Competitive Immunoplatform Based on Antibody-Functionalized Magnetic Nanoparticles for Label-Free Cortisol Detection Using Si3N4 Ion-Sensitive Field-Effect Transistors

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.