Recombinant Histidine-Tagged Nano-protein-based Highly Sensitive Electro-Sensing Device for Salivary Cortisol

We have developed a powerful biosensing strategy for immobilizing histidine-tagged (His-Tag)-oriented recombinant nano-protein immobilization on a chemically modified glassy carbon electrode (GCE) surfaces via (S)-N-(5-amino-1-carboxypentyl)iminodiacetic acid (ANTA) acting as a chelating Ni²⁺ centered interaction. Here, we introduce a label-free electro-sensor to quantify cortisol levels in saliva samples for point-of-care testing (POCT). The high specificity of the chemically modified GCE was established by genetically bio-engineered metal-binding sites on the selected recombinant apoferritin (R-AFTN) nano-protein to impart functionality to its surface and by coating the carbon surface with the self-assembled monolayers of 4-aminobenzoic acid (4-ABA) attached to ANTA groups complexed with Ni²⁺ transition metal ions. Despite the variety of conventional assays available to monitor cortisol levels, they require bulky exterior outfits, which hinders use in the healthcare systems. Therefore, we performed a rapid, easy-to-implement, and low-cost quantitative electro-sensor to enable the real-time detection of cortisol levels in saliva samples. As a result, the cortisol electro-sensor fabricated with high specificity utilizing a GCE could measure cortisol levels with a detection limit of 0.95 ng/ml and sensitivity of 7.91 μA/(ng/mL), which is a practical approach in human saliva. Thus, protein nanoprobe-based cortisol biosensing showed high sensitivity and selectivity for the direct electro-sensing of cortisol for POCT.