Advancements in Cortisol Detection PDF

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This article is licensed under CC-BY 4.0 pubs.acs.org/acssensors Review Advancements in Cortisol Detection: From Conventional Methods to Next-Generation Technologies for Enhanced Hormone Monitoring Visesh Vignesh, Bernardo Castro-Dominguez, Tony D. James, Julie M. Gamble-Turner, Stafford Lightman, and Nuno M. Reis* Cite This: ACS Sens. 2024, 9, 1666−1681 Read Online ACCESS See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles. Metrics & More Article Recommendations Downloaded via FLORIDA STATE UNIV on November 14, 2024 at 23:41:42 (UTC). ABSTRACT: The hormone cortisol, released as the end-product of the hypothalamic-pituitary-adrenal (HPA) axis, has a well- characterized circadian rhythm that enables an allostatic response to external stressors. When the pattern of secretion is disrupted, cortisol levels are chronically elevated, contributing to diseases such as heart attacks, strokes, mental health disorders, and diabetes. The diagnosis of chronic stress and stress related disorders depends upon accurate measurement of cortisol levels; currently, it is quantified using mass spectroscopy or immunoassay, in specialized laboratories with trained personnel. However, these methods are time-consuming, expensive and are unable to capture the dynamic biorhythm of the hormone. This critical review traces the path of cortisol detection from traditional laboratory-based methods to decentralised cortisol monitoring biosensors. A complete picture of cortisol biology and pathophysiology is provided, and the importance of precision medicine style monitoring of cortisol is highlighted. Antibody-based immunoassays still dominate the pipeline of development of point-of-care biosensors; new capture molecules such as aptamers and molecularly imprinted polymers (MIPs) combined with technologies such as microfluidics, wearable electronics, and quantum dots offer improvements to limit of detection (LoD), specificity, and a shift toward rapid or continuous measurements. While a variety of different sensors and devices have been proposed, there still exists a need to produce quantitative tests for cortisol using either rapid or continuous monitoring devices that can enable a personalized medicine approach to stress management. This can be addressed by synergistic combinations of technologies that can leverage low sample volumes, relevant limit of detection and rapid testing time, to better account for cortisol’s shifting biorhythm. Trends in cortisol diagnostics toward rapid and continuous monitoring of hormones are highlighted, along with insights into choice of sample matrix. KEYWORDS: cortisol, stress, immunoassay, continuous, biorhythm, electrochemistry, point-of-care, rapid C ortisol is the primary glucocorticoid hormone released by the body in response to stress,1,2 with chronic cortisol levels leading to various pathophysiologies. stimulated to secrete corticotrophin-releasing hormone (CRH). CRH then stimulates the release of adrenocortico- trophin-releasing hormone (ACTH), which is recognized by This hormone is regulated by the hypothalamic−pituitary− cells in the adrenal cortex, to produce cortisol.6 The cascade is adrenal (HPA) axis, a complex neuroendocrinological system regulated by a negative feedback loop,7 with high cortisol levels (Figure 1A). Most cortisol in blood (90%) is bound to a carrier signaling to the hypothalamus to halt further CRH secretion, as protein, cortisol binding globulin (CBG), with free cortisol well as to the pituitary gland to halt ACTH production. only making up 5−10% of the total cortisol in circulation.3 Cortisol follows a well-characterized diurnal rhythm,8 with a Only free cortisol is biologically active, with bound cortisol peak level observed shortly after awakening, dipping to its being physiologically inactive. Cortisol controls a wide range of lowest point at midnight before starting to rise again during the physiological processes, such as promoting gluconeogenesis, nocturnal hours. This rhythm is composed of multiple reducing inflammation, suppressing the immune system4 and modulating cognitive processes.5 These responses anticipate Received: September 12, 2023 and assist the body’s adaptation to stressful conditions by Revised: January 22, 2024 providing energy for awakening, fuelling a “fight or flight” Accepted: March 8, 2024 response, and diverting resources to deal with a stressor.2 Published: March 29, 2024 Upon detecting a stressor through the central nervous system (CNS), cells situated in the hypothalamus are © 2024 The Authors. Published by American Chemical Society https://doi.org/10.1021/acssensors.3c01912 1666 ACS Sens. 2024, 9, 1666−1681 ACS Sensors pubs.acs.org/acssensors Review Figure 1. (A) Overview of the HPA axis. Created with Biorender. (B) Graph comparing normal cortisol levels versus mild and severe Cushing’s syndrome. Reproduced with permission from ref 112. Copyright 2005, Endocrinology and Metabolism Clinics of North America. (C) Rate of cortisol secretion versus clock time, depicting cortisol rhythm being made up of pulsatile ultradian secretions. Reproduced with permission from ref 113. Copyright 2009, Journal of Medical Engineering and Technology. ultradian cycles,9 each cycle typically lasts 60−120 min and is in glucocorticoid hormone levels and a corresponding upsurge independent of external stressors, with additional cortisol in adrenocorticotropic hormones. The disease can be life secreted in response to stressors. The rhythmicity of cortisol threatening if left unchecked, leading to hypotension and secretion is one of its defining traits, with research showing volume depletion or adrenal crisis.13 The symptoms of these detectable cortisol rhythms in infants as young as one month disorders overlap strongly with other diseases,14 making the of age.10 assessment of an individual’s baseline cortisol levels key to When cortisol production becomes overactivated, baseline diagnosing these conditions in a timely manner. Another factor cortisol levels in the body can become dysregulated, leading to that can influence cortisol measurement is the age of the impairment of normal body functioning and triggering diseases such as diabetes mellitus and depression.11 Excessive cortisol person being sampled. Compared to adults, children show a levels in blood are a diagnostic factor for Cushing’s syndrome, much greater variability in cortisol rhythms through the day.15 where patients may exhibit hyperglycaemia, muscle atrophy, The exact reason for this is unknown, as the HPA axis reaches delayed wound healing and increased susceptibility to adult-level maturity within the first four years of development. infections.12 Conversely, lowered cortisol levels can lead to a Nevertheless, it is important to consider differential cortisol state of adrenal insufficiency, also called Addison’s disease. In expression in adults versus children when conducting this condition, cortisol levels are lowered, leading to a decrease population wide studies. 1667 https://doi.org/10.1021/acssensors.3c01912 ACS Sens. 2024, 9, 1666−1681 ACS Sensors pubs.acs.org/acssensors Review Table 1. Comparison of Various Samples for Cortisol Detection with Their Respective Advantages and Disadvantages4,107,108 Invasive Correlation with circulating Free Ease of Preprocessing Can be collected during Typical cortisol Sample fluid sampling levels cortisol collection required sleep range Saliva √ √ √ √ 5.27 ng/mL109 Sweat √ √ 8−140 ng/mL64 Serum √ √ 50−163ng/mL110 Interstitial √ √ √ √ 1−11 ng/mL111 fluid In addition to physiological insult, chronic psychosocial reflection on advantages and challenges for utilizing various stress has been associated with dysregulation of cortisol levels sample matrices. We then followed with a critical overview of and linked to poorer physical as well as mental health established techniques for cortisol sensing, such as micro- outcomes. In a study on relatives of patients in an intensive dialysis, that were discussed along with their shortcomings for care unit (ICU), higher cortisol levels were linked to avoidance clinical use. We then finished with a critical assessment of the behavior and a depressed state.16 While results from the transformative nature of switching from current lab-based literature are confusing, cortisol levels seem to be elevated in measurement of cortisol to a rapid and/or real-time cortisol some patients suffering from depression.17 Previous research sensing. has also suggested dysregulated cortisol profiles in children with autism, revealing elevated levels in the evening and lowered cortisol in the morning.18,19 Given the negative effects of chronic stress, there is a need to CORTISOL SAMPLING The first aspect that must be considered in terms of cortisol measure cortisol levels accurately and reliably. As early as 1954, measurement is sampling and the challenges presented in studies have revealed the detrimental effects of raised cortisol different matrices. It is well established in literature that levels in mice and patients undergoing corticosteroid cortisol is present in all major bodily fluids, including sweat, therapy.20−22 The effects of cortisol on the human body saliva, interstitial fluid, and blood (serum). This plethora of were particularly scrutinized in the case of elite athletes, where potential samples offers a great deal of flexibility when minor differences in hormonal makeup can mean the designing a detection modality for cortisol, with each offering difference between victory and defeat.23 More recently, a differing advantages and disadvantages, summarized below in growing understanding of stress and the role of cortisol in Table 1 and herein discussed. pivotal processes such as catabolism and sleep hygiene show Serum. Serum cortisol sampling gives a measure of total that there is a need to measure cortisol levels accurately and (free plus bound) cortisol,30 and is used to determine the total reliably, with tangible benefits for a general population.24,25 serum cortisol level of a patient. Serum cortisol can more Nowadays, the most common method of measuring cortisol accurately reflect rapid cortisol changes dynamically, as levels is via immunoassay,26 with a wide range of kits available opposed to a matrix such as saliva which requires time for commercially for centralized diagnostic purposes. The current cortisol diffusion from blood.31 However, there are numerous gold standard for cortisol measurement is mass spectroscopy reported challenges when attempting to use serum as the (MS), a highly sensitive and precise technique for small analyte fluid. Apart from the extraction process being time and molecule detection, which also has the advantage of being able labor intensive, prefiltration steps are often required, using to measure multiple compounds from the same sample.27 techniques such as dialysis, ultrafiltration, and gel filtration to While both immunoassays and MS offer unique advantages separate bound and unbound cortisol fractions. Additionally, and disadvantages, they are both difficult to adopt to point-of- the venepuncture procedure required to collect blood samples care (PoC) detection, due to factors such as cost, requiring is an invasive process, which in some patients can trigger specialized personnel and overhead time loss. Additionally, increased cortisol synthesis,32 potentially providing misleading neither of these modalities can be used for continuous readings. monitoring of cortisol. Saliva. Salivary cortisol offers the opportunity to measure As interest in cortisol biosensing has steadily increased in free cortisol due to passive diffusion. The concentration of recent years, there is now a vast amount of information salivary cortisol is unaffected by flow rate from the salivary regarding various aspects of cortisol sensing. This Review glands and offers the benefits of measuring biologically active summarizes and critically reviews the most remarkable cortisol without resorting to invasive serum sampling.33 Saliva advances in the last 5 years in the area of cortisol sensing is readily (and plentifully) available as a sample fluid and can and highlights key technologies that could facilitate a shift from be rapidly extracted with a swab, passive drool techniques, or current lab-based cortisol measuring to rapid and/or cuvettes.34 continuous sensing at the point-of-need or point-of-care. Having said that, one of the biggest hurdles in measuring Several important reviews have previously discussed certain cortisol from saliva is the presence of salivary cortisone, the specific aspects of cortisol measurements such as immuno- inactive form of cortisol. Cortisone cross reacts with cortisol assays28 or wearables.29 Herein, we present a broader overview specific binding agents in immunoassays and can create of cortisol sensing, from biology and pathophysiology to background noise. Furthermore, cortisol is rapidly converted continuous monitoring and cutting-edge biosensing technolo- into cortisone by the salivary glands due to the presence of an gies, helped by the interdisciplinary expertise of our team, enzyme, 11-β-dehydrogenase isozyme 2, leading to reduced including a health psychologist, endocrinologist, molecular levels of cortisol in saliva than in serum. Oral hydrocortisone chemist and microfluidic and biosensing experts. We provide treatments contaminate the salivary cortisol pool, leading to the reader with a background on cortisol biology and the uninterpretable readings for patients undergoing such treat- difficulties associated with its detection, followed by a ment.35 Finally, patients should fast for 30 min prior to sample 1668 https://doi.org/10.1021/acssensors.3c01912 ACS Sens. 2024, 9, 1666−1681 ACS Sensors pubs.acs.org/acssensors Review collection, making saliva an unwieldy choice for multiple/ hormones (e.g., cortisol, corticosterone, and 11-deoxycortisol) continuous cortisol sampling, or when a subject is asleep. The made the technique redundant. These assays required lower concentration of cortisol present in saliva, approximately expensive reagents, were time-consuming and required trained 1 order of magnitude lower than that of other matrices (Table personnel.43 1) is a major challenge when it comes to limit of detection Immunoassays. To overcome the limitations of specificity (LoD) of the biosensor. and sensitivity, radioimmunoassay’s were developed,43 with the Interstitial Fluid. Interstitial fluid is the liquid solution promise of more convenient measurements and enhanced surrounding tissue cells, providing nutrients that passively assay performance. These assays marked the beginning of diffuse into the cells, while simultaneously removing waste antibody−antigen interactions for hormone detection, specif- products of metabolism such as carbon dioxide. The fluid ically, the use of monoclonal antibodies immobilized to a solid contains free cortisol in detectable concentrations, and is a substrate. These early assays achieved limit of detection (LoD) better indicator of cortisol levels in tissue, as opposed to serum of 8 ng/mL, representing a remarkable improvement compared cortisol.36 One of the key advantages over other matrices such to earlier fluorometric assays.44 as saliva, is the possibility of sampling interstitial fluid in a Modern immunoassays, including ELISAs further improved continuous manner, via methods such as microdialysis,37 also upon radioimmunoassay methods by being easy to perform in reviewed in this article. the lab and by requiring small volumes of sample fluid. These Sweat. Like saliva, sweat is an easily accessible fluid that assays typically combine antigen−antibody binding with exhibits good correlation with serum levels of cortisol. One of fluorescent probes to estimate total cortisol concentrations, the key advantages in using sweat as a detection modality for with the high specificity of antigen−antibody binding allowing cortisol is the option to take advantage of emerging wearable accurate detection of small molecules. A wide range of cortisol technology. Sweat can be collected near-continuously with immunoassays are available in the market, with most kits using microfluidic systems, enabling in situ cortisol detection with a ELISA format for free cortisol detection. A competitive assay wearable biosensors. However, the primary disadvantage of is used instead of the more common sandwich ELISA due to using a sweat based detection system lies in the difficulty of cortisol’s small size of 363 Da; cortisol has a single binding site, obtaining readings when a subject is not perspiring. Intensive while a sandwich ELISA requires a minimum of two binding exercise is often needed to stimulate sweat production, but the sites. act of exercising triggers cortisol secretion, distorting measured Figure 2 demonstrates the schematic for a competitive values.38 Additionally, sweat based readings are not reliable over time due to residual cortisol from past perspiration, ELISA, in which a plastic surface such as a microtiter plate is requiring complete removal of sweat from a wearable coated with anticortisol antibodies. The test sample in which biosensor. cortisol is to be measured (termed as “cold”) is added to the Insights into Choice of Matrix/Sample. Overall, both salivary and sweat show a good correlation with free cortisol levels in serum,39 with the added advantage of being noninvasive. Additionally, salivary cortisol levels are synchro- nous with serum levels for up to 24 h with the important caveat that readings are skewed if the patient is on oral contra- ceptives.40 Thus, both fluids can offer a good detection modality for PoC/rapid diagnostic options for cortisol measurement. Interstitial fluid is another attractive sampling option, although it is more difficult to access compared to sweat or saliva. This can be circumvented by utilizing technologies such as microdialysis37 or microneedles.41 Interstitial fluid also has the unique advantage of being the only fluid that can be collected while a subject is asleep−both saliva and sweat fall short in this regard. The measurement of cortisol levels during sleep is a poorly understood yet critical parameter for a variety of tests such as Cushing’s and Addison’s replacement. Sleep cortisol measurements could also provide insights into psychiatric and inflammatory diseases. OVERVIEW OF ESTABLISHED TECHNOLOGIES FOR CORTISOL DETECTION Throughout the history of cortisol detection, various methods have been deployed, each improving upon the disadvantages and limitations of the last. Currently, a wide range of assays are in use for clinical testing, including methods such as enzyme Figure 2. Schematic of competitive ELISA for cortisol quantitation. Anticortisol antibodies are coated onto a surface, such as a microwell. linked immunosorbent assays (ELISAs), radioimmunoassays A mixture of sample (colored blue, containing unknown cortisol (RIAs), fluoroimmunoassays, colorimetric analysis, biolumi- concentration) and a known quantity of labeled cortisol (colored red, nescent probes, immunoassays, and lateral flow devices. The conjugated to an enzyme, such as HRP) is added to the microwell. earliest reported measurements of cortisol involved fluoro- Upon addition of a chromogenic substrate, a drop in signal is metric analysis experiments.42 However, the nonspecificity of observed if sample cortisol is greater than the quantity of labeled these methods and their inability to distinguish between similar cortisol. 1669 https://doi.org/10.1021/acssensors.3c01912 ACS Sens. 2024, 9, 1666−1681 ACS Sensors pubs.acs.org/acssensors Review Figure 3. Various capture molecules used in cortisol assays. (A) Schematic of monoclonal antibody production via hybridoma technology. (B) Diagram of aptamer undergoing conformation change and binding to form an aptamer-target complex. Created with Biorender. (C) Schematic of MIP synthesis showing polymer molding and resultant cavity formation. Reproduced from ref 114 by Baker et al. Copyright 2015, Iranian Biomedical Journal. Licensed under CC BY 3.0 Deed|Attribution 3.0 Unported|Creative Commons. (D) Schematic overview of cortisol measurement using DPV, via conformation switching aptamers functionalized onto a gold nanowire substrate. Reproduced from ref 58. Copyright 2021, ACS Omega. plate, along with a known concentration of cortisol conjugated the procedure is expensive due to column and solvent costs, to an enzyme (termed “hot”) such as horseradish peroxidase relegating it to the purview of specialized laboratories and (HRP) or alkaline phosphatase (ALP). The two different hospitals that can afford the systems and/or have trained cortisol species (labeled and unlabeled) compete for the same personnel.48 These drawbacks make MS unattractive for use in binding site of the immobilized antibody. Following an PoC or rapid diagnostic applications. incubation period, a substrate is added which is oxidized by Shortcomings of Current Technologies. The rhyth- the enzyme, producing an amplified chromogenic signal. The micity of cortisol secretion is a defining characteristic for intensity of the signal produced is inversely proportional to the diagnostic testing that is difficult to capture with current amount of cortisol present in the sample, due to the “cold” sensing strategies. Due to the oscillating nature of serum cortisol occupying more binding sites than the “hot” cortisol, cortisol levels, there is little diagnostic value obtained from a dampening the signal.45 While immunoassays for cortisol are single point measurement; an observed value is only clinically widely used due to their relatively inexpensive nature and ease relevant when measured in reference to the overall ultradian of use (compared to techniques such as mass spectroscopy), and circadian profile of cortisol levels throughout the day. they can be hampered by issues of cross reactivity with other Currently, the diagnosis for hypercortisolism can be steroid hormones and are thus highly dependent on the quality performed with a pharmacological suppression test or a of the monoclonal antibody. midnight salivary test when cortisol levels reach their nadir Mass Spectroscopy. The current gold standard for clinical or lowest point. Complex conditions such as cyclic hyper- cortisol measurement is mass spectroscopy (MS), a powerful technique routinely used for molecular characterization and cortisolaemia need to be diagnosed with multiple consecutive the current best available tool to characterize free cortisol midnight cortisol readings.49 Cortisol levels in such a condition levels.46 MS is used as a highly specific method to analyze can fluctuate between hyper and normal over a period of bodily fluids for cortisol, with liquid chromatography tandem months the number of tests required can place a large strain mass spectroscopy (LC-MS/MS) able to distinguish between on both the individual and the healthcare system. The logistics various steroid species and even synthetic steroids, allowing of performing multiple, timed cortisol tests produces results clinicians to observe steroid abuse.47 MS offers multiplexing that cannot be clinically interpreted with a high degree of allows simultaneous detection of multiple biomarkers of confidence, in addition to the significant monetary and time interest, a major advantage compared to immunoassays, in cost spent on performing these tests. A rapid cortisol particular, microtiter-plate based assays. MS-based measure- monitoring system could aid in the accurate diagnosis of ments are also unaffected by steroid cross-reactivity. However, adrenal insufficiency or hypercortisolism, while easing the while MS is precise and sensitive, the apparatus is bulky and burden on the healthcare system and the patient.35 1670 https://doi.org/10.1021/acssensors.3c01912 ACS Sens. 2024, 9, 1666−1681 ACS Sensors pubs.acs.org/acssensors Review The benefits of moving to a rapid diagnostic approach method of synthesis, ease of chemical modification, high include frequent monitoring, turn-around time on the scale of stability and overall cost effectiveness.55 Aptamers show minutes (as opposed to hours, as seen in clinical laboratories), excellent biorecognition ability, being able to selectively detect less dependence on skilled technicians/devices and an overall targets in complex fluids such as serum.56 This property is also reduction in cost of sensing per patient. Additionally, the useful in distinguishing cortisol from the many similar technology associated with rapid/real-time sensing tends molecules that routinely interfere in antibody-based capture. toward miniaturization, improving upon the portability and Aptamers can be selectively oriented onto a desired trans- ease of use of the final device. This approach can also lead to ducer,57 making them ideal for coupling to electrochemical multiplexing designs, where multiple streams of data are surfaces. sampled simultaneously.50 Utilizing rapid diagnostics for Additionally, aptamers can be configured to have con- hormones such as cortisol can be invaluable to both clinicians formation switching properties when they bind to cortisol, and patients to better understand hormonal biorhythms. perturbing the charge transfer rate and enabling rapid, reagent Additionally, a distinction between rapid and continuous free detection of cortisol. This is particularly viable when sensing of cortisol should be made, particularly when functionalized onto a conductive substrate such as gold, as measuring the hormones biorhythms. Rapid sensing can refer shown in Figure 3.58 In that study, aptamers specific to cortisol to multiple point measurements made over time, which can were modified to be conformation-switching and were then be collated to produce a picture of overall hormone functionalized onto gold nanowires. The resulting biosensor rhythm. Most PoC sensors would belong to this category of demonstrated a LoD of 0.2 pg/mL while sampling from serum, sensing, as rapid PoC testing is simpler to perform and can be a sample fluid with significant matrix interference. carried out noninvasively. Molecularly Imprinted Polymers (MIPs). Traditional The end-goal of precision medicine style diagnostics would capture molecules used for cortisol detection are monoclonal be a continuous monitoring system, including devices such as antibodies and aptamers. In recent years, molecularly continuous glucose monitoring (CGM) devices, where the imprinted polymers (MIPs) have emerged as an inexpensive device is in continuous contact with the analyte. However, in and versatile tool for cortisol capture,59−61 retaining high comparison to rapid sensing, continuous sensing is far more specificity while being cheaper to synthesize and functionalize. challenging to perform, due to a wide range of constraints. Figure 3 shows the process of MIP synthesis via the process of These can include, but are not limited to, saturation of the molecular imprinting, wherein a template molecule is detection system, size of the device, biofouling, biocompati- incorporated during the polymerization of a chosen monomer. bility, and power source for the device. Each of these problems After polymerization, the template molecule is removed, must be solved before a continuous cortisol biosensor can be leaving behind cavities in the polymer matrix that are developed and used in clinical practice. Some of the complementary in shape, size, and functional groups to the components of a cortisol biosensor that can be improved target molecule. These cavities act as selective recognition sites upon include the capture molecule, the fluid uptake system, for the target molecule. and the sensing transducer. The recognition sites allow high affinity binding to the CAPTURE MOLECULES FOR CORTISOL DETECTION analyte, avoiding the common issue of cross-reactivity with similar targets. By selecting the appropriate monomeric unit of the polymer, recognition sites can be constructed, leading Monoclonal Antibodies. Monoclonal antibody-based MIPs to be called “artificial receptors”.62 Due to their high immunoassays are ubiquitous due to their high specificity customizability and potential for functionalization, MIPs can and affinity toward the target antigen.51 These antibodies can be easily coupled to electrochemical sensing systems in a be reliably generated via hybridoma technology (schematic wearable form.63 This type of sensor achieved rapid (