Shunichi Uchiyama

Enzyme-based catechol sensor based on the cyclic reaction between catechol and 1,2-benzoquinone, using l-ascorbate and tyrosinase

Abstract A cyclic reaction between catechol and 1,2-benzoquinone takes place by combining the tyrosinase reaction and the chemical reduction of 1,2-benzoquinone to catechol by l -ascorbic acid. Catechol is oxidized by the dissolved oxygen catalysed by the enzyme and its consumption is not compensated for by the chemical regeneration of catechol from 1,2-benzoquinone. The dissolved oxygen thus continues to decrease during the cyclic reaction and consequently the decrease in the reduction current of oxygen is amplified. The amplification factor of 1 X 10 −6 M catechol at 0.01 M l -ascorbate and pH 7.0 was found to be 300 when enzyme activity was 20 U ml −1 and the reaction time was 10 min. The calibration graph for catechol with a potato tissue membrane electrode was moved in parallel to a lower concentration range and the detection limit was found to be 5 X 10 −8 M (amplification factor 400) when buffer solution of pH 7.0 containing 0.01 M l -ascorbic acid was used.

Amperometric l-ascorbic acid biosensors equipped with enzyme micelle membrane

Ascorbate oxidase (ASOD) bound to polymaleimidostyrene (PMS) forms stable ASOD micelle structure in polystyrene (PS) membrane. The oxygen permeable hydrophobic ASOD micelle membrane were coated on both aminated glassy carbon electrode (AGCE) and gold electrode (AuE) for the amperometric detections of l-ascorbic acid (AsA) based on the consumption of oxygen. These AsA sensors have good sensitivities with short response time (within 1min.). A good linear relationship was observed in the concentration range of 5muM to 0.4mM when AGCE was used and the applied potential was -0.5V vs. Ag/AgCl. Interferences from the reducing agents can be avoided because the detections were conducted at cathodic potential.

Enzyme-based chemically amplified flow-injection determination of catechol and catecholamines using an immobilized tyrosinase reactor and l-ascorbic acid

Abstract Substrate recycling of polyphenols takes place when the substrate is added to a solution containing tyrosinase and l -ascorbic acid. The current of the oxygen electrode detector in a flow-injection system with an immobilized tyrosinase reactor was significantly amplified when the carrier solution contained l -ascorbic acid, because the o-quinone compounds produced from substrates by the tyrosinase reaction are chemically reconverted to the original polyphenols by l -ascorbic acid. The establishment of a cyclic reaction of substrates in the enzyme reactor leads to amplification of oxygen consumption. The amplification factor increased with decreasing substrate concentration and was found to be larger than 100 when the substrate concentration was below 1 × 10 −7 M. The detection limits of polyphenols (catechol, l -dopa, dopamine, noradrenaline and adrenalin) were 10−7–10−9 M in the presence of 1 × 10−3 M l -ascorbic acid in the carrier (pH 6.5, flow-rate 2.5 ml min−1).

A catechol electrode based on spinach leaves

Abstract Minced spinach leaf (Spinacea oleracea) has a high activity of catechol oxidase (dimerizing) (EC 1.1.3.14), which is utilized for the determination of catechol by coupling the spinach tissue with a Clark oxygen electrode. The calibration graph for catechol is linear over the range 2×10−5–8×10−4 M (RSD 3%). The sensor retains its enzyme activity for at least 18 days. 4-Methylcatechol and glycolate interfere; glucose and ascorbate do not.

Chemically amplified kojic acid responses of tyrosinase-based biosensor, based on inhibitory effect to substrate recycling driven by tyrosinase and l-ascorbic acid.

A tyrosinase-based chemically amplified biosensor, based on the substrate recycling of polyphenols driven by tyrosinase-catalyzed oxidation and chemical reduction by l-ascorbic acid (AsA), has been utilized for the highly sensitive detection of inhibitors of tyrosinase such as kojic acid, benzoic and SCN(-) ion. The amplified current response of immobilized tyrosinase-coupling oxygen electrode due to the recycling was suppressed by the addition of inhibitors, and a highly amplified response to kojic acid over other inhibitors was observed in the presence of 5 mM AsA. The amplification factor (AF) of kojic acid is substantially proportional to the AF of substrates, and the AF for 1 x 10(-7)M kojic acid was increased by up to a factor of 143 when 1 x 10(-5)M dopamine was used as a competitive substrate in the presence of 5 mM AsA. The amplified calibration curve of kojic acid obtained with 5 mM AsA was shifted towards more than a two decades lower concentration range compared with that of the non-amplified response, and the detection limit of kojic acid was lowered to 7 x 10(-8)M.

Electrochemical Introduction of Amino Group to a Glassy Carbon Surface by the Electrolysis of Carbamic Acid

The electrocatalytic activity of a glassy carbon electrode with regard to the oxidation of ammonium carbamate increased with the electrolysis time because of the electrochemical modification of the electrode surface. From X-ray photoelectron spectroscopy data, it was found that a carbon-nitrogen bond was newly formed due to the electrode oxidation of carbamic acid at +1.0 V vs Ag/AgCl. Redox waves of catechol bound to amino group were observed at +0.05 V vs Ag/AgCl when cyclic voltammetry of catechol was carried out by using a glassy carbon electrode pre-electrolyzed in ammonium carbamate solution. This indicates that catechol can be attached to the electrolyzed surface by the reaction of the amino group bound to the pre-electrolyzed electrode surface and 1,2-benzoquinone formed by electrode oxidation. This result supports the concept that an amino group can be introduced by electrolysis in which ammonium carbamate is used as the electrolyte. The electrochemical introduction of the amino group may have occurred due to the decomposition of carbamaic acid attached to the carbon electrode surface.

Mediated glucose sensor using a cylindrical microelectrode

The performance of a cylindrical enzyme electrode with a soluble mediator was analysed theoretically. The normalized current response was calculated at various values of both the electrode radius (a) to enzyme layer thickness (l) ratio, a/l, and the relative catalytic activities, σS and σM { = (kcat[E]l2/KMS,MDS,M)12}. The calculated results demonstrated that at high mediator concentration, larger a/l ratio and increased relative catalytic activity for the substrate, σS, a wider linear range could be obtained for a calculated glucose calibration graph. However, the linearity was found to be less dependent on a/l than σS. Alternatively, at high glucose concentration, the mediator concentration — response curve calculated at low a/l values and a high catalytic activity for the mediator, σM, indicated a wide linear range. Immobilized glucose oxidase (GOD) cylindrical microelectrodes were fabricated and their characteristics were evaluated by using 1,4-benzoquinone as electron mediator. GOD was immobilized in a photo-cross-linkable polymer on two types of cylindrical microelectrodes of 2 and 25 μm diameter. The linear ranges of the observed calibration graphs were wider than that obtained using a disc electrode of 1 mm diameter. This was probably due to the larger σS values obtained with the glucose sensors of 2 and 25 μm diameter. Moreover, the response of the 2-μm glucose sensor based on hydrogen peroxide detection was compared with that using mediators. This result showed that the wider measurable range was obtained using mediators.

Concentration-step amperometric sensor of l-ascorbic acid using cucumber juice

Abstract l -Ascorbic acid was determined by concentration-step amperometry using a thin layer of carbon felt impregnated with cucumber juice as an enzyme solution of ascorbate oxidase. The dilute fruit juice was added on top of the carbon felt and the decreased current peak caused by the enzymatic reaction was measured. The cucumber juice was prepared by filtration and centrifugal separation. The peak current was proportional to the concentration of l -ascorbic acid in the concentration range 2.5 × 10 −4 –1.6 × 10 −3 M. The results obtained were in fairly good agreement with those obtained by liquid chromatography.

Flow-injection determination of L-ascorbate with cucumber juice as carrier

Cucumber (Cucumis sativus L.) juice has a high l-ascorbate oxidase activity and can be used as the carrier solution in an amperometric flow-injection system for the determination of l-ascorbate. The determination time is 1 min. The calibration graph is linear over the range 5×10−4–7×10−3 M (RSD 4%). No enzyme purification is needed. The juice solution retains its activity for 8 days with recycling.

Enzyme-less amperometric biosensor for l-ascorbate using poly-l-histidine-copper complex as an alternative biocatalyst.

A poly-l-histidine(PLH)-copper(II) complex can be used as an alternative biocatalyst in an O(2) detection-type amperometric enzyme-less l-ascorbate (AsA) sensor. The PLH-Cu(II) membrane was simply prepared by entrapping the PLH in polyacrylamide gel and subsequent treatment of the gel with CuCl(2) solution. This enzyme-less biosensor can be used over a relatively wide pH region from 4 to 11 and enables precise determination of AsA (RSD less than 3%, n=10) at pH 7.0. The fundamental performance characteristics (sensitivity, response time, and linear range) of this PLH-Cu(II)-based sensor is comparable to those of a native ascorbate oxidase-based sensor. Unfortunately, the selectivity is inherently rather low and, as a result, the response was degraded in the presence of higher concentrations (more than mM order) of quinones. However, reducing sugars caused no interference and the sensor could be used to detect AsA in some fruits and drinks. This enzyme-less sensor has excellent stability for at least 3 months of repeated analysis (more than 300 samples) without loss of ordinal activity.