Metal–Organic Frameworks Functionalized Porous Screen-Printed Electrodes for Label-Free Ratiometric Electrochemical Immunosensing of Mycotoxin

Ratiometric electrochemical immunosensors offer improved reproducibility and accuracy compared with conventional single-signal platforms, yet their use in label-free mycotoxin detection remains limited due to dependence on signal-molecule labels. Herein, we report a label-free ratiometric electrochemical immunosensor for zearalenone (ZEN), chosen as a representative mycotoxin, constructed on a porous screen-printed carbon-carbon nanotube electrode (porous CNT SPE) functionalized with a nickel metal-organic framework/carbon nanotube composite (Ni-MOF/CNT). The MOF was engineered by partially substituting 2-aminoterephthalic acid (NH₂-BDC) with 2,3-diaminoterephthalic acid ((NH₂)₂-BDC), thereby increasing amino group density and enabling efficient antibody immobilization. Ratiometric detection was achieved by confining a solid-state redox probe within the porous electrode as an internal reference, while a solution-phase probe served as the analytical signal. The MOF layer not only enabled stable biomolecule conjugation but also effectively minimized redox probe leaching. With this concept, the proposed configuration significantly reduced signal fluctuation and improved measurement consistency compared with the non-ratiometric approach. The sensor exhibited high selectivity against common interferents and was validated using real food samples. Fabricated through scalable screen-printing technology, this integrated platform demonstrates strong potential as a universal, low-cost, and practical approach for on-site electrochemical immunosensing of mycotoxins.