Competitive Immunoplatform Based on Antibody-Functionalized Magnetic Nanoparticles for Label-Free Cortisol Detection...

Cortisol is a key biomarker involved in the regulation of the hypothalamic-pituitary-adrenal axis and is widely used for assessing physiological stress and endocrine disorders. However, its small molecular size and low charge make its direct detection using field-effect transistor (FET)-based biosensors challenging. In this work, we present a competitive ImmunoFET biosensing platform combining a Si₃N₄-gated ion-sensitive field-effect transistor (ISFET) with anti-cortisol polyclonal antibody-functionalized magnetic nanoparticles (MNPs) for label-free cortisol detection. The sensing strategy relies on a competitive immunoassay in which cortisol in the sample competes with cortisol immobilized on the ISFET surface for antibody binding sites on the MNPs. Following a 45 min pre-incubation, magnetic separation, and redispersion, the MNPs were brought into contact with the cortisol-functionalized FET and analyzed by electrochemical impedance spectroscopy (EIS). The charge-transfer resistance decreased from 4.012 MΩ to 2.599 MΩ as the cortisol concentration increased from 0.02 to 0.6 ng/mL, confirming the expected competitive behavior. The sensor exhibited a concentration-dependent response over the 0.02-0.6 ng/mL cortisol range, with a limit of detection of 0.006 ng/mL. The platform also showed a stronger response toward cortisol than the investigated non-target biomarkers, including NT-proBNP, TNF-α, and IL-10. Its applicability was further demonstrated in real saliva using the standard addition method, yielding a cortisol concentration of 11.6 ng/mL, compared with an expected value of 10.2 ng/mL, corresponding to a 113.7% recovery. These preliminary results demonstrate the feasibility of the proposed MNP-assisted competitive FET for cortisol detection in biological samples and provide a basis for further development toward point-of-care applications.