Perspective—Electrochemical Bio-wearables for Cortisol Monitoring

Cortisol is a key biomarker, and its measurement has historically relied on intrusive and sporadic techniques like blood or saliva samples. The relatively recent innovation of electrochemical cortisol bio-wearables provides a revolutionary strategy by offering continuous, non-invasive monitoring. This Perspective examines the development, underlying ideas, scientific developments, and possible uses of electrochemical cortisol bio-wearables. The significance of these tools for stress research, clinical application, and individualized healthcare is also highlighted.

Cortisol is a glucocorticoid hormone secreted by the adrenal gland through stimulation by CRH (corticotropin-releasing hormone) and ACTH (adrenocorticotrophic hormone).This steroid is critical to many physiological processes, such as energy metabolism, blood pressure regulation, stress responses, cell proliferation, cell differentiation, immune modulation, and memory and cognitive function regulations. 1,2Cortisol is present in many different biological samples, such as blood (50-230 ng ml −1 ), sweat (8-140 ng ml −1 ), saliva (2-20 ng ml −1 ), and interstitial fluid (9-34 ng ml −1 ). 2,3Monitoring cortisol levels is critical for understanding and managing a variety of health ailments, such as stressrelated disorders, mental health issues, and metabolic diseases. 4he development of analytical chemistry and medical technology has been closely related to the history of cortisol monitoring.Early cortisol assessments required much work and were inaccurate; they frequently used urine samples or animal trials.In the late nineteenth century, scientists used colorimetry to detect cortisol in urine samples. 5This approach was based on color changes that occurred when cortisol reacted with particular reagents.While it gave qualitative information about the presence of cortisol, it lacked precision and quantitative capabilities.Cortisol evaluation revolutionized in the 1960s with the invention of radioimmunoassays (RIA), which allowed for more precise assessments. 6To precisely detect cortisol concentrations, RIA used radiolabeled cortisol and cortisol-specific antibodies.This approach was the gold standard for cortisol assessment for numerous decades due to its superior sensitivity and specificity.Later, sensitivity and specificity were further enhanced using enzyme-linked immunosorbent tests (ELISA).It used enzymes and antibodies to generate colorimetric or fluorescent signals, making it a more secure alternative to RIA as it no longer needed hazardous radioactive labels for testing.Because of its ease of use and dependability, ELISA has become widely utilized in clinical laboratories and research.Microscopy techniques in the early twentieth century enabled researchers to visualize and measure cortisol crystals in urine and blood.This qualitative approach provided limited information regarding cortisol levels and necessitated much technical knowledge.Although RIA and ELISA techniques offered insightful understandings of cortisol fluctuations, they remained intrusive (required invasive biological samples such as blood) and episodic (depended on longitudinal sample collection).
Current methods such as ELISA of measuring cortisol in biological samples such as blood or saliva collection, have drawbacks that restrict their usefulness for thorough stress analysis and real-time monitoring.Continuous monitoring has been transformed by miniaturized wearable devices integrated with cortisol sensors.These devices, often worn as wristbands or patches, allow users to track their cortisol levels throughout the day in a non-invasive and easy-to-use manner.Wearable sensors detect cortisol in noninvasive biological fluid using various technologies, including electrochemical, optical, and impedance-based approaches.They are integrated with highly specialized recognition components, such as cortisolspecific antibodies or aptamers, to minimize cross-reactivity with other compounds. 7The wearable biosensor sensitivity and specificity in continuous cortisol monitoring have improved significantly in the past decade.Modern wearable sensors detect cortisol concentrations in real-time, even at low nanomolar levels, ensuring accurate and exact results.
This perspective provides understanding of the existing state of electrochemical cortisol monitoring wearables with a critical perspective on challenges and gaps that the technology faces.Additionally, we provide insights into what the future entails for electrochemical cortisol biowearables.

Current Status
Electrochemical cortisol bio-wearables are on the rise.-Thefusion of electrochemistry, materials science, and microelectronics has given rise to continuous cortisol monitoring via wearables.Since they provide continuous and non-invasive cortisol level monitoring, electrochemical cortisol wearables 8 represent a substantial paradigm shift.Due to their quick response times, excellent sensitivity, and low cost, electrochemical sensors have emerged as viable options for cortisol monitoring.0][11][12] Apocrine sweat secreted from glands in hair follicles and eccrine sweat secreted from glands on the skin's surface, respectively, are common sources of cortisol.However, the process of developing a sensor system that uses sweat cortisol is difficult because it requires a very high sensitivity for the detection of low sweat cortisol concentrations.A handful of research has also shown promises in detecting cortisol in the interstitial fluids (ISF). 13,14Proteins and metabolites can get to ISF via blood capillaries.ISF typically contains small molecules like cortisol in a proportion comparable to that of blood.Due to this, the periodic calibration that is necessary to correlate the metabolite content of ISF with blood is avoided.For such detection, microscopic needles are needed to puncture the skin's superficial layers in order to collect ISF in order to detect the target analyte, which makes this method minimally or significantly invasive.Numerous painless and less invasive methods for obtaining ISF have emerged in the past years to facilitate cortisol detection. 15,16E-mail: richa.pandey@ucalgary.caAn immobilized recognition component (bioreceptors), usually an enzyme or an antibody, is a fundamental component of electrochemical cortisol sensors. 17In general, a recognition element binds to the cortisol molecules in a biological sample, starting an electrochemical process such as reduction or oxidation, increase in the double layer capacitance or charge transfer resistance which is then measured using a readout.These bioreceptors interact with cortisol, often antibodies or enzymes, are at the heart of these devices and are critical in assuring the specificity and sensitivity of cortisol detection.Through advancements in bioreceptors and electrode materials, electrochemical cortisol wearables' sensitivity and selectivity have significantly increased.High selectivity is ensured by using cortisol-specific affinity based biorecognition elements such as antibodies or aptamers, which reduces interference from other compounds.The biorecognition element chosen is determined by characteristics such as sensitivity, specificity, stability, and cost-effectiveness.Conventional biorecognition elements in electrochemical cortisol biosensors include monoclonal or polyclonal antibodies. 18They are very selective, attaching specifically to cortisol molecules.Cortisol is captured by immobilized antibodies on the sensor surface, allowing for subsequent electrochemical detection. 18However, antibodies suffer from lower stability in various environmental conditions. 19They are produced using time consuming hybridoma technology. 20The electrochemical method for antibodies-based cortisol detection is dependent usually upon non-Faradiac current hence rendering them less sensitive.Enzymes such as glucose oxidase (GOD) and horseradish peroxidase (HRP) are widely utilized in electrochemical cortisol sensors.These enzymes catalyze a reaction upon binding with the cortisol, resulting in electrochemically detectable compounds.For example, glucose oxidase conjugated with cortisol (GOD-c) was realized for the detection of cortisol in biological samples using anti-cortisol antibodies immobilized electrodes. 21In the presence of cortisol, the GOD-c would not bind to the antibody generating a Signal -Off outcome.In the absence of cortisol, a Signal-On outcome will be generated.Different version of this strategy involves using a cortisol-antibody conjugated GOD.This strategy, being directly proportional to the cortisol concentration produces a Signal-On outcome by catalyzing the oxidation of glucose.By replacing the GOD with HRP, similar Signal-On and Signal Off strategies have been used for the cortisol detection. 12Nevertheless, the specific enzyme that can directly catalyze the cortisol in sweat has not yet been demonstrated.Enzyme-based sensors provide high sensitivity, specificity, and regenerative properties that are required for continuous monitoring. 22Hence, a huge benefit would be gained in pursuing protein engineering and synthetic biology approaches to develop cortisol-specific enzymes.Aptamers are emerging bioreceptors that are single-stranded DNA or RNA sequences that can bind to cortisol selectively. 17Because they are very specific, customizable, synthetically produced in less time, and stable, they are continuously gaining trust as biorecognition elements for cortisol monitoring.Aptamers immobilized on electrode surfaces can be employed in label-free Faradaic or non-Faradaic current based electrochemical sensors offering sensitive detection over other receptors. 8,17,23More aptamer-based assay architectures such as concatenated aptamers need to be explored for cortisol detection applications.Molecularly imprinted polymers (MIPs) are synthetic polymers containing molecularly designed cortisol recognition sites. 24These materials are strong, sturdy, and inexpensive. 24,25IP-based sensors mimic antibody selectivity without being susceptible to external influences and hence can be a great alternative to affinity-based cortisol sensing assay that involves antibodies or aptamers.Nanomaterials such as nanoparticles, nanowires, and nanotubes can be used as biorecognition elements in electrochemical cortisol sensors.Their high surface-to-volume ratio and unique features improve sensitivity and allow identification elements to be immobilized. 26lectrochemical cortisol wearables have recently undergone miniaturization, becoming discrete and user-friendly gadgets.
These wearables, worn on the wrist as devices or placed on the skin as patches, are comfortable to incorporate into daily life and provide continuous cortisol monitoring without interfering with routine activities.Developments in microfabrication, which allowed for the creation of small sensors with low power requirements, have been the driving force behind this miniaturization.In addition, improvements in nanomaterials like carbon nanotubes and graphene have greatly improved sensor performance and enabled detection down to the low nanomolar range.The usability has been transformed by integration with smartphone applications and cloud-based systems.

Applications of Electrochemical Cortisol Bio-wearables
One of the primary applications of electrochemical cortisol biowearables is stress management. 23Individuals can employ cortisol biowearable devices to gain insight into their stress levels throughout the day, identify stressors, and develop coping strategies.These wearables empower users to take proactive steps toward stress reduction and overall well-being.Electrochemical cortisol wearables hold immense promise in the field of mental health monitoring.They offer an objective biomarker to complement subjective assessments of conditions such as anxiety and depression.Continuous cortisol monitoring can provide clinicians with valuable data for diagnosing and tailoring treatment strategies.Researchers have adopted electrochemical cortisol wearables in clinical studies to explore the relationship between cortisol levels and various health conditions. 19,27,28Use of wearable electrochemical cortisol monitors has been proposed in recent studies for clinical trials and pharmaceutical research in cancer. 29They provide significant information on the physiological reaction to investigational medications, assisting researchers in determining drug safety and effectiveness.Systemic long-term synthetic corticosteroid medications such as prednisone, deflazacort, and dexamethasone are used to treat chronic inflammation linked to tissue degenerative processes of disease and ageing.Treatment of this kind may significantly alter the HPA-axis and cortisol levels.The therapists will be assisted in analyzing the long-term data of the patients' HPA-axis response to changes in drug type, combinations, regimens, and dosages by a continuous cortisol monitoring device. 30Cortisol sensors can also help with patient categorization and biomarker discovery for patient selection.These wearables enable non-invasive and continuous cortisol data collection, providing a more comprehensive view of cortisol dynamics.In the realm of sports and athletics, electrochemical cortisol wearables can help athletes optimize their training routines. 31Monitoring cortisol levels can assist in identifying the ideal training intensity and recovery periods, thereby enhancing performance and minimizing the risk of overtraining.

Future Needs and Prospects
Despite their potential, the bioassays integrated on electrochemical cortisol wearables suffer calibration, specificity, and long-term stability difficulties.Obtaining high specificity for cortisol is one of the most difficult issues in employing biorecognition elements.Bioreceptors must attach to cortisol molecules preferentially while avoiding cross-reactivity with structurally related substances.Achieving this balance is difficult since the biochemical milieu of human sweat contains multiple steroids, hormones, lactic acid, proteins, creatinine and urea. 32As a result, biorecognition elements may show cross-reactivity, resulting in false cortisol values.Researchers are actively working on producing particular biorecognition elements using monoclonal antibody production and aptamer selection.Furthermore, using MIPs as synthetic recognition elements appears to have promise in minimizing cross-reactivity.Environmental conditions such as temperature, pH, and humidity can affect biorecognition elements' stability and lifespan.These biorecognition elements are subjected to various environments in wearable devices, potentially affecting their function over time.The long-term reliability of electrochemical cortisol wearables depends on the stability of biorecognition elements.High-quality biorecognition elements, particularly antibodies, can be time-consuming and expensive to produce.This cost may limit the availability and affordability of electrochemical cortisol wristbands, preventing their widespread use in professional and consumer settings.Investigating alternate recognition elements, such as aptamers or molecularly imprinted polymers, can reduce production costs while retaining specificity and sensitivity.Furthermore, research towards scalable and cost-effective biorecognition element production technologies are ongoing.Stabilization strategies are being researched, such as immobilizing biorecognition components in protective matrices or encapsulating within nanomaterials.These methods improve the toughness and lifespan of biorecognition elements in wearable devices.The future of electrochemical cortisol wearables seems bright.Sensor technology advancements and progress in data analytics and artificial intelligence will increase the accuracy and usability of sensors.A machine learning model that is trained on the gathered data may be quickly analyzed.Patients may be able to make knowledgeable decisions regarding their health as a result.It is a particularly effective tool for using with biosensors to increase the accuracy of diagnosis and is simple to apply to the categorization and pattern recognition of a variety of output signals generated by biosensors. 33Integration with other wearable health devices, such as heart rate monitors and sleep trackers, may provide a more complete picture of a person's health and stress response.
The clinical application of electrochemical cortisol wearables necessitates regulatory approval and adherence to standardized techniques.Currently, the regulatory bodies around the world categorize these devices as wearable medical devices for diagnosing or screening a disease or wearable devices to monitor lifestyle.The former is more strongly regulated than the latter.Similar to integrated continuous glucose monitoring systems that are wearable in form factor, cortisol biowearables are designed to automatically assess cortisol levels in bodily fluids consistently or at frequent intervals over a specific duration.These systems are crafted to securely and dependably transmit cortisol measurement data to other digitally connected devices.They can be utilized independently or in conjunction with other digitally connected medical devices to effectively manage diseases or conditions associated with cortisol levels.They are currently classified as Class II in North America as per the Food and Drug Administration (FDA).Special controls for this device include robust design verification and validation.This involves providing clinical data showcasing the accuracy of the device in its intended user population.The data must feature a comparison between the cortisol biowearable values and blood cortisol values, obtained through FDA-accepted laboratory-based methods with precision and traceability to higher-order standards.The clinical study supporting the device's performance should comprehensively represent its functionality across the intended user population and measuring range.Furthermore, the study results must exhibit consistent analytical and clinical performance throughout the sensor wear period.Currently, the only cortisol biowearable on the market is developed by Nowatch and Philips which measures a combination of parameters such as skin conductance, heart rate variability, breath rate, activity, and sleep.However, the inclusion of interchangeable accessories, such as stylish "non-watch" faces, distinctly categorizes this as wearable electronic jewelry.
It is imperative that to satisfy regulatory standards for wearable medical devices that detect cortisol, biorecognition elements must go through extensive validation methods to demonstrate the effective binding with the cortisol.On the other hand, the lack of standardized methodologies for measuring biorecognition element performance poses a considerable issue.For example, the gold standard for such validation is still lab-based ELISA tests.This requires the adaptation of ELISA test to cater validation of many new enzyme or aptamerbased bioreceptors.The variability in such validation arises, when the interaction profile of the solution-based assay differs from the device attached assay.There are evidently no standard methods available that can perform head-to-head validation of biorecognition elements.Researchers, healthcare institutions, and regulatory bodies must work together to build standardized validation methodologies for biorecognition elements in wearable devices.Specificity, sensitivity, stability, and reproducibility should all be considered in these methods.The existing landscape reveals a lack of uniformity in methodologies, ranging from sample collection to assay techniques, posing challenges in data interpretation and hindering the advancement of cortisol research.Furthermore, because these devices collect sensitive health information, addressing privacy issues and preserving data security is critical.The requirements of patient data privacy and security contribute to the susceptibility of the current regulatory framework.This is because necessary regulations around efficacy and safety of such devices are yet to be established.Extensive validation and standardization on both the hardware (biosensor components) and the software (data acquisition) are required to acquire universal acceptability of cortisol biowearables in clinical practice.

Conclusions
Electrochemical cortisol wearables will become game changer in stress monitoring, providing continuous, non-invasive cortisol level testing.As of right now, they lack benchmark validation and independent verification.Understanding a big sample of data and using an accurate sampling procedure presents another major challenge.Exact optimization of the sample techniques is necessary to obtain useful data.Because of their miniaturization, increased sensitivity, and wireless connectivity, many of these challenges can be alleviated.As researchers and engineers continue to innovate in biorecognition element design and production, electrochemical cortisol wearables promise to become indispensable tools for monitoring and optimizing wellness.They will play an important role in personalized healthcare, allowing consumers and medical professionals better to understand the complicated interplay between stress and health.However, a critical improvement needs to be made towards.