Masataka Murata

Fish stress become visible: a new attempt to use biosensor for real-time monitoring fish stress.

To avoid fish mortality and improve productivity, the physiological conditions including stress state of the cultured fish must be monitored. As an important indicator of stress, glucose concentrations are monitored using in vitro blood analysis. The physiological processes of fish under environmental conditions are harsher in many ways than those experienced by terrestrial animals. Moreover, the process of anaesthetizing and capturing the fish prior to analysis may produce inaccurate results. To solve these problems, we developed wireless biosensor system to monitor the physiological condition of fish. This system enables artificial stress-free and non-lethal analysis, and allows for reliable real-time monitoring of fish stress. The biosensor comprised Pt-Ir wire as the working electrode and Ag/AgCl paste as the reference electrode. Glucose oxidase was immobilized on the working electrode using glutaraldehyde. We used the eyeball interstitial sclera fluid (EISF) as the in vivo implantation site of the sensor, which component concentration correlates well with that of blood component concentration. In the present study, we investigated stress due to alterations in water chemistry, including dissolved oxygen, pH, and ammonia-nitrogen compounds. Stress perceived from behavioural interactions, including attacking behaviour and visual irritation, was also monitored. Water chemistry alterations induced increases in the glucose concentration (stress) that decreased with removal of the stimulus. For behavioural interactions, stress levels change with avoidance, sensory behaviour and activity. We believe that the proposed biosensor system could be useful for rapid, reliable, and convenient analysis of the fish physiological condition and accurately reflects the stress experienced by fish.

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.

New approach for monitoring fish stress: A novel enzyme-functionalized label-free immunosensor system for detecting cortisol levels in fish

Fishes display a wide variation in their physiological responses to stress, which is clearly evident in the plasma corticosteroid changes, chiefly cortisol levels in fish. As a well-known indicator of fish stress, a simple and rapid method for detecting cortisol changes especially sudden increases is desired. In this study, we describe an enzyme-functionalized label-free immunosensor system for detecting fish cortisol levels. Detection of cortisol using amperometry was achieved by immobilizing both anti-cortisol antibody (selective detection of cortisol) and glucose oxidase (signal amplification and non-toxic measurement) on an Au electrode surface with a self-assembled monolayer. This system is based on the maximum glucose oxidation output current change induced by the generation of a non-conductive antigen-antibody complex, which depends on the levels of cortisol in the sample. The immunosensor responded to cortisol levels with a linear decrease in the current in the range of 1.25-200ngml-1 (R=0.964). Since the dynamic range of the sensor can cover the normal range of plasma cortisol in fish, the samples obtained from the fish did not need to be diluted. Further, electrochemical measurement of one sample required only ~30min. The sensor system was applied to determine the cortisol levels in plasma sampled from Nile tilapia (Oreochromis niloticus), which were then compared with levels of the same samples determined using the conventional method (ELISA). Values determined using both methods were well correlated. These findings suggest that the proposed label-free immunosensor could be useful for rapid and convenient analysis of cortisol levels in fish without sample dilution. We also believe that the proposed system could be integrated in a miniaturized potentiostat for point-of-care cortisol detection and useful as a portable diagnostic in fish farms in the future.

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.

Real-time fish stress visualization came true：A novel multi-stage color-switching wireless biosensor system

An optical communication type biosensor system has been developed which can measure blood glucose concentration, which is a stress indicator of fish, in real-time while fish swimming freely. However, this system is hard to make instant acknowledgment of fish stress level which has to contain an unavoidable delay in the judgment. In this research, we aimed to develop a novel stress visualization system which can quickly judge the levels for fish stress response instantly based on a color changeable LED while another LED was designed to send data. The present system is based on the principle of converting the output current value measured by the glucose biosensor corresponding to the stress response into a voltage value. Then, the color and stress switching points of the LED (Red, Yellow, Green) were decided based on the voltage value gained from the biosensor which mentioned above. Furthermore, we attempted to use our biosensor system to make real-time monitoring of fish stress in vivo. As results, the proposed sensor can make real-time measurement of glucose and shows a great response to those of actual fish sample in the range from 35.36 to 300 mg dl-1 (R = 0.9899). When the glucose concentration in the collected sample was switched to the concentration pre-sett, it was successful to switch the LED color according to the gained voltage value both in vitro and in vivo. Furthermore, when monitoring the stress responses of the fish in vivo, color switching corresponding to the sensor output current value was observed successfully.