Ratiometric Detection of Ochratoxin A Using a Regenerable COF-Au-MB-Apt Signal Probe on a Thermal-Regulated Sensor Module

Ochratoxin A (OTA) frequently contaminates grains and consequently threatens human health. Herein, we develop a regenerable signal probe and apply it to a Au-based screen-printed electrode module (SPE) for OTA determination. The signal probe, containing a structural covalent organic framework, gold nanoparticles (AuNPs), indicative methylene blue (MB), and a highly selective aptamer, is synthesized with hydrothermal and self-assembly methods. The SPE is permanently functionalized with Prussian blue (PB), AuNPs, and semicomplementary ssDNA. The signal probe, absorbed onto this SPE via hybridization, is competitively expelled by OTA, providing a ratiometric readout of ΔI_MB/I_PB. Probe regeneration, to erase expired COF-Au-MB-Apt after each analysis, is established with the synergy of OTA-conducted Apt-ssDNA dissociation and on-chip thermal regulation. This advantage powerfully guarantees reduplicative analyses by avoiding irreversible Apt-OTA combination and accumulation on the sensing interface. Regenerations are performed in repetitive cycles (N = 7) with 98.5% reproduction efficiency, and I_MB and I_PB fluctuations are calculated as 1.45 and 1.12%. This method shows log-linear OTA response in a wide range from 0.2 pg/mL to 0.6 μg/mL, and the limit of detection is 0.12 pg/mL. During natural OTA determinations, recommended readouts match well with HPLC with less than 4.82% relative error.