Point-of-care multiplexed detection of cytokines using a HexaPie electrode array for asthma endotyping

Abstract Accurate multiplexed cytokine quantification in complex biofluids is essential for immune monitoring and respiratory disease phenotyping. We report HexaPie, a label-free, non-faradaic impedance biosensor that enables simultaneous detection of IL-8, IL-10, and IP-10 in human saliva through electrical double-layer (EDL) modulation at spatially multiplexed gold microelectrodes. The platform demonstrated limits of detection (LOD) of 1.17 ng mL −1 (IL-8), 12.5 pg mL −1 (IL-10), and 0.20 ng mL −1 (IP-10) with coefficients of variation below 15% and spike–recovery rates within 80–120%, satisfying CLSI analytical performance criteria. Specificity studies confirmed minimal signal interference from bovine serum albumin, uric acid, and ascorbic acid, validating robust performance in native saliva. Comparative clinical evaluation using ELLA and Luminex xMAP immunoassays revealed strong Bland–Altman concordance for IL-8 and IP-10 (mean bias <0.7; narrow ±1.96 SD limits of agreement), while IL-10 quantification showed greater inter-platform variability, likely due to low endogenous abundance and antibody-affinity differences. Together, these findings position HexaPie as a compact, reagent-free, and scalable point-of-care (POC) platform for rapid detection of clinically relevant cytokine elevations via non-invasive saliva sampling, advancing decentralized immunodiagnostics in respiratory health monitoring.