Tetsuhito Hayashi

Wireless enzyme sensor system for real-time monitoring of blood glucose levels in fish

Periodic checks of fish health and the rapid detection of abnormalities are thus necessary at fish farms. Several studies indicate that blood glucose levels closely correlate to stress levels in fish and represent the state of respiratory or nutritional disturbance. We prepared a wireless enzyme sensor system to determine blood glucose levels in fish. It can be rapidly and conveniently monitored using the newly developed needle-type enzyme sensor, consisting of a Pt-Ir wire, Ag/AgCl paste, and glucose oxidase. To prevent the effects of interfering anionic species, such as uric acid and ascorbic acid, on the sensor response, the Pt-Ir electrode was coated with Nafion, and then glucose oxidase was immobilized on the coated electrode. The calibration curve of the glucose concentration was linear, from 0.18 to 144mg/dl, and the detection limit was 0.18mg/dl. The sensor was used to wirelessly monitor fish glucose levels. The sensor-calibrated glucose levels and actual blood glucose levels were in excellent agreement. The fluid of the inner sclera of the fish eyeball (EISF) was a suitable site for sensor implantation to obtain glucose sample. There was a close correlation between glucose concentrations in the EISF and those in the blood. Glucose concentrations in fish blood could be monitored in free-swimming fish in an aquarium for 3 days.

Biosensor system for continuous glucose monitoring in fish

A biosensor system was developed for continuous estimation of blood glucose in fish. Because it is difficult to measure blood components in real-time due to decreased sensor output resulting from blood coagulation and coalescing blood proteins at the sensor placement site, we used the eyeball scleral interstitial fluid (EISF) as the site of sensor implantation. Evaluation of the relationship between EISF and blood glucose concentrations revealed that the blood glucose concentration correlated closely with the EISF glucose concentration (y=2.2996+0.9438x, R=0.960, n=112). To take advantage of the close correlation between blood and EISF glucose, we prepared a needle-type enzyme sensor for implantation in the fish sclera using a flexible wire electrode. The sensor provided a rapid response, good linearity, and reproducibility. Continuous glucose monitoring could be carried out by implanting this needle-type glucose sensor onto the eye. The findings indicated that the glucose concentration increased with sensor output current over time, and that changes in the blood glucose were continuously reflected in the EISF. The glucose concentration was estimated based on the one-point or two-point calibration methods. The two-point calibration method yielded the most accurate glucose monitoring (blood glucose range of 70-420 mg dL(-1)) over 160 min. Sensor-estimated glucose and whole blood glucose values were highly correlated (y=0.4401+0.8656x, R=0.958).

The superiority of organically cultivated vegetables to general ones regarding antimutagenic activities

We found organically cultivated (OC) vegetables, using a water-soluble chitosan as a soil improvement agent and leaf surface spray, had much longer shelf life and better taste than that of generally cultivated (GC) vegetables. The purpose of this study is to determine the relative antimutagenic activity between OC and GC vegetables. Eleven OC vegetables were harvested in March and April in 1999 and 2000, and GC ones were supplied as a control from nearby farms on the same date. The former vegetables were planted on the field where no pesticide had been used for the last 3 years. Forward mutation test with Salmonella typhimurium TM677 and 8-azaguanine as a detection agent was used to determine the antimutagenic activity of juices prepared from OC and GC vegetables against authentic mutagens, such as 4-nitroquinoline oxide (4NQO), benzo(a)pyrene (BaP), and 3-amino-1-methyl-5H-pyrido[4,3-b]indole acetate (Trp-P-2). This microbiological test is a convenient method to use for the food samples containing free histidine. Antimutagenic activity was evaluated by the difference of mutagenic activities between mutagenecity of authentic compounds and that observed upon incubation at 37 degrees C for 2h with each vegetable juice. OC Chinese cabbage, carrot, Welsh onion, and Qing-gen-cai suppressed 37-93% of the mutagenic activity of 4NQO, while the GC ones were held down to 11-65%. Against BaP, three species of OC vegetables showed 30-57% antimutagenecity, while GC ones did only 5-30%. Similarly, the OC spinach decreased the activity of Trp-P-2 to 78%, and the GC suppressed it by 49%.

Simultaneous determination of hypoxanthine and inosine with an enzyme sensor

Abstract A sensor for the simultaneous determination of hypoxanthine and inosine was prepared by a combination of the enzyme system (shown below) and an oxygen electrode. Xanthine oxidase and nucleoside phosphorylase, respectively, were covalently immobilized on triacetyl cellulose membranes containing 1,8-diamino-4-aminomethyloctane. Xanthine oxidase membrane, three sheets of the triacetyl cellulose membrane described above, and nucleoside phosphorylase membrane were placed in that order on the tip of the oxygen electrode. The optimum conditions for simultaneous determination of hypoxanthine and inosine were pH 7.8, 303 K, and a flow rate of 1.4ml min −1 . Calibration curves for hypoxanthine and inosine were linear up to 0.4 mM and 4 mM, respectively. The relative errors were 6% and 1.5% for hypoxanthine and inosine, respectively, in 24 assays.

Use of a channel biosensor for the assay of paralytic shellfish toxins.

Gonyautoxin (GTX), saxitoxin (STX) and tetrodotoxin (TTX), also known as paralytic shellfish poisons (PSP), block Na+ channels, including those in the frog bladder membrane. A tissue biosensor has been developed, consisting of a Na+ electrode covered with a frog bladder membrane integrated within a flow cell. The direction of Na+ transfer, investigated in the absence of Na+ channel blockers, established that active transport of Na+ occurs across the frogs bladder membrane from the internal to the external face. Transfer was shown to be TTX sensitive. The tissue sensor response to each of the different PSP was recorded and the results compared with toxicities determined by the standard mouse bio-assay. Using high concentrations of TTX from the puffer fish Takifugu niphobles, a linear correlation was found between the results from the two assay systems. However, the tissue biosensor system was also able to detect very low concentrations of TTX in samples from two species of puffer fish (Takifugu niphobles and Takifugu pardalis) at concentrations below the detection limit of the mouse bio-assay.

Development of an ultra high sensitive tissue biosensor for determination of swellfish poisoning, tetrodotoxin

A simple tissue biosensor for measuring Na+ channel blockers such as tetrodotoxin (TTX) and saxitoxin (STX) has been developed. The membrane of frog bladder has Na+ channels which control the passage of Na+. It is well known that TTX blocks Na+ channels. The tissue biosensor consists of a Na+ electrode integrated within a flow cell. The tip of the electrode was covered with frog bladder membrane sandwiched between two sheets of cellulose acetate membrane, and the electrode was set in a flow cell. A solution of 8% NaCl was carried in the cell and the output of the electrode allowed to stabilize. TTX was injected into the sensor system and measured from the inhibition ratio of the sensor peak output. One assay took approximately 5 min. The lower limit of detection was 86 fg. The continuous determination of TTX was feasible for 250 h in the presence of 0.003% NaN3. A Linear correlation was obtained between TTX activities of F-niphobles and F-parudale determined by the methods of TTX sensor and mouse assay.

Novel model experiment for cooking flavor research on crab leg meat

Abstract Fluctuation of extractives upon heating was analyzed and the flavor profile of some amino acid‐enriched model crab leg was studied to elucidate the mechanism of the pleasing flavor of cooked crab. The change in concentration of the extractive components suggested that these nonvolatile compounds took part in the formation of volatile substances characteristic of crab flavor by the heating ranged from 85 to 100°C. The model experiments using specially designed extractive‐enriched crab legs led us to conclude that some amino acids which were distributed in high concentration in the extract might be precursors of the characteristic compounds responsible for cooked crab flavor.

Development of a trimethylamine gas biosensor system

Abstract A trimethylamine (TMA) gas biosensor was developed, based on the immobilized mould, Penicillium decumbens , an oxygen electrode, a flow cell, a peristaltic pump, a recorder and a buffer tank. The optimum conditions for the sensor were: pH 7·6, temperature 30°C, flow rate 0·3 ml/min, and sample volume 50 μl. Upon measurement of TMA concentrations in fish muscle, a good correlation was found between results obtained from the sensor and those obtained from gas-liquid chromatography (GLC). The correlation coefficient was 0·84. Each assay lasted 30 min. The proposed sensor was stable even after 37 days.

Wireless biosensor system for real-time cholesterol monitoring in fish “Nile tilapia”

The rapidly increasing demand for cultured fish as a food resource requires simple, effective methods for controlling fish health in culture conditions. Plasma total cholesterol levels are significantly related to fish mortality following bacterial challenge, and are thus a good indicator of the general health of fish. We developed a wireless biosensor system to continuously monitor the total cholesterol concentration in fish (Nile tilapia, Oreochromis niloticus). The biosensor was constructed with Pt-Ir wire (phi0.178 mm) as the working electrode and Ag/AgCl paste as the reference electrode. Cholesterol oxidase and cholesterol esterase were immobilized on the working electrode using glutaraldehyde. The sensor output was linear and strongly correlated with the cholesterol level (R=0.9970) in the range of 2.65-403 mg dl(-1). This range covers the range of total cholesterol levels in fish. To avoid blood coagulation and proteins coalescing on the sensor, we implanted the sensor in the fluid under the scleral surface of the eyeball (EISF). The EISF is presumed to reflect the levels of most blood components and does not include the substances contained in blood that inhibit sensor measurement. Total cholesterol concentrations in blood and EISF were strongly correlated (R=0.8818, n=72) in the blood total cholesterol range of 74-480 mg dl(-1). Therefore, we used EISF as an alternative to blood and performed continuous in vivo-monitoring of the total cholesterol concentration in fish. We also investigated the application of the calibration method and wireless monitoring system. These applications enabled us to securely monitor total cholesterol levels in free-swimming fish in an aquarium for over 40 h. Thus, our newly developed sensor provided a rapid and convenient method for real-time monitoring of total cholesterol concentrations in free-swimming fish.

Application of flow cytometry for rapid detection of Lactococcus garvieae.

Flow cytometry (FCM) technique was applied to rapid determination of cell number ofLactococcus garvieae. An antiserum againstL. garvieae was prepared and its immunological property was examined. The present antibody would recognize some epitopes ofL. garvieae with a high specificity. The optimum conditions for the FCM assay were as follows: discriminate value, 60; dilution ratio of the antiserum, 1.0 X 104. Calibration curve forL. garvieae cells was linear, in the range of 2.4 X 104-1.5 X 107 cells/mL. The detection ofL. garvieae in cell suspensions contaminated withEscherichia coli was carried out. A good correlation was observed in the range of 20–90% for the mixing ratio ofL. garvieae. One FCM assay could be completed within 2 min, and the total assay time, including the preparation of bacterial sample, was within 3 h.