Electrochemical aptamer biosensor based on reduced graphene oxide modified electrode for cortisol detection

Stress, a common issue faced by almost everyone, is usually associated with absenteeism, lack of motivation and performance. The frequent exposure to stress leads to chronic stress, elevating the risk of other psychological health problems such as anxiety and depression. Regular monitoring of stress level is crucial for health and wellbeing which calls for a need for a simple, convenient and portable stress biosensor. In this study, an electrochemical biosensor for the detection of cortisol as the stress biomarker was fabricated using reduced graphene oxide modified screen printed carbon electrode, rGO-SPCE. Graphene oxide was drop-casted onto the working electrode of the SPCE and electroreduced into rGO using cyclic voltammetry (CV). Amine modified cortisol aptamer was utilised for cortisol specific sensing molecule and immobilised onto the rGO surface using N-ethyl-N′-(3- dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide (EDC-NHS) linker at a ratio of 1:1 (v/v). Activation of carboxylic group on rGO layer, COOH into COO- favoured the attachment of the amine (NH2) group of the cortisol aptamer through amide bond interaction. Bovine serum albumin (1 mg/ml) was added to block non-specific binding of cortisol onto the modified layer. The self-assembled monolayer was then characterised using Field Emission Scanning Electron Microscope (FESEM), Fourier-Transform Infrared Spectroscopy (FTIR) and electrochemically using CV. Using 5 mM potassium ferricyanide (K₃[Fe(CN)₆]) in 0.1 M potassium chloride (KCl) solution as the redox electrolyte, the electrochemical performance of rGO-SPCE was optimised and analysed through differential pulse voltammetry (DPV). Interaction of cortisol with cortisol aptamer hindered the [Fe(CN)6]3−/4− flow across the sensing surface thus resulting in a decrease of current. At rGO concentration of 1.5 mg/ml and 0.1 µM aptamer, C-Apt/rGO-SPCE was able to detect cortisol through the highest peak current reduction obtained within 15 minutes of cortisol incubation time. The electrochemical response exhibited a linear dependence on the cortisol concentration ranging from 0.001 µg/ml to 10 μg/ml, with a detection limit of 1.9836 μg/ml. The highest peak current reduction recorded at 65% (1.54 µA) from the interference study proved that the fabricated biosensor was highly specific towards cortisol amongst other steroid based hormones. This proposed technique demonstrates its potential application in monitoring stress.