Yukihiro Esaka

Electrocatalytic oxidation of carbohydrates at copper(II) -modified electrodes and its application to flow-through detection

Abstract Electrochemical oxidation of carbohydrates at several copper-oxide-modified electrodes in alkaline solution has been studied using cyclic voltammetry and rotating-disk voltammetry. It has been found that Cu(II) oxide on the electrode surface is essential for the electrode process, in which some electrogenerated higher Cu(III) oxide seems to serve substantially as a catalyst. Each mole of xylose, glucose and sorbitol was found to be oxidized to 5 mol, 6 mol and 6 mol of formic acid respectively with the transfer of 10, 12 and 14 electrons. The Cu(II)-oxide-modified electrodes allowed the highly sensitive constant-potential amperometric detection of carbohydrates down to picomole level in flow-through systems.

Hydrogen-bonding Interaction-assisted Micellar Electrokinetic Chromatography Using Mixed Surfactant Systems.

Micellar electrokinetic chromatography was examined using mixed surfactant systems consisting of Brij 35 or Tween 20, non-ionic surfactants with a polyether structure, together with sodium dodecyl sulfate (SDS). Addition of the non-ionic surfactant to a SDS micellar system provides a selective increase in the relative capacity factors of some substituted benzenes having hydrogen-donating substituents such as a hydroxyl, amino and amide groups. This effect can be ascribed to hydrogen bond formation between these solutes and the polyether segments of the non-ionic surfactant. The hydrogen-bonding interaction appears to work additively against the hydrophobic interaction. The separation selectivity can be well controlled by adjusting the mixing ratio of the two surfactants. The thermodynamic aspects of the mixed micellar system are discussed in detail.

Surface characterization and on-line activity measurements of microorganisms by capillary zone electrophoresis

Capillary zone electrophoresis (CZE) was applied to the electrophoretic characterization for microorganisms. The electrophoretic peaks detected using light scattering phenomena were characteristic of the microorganisms used. The electrophoretic mobility (mu) evaluated by CZE was in good agreement with that obtained by classical electrophoresis of microorganisms. The migration time was reproducible and depended on the ionic strength (I). Analysis of the mu vs. I relationship provided information regarding the charge density and the hardness of the microbial cell surface. The redox enzymatic activity of microorganisms was also evaluated by CZE using a running buffer containing a corresponding substrate and an appropriate exogenous electron acceptor. A decrease in the concentration of the electron acceptor due to microbial activity can be simultaneously monitored during the electrophoretic process without significant modification of the CZE instrument. Effects of some chemical treatments of microbial cells were also studied using this technique.

Separation procedures capable of revealing DNA adducts

Detection and quantification of DNA adducts are very important in relation to diseases such as cancer. Both high sensitivity and high selectivity are required for the detection of DNA adducts because the content of adducts in DNA is very small compared with those of normal bases and only small amounts of DNA samples are available for analysis in general cases. In this paper are described separation procedures such as liquid chromatography, gas chromatography and capillary electrophoresis combined with a detection and identification method such as 32P-postlabeling, mass spectrometry, electrochemical detection, fluorescence detection and immunoassay. The merits and demerits of the procedures are also discussed.

Analysis of DNA adducts of acetaldehyde by liquid chromatography-mass spectrometry.

A highly sensitive method using liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) was developed for the analysis of DNA adducts of acetaldehyde (AA). AA, which is the primary oxidative metabolite of ethanol, is considered to possess carcinogenic activity. AA reacts with the exocyclic amino group of guanine in DNA to form N2-ethylguanine (Et-Gua) and 1,N2-propanoguanine (Pr-Gua) adducts. With the present method, such adducts were detected as the base forms from DNA chains using depurination in the pretreatment process. In our measurement with LC-ESI-MS, the limits of detection (LODs) of the Et-Gua and Pr-Gua adducts of the base forms were 3.0 x 10(-10) and 1.0 x 10(-9) M, respectively, and the LODs are about two orders of magnitude lower than those of the nucleoside forms. Calf thymus DNA samples treated with AA and NaBH3CN were analyzed by this method. Et-Gua was clearly detected and, in the absence of NaBH3CN, Pr-Gua was detected predominantly. Furthermore, the method was also applied to study whether or not these two adducts are formed in DNA of cultured HL-60 cells during exposure to AA for 24 h. Pr-Gua was clearly detected and traces of Et-Gua were also detected in the DNA of the cells. Although the sensitivity of this method is lower by at least oneorder of magnitude than the 32P-postlabeling assay, currently the most sensitive method, our method does not involve complex enzymatic reactions for the postlabeling and the use of troublesome radioactive materials. Furthermore, it enables structural identification of guanine adducts. The present method would be a useful tool for studies of Et-Gua and Pr-Gua adducts in connection with carcinogenesis.

Non-aqueous capillary zone electrophoresis using polyethylene glycol as a matrix agent

Non-aqueous capillary zone electrophoresis in acetonitrile containing polyethylene glycol as a matrix agent was investigated. Separation of benzoate anions as model samples were optimized by a control of the concentration and the selection of the chain length of polyethylene glycol. Base line resolution was attained in the presence of 15% (w/v) polyethylene glycol 4000 for the benzoates used. In acetonitrile, polyethylene glycol works as a hydrogen-bonding donor and acceptor; the terminal hydroxyl groups serve as a donor toward the dissociated carboxyl groups of the benzoates, while the ether oxygen atoms serve as an acceptor toward the hydrogen-donating substituents of the benzoates. This property of polyethylene glycol is in marked contrast with that observed in aqueous phase, where polyethylene glycol works predominantly as just a hydrogen acceptor. Quantitative analysis of the interaction is presented.

Enzyme microelectrodes for choline and acetylcholine and their applications

Abstract Hydrogen peroxide based amperometric microsensors have been developed for choline (Ch) and acetylcholine (Ach) by coimmobilization of acetylcholinesterase (AchE) and choline oxidase (ChO) on platinum disk microelectrodes using glutaraldehyde vapor. The sensor was applied in micro flow injection and micro liquid chromatographic analyses. The chromatographic peak heights were linearly proportional to the amounts of Ach and Ch over the range of 0.05 to 10 3 pmol (8 × 10 −7 to 1.5 × 10 −2 M, 0.06 μl) with correlation coefficients > 0.99. Immobilization of ChO was used for the selective determination of Ch with a response time of 5 s. This electrode was used for activity measurements of AchE down to 0.25 U ml −1 in a 1 μl volume in a batch method at a total volume of 5.0 ml.

Analysis of DNA adducts bases by capillary electrophoresis with amperometric detection

We have developed a method for the detection of DNA adducts by combining capillary electrophoresis (CE) with the specificity of amperometric detection. Guanine is the most easily damaged base of the four normal DNA bases and many adducts of guanine have been found in DNA. These guanine adducts are often electrochemically active, while the normal bases with the exception of guanine are not. Therefore, CE with amperometric detection will be a promising method to study DNA damage. The four normal deoxynucleosides and two damaged deoxnucleosides N2‐ethyldeoxyguanosine (N2‐ethyl‐dG) and 8‐hydroxydeoxyguanosine (8‐OH‐dG), were completely separated by micellar electrokinetic chromatography (MEKC). Deoxyguanosine and the two damaged deoxynucleosides were identified using amperometric detection. The sensitivity of our system was comparable to that of UV detection. Analysis of DNA hydrolysis products was also performed briefly using this method.

Hydrogen-bonding interaction in capillary electrophoresis using polyether matrices

Abstract Polyethylene glycol (PEG) serves as a novel matrix in capillary electrophoresis. The purpose of this work was to explore some evidence for hydrogen-bonding complex formation between analytes and PEG in the separation system using benzoic acids as model analytes. An increase in the column temperature resulted in a significant decrease in the interaction between PEG and substituted benzoic acids with hydrogen-donating groups. Addition of urea suppressed the interaction. NMR spectra of phenol and salicyclic acid in the presence of PEG in C 2 HCl 3 showed an obvious electrostatic interaction, probably a hydrogen-bonding interaction, between the hydroxyl protons of the analytes and PEG. These results strongly support the contention that hydrogen-bonding interaction between the polyether segments of PEG and the hydrogen-donating groups of analytes occurs in the separation systems. Some other minor interactions controlling the separation are also described.

Polyacrylamides as hydrophilic selectors in non-aqueous capillary electrophoresis.

Polyacrylamides (PAAms) were investigated as hydrophilic selectors in non-aqueous capillary electrophoresis (CE). Separation of 10 substituted benzoates and unsubstituted benzoate as model samples was greatly improved by the addition of PAAms in acetonitrile-CE. The migration behavior indicates that the carbonyl moiety of PAAms works as a good hydrogen-accepting site toward hydrogen-donating analytes such as 4-hydroxybenzoate anion (4OH-BA) in acetonitrile. PAAms also serve as electron-accepting agents with its amide proton interacting with the dissociated carboxyl groups of the benzoates. The ion-dipole interaction is useful to control the migration behavior of benzoates without hydrogen-donating substituents. The overall mode of the interaction is similar to that of polyethylene glycol (PEG) 20000 reported previously, but the complex formation constant of poly(N-tert.-butyl)acrylamide (PBAAm) with 40H-BA estimated here was 130-fold larger than that of PEG 20000. This would be ascribed to the strong basicity of the carbonyl oxygen atoms of PBAAm as compared with the ether oxygen atoms of PEG. Furthermore, a copolymer of (N-tert.-butyl)acrylamide-acrylamide [70:30 (in feed)] exhibited a complex formation constant of about fourfold larger toward 4OH-BA than PBAAm, most probably due to decrease in steric hindrance from the tert.-butyl groups. Adrenaline and its six precursors have been separated successfully using the PAAms.