Yoshiki Hirata

Photobioreactor with photosynthetic bacteria immobilized on porous glass for hydrogen photoproduction

A photobioreactor was constructed with porous glass as an immobilization matrix. The reactor was a rectangular glass chamber (inner dimensions: 125 × 50 × 2.5 mm) containing a porous glass sheet (125 × 50 × 0.5 mm) on which Rhodobacter sphaeroides RV was immobilized (11.2 mg dry wt./ml porous glass). The maximum rate of hydrogen evolution was 1.3 ml/h/ml porous glass. The conversion efficiency of succinate into hydrogen reached 75%. Stable and efficient hydrogen evolution continued for up to 40 d.

Visualizing water molecule distribution by atomic force microscopy

Hydration structures at biomolecular surfaces are essential for understanding the mechanisms of the various biofunctions and stability of biomolecules. Here, we demonstrate the measurement of local hydration structures using an atomic force microscopy system equipped with a low-noise deflection sensor. We applied this method to the analysis of the muscovite mica/water interface and succeeded in visualizing a hydration structure that is site-specific on a crystal. Furthermore, at the biomolecule/buffer solution interface, we found surface hydration layers that are more packed than those at the muscovite mica/water interface.

Amperometric L-lactate-sensing electrode based on a polyion complex layer containing lactate oxidase. Application to serum and milk samples

Abstract An amperometric enzyme electrode for l -lactic acid was prepared by immobilizing lactate oxidase (LOD) in a polyion complex membrane. An aqueous solution containing poly- l -lysine and LOD was placed on a glassy carbon electrode, and an aqueous solution of poly(4-styrenesulfonate) was added to the polycation/LOD mixture and dried. The anodic current (at 1 V vs. Ag AgCl ) of this enzyme electrode increased immediately after the addition of L -lactic acid and the response time was l -lactic acid was observed up to 0.3 mM with a detection limit of 0.1 μM. The enzyme electrode was applied to the determination of l -lactic acid in sera and sour milk: the polyion complex membrane showed permselectivity based on the solute size with the molecular weight cut-off of ca. 100, which was very effective in suppressing the electrochemical interference by l -ascorbic acid, uric acid and acetaminophen. The electrode could be used for at least two months.

Glucose oxidase/polyion complex-bilayer membrane for elimination of electroactive interferents in amperometric glucose sensor

Abstract An amperometric glucose-sensing electrode was prepared by immobilizing glucose oxidase (GOx) on a polyion complex membrane. First, a monolayer of 3-mercaptopropionic acid (MPA) was made on the surface of a gold electrode by immersing it in an ethanol solution containing MPA. Aqueous solutions of poly- l -lysine and poly-4-styrenesulfonate were successively placed on the electrode surface and allowed to dry. A GOx layer was then formed on the poly- l -lysine/poly-4-styrenesulfonate-complex layer by crosslinking the enzyme by the addition of a glutaraldehyde solution. The polyion complex layer was effective for eliminating electrochemical interferents such as l -ascorbic acid, uric acid and acetaminophen, whereas the hydrogen peroxide produced through the GOx-catalyzed reaction permeated rapidly through the layer. This resulted in a rapid response (100% response in l -ascorbic acid to that for the same concentration of glucose was 0.07). The electrode was applied to the assay of glucose in beverages and sera, and could be used for more than two months.

High-throughput flow-injection analysis of glucose and glutamate in food and biological samples by using enzyme/polyion complex-bilayer membrane-based electrodes as the detectors

The concentration of glucose was determined by a combination of flow injection analysis (FIA) with amperometric enzyme sensor detection. The enzyme sensor was prepared by immobilizing glucose oxidase on an electrode coated with a polyion complex layer consisting of poly-L-lysine and poly(4-styrenesulfonate). The inner, polyion complex layer was useful for preventing electrochemical interferents (e.g., L-ascorbic acid, uric acid and acetaminophen) from reaching the electrode surface, which was effective for reducing the interferential responses upon the injections of biological and food samples. The sensor-based system could be used for the determination of glucose from 10 microM to 3 mM with the sampling rate of 180 h-1, and was stable for more than 2 months. An FIA system for determining L-glutamic acid (3 microM-0.5 mM) was also prepared by using an enzyme electrode based on a glutamate oxidase/polyion complex-bilayer as the detector.

Control of protein orientation in molecular photoelectric devices using Langmuir—Blodgett films of photosynthetic reaction centers from Rhodopseudomonas viridis

A photoelectric device was prepared using Langmuir—Blodgett (LB) films of photosynthetic reaction centers (RCs). Monolayers of RCs from Rhodopseudomonas viridis were deposited on transparent electrodes. The orientation of the RCs on the electrode surface was controlled using various substrates with different surface wettabilities. The degree of alignment was evaluated by measuring the polarities of light-induced electric responses. The orientation was also measured by means of an enzyme-linked immunosorbent assay that uses antibodies to distinguish opposite sites of the RCs. Photocells comprised of RC-LB films showed a steady-state photocurrent. The direction of the current flow was regulated by altering the orientation of the RCs.

Hydrogen peroxide determination based on a glassy carbon electrode covered with polyion complex membrane containing peroxidase and mediator

Abstract Peroxidase (POD)/ferrocene co-immobilized polyion complex membrane was prepared on a glassy carbon electrode by dropping, and then drying, the enzyme solution, ferrocene dispersion, poly- l -lysine and polystyrene sulfonate solutions on the surface. The current response to hydrogen peroxide on the electrode was measured in a phosphate buffer (pH 6.5) solution. The response time was found to be short (15 s). The calibration curve of hydrogen peroxide was obtained. The response to hydrogen peroxide on the electrode was found to be linear to its concentration up to 10 μM, and the lower detection limit was 0.5 μM (S/N=5). The results indicate that a sensitive detection of hydrogen peroxide was accomplished. Influence of an electrochemical interfering material on the current response was examined. The ratio of the current response to hydrogen peroxide/ l -ascorbate was found to be ca. 10 when these substances of the same concentration (10 μM) were injected to the test solution. Highly-sensitive biosensors were obtained by immobilizing oxidases on the surface of the POD-based electrode.

Molecular Resolution Imaging of Protein Molecules in Liquid Using Frequency Modulation Atomic Force Microscopy

We demonstrated molecular resolution imaging of biological samples such as bacteriorhodopsin protein molecules in purple membrane and isolated chaperonin (GroEL) protein molecules, both adsorbed on mica using frequency modulation atomic force microscope (FM-AFM) in liquid. We also showed that the frequency noise of FM-AFM in liquid can be greatly reduced by the reduction of the noise-equivalent deflection of an optical beam deflection sensor.

High-Resolution Frequency-Modulation Atomic Force Microscopy in Liquids Using Electrostatic Excitation Method

We developed a novel method to drive the cantilever oscillation for frequency modulation atomic force microscopy (FM-AFM) in liquid environments using electrostatic excitation. The cantilever with a gold backside coating was vibrated by applying an oscillating bias voltage between the cantilever backside and an optically transparent electrode used as a liquid cell window. The frequency spectrum of the oscillation shows a simple resonance curve without spurious peaks. The method does not require electrical conductivity of samples at all. In fact, both muscovite mica and potassium chloride surfaces in aqueous solutions were successfully imaged on an atomic scale.

Glucose-Sensing Electrode Based on Glucose Oxidase-Attached Polyion Complex Membrane Containing Peroxidaseand Ferrocene

A glucose oxidase (GOD)-attached polyion complex membrane containing peroxidase (POD) and ferrocene was prepared by two steps on a glassy carbon electrode. First, a polyion complex membrane containing POD and ferrocene was prepared: a poly-L-lysine solution, a POD solution, a ferrocene dispersion and a poly(styrenesulfonate) solution were successively dropped on an electrode, and the electrode was allowed to dry. Then GOD was attached by the use of glutaraldehyde: GOD and glutaraldehyde solutions were dropped on the membrane, and the electrode was dried. Current response to glucose for the enzyme electrode was measured at a potential of −0.2 V (vs. Ag/AgCl). The 100 % response time was ca. 20 s. The current was proportional to the glucose concentration up to 0.1 mM. The lower detection limit was 1 µM (signal-to-noise ratio, S/N=5). The enzyme electrode was used for the glucose measurements in beverages. The results obtained agreed well with those obtained by Boehringer F-kit method. The electrode would be useful as a glucose biosensor for beverages.