Herein, a delicate bifunctional reagent regulated ratiometric electrochemiluminescence (ECL) biosensor was developed for the detection of deoxynivalenol (DON) by virtue of the ratio of two ECL signals from Luminol and tris(4,4'-dicarboxylicacid-2,2'-bipyridyl) ruthenium(II) dichloride (Ru(dcbpy)). Initially, surfactant-assisted synthesis of TiO mesocrystals dispersing in Nafion and ionic liquid (IL) complex film held great promises in loading Ru(dcbpy) and amplifying the ECL signal. Additionally, helical carbon nanotube (HCNTs) with superior specific area and good conductivity emerging as a scaffold not only realized the high fixing of Luminol, ferrocenecarboxylic acid (FCA) and secondary antibody (Ab), but accelerated the electron transfer. It's noteworthy that FCA was first utilized as a bifunctional reagent to regulate the ratiometric ECL sensing mode on account of the influences in enhancing the ECL response of Luminol and quenching the ECL emission of Ru(dcbpy). On the basis of the sandwich-type immunoreactions between Ab, DON and Ab, an accurate and sensitive ratiometric ECL immunosensor was established for the detection of DON in a wide range from 0.05 pg/mL to 5 ng/mL with the detection limit of 1.67 × 10 pg/mL.
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