Development of a Novel Enhanced Biosensor System for Real-Time Monitoring of Fish Stress Using a Self-Assembled Monolayer.

Wireless biosensor systems were developed in our lab for monitoring blood glucose concentrations in fish as an indicator of fish stress. However, uniform immobilization of the enzyme on the surface of the electrode is difficult, so the sensor response is typically reduced at a range of high glucose concentrations during the stress monitoring. In this study, we attempted to enhance sensor response by using a self-assembled monolayer-immobilized enzyme. Glucose oxidase was immobilized on a working electrode modified with a self-assembled monolayer. The proposed biosensor showed a good correlation between the output current and the glucose concentration range of 10⁻3500 mg dL-1 under an optimized working condition. The dynamic measurement range of this newly developed sensor is significantly improved, especially over a high concentration range, which helps the sensor to achieve better performance in dramatic changes in the stress response of fish. In addition, we used biological samples from test fish and obtained a good correlation coefficient between the sensor output current and the glucose concentration using a conventional method. The proposed wireless biosensor system was also applied to monitor fish stress responses in real time through different stressors and to obtain some precise data that reflect real fish stress responses.

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.

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.

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.

Development of mediator-type biosensor to wirelessly monitor whole cholesterol concentration in fish.

We developed a wireless monitoring system to monitor fish condition by tracking the change in whole cholesterol concentration. The whole cholesterol concentration of fish is a source of steroid hormones or indicator of immunity level, which makes its detection important for tracking physiological condition of fish. Wireless monitoring system comprises of mediator-type biosensor and wireless transmission device. Biosensor is implantable to fish body, and transmission device is so light, in that fish is allowed to swim freely during monitoring. Cholesterol esterase and oxidase were fixated on to the detection site of biosensor and used to detect the whole cholesterol concentration. However, cholesterol oxidase incorporates oxidation-reduction reaction of oxygen for detection, which concentration fluctuates easily due to change in environmental condition. Meanwhile, mediator-type biosensor enables monitoring of whole cholesterol concentration by using mediator to substitute that oxidation-reduction reaction of oxygen. Characteristic of fabricated mediator-type biosensor was tested. The sensor output current of mediator-type biosensor remained stable compared to output current of non-mediator-type biosensor under fluctuating oxygen concentration of 0-8 ppm, which implied that this sensor is less affected by change in dissolved oxygen concentration. That biosensor was then implanted into fish for wireless monitoring. As a result, approximately 48 h of real-time monitoring was successful.

Carbon nanotube enhanced mediator-type biosensor for real-time monitoring of glucose concentrations in fish.

We have developed a mediator-type biosensor to rapidly monitor blood glucose concentrations in fish, which are an indicator of stress. Glucose oxidase was used to detect glucose concentrations and ferrocene was used to limit the effect of oxygen. We also improved the sensitivity and durability of the sensor for better performance. Single-walled carbon nanotubes were used to enhance sensor sensitivity. Affixing the carbon nanotubes (30 mg ml(-1)) to the working electrode increased the sensor sensitivity to 61.9 mM nA(-1) mm(-2), twice the value for the sensor without single-walled carbon nanotubes. A fabricated mediator-type biosensor sensor was used to perform real-time in vivo measurements. The sensor was implanted into the interstitial fluid of a fish eyeball, and detection was transmitted to a personal computer by a wireless potentiostat. Continuous measurement of the glucose concentration was possible for 78 hours. Stress was artificially applied to the fish during the measurement, and the change of blood glucose concentrations were observed. Our proposed sensor is applicable for effectively monitoring stress in free-swimming 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.