Immunoassays based on the antigen antibody specific reactions are considered

Immunoassays based on the antigen–antibody-specific reactions are considered major analytical tools in clinical diagnoses, and in environmental and biochemical studies [17,18]. Various immunoassay protocols, such as surface plasmon resonance [19], quartz crystal microbalance [20], chemiluminescence [21], and electrochemical methods [22,23], have been extensively developed for detecting biomarkers like cortisol. Among these methods, electrochemistry with high sensitivity, low cost, low power requirements, and high compatibility is the preferred approach for clinical and environmental immunoassays. We developed some label-free immunosensor systems using cyclic voltammetry (CV) that allow for easy and rapid assay of various steroid hormones in fish [24,25], and we believe these systems will be as effective as the conventional detection methods used in fish farms while being simpler and more rapid to use. Though the sensors have a relatively high sensitivity, they require frequent electrode replacement and the measurement is performed by CV, which requires a large device and time-consuming cyclic scans. On the other hand, we also developed a biosensor for Merimepodib based on a flow injection analysis, which allows for rapid and convenient cortisol detection [26]. This system, however, requires a complex competitive immunologic reaction and immunomagnetic separation. Therefore, based on the knowledge gained from our previous studies, here we aimed to develop a cortisol measurement system in fish plasma based on flow analysis with easy electrode replacement via an electrode replacement unit and electrochemical measurement. We evaluated analytic parameters (pH and incubation temperature) and evaluated the sensitivity and specificity of this new sensor system. Finally, the proposed system was applied to the measurement of cortisol in fish plasma samples. The results were compared with those obtained using a conventional enzyme-linked immunosorbent assay (ELISA).

Materials and methods

Results

Discussion

Conclusion
Here we described a newly designed flow immunobiosensor system with an electrode replacement unit for continuous cortisol monitoring for fish. Highlights of the immunoassay system are that it is direct, rapid, and simple without extra labeling and separation steps. And the measurement can be finished in ~30min. Furthermore, because the electrode replacement unit allows the electrode change to be completed within several seconds, it is possible to continuously monitor cortisol levels for fish. The results Merimepodib obtained by this method may also provide new knowledge in the field of fish stress, aquaculture, and fish physiology. Importantly, the developed immunoassay method can be further automated and miniaturized for more rapid and high-throughput electrochemical immunoassay of field work. This system will be useful for rapid, reliable, and convenient analysis of fish health in combination with other indicators of fish health, such as blood glucose. Based on this knowledge, improvements to the aquatic environment and farming technology can be implemented to enhance fish-friendly fisheries.

Acknowledgments
This research was supported in part by a Grant-in-Aid for Scientific Research (B) (No. 26292114) from the Ministry of Education, Culture, Sports, Science and Technology in Japan. We wish to thank Dr. Huifeng Ren (Tokyo University of Marine Science and Technology, Japan), Prof. Pierre Morizet-Mahoudeaux (University of Technology of Compiègne, France) and Dr. Chiharu Ichioka (Ichioka Dental Office, Japan) for their helpful discussions.

Introduction
Bio and chemical sensors based on SPR in optical fibers have been shown to be able to play an important role in numerous relevant fields, including pharmaceutical researches, medical diagnostics, industrial applications, environmental monitoring, food safety and security, where fast, portable, low cost and rugged units are needed for early detection and identification [1–5]. Here the fiber core replaces the glass prism and couples the entrance light to the sensing element, giving a sensor which works in the wavelength interrogation mode and has advantages over the prism configuration such as miniaturization and integration, remote sensing, real time and in situ monitoring [6]. In general, the optical fiber is either a glass or a plastic one (POF). POFs are practically advantageous due to their excellent flexibility, easy manipulation, great numerical aperture, large diameter, and the fact that plastic is able to withstand smaller bend radii than glass. The advantage of using POFs is that the main features of POFs, that have increased their popularity and competitiveness for telecommunications, are exactly those that are important for optical sensors based on glass optical fibers, with the addition of simpler manufacturing and handling procedures. POF with a large diameter are suited for sensors also for the great number of modes, the relatively easy splitting of the light beam and connection with other fibers. Also, experimental results on POF sensor indicate that the configuration with a fiber of large diameter exhibits better performance, with respect to a fiber with minor diameter, in terms of sensitivity and resolution even if not in terms of signal-to-noise ratio [7,8].