Vol. 53 No. 5
May, 2004
Yu-suke Torisawa1, Hitoshi Shiku1, Tomoyuki Yasukawa1 and Tomokazu Matsue1
1 Graduate School of Environmental Studies, Tohoku University, Aramaki, Aoba-ku, Sendai-shi, Miyagi 980-8579(Received 19 January 2004, Accepted 9 February 2004)
A living cell-based assay is widely used for the characterization of various cellular functions, screening of drugs and environmental monitoring. Recent progress in cell culture and microfabrication technologies has attracted the integration of cell culture and sensors on a chip. A bioassay using a cell chip makes it possible to evaluate small-volume samples with multitudinous characterizations, and is applicable for a rapid and simple detection system. This paper reviews cell-based bioassay using living cells integrated on a chip. The cell patterning and three-dimensional cell culture facilitates the integration of cell culture technology. These in vitro culture technologies are appropriate for the recapitulation of in vivo-like cell behavior, because of the potential to control the cell differentiation and polarity. Alternatively, the evaluation methods, such as cellular acidification, oxygen consumption and impedance measurements, are principally non-invasive, and are especially expected in the field of anticancer drug sensitivity test using a biopsy tumor.
Keywords : cell-based assay; cell chip; cell patterning; 3-dimensional cell culture; anticancer drug sensitivity test; microphysiometer; LAPS; ISFET.
Seiichi Nishizawa1, Keitaro Yoshimoto1, Takehiro Seino1, Chun-Yan Xu1 and Norio Teramae1
1 Department of Chemistry, Graduate School of Science, Tohoku University, Aramaki, Aoba-ku, Sendai-shi, Miyagi 980-8578(Received 6 January 2004, Accepted 5 February 2004)
In combination with abasic site (AP site)-containing oligodeoxynucleotides (ODNs), we demonstrate the potential use of hydrogen bond-forming ligands, 2-amino-7-methyl-1,8-naphthyridine (AMND) and 2-amino-4-oxopteridine (pterin), for the recognition of nucleobases in the aqueous phase. Our strategy is based on the construction of an AP site in ODN duplexes, which allows small synthetic and/or biotic ligands to strongly bind to target nucleobases; an AP site-containing ODN is hybridized with a target ODN so as to place the AP site toward a target nucleobase, by which hydrophobic microenvironments are provided for ligands to recognize target nucleobases through hydrogen-bonding. From an examination of the binding behaviors by melting temperature (Tm), fluorescence and circular dichroism spectra measurements, it has been found that AMND selectively binds to a cytosine base with a 1:1 binding constant of>106 M-1, while pterin shows selectivity for a guanine base (K11=1.2×104 M-1) over other nucleobases. The observed high stabilities of 1:1 complexes can be explained by a cooperative binding event, i.e., hydrogen-bonding with target nucleobases and stacking with nucleobases flanking the AP site. The potential use of these ligands is also presented for the fluorescence detection of single-nucleotide polymorphisms (SNPs) of the cancer repression gene p53.
Keywords : ligand; hydrogen bond; stacking; abasic site; fluorescence detection; nucleobase; single-nucleotide polymorphism.
Hitoshi Muguruma1 and Atsunori Hiratsuka2
1 Department of Electronic Engineering, Shibaura Institute of Technology, Shibaura 3-9-14, Minato-ku, Tokyo 108-8548(Received 23 January 2004, Accepted 5 March 2004)
A biosensor is a device that converts information on the concentration of chemical or biochemical substances into a quantifiable electronic signal. The typical structure of a biosensor is an integrated product utilizing biological components and transducers. We have indicated that plasma-polymerized thin films have potential for use in the interfacial design between these two components for biosensors. A plasma-polymerized film was achieved in a plasma in the vapor phase. The properties of the film are that they: (i) are extremely thin (<1 µm), (ii) provide good adhesion to the substrate, (iii) are pin-hole free and present a flat surface structure, (iv) are mechanically and chemically stable because of the highly branched and cross-linked structure in the polymer, and (v) allow for large amount of biological components, such as enzymes and antibodies, to be loaded onto the surface of the film. Recently, the trend in biosensors can be addressed concerning two main aspects. One is a micro total analytical system and/or a micro electro mechanical system, which is an analytical system integrated into one chip in order to reduce the solvent, the amount of the sample, and the time and cost of analysis, The other is a post genome research tool, such as a DNA or a protein chip; the former allows an easy and rapid analysis for gene expression and single-nucleotide polymorphism; the latter allows proteome analysis e.g., a capillary electrophoresis chip and analysis for biospecific interaction. In this article, we stress the usefulness of plasma-polymerized films and/or a process for a new generation of biosensers.
Keywords : plasma-polymerized film; immobilization matrix; biosensor; micro total analytical systems; micro electro mechanical systems; surface plasmon resonance; mediator; DNA chip; protein chip; capillary electrophoresis chip.
Takashi Ishiyama1,2, Teruyuki Shimadu1, Chieko Nudeshima1, Junya Hoshi1 and Yuko Sasaki1
1 The Tokyo Metropolitan Research Institute for Environmental Protection, 1-7-5, Shinsuna, Koto-ku, Tokyo 136-0075(Received 27 October 2003, Accepted 25 January 2004)
A selective and sensitive method was developed for the determination of estrogens and bisphenol A by high-performance liquid chromatography with electrochemical detection. A mixing solution consisting 45 vol% acetonitrile and 55 vol% water containing 0.07 mol l-1 sodium perchlorate at pH 2.7 was used as a mobile phase. The electrolytic oxidation of estrogens and bisphenol A was performed on a glassy carbon working electrode at 1.05 V vs. Ag/AgCl. The calibration curves (peak area vs. concentration) were linear (correlation coefficient ≥0.999) in the range from 10 ng ml-1 to at least 1000 ng ml-1, and passed through the origin with a relative standard deviation of 0.6 to 2% for 100 ng ml-1 (n=5). This method was applied to the simultaneous determination of these compounds at ng l-1 level in river water and the discharge water of a sewage treatment plant. Interference from humic compounds could be avoided by using a clean-up technique with a 45 vol% methanol solution at pH 9 to 10, and a stable background current was obtained. The detection limits calculated from the S/N ratio (S/N=3) were ca. 5 ng l-1. The time required for an analysis after solid-phase extraction was within 40 min. The proposed method without any troublesome and time-consuming pretreatment process, such as derivatization, is more simple and rapid compared with conventional methods.
Keywords : high-performance liquid chromatography; electrochemical detection; determination of estrogens and bisphenol A; river water; discharge water of sewage treatment plant.
(Received 24 December 2003, Accepted 3 February 2004)
Utilizing the adhesion of a precipitate of ammonium phosphomolybdate onto a piezoelectric quartz crystal (PQC), phosphate ions in water could be determined as a frequency change of the PQC. The precipitate was formed in the tubing of the flow system by a precipitation reaction between phosphate and hexaammonium heptamolybdate ions, and adhered onto the PQC. The adhesion of the precipitate was largely dependent on a surface condition of the PQC. That is, the adhesion proceeded smoothly with time on a surface pretreated by coating with the precipitate of ammonium phosphomolybdate, although not smoothly on a clean surface. In order to obtain optimal conditions for the determination of phosphate ions, the effects of the composition, acid concentration and flow rate of the carrier solution, and the length of the reaction coil on the frequency change were examined by using a PQC pretreated by coating with the precipitate. Under the optimal conditions, the frequency change was proportional to the concentration of phosphate ions to 30 µmol dm-3. Phosphate ions in river and lake water samples were actually determined by the proposed method using a PQC. Results for the determination by the standard addition method were in fairly good agreement with those determined by spectrophotometry, i.e. a molybdenum-blue method. It is therefore suggested that the proposed method is applicable to the determination of phosphate ions in natural water.
Keywords : determination of phosphate ion; piezoelectric quartz crystal; ammonium phosphomolybdate; adhesion of a precipitate; flow analysis.
Tetsuo Uchida1, Eri Tsuzuki1, Yusuke Takahashi1 and Utako Inoue1
1 Laboratory of Analytical Chemistry, Department of Applied Chemistry, Nagoya Institute of Technology, Gokiso-cho, Showa-ku, Nagoya-shi, Aichi 466-8555(Received 15 December 2003, Accepted 1 March 2004)
A simple, rapid and contamination-free method of iron removal was developed for the determination of trace elements in steel by ICP-AES. A 200 mg sample was dissolved in HCl, HNO3 and HF containing Y as an internal standard, and evaporated to dryness. The residue was dissolved in 10 ml of 6 M HCl, then transferred to a Teflon test tube. Under vigorous stirring, MIBK was introduced at a flow rate of 1 ml/min through a Teflon tube inserted in the bottom, and the upper MIBK layer was suctioned off. This continuous extraction enabled the removal of 200 mg of iron using only 6 ml of MIBK. After evaporating the resulting aqueous layer(~8 ml) in an evaporation chamber, the residue was dissolved in 12 ml of 0.1 M HCl, and then used for an ICP-AES measurement of trace elements. The proposed method was applied to analyses of Certified Reference Materials prepared by the Japan Iron and Steel Federation. The analytical results for Mn, Ni, Cr, Cu, V, Co, Ti, Al and Ca agreed with the certified values.
Keywords : continuous extraction with MIBK; ICP-AES; trace elements in steels; internal standard; certified reference samples.
Tomoko Tamada1
1 Graduate School of Human and Environmental Studies, Kyoto University, Yoshida nihonmatsu-cho, Sakyo-ku, Kyoto-shi, Kyoto 606-8501(Received 25 November 2003, Accepted 7 February 2004)
The determination of trace amounts of aluminum in blood is problematic due to the great amount and complicated composition of organic and inorganic matrices. Therefore, most previous studies have investigated only serum or plasma samples, and there have been few studies on whole blood samples. In this study, organic matter in serum and whole blood samples was digested by microwave heating with acid, and then inorganic matter, such as iron, was removed using solvent extraction; finally, aluminum was fluorimetrically determined by the lumogallion method. Because the interference from matrices was markedly reduced, especially in whole blood samples, it enabled the determination of aluminum in serum and whole blood samples by the same operation. From the values in healthy human serum and whole blood, it also allowed an estimation of the aluminum concentration in erythrocytes; the distribution ratio of aluminum between serum and erythrocytes was also obtained.
Keywords : aluminum in blood; serum; whole blood; matrix interference; fluorimetry.
Daijiro Kobori1
1 Organo Corporation, 1-4-9, Kawagishi, Toda-shi, Saitama 335-0015(Received 11 December 2003, Accepted 21 February 2004)
The quantitative determination and depth profiling of polystyrene sulfonic acid(PSS), which was adsorbed to the surface of anion-exchange resins, were examined by attenuated total-reflection Fourier-transform infrared spectroscopy (ATR). A calibration curve that was calculated from the amount of adsorption of PSS, and the relative intensity of PSS to the resin by micro ATR showed a linear relationship within 58~718 mg, proving that it was possible to quantify PSS in this range. The depth profile of PSS was assumed an exponential function by secondary ion mass spectrometry (SIMS), and the relational expression of the relative intensity of PSS to resin by ATR and penetration depth(dp) of infrared was deduced. The depth profile that was calculated from the relative intensity and dp by variable-angle ATR was coincident with that of SIMS.
Keywords : ion exchange resin; ATR; depth profiling; polystyrene sulfonic acid.
Toru Fujimura1, Akira Yamamoto1,2 and Kazuaki Wagatsuma3
1 Steel Research Laboratory, JFE Steel Corporation, 1 Kawasakidori, Mizushima, Kurashiki-shi, Okayama 712-8511(Received 15 December 2003, Accepted 2 February 2004)
In an r.f. glow-discharge plasma, a d.c. bias current can be introduced by connecting an electric circuit comprising a low-pass filter and a variable resistor. The bias current promotes the emission excitations in the plasma, leading to an improvement of the detection power in the optical-emission spectrometry. By conducting a bias current of 27 mA, the emission intensities of the atomic resonance lines were several-times larger than those obtained with conventional r.f.-powered plasmas. The detection limits for the determination of alloyed elements in Fe-based low-alloyed standard samples were estimated to be 2 ppm Mn for Mn I 403.08 nm, 2 ppm Al for Al I 396.15 nm, 7 ppm Si for Si I 288.158 nm, and 8 ppm Cr for Cr I 425.43 nm.
Keywords : optical emission spectrometry; radio frequency-powered glow discharge plasma; conduction of a d.c. bias current; self-bias voltage; detection limit.
Tsutomu Miura
(Award by Tokyo Metropolitan University dated February 20, 2003)
A separation and preconcentration method of alpha emitting nuclides using an anion exchange resin, extraction chromatographic resin, chelating resin disk and anion exchange resin disk was studied for the determination of nuclides in environmental samples. The target alpha emitting nuclides were Po (210Po), U (238U, 235U and 234U), Th (232Th, 230Th and 228Th), Pu (238Pu, 239Pu and 240Pu) and Am (241Am). The aim of this study was to develop a rapid and efficient analytical method of these nuclides in environmental samples for environmental monitoring. In preliminary experiments, the adsorption and elution behaviors of the target nuclides and interference elements were investigated on an anion exchange resin (Th), crown ether impregnated extraction chromatographic resin (Po), organophosphorus extractant impregnated extraction chromatographic resin (Pu, Am), chelating resin disk (U) and anion exchange resin disk (Pu). The decontamination factors of interference elements were measured. The preparation procedures (alpha ray counting sample), including electrodeposition (Th, Po), spontaneous deposition (Po), coprecipitation onto micro-quantity NdF3 precipitate (Pu, Am) were studied. The developed methods were successfully applied to certified reference materials and environmental samples. As a result, the developed methods are useful for the monitoring of alpha emitting nuclides in environmental samples.
(Received February 2, 2004)
Keywords : alpha emitting nuclides; environmental samples; preconcentration; ion exchange chromatography; extraction chromatography; certified reference materials.
Go to the page of "Bunseki
Kagaku"
Go to the top page of JSAC©2004 The Japan Society for Analytical Chemistry
All rights reserved.
26-2, Nishigotanda 1-chome, Shinagawa-ku, Tokyo 141-0031, JAPAN