A novel colorimetric immunoassay based on enzyme-regulated instant generation of Turnbull's blue for the sensitive determination of ochratoxin A

Abstract

The aim of this study was to develop a novel colorimetric sensing method based on enzyme-regulated instant generation of Turnbull's blue, serving as a chromogenic agent, for a sensitive immunoassay for the determination of ochratoxin A (OTA). Unlike the traditional enzyme-linked immunosorbent assay (ELISA), the chromogenic reaction reported herein relies on the immediate formation of Turnbull's blue. K₃[Fe(CN)₆] rapidly forms a coordinate bond with iron(II), yielding a blue product. Meanwhile, glucose oxidase (GOx) catalyzes glucose hydrolysis to produce hydrogen peroxide (H₂O₂), which was used to inhibit the formation of Turnbull's blue by oxidizing iron(II) to iron(III). Thus, Turnbull's blue was generated in an enzyme-regulated manner. Accordingly, a competitive-type colorimetric enzyme immunoassay was established using a GOx based nanolabel. Under optimal conditions, the absorbance increased upon increasing the target OTA concentration in the range of 0.01–10 ng mL⁻¹ with a detection limit of 8.3 pg mL⁻¹ estimated at the 3S_blank level. The assay accuracy was validated by analyzing spiked wine samples. The present results potentially provide novel insights into the development of Turnbull's blue-based biological detection methods and colorimetric immunoassay strategies.