An engineered chimeric DNA probe for signal-on electrochemical cortisol detection via Hairpin-to-G-quadruplex switching

Sensitive and selective cortisol detection in biological fluids remains challenging because of matrix effects and non-specific interfacial interactions. Herein, a label-free signal-on electrochemical aptasensor was developed using an engineered cortisol-responsive chimeric DNA probe that integrates target-induced hairpin-to-G-quadruplex (G4) switching with hemin/G4 DNAzyme-mediated signal amplification. The probe was covalently immobilized onto a chitosan/glutaraldehyde-modified screen-printed graphene electrode (SPGE). In absence of cortisol, the G4-forming domain was constrained within the hairpin configuration, resulting in minimal hemin/G4 DNAzyme activity and a low background signal. During co-incubation with cortisol and hemin, cortisol recognition shifted the probe toward a more open, G4-accessible conformation, facilitating G4 folding and formation of the catalytically active hemin/G4 DNAzyme. The resulting complex catalyzed the H₂O₂-mediated oxidation of hydroquinone to electroactive benzoquinone, generating an amplified square-wave voltammetric response. Under optimal conditions, the aptasensor exhibited a linear response from 5 pM to 5 nM and an experimentally determined limit of detection of 1 pM. The high sensitivity enabled cortisol determination after 25-fold dilution of biological samples, thereby reducing matrix interference while maintaining quantitative performance. The analytical selectivity of the platform was evaluated against structurally related corticosteroids, while molecular docking was used to examine plausible cortisol-probe interactions within the modeled chimeric DNA architecture. The aptasensor was further evaluated using spiked pooled human serum and saliva samples, affording satisfactory recoveries and good tolerance toward complex biological matrices. This work demonstrates an engineered, surface-confined electrochemical sensing format for sensitive cortisol analysis and provides a basis for future disposable cortisol monitoring platforms.