciferase. When N- and C-terminal ends of Renilla luciferase 8 were flanked by the ligand-binding domain of human estrogen receptor alpha (ER LBD) and SH2 domain of Src, named ERS, this simple probe was surprisingly sensitive to estrogens. The luminescence spectra by ERS were largely enhanced by an addition of 4-hydroxytamoxifen (OHT), 17beta-estradiol, and genistein. The detection limit of ERS reached 1 nM OHT. Quantum yield (QY) and Michaelis-Menten constant of ERS were found to be 6.3% and 94.3 muM, respectively. The enzymatic activities of ERS are also governed by different types of coelenterazine (CTZ) variants. The two hydroxy groups in CTZ are critical for the enzymatic activities of ERS. This study is the first example that an artificially appended molecular tension to a full-length luciferase can be taken as an optical signature upon molecular imaging. This study also provides new insight into the construction of a new lineage of bioluminescent probes for estimating protein-protein interactions.

This study demonstrates bioluminescent indicators for determining stress hormones in mammalian cells. A genetically encoded bioluminescent probe for stress sensing was first synthesized with a LXXLL motif-linked ligand binding domain of the glucocorticoid receptor (GR LBD), which was then sandwiched between the fragments of Gaussia luciferase (GLuc). This prototype of the bioluminescent indicators was carefully modified with a circular permutation (CP) and/or a corepressor motif. The first notable appearance by cofusion of a corepressor motif to the probe was the biphasic dose-response curves of the indicator to cortisol. A CP largely improved the detection limit of the indicator to cortisol up to 100 times. Fabrication of both CP and the attachment of a corepressor motif in the indicator synergistically contributes to (i) the lower detection limit and wider dynamic range and (ii) the enhanced absolute luminescence and ligand selectivity. This study is the first example that contribution of corepressor motifs to single-chain probes was investigated. This study also provides new insight into improving the sensorial properties of single-chain probes with CP.

In surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS), a chemical background signal, arising from organic contaminants such as plasticizers, is frequently observed mainly under m/z ca. 600, which impairs the advantages of the matrix-free approach. Silver salts, which are used for the cationization of aromatic compounds, are also difficult to remove completely after the measurements. In this study, surface cleaning techniques used in semiconductor processing were used to clean our developed silicon-based SALDI substrate on which self-assembled germanium nanodots (GeNDs) had been deposited (termed a GeND chip). An immersion cleaning method using acetone with sonication, and a sulfuric-peroxide mixture (SPM) cleaning method using a mixture of H(2)SO(4)/H(2)O(2)/deionized water, were examined for their effectiveness in removing organic compounds and residual silver salts. Removal of both types of contaminants was successfully performed by SPM cleaning. The limit of detection for glutathione was improved from ca. 5 pmol without cleaning to ca. 50 fmol after the SPM cleaning. Since GeND chips can tolerate acidic cleaning and sonication due to their chemical inertness and rigid nanodot structures, they appear to be an ideal reusable SALDI substrate., (John Wiley & Sons, Ltd)

The goal of the present study is to develop a small luminescence template, in which any protein may be incorporated, for illuminating the dynamics of protein signaling. We first determined optimal fragmentation sites of Gaussia princeps-derived luciferase (GLuc). The utility of the template was demonstrated with calmodulin (CaM) and a peptide-linked CaM, which were sandwiched between the fragments of split GLuc dissected at Q105. Living mammalian cells with the probe quickly emitted luminescence in response to endo- and exogenous Ca2+ levels. The applicability of the template was expanded to the visualization of the phosphorylation of the estrogen receptor and interaction of the androgen receptor with a peptide via an intramolecular complementation between GLuc fragments. It also provides the smallest bioluminescence template ever synthesized. Considering that conformational changes of proteins generally occur for signal transductions, the present luminescence template will contribute to the exploration of intracellular molecular events with signaling proteins.

The drift in sensitivity due to carbon deposition on the sampling cone, skimmer cone and ion lenses has been a serious problem in gas chromatography/inductively coupled plasma mass spectrometry (GC/ICP-MS). To overcome this problem, a high-speed switching method between a mixed-gas plasma and a pure-argon plasma (named plasma gas-switching method) using an oxygen permeation tube and a switching valve was developed. This enabled both the cleaning of deposited carbon and an enhancement of the sensitivity; as a consequence, both the repeatability and the sensitivity of polybrominated diphenylether (PBDE) were improved by more than 3 and 4 times, respectively. The drifts of sensitivity over a period of 8 h were less than 5% in most cases. Concerning the analytical performance of thermally labile congeners from octa- to deca-BDE, the detection limits, dynamic ranges of the calibration graphs and unequivalent sensitivities were remarkably improved by using a metal capillary separation column coated with a very thin (0.05 micromm) film of immobilized-polydimethylsiloxane. The detection limits ranged from 0.014 pg (BDE-154) to 0.093 pg (BDE-209), which were equal or superior to the lowest values reported hitherto by GC/MS (high resolution). A remarkable loss of sensitivity for highly-brominated congeners, such as nona- and deca-BDE, was observed in an analysis of PBDE technical mixtures when the solvent was methanol. The loss of sensitivity turned out to be due to an activation of the retention gap used for on-column injection; this problem was solved by changing methanol to isooctane in the sample-preparation step before analysis.

Electrochemically enhanced DNA detection was demonstrated by utilizing the couple of a synthesized ferrocene-terminated peptide nucleic acid (PNA) with a cysteine anchor and a sacrificial electron donor [Fe(CN)(6)](4-). DNA detection sensors were prepared by modifying a gold electrode surface with a mixed monolayer of the probe PNA and 11-hydroxy-1-undecanethiol (11-HUT), protecting [Fe(CN)(6)](4-) from any unexpected redox reaction. Before hybridization, the terminal ferrocene moiety of the probe was subject to a redox reaction due to the flexible probe structure and, in the presence of [Fe(CN)(6)](4-), the observed current was amplified based on regeneration of the ferrocene moiety. Hybridization decreased the redox current of the ferrocene. This occurred because hybridization rigidified the probe structure: the ferrocene moiety was then removed from the electrode surface, and the redox reaction of [Fe(CN)(6)](4-) was again prevented. The change in the anodic current before and after hybridization was enhanced 1.75-fold by using the electron donor [Fe(CN)(6)](4-). Sequence-specific detection of the complementary target DNA was also demonstrated.

We have developed an 8-ch capillary-based dispensing workstation with a variable capillary pitch mechanism. The capillary intervals can be varied from 1 to 9 mm to dispense different solutions simultaneously at an arbitrary dispensing pitch, allowing direct dispensing from microplates to integrated analytical systems. To evaluate the precision of its dispensing performance, droplets of Rhodamine G dye were dispensed onto glass slides and the values of the optical volume were analyzed. The error in the dispensed volume proved to be 0.54 nL when dispensing 20 nL. In dispensing small volumes, the volume error for this workstation was found to be about 100-fold less than that seen in conventional dispensers. Even highly viscous solutions containing 50% glycerol could be dispensed with precision. Rapid dispensing was also achieved. Moreover, the application of the workstation to preparing addressable 8 x 12 microsensor array chips was demonstrated, providing an independent and reproducible spot array.

Rapid identification of bacteria by a bioinformatics-based approach, which processes the mass spectra observed by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS), relies on the calculated masses of ribosomal subunit proteins as biomarkers predicted from amino acid sequences found in protein sequence databases. To verify the actual state of the registered sequence information, a simple intact protein analysis by MALDI-MS using cell lysates as samples was applied to the characterization of ribosomal proteins from genome-sequenced Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus strains. This method avoided the risk of loss of some subunit proteins and the formation of disulfide bonds during the purification of ribosomal proteins. By comparing this with the MALDI mass spectra of different strains and carrying out manual inspection of sequence information, a total of five errors in N-terminal amino acid sequences were identified. After sequence correction, approximately 40 out of 53 subunit proteins could be assigned, considering N-terminal methionine loss only as a post-translational modification. These show promise for use as practical biomarkers for the rapid identification of S. thermophilus and L. bulgaricus. After verification of these amino acid sequences, mass differences relative to those of genome-sequenced strains have the potential for distinguishing bacteria at the strain level.

A microwave-assisted continuous-flow sequential extraction system was developed for rapid fractionation analysis of heavy metals in soil. Insertion of pressure-adjusted air between the extractants provided stable flows of the extractants without mutual mixing and back-pressure influence of a column packed with soil, thereby facilitating reliable continuous-flow extractions. In addition, use of pure water as a pumping solvent removed metal contamination because of direct contact between corrosive extractants and the pump containing metallic materials. Focused microwave irradiation to the soil accelerated the selective extractions of the acid-soluble and reducible fractions of heavy metals in soil in the first and second steps of the sequential extraction conditions, as defined by the Commission of the European Bureau of Reference (BCR). The microwave-assisted continuous-flow extraction provided high correlations in amounts of six heavy metals except Zn in the first step and Cu in the second step extracted from a reference sludge soil, BCR CRM 483, with a conventional batchwise extraction proposed by BCR; continuous-flow extraction assisted by conductive heating provided lower correlations for all the six metals. The proposed method drastically reduced the time required for the sequence extraction to ca. 65 min without losing accuracy and precision of the fractionation analysis of heavy metals in soil, whereas the BCR batchwise method requires ca. 33 h.

An on-line column preconcentration technique coupled with inductively coupled plasma-mass spectrometry (ICP-MS) and -atomic emission spectrometry (ICP-AES) was developed using a mini-column (ca. 3 mm i.d., 40 mm length), that was packed with chelating resin (0.2 g) of iminodiacetic acid groups, Muromac A-1. After the preconcentration step, the column was washed with ammonium acetate buffer (pH 5.5) and water to remove major elements, such as Ca and Mg, and then eluted with 4 ml of 2 mol l(-1) nitric acid. Eleven trace elements (Al, V, Fe, Co, Ni, Cu, Zn, Cd, Pb, Th and U) in seawater were determined by ICP-MS/AES. Recoveries for most of the elements tested were over 90%, although those for Al, V and Th were around 70%. The accuracy of the proposed method was evaluated by analyzing a standard reference material of seawater (NASS-4, NRC Canada). The values of Fe, Co, Ni, Cu, Zn, Cd and Pb obtained with the present method showed good agreement with the certified values as judged from the standard deviation. The method was successfully applied to characterize seasonal variations of trace elements in deep seawater (DSW) and surface seawater (SSW). In addition, no serious decrease in analytical performance of the present column system was observed during the experimental period of about 1 year.

A novel postcolumn photooxidation reactor with remarkably high efficiency was developed. It was installed into a system of liquid chromatography (LC) combined with inductively coupled plasma mass spectrometry (ICPMS) using a hydride generation (HG) technique to enable sensitive speciation of organic arsenicals. An LC eluent was passed into a reaction tube that runs through a low-pressure mercury lamp of a high-efficiency photooxidation (HEPO) reactor. It provided efficient irradiation with vacuum ultraviolet light at 185 nm, which induced oxidative decomposition of persistent organic arsenicals, for example, arsenobetaine, to the final product, arsenate, for an extremely short time, 3.5 s, without using any oxidizing agents. That arsenate was subsequently detectable by HG-ICPMS. In contrast, conventional photooxidation reactors require more than 100 s for completion, even when using a peroxodisulfate solution as an oxidizing agent. Because of the absence of broadening and dilution of the LC-separated peaks caused by mixing with the oxidant solution, the detection limits for 10 arsenic species including the persistent organic arsenicals by the proposed LC-HEPO-HG-ICPMS ranged from 2.3 to 18 ng of As/L, which were about one-tenth as low as those by LC-ICPMS. The proposed method revealed for the first time the occurrence of 20 arsenic species, including the unknown trace species, which were undetectable by LC-ICPMS and LC-HG-ICPMS, in human urine.

A microwave digestion method with HNO(3) alone was conducted at a temperature as high as 250 degrees C for determination of 19 trace elements (Li, Be, V, Cr, Mn, Co, Ni, Cu, Zn, Ga, As, Se, Rb, Sr, Cd, Cs, Ba, Hg, and Pb) in coal jointly by inductively coupled plasma optical emission spectrometry (ICP-OES), inductively coupled plasma mass spectrometry (ICP-MS), and flow injection ICP-MS (FI-ICP-MS). The validity of determination was assessed by using three standard coals, SRM 1632c, BCR 180, and SARM 19. It was found that the high-temperature digestion led to an extensive decomposition of the organic matrix and clay in coal, and no dissolved and solid carbon remained in the final solution after evaporation. Good recoveries were observed for all trace elements in three coals, with the exception of V, Rb, and Cs in high-ash SARM 19. Additionally, FI-ICP-MS combined with the present digestion without evaporation pretreatment was proved to be a rapid and efficient approach for determination of ultra-trace elements such as Se, Cd, and Hg in coal.

Desorption/ionization on porous silicon-mass spectrometry (DIOS-MS) is a novel soft ionization MS technique that does not require any matrix reagent, ideally resulting in fewer obstructive peaks in the lower mass region. In this study, the etching conditions of porous silicon spots as an ionization platform of DIOS-MS were investigated for determining the molecular weight distribution (MWD) of polymers. To evaluate the accuracy of DIOS mass spectra observed using porous silicon spots prepared under various etching conditions, a certified polystyrene (PS) standard sample with an average molecular weight of ca. 2400 was used as a model sample. By optimizing the etching conditions, the MWD of the PS sample could be accurately observed by DIOS-MS using both p-type and n-type porous silicon spots. Especially, in the case of a suitable n-type spot, an accurate peak distribution with very fewer obstructive background peaks could be observed using the minimum laser power, comparable to the conventional matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS).

We developed and optimized a system coupling microchip capillary electrophoresis (MCE) and laser-induced fluorescence (LIF) detection for the analysis of microorganisms. The MCE-LIF system successfully separated pure cultures of lactic acid bacteria and Saccharomyces cerevisiae within 200 s. The results indicate that the MCE system can be conveniently used for the rapid and highly sensitive detection of microorganisms. Thus, MCE can provide a cheap and simple method for the on-line detection of microbial contamination.

A novel data processing method for a hyphenated technique, size exclusion chromatography/matrix-assisted laser desorption/ionization-mass spectrometry (SEC/MALDI-MS), has been proposed to determine accurate molecular weight distributions on the basis of the individual oligomer species of a polymer. This method is based on the concept that the individual peak intensities of MALDI mass spectrum observed for every SEC fraction with narrow molecular weight distribution could be adjusted to the quantified values to reveal the accurate molecular weight distribution using the signal intensity of the corresponding fraction on the SEC chromatogram observed with a refractive index detector. At first, the theory of the proposed date processing is described in detail. Then, experimental verification of the method is described. This was performed through the characterization of mixtures of three kinds of monodispersed polystyrene reference materials (weight average molecular weight = ca. 6000, 10000, and 18000) as model samples. An accurate trimodal molecular weight distribution for the individual oligomer species of the sample was obtained without any influence of the chromatographic band broadening observed in the original SEC chromatogram. Moreover, the method for depicting the elution profiles of individual oligomer species during SEC separation was also obtained as a "mass chromatogram" using the data processing procedure.

The coupling of a high-power nitrogen (N2) microwave-induced plasma (MIP) mass spectrometry--(MS) (1.3 kW) with high-performance liquid chromatography, connected with concentric nebulizer (CN), ultrasonic nebulizer (USN) and a hydride generation (HG) systems, for the optimization and determination of selenium compounds, has been carried out. The MIP-MS system fulfils the ideal requirement being an on-line real-time chromatographic detector for Se speciation analysis. Interchanging of MIP-MS system fabricated nebulizer (concentric) with an ultrasonic nebulizer increases about 3.4-12 (peak height) and 6.5-10 (peak area) times ion signals for the selenium compounds. The detection limits for selenate, selenite, trimethylselenonium ion (TmSe), selenomethionine (Semet) and selenoethionine (Seet) (in Milli-Q-water) obtained with the optimized HPLC-USN-N2MIP-MS system are 0.11, 0.14, 0.09, 0.14 and 0.10 microg L(-1), respectively, about 12-48 times lower than the HPLC-CN-MIP-MS and 1.5-4.4 (peak height) times lower compared to the HPLC-CN-inductively coupled plasma (ICP)-MS coupling. Considering peak area, the repeatability (R.S.D. for three successive analyses) and intermediate precision (R.S.D. for three successive analyses performed on three different days), achieved for five Se compounds are 0.8-5.6, and 1.1-5.9%, comparable with the HPLC-CN-ICP-MS, HPLC-HG-MIP-MS and HPLC-CN-MIP-MS systems. The combined HPLC-USN-N2MIP-MS has been adequately applied for the determination of Se compounds in certified National Institute for Environmental studies human urine CRM No. 18. The results reasonably agree with the HPLC-CN-ICP-MS values. This encouraging combination may be an alternative ion source of mass spectrometry for coming generation in regard to the selenium speciation analysis.

Uwakai of Japan is famous for pearl and yellowtail fish culture. Recently, pearl culture farming in that region has suffered from a low production of pearls. An illegal use of organotin antifouling paints on fishing nets was reported. In the line of pollution studies, thus, the present investigation was carried out to examine the contamination status and fate of organotin compounds. Totally, 23 water, 10 sediment and 8 pearl oyster tissue samples were analyzed for tributyltin (TBT), triphenyltin (TPT), and their breakdown products (di- and mono compounds) by gas chromatography combined with inductively coupled plasma mass spectrometry (GC/ICP-MS). The results show that the TBT concentrations in water, sediment and biota were in the range from 0.11 to 10.6 ng Sn l(-1), 0.35 to 2500 ng Sn g(-1), and 50.4 to 181 ng Sn g(-1), respectively. The values for sediment and biota are expressed on the dry-weight basis. Triphenyltin in water, sediment and biota were in the range from 0.009 to 0.108 ng l(-1), non-detect to 12.7 ng g(-1), and non-detect to 6.83 ng g(-1), respectively. Although the TBT concentration in seawater is below the tentative assessment level of 10 ng l(-1) set by the Japanese Environment Agency in 1992, it may cause endocrine disruption/other effects in aquatic organisms. Octyltin compounds (mono-, di- and trioctyltin) were also quantified in seawater and sediment. The detection of dibutyldimethyltin (DBDMT) and tributylmonomethyltin (TBMMT) in sediment (methylated butyltins comprised 2.8-31% of total butyltins), and TBMMT in seawater suggested that biomethylation of anthropogenic tributyltins is a significant transformation pathway in the coastal environment.

Selenium species, selenite, selenate, selenomethionine (Semet), seneloethionine (Seet) and trimethylselenonium ion (TmSe) were separated in aqueous solution using a gel-permeation (polyvinyl alcohol-based resin) GS-220 column by eluting with 25 mM tetramethylammonium hydroxide and 25 mM malonic acid at pH 7.9. The GS-220 column coupled with inductively coupled plasma mass spectrometry was used for the separation, identification, and quantification of selenium compounds present in certified reference material (CRM) No. 18 human urine from the National Institute for Environmental Studies in Japan (NIES). Spiking of the authentic standard to the urine and use of a silica-based LC-SCX cation-exchange column validated the peak of selenium compounds. High concentrations of chloride and bromide in the urine eluted from the GS-220 column formed molecular ions 40Ar37Cl+ and 81Br1H+ in the plasma, and these molecular ions created additional peaks in the chromatograms when 77Se and 82Se isotopes were monitored respectively. Thus, both the isotopes were selected concurrently for signal monitoring to eliminate the interfering signals. On the LC-SCX column, chloride and bromide were eluted with selenate and complicated its determination, but the peak of TmSe was baseline separated from rest of the Se compounds. Two unknown Se compounds were detected in both the columns. An additional Se compound having the same retention time as that of Semet was detected on the LC-SCX column. Peaks of selenite, selenate, TmSe and unknown selenium compounds in the urine were baseline separated on the GS-220 column, and were free from interferences. Therefore, the GS-220 column was used for the determination of selenium compounds in NIES CRM No. 18. Unknown Se compounds were the predominant selenium species followed by selenite, TmSe and selenate. The estimated value of TmSe as Se, by the standard additions method using the GS-220 column, was 3.42 +/- 0.17 microg l(-1) and was in good agreement with the LC-SCX value [3.38 +/- 0.21 (n=5) microg l(-1)].

This paper describes a robust and sensitive method for the determination of arsenic species in seawater by ion-exclusion liquid chromatography (LC) combined with inductively coupled plasma mass spectrometry (ICP-MS) using reaction cell and hydride generation (HG) techniques. A good separation of arsenite, arsenate, and monomethylarsonic acid was achieved using an ion-exclusion column packed with a sulfonated polystyrene resin and a dilute nitric acid at pH 2.0 as the eluent, even when a large volume, i.e. 200 mul, of seawater samples containing a large amount of matrix was repeatedly injected. Separations of the chloride ion due to the matrix and arsenic species were partially performed; however, the extensive peak of ArCl due to high content of Cl(-) in a sample overlapped peaks of the three arsenic species on (75)As measurement by ICP-MS. This ArCl polyatomic interference was efficiently eliminated by collision of ArCl molecules with helium in an octopole reaction cell which was introduced prior to a mass spectrometer. Detection limits of the three arsenic species in a sample containing 2% Cl(-), the concentration of which is comparable to that in a seawater sample, by LC-ICP-MS with the octopole reaction system (ORS), ranged from 21 to 25 pg As ml(-1); these values were three-six times lower than those by LC-ICP-MS without ORS. As another technique for ArCl interference elimination, HG prior to ICP-MS was also successfully used not only to reduce the interference but also to improve the detection limits to 3.4-4.5 pg As ml(-1). The developed LC-ICP-ORS-MS and LC-HG-ICP-MS were validated by analyzing a certified reference material (CRM) of seawater. In addition, no serious decrease in analytical performance of present methods was observed in the experimental periods of half a year for LC-ICP-ORS-MS and 1 year for LC-HG-ICP-MS, respectively. The latter method was successfully applied to characterize seasonal variations of three arsenic species in deep seawater and surface seawater.