Single‐Response Duplexing of Electrochemical Label‐Free Biosensor from the Same Tag

Abstract Multiplexing is a valuable strategy to boost throughput and improve clinical accuracy. Exploiting the vertical, meshed design of reproducible and low‐cost ultra‐dense electrochemical chips, the unprecedented single‐response multiplexing of typical label‐free biosensors is reported. Using a cheap, handheld one‐channel workstation and a single redox probe, that is, ferro/ferricyanide, the recognition events taking place on two spatially resolved locations of the same working electrode can be tracked along a single voltammetry scan by collecting the electrochemical signatures of the probe in relation to different quasi‐reference electrodes, Au (0 V) and Ag/AgCl ink (+0.2 V). This spatial isolation prevents crosstalk between the redox tags and interferences over functionalization and binding steps, representing an advantage over the existing non‐spatially resolved single‐response multiplex strategies. As proof of concept, peptide‐tethered immunosensors are demonstrated to provide the duplex detection of COVID‐19 antibodies, thereby doubling the throughput while achieving 100% accuracy in serum samples. The approach is envisioned to enable broad applications in high‐throughput and multi‐analyte platforms, as it can be tailored to other biosensing devices and formats.