key: cord-0962658-hsthkbah
authors: Giacomello, Ginevra; Scholten, Andreas; Parr, Maria Kristina
title: Current Methods for Stress Marker Detection in Saliva
date: 2020-09-06
journal: J Pharm Biomed Anal
DOI: 10.1016/j.jpba.2020.113604
sha: ce0faabb353a0a0159e6d012fc186fbf68c7c3da
doc_id: 962658
cord_uid: hsthkbah

Stress and stress-related diseases are leading to drastic consequences in private and professional life. Therefore, the need for stress prevention strategies is of personal and economic interest. Especially during the recent period related to covid-19 outbreak and lock-down, an ongoing discussion of increasing stress etiology is reported. Biomarker analysis may help to assist diagnosis and classification of stress-related diseases and therefore support therapeutical decisions. Due to its non-invasive sampling, the analysis of saliva has become highly attractive compared to the detection methods in other specimen. This review article summarizes the status of research, innovative approaches, and trends. Scientific literature published since 2011 was excerpted with concentration on the detection of up to seven promising marker substances. Most often reported cortisol represents the currently best evaluated stress marker, while norepinephrine (noradrenaline) or its metabolite 3-methoxy-4-hydroxyphenylglycol is also a quite commonly considered stress marker. Other complementary stress marker candidates are testosterone, dehydroepiandrosterone (DHEA) and its sulfonated analogue DHEA-S, alpha-amylase, secretory immunoglobulin A, and chromogranin A. Several working groups are researching in the field of stress marker detection to develop reliable, fast, and affordable methods. Analytical methods reported mainly focused on immunological and electrochemical as well as chromatographic methods hyphenated to mass spectrometric detection to yield the required detection limits.

According to the endocrinologist Hans Selye, stress is the non-specific response of the body to any demand.

While generally connected with adaptations of the body by the activation of the sympathetic nervous system, psychological stress in psychology is often separated as positive and negative stress. Positive stress (eustress) helps to improve performance and motivation (either mental or physical), whereas distress is considered as excessive amounts of stress, which may lead to health risks. In public language, stress is most often connected with distress. Thus, in psychology, stress is mainly related to emotional strain and pressure.

Prolonged emotional pressure of distressing periods, or chronic stress, may lead to a broad spectrum of physical and psychological diseases [1] [2] [3] [4] [5] . Whether an experience is perceived or not as distressing, strongly depends on individual aspects (e.g., environment, socioeconomic stability, passed personal history, or mental health) and is physiologically hard to estimate. Several factors like persisting liability, ongoing anxiety, desperateness, or a lack of prospects are tightening this experience. Hence, there is an increased risk for people who are working in fields with ongoing emotional pressure, like physicians, caregivers, nurses, social workers or teachers, etc. [6] [7] [8] [9] [10] . Especially during nowadays coronavirus pandemic, an ongoing discussion of increasing stress etiology in health caregivers, but also in the general public is reported [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] . In public perception, increasing stress load leads to symptoms summarized as burnout. Chronic stress and burnout also influence how people will physiologically react, cope, and adapt to a future acute stress event. Thus, the diagnosis of chronic stress is becoming more and more important in health protection [22] . Methods often rely on psychological scales achieved by questionnaires, that strongly depend on the individual patient [23] .

Additionally, the physiological response to mental and physical stress involves various compounds that may, therefore, be used as biomarkers in biological signal-based methods.

The most commonly considered biomarker for stress determination in humans is cortisol. This wellexamined hormone is part of the stress response, and its concentration varies in acute and chronic stressful situations. In addition, there are a lot of other biomarkers with a strong correlation to the perceived stress load. Their detection methods are as multifarious as their amount. Many research groups are working in the field of stress marker detection. Therefore, the number of reported detection methods is increasing too. Most of the manuscripts published in the scientific literature are dealing with the detection of a few stress markers in different matrices. Due to its non-invasive and stress-free sampling, the analysis of saliva has become highly attractive compared to the detection methods in blood, liquor, or hair. Therefore, the aim of this review is to give an overview of state-of-the-art analysis, to summarize the different detection approaches, and to provide a lookout for prospective methods.

J o u r n a l P r e -p r o o f 5 

Stress, caused by exceptional circumstances, is leading to a dysregulation of biological homeostasis.

Thereby, these dysfunctions are concerning all hormonal, neurohumoral, and physiological axes. The measurement of marker substances levels of these axes can be used for estimating the stress response level of an individual. The interactions of these substances and the physiology of the axes will be briefly introduced in the following.

The immediate stress reaction, or the fight-or-flight response, involves the rapid activation of the autonomic nervous system (adrenal medulla, ANS), which leads to an increased concentration of epinephrine and norepinephrine (chemical structure in Figure 1 ) in blood. Only limited research focused on the detection of the parent compounds themselves as they display limited chemical stability and are rapidly metabolized to yield 3-methoxylated metabolites. Therefore, the level of their metabolite 3-methoxy-4-hydroxyphenylglycol (MHPG, Figure 1 ) is also associated with a stress response. Its reference value from 12.85 ng/ml was determined by Reuster et al. [24] , while Okumura et al. reported a method able to quantify norepinephrine at about 0.17 pmol/ml (i.e., 30 pg/ml) and epinephrine at about 0.1 pmol/ml (20 pg/ml) in saliva [25] .

Furthermore, they mentioned a stress-related increase of the salivary neurotransmitter dopamine in some cases, most likely related to strong fear or anxiety. As increasing catecholamine levels are also associated with an induction of an elevated metabolic rate, metabolomics may also be considered an interesting future approach. However, at present, no publications related to metabolomics investigations on human saliva in psychological stress were retrieved within our database search.

As hypothesized, the hypothalamus-pituitary-adrenal (HPA) axis responds deferred. Limbic and hypothalamic structures are coordinating the emotional, cognitive, neuroendocrine, and autonomic inputs.

On its activation, corticotropin-releasing-hormone (CRH) is excreted. This leads to a release of adrenocorticotropic hormone (ACTH) in the pituitary, which triggers the release of glucocorticoids in the adrenal gland. While ACTH is reported to increase as a stress-related response by Ramos et al., others found ACTH levels varying with age, sex and type of stress [26] [27] [28] . Salivary ACTH levels are only rarely used in stress marker research.

Triggered by the HPA signaling the most important stress-related glucocorticoid is cortisol. The normal ranges of cortisol in blood and saliva for healthy individuals are around 30-160 ng/mL (blood) and 1-1.6 ng/mL (saliva), respectively [29] . It is crucial to notice, though, that cortisol concentration is influenced by several factors such as sex, age, population, circumstances, and circadian rhythm [30] . In the case of an acute stress situation, the cortisol level is increasing and reaches the concentration peak around 20-30 minutes after the stressor event. If the stressor is still ongoing over a long-term period, cortisol levels are decreasing. If stress becomes chronic, the response of the HPA axis is attenuated. Therefore, cortisol ( Figure 1) is also considered as a marker of long-term response in humans.

Reflexively, a high cortisol concentration influences the testosterone (Figure 1 ) level in blood, which is reported to decrease from its normal range (0.35 nmol/L [31] ). The pre-stages of testosterone are dehydroepiandrosterone (DHEA) and its sulfated form DHEA-S ( Figure 1 ). Concomitant with an altered level of cortisol, the level of DHEA and DHEA-S change as well [32] . This means that its average concentration in saliva (0.2 -2.7 ng/ml) also depends strongly on age, sex, and daytime [33] . While the acute stage of stress, the level of DHEA (-S) increases, while its level decreases in a long-term situation.

In parallel with the HPA axis, further paths are activated: gonadal axis, adipose axis, and immune system. Thereby, several other biological marker-substances are associated with acute and chronic stress. Thus, in addition to epinephrine, norepinephrine, cortisol, and testosterone, there are further proteins and enzymes correlated with stress.

The increase in catecholamines leads to a greater need for energy. Alpha-amylase (AA, Figure 2 ) is an enzyme, which also occurs in saliva (salivary AA (sAA)) with a normal range between 90 -250 U/mL [29] .

Its task is to split carbohydrates into digestible oligosaccharides, which are very important during a fight-orflight response situation. The increase of AA activity is, therefore, also associated with acute stress.

Another stress-related protein is the secretory immunoglobulin A (sIgA), which is secreted by B cells from the immune system. It plays a significant role in mucosal defense against pathogens and occurs in healthy individuals and normal conditions in a range of ca. 60.3 ± 3.46 µg/mL in saliva [34] . B cells have a high density of β2-adrenergic receptors. In case of a stress reaction, the increased level of epinephrine and cortisol works along this pathway.

Another stress response marker is chromogranin A (CgA, Figure 3 ), which is part of the pathway of the autonomic nervous system. It is the precursor to several functional peptides like vasostatin-1, vasostatin-2, pancreastatin, catestatin, or parastatin. These peptides negatively modulate the neuroendocrine function of releasing cells, like cells of the adrenal medulla, which releases adrenaline to the blood. In this way, the chromogranin A level is associated with stress.

In some papers, serotonin is considered as a stress biomarker, but mainly in blood or urine, where the concentration is higher [29] . Moreover, Egri et al. found out that in children the salivary serotonin levels do not correlate well with plasma levels and Leung et al. highlighted that they do not correlate with central serotonin turnover either, making serotonin a non-suitable salivary biomarker for evaluation of serotonergic system functioning [35, 36] .

Based upon two publications from Danhof-Pont et al. [37] and Yamaguchi et al. [38] , we have searched the databases of Pubmed and ISI Web of Science for articles published since 2011, which are dealing with stress marker detection in saliva. According to the described stress markers (x), we have worked with the Boolean J o u r n a l P r e -p r o o f search terms (x) AND detection AND saliva. The received articles were then excerpted given their relation to stress research, developed or used method, the achieved detection limits, and feasibility.

Well established and innovative analytical techniques are both used for biomarker determination. Initially, radioimmunoassays (RIAs) were very common, but other immunoassays (IA) were developed and optimized later to avoid the use of radioactive reagents. Currently, the enzyme-linked immunosorbent assay (ELISA) and immune-based biosensors are of more frequent use [39] [40] [41] [42] .

In general, IA are susceptible to cross-reactivity dependent on the selectivity of antibodies used therein.

Molecularly imprinted polymers (MIPs) are recently studied as alternatives and used in assays to overcome the economic downside of antibody-based assays [43] . Similarly, bio-or chemiluminescent assays may serve as alternatives [44] [45] [46] [47] [48] [49] .

Major advantages of IA include their effective usability in the clinical routine with several commercially available kits. However, classical IAs represent single analyte assays only. Furthermore, cross-reactivities with structurally related compounds may confound the analytical results, especially if differences in concentration between the analyte and cross-reacting agent are very high.

In addition to classical immunoassays, immunological methods and electrochemical analysis are often combined in stress marker analyses [50] [51] [52] [53] [54] [55] . Bio-and immunosensors are widespread thanks to their versatility, easy use, sensitivity, and short analysis time [45, 46, [50] [51] [52] [53] [54] [55] [56] . Furthermore, due to their possibility of miniaturization, they offer an excellent opportunity for developing point of care (POC) devices.

The majority of steroidal stress biomarkers in saliva are still analyzed with IAs because they are technically easy to use, rapid, and relatively cheap. There are, though, some downside not always negligible. IAs are based on the chemical binding reaction between antibodies and a specific analyte, but sometimes the assay selectivity can be mined by the antibodies cross-reactivity with structurally similar compounds. As reported in literature, this is the case of cortisol and cortisone in saliva [57] [58] [59] . Similarly, in plasma sample analyses cross reactivities for DHEA-S and testosterone were reported [60, 61] . Often overlooked high differences in the concentration levels (even in order of magnitude) may require cross-reactivities far below 1 % which are often stated as "no cross reactivity" in IA kits. Furthermore, cross-reactivity is not the only variable that can influence the reliability of an IA. Since they might be designed differently, also the resulting interaction antibodies-analytes might change. Therefore, it is often not possible to compare different IAs results [57] . Therefore, some conversion tables have been developed to obtain comparable factor scores between results obtained with the most common IAs [57] . Buttler et al. [62] [9, 63] . To extend the scope of analytes, we have tested a GC-MS method for the simultaneous determination of stress related small molecule biomarkers. First experiments had shown that one could combine cortisol, testosterone, DHEA, and MHPG. These substances were well detectable as trimethylsilyl (TMS) derivatives by GC-MS. Currently, method validation is in progress.

However, the analysis of sulfoconjugates is rather complicated by GC-MS. Thus, HPLC based methods were also tested for combined analysis of the above-mentioned small molecule stress biomarkers. In bioanalysis, HPLC is often coupled with (tandem) mass spectrometry (LC-MS(/MS)), which leads to higher selectivity and sensitivity and concomitantly a much lower limit of detection (LOD). It offers a fast, reliable, highly selective, and robust determination of a wide variety of biomarkers for use in multi-analyte designs [64] [65] [66] .

The product ion spectra of cortisol, testosterone, and norepinephrine, DHEA-S, and MHPG are displayed in More specific details of analytical methods are given in the individual sections of specific biomarkers.

Saliva represents a highly attractive specimen in bioanalysis due to its non-invasive sampling. It has the undeniable advantage of being easy to get, even from newborns and elderly, non-invasive, and non-stressful, and is therefore frequently used in stress-related research. The sampling procedure, though, requires some precautions to obtain an accurate and reliable results. For several analytes concentrations in saliva are highly correlated to serum concentrations [71, 72] . As shown in this review, there are different collection methods resulting in data that are not always interchangeable: comparing analytical results with different procedures of sampling might lead to incorrect deductions. Sampling is most often performed using easy collection devices. Standard protocols are developed and reported for many fields of analysis and adaptation to field conditions was achieved already many years ago [73] .

However, several potentially confounding factors need to be considered to obtain significant and reliable data. Lipson and Ellison reported no influence of the selection of the type of sampling tubes (glass or polystyrene) on salivary steroid concentrations [73] .

For all the biomarkers considered, some factors can influence the analysis, it is, therefore, recommended not to eat, smoke, drink, or brush the teeth for at least one hour before the collection, and to avoid blood contamination while collecting (i.e., by scratching the gum) [74] [75] [76] [77] .

Whetzel et al. and Shirtcliff et al. compared cotton swab (Salivette) and passive droll collection methods [33, 78] . No significant differences in the two approaches were detected in case of cortisol and DHEA-S. Similar findings for cortisol were obtained by Gallagher et al., and Hodgson and Granger in the comparison of passive droll and Salivette swab collection [79, 80] . The comparison of passive droll collection with two commercial kits, OraSure and Oracol reported by Nurkka et al. also resulted in non-significant influences for sIgA [81] . In contrast, Shirtcliff et al. reported significantly different concentrations for testosterone and sIgA when comparing Salivette and passive droll collection. The authors hypothesized that this effect might be caused by a non-specific binding or cross-linking reaction of the antibody used in the immunoassay, or that an interfering substance is filtered out by the cotton [78] . Lipson and Ellison found a high correlation of salivary steroids (progesterone, androstenedione, testosterone and cortisol) in non-stimulated collection and stimulated collection utilizing gum chewing (with or without sugar additives), candy, or lemon juice [73] .

However, significantly different concentrations compared to unstimulated collection were determined for cortisol (lemon juice, lower concentrations), testosterone and androstenedione (sugarless gum, lower concentrations), as well as for progesterone (stimulation by sugared gum, higher concentrations).

Additionally, interindividual variances in the slope of the correlation equation were found for progesterone concentrations comparing unstimulated and sugarless gum stimulated collection (higher or lower concentrations depending on the individuals) [73] .

For evaluation of sAA activity Beltzer et al., and Rohleder et al. highlighted that the salivary flow rate has a non-critical impact [82, 83] . However, it is observed that the activity of sAA strongly depends on the sampling area of the mouth [82, 84] . Thus, differences in the sampling devices impact the results even if they do not result in different flows of saliva. Robles et al. hypothesized that parotid and submandibular glands produce a higher level of sAA in comparison with sublingual glands [84] . On the analysis of MHPG and CgA, there are only a few studies. To our knowledge, only Higashi et al. studied the MHPG concentration with (stimulated) and without (unstimulated) gum-mastication, resulting in a significant decrement in MHPG amount caused by salivary stimulation [85] .

Additionally, the correct time for sampling is crucial for later result interpretation. Analyses of biomarkers have different time windows in case of chronic or acute stress. For example, a specific stress event causes an increase of cortisol after 15-20 minutes from the exposure; then, the level begins to decrease even if the stressor persists for some time, therefore, taking a sample of saliva after this window will not be useful [86] .

Especially in monitoring of chronic stress a longitudinal evaluation is often performed and needs careful experimental design. Most of the biomarkers taken into consideration in this review are secreted with a trend, or circadian cycle, regulated mainly by day/night alternation and their amounts in saliva change within 24 hours [33, [86] [87] [88] [89] [90] [91] [92] [93] [94] [95] [96] . The concentration of cortisol, for instance, starts rising in the morning and reaches its peak within 40 minutes after awakening. Then, the level begins to decrease until the minimum, or nadir, between J o u r n a l P r e -p r o o f habits or behavior. Intense training, lack of sleep, or fasting disrupt only momentarily the circadian cycle that will quickly return to homeostasis. In the case of chronic stress, instead, the level of cortisol remains high during the 24 hours resulting in an attenuated circadian cycle and a blunted response to future stressors [86] [87] [88] [89] [90] 96] .

It is also critical to consider the medical history of the subject examined because some diseases can cause an alteration of the basal level of the biomarker. The Cushing's and the Chronic fatigue syndromes, i.e., create a blunted cortisol wake/sleep variation similar to the one due to chronic stress [86, 87, 96] .

Another aspect to consider is the storage of the salivary samples prior to the analytical determination.

The steroids are reported as quite stable biomarkers in saliva. Clements and Parker demonstrated that saliva samples for the analysis of cortisol resist at least five days with temperature variations between 15°C and 38°C [97] . Moreover, cortisol concentrations are stable at 5°C for up to 3 months or at -20°C and -80°C for at least one year. The only variation registered was after one month of storage at room temperature [77] .

Durdiaková et al. studied the stability of testosterone at room temperature, at 4°C, at -20°C, and at -80°C. In each of these cases, no significant variations were found at least one month [98] . To our knowledge, there are no stability studies for DHEA-S and MHPG in saliva.

In contrast, sample storage for the analysis of the protein biomarkers required more attention. Bellagambi et al. studied the stability of sAA for short and long term storage. The enzymatic activity remained stable for at least 8 hours at room temperature (analysis at ten different time points) and after four weeks at -80°C. the activity showed a decrease of about 15% at 4°C, always after four weeks [99] . In IgA analyses, snap-freezing with glycerol in liquid nitrogen resulted in significantly higher concentrations in comparison to those detected after storage for 4 to 8 hours at 4°C with and without the addition of protease enzyme inhibitors before freezing at -70°C [81] . In contrast, Ng candidates for biomarkers in case of burnout [37] . These biomarkers concerned the HPA axis, the steroid hormones, the autonomic nervous system (ANS), the immune system, different metabolic processes, and the antioxidant defense system. In the following sections, we itemize the most relevant biomarkers and their analytical methods for determination.

Cortisol (chemical structure in Figure 1 ) is the most commonly utilized stress biomarker. It is reported to show a strong correlation between serum and salivary concentrations [102] . This correlation allows the detection of active cortisol in saliva, with a non-invasive, stress-free sampling.

Since 1979, when Kabra et al. reported the first HPLC method using UV detection for the detection of cortisol in serum/plasma [103] , the variety of approaches has drastically increased. realized a selective sensor platform for testosterone [43] .

There are some additional methods published, diff erent in sample pre-treatment and detection. To resume the results, table 2 gives an overview. 

The detection of alpha-amylase is well investigated (structure in Figure 2 ). There are already commercial easy-to-use, quick tests, and POC devices available. The most common detection method for this marker is by measuring its activity. Several different assay methods and unit definitions make it almost impossible to compare reported activities. As one example, one NU (Novo Unit) is defined as the amount of the enzyme that breaks down 0.00526 g of starch per hour. Alternatively, its activity is given as unit that liberates 1.0 mg of maltose from starch in 3 minutes at pH 6.9 at 20 °C [126] .

Ohtomo et al. developed a flow injection spectrophotometric analysis for analyzing alpha-amylase activity in saliva [127] . The principle of this method was the degradation of a starch-iodine complex by alpha-amylase activity. The reached LOD was 60 NU/mL. Another spectrophotometric method is described by Fuentes et J o u r n a l P r e -p r o o f al. [128] for the determination of AA in porcine saliva. The LOD was 11.65 IU/L. Unfortunately, they did not share any information on their calculations, so that both LODs are even harder to compare.

IFMA) for the quantitation of sAA in sheep [129] and horses [130] . They also compared the results obtained for the equine saliva with a commercial enzymatic assay. In both cases, the assay is based on an indirect, non-competitive sandwich method. As capture reagent, they used the anti-alpha-amylase polyclonal antibody and as a detector, the Eu 3+ -chelate labeled anti-alpha-amylase polyclonal antibody.

Alternatively, electrochemistry is often used also to determine sAA. Indeed, electrochemical techniques allow the realization of simple, not expensive and portable assays suitable for point-of-care testing. an application quantifies sAA concentration through a calibration curve [132] .

Another device for in situ testing was developed by Della Ventura et al. [133] . They functionalized the gold surface of the electrodes of a quartz-crystal microbalance (QCM) with antibodies by the photochemical immobilization technique (PIT). This simple method immobilizes and activates the antibodies with UV radiation.

Lastly, Tsyrulneva et al. developed a simple, colorimetric method with a paper membrane strip that also can be used without a qualified analyst or expensive instrument [131] . They exploit the colored byproduct formed during the cleavage of the alpha-bond of 2-chloro-4-nitrophenyl-α-D-maltotrioside by sAA, to obtain a semi-quantitative analysis. J o u r n a l P r e -p r o o f nanocomposite with immobilized Anti-sIgA monoclonal antibody (mAbs) deposited on modified carbon nanofiber electrodes (CNF-SPE) [140] . With this electrochemical immunosensor, they obtained an extremely low LOD (500 fg/mL).

Another electrochemical immunosensor was developed by Lim et al. [141] . They covalently immobilized sIgA to magnetic beads and then incubated with biotin-conjugated secondary antibody and with streptavidinhydrogen peroxidase. Finally, they immobilized the obtained magnetic beads on a single-walled carbon nanotube working electrode for amperometric measurements. This immunosensor reaches a LOD 5 pg/mL and has a linear range of 5 pg/mL-10 ng/mL.

According to your database search, the most recent paper about sIgA in salivary matrix is published by [142] . Here, they used polyclonal antibodies against CgA (Catestatin) and combined them with the commercially available kit (DELFIA, Perkin Elmer). The relevant amino acid sequence used for detection in this assay is displayed in Figure 3 . This kit utilizes the lanthanide Europium, which is liberated after antibody-reaction. The concentration of liberated Eu is proportional to the concentration of CgA. The reached LOD was 4.27 ng/mL. This method was also used by Casal et al. in their study about the effects of an environmental enrichment and herbal supplement on physiological stress indicators in pigs [143] .

Two more studies on the stress biomarkers in porcine saliva were carried out. The first by Huang et al.

analyzed CgA with western blot [144] . The other, by Tecles et al., reports the development and validation of a time-resolved immunofluorometric assay (TR-IFMA). They obtained a 4.27 ng/mL LOD and a CV <10%

for intra and inter-assay [145] .

CgA was tested as a salivary stress biomarker also in humans. Rai et al. tested a commercially available ELISA. However, the sensitivity of the kit was not in the focus of the work and thus not reported [146] .

Abekura et al. used a commercial ELISA kit as well. They studied the correlation between sleep bruxism and psychological stress by combining objective (CgA) and subjective (ten-division visual analog scale) parameters [147] . In 2019 Lihala et al. correlated CgA concentrations and personally perceived stress levels before and after non-surgical periodontal therapy [148] .

It is essential to consider when to collect a sample of saliva to quantitate CgA. Its concentration is not constant during the day. Salivary CgA has higher levels during the night, reaches the highest peak just after awakening, then decreases rapidly, and remains low through the day [94] .

With ongoing discussions on psychological (dis-)stress, reliable and objective diagnostic tools are highly desired to categorize the stress level. An event of acute stress has a disrupting effect on the physiological homeostasis and it causes, for example, a momentary increase in sAA or cortisol levels. If the distressing event is prolonged or repeated over-time, the ability of hormonal, neurohumoral, and physiological axes J o u r n a l P r e -p r o o f might result blunted and not adequate. Therefore, chronic stress not only leads to a feeling of exhaustion, mental distance from society and work, loss of productivity, but also to secondary psychological and physical diseases.

Thus, research on stress-related biomarkers is of persevering interest. In view of the fact that stress and its consequences are very multifaceted, harmless, comfortable, and economical detection devices are of increasing interest.

Classical clinical assays like IA offer easy analytical approaches for single analytes that may be designed for POC testing. Provided a sufficient sensitivity, economic efficiency is becoming more critical. Therefore, test methods, which are lower priced and easy to use, are of major interest, especially in research fields like stress prophylaxis and personalized medicine. To prevent risks related to stressful jobs or other conditions, the necessity of fast, sufficiently sensitive, and portable devices is of primary importance. This goal may also be met utilizing suitable for electrochemical developments. Dependent on the selectivity of the antibody utilized cross-reactivity may result in the overestimation of analytes when compared with MS data, particularly at lower concentrations. When comparing the data obtained with MS and IAs, there is often no linear correlation between the results [57, 149, 150] . To overcome this issue and produce more reliable kits, IAs manufacturers are nowadays often using MS based techniques to validate and calibrate their kits more accurately [59] .

To supplement clinical testing and confirmation of the POC device results exact and highly sensitive detection methods are developed meanwhile. In this field, mainly sophisticated methods based on chromatography coupled to mass spectrometric detection appear to be the methods of choice. While many detection methods reported in literature are designed for detecting only one or two markers simultaneously, LC-MS/MS or GC-MS(/MS) based methods offer the possibility for multi-analyte methods. After successful optimization, they may cover several stress-related biomarkers in one single analytical method. This allows for an efficient simultaneous measurement and helps to reliably assess of possible stress-related risks in a multifactorial design. As one example, Gaudl et al. report the simultaneous determination of 17-OHP, aldosterone, AED, cortisol, cortisone, DHEAS, estradiol, progesterone, and testosterone in saliva by one LC-MS/MS method [66] .

The capability of LC-MS/MS based methods to cover multiple analytes is most persuasively demonstrated in metabolomics studies. Only recently, metabolomics investigations are also reported for stress-related conditions. For further reading on this topic, we would like to refer to the review paper of Mellon et al. [151] . Complementary, Nathalie Michels recently reviewed multi-omics approaches in the context of psychological stress [152] . Using gene expression analysis Le-Niculescu et al. discovered and validated NUB1, APOL3, MAD1L1, and NKTR as predictive biomarkers in addition to FKBP5, DDX6, B2M, LAIR1, and RTN4 [153] . Additionally, Dean et al. recently reported the use of multi-omics for the identification of biomarkers for the diagnosis of post-traumatic stress disorders [154] . They finally reported a set of 28 markers, that yielded 81% accuracy. Using three out of these markers, namely gamma glutamyl tyrosine, J o u r n a l P r e -p r o o f insulin, and the methylation marker cg01208318, they still achieved 60% accuracy in the validation group.

Unfortunately, the read-out for most of the multi-analyte methods is still challenging and multivariate statistics are often required. However, the discovery of further suitable biomarkers may be achieved by these designs. Their suitability needs further validation. Furthermore, their recovery in saliva needs to be evaluated in future studies as well.

Further innovative approaches may off er attractive solutions for developing cost and sensitivity efficient devices. Martin et al. recently showed that the use of aptamers is leading to promising results in the detection of small molecules, especially in terms of selectivity [155] . They did not report the LOD of the methods but opened a new perspective for combining the detection of small and large molecule marker substances [156] .

This approach shows another potential future trend.

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Giacomello) was received. 

Salivary cortisol and αamylase: subclinical indicators of stress as cardiometabolic risk

Molecular Signatures of Biomarkers with a Special Reference to Stress and Related Diseases/Disorders: Diagnostic, Prognostic and Therapeutic Values-Current Progress and Futuristic Vision

The human stress response

Chronic psychological stress and the regulation of pro-inflammatory cytokines: a glucocorticoid-resistance model

Circadian misalignment and health

The change in the amount of immunoglobulins as a response to stress experienced by soldiers on a peacekeeping mission

Stress-induced cortisol facilitates threat-related decision making among police officers

Negative affect, perceived health, and endocrine and immunological levels in caregivers of offspring with schizophrenia

Self-perceived work-related stress and its relation to salivary IgA, cortisol and 3-methoxy-4-hydroxyphenyl glycol levels among neonatal intensive care nurses

Chemiluminescence-based biosensor for monitoring astronauts' health status during space missions: Results from the International Space Station

Acute stress, behavioural symptoms and mood states among school-age children with attention-deficit/hyperactive disorder during the COVID-19 outbreak

Examining the impact of COVID-19 on stress and coping strategies in individuals with disabilities and chronic conditions

COVID-19 Diary From a Psychiatry Department in Italy

The outbreak of COVID-19 coronavirus and its impact on global mental health

COVID-19 and mental health: A review of the existing literature

Posttraumatic stress symptoms and attitude toward crisis mental health services among clinically stable patients with COVID-19 in China

Traumatic stress in the age of COVID-19: A call to close critical gaps and adapt to new realities

Immediate Psychological Responses and Associated Factors during the Initial Stage of the 2019 Coronavirus Disease (COVID-19) Epidemic among the General Population in China

Indoor) isolation, stress and physical inactivity: vicious circles accelerated by Covid-19?

COVID-19 Pandemic and Impending Global Mental Health Implications

COVID-19 From a Psychiatry Perspective: Meeting the Challenges

Role of common mental and physical disorders in partial disability around the world

User-level psychological stress detection from social media using deep neural network

Influence of reboxetine on salivary MHPG concentration and cognitive symptoms among patients with alcohol-related Korsakoff's syndrome

Study of salivary catecholamines using fully automated column-switching high-performance liquid chromatography

Age-dependent effects of chronic stress on ACTH and corticosterone responses to an acute novel stress

Chronic stress alters adrenal clock function in a sexually dimorphic manner

Control of Stress-Induced ACTH Secretion by Vasopressin and CRH: Additional Evidence

Stress Biomarkers in Biological Fluids and Their Point-of-Use Detection

Spectral effect: each population must have its own normal midnight salivary cortisol reference values determined

Reference ranges for serum and salivary testosterone in young men of Mediterranean region

Attenuated DHEA and DHEA-S response to acute psychosocial stress in individuals with depressive disorders

Measuring DHEA-S in saliva: time of day differences and positive correlations between two different types of collection methods

Serum and salivary IgA levels in normal subjects: comparison between tonsillectomized and non-tonsillectomized subjects

Correlation Between Salivary

Salivary serotonin does not correlate with central serotonin turnover in adult phenylketonuria (PKU) patients, Molecular genetics and metabolism reports

Biomarkers in burnout: a systematic review

Salivary Sensors for Quantification of Stress Response Biomarker

Validation and application of a highly specific and sensitive ELISA for the estimation of cortisone in saliva, urine and in vitro cell-culture media by using a novel antibody

The detection of cortisol in human sweat: implications for measurement of cortisol in hair

Development of a biomimetic enzyme-linked immunosorbent assay based on a molecularly imprinted polymer for the detection of cortisol in human saliva

Development of a competitive immunoassay for the determination of cortisol in human saliva

Micro-patterned molecularly imprinted polymer structures on functionalized diamond-coated substrates for testosterone detection

A bioluminescent probe for salivary cortisol

Label-free, chemiresistor immunosensor for stress biomarker cortisol in saliva

Direct immune-detection of cortisol by chemiresistor graphene oxide sensor

Measurement of salivary cortisol by a chemiluminescent organic-based immunosensor

Salivary testosterone measurements in growing pigs: validation of an automated chemiluminescent immunoassay and its possible use as an acute stress marker

Use of Saliva in Alternative to Serum Sampling to Monitor Biomarkers Modifications in Professional Soccer Players

Electrochemical Immunosensing of Saliva Cortisol

Polyaniline protected gold nanoparticles based mediator and label free electrochemical cortisol biosensor

Electrochemical immunoassay for the detection of stress biomarkers

Electrochemical cortisol immunosensors based on sonochemically synthesized zinc oxide 1D nanorods and 2D nanoflakes

Disposable electrochemical immunosensor for cortisol determination in human saliva

Immunosensor with fluid control mechanism for salivary cortisol analysis

Plasmonic sensors for the competitive detection of testosterone

Comparison of salivary cortisol as measured by different immunoassays and tandem mass spectrometry

Measurement of salivary cortisol in 2012 -laboratory techniques and clinical indications

The impact of neuromuscular electrical stimulation on recovery after intensive, muscle damaging, maximal speed training in professional team sports players

Dehydroepiandrostenedione sulphate interferes in many direct immunoassays for testosterone

Position statement: Utility, limitations, and pitfalls in measuring testosterone: an Endocrine Society position statement

Measurement of dehydroepiandrosterone sulphate (DHEAS): a comparison of Isotope-Dilution Liquid Chromatography Tandem Mass Spectrometry (ID-LC-MS/MS) and seven currently available immunoassays

Saliva level of free 3-methoxy-4-hydroxyphenylglycol (MHPG) as a biological index of anxiety disorders

Automated analysis of salivary stress-related steroid hormones by online in-tube solid-phase microextraction coupled with liquid chromatography-tandem mass spectrometry

Noninvasive determination of human cortisol and dehydroepiandrosterone sulfate using liquid chromatography-tandem mass spectrometry

Liquid chromatography quadrupole linear ion trap mass spectrometry for quantitative steroid hormone analysis in plasma, urine, saliva and hair

Bioanalytical separation and preconcentration using ionic liquids

Simultaneous determination of testosterone, cortisol, and dehydroepiandrosterone in saliva by stable isotope dilution on-line in-tube solid-phase microextraction coupled with liquid chromatography-tandem mass spectrometry

Multi-matrix assay of cortisol, cortisone and corticosterone using a combined MEPS-HPLC procedure

Ionic liquid dispersive liquid-liquid microextraction combined with LC-UV-Vis for the fast and simultaneous determination of cortisone and cortisol in human saliva samples

Correlations between serum and salivary hormonal concentrations in response to resistance exercise

The correlation between serum and salivary melatonin concentrations and urinary 6-hydroxymelatonin sulphate excretion rates: two non-invasive techniques for monitoring human circadian rhythmicity

Development of protocols for the application of salivary steroid analysis to field conditions

Salivary mental stress proteins

Estimating intra-and inter-assay variability in salivary cortisol

Effect of snack eating on sensitive salivary stress markers cortisol and chromogranin A

Effects of lifestyle factors on concentrations of salivary cortisol in healthy individuals

Use of salivary biomarkers in biobehavioral research: cotton-based sample collection methods can interfere with salivary immunoassay results

Collecting saliva and measuring salivary cortisol and alpha-amylase in frail community residing older adults via family caregivers

Assessing cortisol and dehydroepiandrosterone (DHEA) in saliva: effects of collection method

Effects of sample collection and storage methods on antipneumococcal immunoglobulin A in saliva

Salivary flow and alpha-amylase: collection technique, duration, and oral fluid type

The psychosocial stress-induced increase in salivary alpha-amylase is independent of saliva flow rate

Saliva sampling method affects performance of a salivary alpha-amylase biosensor

Influence of saliva flow rate stimulated by gum-chewing on salivary concentrations of catecholamine metabolites

The Functional and Clinical Significance of the 24-Hour Rhythm of Circulating Glucocorticoids

Circadian rhythm of adrenal glucocorticoid: its regulation and clinical implications

Altered diurnal pattern of steroid hormones in relation to various behaviors, external factors and pathologies: A review

Adverse effects of two nights of sleep restriction on the hypothalamic-pituitary-adrenal axis in healthy men

Greater lifetime stress exposure predicts blunted cortisol but heightened DHEA responses to acute stress

Circadian variation of salivary immunoglobin A, alpha-amylase activity and mood in response to repeated double-poling sprints in hypoxia

Time course of saliva and serum melatonin levels after ingestion of melatonin

Salivary testosterone measurements: reliability across hours, days, and weeks

Circadian rhythm of human salivary chromogranin A

Modeling of circadian testosterone in healthy men and hypogonadal men

Endocrine Rhythms, the Sleep-Wake Cycle, and Biological Clocks

The relationship between salivary cortisol concentrations in frozen versus mailed samples

The effects of saliva collection, handling and storage on salivary testosterone measurement

Determination of salivary α-amylase and cortisol in psoriatic subjects undergoing the Trier Social Stress Test

Effects of storage time on stability of salivary immunoglobulin A and lysozyme

Saliva chromogranin A in growing pigs: a study of circadian patterns during daytime and stability under different storage conditions

The Relationship Between Serum and Salivary Cortisol Levels in Response to Different Intensities of Exercise

Improved liquid-chromatographic method for determination os serum cortisol

Methods in endogenous steroid profiling -A comparison of gas chromatography mass spectrometry (GC-MS) with supercritical fluid chromatography tandem mass spectrometry (SFC-MS/MS)

Classification criteria for distinguishing cortisol responders from nonresponders to psychosocial stress: evaluation of salivary cortisol pulse detection in panel designs

Recent advances in cortisol sensing technologies for point-of-care application

Development of Indirect Competitive Immuno-Assay Method Using SPR Detection for Rapid and Highly Sensitive Measurement of Salivary Cortisol Levels

A contemporary approach for design and characterization of fiber-optic-cortisol sensor tailoring LMR and ZnO/PPY molecularly imprinted film

On The Application of SiO2/SiC Grating on Ag for High-Performance Fiber Optic Plasmonic Sensing of Cortisol Concentration

Validation of an automated chemiluminescent immunoassay for salivary cortisol measurements in pigs

A sensitive chemiluminescence based immunoassay for the detection of cortisol and cortisone as stress biomarkers

Development of competitive lateral flow immunoassay coupled with silver enhancement for simple and sensitive salivary cortisol detection, Excli j

A simple and compact smartphone accessory for quantitative chemiluminescence-based lateral flow immunoassay for salivary cortisol detection

Development and evaluation of a liquid chromatography tandem mass spectrometry method for simultaneous determination of salivary melatonin, cortisol and testosterone

High-throughput determination of cortisol, cortisone, and melatonin in oral fluid by on-line turbulent flow liquid chromatography interfaced with liquid chromatography/tandem mass spectrometry

Simultaneous analysis of cortisol and cortisone in saliva using XLC-MS/MS for fully automated online solid phase extraction

Ultra high performance liquid chromatography tandem mass spectrometry determination and profiling of prohibited steroids in human biological matrices. A review

Simple Measurement of Testosterone in Male Saliva Samples Using Dispersive Liquid-Liquid Microextraction Followed by Liquid Chromatography-Tandem Mass Spectrometry Detection

Determination of the steroid profile in alternative matrices by liquid chromatography tandem mass spectrometry

Development of a Derivatization Method for Investigating Testosterone and Dehydroepiandrosterone Using Tandem Mass Spectrometry in Saliva Samples from Young Professional Soccer Players Pre-and Post-Training

Electrochemical Investigation of Testosterone Using a AuNPs Modified Electrode

Analysis of steroid hormones in human saliva by matrix-assisted laser desorption/ionization mass spectrometry

Multimode sensors as new tools for molecular recognition of testosterone, dihydrotestosterone and estradiol in children's saliva

Diurnal and stressreactive dehydroepiandrosterone levels and telomere length in youth

Free epinephrine, norepinephrine and dopamine in saliva and plasma of healthy adults, in: European journal of clinical chemistry and clinical biochemistry: journal of the Forum of European Clinical Chemistry Societies

Comparison of α-amylase activities from different assay methods

Flow injection spectrophotometric analysis of human salivary alpha-amylase activity using an enzyme degradation of starch-iodine complexes in flow channel and its application to human stress testing

Validation of an automated method for salivary alpha-amylase measurements in pigs (Sus scrofa domesticus) and its application as a stress biomarker

Validation of an assay for quantification of alpha-amylase in saliva of sheep

Measurements of salivary alpha-amylase in horse: Comparison of 2 different assays

Amperometric detection of salivary alpha-amylase on screen-printed carbon electrodes as a simple and inexpensive alternative for point-ofcare testing

Smartphone-based point-of-care testing of salivary alphaamylase for personal psychological measurement

Flexible immunosensor for the detection of salivary alpha-amylase in body fluids

An Aggregation-induced Emission Probe Based on Host-Guest Inclusion Composed of the Tetraphenylethylene Motif and gamma-Cyclodextrin for the Detection of alpha-Amylase

Colorimetric Detection of Salivary alpha-Amylase Using Maltose as a Noncompetitive Inhibitor for Polysaccharide Cleavage

The evaluation and validation of Phadebas((R)) paper as a presumptive screening tool for saliva on forensic exhibits

Developmental validation of a point-ofcare, salivary alpha-amylase biosensor

Capillary isoelectric focusing with whole column imaging detection (iCIEF): A new approach to the characterization and quantification of salivary alpha-amylase

Single-step turn-on homogeneous fluorescent immunosensor for rapid, sensitive, and selective detection of proteins

Combining a gold nanoparticle-polyethylene glycol nanocomposite and carbon nanofiber electrodes to develop a highly sensitive salivary secretory immunoglobulin A immunosensor

Single Wall Carbon Nanotube and Magnetic Bead Based Electrochemical Immunosensor for Sensitive Detection of Salivary Secretory Immuno-globulin A

Measurement of chromogranin A in porcine saliva: validation of a time-resolved immunofluorometric assay and evaluation of its application as a marker of acute stress

Effect of environmental enrichment and herbal compound supplementation on physiological stress indicators (chromogranin A, cortisol and tumour necrosis factor-alpha) in growing pigs

Short communication: Salivary haptoglobin and chromogranin A as non-invasive markers during restraint stress in pigs

Cholinesterase in porcine saliva: Analytical characterization and behavior after experimental stress

Salivary stress markers and psychological stress in simulated microgravity: 21 days in 6 degrees head-down tilt

Association between sleep bruxism and stress sensitivity in an experimental psychological stress task

Effect of non-surgical periodontal therapy on stress and salivary Chromogranin-A levels: A clinico-biochemical study

Salivary steroid assays -research or routine?

Measuring cortisol in serum, urine and saliva -are our assays good enough?

Metabolomics, and Inflammation in Posttraumatic Stress Disorder

Biological underpinnings from psychosocial stress towards appetite and obesity during youth: research implications towards metagenomics, epigenomics and metabolomics

Towards precision medicine for stress disorders: diagnostic biomarkers and targeted drugs

Multi-omic biomarker identification and validation for diagnosing warzone

Tunable stringency aptamer selection and gold nanoparticle assay for detection of cortisol

Therapeutic RNA aptamers in clinical trials

Structure of human salivary α-amylase at

List of Figures Figure 1: Chemical structures of the small molecule stress-related biomarkers cortisol, norepinephrine, 3-methoxy-4-hydroxyphenylglycol (MHPG), testosterone, dehydroepiandrosterone (DHEA)

Product ion spectra (LC-QTOF-MS) of cortisol (upper), testosterone (middle), norepinephrine (lower), all positive ionization (ESI+), collision energy CE = 20V

The State of Berlin, Germany, is acknowledged for granting the Elsa-Neumann PhD scholarship of Ginevra Giacomello. The authors thank Bernhard Wuest, Agilent Technologies Inc. for his assistance in mass spectrometry.J o u r n a l P r e -p r o o f