Soichi Yabuki

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.

Amperometric determination of pyruvate, phosphate and urea using enzyme electrodes based on pyruvate oxidase-containing poly(vinyl alcohol)/polyion complex-bilayer membrane

Abstract An amperometric pyruvate-sensing electrode was prepared by immobilizing pyruvate oxidase (PyOx) on a polyion complex membrane. First, aqueous solutions of poly- l -lysine and poly(4-styrenesulfonate) were successively placed on a mercaptopropionic acid-modified gold surface and allowed to dry. A photo-crosslinked poly(vinyl alcohol) layer containing PyOx was then formed on the poly- l -lysine/poly(4-styrenesulfonate)-complex layer. The polyion complex layer was effective for eliminating electrochemical interferents such as l -ascorbic acid, uric acid, l -cysteine and acetaminophen, whereas the hydrogen peroxide produced through the PyOx-catalyzed reaction permeated easily through the layer. This resulted in a high sensitivity (detection limit, 50 nM) and a low interference level (e.g. the ratio of response for l -ascorbic acid to that for the same concentration of pyruvic acid, 0.18). The electrode could be used for determining phosphoric acid (detection limit, 0.2 μM), since PyOx consumes phospholic acid as the co-substrate during the course of pyruvate oxidation. Further, an amperometric urea-sensing electrode (detection limit, 0.5 μM) was prepared by coupling the phosphate-sensing system with urea amidolyase which catalyzes an ATP-consuming urea hydrolyzation.

RAPID MEASUREMENT OF TRANSAMINASE ACTIVITIES USING AN AMPEROMETRIC L-GLUTAMATE-SENSING ELECTRODE BASED ON A GLUTAMATE OXIDASE-POLYION COMPLEX-BILAYER MEMBRANE

Abstract An amperometric l -glutamate-sensing electrode was prepared by immobilizing glutamate oxidase (GlOx) on a polyion complex layer-modified electrode. First, a monolayer of 3-mercaptopropionic acid was made on the surface of a gold electrode by immersing it in an ethanol solution containing the modifier. Next, aqueous solutions of poly- l - lysine and poly(4-styrenesulfonate) were successively placed on the electrode surface and allowed to dry. Finally, a GlOx layer was formed on the poly- l- lysine/poly(4-styrenesulfonate)-complex layer by crosslinking the enzyme by the addition of a glutaraldehyde solution. The use of thin bilayer system with the inner, polyion complex membrane, which showed permselectivity based on the solute size with the molecular cut-off of ≈100, brought high performance characteristics to the l- glutamate-sensing electrode; it showed high sensitivity (detection limit, 20 nM), rapid response (100% response time, 3 s), low interferential level (the ratio of response for l -ascorbic acid to that for the same concentration of l -glutamic acid, 8×10−2), and high stability (usable for more than a month). The bilayer-based electrode was useful for the rapid measurement of glutamate–oxaloacetate transaminase (GOT) and glutamate–pyruvate transaminase (GPT) in serum sample: each transaminase (0.2–1000 U l−1) could be determined within 10 s.

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.

Amperometric enzyme electrode with fast response to glucose using a layer of lipid-modified glucose oxidase and Nafion anionic polymer

Abstract An amperometric enzyme electrode for glucose was prepared by using a lipid-modified glucose oxidase and a perfluorinated ionomer (Nafion). First, a glassy carbon base electrode was dipped into a benzene solution of the modified enzyme and dried, then the electrode was dipped into a Nafion solution and dried. The water-insoluble, modified enzyme was thus immobilized on the electrode surface with a strongly adhering, thin polymer coating. The anodic current (at 0.9 V vs. Ag/Agcl) of this electrode increased immediately after the addition of glucose and the response time was shorter than 2 s. A linear response to glucose was observed up to 3 mM with a detection limit of 0.2 μM. The enzyme electrode was applied to the determination of glucose in fruit juices; the Nafion coating was effective in suppressing the electrochemical interference by l -ascorbate in the samples. The electrode could be used for at least 6 weeks.

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.

Highly-sensitive measurement of hydroquinone with an enzyme electrode

Abstract An enzyme electrode with a chemically amplified response for hydroquinone and other dihydroxyphenols, such as catechol, dopamine, norepinephrine, and epinephrine, is constructed from a glassy carbon electrode and a layer containing immobilized glucose oxidase. Dihydroxyphenol molecules are consumed at the electrode surface but are regenerated by the glucose oxidase reaction in the layer. The consumption/regeneration cycle for dihydroxyphenols results in an electrode response amplified 2–210 times, and, accordingly, in an increased sensitivity. The detection limit of hydroquinone, catechol, or dopamine is as low as 1 n m .

Amperometric glucose-sensing electrode based on carbon paste containing poly (ethylene glycol)-modified glucose oxidase and cobalt octaethoxyphthalocyanine

Abstract An amperometric enzyme electrode for glucose was prepared by incorporating poly (ethylene glycol)-modified glucose oxidase and cobalt octaethoxyphthalocyanine [CoPc(OEt)8], a new mediator, into a carbon paste matrix. The polymer-modified enzyme exhibited a higher activity than the native enzyme in the hydrophobic carbon paste medium. CoPc(OEt)8 could oxidize the enzyme at more negative potentials than unsubstituted cobalt phthalocyanine (CoPc). Further, the CoPc(OEt)8-mediated enzyme electrode showed a high stability: the electrode response to glucose did not decrease for at least 4 weeks (or for 700 assays), whereas the glucose response from a CoPc-mediated enzyme electrode fell to half of the initial value within 2 weeks (or after 300 assays). CoPc(OEt)8, a paraffin-oil soluble derivative of CoPc, incorporated in the bulk of carbon paste, diffused towards the electrode surface so as to renew continuously the electrode surface, which resulted in the high stability of the new mediator-based enzyme electrode.

L-Malate-sensing electrode based on malate dehydrogenase and NADH oxidase

Abstract An L -malate-sensing electrode was constructed from an oxygen electrode and a layer containing immobilized malate dehydrogenase (MDH) and NADH oxidase. MDH catalyses the dehydrogenation of L -malate by NAD+ and NADH oxidase catalyses the regeneration of NAD+ with the use of oxygen. The regeneration enables the L -malate oxidation to proceed efficiently even in a medium of neutral pH. At pH 8.0, L -malate in the concentration range 5 μM–1.5 mM can be measured. The relative standard deviation for the measurement is 1.2% ( L -malate concentration, 0.2 mM; n=10). The present L -malate-sensing electrode is stable for 8 weeks. A two-electrode sensor system consisting of the L -malate-sensing electrode and an L -lactate-sensing electrode based on lactate oxidase was prepared and applied to the simultaneous determination of the two components in wines.