BUNSEKI KAGAKU Abstracts

Protein thin films containing avidin and lectin have been prepared on solid surfaces to develop devices sensitive to pH and biomolecules. Concanavalin A (Con A), one of the glucose- and mannose-binding lectins, and glycogen were alternately deposited onto the surface of a quartz slide to prepare layer-by-layer (LbL) thin films, through strong Con A-glycogen binding. Con A-glycogen LbL films are sensitive to sugars, and can be disintegrated in response to sugars, depending on the type of sugars and the concentration. Avidin and biotin (or iminobiotin)-modified polymers were used for constructing LbL thin films and the response to the pH and biotin analogues was evaluated. LbL films composed of avidin and iminobiotin-modified polymer can be disintegrated upon changing the pH of the media in which the films are immersed. LbL films were decomposed when the solution was acidified, while in basic solutions the films were stable. In a similar way, LbL films deposited onto the surface of electrodes that were sensitive to an electrode potential. LbL films were disintegrated upon applying positive potentials (1 V or lower) to the electrode due to acidification of the local pH at the electrode-film interface, arising from the electrolysis of water. LbL films composed of avidin and iminobiotin-modified polymer were also decomposed by adding biotin and analogues in solution. Thus, a possible use of LbL films containing Con A and avidin in biosensing and delivery was suggested.

We have been investigating a new concept of chromatography, an environmental-responsive chromatography, using these functional polymers modified packing materials for HPLC. We designed and synthesized thermo-responsive copolymers with N-isopropylacrylamide (NIPAAm), butyl methacrylate (BMA) and other comonomers. These polymers underwent a reversible phase transition from water-soluble forms into aggregates by changing the temperature. The copolymers were grafted onto aminopropyl silica beads, and the products were evaluated as HPLC packing materials. In a chromatographic system using the thermo-responsive polymers, we could separate steroids, amino acids and proteins with a variety of hydrophobicities using a sole aqueous mobile phase. The retention on the polymer-modified stationary phase remarkably changed upon changing the temperature. With increasing temperature, an increased interaction between analytes and polymer-grafted surfaces of the stationary phases was observed. The drastic and reversible surface hydrophilic-hydrophobic property alteration should be due to rapid changes in the polymer hydration state around the polymer’s transition temperature. A solvent gradient elution-like effect could be achieved with a single mobile phase by programmed temperature changes during chromatographic runs. An environmental-responsive analytical system using a functional polymer-modified surface would be highly useful to control the function and property of the stationary phase for HPLC only by changing the temperature with an aqueous solvent. These systems have advantages over conventional HPLC, including improved maintenance of recovered solute bioactivity and the elimination of organic solvent use.

We constructed a high-resolution, high-throughput photoemission spectroscopy (PES) system combined with a combinatorial laser molecular-beam epitaxy thin-film growth system in order to realize in-situ characterization for electronic structures of transition-metal oxide (TMO) heterointerfaces. The elemental selectivity of the PES technique using synchrotron radiation enables us to extract the electronic structure of constituent layers in the vicinity of the heterointerface. Direct observation of the interfacial electronic structures is a key to understanding the physical and chemical properties of junctions based on TMO. The capabilities of the technique have been demonstrated by the in-situ resonant PES analysis of La_0.6Sr_0.4MnO₃ layers buried in insulating perovskite oxides, such as La_0.6Sr_0.4FeO₃ and SrTiO₃ (STO).

The dependence of the bioelectrocatalytic current on the thickness of an enzyme-mediator modified layer thickness was investigated. The layer was fabricated by an electrostatic layer-by-layer assembly method. Positively charged enzyme (PQQ-dependant glucose dehydrogenase) and polymer-bounded mediator {Os complex-coordinated poly(N-vinylimidazole)} are successively entrapped on the electrode surface with negatively charged polymer (κ-carrageenan). The steady-state catalytic current value increased linearly with increasing the layer thickness, and reached the saturated value. The behavior was successfully interpreted based on the theory of the reaction layer with the enzymatic kinetic parameters, the concentrations of the enzyme and the mediator, and the diffusion coefficient of the mediator.

Of reactive oxygen species (superoxide ion O₂^-•, hydrogen peroxide H₂O₂, singlet oxygen ¹O₂, hydroxyl radical • O₂^-• was sensitively and specifically detected by means of lucigenin chemiluminescence (CL) in ordered surfactant molecular assemblies (mixed vesicles) which aqueous mixtures of octyltrimethylammonium chloride (OTAC) and sodium dodecanoate (SD) formed. The CL signal for O₂^-• was greatly enhanced in a mixed vesicular solution, e.g., by a factor of 1400 in a 1/1 mixture (due to the hydrolysis of SD, at pH 9.2) of 0.50 M OTAC and 0.50 M SD, compared with that in an aqueous solution (pH 9.2) with no surfactant. The relative CL signals in the mixed vesicular solution were 100, 4, 4 and 0.2 for O₂^-•, ¹O₂, H₂O₂ and , respectively, while those in the aqueous solution were 100, 120, 3 and 18, respectively. Specific detection of O₂^-• was realized in the neutralized mixed vesicular solution of 5.0 M OTAC/1.0 M SD (1/1), the relative CL signals being 100, 0.011, 0.021 and 0.037, respectively. In both mixed vesicular solutions, i.e., 0.50 M OTAC/0.50 M SD (1/1) at pH 9.2 and 0.83 M OTAC/0.17 M SD (1/1) at pH 7.0, log-log plots of the CL signal for O₂^-• vs. the concentration of hypoxanthine exhibited straight lines with slopes of 1.6. The linear ranges were 2.6×10^-8 (lower detection limit) 〜1.0×10^-6 M and 1.8×10^-7 (lower detection limit) 〜1×10^-5 M, respectively. From an analysis of the CL signal profiles and the HPLC determinations of lucigenin and N-methylacridone (emitter), it can be said that the vesicles alter the lucigenin - O₂^-• reaction path favorably, resulting in a great improvement of the CL quantum yield. The proposed lucigenin CL method was applied to new CL reaction systems, in which the formation of O₂^-• has been speculated.

Amino groups were electrochemically introduced to the glassy carbon electrode surface by the electrode oxidation of an ammonium carbamate solution. This aminated glassy carbon electrode was used for selective amperometric and coulometric determinations of hypochlorite. The reduction wave of hypochlorite obtained by an aminated electrode was moved by as much as 0.4 V toward the positive direction in a neutral pH range, compared with that obtained by an unaminated electrode. It was found that the reduction wave of hypochlorite without oxygen interference began to appear at ＋0.6 V vs. Ag/AgCl. The reason why the reduction wave moved toward the positive direction is considered to be because the activation energy of electron transfer of hypochlorite significantly decreases due to an electrochemical introduction of active sites containing nitrogen atoms. The determination of hypochlorite without oxygen intereference in drinking water was carried out by a flow injection method using an aminated flow-through porous carbon felt electrode. This work is the first research to report on the electroanalytical usefulness of an aminated carbon electrode that can be expected to be applied to other analytical species.

This paper reports formation of a bimolecular layer by novel surfactants, where a hydrogen-bonding site is located into an alkyl chain, on sulfonyl group-bound supports and its hydrophobic nature observed with liquid chromatography. The surfactants used are N-{6-(nonanoyl-L-valylamino)hexyl}-N-octadecanyl-N,N-dimethylammonium bromide and N-{3-(dodecanoyl-L-valylamino)propyl}-N-octadecanyl-N,N-dimethylammonium bromide. Each surfactant possesses a quaternary ammonium head group and a valine diamide moiety inserted into one long alkyl chain as the hydrogen-bonding site. The two surfactants formed vesicles, which were observed with low angle light scattering technique. When the latter surfactant was casted on both DIAION SK 110 (SK) and NUCLEOSIL 100-5 SA (SA), having sulfonyl groups as cation-exchange sites, a bimolecular layer was formed spontaneously on the support surface in each. This was indicated by the entrapment of a fluorescein marked dextran (FTIC) on the surfactant-casted SK, and its fluorescence was observed along the outline of the support by confocal laser scanning microscopy. When the surfactant-casted SA was applied for chromatography, solute retention was affected by hydrogen-bonding affinity of the valine diamide moiety buried into the hydrophobic bimolecular layer.

The structures of water in polycrystalline gold electrode | electrolyte interphases was investigated in terms of the strength of hydrogen bonding by means of in situ Fourier transform infrared reflection absorption spectroscopy (FT-IRAS). The structures strongly depended on hydration force of electrolyte ions, i.e., the structure making or structure breaking characters. ClO₄^-, which shows the structure breaking character, caused the significant disruption of the structure of water in the interphase, as compared to F^-, which is the structure maker. Furthermore, we have investigated the influence of adlayers of pyridine and pyridine derivatives on the structures of water in the interphases. The adsorption of pyridine and alkylpyridines in aqueous solutions containing F^- increased the number of strongly hydrogen-bonded water molecules, and we conclude that hydrophobic hydration is formed outside the adlayers. The structure of water also depended on the bulk concentration of the aromatic compound. No significant hydrophobic hydration is observed in the solution containing ClO₄^-.

Adsorption behavior of three isomeric pyridinecarboxylic acids (picolinic acid: 2-PCA, nicotinic acid: 3-PCA, and isonicotinic acid: 4-PCA) on a smooth polycrystalline gold electrode surface was investigated in three electrolytic solutions, 0.1 mol dm^-3 HClO₄, NaClO₄, and NaOH aqueous solutions, by in situ Fourier transform infrared reflection absorption spectroscopy (FT-IRAS). The reversible adsorption/desorption and reorientation of the PCAs were observed over the potential range investigated. The adsorbed PCAs existed as either zwitterions or anions, i.e., the carboxylate group of the PCA molecule was not protonated. In the HClO₄ aqueous solution, it was found that as the electrode potential became more positive, adsorbed 3- and 4-PCAs reoriented from a flat (parallel) configuration to a vertical one that binds to the surface through lone pairs of electrons on the two oxygen atoms of the carboxylate group. In the NaClO₄ aqueous solution, adsorbed 3-PCA and 4-PCA anions showed the flat configurations. In the NaOH aqueous solution, 3-PCA and 4-PCA anions adsorbed in the flat and the vertical configuration, respectively. No significant adsorption of 2-PCA was observed in the HClO₄ and the NaOH aqueous solution. 2-PCA anion adsorbed in the flat configuration only in the NaClO₄ aqueous solution. These findings indicate the ortho effect on adsorption.

Suppressing such failures as electromigration (EM) and stress-induced voiding (SIV), which tend to occur around vias, is a serious issue concerning the reliability of Cu interconnects. Alloying Cu using a Cu-alloy seed is a suitable method to improve the reliability of Cu interconnects. In this study, we characterized electrochemically plated (ECP) Cu films formed on a Cu-Al alloy seed layer using combined physical analysis techniques. According to these physical analysis data, it is supposed that precipitated Al atoms along the grain boundaries in Cu film suppress the diffusion of Cu atoms and vacancies, and prevent the migration of Cu during reliability tests. Consequently, EM and SIV endurance is achieved using a Cu-Al alloy seed.

A quantitative electrochemical detection of testosterone was performed using a nitrocellulose membrane as a support for antibodies. Testosterone competitively reacted with horseradish peroxidase (HRP)-labeled testosterone, and was captured with an anti-testosterone antibody immobilized on the membrane. The activity of the labeled HRP was electrochemically estimated with a gold-disk electrode (diameter, 300 μm) by detecting the oxidized form of ferrocenemethanol (FcOH), which was produced by the HRP-catalyzed oxidation of FcOH with H₂O₂. The electrochemical detection chamber was fabricated in order to maintain a constant distance between the electrode and the membrane surface. We optimized some factors, especially nitrocellulose membranes and the concentration of bovine serum albumin (BSA) and HRP-labeled testosterone for the electrochemical analysis of testosterone with a high reliability. Testosterone was detected by a competitive amperometric measurement under the optimized conditions. As a result, the current response decreased with increasing testosterone concentration, and a linear current response was observed in the range of 0.5〜20 ng/mL on a semi-log graph. This method provides high sensitivity by one order of magnitude, compared with the conventional well plate assay.

The separation and quantification of aluminum ion in water samples by capillary electrophoresis (CE) have been performed with various analyses since the 1990s when analysis equipment was marketed. However, to date, the inductively coupled plasma mass spectrometer (ICP-MS) and the graphite furnace atomic absorption spectrophotometer (GFAAS) have often been used for the analysis of trace elements, including aluminum, because CE has low sensitivity. This study suggests that aluminum in water samples (rainfall, soil solution and streamwater) can be effectively determined by CE employing an on-line preconcentration method involving field-amplified sample stacking. This method can measure the total dissolved aluminum with a high sensitivity (detection limit: 1.59 ppb) and a small sample volume requirement (＜100 μL). A linear calibration curve of an Al standard solution was obtained with a good determination coefficient of 0.999, ranging from 0 to 1000 ppb

The formation of avidin sites on the surface of glass slips by chemical modification and an avidin-biotin technique was investigated. In a chemical modification approach, a glass surface was treated with 3-mercaptopropyltrimethoxysilane, on which avidin was introduced by an amine coupling method. In the avidin-biotin technique, biotin sites were formed on glass slips by the patterning of biotin-4-fluorescein on bovine serum albumin (BSA)-modified and BSA-adsorbed glass slips by UV irradiation. Avidin-modified glass slips were prepared by treating the biotin sites with an avidin solution. The avidin slips prepared by UV patterning were able to be kept in the dark at 4℃ for at least 5 days, while the chemically modified slips were able to be kept for one day. The preparation of avidin arrays by UV patterning was also demonstrated. The avidin slips appeared to be useful for introducing biomolecules to design a variety of sensing systems.

The orientation of cations at the interface between water and a room-temperature ionic liquid (RTIL) has been analyzed using optical second harmonic generation. To detect second harmonic light from the RTIL|water interface with high sensitivity, a RTIL, N-dodecylisoquinolinium bis(pentafluoroethylsulfonyl)imide, which is based on N-dodecylisoquinolinium (C₁₂Iq^＋) having strong hydrophobicity and high hyperpolarizability, has been used. A polarization-dependence measurement of the light intensity of the second harmonic light has suggested that C₁₂Iq^＋ is orientated at the RTIL|water interface with the isoquinolinium moiety tilting at 50° with respect to the surface normal, and with the dodecyl moiety protruding to the RTIL phase. 

We proposed and investigated an X-ray fluorescence analysis that enables direct measurements at solid-liquid interfaces, which is generally used in the laboratory. A newly developed XRF device can be inserted into the solution. The top of the XRF device was covered with a polyimide film to prevent the solution from entering into the device. A quantitative analysis of the method was evaluated by using metal ionic solutions. The lower detection limit of the method was obtained in the range of ppm order. The analysis depth in the liquid phase was about 1 mm, by measuring the solid sample in liquid phase. It enables simultaneous measurements of both liquid samples and solid samples. A time-resolved measurement of Ni electroless plating was performed for the application of solid-liquid interfaces. It was confirmed that this method was able to be applied to monitoring of the plating processes.

In a surface-tension measurement of a solution by a drop volume method, a magnetic force was applied by a pulsed electromagnet (＜15T) and a static permanent magnet (0.55T) in order to investigate the effect of a magnetic force. In the drop volume method, a drop of MnCl₂ solution was formed at the end of a glass capillary that had 0.73 mmφ in outer diameter, using a syringe pump. From the droped volume of the aqueous solution, the surface tension was measured. When a magnetic field gradient was applied to the droplet formed at the capillary end, a smaller droplet was droped than that of the volume in the absence of a magnetic field. In case of a static magnetic field, the effect was well explained by the magnetic gravity force added to the original garavity force. On the other hand, a pulsed magnetic force of 0.43 ms or 0.80 ms was not enough to make the drop fall, because the pulse duration was too short to pull down the droplet. This result required the introduction of an effciency factor of 0.01〜0.97, depending on the pulse duration and the magnetic susceptibility of the sample solution. Furthermore, the pulsed magnetic-field experiment allowed to esitimate the relaxation time of a droplet as being 7.8 ms, which implied that the magnetic filed had to continue for at least this time to be droped. Based on the present study, it has been suggested that a simultaneous measurment of surface tention and magnetic susceptivility was plausible by using a drop-volume method under a static magnetic field, or a pulsed magnetic field.

Spherical porous titania particles were synthesized by a sol-gel process from titanium tetraisopropoxide (TTIP) in a W/O emulsion. These particles were obtained by the addition of diethanolamine (DEA), methanol and colloidal silica particles. The molar ratio of TTIP, DEA, methanol and H₂O was 1 : 1.5 : 4 : 20. Spherical titania particles could be synthesized by a stepwise increasing-temperature process. This process was carried out by stepwise emulsification at 30℃ for 30 min, at 40℃ for 30 min and at 50℃ for 2 h. After calcination at 500℃, the SiO₂ parts in the particles were extracted by a 3 M NaOH aqueous solution. The specific surface area of the particles was 362 m² g^-1. The peak pore-diameter of the particles was 6 nm.