Innovative Bio(Nano)Sensor Designs for Cortisol Stress Hormone Detection: A Continuous Progress

Abstract

Nowadays, the population is subject to a lot of stress, being one of society’s most encountered problems affecting people all over the world. Being under a lot of stress for prolonged periods of time impacts the physical and mental health of individuals with effects on society as an economic burden. Cortisol is one of the main indicators of stress. Long-term exposure to this stress hormone can lead to severe medical conditions such as heart disease, lung issues, obesity, anxiety, or depression. In this context, the current review aims to provide a comprehensive overview of the most recent advances made in the development of versatile and efficient cortisol devices and biosensors capable of monitoring the cortisol levels in biofluids. Lately, both non-plasmonic (polymer-based sensors, optical sensors, electrochemical sensors) and plasmonic sensors (mono- and multiple-metallic nanoparticles-based sensors) have shown great results in cortisol detection. The work focuses on the advantages, remaining restrictions, and limitations in the field of cortisol biosensors from solution-based immunosensors to wearable and Lab-on-Skin monitoring devices, providing a better understanding of the fulfilled requirements and persisting challenges in the accurate detection and monitoring of the cortisol stress hormone.

1. Introduction

One of the major health problems of modern society is associated with stress, a risk factor with high impact on quality of life and mental health. The factors of increased levels of stress can be classified depending on their source, however a high standard lifestyle or the lack of time in our daily activities are the main factors which affect our nervous and hormonal systems [1]. Stressful life events or experiencing traumatic life events have also been associated with poor outcomes in our biological system, weight gain, sleep problems and mental disorders [2,3], as illustrated in Figure 1. This figure shows the impact of high cortisol levels on our body and the possible effects of these values on human life. Biosensors based on cortisol detection can improve patients’ lives by allowing the identification of a pathology with facile devices able to investigate also non-conventional body fluids.

Figure 1. Cortisol production and the impact of high cortisol levels on human body. Innovative and facile detection of cortisol using biosensors from non-conventional biological fluids.

1.1. Cortisol—The Biomarker of Stress

Cortisol is the main glucocorticoid hormone found in the adrenal cortex. Under normal physiological conditions, cortisol secretion exhibits a well-defined rhythm [4]. Levels begin to rise in the early morning hours, peaking approximately 30 min after awakening. Following this peak, cortisol concentrations gradually decline throughout the day, reaching their lowest levels around midnight. This diurnal variation is essential for maintaining metabolic balance, immune function, and overall homeostasis [4]. Cortisol production and secretion are constantly occurring in the hypothalamus–pituitary–adrenal (HPA) axis, as observed in Figure 1, where loss regulation can lead to high blood glucose, inflammation of the immune system, and various diseases or syndromes, such as Cushing syndrome or Addison disease [5]. In Cushing syndrome, an excess level of cortisol is considered the trigger factor, while in case of Addison’s diseases, lower levels of cortisol are recorded [6]. The key mechanism of cortisol production is based on HPA function, an important part of the neuroendocrine system. The stress perceived by the organs leads to the release of a corticotropin-releasing hormone by the implication of hypothalamus. This hormone triggers the anterior pituitary gland to produce adrenocorticotropic hormone (ACTH), the main source of cortisol release in the body [7]. Upon production, cortisol is released in the blood circulatory system. In circulation, approximately 90–95% of cortisol is bound to plasma proteins, mainly to corticosteroid-binding globulin. Only the unbound fraction, representing around 5% of total cortisol, is biologically active and capable of crossing cell membranes to interact with intracellular glucocorticoid receptors. This free cortisol fraction mediates the interaction within the HPA axis, thereby influencing critical physiological functions such as inflammation control and glucose homeostasis. As such, accurate assessment of free cortisol levels is essential for evaluating stress-related endocrine dysfunctions and guiding appropriate therapeutic interventions [7,8].

Cortisol can be found in various parts of the human body, especially in blood, plasma, serum, sweat, urine, saliva, or hair [9]. The main actual detection method for most patients is based on the blood tests performed, usually at 8 AM. However, in time, various facile strategies were proposed as alternatives to this invasion approach, since cortisol can be detected in other biological fluids as well. Indeed, due to its passive diffusion across biological membranes, the concentration of free cortisol in peripheral biological fluids is significantly lower than in blood [9]. As a result, cortisol levels detected in matrices such as saliva, sweat, or interstitial fluid (ISF) are typically reduced compared to those in serum or plasma, as can be observed in Table 1, in which various cortisol levels are mentioned.

Table 1. Cortisol levels corresponding to their biological availability.

Biological Availability	Cortisol Level	Refs.
Blood	in the morning: 25 µg/mL at midnight: 2 µg/mL	[10,11]
Serum and Plasma	in the morning: 45–227 ng/mL at midnight: 17–141 ng/mL	[12]
Urine	36–137 µg/24 h	[13]
Sweat	8–142 ng/mL	[14]
ISF	in the morning: 12–34 ng/mL at midnight: 9–13 ng/mL	[15]
Hair	1.7–153.2 pg/mL	[16]
Saliva	0.5–0.05 µg/dL	[17]

The major drawback to testing cortisol from blood is that both bound and free forms of the hormone are present in the bloodstream; thus, the total cortisol is determined [18], while the active fraction, known as Cortisol Free Index (CFI), is deduced using Coolen’s equation [19]. In addition, the sampling process is complex and time-consuming and implies high costs for the collection of the sample by specialized personnel, manipulation, storage, and testing. The main detection tools for blood cortisol determinations are immunoassays [20]. Ultimately, it is painful for the patients as it requires repeated vein punctures, which could cause elevated cortisol levels in patients considering they are exposed to a stressful experience. Lately, various biological fluids are used for cortisol investigation using conventional techniques. Human hair has become an increasingly important biological sample in a variety of domains such as forensic science, toxicology, doping control, and clinical diagnostics [21]. Because hair grows at a relatively constant rate, the cortisol embedded within the hair shaft provides a chronological record of cortisol production. Early feasibility of detecting cortisol in hair was demonstrated by Koren et al., using a modified salivary enzyme-linked immunoassay (ELISA) [22]. Sauve et al. reported hair cortisol concentrations ranging from 1.7 to 153.2 pg/mL and found a positive correlation with 24 h urinary cortisol [16]. Van Ockenburg et al. have shown that hair cortisol showed high test–retest reliability, demonstrating strong reproducibility [23]. Another interesting source of cortisol is represented by the saliva. Cortisol is present in the saliva in its free, active form, which is the biologically relevant form of cortisol [24], which simplifies the assessment of cortisol levels by reducing the influence of the bound form of the hormone. Furthermore, the analysis of saliva is easy, non-invasive, and patient-friendly—the sample collection does not induce any stress on the patient, nor does it inflict any harm, which might decrease the patient’s willingness to undergo successive monitoring procedures—and it can be realized in clinical settings as well as in the comfort of the patient’s home. The determination of salivary cortisol is already employed as a screening test for Cushing’s [25]. Saliva testing for cortisol has been proven efficient for the diagnosis of adrenal insufficiency as well [26], proving its versatility in the prevention and diagnosis of medical conditions associated with unhealthy cortisol levels. Russel et al. have shown that the salivary cortisol concentrations are well correlated with the cortisol levels detected in sweat [14].

Even if human hair and saliva present numerous advantages, the other sources have shown limitations in the identification of cortisol levels. The lack of reproducibility and the conditions in which the extraction can be obtained are mainly the issues described lately. In Table 2, the main limitations of various non-conventional fluids are presented. These drawbacks encourage researchers to focus their attention on designing ingenious solutions for facile detection.

Table 2. Limitations in cortisol extraction from various non-conventional biological fluids.

Non-Conventional Biological Fluids as Cortisol Source	Limitations of Classical Extraction Techniques	Refs.
Urine	Collection and testing of urine samples over the course of 24 h Maintaining the urine in a refrigerator for 24 h The need of multiples techniques for further investigations: protein-binding assays, thin-layer chromatography (TLC), fluorimetry	[27,28,29,30]
Sweat	Collection using sweat patches that need further investigations: ELISA, Liquid Chromatography–Mass Spectrometry (LC-MS), TLC, or immunosensors The influence of external factors on the cortisol hormone in sweat samples	[31]
ISF	Extraction area: right below the skin as ISF does not permeate through the skin due to the epidermal keratinized layer Concerns about the infection risks of the collection devices Very slow speed at which ISF needs to be extracted	[13,15]
Human hair	Moderate results thus far	[14,16,21,23]
Saliva	Discomfort in measuring cortisol level	[25,26]

Recent approaches rely on innovations in nanotechnology and biomedical sciences to overcome these disadvantages. For instance, the implementation of (nano)biosensors led to the development of excellent miniaturized tools for a highly sensitive cortisol detection offering new horizons in cortisol detection and in the detection of medical conditions associated with this stress hormone [32,33]. In a recent study, Ahmed et al. reported a study in which 22 healthy people were introduced in order to analyze their stress level by using saliva and a Maastricht Acute Stress Test modern kit based on blue tetrazolium [34]. Using optical approaches and redox reactions, the researchers recorded spectrometry absorbances at 510 nm as a function of cortisol levels. Since the reaction is based on chemical changes in color, the results showed a stronger magenta color for higher cortisol levels from saliva; therefore, a non-invasive method was established as an alternative for blood cortisol determinations. Kim et al. also studied the detection of cortisol saliva using an innovative technique based on biosensors [35]. For precise measurements, the group realized a biosensor using a cortisol monoclonal antibody covalently immobilized on reduced graphene oxide. The biosensor showed excellent detection, achieving a limit of detection of just 10 pg/mL in a very short time and under simple conditions.

Cortisol detection involves the implementation of many techniques to analyze the samples extracted from patients. In the literature, the techniques are classified into conventional and non-conventional methods, depending on the novelty and the technique applied. Therefore, conventional methods nowadays are considered mainly chromatographic techniques and enzymes-based reactions for cortisol detection, methods which are further described in this review. In contrast, the latest studies regarding cortisol detection are related to the development of biosensors and nanosensors, the main focus of this review.

1.2. Conventional Analytical Tools for Cortisol Monitoring

Current strategies for assessing human stress levels typically rely on physiological, behavioral, or psychological indicators. Several cortisol detection techniques have been described in the literature; however, most of them are based on conventional methods that require invasive sample collection [36]. These standardized invasive approaches may not always be appropriate, especially given the added stress imposed on patients during sample collection, which can alter the physiological parameters under investigation. Repeated testing, a process often necessary for hormonal monitoring, may discourage individuals from seeking medical evaluation due to the discomfort and inconvenience associated with such procedures. Furthermore, psychological stress related to sample collection may artificially elevate cortisol levels, potentially leading to inaccurate or inconclusive diagnostic outcomes. Additional limitations of conventional methods include complex sample preparation protocols, reduced analytical precision, the need for large sample volumes, reliance on costly instrumentation, and long processing times. A promising alternative lies in the development of biosensors based on nanotechnology for efficient and fast detection. Portable and miniaturized sensing devices using plasmonic or non-plasmonic nanoparticles increase the detection sensitivity and efficiency, while offering a more patient-friendly solution for cortisol monitoring.

LC-MS and High-Precision Liquid Chromatography (HPLC) are the main analytical techniques used for free cortisol detection. LC-MS and HPLC are powerful and precise techniques used for very small molecules and particles able to distinguish between similar compounds from a qualitative and quantitative approach [37]. Both have high selectivity and specificity in detection of multiple analytes, including various hormones and proteins together with their isomers [38]. Jia et al. developed a precise HPLC method to investigate cortisol from human eccrine sweat [39]. The study relies on the analyzation of samples extracted artificially in two moments of the day, 1 and 4 pm, with the aim of obtaining information only about pure cortisol. The main advantage of the method proposed was the ability to separate cortisol from other substances or other isomers that could interfere with it. The concentration obtained was between 1.832 and 1.307 ng mL⁻¹ at 1 pm, while in the evening, less cortisol levels were identified, as it is expected to happen. Lee et al. used LC-MS to determine both cortisol and cortisone from serum samples [11]. The precision of the method was sustained by the low limit detection obtained during measurements: 0.2 ng/mL for cortisol and 1 ng/mL for cortisone. Similar results were obtained by Chen et al. who quantified cortisol and cortisone levels from serum and saliva extracted from depressive patients [40]. HPLC was chosen as separation technique, and the method was validated recording high selectivity and accuracy within the 28 patients studied. The authors exposed very low limit of detection of 100 pg/mL and 50 pg/mL for cortisol and cortisone, suggesting the accuracy of the method regarding FDA guidelines.

Besides chromatographic methods, ELISA is another type of conventional method to study cortisol [13]. ELISA is a technique based on antigen–antibody recognition in which quantitative and qualitative detection of antigen is possible based on a color change [41]. To identify the biological molecules, an enzyme is usually attached to the antibody, which will further target the antigen. To be very specific and to have precise results, a second enzyme-labeled antibody is linked to the first antigen-primary antibody complex. The reaction results are identified using sensitive instrumentation depending on the antigen used, its absorption in various wavelengths, and on the detection method chosen [42]. Over the years, many studies for cortisol identification using ELISA were addressed.

Mizuhata et al. conducted a study in which the effect of breastfeeding on cortisol was assessed using saliva as sample source [43]. The study involved 79 women and perceived stress scale and Edinburgh postnatal depression scale were chosen as references for cortisol detection. Based on an ELISA kit, the authors proved that breastfeeding decreases stress since an impressive decrease was observed in the salivary cortisol levels. Butts et al. investigated the association of urinary cortisol with the stress that appeared usually during in vitro fertilization (IVF) [44]. Based on ELISA kit, the study involving 28 males and 52 women could not indicate a clear association between the stress accumulated and urinary cortisol. Albahli et al. studied the association between schizophrenia and periodontal disease in relation to cortisol level using an ELISA kit [45]. The study involved 40 subjects divided into two groups: 20 schizophrenic patients and another group with 20 non-schizophrenic healthy patients. Lower cortisol levels were associated with schizophrenic patients, most likely due to their antipsychotic treatment. No correlation between cortisol and periodontal disease was identified [45].

ELISA has the advantages of being a simple procedure that is easy to perform, has high specificity and efficiency, the reagents are relatively low-cost, and is generally safe. However, antibody instability, the restrictive conditions in which the reagents must be kept, the high possibility of false results, or the sophisticated techniques applied during the procedure [46] have led to the development of alternative solutions for cortisol detection. The need for miniaturized devices for facile hormone detection is extremely important for society. The evolution and the comfort offered by these small devices can reduce the side effects in the long term since the patients will be encouraged to use them and to regularly check cortisol levels. Cortisol is also very important for athletes, who spend many hours in gyms where this miniaturized device can be easily used. All these aspects have a high social and economic impact, especially since the lack of such small devices on the market is felt by patients with multiple medical conditions.

1.3. Societal and Economic Impact

As society is faced with increasing amounts of stress on a daily basis [47], the accurate real-time detection and monitoring of cortisol become imperative for the prevention of a great variety of diseases. Cortisol level variations are normal to an extent; however, chronic stress and prolonged exposure to high levels of cortisol lead to severe health problems with life-threating outcomes. Abnormal levels of cortisol affect the regulation of various human organ processes, thus having implications in the immune [48], cardiovascular [49], nervous [7], respiratory [50], reproductive [51], musculoskeletal [52], integumentary [53], and visual systems [54]. With rising stress levels, the incidence of chronic diseases is on the rise, impacting the citizen’s health as well healthcare systems in a major economic way. The symptoms of high cortisol levels can be easily misinterpreted as they are generally non-specific, such as fatigue, weight gain, headaches, bone fragility, or irritability [10]. Being under a lot of stress and feeling unwell may also lead to the development of mental health issues, which first affect the everyday life of citizens by gradually decreasing their personal and professional performance, in turn impacting both their life quality and job performance—a significant economic impact on the individual and employer. If remained untreated, mental health disorders can become a societal and economic burden, as low performances at work lead to layoffs and difficulties in maintaining a job; thus, poverty, substance abuse, homelessness, criminal activity, and suicide rates might increase [55,56]. Therefore, increasing stress levels have a strong influence on individuals and society as a whole, with the economic burden relating to higher costs for healthcare, disability expenses, and low productivity of industries [57,58]. Addressing the effects of stress and, implicitly, the prolonged exposure to high cortisol levels supports individual health and well-being by reducing the prevalence of diseases, as well as favoring safe social environments, allowing society’s further economic evolution.

2. Non-Plasmonic Sensor Designs for Cortisol Detection

In response to these limitations of conventional methods, the development of innovative biosensors for cortisol detection has garnered significant attention. Various plasmonic or non-plasmonic sensors were addressed lately as alternative solutions for facile hormone detection. Among these, non-plasmonic sensor designs have emerged as promising alternatives due to their versatility, sensitivity, and potential for miniaturization [59]. This section delves into various non-plasmonic sensor architectures, focusing on polymeric nanosystems, optical (nano)fiber-based sensors, electrochemical sensor designs, and aptamer-based detection methods.

2.1. Polymeric Nanosystems for Cortisol Detection

Polymeric nanosystems have emerged as a cornerstone in the development of non-plasmonic biosensors for cortisol detection. These systems harness the unique features of polymers, such as biocompatibility, chemical stability, and easy functionalization, to create sensitive and selective detection platforms. By engineering polymers at the nanoscale, researchers have developed various sensor configurations, including molecularly imprinted polymers (MIPs), conductive polymer matrices, and polymer-based nanocomposites, each offering distinct advantages in cortisol sensing applications [60,61].

MIPs are synthetic polymers engineered with specific cavities that match the size, shape, and functional groups of target molecules, enabling selective recognition. In the context of cortisol detection, MIPs serve as artificial receptors that bind cortisol molecules with high specificity. The fabrication process typically involves polymerizing functional monomers in the presence of the cortisol template, followed by template removal to create the imprinted sites [62]. This approach has been employed to develop electrochemical sensors where the binding of cortisol induces measurable changes in electrical signals, facilitating quantification [63]. For instance, the study reported by Yulianti et al. revealed the use of a MIP-based electrochemical sensor that exhibited a 200 fM detection limit and selective detection against other steroid hormones, showcasing the method’s sensitivity and potential for clinical application [64].

Integrating MIPs into wearable platforms offers a robust, cost-effective, and selective approach to cortisol detection in biofluids. MIPs offer unique advantages for wearable devices: they provide high selectivity toward cortisol through template-based imprinting, exhibit excellent mechanical and chemical stability, and can be integrated into diverse transduction mechanisms such as electrochemical, capacitive, and optical sensors. Their synthetic origin eliminates the limitations associated with biological recognition elements (e.g., antibodies), such as denaturation and high cost. For instance, in a recent study, cortisol sensors, as observed in Figure 2, have been successfully integrated onto flexible substrates such as polydimethylsiloxane (PDMS), polyimide (PI), and textile fibers, enabling close contact with skin and reliable sampling of sweat or ISF [65].

Figure 2. Microfluidic systems for cortisol detection: (A) Structure of the device; (B) magnified view of the main serpentine skeletal channel for cortisol tracking; (C) cross-sectional view of the main channel; (D) microfluidic structures with the optical image of the system; (E) optical (Top), mechanical twisting (Middle), and bending (Bottom) mechanisms; (F) 3D modeling of the system. Reprinted from Kim et al.’s open-source article [65].

2.2. Optical (Nano)Fiber-Based Sensors for Cortisol Detection

Optical biosensors have gained significant interest for real-time, label-free, and non-invasive hormone monitoring. These sensors are particularly advantageous for detecting cortisol, due to their high sensitivity, compactness, and compatibility with wearable platforms. Unlike plasmonic designs, these sensors rely on evanescent field perturbations within guided light modes and do not depend on surface plasmon resonance (SPR).

The fundamental detection mechanism involves the interaction between light guided through the fiber core and the external medium via the evanescent field. This interaction is especially pronounced in modified fiber geometries which expose the guided mode to the surrounding analyte. When cortisol binds to recognition elements on the fiber surface, it induces refractive index changes, physical swelling, or surface mass changes, which are detected through alterations in light intensity, wavelength, or phase [66]. Several designs and materials have been reported for cortisol detection using optical fiber sensors using alternative biorecognition strategies instead of aptamers, some of them being exposed in Table 3.

Table 3. Classification of non-plasmonic optical sensors for cortisol detection.

Sensor Category	Principle	Materials	Sample/ Application	Limit of Detection (LOD)	Refs.
Hydrogel-based photonic sensors	Cortisol binding induces volumetric and refractive index changes in a photonic crystal hydrogel, leading to a visible colorimetric shift	Photonic crystal hydrogel composed of cortisol-responsive polymer network	Human sweat (artificial sweat and on-body testing)	1.7 nM	[67]
Polymeric Bragg grating sensors	Cortisol interaction alters the effective refractive index of the Bragg fiber, producing a measurable Bragg wavelength or transmission spectrum shift	Polished hollow-core Bragg fiber functionalized with cortisol-specific aptamer	Artificial saliva/biofluids (proof-of-concept wearable sensing)	4.3 pM	[68]
Fluorescence-based optical sensors	Cortisol binding to selective molecularly imprinted sites modulates fluorescence polarization/anisotropy	Core–shell MIP nanoparticles using cortisol-21-monomethacrylate template	Aqueous solutions (laboratory assays)	80 nM	[69]

The inherent flexibility and light-guiding capabilities of optical fibers make them suitable for wearable health-monitoring systems, such as skin patches, textile-embedded sensors, or headbands. Their main advantages of optical readout with wireless transmission and integration with soft materials have enabled the development of fully stretchable or skin-conformal sensors for stress management applications.

2.3. Electrochemical Sensor Designs for Cortisol Detection

Electrochemical sensors have emerged as a promising class of analytical tools for the detection of cortisol. They offer advantages such as high sensitivity, rapid response, low cost, and compatibility with miniaturized and wearable formats. Unlike optical sensors, electrochemical sensors detect cortisol through changes in electrical properties, such as current, potential, or impedance, resulting from specific interactions between cortisol molecules and recognition elements immobilized on the electrode surface.

For instance, enzymatic electrochemical immunosensors employ antibodies as biorecognition elements to achieve high specificity for cortisol detection. These sensors typically utilize a competitive binding assay, where cortisol in the sample competes with a labeled cortisol analog for binding sites on the immobilized antibody. The resulting change in signal correlates with the cortisol concentration. Sun et al. report the successful development of an electrochemical immunosensor based on magnetic functionalized reduced graphene oxide [70]. The sensor exhibited a detection range from 0.1 to 1000 ng/mL, proving a detection limit of 0.05 ng/mL, demonstrating excellent analytical performance for cortisol detection in real samples. Similarly, a disposable electrochemical immunosensor utilizing a direct competitive ELISA was reported by Kamarainen et al. [71], achieving a detection limit of 0.6 ng/mL in buffer solution and 1.7 ng/mL in saliva, successfully covering the physiological range of salivary cortisol.

Non-enzymatic electrochemical sensors offer an alternative approach by eliminating the need for biological recognition elements, thus enhancing stability and shelf-life. These sensors rely on the direct electrochemical oxidation or reduction of cortisol at the electrode surface. Such an example was reported by Rison et al., who described a non-enzymatic electrochemical sensor using zinc oxide nanoparticles deposited on a graphene-coated pencil graphite electrode [72]. The sensor achieved a detection limit of 0.15 nM and demonstrated excellent selectivity for cortisol in the presence of interfering substances. In this study, it was shown that with the increase in electroactive surface area of the final assembly, the electrocatalytic activity of the zinc oxide nanoparticles immobilized on graphene-coated pencil graphite electrode was improved. Furthermore, the immobilized nanoparticles help generate high-energy electroactive surface sites that promote the capture of cortisol molecules by the electrode surface [72].

Electrochemical sensors exhibit advantages such as miniaturization and flexibility, which make them suitable for integration into wearable devices for real-time, non-invasive monitoring of cortisol levels. Advancements in materials science and microfabrication techniques have facilitated the development of skin-conformal sensors capable of detecting cortisol in sweat or ISF. For example, Hossain et al. report on developing a wearable electrochemical sensor capable of real-time monitoring of cortisol in human sweat [73]. The sensor demonstrated accurate detection of cortisol levels, aligning with circadian rhythms and stress-related changes. Significant progress has been made in the development of both enzymatic and non-enzymatic electrochemical sensors; however, further improvements are required to ensure selectivity in complex biological matrices. In spite of the remaining challenges, the integration of electrochemical sensors into user-friendly, wearable platforms holds great promise for personalized stress monitoring and health management.

2.4. Capacitive and Piezoelectric Sensors for Cortisol Detection

Other promising non-plasmonic platforms for cortisol detection are capacitive and piezoelectric sensors, offering advantages such as label-free operation, real-time monitoring, miniaturization, and compatibility with flexible substrates. These sensors convert biochemical recognition events into measurable electrical signals, such as changes in capacitance or mechanical-to-electrical conversion, making them attractive for wearable and point-of-care devices [74,75].

Capacitive sensors operate by detecting variations in the dielectric properties of the sensor interface caused by the presence of target molecules. When cortisol binds to a chemically functionalized surface, it alters the local permittivity, leading to a measurable change in capacitance between the electrodes. A representative example is a cyclodextrin-based capacitive sensor where β-cyclodextrin (β-CD) functionalization provided selective binding sites for cortisol [76]. The interaction induced a change in dielectric constant and, consequently, the capacitance, enabling quantitative detection with high sensitivity and specificity. The sensor demonstrated a detection limit of 2.13 nM and was successfully used to measure cortisol in saliva and urine samples, as reported by Panahi et al. In another approach, a paper-based capacitive biosensor fabricated from a graphene-nanoplatelet and amphiphilic diblock copolymer composite was employed for cortisol detection in human saliva. This platform reached an impressive detection limit of 3 pg/mL and covered a wide linear detection range (3 pg/mL to 10 μg/mL), highlighting its feasibility for low-cost, portable diagnostic systems [77]. Capacitive sensors have also been incorporated into wearable systems for sweat analysis. A microfluidic sensor incorporating inkjet-printed capacitive electrodes and a sweat collection channel was developed to monitor cortisol and glucose simultaneously [78].

On the other hand, piezoelectric biosensors leverage the property of certain materials to generate an electric charge in response to applied mechanical stress. When cortisol binds to the sensor surface, the mass change perturbs the resonance frequency or voltage output of the piezoelectric element. While direct reports of piezoelectric cortisol biosensors are scarce, several foundational technologies offer promising avenues. For example, wearable piezoelectric nanofabrics made of polyvinylidene fluoride nanoyarns have demonstrated excellent mechanical-to-electrical energy conversion capabilities [79]. Though these were not directly configured for cortisol detection, their mechanical flexibility and biocompatibility make them promising scaffolds for future cortisol sensor designs. The inherent flexibility and low power requirements of both capacitive and piezoelectric sensors make them well-suited for integration into wearable platforms for continuous stress hormone monitoring. Nonetheless, several key challenges still need to be addressed in the development of efficient and accurate capacitive and piezoelectric cortisol sensors. Ensuring specific recognition of cortisol and structurally similar steroids like cortisone, maintaining sensor response consistency under variable temperature and humidity in wearable settings, as well as normalizing sensor response for inter-individual variability in sweat composition and skin physiology are challenges to be addressed in the future.

2.5. Aptamer-Based Sensors for Cortisol Detection

Aptamers are single-stranded DNA or RNA oligonucleotides selected in vitro for their high affinity and specificity towards a target molecule. Lately, they have emerged as powerful biorecognition elements in biosensor development [80]. Their inherent advantages, including high specificity, chemical stability, and the possibility of tailoring their properties through modification, make them attractive alternatives to antibodies in cortisol sensing. While many aptasensors leverage optical, electrochemical, or electrical transduction mechanisms, innovative approaches are continuously being explored to harness the unique binding capabilities of cortisol-specific aptamers.

One such approach involves microcantilever-based sensing. In this method, a cortisol-specific aptamer is immobilized on the surface of a microcantilever. Upon binding of cortisol to the aptamer, the resulting conformational changes and the added mass induce a measurable deflection or a shift in the resonant frequency of the cantilever [81]. This label-free technique offers the potential for real-time monitoring of cortisol levels with high sensitivity. For instance, Kim et al. have demonstrated the feasibility of using aptamer-functionalized microcantilevers for the detection of small molecules, paving the way for their application in cortisol assays [82]. The sensitivity of these sensors can be further enhanced by optimizing the aptamer density and the mechanical properties of the microcantilever.

Quartz crystal microbalance (QCM)-based sensors also present a label-free alternative. Similarly to microcantilevers, QCM sensors rely on the change in resonant frequency of a quartz crystal upon mass loading. Immobilizing a cortisol-specific aptamer on the QCM sensor surface allows for the detection of cortisol binding events through the measured frequency shift [83]. The sensitivity of QCM sensors can be improved by employing techniques such as acoustic impedance matching and by using high-overtone crystals [84]. Furthermore, the integration of microfluidic systems with aptamer-functionalized QCM sensors enables continuous flow analysis and reduces sample consumption.

Another strategy is based on nanomaterials coupled with aptamers for improved sensing abilities. For example, aptamers conjugated to magnetic nanoparticles can be used for the magnetic separation and subsequent detection of cortisol, using techniques like nuclear magnetic resonance (NMR) relaxometry [85]. The binding of cortisol to the aptamer on the nanoparticle surface can induce changes in the magnetic properties of the surrounding environment, leading to detectable shifts in NMR relaxation times. Continued research and development in this area will contribute to the advancement of cortisol monitoring in various applications, ranging from clinical diagnostics to stress management.

Lately, the development of wearable devices incorporating aptamer-based sensors, as presented in Figure 3, has opened new avenues for non-invasive cortisol monitoring. Flexible substrates functionalized with cortisol-specific aptamers can be integrated into skin patches or textiles, allowing for real-time monitoring of cortisol levels in sweat and achieving very low detection limits, even at 0.2 pM [86]. These platforms can utilize colorimetric changes or mechanical responses to indicate cortisol concentration, eliminating the need for complex instrumentation.

Figure 3. In vitro cortisol detection using the wearable integrated microfluidic patch: (A) buffer without microfluidics and (B) with microfluidics; (C) artificial sweat without microfluidics and (D) with microfluidics. Reprinted from Singh et al.’s open-source article [86].

3. Plasmonic Sensor Designs

The development of plasmonic sensors has attracted significant interest due to their ability to detect changes through the excitation of surface plasmons. This chapter examines various plasmonic sensor design strategies, as illustrated in Figure 4, with an emphasis on the structures, materials, and optimization criteria that determine sensor sensitivity.

Figure 4. The main actual cortisol plasmonic biosensors designs.

3.1. Plasmonic Monometallic Nanoparticles

Plasmonic sensors are the main type of biosensors used for cortisol detection lately. Nanoparticles are one of the smallest entities related to nanomaterials classification, being an intermediate between an atom and a bulk material. Their small dimensions of maximum 100 nm have led to increased reactivity and better surfaces exchanges compared to other nanomaterials. Metal nanoparticles, also known as plasmonic nanoparticles [87], exhibit unique physicochemical properties that make them highly suitable for biosensing applications, particularly in the detection of biomolecules such as cortisol. Their outstanding optical, electrical, mechanical, and magnetic characteristics render them excellent candidates for integration into advanced electronic and sensing devices. Noble plasmonic metals, including silver (Ag), gold (Au), copper (Cu), and platinum (Pt), designed with innovative fabrication techniques such as nanotechnology, provide a promising platform for developing smart diagnostic tools aimed at monitoring physiological markers like cortisol [88]. Their remarkable plasmonic activity [89] enables strong light–matter interactions, which are essential in photonic and biosensing applications, such as fluid and biomolecule detection. Among them, Au and Ag are often described in the literature, proving that medical devices for cortisol detection is a new approach in medical science. The ability of these nanoparticles to manipulate light, coupled with their biocompatibility, creates a synergistic basis for optical energy modulation and molecular recognition.

3.1.1. Gold Nanoparticle (Au NPs)-Based Biosensors

Au NPs have been intensively studied lately due to their great response in biosensing applications and due to their biocompatibility. Their excellent interaction with light via SPR [90] has led to the development of multiple optical devises for biomedical and biosensing applications. Au NPs are predominately studied in chemistry, materials sciences, physics, or medicine due to their advantages in detection, imaging, or therapeutics. In medical applications, they are mainly used in cancer therapy by photothermal therapy or in drug delivery systems since they are stable, they have high surface energy, and their surface can be easily functionalized [91]. Lately, various studies have reported the implementation of Au NPs in biosensor designs as well [92,93]. The good yield of detection, their stability, and their facile-binding capabilities to small biological or chemical molecules [94] have increased the researchers’ attention. Depending on the detection method, the biosensors based on Au NPs rely on SPR detection, surface-enhanced Raman scattering (SERS), localized surface plasmon resonance (LSPR), and fluorescent or electrochemistry methods [95]. In cortisol detection, highly sensitive Au NPs-based biosensors are predominantly studied among all the noble metals used in nanoscience [96,97,98].

Yilmaz et al. developed a sensor based on Au NPs-modified cortisol—imprinted for signal amplification and real-time cortisol detection [99]. The study confirmed the morphological aspect of Au NPs as spherical nanoparticles with an average size around 59 nm using transmission electron microscopy (TEM) and dynamic light scattering (DLS) analysis. The authors tested the kinetic analyses of the proposed material and revealed optimal detection in real-time monitoring, leading to a low detection limit of 0.0087 ppb for cortisol. Also, the Au NPs-based sensor proved to have high performance in various solutions such as artificial plasma and urine, leading to the conclusion that the proof-of-concept device can be used in multiple matrices without losing its performance. The high stability of Au NPs was also revealed in the research made by Mani et al. [100]. The work describes the development of an electrochemical biosensor for cortisol detection by attaching a MIP cortisol on the surface of Au NPs-incorporated carboxylated graphene oxide. The work compared various strategies for biosensor design, leading to the conclusion that the addition of Au NPs and graphene oxide will enhance the electronic mobility and the detection level by increasing the final biosensor’ surface reactivity. The sensor proved to have a very low detection limit of 0.61 × 10⁻¹⁴ M for cortisol, with high selectivity and sensitivity in the human blood serum samples tested. Jing et al. also proved the enhanced effect of Au NPs in a recent paper by fabricating a microneedle-based electrochemical biosensor with dendritic Au NPs for cortisol detection from skin [101]. As a transdermal device, the biosensor proved high electrochemical and mechanical performances. The addition of Au NPs has increased the surface reactivity and the conductivity of the electrode, from 73.87 ± 1.33 Ω to 6.76 ± 0.22 Ω, confirming that Au NPs’ addition can enhance the sensibility and the detection limit for cortisol measurements. The electrochemical tests have established a wide detection range of 1–1000 nM, featuring low detection limits of 0.17 nM and 0.22 nM. In the study, the optimization of the biosensor was clinically confirmed on volunteers. The analysis revealed that for all patients, the highest cortisol level is identified in the morning [101], thus confirming the hypotheses described in the literature.

3.1.2. Silver Nanoparticles (Ag NPs)-Based Biosensors

Ag NPs are metallic nanoparticles with enhanced optical and electrical properties. Their assets derived from the physical–chemical properties have encouraged their integration into electric devices for automotive industry or electrical engineering. In addition, due to their versatility, Ag NPs also found applications in biomedical applications. In biological systems, they are used as antibacterial agents against microorganisms or in biosensors, where detection can be improved once they are integrated into a material [102]. The stability of Ag NPs is considered to be lower than other noble metals in biosensing; therefore, for cortisol detection, less reports have been described in the literature thus far.

Shama et al. proposed the development of a sensitive device based on Ag NPs-doped MIPs for cortisol detection in aqueous and biological samples [103]. The optical-morphological characterization for AgNPs exhibited a spherical shape with an average size of 40 nm. For proper optimization, the authors tested five different cortisol concentrations (0.395, 0.791, 1.32, 2.64, and 3.96 nM) in phosphate-buffered saline (PBS) using differential pulse voltammetry (DPV) techniques, thus obtaining a calibration curve for further cortisol quantification, which was proven to offer accurate and reproductible results, as can be observed in Figure 5. The study revealed that the addition of Ag NPs enhances the biosensor’s performance in terms of sensitivity and selectivity, mostly due to the high surface-to-volume area of the nanoparticles. Cortisol detection proved high yield in human plasma samples, leading to a very low detection limit of 0.214 nM, which could hardly be achieved through classical detection methods [103].

Figure 5. Cortisol detection and kinetic parameters: (A) Ag NPs-based biosensor response for various cortisol concentrations and (B) the calibration curve based on these cortisol concentrations. Reprinted from Shama et al.’s open-source article [103].

Another potential study, which is proving the enhanced effects of cortisol-based biosensor due to Ag⁺ addition, was performed by Moghadam et al. [104]. The research from this work was mainly based on the development of a Target-Induced Structural Switching (TISS) mode Aptamer nanobiosensor for cortisol detection from saliva. In this study, Ag plays a pivotal role in enhancing the optical performance of the aptasensor through the formation of DNA-templated Ag nanocluster supraparticles. The cytosine-rich regions at both the 3′ and 5′ ends of the single-stranded DNA act as nucleation sites for Ag⁺ reduction, leading to the formation of ordered Ag nanocluster arrays. These Ag nanocluster supraparticles exhibit fluorescence enhancement via Aggregation-Induced Emission and Self-Assembly Induced Emission mechanisms, which arise from the close spatial arrangement of clusters in the presence of cortisol. The incorporation of Ag nanoclusters not only amplifies photoluminescence efficiency compared to homogeneously dispersed nanoclusters, but also improves the sensor’s signal stability and sensitivity, making silver a crucial component in the fluorescence-based detection mechanism. The developed TISS aptamer-based nanobiosensor demonstrated a low detection limit of 1 nmol/L and a linear response range of 1–900 nmol/L, effectively covering the physiological concentration range of salivary cortisol in healthy individuals. Its successful performance in both artificial and real saliva samples confirms the sensor’s accuracy, repeatability, and potential as a rapid and reliable photometric tool for small-molecule quantification such as cortisol [104].

3.2. Plasmonic Composite Sensors

Plasmonic composite sensors based on metallic nanoparticles have emerged as powerful analytical tools due to their exceptional optical, electrical, and catalytic properties derived from LSPR. To overcome these challenges, mono- or multi-metallic nanoparticle-based composite sensors have been developed, offering synergistic effects that arise from the combination of different electronic configurations, crystal structures, and surface chemistries [105,106]. The incorporation of multiple materials within a single nanostructure enables enhanced charge transfer, broader plasmonic resonance spectra, and improved catalytic and photothermal responses. In composite sensor systems, plasmonic nanoparticles are often integrated with supporting materials, such as graphene, metal–organic frameworks, or polymers, to enhance surface area, biocompatibility, mechanical stability, and signal stability [107]. Such hybrid plasmonic composites exhibit superior performance in biosensing applications, providing ultralow detection limits, rapid signal transduction, and improved selectivity for biomarkers and small molecules. The combination of plasmonic enhancement and multifunctional material integration positions composite plasmonic sensors as a next-generation platform for precise, reliable, and real-time detection of cortisol from biological fluids. Some examples are described in Table 4.

Table 4. Nanocomposite-based biosensors for cortisol detection.

Composite Materials	Detection Mechanism	Biofluid	Limit of Detection (LOD)	Key Features	Refs.
MIP-based biosensor with Ag core–Au NPs	Surface-enhanced Raman scattering (SERS)	Saliva	10 pM	Electrodeposition and self-polymerization; enhanced electromagnetic field due to metallic nanoparticles; high selectivity against interferents; stable in artificial saliva; wide linear response	[108]
Papain-stabilized bimetallic Au–Cu nanoclusters	Fluorescence-based sensing	Urine and plasma	1.89 nM	High analytical sensitivity; negligible interference from other biomolecules; validated for stability and reproducibility; selective toward cortisone	[109]
Nanocomposite: MWCNT + ordered mesoporous carbon CMK-3 + Ag NPs + aptamer–antibody sandwich on glassy carbon electrode	Electrochemical signal amplification/sandwich detection	Human saliva	0.09 pg/mL	Non-invasive; aptamer attached via amide bonds; signal amplification from metallic nanoparticles; aptamer–antibody sandwich design; demonstrates stress-level differentiation between participants	[110]
MIP-Au NPs	Electrochemical	Spiked saliva	0.09 pg/mL	High affinity; antibody-free; large surface area	[64]
AuNP-incorporated carboxylated graphene oxide-based MIP	Electrochemical	Blood serum	0.61 × 10−14 M	Enhanced electron transfer; electrocatalysis	[100]
MXene-Au NPs	Electrochemical	Sweat	~pM	Real-time monitoring; wearable, non-invasive	[111]
Au-decorated SiC/β-cyclodextrin nanocomposite	Electrochemical	Sweat	~nM	High selectivity; wearable compatible	[112]

Most of the studies rely on cortisol determination from saliva or plasma; however, the most non-invasive approach for nanocomposites-based sensors is based on the analysis of sweat [110]. For instance, monitoring physiological biomarkers is very important for evaluating athletic performance and guiding training and recovery. Cortisol serves also as an important indicator of both acute exercise-induced stress and long-term training adaptations. Because changes in cortisol levels provide insights into an athlete’s physical load, stress state, and overall well-being, real-time tracking is highly valuable for personalized training and prevention of overtraining [110]. Hu et al. revealed the fabrication of such devices for cortisol quantification in human sweat [112]. The work describes the fabrication of a new material based on Au-decorated SiC/β-CD nanocomposite with a very narrow limit of detection of just 2.8 ng/mL. The results revealed that the device proposed show strong mechanical durability, exhibiting only a 4% decrease in capacitance after 20 bending cycles and maintaining stable performance for 30 days, demonstrating excellent long-term stability [112].

3.3. Microfluidic Cortisol Biosensors

Microfluidics plays a pivotal role in the design and development of modern biosensors by enabling precise manipulation of extremely small sample volumes within microscale channels, which enhances reaction efficiency and reduces reagent consumption [113]. The ability to integrate multiple functions, such as sample preparation, mixing, separation, and detection, into a single compact device makes microfluidic systems particularly attractive for point-of-care diagnostics [114,115]. Microfluidic platforms also facilitate sensitive and specific analysis of complex biological fluids with minimal preprocessing, supporting applications ranging from clinical diagnostics to environmental monitoring [116,117]. Advances in integrated microfluidic architectures have further expanded the potential for multiplexed biomarker detection and wearable sensing technologies [118]. As a result, microfluidics continue to transform biosensor technology by enabling miniaturized, automated, and highly efficient analytical systems with broad biomedical relevance [119,120]. For instance, Xiong et al. have developed a pump-free microfluidic chip based on a solid-state Au-nanostructured SERS substrate that is able to detect trace amounts of sweat cortisol [121]. The detection relies on an immunoassay, more specifically, on the competitive interaction between cortisol and SERS tags with cortisol antigens anchored on the Au-nanostructured substrate within the microfluidic system. The loading of the sample inside the microfluidic chip takes up to 60 s, while the detection process is realized in less than 10 min. Thus, the authors report a fast and high-throughput detection method, which reaches a detection limit of 10 pg/mL, showing great promise for fast and sensitive cortisol detection and monitoring technological transfer. Similar results were obtained by Sanghavi et al., who have designed a quantitative aptamer-based microfluidic assay for cortisol that requires no probe immobilization, target labeling, or washing steps [122]. The assay showed linear detection over five orders of magnitude (30 pg/mL–10 μg/mL) with rapid, cortisol-specific binding, accelerated at low concentrations. Minimal interference was observed from estradiol, testosterone, and progesterone, except at 0.06 μg/mL cortisol with 1 μg/mL progesterone, where an 18% signal increase occurred. Results correlated well with ELISA and radiolabeling, while requiring only 2.5 min and <1 μL sample volume [122].

Lateral Flow Assays (LFAs) are a class of diagnostic devices that leverage capillary action to transport liquid samples along a porous strip (in general, nitrocellulose strip) and can be considered simple, passive microfluidic systems. For example, Kim et al. have developed a sandwich-type chemiluminescent (CL) immunoassay using a luminescent label enzyme-conjugated Au NPs integrated into a LFA platform, as observed in Figure 6, which was tested for the quantitative detection of serum cortisol [123]. The proposed plasmonic LFA platform was demonstrated to achieve a limit of detection of 0.343 µg/dL, thus showcasing its high sensitivity towards serum cortisol detection.

Figure 6. Illustration of the developed sandwich-type cortisol CL-based LFA design. Reprinted from Kim et al.’s open-source article [123].

3.4. Wearable and Lab-on-Skin Sensors

Recent advances in wearable and non-invasive cortisol sensing technologies have accelerated the translation of cortisol from a classical laboratory biomarker into a real-time stress monitoring tool. Balasamy et al. provide a comprehensive review of emerging wearable cortisol sensors, highlighting electrochemical, immunosensor, aptamer-based, and FET-based platforms, coupled with flexible substrates, microfluidics, and wireless data transmission [124]. These innovations are lowering costs and improving usability, paving the way for broader adoption in personalized health and preventive monitoring. Wearable cortisol sensors are increasingly being integrated into smart devices. For example, a recent study demonstrated a smartwatch combining a sweat cortisol sensor and a heart-rate variability (HRV) sensor, enabling continuous, non-invasive stress monitoring under real-world conditions [125]. Meanwhile, implantable or skin-interfaced bioelectronics for cortisol monitoring are being explored, expanding potential use cases in both clinical and consumer domains [126]. Nan et al. assembled a simple LSPR-based wearable sensor by integrating an aptamer-based immunosensor onto a PDMS flexible substrate [127]. After the exposure to cortisol, the LSPR response of the Au NPs deposited on the PDMS was evaluated, showing that the cortisol-specific aptamer used as recognition element was able to detect cortisol with a limit of detection as low as 0.1 nM. A hybrid wearable physicochemical sensor suite was developed by Hossain et al., that simultaneously monitors sweat cortisol and electromyogram (EMG) signals, along with skin temperature and pH, using an integrated non-invasive sensor patch [73]. The sensor patch integrates a three-electrode electrochemical cortisol sensor with pH and temperature sensors for calibration, and EMG electrodes placed opposite the sensing area. The cortisol electrode is functionalized with a PANI–Au NPs-doped MWCNT nanocomposite, followed by anti-cortisol antibody immobilization. The device, optimized for durability and signal accuracy on curved skin surfaces, includes on-board processing and Internet of Things (IoT) connectivity for real-time data acquisition and wireless transmission. During cycling tests, the patch captured rising sweat cortisol levels and reduced EMG power spectral density, demonstrating its potential for real-time stress assessment and early detection of physiological changes.

Hydrogels are one of the latest approaches in biosensor platforms for cortisol detection. Hydrogels are soft, water-rich materials composed of three-dimensional polymer networks capable of holding substantial amounts of liquid without losing their shape, thanks to either chemical or physical cross-links. Their combination of flexibility, high hydration, and adjustable mechanical behavior has made them promising candidates for use in wearable electronic technologies, where comfort, biocompatibility, and adaptability are crucial [128]. For hydrogels to function effectively in wearable systems, they must satisfy a range of performance requirements to tolerate repeated bending, stretching, and external stress, strong adhesion to diverse surfaces such as human skin, stability under long-term exposure to environmental factors, and reliable conductivity for transferring electrical signals, as Figure 7 is suggesting [129].

Figure 7. (A) The mechanical invisibility for skin-conformal integration; (B) optical transparency for information identification and wearable components blending; (C) the combination of mechanical and optical imperceptibility, which will enhance the performance of wearable devices for extended reality applications. Reprinted from Won et al.’s open-source article [129].

Equally important are the electronic characteristics, which involve maintaining high ionic or electronic conductivity, low impedance, and long-term signal stability, thus ensuring precise signal detection for sensing applications [128,130]. To improve their electrical performance while preserving softness, many modern hydrogel systems incorporate conductive additives, including metal plasmonic nanoparticles. Ideally, hydrogels designed for wearable platforms achieve a careful balance between structural strength and electronic efficiency [131], allowing them to serve as compliant, skin-like components for cortisol monitoring, therapeutic actuation, and interactive electronic interfaces. For instance, Qin et al. have designed a battery-free, wearable hydrogel sensor for non-invasive cortisol monitoring [67]. Two photonic hydrogels, based on molecular imprinting and antibody–antigen competitive binding, were integrated into flexible devices, offering complementary stability and sensitivity for naked-eye and smartphone-assisted detection at nM levels. The sandwich-structured device enables real-time cortisol measurement in under 15 min, with robust stability suitable for storage and transport. Similarly, a reagent-free aptasensor with a hybrid hydrogel network was developed for detecting salivary cortisol by Karuppaiah et al. [132]. The developed electrochemical hydrogel sensor consists of hydrogel nanocomposite layer of gold nanocubes (Au NCs) and cortisol-specific aptamers–chitosan (CS). The hydroxyethyl cellulose (HEC) hydrogel provides structural support, while Au NCs enhance conductivity and enable aptamer immobilization. The hydrogel minimized non-specific binding, enabling accurate measurement of physiological cortisol levels (0.1–50 ng/mL) and proving a detection limit of 0.1 ng/mL, results well correlated with ELISA [132].

Another innovative approach leading to versatile, continuous, real-time monitoring devices is the Lab-on-Skin technology, representing an emerging class of wearable bioanalytical systems designed to interface intimately with the epidermis. Through the integration of soft, stretchable materials and advanced microfluidic routing, these platforms enable microfluidic sampling and in situ processing of sweat or ISF with minimal skin irritation [133,134]. Innovations in epidermal electronics further allow incorporation of signal amplification and wireless communication within devices [135,136]. Such designs make possible high-temporal-resolution tracking of biomarkers, supporting applications ranging from stress monitoring to personalized healthcare [137]. Hu et al. developed a wearable microfluidic patch, which enabled the non-invasive detection of cortisol from sweat by SERS [138]. The design of the wearable patch incorporates the following: (i) a silk epidermal adhesive layer, (ii) PDMS microfluidics to collect sweat, (iii) two SERS substrates composed of a monolayer of polystyrene opal structure coated with chromium (Cr) and Au thin films and a polyvinylpyrrolidone-coated Ag NPs layer, and (iv) medical tape for encapsulation purposes. The developed wearable sensor was demonstrated to detect the cortisol levels falling within the range of 0.1 to 1000 nmol/L in simulated conditions, while for on-skin testing, cortisol concentrations comparable with relevant sweat cortisol values were obtained. The results have been well correlated with ELISA measurements, proving the efficiency of the SERS patch for cortisol monitoring. Thus, Lab-on-Skin systems enabled by microfluidics and soft materials are poised to transform digital health by offering unobtrusive, real-time biochemical sensing directly on the skin.

4. Recent Developments in Cortisol Continuous Monitoring Devices

Traditional measurements often fail to capture the dynamic fluctuations of cortisol in response to various physiological and psychological stimuli, limiting their utility in understanding stress patterns and related health conditions. Thus, the growing demand for continuous, real-time cortisol monitoring has driven significant advances in biosensor technologies and the integration of wearable devices [139]. In the following, as Figure 8 is suggesting, the latest breakthroughs in continuous cortisol monitoring devices are described, encompassing diverse sensing modalities, integration strategies, and ongoing research efforts.

Figure 8. Recent technology in cortisol monitoring biosensors.

Building upon the minimally invasive nature of microneedles, recent research has focused on enhancing their capabilities for continuous cortisol monitoring [140]. Significant advancements have been made in hydrogel-based microneedle patches that allow the sustained extraction of ISF and integration with various sensing mechanisms. For example, Aroche et al. present a hydrogel microneedle array for continuous monitoring [141]. The hydrogel matrix facilitated continuous ISF extraction, while the aptamer-based sensor provided selective and reversible binding of cortisol, enabling real-time fluorescence intensity measurements via a portable reader. The reversibility of aptamer binding offers the potential for prolonged monitoring without sensor saturation. Furthermore, researchers are exploring electrochemical detection integrated with microneedles. The results reported by Mugo et al. demonstrated a microneedle array coated with a conductive polymer and functionalized with a high-affinity anti-cortisol antibody [142]. Upon insertion, the antibody captured cortisol in the ISF, leading to a change in the electrochemical impedance measured by the underlying electrodes. The device exhibited good sensitivity and selectivity for cortisol and was successfully tested in vivo for continuous monitoring during stress induction. The small size and low power requirements of electrochemical sensors make them particularly attractive for wearable integration.

Nonetheless, continuous monitoring of cortisol presents unique challenges due to the lower analyte concentrations and the complex environment. Recent efforts are focusing on developing miniaturized and robust biosensors that can be integrated into oral appliances or wearable devices. The study realized by Wang et al. reports on successfully developing a smartwatch for non-invasive stress biomarker data acquisition that provides real-time feedback to the wearer [143]. The sensor can continuously monitor cortisol levels, with data transmitted wirelessly to a smartphone.

Another innovative approach involves optical detection in salivary cortisol monitoring. Ahmed et al. presents the blue tetrazolium method as an effective and rapid approach for measuring cortisol in saliva, showing strong agreement with the gold-standard ELISA, as indicated by a high coefficient of determination (R² = 0.997) [34]. These findings highlight the method’s potential for point-of-care optical sensing, integrated with a mobile application aimed at cortisol monitoring and stress assessment in adults. Similarly, SPR sensors, miniaturized and integrated into wearable platforms, offer label-free and real-time detection of cortisol binding to immobilized antibodies or aptamers [127].

Acoustic wave sensors, such as surface acoustic wave (SAW) devices and QCM sensors, are also considered an effective alternative. Functionalizing the surface of these sensors with cortisol-specific bioreceptors allows for the detection of mass changes upon cortisol binding, which can be correlated to the concentration [144]. Recent research focuses on enhancing the sensitivity and stability of these sensors for continuous monitoring in biological fluids.

As artificial intelligence (AI) and machine learning (ML) are developing, showing great advantages in various domains, their integration with continuous cortisol monitoring devices is becoming increasingly attractive. These algorithms can analyze the continuous data streams, identify patterns and trends, and correlate cortisol fluctuations with other physiological and contextual information (e.g., activity levels, sleep patterns, self-reported stress, etc.). The successful coupling of wearable sensing devices and AI and/or ML can lead to personalized stress profiles and early detection of abnormal cortisol patterns offering the breakthrough for accurate and early diagnosis of stress-related medical conditions [145].

5. Challenges and Future Directions

Both non-plasmonic and plasmonic biosensors have become increasingly attractive for cortisol detection due to their ability to provide real-time biomolecular analysis with high sensitivity while maintaining a compact and versatile design. Recent developments such as immunosensors, plasmonic composite biosensors, flexible LSPR patches, polymer-optical-fiber SPR immunosensors and integrated plasmonic microfluidic systems as well as wearable devices, demonstrate the high efficiency and accuracy of plasmonic transducers to provide signal enhancements that allow the detection and monitoring of cortisol in biomedical applications.

A major advantage of plasmonic biosensors lies in their real-time detection [146], whereby refractive index shifts caused by cortisol binding can be monitored continuously, which is particularly relevant for cortisol, whose physiological fluctuations require dynamic readout. Additionally, the sensor fabrication allows miniaturized designs without affecting their sensitivity. Another important advantage is their compatibility with flexible substrates and microfluidic components. Furthermore, plasmonic platforms exhibit multiplexing capabilities, allowing the simultaneous measurement of cortisol alongside secondary biomarkers or environmental parameters, which improves signal interpretation and robustness of the developed biosensors.

Nonetheless, despite these strengths and the constant advancement of the technologies, cortisol biosensors still face important challenges. First, biofouling and matrix complexity remain a challenge to be overcome as non-specific adsorption onto surfaces of other components of the biofluids can appear. One way to overcome this limitation is the use of cortisol-specific recognition elements, which leads to a subsequent challenge: the stability of molecular recognition, since antibodies may lose activity over time or under variable environmental conditions [147]. In case of aptamers and MIPs, despite offering improved stability, cross-reactivity can also appear [148]. Encapsulation strategies, self-calibration mechanisms, and robust anti-fouling coatings are essential [110]. Moreover, for wearable and Lab-on-Skin monitoring devices, the quantitative accuracy and calibration can be challenging due to biofluid composition, secretion rate, and skin properties, all of them being particular for each individual according to their dietary and daily activities. In particular for wearable and Lab-on-Skin sensing devices, the mechanical durability still poses as a limiting factor, as nanoparticle reorganization, surface oxidation, or bending-induced optical drift during prolonged wear might occur [124].

While recent advances in the development of biosensors, from solution-based to microfluidic and wearable sensing devices, highlight the strong potential and low detection limits of plasmonic platforms for continuous cortisol monitoring, several key limitations must be addressed before these technologies can achieve widespread clinical and wearable use. Continued progress in anti-fouling strategies, recognition element engineering, device miniaturization, and clinical validation will be essential for translating cortisol biosensors into reliable tools for stress monitoring and health assessment.

Other challenges refer to robustness and long-term performance. Ensuring the long-term stability, accuracy, and reliability of sensors under continuous exposure to biological matrices and varying environmental conditions is paramount [149]. Designing comfortable, unobtrusive, and user-friendly wearable devices that can be seamlessly integrated into daily life is crucial for user compliance. This includes considerations for size, weight, flexibility, and esthetics [80]. Moreover, given the sensitive nature of continuous physiological data, robust data security and privacy measures must be implemented to protect user information. Even more, further crucial steps consist of demonstrating the clinical utility of continuous cortisol monitoring in various applications and navigating the regulatory approval processes for medical devices.

Future research will undoubtedly focus on overcoming these challenges through multidisciplinary collaborations spanning materials science, bioengineering, microelectronics, data science, and clinical medicine. Future directions include the development of multi-analyte photonic systems, responsive smart coatings, and integrated photonic chips for full wearable deployment. The combination of nanophotonics, biomimetic interfaces, and real-time analytics may soon position these devices as key tools for precision medicine. The development of next-generation continuous cortisol monitoring devices promises to provide unprecedented insights into the dynamics of stress and their impact on health, paving the way for personalized interventions and improved well-being.

6. Conclusions

Cortisol, the main stress hormone from the human body, has become an important analyte in biosensors world. In this review, we summarized the most valuable developments realized throughout the recent progress of cortisol biosensors, highlighting their strengths and limitations compared to classical strategies. Non-plasmonic and plasmonic biosensors have shown great results lately in cortisol investigation, increasing the quality of life due to their main advantages. Real-time monitoring, miniaturized devices, and the ability to offer support while still recording the biological modifications in cortisol levels can encourage the use of these wearable biosensors in children and adults. Considerable amounts of effort are invested in the development of highly efficient, sensitive, and accurate non-invasive cortisol biosensors. Nonetheless, few challenges still remain to be overcome despite continuous progress. Advancing cortisol monitoring still requires solutions for reduced biofouling and improved mechanical stability as well as standardized calibration and validation protocols across biofluids prior to their translation to clinical settings. Overcoming these limitations could take the developed biosensors one step closer to large-scale production and widespread user implementation. Nonetheless, current advances are laying the groundwork for innovative technologies, including highly sought-after wearable and Lab-on-Skin biosensors, which enable user-friendly continuous cortisol monitoring and, implicitly, contribute to the progress of early diagnosis and personalized medicine.