Development of Selective Cortisol Biosensors in the Control of Fatigue and Related Diseases
Authors: de Arruda Milena Nakagawa , Palley Bianca Fortes , Souza Gustavo Freitas de , Goncalves Emerson Sarmento 
Published: November 2022

The study of fatigue becomes complex due to several subjective factors related to its causality, making the relationship between the level of fatigue and the concentration of cortisol in an individual characteristic. However, cortisol is identified as the main element released in response to fatigue, and as such, it can be used as a biomarker for its determination and other associated diseases. Inadequate amounts of cortisol are related to non-specific diseases such as diabetes and osteoporosis. The body's cortisol levels fluctuate throughout the day, with the greatest levels in the morning and the lowest in the evening. To correlate these various fluctuations to the degree of fatigue, is required a real-time determination of cortisol levels with an effective sensory device, which overcomes the demand of costly and time-consuming laboratory analysis. Cortisol, as expressed in sweat, has a wide dynamic concentration range that can be used to monitor fatigue during different activities, being useful in preventing diseases and accidents. Currently, cortisol evaluation may be critical as one of the prospective biomarkers for Covid-19-induced significant inflammatory consequences. Some studies show possible severity between the production of pro-inflammatory cytokines related to viral infection by the new Sars-Cov-2 in people with above-average serum cortisol levels. In this regard, the establishment of a simple and low-cost biosensor with rapid diagnosis and in loco application would be beneficial due to the analytical center's independence and the simplicity with patients positive for the Sars-Cov-2 virus or others may be followed and treated. For the cortisol biosensor development, electrochemically exfoliated graphene (EEG) was used as a current collector nanomaterial in combination with chitosan (CS), a biomaterial derived from discarded crustacean shells. The carbon DropSens were modified with nanocomposite containing CS and EEG prepared using an ultrasonication. 21µL of CS_EEG dispersion by drop casting and exposed for 4 hours by white light, in order to dry the nanocomposite and recover all the working electrode. The material not bounded to electrode was then removed after 3 washes of 50µL of Milli-Q water. 10 µL of glutaraldehyde 1 % solution (GA) was dripped on the electrodes and allowed to react for 15 minutes to guarantee greater interaction of the nanocomposite with the antibody. The wet surface of electrodes was then evaluated for the incorporation of monoclonal antibody solution by IgG type specific for cortisol at varying concentrations. A total of 7.5 µL of diluted IgG solution (3.33 ng mL-1) and pure IgG solution were added to the biosensor immune complex (100 ng mL-1) and allowed to act for 30 minutes. Sensory measurements are performed in standard cortisol dilutions in PBS 7.4 in the following concentrations: 1 ng mL-1, 10ng mL -1, 100ng mL-1, 200ng mL-1, 500ng mL-1 and 1 µg. Then, the electrodes with and without antibody were characterized, via electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV), FEG microscopy, Goniometry and Zeta Potential. Goniometry assays showed an increase of hydrophilic profile of the nanocomposites after antibody modification conferring a suit surface for biological elements deposition, Fig 1. FTIR and FEG Microscopy provided the interaction of antibody-nanocomposite and antibody- antigen complex, Fig 2 and 3. Infrared analysis showed bands assigned to methyl groups disappeared after antibody adhesion and this condition may relates to the recovery of heavy chains of IgG on the nanocomposite. The increase in capacitance value after each sensory electrochemical measurement provided a cortisol curve calibration, Fig 4. The zeta potential of the immune complex formation showed an increase of values indicating a high interaction of immune system, mainly for GA condition, Fig 5. The cortisol calibration curve was improved by GA intercalation and the detections were enhanced in concentrated antibody condition, Fig 6.

Figure 1