A vertical-flow O-ring electrochemical immunosensor with self-washing for glyphosate detection

Conventional label-free electrochemical immunosensors require manual washing to remove unbound species and sample matrices, which increases assay time and handling complexity. Herein, we report a self-washing vertical-flow O-ring electrochemical immunosensor for rapid glyphosate detection. The device integrates a poly(L-cysteine)-modified paper-based graphene electrode for covalent anti-glyphosate antibody immobilization with a filter membrane and absorbent pad positioned beneath an O-ring opening at the working electrode. This configuration controls vertical sample transport, provides sufficient residence time for immunocomplex formation, and enables ferri/ferrocyanide to act simultaneously as the redox probe and washing solution. Under optimized conditions, the sensor exhibited a linear range of 6.25 ng/mL to 5 μg/mL with a detection limit of 1.54 ng/mL. Compared with the conventional format, the O-ring platform lowered the detection limit by approximately four-fold, reduced the workflow from five to two steps, and completed detection within 15 min. Real food sample analysis showed satisfactory agreement with LC-MS/MS, demonstrating its potential for practical food safety monitoring.