title: New approach for monitoring fish stress: A novel enzyme-functionalized label-free immunosensor system for detecting cortisol levels in fish.
abstract: 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.
authors: Graduate School of Marine Science and Technology, Tokyo University of Marine Science and Technology, 4-5-7, Konan, Minato-ku, Tokyo 108-8477, Japan.
authors: Haiyun
authors: H
authors: Wu
authors: Graduate School of Marine Science and Technology, Tokyo University of Marine Science and Technology, 2-1-6, Etchujima, Koto-ku, Tokyo 135-8533, Japan.
authors: Hitoshi
authors: H
authors: Ohnuki
authors: Graduate School of Marine Science and Technology, Tokyo University of Marine Science and Technology, 4-5-7, Konan, Minato-ku, Tokyo 108-8477, Japan.
authors: Shirei
authors: S
authors: Ota
authors: Hokkaido Industrial Technology Center, 379 Kikyo-cho, Hakodate, Hokkaido 041-0801, Japan.
authors: Masataka
authors: M
authors: Murata
authors: Yashima station, Stock Enhancement and Management Department, National Research Institute of Fisheries and Enhancement of Inland Sea, Japan Fisheries Research and Education Agency, 243 Yashima-higashi, Takamatsu, Kagawa 761-0111, Japan.
authors: Yasutoshi
authors: Y
authors: Yoshiura
authors: Graduate School of Marine Science and Technology, Tokyo University of Marine Science and Technology, 4-5-7, Konan, Minato-ku, Tokyo 108-8477, Japan. Electronic address: endo@kaiyodai.ac.jp.
authors: Hideaki
authors: H
authors: Endo
mesh_terms: D000818:Animals; D000936:Antigen-Antibody Complex / Q000737:chemistry / Q000276:immunology; D015374:Biosensing Techniques; D023681:Cichlids / Q000502:physiology; D055664:Electrochemical Techniques; D004566:Electrodes; D005949:Glucose Oxidase / Q000737:chemistry; D006046:Gold / Q000737:chemistry; D006801:Humans; D006854:Hydrocortisone / Q000737:chemistry / Q000302:isolation & purification*; D013312:Stress, Physiological
chemical_list: D000936:Antigen-Antibody Complex; D006046:Gold; D005949:Glucose Oxidase; D006854:Hydrocortisone
keywords: Amperometry; Cortisol; Enzyme-functionalized biosensor; Fish stress; SAM