Highly sensitive, real-time cortisol sensing with molecularly imprinted polymer nanoparticles on graphene electrodes in a microfluidic biosensor

We report the development of a highly sensitive, and selective electrochemical biosensor for real-time cortisol detection, integrating laser-induced graphene (LIG) electrodes with electropolymerized molecularly imprinted polymer nanoparticles (nanoMIPs). These synthetic recognition nanomaterials, approximately 65 nm in diameter, were synthesized directly on porous LIG electrodes via cyclic voltammetry, eliminating the need for antibodies and improving sensor stability. Under static conditions in spiked human serum, nanoMIP-based sensors demonstrated sub-nanomolar affinity (K_D = 0.79 nM) and a limit of detection (LOD) of 0.27 ng mL^-1 covering clinically relevant cortisol concentrations (50-250 ng mL^-1). To assess real-time performance under physiologically relevant flow, we developed a microfluidic platform for continuous chronoamperometric monitoring. Operation at 10 μL min^-1 yielded recovery rates above 95 % over 40-150 ng mL^-1 within an 8-min assay, supported by transport analysis indicating convection-enhanced, kinetically coupled binding (Pe ≈ 2.1 × 10⁴, Da ≈3). This flow-based format achieved an LOD of 0.28 ng mL^-1 and demonstrated reproducible performance in complex biological matrices. By combining synthetic nanoMIP recognition, scalable LIG electrode fabrication, and automated microfluidics, this modular lab-on-a-chip platform provides a robust and scalable approach for decentralized cortisol sensing, with strong potential for point-of-care diagnostics and stress-related health monitoring.