Disposable Electrochemical Biosensor for Ultrasensitive Detection of Salivary Cortisol on a Non-Covalently Modified Laser-Induced Graphene Surface for Stress Monitoring

Monitoring stress indicators (such as cortisol) is crucial for understanding and managing the root cause of several cognitive, neurological, cardiovascular and endocrinological diseases. Cortisol negatively affects the regulation of various physiological processes such as carbohydrate metabolism and blood glucose levels. Long-term stress can disrupt homeostasis in the cardiovascular, renal, skeletal, and endocrine systems, leading to the development of chronic diseases [1,2]. Because of its central role, continuous monitoring of cortisol levels in the human body is critical for health and wellness. Current diagnostic techniques are complex and performed at centralized facilities by qualified specialists rather than at the site of therapy, which is critical for real-time monitoring. Electrochemical biosensors based on graphene's conductivity and flexibility are appealing in such contexts. Towards the goal of routine stress-level monitoring, this work realizes the design and development of an ultrasensitive and disposable immunosensor based on vertically aligned laser-induced-graphene (LIG) electrodes as a transducer surface for non-invasive salivary cortisol monitoring via differential pulse voltammetry (DPV) signal transduction mechanism. The LIG electrode was used due to its wide scalability, high electrochemical stability, increased number of reaction sites, efficient electrical conduction owing to the increased surface area and structurally more uniform alignment of the graphene layer conductivity, and low cost [3]. The LIG electrodes were fabricated using a laser ablation technique (in-house). Before use, the surface of the electrode was cleaned by cyclic voltammetry in 0.50 M sulphuric acid containing 0.10 M KCl as electrolyte. Later, the LIG electrode surface was chemically functionalized with a self-assembled monolayer of pyrene-NHS (1-Pyrenebutyric acid N-hydroxy succinimide) ester incorporating an NHS-activated functional group for the receptor (antibody) immobilization. Furthermore, a monoclonal antibody to salivary cortisol (mAb) was incubated on the Pyr-NHS-LIG electrode surface and immobilized via peptide bond formation between the carboxyl terminal of the crosslinker and the amine terminal of the antibody. This step is followed by electrode passivation to incorporate an antifouling layer. LIG's structure and electrical conductivity were preserved by using the non-covalent functionalization of Pyr-NHS ester. Cortisol complexation with immobilized mAb-Cort resulted in ferri/ferro redox electrochemistry-mediated measurement. Under optimum experimental settings, the immunosensor performance exhibited a dynamic working range from 100 fgmL^-1 to 10.0 ngmL^-1 (R² = 0.9982) with a detection limit of 10.0 fgmL^-1for cortisol (n=3). The developed immunosensor was successfully deployed to assay human salivary cortisol and showed a strong correlation with established laboratory methods (e.g., ELISA). This offers great promise for point of care monitoring of diseases.<br/><br/><b>KEYWORDS: </b>Laser induced graphene<b>, </b>Cortisol, Human Saliva, Electrochemical- Immunosensor, Pyrene-NHS ester, Stress monitoring