Yasushi Hasebe

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.

Layer-by-layer deposited nano- and micro-assemblies for insulin delivery: a review.

We present an overview of the recent progress in the development of layer-by-layer (LbL) assembled thin films and microcapsules for insulin delivery. The LbL deposition of insulin-containing thin films on the surfaces of flat substrates or microparticles has been investigated for orally administered insulin formulations. The amount of insulin in the LbL films can be precisely controlled by altering the number of layers in the films. As-prepared LbL films and microcapsules can be loaded with insulin by exposing the films and microcapsules to an insulin solution. The insulin can be released by pH-induced decomposition or permeability changes in the LbL films and microcapsules. Closed-loop insulin delivery systems that can release insulin in response to changes in glucose concentration have also been constructed with LbL films and microcapsules. Glucose-sensitive materials, such as glucose oxidase, concanavalin A, and phenylboronic acid, have been incorporated into insulin-containing LbL assemblies. In addition, LbL film-coated pancreatic islet cells have recently been developed as a bio-artificial pancreas, in which the islet cells are isolated from the recipients immune system by the LbL coatings. Thus, LbL films and microcapsules could make a significant contribution to the further development of patient-friendly insulin delivery systems.

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).

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.

Microbial cyanide sensor for monitoring river water.

A microbial cyanide sensor using Saccharomyces cerevisiae for monitoring a river water is described. This sensor is based on the inhibition of S. cerevisiaes respiration by cyanide. This sensor is a reactor type flow system and composed of two oxygen electrodes and a reactor which contains S. cerevisiae immobilized beads. The S. cerevisiaes respiration activity is measured using the oxygen electrodes. The sensor showed a linear response in the range from 0 to 15 microM and maintained stable response for 9 days at ambient temperature. The sensor was optimized for the monitoring of river water and was applied to river water analysis.

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.

Insulin-containing layer-by-layer films deposited on poly(lactic acid) microbeads for pH-controlled release of insulin

Layer-by-layer (LbL) thin films containing insulin were deposited on the surface of biodegradable poly(lactic acid) (PLA) microbeads and the pH-triggered release of insulin was studied. The LbL films were successfully prepared by the alternate deposition of insulin and poly(vinyl sulfate) (PVS) or dextran sulfate (DS) at pH 4.0 through the electrostatic force of attraction between positively charged insulin and polyanions. The loading of insulin on the microbeads was dependent on the number of insulin layers and the type of polyanions used; higher insulin loading was observed for thicker films and when PVS was used as the polyanion. Insulin was released from the microbeads when they were exposed to neutral solution (pH 7.4) due to a loss of electrostatic attraction between the insulin and polyanions in the films, which in turn was caused by the charge reversal of insulin from positive to negative in the neutral medium. The pH threshold for insulin release was found to be pH 5.0-6.0. The released insulin retained its original secondary structure as evidenced by circular dichroism spectra. The insulin loaded on the microbeads was satisfactorily stable even in the presence of a digestive enzyme (pepsin) at pH 1.5. These results suggest a potential future use for insulin-loaded microbeads in the oral delivery of insulin.

Carbon felt-based biocatalytic enzymatic flow-through detectors: Chemical modification of tyrosinase onto amino-functionalized carbon felt using various coupling reagents

Tyrosinase (TYR) was covalently immobilized onto amino-functionalized carbon felt (CF) surface via eight different coupling reagents. Prior to the TYR-immobilization, primary amino group was introduced to the CF surface by the treatment with 3-aminopropyltriethoxysilane (APTES). The APTES modification of the CF surface was confirmed by XPS and SEM measurements. The terminal amino groups on the CF surface were cross-linked with protein lysine group (or cysteine group) using various coupling reagents. The resulting TYR-immobilized CF (TYR-CF) was utilized as a working electrode unit of a biocatalytic enzymatic flow-through detector. Catechol and 4-chlorophenol (4-CP) were used as model analytes for the evaluation of catecholase activity and phenolase activity, respectively, and flow injection peaks based on the electro-reduction of the enzymatically produced o-quinone species were monitored at -0.05 V vs. Ag/AgCl. Among eight coupling reagents, glutaraldehyde (GA) exhibited the best results on the sensitivity, the operational stability and the storage stability. The detection limits of catechol and 4-CP obtained by the GA-coupling method were found to be 6.0 x 10(-9)M and 1.5 x 10(-8)M, respectively with the sample through-put of 36 samples/h. No serious degradation of the peak current was observed over 30 consecutive samples injections on the GA-coupling method, while gradual decrease in the peak currents was observed on other seven coupling reagents. The GA-coupling method showed the best results on the storage stability, and 85% of original activity for catechol oxidation remained after 25 days storage.

Highly sensitive flow detection of uric acid based on an intermediate regeneration of uricase.

The principle of the signal amplification of a uric acid sensor based on dithiothreitol (DTT)-mediated intermediate regeneration of uricase was applied to a flow-injection system with an immobilized uricase reactor and a DTT-containing carrier. Highly sensitive detection for nM to microM order of uric acid was achieved when 10 mM TRIS-HCl buffer (pH 10.0) containing 20 mM DTT was used as a carrier at 0.6 ml min-1 and 37 degrees C. The sensitivity of the uric acid was much improved over a batch method using a uricase membrane-coupling electrode, and the detection limit (ca. peak current 8 nA) of uric acid was found to be down to 3 x 10(-10) M (amplification factor; more than 10,000). This chemically amplified flow-system is very useful for the direct assay of uric acid in highly diluted biological fluids (urine and serum) without complicated pretreatment of the samples, because this sensor has the potential to detect trace amounts (nM to microM) of uric acid in highly diluted body fluids in which the concentration of interfering constituents was decreased to negligible levels. Good correlation was observed between this system and conventional spectrophotometry. The immobilized uricase reactor could be re-used for at least 4 months of repeated analysis without loss of activity and was stable if stored at 4 degrees C in 10 mM TRIS-HCl buffer, pH 9.0.

Highly amplified spectrophotometry of polyphenols based on a cyclic reaction between polyphenols and o-quinone compounds using tyrosinase and L-ascorbic acid

Abstract Highly sensitive spectrophotometry for polyphenols has been proposed based on amplified pH changes during the substrate recycling of polyphenols driven by tyrosinase and L-ascorbic acid, which can be monitored as a spectral change in BTB (bromothymol blue). Polyphenolic compounds are enzymatically oxidized to o-quinone compounds catalyzed by tyrosinase, and are chemically regenerated by L-ascorbic acid, followed by substrate recycling. During this cyclic redox reaction, the pH continuously increases because a proton in the solution is consumed when o-quinones react with L-ascorbic acid. The magnitude of this pH change is significantly dependent on the concentration of the polyphenols, so a highly sensitive determination was possible by monitoring the absorbance of BTB in the enzyme solution with a reaction time of 5-10 min. The detection limits of catechol and catecholamines (L-dopa, dopamine, noradrenaline and adrenaline) were found to be on the order of 1x10−7 M-1x10−9 M using tyrosinase (catec...