Dual fluorescence output aptamer sensing of cortisol in Cushing syndrome samples assisted by tandem hybridization chain reaction

Accurate and sensitive detection of serum free cortisol has been a challenge for precise diagnosis and assessment of Cushing syndrome (CS). The present study described a dual fluorescent homogeneous cortisol aptasensor based on the tandem hybridization chain reaction (HCR) amplification strategy. The system used split G-quadruplex (G4)/thioflavin T (ThT) complexes and dsDNA-templated copper nanoparticles (Cu NPs) as signal reporters. The cortisol binds its aptamer, inhibiting the triggering of HCRs between the aptamer and the four hairpin structures (H1-H4), thereby inhibiting the generation of the dsDNA template and G4 structure. Under optimized conditions, the limit of detection (LOD) was 3 fg/mL for G4/ThT and 2.5 fg/mL for Cu NPs, with a linear range of 1 fg/mL-100 pg/mL, indicating high specificity to various potential interfering substances. This method was used to determine cortisol levels in 54 clinical blood samples. Cortisol levels in clinical samples detected by this method agreed well with those obtained by the electrochemiluminescence immunoassay (ECL-IA) kit and radiological findings. We could distinguish CS from healthy controls and other patients using this approach, with 0.94 of the area under the receiver operating characteristic curve (AUC). This cortisol quantification method was 100% specific and 83.3% sensitive. This sensing strategy has shown great potential as an additional diagnostic tool for cortisol-related disorders.