Ultrasensitive electrochemical sensor for detection of salivary cortisol in stress conditions
Highlights
- This label-free electrochemical immunosensor based on Gii-Sens uses the bioreceptor (mAb-cort) and analyte (cortisol) in their natural forms without any modification to study their biomolecular interaction with high affinity by decreasing non-specific adsorption.
- Some salient features of the proposed graphene-based (Gii) cortisol immunosensor are its simple fabrication, low cost, higher selectivity, reproducibility, and superior sensitivity (0.24 fg mL−1) in actual saliva samples, making this an ideal platform for point-of-care diagnostics and health monitoring.
- The developed sensor was successfully deployed for detecting cortisol in human saliva in subjects (n=16) exposed to different stress levels and demonstrated a strong correlation upon validation against the on-site ELISA and Standard Salimetric assay.
- The impressive performance of a disposable cortisol sensor
within a laboratory-based environment suggests a promising
substitute for traditional methods involving complex procedures such as freezing, transporting, processing, and analysing samples.
Abstract
A natural stress response induces elevated cortisol levels in biological fluids, such as saliva. While current sensor technologies can detect cortisol in real time, their sensitivity and reliability for human subjects have not been assured. This is due to relatively low concentrations of salivary cortisol, which fluctuate throughout the day and vary significantly between individuals. To address these challenges, we present an improved electrochemical biosensor leveraging graphene’s exceptional conductivity and physicochemical properties. A 1-pyrenebutyric acid N-hydroxysuccinimide ester (PBASE-NHS)-modified commercial graphene foam (GF) electrode is presented to realize an ultra-sensitive biosensor for cortisol detection directly in human saliva.
The biosensor fabrication process entails the attachment of anti-cortisol monoclonal antibodies (mAb-cort) onto a PBASE-NHS/GF electrode through noncovalent immobilization on the vertically stratified graphene foam electrode surface. This unique immobilization strategy preserves graphene’s structural integrity and electrical conductivity while facilitating antibody immobilization. The binding of cortisol to immobilized mAb-cort is read out via diferential pulse voltammetry using ferri/ferro redox reactions. The immunosensor demonstrates an exceptional dynamic range of 1.0 fg mL−1 to 10,000 pg mL−1 (R2=0.9914) with a detection limit of 0.24 fg mL−1 (n=3) for cortisol. Furthermore, we have established the reliability of cortisol sensors in monitoring human saliva.
We have also performed multiple modes of validation, one against the established enzyme-linked immunosorbent assay (ELISA) and a second by a third-party service Salimetric on 16 student volunteers exposed to diferent stress levels, showing excellent correlation (r=0.9961). These fndings suggest the potential for using mAb-cort/PBASE-NHS/GF-based cortisol electrodes for monitoring salivary cortisol in the general population.
Introduction
People living in modern, fast-paced lifestyles inevitably suffer from long-term psychological and physical stress [1, 2]. The experience of stress varies from person to person and is influenced by a wide range of factors, including mental health, environment, medical history, and socioeconomic status. This complexity makes it challenging to accurately assess its biophysical effects [3]. Persistent anxiety, mental well-being, and susceptibility to other mental illnesses can further intensify this emotional burden. Furthermore, as stress accumulates over time, it often leads to a set of symptoms known as chronic stress including depressive and post-traumatic stress syndromes, Alzheimer’s and Parkinson’s diseases, inflammatory conditions, as well as diabetes resulting from dysfunctions within the cardiovascular, immune, nervous, and endocrine systems [4–6].
For stress monitoring, POC diagnostics based on non-invasive biological fluids such as saliva are favoured, particularly in chronic stress conditions [14]. This is owing to a significant link between salivary and blood cortisol levels caused by the passive diffusion of free cortisol into saliva via ultra-filtration [15]. Recently, electrochemical cortisol biosensors [5, 7, 16–19], which are established on the specific molecular recognition between cortisol and receptor (antibodies/aptamer/molecularly imprinted polymers (MIPs) [20–22], have emerged as a promising technology due to their sensitivity to quantify cortisol in the range from 1.0 pM to 1.0 M. However, cortisol levels in saliva (morning, 1–12 ng mL−1, and evening, 0.1–3.0 ng mL−1) are more than 100-fold lower than in blood (morning, 25 µg mL−1, and midnight, 2 µg mL−1) [23].
While aforementioned methods are capable of detecting cortisol in saliva, there is always a question about their reliability and performance when dealing with diluted samples, particularly in terms of their clinical relevance, where concentrations are low. By choosing the right sensing structure and electrode material that facilitates the immobilization of biorecognition components, it is possible to enhance sensitivity and performance when working with actual samples reaching sub-attomolar concentrations.