We have developed a fluorescence resonance energy transfer (FRET) probe based on the conjugation of a quantum dot (QD) with dye (YOYO-3) intercalated DNA. The FRET-inducing electrostatic coupling of DNA and the QD made structural changes to the QD-DNA conjugates, which significantly prevented an enzymatic reaction between the DNA and a conventional restriction endonuclease (EcoRI).

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Recent progress in analytical terahertz (THz) spectroscopy is reviewed with illustrative examples showing that it is an effective method for detecting and identifying intermolecular interactions in chemical compounds, such as hydrogen bonds. The unique and characteristic properties of THz waves, their significance to both science and industry, and the bases of one of the successful fields of analytical THz spectroscopy, namely THz time-domain spectroscopy and THz imaging for chemical analysis, are described. Preliminary quantitative studies are presented to show the potential of THz spectroscopy for the detection and identification of intermolecular hydrogen bonds in unknown mixture samples. The selective detection of intramolecular hydrogen bonds and the detection of intramolecular interactions in ice are also introduced. Some brief remarks are provided on future developments, the main issues, and the prospects for analytical THz spectroscopy.

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Micromarkers with five photoluminescence colors were developed as tracers for shadowing pursuits. The markers are colorless powders with particle diameters of several tens to several hundreds of micrometers, prepared using a cryogenic sample crusher. They were visualized using red, green, yellow, magenta or cyan photoluminescence under ultraviolet light at approximately 365 nm. The markers were composed of photoluminescent compounds dispersed in polyvinyl butyral. The photoluminescent compounds in the polyvinyl butyral were stable under ambient conditions for more than one year after application. The compounds with the red, green, yellow, magenta and cyan photoluminescence contained a europium (Eu³⁺) complex, a terbium (Tb³⁺) complex, a mixture of Tb³⁺ and Eu³⁺ complexes, a mixture of Eu³⁺ complex and o-coumaric acid, and a mixture of Tb³⁺ complex and 7-hydroxycoumarin along with a few drops of a sodium bicarbonate aqueous solution, respectively. Neodymium (Nd³⁺) and ytterbium (Yb³⁺) complexes with photoluminescence in the near-IR wavelength region can also be added to these visible photoluminescent compounds as secret markers for discrimination. The markers were non-destructively identified using a microscopic FT-IR spectrometer and a microscopic spectrometer equipped with a fluorescence detector.

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In this paper, a novel flow injection chemiluminescence (FI-CL) method is proposed for the determination of picomolar L^-1 levels of hydrogen peroxide (H₂O₂) in exhaled breath condensate (EBC). This method is based on the oxidation of a low concentration of luminol (10^-7 M) by H₂O₂ at a low concentration level (< 10^-8 M) in an alkaline medium catalyzed by a complex, K₅[Cu(HIO₆)₂] (DPC), which is not interfered by other metal ions or horseradish peroxidase (HRP). Under the optimum conditions, H₂O₂ was determined over the range of 1.0 × 10^-10 to 1.0 × 10^-8 mol L^-1 with a detection limit of (3σ) of 4.1 × 10^-11 mol L^-1. The relative standard deviation (RSD) was 3.2% for 5 nmol L^-1 H₂O₂ (n = 7). The proposed method offers the advantages of ultra-sensitivity, selectivity, simplicity and rapidity for H₂O₂ determination. It was successfully applied to directly determine trace amounts of H₂O₂ (nmol L^-1) in human's EBC of both rheum and healthy volunteers. A statistically significant difference was found between patients with rheum (n = 11) and control subjects without rheum (n = 11).

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An inclusion complex consisting of a fluorescent phenylboronic acid (C1-APB) and β-cyclodextrin (β-CD) acts as a supramolecular saccharide sensor whose response mechanism is based on photoinduced electron transfer (PET). This study evaluated four kinds of cyclodextrins (α-CD, β-CD, γ-CD, and NH₂-β-CD) by comparing their pH profiles, and confirmed that β-CD was the best host for C1-APB because the C1-APB/β-CD complex exhibited high affinity for saccharides as well as high fluorescent recovery upon saccharide binding. An investigation of the β-CD concentration effect revealed the formation of a 1:1 inclusion complex of C1-APB with β-CD. The observed saccharide selectivity of the C1-APB/β-CD complex is in the following order: D-fructose (4039 ± 69 M^-1) > D-ribose (1083 ± 26 M^-1) > L-arabinose (474 ± 11 M^-1) > D-galactose (318 ± 3 M^-1) > maltotoriose (135 ± 5 M^-1) > D-glucose (114 ± 2 M^-1) > maltose (81 ± 2 M^-1). In addition to monomer emission, dimer emission from pyrene dimers was observed in the spectra for the C1-APB/γ-CD complex, which allowed a ratiometric analysis. This study shows that the combination of a simple fluorescent probe, C1-APB, with various CDs diversifies the response systems for saccharide recognition.

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Water-soluble near-infrared (NIR) fluorescent labeling probes, named KSQ-3 and -4, which are based on a squaraine backbone, were synthesized and applied to biological labeling. The presented results demonstrate that the large, planar and hydrophobic squaraine dye becomes fully soluble in aqueous solution by the introduction of several sulfo group terminated alkyl substituents. Especially KSQ-4, which is substituted with four sulfo groups, exhibited perfect water solubility and significant fluorescence emission at the NIR region (817 nm) in the presence of bovine serum albumin (BSA). BSA was covalently labeled with KSQ-4, and the conjugate showed a strong absorption peak at 787 nm, which indicates compatibility with commercially available NIR laser diodes used for exciting the fluorophore. Furthermore, strong fluorescence emission was observed at 812 nm (Φ = 0.08).

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A new visual colorimetry for trace antimony(V) based on ion-pair solid-phase extraction to a PTFE-type membrane filter with bis[2-(5-chloro-2-pyridylazo)-5-diethylaminophenolato]cobalt(III) ion ([Co(5-Cl-PADAP)₂]⁺) has been developed. Experiments showed that hexachloroantimonate(V) ion (SbCl₆^-) was adsorbed with [Co(5-Cl-PADAP)₂]⁺ to the front surface of the PTFE filter. The adsorption of antimony(V) ion was promoted by the addition of lithium chloride as a source of chloride ion. The excess reagent of [Co(5-Cl-PADAP)₂]⁺ was eluted by rinsing with a 10 wt% methanol aqueous solution. In this case, the slow rate of the hydrolysis reaction of SbCl₆^- and the difference of the hydrophobicity of the ion pairs were important for adsorption and separation with a PTFE-type membrane filter. The antimony(V) concentration was determined through a visual comparison with a standard series. The visual detection limit was 0.10 µg. The calibration curve assessed with the reflection spectrometric responses at 580 nm was linear in the concentration range of 0.10 - 1.2 µg (r = 0.996). The proposed method has been applied to the determination of sub-microgram levels of antimony(V) ion in water samples.

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Novel 4-acyl-5-pyrazolones having aza-15-crown-5 (HPMP-A15C5) and aza-18-crown-6 (HPMP-A18C6) moieties as an intramolecular synergist have been synthesized by simple coupling reactions between 1-phenyl-3-methyl-4-chloroacetyl-5-pyrazolone and the corresponding azacrown ethers. The solvent extraction of the divalent metal ions (Mn²⁺, Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺, Cd²⁺ and Pb²⁺) were examined. Synergistic extractions with 1-phenyl-3-methyl-4-benzoyl-5-pyrazolone (HPMBP) and benzocrown ethers were also examined for a comparison. Extractions with the novel acylpyrazolones were unique and quite different from those with HPMBP and benzocrown ethers. The synergistic effect with benzocrown ethers was low, and an obvious difference brought by the ring size was not observed. The extractions of the divalent metal ions with HPMP-A18C6 were generally enhanced, as compared to those alone with HPMBP; on the contrary, the extractions with HPMP-A15C5 were relatively poor.

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A new extraction and cleanup procedure with gas chromatography was developed for the sensitive determination of acephate, dimethoate, malathion, diazinon, quinalphos, chlorpyrifos, profenofos, α-endosulfan, β-endosulfan, chlorothalonil and carbaryl using 1-chloro-4-fluorobenzene as an internal standard in fruits and vegetables. Several extracting and eluting solvents for solid-phase extraction were investigated. The overall extracting solvent with a mixture of acetone:ethyl acetate:hexane (10:80:10, v/v/v) and a eluting solvent of 5% acetone in hexane used with the RPC18 cartridge gave the best recovery for all of the investigated pesticides, and minimized the interference from co-extractants. Under the optimal extraction and clean-up conditions, recoveries of 85 - 99% with RSD < 5.0% (n = 3) for most of the pesticides at the 0.02 - 0.5 mg/kg level were obtained. The limit of detection was between 0.005 - 0.01 mg/kg and the limit of quantification was 0.01 mg/kg. This analytical procedure was characterized with high accuracy and acceptable sensitivity to meet requirements for monitoring pesticides in crops.

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The thermal stability of a redox enzyme, bilirubin oxidase (BOD), has been quantitatively evaluated by measuring the inactivation kinetics of BOD at several temperatures. The enzyme activity is directly related to the mediated bioelectrocatalytic current for the BOD-catalyzed reduction of O₂. Thus, the inactivation process is measured by the time-dependent decrease in the bioelectrocatalytic current. The results reveal that the inactivation obeys first-order kinetics, whose rate constants (k) are determined at pH 7.0 and at 50 - 70°C. The half life of BOD activity, calculated from the k value at 50°C is 114 min, which is in harmony with the thermal-stability data given in a catalog by Amano Enzyme Inc. The bioelectrocatalysis method allows in situ measurements of the inactivation kinetics in the period of a few minutes at relatively high temperatures. The rate constants show a large temperature dependence, leading to a large Arrhenius activation energy (E_A) of 221 kJ mol^-1. The activation Gibbs energy (ΔG^≠), activation enthalpy (ΔH^≠), and activation entropy (ΔS^≠) are also determined.

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A living cell has numerous proteins, only a few thousand of which have been identified to date. Cell-free protein synthesis is a useful and promising technique to discover and produce various proteins that might be beneficial for biotechnological, pharmaceutical, and medical applications. For this study, we evaluated the performance and the general applicability of our previously developed microreactor array chip to cell-free protein synthesis by comparisons with a commercially available system. The microreactor array chip comprises a temperature control chip made of glass and a disposable reaction chamber chip made of polydimethylsiloxane (PDMS). For evaluation of the microreactor array chip, rat adipose-type fatty acid binding protein, glyceraldehyde-3-phosphate dehydrogenase, cyclophilin, and firefly luciferase were synthesized from their respective DNA templates using a cell-free extract prepared from Escherichia coli. All these proteins were synthesized in the microreactor array chip, and their respective amounts and yields were investigated quantitatively.

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A stable solid spike for the measurement of uranium and plutonium content in nitric acid solutions of spent nuclear fuel by isotope dilution mass spectrometry has been prepared at the European Commission Institute for Reference Materials and Measurements in Belgium. The spike contains about 50 mg of uranium with a 19.838% ²³⁵U enrichment and 2 mg of plutonium with a 97.766% ²³⁹Pu abundance in each individual ampoule. The dried materials were covered with a thin film of cellulose acetate butyrate as a protective organic stabilizer to resist shocks encountered during transportation and to eliminate flaking-off during long-term storage. It was found that the cellulose acetate butyrate has good characteristics, maintaining a thin film for a long time, but readily dissolving on heating with nitric acid solution. The solid spike containing cellulose acetate butyrate was certified as a reference material with certified quantities: ²³⁵U and ²³⁹Pu amounts and uranium and plutonium amount ratios, and was validated by analyzing spent fuel dissolver solutions of the Tokai reprocessing plant in Japan. This paper describes the preparation, certification and validation of the solid spike coated with a cellulose derivative.

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The removal properties of diesel exhaust particles (DEP) were investigated using an engine exhaust particle size spectrometer (EEPS), field emission-type scanning electron microscopy (FE-SEM) and time-of-flight secondary ion mass spectrometry (TOF-SIMS). DEP were treated using a dielectric barrier discharge (DBD) reactor installed in the tail pipe of a diesel engine, and a model DBD reactor fed with DEP in the mixture of N₂ and O₂. When changing the experimental parameters of both the plasma conditions and the engine load conditions, we obtained characteristic information of DEP treated with plasma discharges from the particle diameter and the composition. In evaluating the model DBD reactor, it became clear that there were two types of plasma processes (reactions with active oxygen species to yield CO₂ and reactions with active nitrogen species to yield nitrogen containing compounds). Moreover, from the result of a TOF-SIMS analysis, the characteristic secondary ions, such as C₂H₆N⁺, C₄H₁₂N⁺, and C₁₀H₂₀N₂⁺, were strongly detected from the DEP surfaces during the plasma discharges. This indicates that the nitrogen contained hydrocarbons were generated by plasma reactions.

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In order to overcome the difficulties with existing methods for sample immobilization in imaging Halobacterium salinarum (H. salinarum) living in a highly salty medium by atomic force microscopy (AFM), a heat-fixation method was, for the first time, used to overcome existing problems in preparing samples for AFM. The effect on the cell morphology of the heat-fixation method was studied by MAC mode AFM, and was compared with the drop-and-dry and the polylysine-adhesion methods. It was found that the heat-fixation method can be successfully used for preparing Gram-negative and Gram-positive bacteria samples for AFM studies. Using this method, high-resolution AFM images of H. salinarum were obtained. Round protrusions on the cell surface and horn-like protrusions only at one pole of H. salinarum were observed.

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Simultaneous determination of hydrazine (HZ) and phenylhydrazine (PHZ) by H-point standard addition method (HPSAM) and partial least squares (PLS) regression was carried out based on kinetic data from novel potentiometry methods. The rate of chloride ion production in the reaction of HZ and PHZ with N-chlorosuccinimide (NCS) was monitored by a chloride ion-selective electrode. The experimental data show the good ability of ion-selective electrodes (ISEs) as detectors not only for the direct determination of chloride ion but also for simultaneous kinetic-potentiometric analysis using HPSAM and PLS methods. The methods are based on the differences observed in the production rate of chloride ions. The results show that simultaneous determination of HZ and PHZ can be performed in concentration ranges of 0.5 - 20.0 and 0.8 - 25.0 µg mL^-1, respectively. The total relative standard error for applying the PLS method to 8 synthetic samples in the concentration ranges of 1.0 - 16.0 µg mL^-1 for HZ and 2.0 - 16.0 µg mL^-1 for PHZ was 3.96. In order for the selectivity of the method to be assessed, we evaluated the effects of certain foreign ions upon the reaction rate and assessed the selectivity of the method. Both methods (PLS and HPSAM) were evaluated using a set of synthetic sample mixtures and then applied for simultaneous determination of HZ and PHZ in water samples.

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A 5-amino-8-hydroxyquinoline (AHQ)-modified gold nanoparticle (GNP) layer was fabricated on an inner wall of a silica capillary column by alternatively passing a citrate-stabilized GNP solution and an AHQ solution in a repeating fashion. The observations by a field emission scanning electron microscope showed that the thickness of the resulting GNP layer was about 0.15 µm. This column was then used as an open tubular solid-phase extraction column for cadmium, followed by electrothermal atomic absorption spectrometric determination. The detection limit of 0.009 ng ml^-1 was obtained.

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A headspace solid-phase microextraction method has been developed for the determination of 8 pesticides in vegetables and fruits by using gas chromatography with an electron capture detector. Two types of fibers (polyacrylate, 85 µm and polydimethylsiloxane, 100 µm) have been assayed and compared. The main factors: extraction and desorption parameters, ionic strength, and the effects of dilution and organic solvents, were studied and optimized. The optimized procedures resulted in more than 80% recovery for all the investigated vegetable and fruit samples with RSD values below 10%.

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The interactions between Cd²⁺ and the C-terminal region of phytochelatin (PC) synthase using recombinant wild-type and mutant PC synthase were studied. We show that site-directed mutagenesis of Cys residues at C³⁵⁸C³⁵⁹XXXC³⁶³XXC³⁶⁶ motif decreases the number of Cd²⁺ and other heavy metal ions interacting with the enzyme, and that the motif binds the metals discriminatingly. The optimum binding ratio of PC synthase to Cd²⁺ was also determined. The findings indicate that Cys exists as a free SH residue and that it is involved in the regulation of PC enzyme activity by transferring the metals into closer proximity with the catalytic domain. These results are important in understanding heavy metal detoxification mechanisms in higher plants, a step towards phytoremediated-applications.

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A robust oxygen-flask combustion system was developed using light beam ignition. An appropriate amount of environmental sample was wrapped with a small piece of filter paper and held in a basket made of platinum-zirconium spiral. After the sample was inserted into an oxygen-filled flask containing an acid mixture, the light beam from an overhead projector lamp was focused to ignite the sample. The combusted products were absorbed into the acid mixture by shaking the flask and then used for the ICP-AES measurement of metals. The proposed method was successfully applied to the determination of metals in Environmental Standard Reference Materials and Artemia salina. The present method is also applicable to ion chromatographic measurements of chlorine in polyvinyl chloride wall paper by using hydrogen peroxide as absorbent.

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A quantitative analysis of trace lead in tin-base lead-free solder was carried out with laser-induced plasma spectroscopy (LIPS). In order to evaluate the applicability of the technique for rapid in situ analytical purposes, measurements were performed in air at atmospheric pressure, and the emission characteristics of the plasma produced by a Q-switched Nd:YAG laser over a laser energy range of 10 - 90 mJ were investigated using time-resolved spectroscopy. The experimental results showed that the emission intensity of the analysis line (Pb I 405.78 nm) was maximized at a laser energy of around 30 mJ, and a time-resolved measurement of a spectrum with a delay time of 0.4 µs after the laser pulse was effective for reducing the background continuum. Based on the results, lead-free solder certified reference materials were analyzed for trace lead (concentration 174 - 1940 ppm), and a linear calibration curve was obtained with a detection limit of several tens ppm.

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A fluorescent probe, DPPEC (1,2-dipalmitoylglycerophosphorylethanolamine labeled with coumarin) was developed for detecting hydroxyl radical (·OH) in lipid membranes. The coumarin moiety contributes to the fluorescent detection of ·OH and the phospholipids moiety gives a driving force to localize the probe in lipid membranes. DPPEC in liposomal membranes rapidly reacted with ·OH and increased the fluorescence intensity, depending on the concentration of ·OH. The increase in the fluorescence intensity induced by ·OH was effectively suppressed by the addition of DMSO. The probe exhibited a higher fluorescence response to ·OH over other reactive oxygen species, such as hydrogen peroxide, nitric oxide, peroxynitrite, alkylperoxyl radical, and hypochlorite. DPPEC would be useful as a new type of fluorescent probe that can localize in lipid membranes and detect ·OH efficiently.

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A microscope device to measure the circular dichroism (CD) spectra of a specified microscopic region of chiral samples was constructed by combining of a couple of objective lenses and a CCD camera, which was installed in a sample chamber of a commercially available CD spectropolarimeter. By using this apparatus, high quality micro-CD spectra in the 60 × 60 µm region of samples could be measured. Micro-CD spectra of thin film of chiral DNA samples on glass and a natural kidney bean leaf were measured, and the potential of the micro-CD apparatus was successfully demonstrated.

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