Kyoko Hibi

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.

Bioelectronic detector with monoamine oxidase for halitosis monitoring

Methyl mercaptan (MM) is known as one of the major chemicals of halitosis (bad breath). In this study, a bioelectronic gas sensor (bio-detector) for gaseous MM was developed and was applied to measure halitosis in breath. The bio-detector consisted of a Clark-type dissolved oxygen electrode, a monoamine oxidase type-A (MAO-A) immobilized membrane and a reaction unit that had liquid and gaseous compartments separated by a hydrophobic porous polytetrafluoroethylene (PTFE) diaphragm membrane. The tip of the electrode covered with MAO-A membrane was placed into the liquid compartment as touching to the PTFE diaphragm membrane. In order to amplify the bio-detector output, a substrate regeneration cycle caused by coupling the monooxygenase with l-ascorbic acid as reducing reaction with reagent system, was applied. The results of MM vapor measurements showed the calibration range of the bio-detector for MM vapor was from 0.087 to 11.5 ppm (correlation coefficient: 0.993) and included the human sense of smell level 5 (0.2 ppm). The bio-detector had good selectivity being attributed to enzyme specificity was obtained for several substances (trimethyl amine, ammonia, dimethyl sulfide, etc.). The bio-detector was applied for halitosis measurement. Expired gases in five subjects were sampled every hour and the concentrations of MM in the expired gases were monitored. The output of bio-detector showed behaviour of halitosis level changes in a day such as increasing with passage of time and decreasing after eating.

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.

Wireless Biosensor System for Real-Time L-Lactic Acid Monitoring in Fish

We have developed a wireless biosensor system to continuously monitor l-lactic acid concentrations in fish. The blood l-lactic acid level of fish is a barometer of stress. The biosensor comprised Pt-Ir wire (φ0.178 mm) as the working electrode and Ag/AgCl paste as the reference electrode. Lactate oxidase was immobilized on the working electrode using glutaraldehyde. The sensor calibration was linear and good correlated with l-lactic acid levels (R = 0.9959) in the range of 0.04 to 6.0 mg·dL−1. We used the eyeball interstitial sclera fluid (EISF) as the site of sensor implantation. The blood l-lactic acid levels correlated closely with the EISF l-lactic acid levels in the range of 3 to 13 mg·dL−1 (R = 0.8173, n = 26). Wireless monitoring of l-lactic acid was performed using the sensor system in free-swimming fish in an aquarium. The sensor response was stable for over 60 h. Thus, our biosensor provided a rapid and convenient method for real-time monitoring of l-lactic acid levels in fish.

Electrochemical flow injection immunoassay for cortisol using magnetic microbeads

We developed a novel flow injection assay for cortisol based on competitive immunologic reactions, magnetic separation, and electrochemical measurement. The proposed flow assay system was composed of two reaction units. An anti-cortisol antibody was immobilised on magnetic beads and injected into the reaction coil of a competitive reaction unit with a blood sample and a specific quantity of acetylcholinesterase-labelled cortisol (cort-AChE). After reacting in the reaction coil, the sample was separated magnetically using a neodymium magnet. The cort-AChE was detached from the magnetic beads and transferred into the enzyme reaction unit with acetylthiocholine (ATCh). ATCh was hydrolysed by the cort-AChE to produce thiocholine. The thiocholine was quantified downstream by electrochemical detection using a Pt-Ir electrode. The performance of the proposed flow assay system was optimised under the following conditions: pH 7.5, temperature 25°C, flow rate 170 µl min−1, ATCh concentration in the substrate buffer 5 mmol L−1. The output current was well correlated with the concentration of the cortisol standard solution (range: 7.8–500 pg mL−1). The results obtained using the proposed flow method were compared with those obtained using conventional ELISA (correlation coefficient 0.9585 [y = −0.9797 + 1.173(x), n = 11]). These findings suggest that the EFIIA system can be used to analyse cortisol in fish plasma samples.

Development of a biocompatible glucose biosensor for wireless and real time blood glucose monitoring of fish

We developed glucose biosensors coated with biocompatible polymers to rapidly monitor glucose levels in free-swimming fish. Biocompatible polymers have a similar structure to living organisms and are thus used to make metallic materials more compatible with the living body. We focused on three widely used biocompatible polymers, 2-methacryloyloxyethyl phosphorlycholine (MPC) polymers, polypyrroles, and polyurethanes, to achieve biocompatibility of our glucose biosensor. The developed glucose biosensor has a Pt-Ir wire (φ0.178 mm) as the working electrode and Ag/AgCl paste as the reference electrode. The biosensor was first coated with Nafion to prevent coexisting substances such as ascorbic acid and uric acid from interfering with the sensor output current, and then glucose oxidase (GOx) was fixed on top of the Nafion layer along with biocompatible polymers. The sensor was inserted into the fish eyeball interstitial scleral fluid (EISF), which contains low levels of proteins and correlates well with the glucose levels in the whole blood. Those three sensors were tested for durability and sensors coated with MPC polymers (Nafion/GOx/MPC sensor) proved to be most durable: the sensor output current maintained 93% output for 15 h in standard glucose solution, and 80% in EISF for 8 h, whereas the output current of the other sensors decreased more rapidly overtime. We then inserted Nafion/GOx/MPC sensor to wirelessly monitor EISF glucose levels in free-swimming fish. One-point calibration method was used to calibrate the sensor output current. As a result, 24 h of wireless monitoring was successfully achieved.

A Fiber Optic Immunosensor for Rapid Bacteria Determination

Attention is currently focused on fiber optic immunosensor as sensitive and nearly real time protein detector. This kind of sensor is expected to detect bacteria in foods directly by dipping the thin optical fiber dominant area into foods. In the study, an antibody based fiber optic immunosensor to detect Escherichia coli O157:H7 (E.coli O157:H7) was constructed. The principle of the sensor was a sandwich immunoassay on the optical fiber surface. A goat polyclonal antibody was first immobilized on polystyrene optical fiber. E.coli O157:H7 and a cyanine 5 (Cy5) -labeled goat polyclonal antibody were used to generate a specific fluorescent signal. An excitation light (λ = 635 nm) was illuminated into the optical fiber, and the Cy5 florescent molecules near the optical fiber (approximately 100 nm) were excited by evanescent wave emitted from the optical fiber. The fluorescent light (λ = 670 nm) collected by the optical fiber was measured using a photodiode. The measurement range for E.coli O157:H7 diluted with phosphate buffer (PB) was from 1×102 to 1×107 cells/ml. This method could also detect E.coli O157:H7 in milk artificially inoculated with 1×102 to 1×107 cells/ml. This immunosensor was specific for E.coli O157:H7 and showed significantly higher signal strength than for nonpathogenic E.coli or other bacteria, including Listeria monocytogenes and Vibrio s.p., in pure or in mixed-culture setup. The results could be obtained within about 15 min of sampling.