Electrochemical flow injection immunoassay for cortisol using magnetic microbeads

Abstract

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.

Keywords:

• cortisol
• FIA
• magnetic microbeads
• immunoassay
• fish
• monitoring

Acknowledgements

This research was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology.