キャピラリー電気泳動法の高精度化･高感度化

研究期間:平成14-15年度 ; 研究種目:基盤研究C2 ; 課題番号:14550782原著には既発表論文の別刷を含む

High-sensitive analysis by capillary electrophoresis and microchip electrophoresis using on-line preconcentration methods

キャピラリー電気泳動(CE)やマイクロチップ電気泳動(MCE)の濃度検出下限(LOD)は試料負荷に対する構造上の制約から他の分離分析法と比べてかなり高い.この欠点はレーザー励起蛍光検出のような高感度検出器や適切なオンライン前濃縮法の適用により改善できるが,後者は紫外線検出器のみを備えた通常のCE装置においても有用であるため,各種のオンライン前濃縮法が考案されている.本論文では過渡的等速電気泳動(tIPT)及び試料の電気的導入を併用するtIPT(electrokinetic supercharging)に重点を置き,CE及びMCEのLOD向上の戦略を解説し,数種のカチオン,海水中のアニオン及びDNA断片やSDSタンパク質への応用例を示した.最適化された条件ではCEのLODはサブppbレベルに達し,これは金属カチオンやアニオンについてイオンクロマトグラフィーや誘導結合プラズマ原子発光分析法に比肩する濃度感度である.MCEではDNA断片について0.02 μg/ml,SDSタンパク質について0.3 μg/mlと通常法に比べ10～40倍良好な結果を得た.The concentration detection limit (DL) of capillary electrophoresis (CE) and microchip electrophoresis (MCE) is rather high due to geometrical restrictions of sample load. This drawback can be improved by applying high sensitivity detectors, such as a laser-induced fluorescence detector, and/or appropriate on-line preconcentration. Since the latter approach is useful even when using conventional instruments equipped with a UV detector, several on-line preconcentration methods have been devised. This paper emphasizes the effectiveness of transient isotachophoresis (tITP) and tITP with electrokinetic injection of sample (electrokinetic supercharging). Such advancing strategies for improving DL in CE and MCE were illustrated by applications to several metal cations and anions in seawater and biopolymers, such as DNA fragments and SDS-proteins. Under the optimized operational conditions, DLs of CE reached a sub-ppb level comparable with those of IC and ICP-AES for anions and metal cations, respectively. The DL of MCE was ca. 0.02 μg/ml for DNA fragments and ca. 0.3 μg/ml for SDS-proteins, which are 10∼40 times better than of the conventional methods

High-sensitive analysis of DNA fragments by capillary gel electrophoresis using transient isotachophoresis preconcentration and fluorescence detection

In this report aimed on further development of a high-sensitivity capillary gel electrophoresis (CCE) method for analysis of DNA fragments, we firstly explored online transient isotachophoresis (tITP) preconcentration combined with fluorescence detection (FD). The fluorescence signal (excitation: 488 nm; emission: 590 nm) was generated using the intercalating dye of ethidium bromide (EB). It was found when the leading electrolyte (LE) was injected behind the sample zone, such a special tITP mode has significant advantages to solve the bubble formation issue and to improve the analytical performance stability. Two standard DNA samples, a 50 bp DNA step ladder and the phi X174/HaeIII digest. were used to evaluate the qualitative and quantitative abilities of the tITP-FD approach. A highly diluted sample (10,000-fold in the water, e.g. the phi X174/HaeIII digest diluted from 500 mu g/ml to the 50 ng/ml level) was enriched and detected; the LOD was down to 0.09 ng/ml for the 72 bp fragment, apparently improved more than 1000-fold in comparison with UV detection. Although the RSD of peak areas (n = 3) was around 15.5% for the sample was electrokinetically injected, good linearity of peak area response showed that the proposed method is suitable for quantitative analysis

experimental methods and techniques 328 Generation of an X-ray microbeam for spectromicroscopy at SPring-8 BL39XU

A pair of elliptical mirrors (KB mirror) was designed and fabricated to realize an energy tunable x-ray microbeam for spectromicroscopy at SPring-8 BL39XU. As is commonly recognized, the obtainable beam size with the aspherical total reflection mirrors is strongly affected with the slope error of the mirror. Considering that the extremely high brilliance of the undulator radiation from the SPring-8, the small mirror size and the small mirror-to-focus distance were employed to minimize effects of the slope error. Preliminary evaluation of the KB mirror was carried out using 10 keV monochromatized undulator radiation. Alignment of the mirror was assisted by the beam monitor system composed of a scintillator and a CCD, and the beam size less than 5 µm can be easily achieved even when the source was fully used. The beam size obtained with this experiment was 2 x 4 µm 2 with the photon flux of 1 x 10 10 photons/s. Smaller beam size may be expected with the use of intermediate slits. Characterization of trace elements with the spatial resolution will be carried out by using x-ray fluorescence (XRF) analysis and x-ray absorption fine structure (XAFS) measurements with XRF yield method

Standardization of electropherograms of CZE

Although chromatography is widely utilized, it is behind the other analytical methods from the viewpoint of the standardization of the data. This is because usual chromatograms are strongly dependent on the used hardware. Capillary electrophoresis is not either the exception: The obtained electropherograms are also dependent on the hardware such as capillary length, capillary inner surface, applied voltage, and thermostatting capacity etc, even if the same background electrolyte and the same sample is used. We have developed a conversion method of the time-based electropherograms of capillary zone electrophoresis (CZE) into the mobility-based ones by removing contribution of electroosmotic flow considering temperature rise caused by Joule heating. The conversion method also contained correction for delay of migration time caused by relaxation of potential gradient at sample plug. In this paper, after discussing the factors affecting migration time of CZE, electropherograms of rare-earth ions obtained using different migration voltage were converted to demonstrate utility of our method proposed for standardization (data transfer). The conversion method was also successfully applied to electropherograms of several rations obtained by using field enhanced sample stacking.第51回日本電気泳動学会総会・招待講

Effects of Dietary Forage and Calf Starter Diet on Ruminal pH and Bacteria in Holstein Calves during Weaning Transition

We investigated the relationship between ruminal pH and bacteria in calves fed calf starter with and without forage during weaning transition. First, 16 Holstein bull calves were obtained from dairy farms and equipped with rumen cannulas by cannulation surgery. Then, calves (73.5 ± 4.2 kg; mean ± SE) were assigned to groups fed calf starter either with forage (HAY, n = 8) or without forage (CON, n = 8), and all calves were weaned at 8 weeks of age. Ruminal pH was measured continuously, and rumen fluid samples were collected at 7, 8, 9, and 11 weeks of age, namely 1, 0, 1, and 3 weeks after weaning, respectively, to assess volatile fatty acid concentrations and bacterial DNA. The 24-h mean ruminal pH was significantly (P < 0.05) different between the two groups. Diurnal changes in the 1-h mean ruminal pH were observed throughout the study in the HAY group; however, they were not observed at 0 and 1 weeks after weaning in the CON group. Moreover, the HAY group had significantly (P < 0.05) higher proportions of acetate and butyrate and lower proportion of propionate, and significantly (P < 0.05) lower ruminal acetate-to-propionate ratios were observed in the CON group. The ruminal bacterial diversity indices decreased after -1 week in both groups and increased at 0 and 1 weeks after weaning in the HAY and CON groups, respectively. From the 454 pyrosequencing analysis, significant differences (P < 0.05) were observed in the relative abundance of several phyla (Bacteroidetes, Actinobacteria, and Tenericutes) and one genus (Prevotella) between the two groups. From quantitative real-time PCR analysis, the HAY group had the higher copy numbers of cellulolytic bacteria (Ruminococcus flavefaciens and Ruminococcus albus) compared with the CON group. This study demonstrated that feeding of dietary forage alleviates subacute ruminal acidosis due to diurnal changes in ruminal pH. Furthermore, changes in ruminal pH affect the ruminal bacterial diversity and relative abundance, and these changes might have influenced the establishment of fermentative ruminal functions during weaning transition