Systematic optimisation of an integrated electrochemical aptamer-based sensor for cortisol detection

This paper presents the development of a self-contained electrochemical aptamer-based (EAB) sensor for cortisol detection, addressing the growing need for rapid, reliable stress hormone monitoring. Built on an integrated three-electrode system, our sensor employs a 14-mer truncated cortisol aptamer, strategically selected for its enhanced binding affinity and structure-switching capability upon cortisol interaction, offering significant advantages over its 61-mer parent sequence. The sensor design combines computational analysis of aptamer-cortisol interactions with experimental validation using methylene blue (MB)-labelled aptamers on a thin-layer gold electrode platform. Notably, we implemented a systematic two-stage design of experiments (DOE) approach - optimising critical factors including aptamer density, and fabrication conditions - a methodology rarely applied in EAB sensor development. Under optimised conditions, cortisol quantification was achieved through differential pulse voltammetry (DPV) peak current measurements, yielding a practical detection range of 10-500 nM with a remarkable limit of detection of 4.6 nM, suitable for physiologically relevant cortisol monitoring. The successful integration of computational design, systematic optimisation, and a simplified sensor architecture demonstrates a robust approach for developing high-performance, ready-to-use biosensors, providing a valuable template for future point-of-care diagnostic platforms. • Computational study evaluates truncated aptamer performance for cortisol sensing. • Integrated thin-layer gold electrodes enable disposable cortisol EAB sensors. • First systematic DOE optimisation approach for cortisol EAB sensor development.