Hitoshi Ohnuki

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.

Impedimetric and amperometric bifunctional glucose biosensor based on hybrid organic–inorganic thin films

A novel glucose biosensor with an immobilized mediator was studied using electrochemical impedance spectroscopy (EIS) and amperometry measurements. The biosensor has a characteristic ultrathin form and is composed of a self-assembled monolayer anchoring glucose oxidase (GOx) covered with Langmuir-Blodgett (LB) films of Prussian blue (PB). The immobilized PB in the LB films acts as a mediator and enables the biosensor to work under a low potential (0.0V vs. Ag/AgCl). In the EIS measurements, a dramatic decrease in charge transfer resistance (Rct) was observed with sequential addition of glucose, which can be attributed to enzymatic activity. The linearity of the biosensor response was observed by the variation of the sensor response (1/Rct) as a function of glucose concentration in the range 0 to 25mM. The sensor also showed linear amperometric response below 130mM glucose. The organic-inorganic system of GOx and PB nanoclusters demonstrated bifunctional sensing action, both amperometry and EIS modes, as well as long sensing stability for 4 days.

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.

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.

Oriented antibody immobilization on self-assembled monolayers applied as impedance biosensors

Oriented immobilization of antibodies on a sensor chip is crucial for enhancing both the sensitivity and antigen-binding capacity of immunosensors. Here, we report a comparative study of the effect of oriented and random antibody immobilization on the binding efficiency by electrochemical impedance spectroscopy (EIS). Oriented immobilization of anti-myoglobin immunoglobulin G (anti-Myo IgG) was achieved by bonding to an Fc receptor of protein G (PrG) on a self-assembled monolayer (SAM), which results in the myoglobin (Myo) binding sites being exposed outside the sensing surface. Random immobilization of anti-Myo IgG was achieved by direct covalent attachment to the SAM surface. Both immobilizations were applied to interdigitated electrodes to enhance the electrochemical signal, and the Myo biosensor performance was then evaluated by a series of EIS measurements. We found that (i) the rate of the normalized charge transfer resistance for the oriented sample was 3 times higher than that for the random sample and (ii) the detection limit was 0.001 ng/mL, which is the lowest recorded detection limit among Myo immunosensors based on EIS. These findings indicate that oriented antibody immobilization is crucial for preparing highly sensitive EIS-based biosensors.