==== Front Int J Oral Sci Int J Oral Sci International Journal of Oral Science 1674-2818 2049-3169 Nature Publishing Group UK London 37386003 231 10.1038/s41368-023-00231-6 Review Article Salivary biomarkers: novel noninvasive tools to diagnose chronic inflammation http://orcid.org/0000-0003-4343-7213 Dongiovanni Paola 1 Meroni Marica 1 http://orcid.org/0000-0002-5049-7321 Casati Sara sara.casati@unimi.it 2 Goldoni Riccardo 34 http://orcid.org/0000-0003-0000-3466 Thomaz Douglas Vieira 5 Kehr Nermin Seda 26 http://orcid.org/0000-0002-9284-5953 Galimberti Daniela 27 http://orcid.org/0000-0001-7144-0984 Del Fabbro Massimo 28 Tartaglia Gianluca M. 28 1 grid.414818.0 0000 0004 1757 8749 Medicine and Metabolic Diseases, Fondazione IRCCS Cà Granda Ospedale Maggiore Policlinico, Milan, Italy 2 grid.4708.b 0000 0004 1757 2822 Department of Biomedical, Surgical and Dental Sciences, University of Milan, Milan, Italy 3 grid.4643.5 0000 0004 1937 0327 Department of Electronics, Information and Bioengineering (DEIB), Politecnico di Milano, Milan, Italy 4 grid.5326.2 0000 0001 1940 4177 Istituto di Elettronica e di Ingegneria dell’Informazione e delle Telecomunicazioni, CNR, Pisa, Italy 5 grid.411195.9 0000 0001 2192 5801 Laboratory of Medicinal Pharmaceutical Chemistry, Faculty of Pharmacy, Federal University of Goiás, Goiânia, GO Brazil 6 grid.419609.3 0000 0000 9261 240X Department of Chemistry, İzmir Institute of Technology, Gülbahçe Kampüsü, Urla İzmir, Turkey 7 grid.414818.0 0000 0004 1757 8749 Neurology-Neurodegenerative Diseases, Fondazione IRCCS Cà Granda, Ospedale Maggiore Policlinico, Milan, Italy 8 grid.414818.0 0000 0004 1757 8749 UOC Maxillo-Facial Surgery and Dentistry Fondazione IRCCS Cà Granda, Ospedale Maggiore Policlinico, Milan, Italy 29 6 2023 29 6 2023 2023 15 2724 11 2022 23 5 2023 5 6 2023 © The Author(s) 2023 https://creativecommons.org/licenses/by/4.0/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Several chronic disorders including type 2 diabetes (T2D), obesity, heart disease and cancer are preceded by a state of chronic low-grade inflammation. Biomarkers for the early assessment of chronic disorders encompass acute phase proteins (APP), cytokines and chemokines, pro-inflammatory enzymes, lipids and oxidative stress mediators. These substances enter saliva through the blood flow and, in some cases, there is a close relation between their salivary and serum concentration. Saliva can be easily collected and stored with non-invasive and cost-saving procedures, and it is emerging the concept to use it for the detection of inflammatory biomarkers. To this purpose, the present review aims to discuss the advantages and challenges of using standard and cutting-edge techniques to discover salivary biomarkers which may be used in diagnosis/therapy of several chronic diseases with inflammatory consequences with the pursuit to possibly replace conventional paths with detectable soluble mediators in saliva. Specifically, the review describes the procedures used for saliva collection, the standard approaches for the measurement of salivary biomarkers and the novel methodological strategies such as biosensors to improve the quality of care for chronically affected patients. Subject terms Diagnostic markers Biological techniques Predictive markers issue-copyright-statement© West China School of Stomatology Sichuan University 2023 ==== Body pmcIntroduction Inflammation is a process that the host enacts to defend itself against toxins, bacteria, viruses, tissue damage, metabolic stress by recruiting immune and non-immune cells. Thus, in a physiological context inflammation is protective and once the insult is eradicated several mechanisms intervene and lead to a process named “resolution of inflammation”.1 A prolonged inflammatory status may become chronic and pathological when the regulatory events which promote the resolution are lost. Both acute and chronic inflammation share several phases: increased blood flow to the site of inflammation, higher capillary permeability to allow even larger molecules to cross the endothelium, recruitment of leukocytes from the capillaries to the surrounding tissue and finally the release of mediators by the latter, including cytokines, chemokines, markers of oxidative stress (as superoxide), enzymes (i.e. metalloprotease) and lipid mediators as prostaglandins and leukotrienes.2 The majority of chronic diseases are preceded by a chronic low-grade inflammation. Hence, conceivable biomarkers for the early assessment of disorders encompass acute phase proteins (APP), cytokines and chemokines, pro-inflammatory enzymes, and oxidative stress mediators. Among the most common chronic diseases, heart disease, cancer, obesity, and type 2 diabetes (T2D) constitute the leading causes of disability and death in the United States.3,4 Given the widespread manifestations, the number of hospitalization and the mortality rate, these conditions represent a huge socio-economic burden. Indeed, the affected subjects require ongoing medical attention from the first symptoms to the management of therapeutic options. Therefore, the identification of forefront diagnostic tools is essential to establish preventive approaches and targeted pharmacological interventions with the purpose of minimizing the risks, distress and ultimately the costs of chronic diseases. In this context, the possibility to exploit salivary biomarkers as tool to detect systemic disorders may constitute an intriguing opportunity to implement the strategies to diagnose and follow-up patients affected by chronic disorders, limiting the risks related to more invasive procedures. Saliva is an exocrine secretion of the salivary glands mainly composed of water (99%), but it also contains electrolytes, proteins, lipids, and enzymes. Contaminants such as bacteria, epithelial cells, gingival crevicular fluid and food debris are also detectable in saliva.5 Proteins and other substances enter saliva through the blood flow and in some case, there is a close relation between their salivary and serum concentration. Indeed, salivary glands are highly vascularized and there is an exchange of compounds which pass by passive diffusion or active transport from blood to saliva and viceversa. To this regard, recent studies have reported the diagnostic utility of saliva to detect cardiovascular diseases (CVD), systemic and local inflammation, endocrinological and metabolic disorders.6 Salivary lipids are mostly secreted by the major salivary glands, but some lipids like cholesterol and some fatty acids (FAs) diffuse directly from serum into saliva.7 Therefore, the use of saliva as an alternative diagnostic tool is advisable since its collection is non-invasive and possibly stress-free. In addition, no differences were found between men and women in salivary composition although the literature is still contradictory.6,8–11 Furthermore, a large amount of saliva can be easily collected and stored with non-invasive and cost-saving procedures.12,13 Therefore, the opportunity to detect and quantify biomarkers in salivary samples becomes highly attractive for research, clinical, and unobtrusive proactive healthcare applications, with the purpose to early diagnose chronic diseases and to allow a continuous disease monitoring. To this purpose, the present review aims to discuss the advantages and challenges of using cutting-edge techniques to discover salivary biomarkers which may be used in diagnosis/therapy of several chronic diseases with inflammatory consequences, with the pursuit to possibly replace conventional paths with detectable soluble mediators in saliva. Modulators of the inflammatory status The inflammatory milieu is firstly affected by the triggering event which in turn may determine the recruitment of different numbers and types of immune cells and, accordingly, a diversified release of proinflammatory mediators. However, several factors can cause dynamic alterations in serum biomarkers of inflammation including age, presence of obesity, gender, diet, smoking, genetics, drug consumption and gut microbiota. Age Aging is featured by an increase in serum levels of cytokines and acute phase proteins (APPs) due to a low-grade inflammatory status which may be induced by increased visceral adiposity, declined function of sex hormones, genetics, neurodegenerative disorders as Alzheimer’s disease (AD) or cardiovascular complications. Interleukin-6 (IL-6) levels are increased in elderly subjects to the point that it has been dubbed a cytokine for gerontologists.14 Similarly, it has been reported that circulating tumor necrosis factor alpha (TNFα), IL-1 as well as C-reactive protein (CRP), α1-acid glycoprotein and fibrinogen increase with age.15 In healthy, elderly populations, high circulating levels of TNFα and IL-6 predict mortality, regardless of comorbidities, whereas in cohorts of frail, older individuals, these cytokines may represent risk factors for atherosclerosis, T2D, AD, thromboembolic complications, and are associated with sarcopenia and muscle loss.16,17 Higher levels of IL-18, a linked IL-1 pro-inflammatory cytokine, IL-2, IL-17 and IL-12 have been found in elderly, associated with CVD, stroke, type 1 diabetes, AD and osteoarthritis.18–21 Finally, IL-17 and IL-8 (CXCL8) promote inflammation, recruitment and activation of neutrophils and increased levels have been found in autoimmune diseases such as systemic lupus erythematosus, inflammatory bowel disease and psoriasis.22 Moreover, lipopolysaccharide (LPS)-stimulation of leukocytes from elderly individuals induces IL-8 release and the latter has been also suggested as a possible longevity factor in centenarians.23 Adiposity Obesity is associated with low-grade chronic inflammation. Visceral fat produces several proinflammatory cytokines (TNFα, IL-1, IL-6) and chemokines (MCP-1) and it has been described that also ectopic sites of adipose tissue such as those localized in liver, heart or muscle may contribute to the release of pro-inflammatory mediators also independently of body fatness.24 Elevated CRP levels were found in overweight (body mass index [BMI], 25–29.9 kg·m−2) and obese (BMI, ≥30 kg·m−2) individuals compared to normal weight ones and regardless of age.25 Moreover, it has been suggested that TNFα pathway could be involved in the regulation of circulating leptin, whose levels are elevated in obese subjects.26 Conversely, it has been described a tendency for reduced adiponectin levels in obese subjects possibly mediated by TNFα.27 The state of adiposity is closely correlated with physical inactivity, which leads to an increase in visceral fat. It has been demonstrated that aerobic exercise leads to reduced circulating IL-6 and CRP, as shown in a study on 2 120 Finnish participants where the levels of the latter were positively associated with obesity indices and inversely related to physical activity.28 Finally, in a large intervention study CRP concentration diminished by 41% in subjects who performed physical activity compared to inactive ones. Gender Clinical findings indicate that inflammatory responses differ across sexes, although data are still conflicting. In the CoLaus study which assessed the determinants of cytokines and APPs levels in a Caucasian population made of 2 884 men and 3 201 women, male gender was independently and positively related to IL-6 and TNFα levels whereas for IL-1 and CRP no associations were found.29 In another small study, which enrolled 15 healthy women and 20 healthy men, higher concentrations of IL-12, IL-1β, and TNFα and lower levels of IL-2 were found in males compared to women.30 Differences in lipid mediator levels have been observed between males and females in multiple diseases, such as prostaglandins (PGs) in T2D,31 linoleic acid-derived lipid mediators in chronic obstructive pulmonary disease,32 docosahexaenoic acid (DHA) in AD,33 and lipoxin A4 (LXA4) in metabolic syndrome.34 Smoking Several studies have assessed a close relationship between smoking and chronic inflammation. Cigarettes contain oxidative molecules (superoxide, hydrogen peroxide, nitrogen oxides) which drive oxidative stress thus leading to an inflammatory response, several toxins with immunomodulatory effects and also trace amounts of microbial cell components, including bacterial LPS.35 It has been definitively established that IL-1β, IL-6, CRP, and fibrinogen are sensitive biomarkers for cigarette smoke-induced inflammation.36 In addition, it has been shown that the imbalance between oxidants and antioxidants resulting from exposure to tobacco smoke leads to oxidative stress, increased mucosal inflammation, enhanced release of IL-8, IL-6, and TNFα and to the recruitment of macrophages and neutrophils.37 Concerning lipid biomarkers related to smoking status, arachidonic acid (AA) derived lipoxygenase (LOX)-metabolites, which are potent pro-inflammatory mediators leading to tissue destruction in periodontal inflammation,38 are significantly increased in smokers vs. non-smokers. In addition, 8-iso-prostaglandin F2α (8-iso-PGF2α) excretion exhibits dose-dependent increments in individuals who smoke cigarettes or consume alcohol.39,40 In contrast, 6-oxo- prostaglandin F1a (PGF1a), prostaglandin I2 (PGI2) and prostaglandin F2a (PGF2a) were significantly decreased in smokers vs. non-smokers. There are contradictory results in prostaglandin E2 (PGE2) synthetic rate, cigarette smoking and bone loss.41,42 Epigenetics and genetics Emerging evidence suggests that epigenetics (DNA methylation, histone acetylation/deacetylation and microRNA (miRNA) expression) may contribute to the pathophysiology of inflammatory processes.43 Epigenome-wide association studies (EWAS) have reported several epigenetic changes related to serum inflammatory markers suggesting a global hypomethylation of the genome during inflammation.44 Indeed, miR-126, miR-132, miR-146, miR-155, and miR-221 have recently emerged as important transcriptional regulators of TNFα, IL-8, MCP-1, IL-6, and adhesion molecules.45 Epigenetic changes may also affect the risk of chronic inflammatory diseases, including obesity, T2D, CVD, cancer, and neurological disorders. Prats-Puig et al. reported that 15 specific circulating miRNAs were significantly deregulated in prepubertal obesity, including a downregulation of miR-221 and miR-28-3p and an upregulation of miR-486, miR-142-3p, miR-130b, and miR-423-5p in plasma.46 It has been demonstrated that the inhibition of miR-153 prevents hyperglycemia in db/db mice, thus suggesting that it may be a promising therapeutic target for the treatment of inflammation-associated diabetes.47 Jiang and colleagues demonstrated that steatotic hepatocyte-derived extracellular vesicles (EVs) promote endothelial inflammation and facilitate atherogenesis by miR-1 delivery, KLF4 suppression and NF-κB activation by exploiting apolipoprotein E (ApoE)-deficient mice.48 Finally, several miRNAs regulate the expression of genes involved in AD-related oxidative stress49 and their levels may be influenced by transcription factors, among which NF-kB thus modulating inflammation and cancer.50 Genetic polymorphisms, especially in genes encoding molecules of the host defense system, such as cytokines, influence susceptibility to chronic inflammation. The −174 G/C genetic variant in the promoter region of IL-6 gene has been related to reduced gene expression and circulating levels of IL-6 and it has been described an association between this polymorphism, AD and also coronary artery disease (CAD).51,52 The G > A nucleotide substitution at position 308 in the TNFα promoter directly affects TNFα expression and has been associated with several inflammatory conditions as liver disease, primary sclerosing cholangitis, biliary cirrhosis, Crohn’s disease, rheumatoid arthritis and CAD.53 Finally, the 1082 G > A IL-10 and −308G > A TNFα variations have been associated with lower circulating levels of the anti-inflammatory IL-10 and with higher serum TNFα in 72 centenarians compared to controls, respectively.54 Methodological approaches for the detection of salivary biomarkers Methods for saliva sampling and collection Saliva has largely been disregarded in the past due to the presence of several food contaminants in its biochemical profile, as well as paucity of demonstration of correlations between salivary and blood markers.55 However, several studies have recently highlighted how saliva well correlates with blood markers, thus representing a promising alternative for noninvasive diagnostics,56 as it is the case with glucose57 and cortisol.58 Among others, IL-6, an inflammatory marker, also shows a significant correlation between blood and saliva59 (Table 1).Table 1 Inflammatory biomarkers detected in serum and saliva in healthy subjects Biomarkers Serum concentration Saliva concentration Cytokines/chemokines TNFα 60–90 pg·mL−1 20–45 pg·mL−1 IL-6 0–44 pg·mL−1 0.5–34 pg·mL−1 IL-1α 31.4 pg·mL−1 361 pg·mL−1 IL-1β 0.5–20 pg·mL−1 40.5–494 pg·mL−1 IL-2 21 pg·mL−1 4.3–10.3 pg·mL−1 IL-4 10–20 ng·L−1 15–25 ng·L−1 IL-5 2.6–13 pg·mL−1 0–3.5 pg·mL−1 IL-7 6.5 pg·mL−1 8.3 pg·mL−1 IL-8 6.8–39 pg·mL−1 150–400 pg·mL−1 IL-10 0.5–2.9 pg·mL−1 0.5–5.1 pg·mL−1 IL12p70 2–10.5 pg·mL−1 19.2 pg·mL−1 IL-13 17 pg·mL−1 0.7 pg·mL−1 IL-15 65.5–170 ng·L−1 0–8.7 pg·mL−1 IL-17a 15–40 pg·mL−1 5–10 pg·mL−1 IFNγ 20–42 pg·mL−1 28 pg·mL−1 CCL2 (MCP1) 6–70 pg·mL−1 125 pg·mL−1 CCL3 (MIP1α) 5.2 pg·mL−1 2.3 pg·mL−1 eotaxin (CCL11) 35–50 pg·mL−1 5.2–6.2 pg·mL−1 TGFβ 1.8–26 ng·mL−1 5.4–30 ng·mL−1 Proinflammatory enzymes MMP8 5.7–19 ng·mL−1 2.5–309 pg·mL−1 MMP9 215–608 ng·mL−1 50–100 ng·mL−1 TIMP1 305–342 g·L−1 1.5–3 pg·mL−1 TIMP2 100–200 ng·mL−1 2–3.5 ng·mL−1 Carboxyterminal telopeptide of type I collagen (ICTP) 3.5–4.5 ng·mL−1 15.2 ng·mL−1 Antioxidant markers 8-Hydroxy-2′-deoxyguanosine (8-OHdG) 121–200 ng·L−1 6.5–7.5 ng per 1 mg albumin Malondialdehyde (MDA) 0-29-0.98 mmol·L−1 0.85–4.31 mmol·L−1 Uric acid 0.5–1.5 mg·dL−1 2.8–4 mg per 1 mg albumin Glutatione peroxidase (GPX) 196–477 U·L−1 17–39 U per 1 mg albumin TAC (total antioxidant capacity) (1.92 ± 0.34) mmol trolox equivper L 1.1–1.5 nnol per 1 mg albumin Superoxide dismutase (SOD) 0.78–1.48 U·mL−1 0.6–1.53 U·mL−1 Glutathione (GSH) 1.91–4.41 μmol·L−1 1.1 (0.1–3.3) μmol·L−1 Mieloperossidase (MPO) 30–40 ng·mL−1 (0.40 ± 0.16) μmol·L−1 4-hydroxynonenal (4-HNE) 0.5–2 μg·mL−1 0–0.15 μg·mL−1 Acute phase proteins C-reactive protein (CRP) 0.1–10 mg·L−1 0–472 pg·mL−1 Serum amyloid A (SAA) 15–35 mg·L−1 3.1–423 U·mL−1 Haptoglobin (Hp) 50–220 mg·dL−1 451–1 457 μg·L−1 C3 1–3 mg·L−1 0–2 mg·mL−1 Alpha1antitrypsin (AAT) 0.9–1.75 g·L−1 2–2 271 ng·mL−1 IL-1ra 350–700 ng·L−1 3 700 pg·mL−1 Ferritin 10–250 ng·mL−1 147–191 mg·L−1 Cortisol 5–23 mg·dL−1 3–19 mg·L−1 Lipopolysaccaride (LPS) 0.1–10 mg·mL−1 4.2–10.1 mg·mL−1 Adipokines Leptin 10 ng·mL−1 21–42 pg·mL−1 Adiponectin 0-5-30 μg·mL−1 29 mg·dL−1 Although the procedures for saliva collection and storage have been already described in other reviews,60,61 it is important to contextualize these methods in the field of inflammatory markers, delineating best practices for a correct collection, handling, and biobanking. Moreover, although saliva receives increasing consideration as a potential sample for target diseases,62 it is still warranted a comparison with the current diagnostic methodologies to evaluate inflammatory markers in blood. The gold standard method of saliva collection is “passive drool”, that consists in saliva accumulation in the mouth and then let it flow through specific straws that collect the pooled saliva for a predetermined amount of time.61 Saliva can be easily sampled by either health professionals or people without previous medical training.63,64 Moreover, there are numerous standardized protocols for oral sampling, and saliva collection can be easily automated by means of wearable modules.65,66 Nevertheless, it must be remarked that saliva is a complex mixture of fluids from distinct microenvironments, such as gingival crevices,67 which may present a particular microbial population that often differs from that of other oropharyngeal regions.68 Other collection procedures include spitting, chewing, and swab-based methods. Saliva collected through spitting only involves specific salivary glands such as the submandibular and minor salivary ones and it is thus less preferred, whereas chewing, usually performed through the use of wax, allows the collection of stimulated saliva mainly from parotid glands. The latter is currently the recommended medium to measure the concentration of CRP in saliva,69 while as concern the other inflammatory markers, unstimulated saliva remains the most preferred fluid. Lastly, swab-based methods have been largely used in SARS COVID-19 tests and different studies report how this method can be particularly suited to perform salivary analysis in children.70 However, it has also been demonstrated how immunoassay tests showed a high deviation in the concentration of salivary markers due to the use of cotton-based swabs71 (Figs. 1–2).Fig. 1 Pros and Cons of the use of saliva or blood for the detection of clinically relevant analytes Fig. 2 Possible applications and clinical utility of saliva, as biological fluid for omics-based studies Standard approaches for the detection of salivary biomarkers The assays that are currently used for salivary analysis of inflammatory markers require the samples to be sent to centralized laboratories, while there are only few examples of commercial products that allow for point of care salivary diagnostics. Costs and time to response are the two parameters mainly taken into consideration in evaluating these procedures. The most widespread immunoassay, possibly representing the gold standard for many examinations is the enzyme linked immunosorbent assays (ELISA), which allows the detection of target antigens through the use of plates functionalized with antibodies. ELISA kits are available for the detection of several targets, including inflammatory markers such as cytokines,72 both in centralized laboratories and on the market (i.e. Salimetrics). A panel of biomarkers can be screened by using multiplexed technologies where their concentration is simultaneously evaluated, providing more comprehensive results and faster lead times. Since ELISA is used for both blood and saliva tests, it is of utmost importance to compare results coming from a novel tool with it. Studies which describe the use of sensors and innovative devices for salivary screening, do not always report a validation against standardized procedures,66,73,74 thus limiting the applicability of the obtained results for clinical trials. Novel perspectives to analyze salivary biomarkers To overcome salivary sample complexity, many researchers have investigated protocols to separate non-biomarker proteins and carbohydrates from raw saliva, thereby yielding pre-treated testing material that could be easier to analyze.75–77 This strategy has been nonetheless pursued due to the issue of viscosity, that could hinder the application of microfluidic arrays on raw untreated saliva. However, even if this treatment may minimize the effect of potential interferents, it might also compromise the concentration of target biomarkers78 as mucin which is an inflammatory biomarkers for periodontitis79 and matrix metalloproteinases,80 which can be lost from the sample upon membrane filtration procedures. Moreover, the removal of biomarkers from the sample poses a major hindrance if the molecules to be detected form complexes with other macromolecules and precipitate, thus decreasing their final concentrations in pre-treated samples.81 The opportunity to employ untreated or undiluted saliva is highly sought after by scientists worldwide, and great attention has been given to the development of highly selective and sensible biosensing platforms. In order to increase the specificity and sensibility of analytical strategies to determine inflammatory biomarkers, several researchers have highlighted the possibility of using surface-modified substrates as biorecognition elements.82,83 In this regard, several materials (carbon-or gold-based materials) have been discussed as promising alternatives for biosensor development.84 The benefits of carbon-based materials in biosensing are numerous, owing to many allotropic forms bearing highly conductive sp2 lattices,85 as well as offering adequate stability for the anchoring of coordination complexes and construction of metalorganic frameworks that are capable of enhancing analytic signal acquisition.86 Moreover, even though advanced carbon materials such as graphene may be expensive, the methods for their production are in constant development, and this resource is abundant in nature as opposed to precious metals. On the other hand, gold-based substrates offer simplicity and an easier function as their main benefit.87 It is well reported that thiolate compounds show thermodynamic feasibility to spontaneously assemble monolayers on gold substrates.88 This phenomenon, named self-assembled monolayer formation has been extensively explored in the crafting of biosensing strategies to determine inflammatory biomarkers in saliva and other biological fluids, and can be easily performed on room temperature without the need of extreme conditions.89,90 Electrochemical,91 optical92 and acoustic transduction93 represent the most diffuse signal transduction technologies used for the development of biosensors for the detection of salivary inflammatory biomarkers. Electrochemical technologies have as analytical principle the inherent electrical properties of matter, by measuring changes in electrical charge, current, potential and resistance.91 Overall, this method has been the most used by researchers in the development of label-free biosensing platforms for oral applications, due to the versatility, low cost and high portability.91 On the other hand, methods based on optical signal transduction rely on shifts in spectra absorption, reflection and refraction and they often employ spectrophotometry, spectroscopy, colorimetry and surface-plasmon resonance.83 These techniques offer high sensibility and selectivity, and similarly to electrochemical transduction, also allow the development of either label-free or sandwiched biosensing strategies. In regard to acoustic signal transduction, this approach is a recent trend in biosensing technologies, and has attracted much attention due to the possibility of refining the signal acquisition and attaining very low limits-of-detection.94 This technology mostly bases on the dependence of frequency and dissipation of mechanical disturbances, which can be generated by piezoelectric materials serving as sensing substrates.94 However, owing to its novelty and reliance on high frequencies, there are still limitations regarding its point-of-care application.94 In order to improve the specificity and the sensitivity of biosensors, molecularly imprinted polymer (MIP) technology is implemented onto the biomaterial surfaces. In MIP, specific functional monomers are polymerized in the presence of the target molecule, e.g., a biomarker. The target molecule is then removed, leaving a polymer matrix that has recognition cavities that are complementary to the target molecule in terms of size, shape, and functionality. In this way, MIP specifically rebinds the target molecule and reduces the effect of potential interferents on false positives and thus enhance safe data generation.95 MIPs have been successfully applied not only for small molecules recognition but also for biomacromolecules, such as proteins.96 MIPs are used in the design of MIPs-based biosensors, due to their higher stability, specificity, and reusability than biological receptors. However, industrial application of MIP-based biosensors is limited due to their lack of reproducible preparation and stability on the sensor substrate, as well as the limited slow diffusion of analyte into the cavities and binding sites (Fig. 3).Fig. 3 Saliva and blood as biological fluids to exploit biosensors to detect peptides, DNA, RNA and proteins. Schematic representation of the components of biosensors and workflow The rapid advances in nanotechnology, microelectronics, and Internet of Things enable the development of wearable biosensors that can be positioned in the oral cavity for the detection of salivary biomarkers.74 Such biosensors communicate with computers/smartphones wirelessly and enables the online data analysis. Data transfer is generated through WiFi and BlueTooth Low Energy. For example, Kim et al. reported an integrated wireless mouthguard amperometric biosensor that enables the non-invasive monitoring of salivary uric acid levels.97 Lee at al. described a wireless intraoral device capable of a real-time recording of sodium detection.98 Mannoor et al. fabricated a graphene-based wireless biosensor for remote monitoring of respiration and bacteria detection in saliva.99 Tseng at al. demontrated the wireless monitoring of oral cavity and food consumption by a radiofrequency-trilayer dielectric sensor100 (Fig. 4).Fig. 4 Summary of wearable intraoral biosensing platforms for non-invasive salivary analysis. Lactate biosensor on a mouthguard (a, on the left). Reproduced with permission from Kim et al..86 Mouthguard with biosensor and integrated electronics for real-time uric acid detection (a, on the right). Reproduced with permission from Kim et al..86 Mouthguard with screen printed electrodes for N-Carboxymethyl lysine detection (b). Reproduced with permission from Ciui et al..177 Glucose biosensing telemetry system (c, on the left), reproduced with permission from Arakawa et al.,178 and a hybrid flexible bioelectronic platform for sodium monitoring (c, on the right). Tooth mounted hydrogel radiofrequency biosensor (d, on the left), reproduced with permission from Tseng et al.179 and graphene-based biosensor for pathogen’s detection (d, on the right), reproduced with permission from Mannoor et al.99 Technological developments have provided a growing improvement in methods that may be exploited to measure disease biomarkers although the detection of the latter is challenging due to the high inter-individual variability. Therefore, machine learning models have been developed to identify signatures in multiple circulating biomarkers for specific diseases. Machine learning algorithms enable full automation and allow to analyze large datasets in a short period of time by reducing false positives that lead to incorrect diagnosis, and thus saving clinicians’ time for data analysis. Currently, machine learning methods including Random Forests or Gradient Boosted Trees to deep learning have been increasingly applied to identify biomarkers from body fluids for non-invasive disease diagnosis.101–104 Despite the scientific and technological advances, an early and non-invasive biomarker detection is still limited by current biosensors. In particular, the co-detection of different biomarkers that characterize a specific disease, by a single biosensor, is still unripe. Therefore, multiplex technologies for advanced biosensor manufacturing and biomarker detection are needed to improve the quality of patient care and to reduce the costs through the early assessment and diagnosis of chronic diseases. Salivary biomarkers: an attractive way to approach screening and monitoring To date, soluble markers of chronic inflammation have been assessed in blood. However, recent findings have demonstrated that inflammatory mediators may be detectable also in saliva which has drawn a growing attention as biological fluid especially due to its stress-free and non-invasive collection. Acute phase proteins (APPs) as salivary biomarkers Chronic subclinical inflammation and tissue injuries are associated with the secretion of acute phase proteins, such as haptoglobin (Hp), CRP, Alpha 1-antitrypsin (A1AT), fibrinogen and ferritin, mainly from the liver into the bloodstream.105 Their concentrations progressively increase with the tissue damage and then, these substances passively diffuse through the porous capillaries, are actively transported into saliva or may also diffuse between acinar cells.106 Some of them also directly derive from salivary glands.107 Abnormal serum levels of APPs are reported in several disorders, among which myocardial infarction, T2D, insulin resistance (IR), inflammatory bowel disease, psoriasis, and cancer. In detail, APPs are stratified in positive and negative ones, according to their up- or downregulation during inflammation.108 Among the positively regulated, CRP has been indicated as a predictor of primary and secondary adverse cardiovascular events, participating in atherogenesis whereby mediating the recruitment of innate immunity and the activation of complement pathway.109 Although it has a high molecular weight, CRP can enter into the oral cavity, through plasma exudates of systemic origin from gingival crevicular fluid (GCF).110,111 It has been demonstrated that CRP levels in saliva moderately correlated with those in serum samples and with systemic inflammation, postulating this via as a useful strategy to assess various inflammatory conditions.112,113 Indeed, CRP is synthetized only by the liver, and it is not produced locally in the mouth. Thus, its salivary levels may more accurately reflect the systemic inflammatory status, compared to cytokines and chemokines, that are modulated also by oral pathologies.114 For the same reason, fibrinogen concentrations assessed in saliva are representative of the blood protein content albeit with low levels and possible contaminations by ulcerated gingival epithelium.114 However, it seems to own reliable clinical utility to detect tuberculosis and polycystic ovarian syndrome (PCOS).115,116 Another molecule that has been pointed out as possible salivary biomarker of subclinical inflammation is haptoglobin (Hp),117 and it has been studied together with the cortisol hormone. Indeed, in stressful situations, the activation of the hypothalamic-pituitary-adrenal (HPA) axis is proven by the release of cortisol by adrenal cortex into the bloodstream within few minutes. A significant elevation of both Hp and cortisol has been identified in a model of experimentally induced systemic inflammation by an LPS challenge117 and salivary and blood concentrations of the latter have been found to be strictly correlated.118,119 Finally, the assessment of salivary A1AT levels may be used for monitoring effectiveness of oncological interventions.120 Salivary biomarkers of inflammation The feasibility to estimate the individual inflammatory status from saliva opens the route to unraveled approaches to the diagnosis and management of several inflammatory disorders and the possibility to non-invasively assess the response to acute stressors.121 However, salivary measurements often show inconsistencies, mainly due to various methodological applications, handling technique and timing of collection. Moreover, the degree of translatability of blood-based inflammatory markers on saliva-based ones and the pathological range of concentration for each salivary biomarker remain to be defined. Indeed, the impact of the fluctuations, due to local inflammatory milieu, oral mucosal immunity, and interruptions in gingival integrity on the content of these mediators in saliva needs to be taken into account. Therefore, standard procedures of collection and preservation are largely recommended for the utilization of saliva as a reliable diagnostic medium.59 Nevertheless, it has been established that a broad variety of interleukins (IL-1β, IL-2, IL-4, IL-6, IL-10 etc.), TNFα and pro-inflammatory enzymes, involved in matrix remodeling such as metalloproteinases and their inhibitors, may be quantified both in saliva and serum samples. For example, IL-1β, a cytokine released from macrophages and non-immune cells in the context of inflammation, participates to innate immune response, whereby promoting the secretion of IL-6 and TNFα. The comparison between the circulating and salivary levels of these three cytokines has been widely explored,122–125 showing overall modest reliability of saliva and low consistency. The only one marker that may more precisely reflects blood distribution is IL-6.126,127 Indeed, a significant correlation between plasma and saliva has been reported in patients affected by IBD and oral lichen planus (OLP).56,126 Furthermore, IL-8 and MMP-8 were found to be increased in patients with head and neck squamous cell cancer or bowel diseases and in those suffering of diabetes or who underwent cardiac surgery, respectively.128 In detail, a population study across 441 adults described that diabetic patients have a three times higher ratio of MMP-8/TIMP-1 and twice as high concentration of MMP-8, as a consequence of the elevated inflammatory status in these patients.128 According to this notion, MMP-8 has been found to be up-regulated in patients with elevated risk of CVD.128,129 Among salivary biomarkers, miRNAs seem very promising, both for the early diagnosis and for understanding the pathogenesis of some diseases (e.g., oral cancer, salivary glands cancer, neurological or psychiatric deficiencies).130 Moreover, it has been demonstrated that salivary transcriptome is very abundant, consisting of thousands of mRNAs and miRNAs.131,132 In saliva samples of patients with oral squamous cell carcinoma (OSCC), miRNA-125a and miR-200a were significantly decreased and miR-31 was over-expressed. Aberrant methylation and atypical expression were observed for miR-200c/miR-141 and miR-375/miR-200a, respectively.133 In addition, promising biomarkers were represented by miR-768-3p and miR-574 for salivary gland inflammation and by miR-5100 for Sjögren’s syndrome.134,135 Other miRNAs whose expression is deregulated in saliva include miR-101 in Crohn’s disease and miR-21, miR-31, miR-142-3p/5p in ulcerative colitis.136 Evidence exists that salivary miR-940 and miR-3679-5p are reliable markers for pancreatic cancer whereas miR140-5p and miR301a are attractive molecules for the salivary diagnosis of gastric cancer.137 In addition to the more traditional inflammatory biomarkers, salivary levels of lipid mediators can also be used for diagnostic and prognostic purposes despite their investigation has been given little scientific attention and remains poorly understood.138 A study of serum and saliva lipid profile levels in about 100 healthy subjects showed that there is a reasonable correlation between their concentration of total cholesterol and triglycerides.139 The measurement of short-chain fatty acids (SCFAs) in saliva, produced by causal bacteria, may be an indicator of the inflammation degree closely related to the onset and progression of periodontal disease.140,141 Elevated salivary leukotriene B4 (LTB4) and PGE2 are correlated with arterial stiffness.142 Asthmatic patients exhibit elevated Cys-leukotriens (LTs) levels in saliva.143 Elevated salivary levels of PGE2 were found to be correlated with gingivitis144,145 and periodontitis.146 In chronic periodontitis patients, salivary LTB4 levels were correlated with the severity of alveolar bone loss.147 Regarding the primary Sjögren’s syndrome (SS), Slomiany et al. demonstrated a general increase in total salivary lipid count in SS patients as well as an increased proportion of glycolipids, phospholipids and some neutral lipids.148 Another study analyzing eicosanoids reported an increase in PGE2 and thromboxane B2 (TXB2) in SS patients when compared to healthy controls.149 Fineide et al. revealed several significant differences in the lipidomic profiles of saliva in human patients suffering from SS compared to healthy controls showing increasing levels of sphingomyelins and diacylglycerophosphocholines and decreasing levels of diacylglycerols and ceramides in unstimulated saliva from SS patients.150 Salivary biomarkers of oxidative stress Persistent inflammation and blunted antioxidant capacities resulted in the exaggerate generation of free radicals which propagate injuries, precipitating cell death. The release of these harmful radicals, along with reactive oxygen and nitrogen species (ROS/RNS) boost the activation of signaling molecules and transcription factors, that may be particularly useful to pinpoint the disorders. In detail, free radicals are responsible for the oxidation of cellular components, such as membrane lipids, proteins, and nucleic acids, contributing to mitochondrial dysfunction, antioxidant systems impairment, ageing and chronic diseases.151–153 Products derived from lipid peroxidation, protein oxidation and DNA damage can be directly assessed in saliva, possibly paving the way to diagnose systemic disorders associated with oxidative stress, by using this mean.152,154,155 Nonetheless, local oral status and oral cavity-related pathologies (i.e., periodontitis and dental caries) may also modulate the redox balance of saliva, interfering with its widespread routine clinical use.156 A broad number of studies indicate that an imbalance in oxidant/antioxidant mediators may exert a crucial role in the pathogenesis and progression of metabolic syndrome, T2D and CVD.157 However, the majority of the research is focused on tissue and blood distribution of these indicators, and less is known regarding their impact on saliva composition. A preclinical study in insulin resistant rats compared salivary antioxidants and oxidative stress products to those in plasma samples, exploring their diagnostic utility. The authors showed an impairment in antioxidant barriers and in ROS scavengers both in plasma and in saliva, proven by the reduction in superoxide dismutase, ascorbic acid and glutathione (GSH) levels in IR mice.158 These alterations were paralleled by strengthened lipid/protein oxidation and advanced glycation end products in both biological fluids, showing an elevated coefficient of correlation between the two. The assessment of salivary redox biomarkers seems to be applicable also for diagnosis and monitoring of obesity,159,160 diabetes,161 hypertensive disorders,162 chronic kidney disease,163 heart failure,164,165 neurodegenerative diseases166 and cancer,167,168 in which molecules and enzymes with antioxidant properties are pathologically depleted in saliva, whereas oxidative and nitrosative by-products are favored. For instance, it has been demonstrated that salivary oxidative biomarkers, among which 4-hydroxynonenal (4-HNE) and 8-isoprostanes (8-isoP), advanced oxidation protein products (AOPP) and protein carbonyl groups (PC), 8-hydroxy-D-guanosine (8-OHdG), derived from lipoperoxidation of cell membranes, protein oxidation and DNA aberrancies respectively, were increased in 47 subjects with morbid obesity compared to controls (BMI < 25 kg·m−2) and that bariatric surgery reduced their salivary concentrations.160 Similar findings have been reported by Zalewska and colleagues, which yielded an enhanced total oxidative status accompanied by reduced glutathione levels in saliva and in plasma from 40 young obese subjects.159 Other important markers of oxidative damage are mitochondrial DNA mutations, which has been evaluated in blood and gingival tissues.153 Higher levels of glutathione peroxidase (GSHPx), an antioxidant enzyme, and malondialdehyde (MDA), a biomarker for lipid peroxidation, were observed in the saliva of patients that had periodontitis and that were smokers compared to the non-smoking control group.169,170 Therefore, increased levels of GSHPx and MDA can indicate increased lipid peroxidation in patients with periodontal disease which is further elevated by smoking.169,170 The results of Wolfram et al.171 and Morrow et al.172 indicate that salivary lipid isoprostanes (IPs) can reliably assess the degree of oxidative stress. In detail, elevated salivary 8-iso-PGF2α levels were determined by oxidative damage associated with the extent of periodontal disease and significantly aggravated by concomitant tobacco use. Likewise, the levels of salivary PGE2, PGF2α and prostaglandin D2 (PGD2) have been successfully used as biomarkers for chronic inflammatory processes and to assess the degree of oxidative stress caused by smoking and periodontitis. The results of Huang et al. demonstrate that a local redox alteration contributes significantly to periodontitis through the modulation of fatty acid metabolism in response to inflammation and oxidative stress.173 In addition, smoking induces specific structural alterations in the lipid A-derived 3-hydroxy (OH) fatty acid profile in saliva of individuals with chronic periodontitis that are consistent with an altered oral microflora.174 The most prominent shifts in smokers, compared to non-smokers, occurred in the short, straight-chain pro-inflammatory lipid A fatty acids, 3-OH C12, C13, and C14. Furthermore, the concentration of salivary redox biomarkers progressively increases according to the disease progression, mirroring their presence at the serum level.175,176 For instance, the salivary content of 8-OHdG, MDA, and PC was significantly higher in patients affected by CAD compared to healthy individuals, supporting the paramount role of lipid oxidative damage in the etiology of CVD. Even more, in these subjects, MDA levels were associated with serum high sensitivity CRP (hsCRP) and with plasma fibrinogen, that are strong predictors of cardiovascular events.176 Conclusions The measurement of soluble mediators outlines the entire therapeutic route from diagnosis to therapy and follow-up. In detail, a large series of acute phase proteins, cytokines and chemokines, lipids, pro-inflammatory enzymes, and oxidative stress indicators have been pointed out as trustworthy biomarkers in biological fluids. Since chronic disorders, among which cardiovascular failure, obesity, diabetes, and cancer, have been progressively spread in the last century, there is an urgent need to identify novel diagnostic strategies to tailor the management of patients and to stage the diseases. In this regard, ever increasing number of studies have suggested that salivary biomarkers modulation will be an innovative and minimally invasive option in the care of chronically affected patients. Hence, addressing the efforts to the research of peculiar salivary molecules will provide clinicians an unprecedented opportunity to soften the painful path of the disease. Furthermore, technological advances enable the implementation of wearable biosensors in the oral cavity that automates the detection and quantification of biomarkers in saliva. That allows early, non-invasive and unobtrusive diagnosis, continuous monitoring of chronic disease and early and continuous communication with physicians and thus improve the quality of patient care while reducing the cost of care. The possibility to assign a specific spectrum of candidate molecules and detecting salivary technologies to discriminate each stage of chronic disorders and to formulate panels of salivary mediators as suitable molecular biomarkers to be combined with the demographic, genetic and anthropometric features of patients, might represent a novel tool to improve the diagnosis and more accurately evaluate the prognosis (Fig. 5).Fig. 5 Schematic illustration of possible modifiers of chronic inflammation, analytes enchanged between blood and oral cavity, and dosable compounds in biological fluids Acknowledgements This study was (partially) supported by the Italian Ministry of Health (Ricerca Corrente 2022 - Fondazione IRCCS Cà Granda Ospedale Maggiore Policlinico) and by the Italian Ministry of Health (Ricerca Finalizzata - GR-2019-12370172). Author contributions The authors’ responsibilities were as follows: P.D. and M.Me manuscript drafting and figures preparation; S.C. manuscript drafting and figures preparation; R.G. figures preparation, D.V.T., N.S.K., D.G., and M.D.F. critical revision of the manuscript. G.T. study funding, supervision and has primary responsibility for final content. All authors read and approved the final manuscript. Competing interests The authors declare no competing interests. These authors contributed equally: Paola Dongiovanni, Marica Meroni ==== Refs References 1. Furman D Chronic inflammation in the etiology of disease across the life span Nat. Med. 2019 25 1822 1832 10.1038/s41591-019-0675-0 31806905 2. Calder PC A consideration of biomarkers to be used for evaluation of inflammation in human nutritional studies Br. J. Nutr. 2013 109 S1 S34 10.1017/S0007114512005119 23343744 3. Raghupathi W Raghupathi V An Empirical Study of Chronic Diseases in the United States: A Visual Analytics Approach Int. J. Environ. Res. Public Health 2018 15 431 10.3390/ijerph15030431 29494555 4. Prasad S Tyagi AK Aggarwal BB Detection of inflammatory biomarkers in saliva and urine: potential in diagnosis, prevention, and treatment for chronic diseases Exp. Biol. Med. 2016 241 783 799 10.1177/1535370216638770 5. Edgar WM Saliva: its secretion, composition and functions Br. Dent. J. 1992 172 305 312 10.1038/sj.bdj.4807861 1591115 6. Pfaffe T Diagnostic potential of saliva: current state and future applications Clin. Chem. 2011 57 675 687 10.1373/clinchem.2010.153767 21383043 7. Karjalainen S Salivary cholesterol of healthy adults in relation to serum cholesterol concentration and oral health J. Dent. Res. 1997 76 1637 1643 10.1177/00220345970760100401 9326895 8. D RM Evaluation of salivary flow rate, pH and buffer in pre, post & post menopausal women on HRT J. Clin. Diagn. Res. 2014 8 233 236 24701542 9. Inoue H Gender difference in unstimulated whole saliva flow rate and salivary gland sizes Arch. Oral. Biol. 2006 51 1055 1060 10.1016/j.archoralbio.2006.06.010 16919593 10. Srivastava A Age and gender related differences in human parotid gland gene expression Arch. Oral. Biol. 2008 53 1058 1070 10.1016/j.archoralbio.2008.05.006 18571147 11. Prodan A Interindividual variation, correlations, and sex-related differences in the salivary biochemistry of young healthy adults Eur. J. Oral. Sci. 2015 123 149 157 10.1111/eos.12182 25809904 12. Schepici G Silvestro S Trubiani O Bramanti P Mazzon E Salivary biomarkers: future approaches for early diagnosis of neurodegenerative diseases Brain Sci. 2020 10 245 10.3390/brainsci10040245 32326227 13. Pawlik P Blochowiak K The role of salivary biomarkers in the early diagnosis of Alzheimer’s disease and Parkinson’s disease Diagnostics (Basel) 2021 11 371 10.3390/diagnostics11020371 33671562 14. Ershler WB Interleukin-6: a cytokine for gerontologists J. Am. Geriatr. Soc. 1993 41 176 181 10.1111/j.1532-5415.1993.tb02054.x 8426042 15. Ferrucci L The origins of age-related proinflammatory state Blood 2005 105 2294 2299 10.1182/blood-2004-07-2599 15572589 16. Brüünsgaard H Pedersen BK Age-related inflammatory cytokines and disease Immunol. Allergy Clin. North Am. 2003 23 15 39 10.1016/S0889-8561(02)00056-5 12645876 17. Ridker PM Plasma concentration of interleukin-6 and the risk of future myocardial infarction among apparently healthy men Circulation 2000 101 1767 1772 10.1161/01.CIR.101.15.1767 10769275 18. Dinarello CA Interleukin 1 and interleukin 18 as mediators of inflammation and the aging process Am. J. Clin. Nutr. 2006 83 447s 455s 10.1093/ajcn/83.2.447S 16470011 19. Rea IM Changes in lymphocyte subsets, interleukin 2, and soluble interleukin 2 receptor in old and very old age Gerontology 1996 42 69 78 10.1159/000213775 9138976 20. Rübenhagen R Interleukin-7 levels in synovial fluid increase with age and MMP-1 levels decrease with progression of osteoarthritis Acta Orthop. 2012 83 59 64 10.3109/17453674.2011.645195 22206448 21. Zykov MV Interleukin-12 serum level has prognostic value in patients with ST-segment elevation myocardial infarction Heart Lung 2016 45 336 340 10.1016/j.hrtlng.2016.03.007 27094852 22. Garrett-Sinha LA John S Gaffen SL IL-17 and the Th17 lineage in systemic lupus erythematosus Curr. Opin. Rheumatol. 2008 20 519 525 10.1097/BOR.0b013e328304b6b5 18698171 23. Rink L Cakman I Kirchner H Altered cytokine production in the elderly Mech. Ageing Dev. 1998 102 199 209 10.1016/S0047-6374(97)00153-X 9720652 24. Greenfield JR Campbell LV Relationship between inflammation, insulin resistance and type 2 diabetes: ‘cause or effect’? Curr. Diabetes Rev. 2006 2 195 211 10.2174/157339906776818532 18220627 25. Visser M Elevated C-reactive protein levels in overweight and obese adults Jama 1999 282 2131 2135 10.1001/jama.282.22.2131 10591334 26. Corica F Relationship between plasma leptin levels and the tumor necrosis factor-alpha system in obese subjects Int. J. Obes. Relat. Metab. Disord. 1999 23 355 360 10.1038/sj.ijo.0800826 10340812 27. Fantuzzi G Adipose tissue, adipokines, and inflammation J. Allergy Clin. Immunol. 2005 115 911 919 10.1016/j.jaci.2005.02.023 15867843 28. Raitakari M Distribution and determinants of serum high-sensitive C-reactive protein in a population of young adults: the Cardiovascular Risk in Young Finns Study J. Intern. Med. 2005 258 428 434 10.1111/j.1365-2796.2005.01563.x 16238678 29. Marques-Vidal P Levels and determinants of inflammatory biomarkers in a Swiss population-based sample (CoLaus study) PLoS ONE 2011 6 e21002 10.1371/journal.pone.0021002 21695270 30. Bouman A Gender difference in the non-specific and specific immune response in humans Am. J. Reprod. Immunol. 2004 52 19 26 10.1111/j.1600-0897.2004.00177.x 15214938 31. Tessaro FH Ayala TS Martins JO Lipid mediators are critical in resolving inflammation: a review of the emerging roles of eicosanoids in diabetes mellitus Biomed. Res. Int. 2015 2015 568408 10.1155/2015/568408 25866794 32. Balgoma D Linoleic acid-derived lipid mediators increase in a female-dominated subphenotype of COPD Eur. Respir. J. 2016 47 1645 1656 10.1183/13993003.01080-2015 26965288 33. Pomponi MF Why docosahexaenoic acid and aspirin supplementation could be useful in women as a primary prevention therapy against Alzheimer’s disease? Ageing Res. Rev. 2011 10 124 131 10.1016/j.arr.2010.09.003 20920611 34. Yu D Inverse relationship between serum lipoxin A4 level and the risk of metabolic syndrome in a middle-aged Chinese population PLoS ONE 2015 10 e0142848 10.1371/journal.pone.0142848 26565966 35. Lee J Taneja V Vassallo R Cigarette smoking and inflammation: cellular and molecular mechanisms J. Dent. Res. 2012 91 142 149 10.1177/0022034511421200 21876032 36. Elisia I The effect of smoking on chronic inflammation, immune function and blood cell composition Sci. Rep. 2020 10 19480 10.1038/s41598-020-76556-7 33173057 37. Strzelak A Ratajczak A Adamiec A Feleszko W Tobacco smoke induces and alters immune responses in the lung triggering inflammation, allergy, asthma and other lung diseases: a mechanistic review Int. J. Environ. Res. Public Health. 2018 15 1033 10.3390/ijerph15051033 29883409 38. Van Dyke TE Control of inflammation and periodontitis Periodontol 2000 2007 45 158 166 10.1111/j.1600-0757.2007.00229.x 17850455 39. Delanty N 8-Epi PGF2 alpha: specific analysis of an isoeicosanoid as an index of oxidant stress in vivo Br. J. Clin. Pharm. 1996 42 15 19 10.1046/j.1365-2125.1996.03804.x 40. Reilly M Modulation of oxidant stress in vivo in chronic cigarette smokers Circulation 1996 94 19 25 10.1161/01.CIR.94.1.19 8964113 41. Alpagot T Longitudinal evaluation of prostaglandin E2 (PGE2) and periodontal status in HIV+ patients Arch. Oral. Biol. 2007 52 1102 1108 10.1016/j.archoralbio.2007.04.013 17586460 42. Ng PY Candidate salivary biomarkers associated with alveolar bone loss: cross-sectional and in vitro studies FEMS Immunol. Med. Microbiol. 2007 49 252 260 10.1111/j.1574-695X.2006.00187.x 17328758 43. Shanmugam MK Sethi G Role of epigenetics in inflammation-associated diseases Subcell. Biochem. 2013 61 627 657 10.1007/978-94-007-4525-4_27 23150270 44. Gonzalez-Jaramillo V Epigenetics and inflammatory markers: a systematic review of the current evidence Int. J. Inflam. 2019 2019 6273680 31205673 45. Marques-Rocha JL Noncoding RNAs, cytokines, and inflammation-related diseases FASEB J. 2015 29 3595 3611 10.1096/fj.14-260323 26065857 46. Prats-Puig A Changes in circulating microRNAs are associated with childhood obesity J. Clin. Endocrinol. Metab. 2013 98 E1655 E1660 10.1210/jc.2013-1496 23928666 47. Sun Y Inhibition of miR-153, an IL-1β-responsive miRNA, prevents beta cell failure and inflammation-associated diabetes Metabolism 2020 111 154335 10.1016/j.metabol.2020.154335 32795559 48. Jiang F Hepatocyte-derived extracellular vesicles promote endothelial inflammation and atherogenesis via microRNA-1 J. Hepatol. 2020 72 156 166 10.1016/j.jhep.2019.09.014 31568800 49. Nunomura A Perry G RNA and oxidative stress in Alzheimer’s disease: focus on microRNAs Oxid. Med. Cell Longev. 2020 2020 2638130 10.1155/2020/2638130 33312335 50. Markopoulos GS Roles of NF-κB Signaling in the Regulation of miRNAs Impacting on Inflammation in Cancer Biomedicines 2018 6 40 10.3390/biomedicines6020040 29601548 51. Capurso C Interleukin 6-174 G/C promoter gene polymorphism and sporadic Alzheimer’s disease: geographic allele and genotype variations in Europe Exp. Gerontol. 2004 39 1567 1573 10.1016/j.exger.2004.07.006 15501028 52. Humphries SE The interleukin-6 -174 G/C promoter polymorphism is associated with risk of coronary heart disease and systolic blood pressure in healthy men Eur. Heart J. 2001 22 2243 2252 10.1053/euhj.2001.2678 11728144 53. Elahi MM Tumor necrosis factor alpha -308 gene locus promoter polymorphism: an analysis of association with health and disease Biochim. Biophys. Acta 2009 1792 163 172 10.1016/j.bbadis.2009.01.007 19708125 54. Lio D Inflammation, genetics, and longevity: further studies on the protective effects in men of IL-10 -1082 promoter SNP and its interaction with TNF-alpha -308 promoter SNP J. Med. Genet. 2003 40 296 299 10.1136/jmg.40.4.296 12676903 55. Wilson, S. J., Woody, A. & Kiecolt-Glaser, J. K. Inflammation As a Biomarker Method in Lifespan Developmental Methodology (Oxford University Press, 2018). 56. Williamson S Comparison of biomarkers in blood and saliva in healthy adults Nurs. Res. Pr. 2012 2012 246178 57. Kumar S Padmashree S Jayalekshmi R Correlation of salivary glucose, blood glucose and oral candidal carriage in the saliva of type 2 diabetics: a case-control study Contemp. Clin. Dent. 2014 5 312 317 10.4103/0976-237X.137925 25191065 58. Adebero T Salivary and serum concentrations of cortisol and testosterone at rest and in response to intense exercise in boys versus men Pediatr. Exerc. Sci. 2020 32 65 72 10.1123/pes.2019-0091 31770720 59. Nam Y Salivary biomarkers of inflammation and oxidative stress in healthy adults Arch. Oral. Biol. 2019 97 215 222 10.1016/j.archoralbio.2018.10.026 30399508 60. Granger DA Incorporating salivary biomarkers into nursing research: an overview and review of best practices Biol. Res. Nurs. 2012 14 347 356 10.1177/1099800412443892 22593229 61. Padilla, G. A. et al. Saliva Collection, Handling, Transport, and Storage: Special Considerations and Best Practices for Interdisciplinary Salivary Bioscience Research, in Salivary Bioscience: Foundations of Interdisciplinary Saliva Research and Applications (eds Granger, D. A. & Taylor, M. K.). 21–47 (Springer International Publishing, Cham, 2020). 62. Orive G Lopera F Carro E Saliva is a good candidate to be the new gold-standard sample for neurodegenerative diseases J. Alzheimers Dis. 2022 87 1497 1501 10.3233/JAD-220144 35491793 63. Lakshmi K Oral fluid-based biosensors: a novel method for rapid and noninvasive diagnosis Indian J. Dent. Sci. 2017 9 60 66 10.4103/IJDS.IJDS_6_17 64. Eftekhari A Bioassay of saliva proteins: the best alternative for conventional methods in non-invasive diagnosis of cancer Int. J. Biol. Macromol. 2019 124 1246 1255 10.1016/j.ijbiomac.2018.11.277 30513307 65. García-Carmona, L. et al. Pacifier Biosensor: toward noninvasive saliva biomarker monitoring. Anal. Chem. 91, 13883–13891 (2019). 66. Goldoni R Salivary biomarkers of neurodegenerative and demyelinating diseases and biosensors for their detection Ageing Res. Rev. 2022 76 101587 10.1016/j.arr.2022.101587 35151849 67. Simón-Soro A Microbial geography of the oral cavity J. Dent. Res. 2013 92 616 621 10.1177/0022034513488119 23674263 68. Gug IT Salivary biomarkers detection: analytical and immunological methods overview TrAC Trends Anal. Chem. 2019 113 301 316 10.1016/j.trac.2019.02.020 69. Mohamed R The impact of saliva collection and processing methods on CRP, IgE, and Myoglobin immunoassays Clin. Transl. Med. 2012 1 19 10.1186/2001-1326-1-19 23369566 70. Pappa, E. & Kousvelari E. Saliva in the “Omics” era: a promising tool in paediatrics. Oral Dis. 25, 16–25 (2019). 71. Shirtcliff EA Use of salivary biomarkers in biobehavioral research: cotton-based sample collection methods can interfere with salivary immunoassay results Psychoneuroendocrinology 2001 26 165 173 10.1016/S0306-4530(00)00042-1 11087962 72. Minetto MA Influence of the sample collection method on salivary interleukin–6 levels in resting and post-exercise conditions Eur. J. Appl. Physiol. 2007 101 249 256 10.1007/s00421-007-0484-x 17569075 73. Goldoni R Recent advances in graphene-based nanobiosensors for salivary biomarker detection Biosens. Bioelectron. 2021 171 112723 10.1016/j.bios.2020.112723 33096432 74. Goldoni R Malignancies and Biosensors: A Focus on Oral Cancer Detection through Salivary Biomarkers Biosensors (Basel) 2021 11 396 10.3390/bios11100396 34677352 75. Herr AE Microfluidic immunoassays as rapid saliva-based clinical diagnostics Proc. Natl Acad. Sci. USA 2007 104 5268 5273 10.1073/pnas.0607254104 17374724 76. Yee EH Detection of biomarkers of periodontal disease in human saliva using stabilized, vertical flow immunoassays ACS Sens. 2017 2 1589 1593 10.1021/acssensors.7b00745 29090909 77. Jung DG Jung D Kong SH A lab-on-a-chip-based non-invasive optical sensor for measuring glucose in saliva Sensors 2017 17 2607 10.3390/s17112607 29137200 78. Rossini EL Paper microfluidic device using carbon dots to detect glucose and lactate in saliva samples Spectrochim. Acta Part A: Mol. Biomol. Spectrosc. 2021 248 119285 10.1016/j.saa.2020.119285 79. Helton KL Conditioning saliva for use in a microfluidic biosensor Lab Chip 2008 8 1847 1851 10.1039/b811150b 18941684 80. Herr AE Microfluidic immunoassays as rapid saliva-based clinical diagnostics Proc. Natl Acad. Sci. USA 2007 104 5268 5273 10.1073/pnas.0607254104 17374724 81. Lee YJ Optimization of Saliva Collection and Immunochromatographic Detection of Salivary Pepsin for Point-of-Care Testing of Laryngopharyngeal Reflux Sensors (Basel) 2020 20 325 10.3390/s20010325 31935973 82. Malon RSP Saliva-based biosensors: noninvasive monitoring tool for clinical diagnostics BioMed. Res. Int. 2014 2014 962903 10.1155/2014/962903 25276835 83. Lukose, J. et al. Photonics of human saliva: potential optical methods for the screening of abnormal health conditions and infections. Biophys. Rev. 13, 359–385 (2021). 84. Suni II Substrate materials for biomolecular immobilization within electrochemical biosensors Biosensors (Basel) 2021 11 239 10.3390/bios11070239 34356710 85. Liang X Carbon-based SERS biosensor: from substrate design to sensing and bioapplication NPG Asia Mater. 2021 13 8 10.1038/s41427-020-00278-5 86. Kim J Non-invasive mouthguard biosensor for continuous salivary monitoring of metabolites Analyst 2014 139 1632 1636 10.1039/C3AN02359A 24496180 87. Chen, Y., Xianyu, Y. & Jiang, X. Surface modification of gold nanoparticles with small molecules for biochemical analysis. Acc. Chem. Res. 50, 310–319 (2017). 88. Guo Q Li F Self-assembled alkanethiol monolayers on gold surfaces: resolving the complex structure at the interface by STM Phys. Chem. Chem. Phys. 2014 16 19074 19090 10.1039/C4CP00596A 24777119 89. Zamani, M. et al. Surface requirements for optimal biosensing with disposable gold electrodes. ACS Meas. Sci. Au. 2, 91–95 (2022) 90. Samanta D Sarkar A Immobilization of bio-macromolecules on self-assembled monolayers: methods and sensor applications Chem. Soc. Rev. 2011 40 2567 2592 10.1039/c0cs00056f 21264402 91. Mani V Electrochemical sensors targeting salivary biomarkers: a comprehensive review TrAC Trends Anal. Chem. 2021 135 116164 10.1016/j.trac.2020.116164 92. Lukose J Photonics of human saliva: potential optical methods for the screening of abnormal health conditions and infections Biophys. Rev. 2021 13 359 385 10.1007/s12551-021-00807-8 34093888 93. Taylor JJ A prototype antibody-based biosensor for measurement of salivary MMP-8 in periodontitis using surface acoustic wave technology Sci. Rep. 2019 9 11034 10.1038/s41598-019-47513-w 31363141 94. Huang Y Das PK Bhethanabotla VR Surface acoustic waves in biosensing applications Sens. Actuators Rep. 2021 3 100041 10.1016/j.snr.2021.100041 95. BelBruno JJ Molecularly imprinted polymers Chem. Rev. 2019 119 94 119 10.1021/acs.chemrev.8b00171 30246529 96. Fedorenko V Application of polydopamine functionalized zinc oxide for glucose biosensor design Polymers (Basel) 2021 13 2918 10.3390/polym13172918 34502958 97. Kim J Wearable salivary uric acid mouthguard biosensor with integrated wireless electronics Biosens. Bioelectron. 2015 74 1061 1068 10.1016/j.bios.2015.07.039 26276541 98. Lee, Y. et al. Wireless, intraoral hybrid electronics for real-time quantification of sodium intake toward hypertension management. Proc. Natl Acad. Sci. USA 115, 5377–5382 (2018). 99. Mannoor MS Graphene-based wireless bacteria detection on tooth enamel Nat. Commun. 2012 3 763 10.1038/ncomms1767 22453836 100. Tseng P Functional, RF-trilayer sensors for tooth-mounted, wireless monitoring of the oral cavity and food consumption Adv. Mater. 2018 30 1703257 10.1002/adma.201703257 101. Ludwig N Machine learning to detect Alzheimer’s disease from circulating non-coding RNAs Genomics Proteom. Bioinforma. 2019 17 430 440 10.1016/j.gpb.2019.09.004 102. Li Z Identifying key microRNA signatures for neurodegenerative diseases with machine learning methods Front. Genet. 2022 13 880997 10.3389/fgene.2022.880997 35528544 103. Ko J Machine learning to detect signatures of disease in liquid biopsies – a user’s guide Lab a Chip 2018 18 395 405 10.1039/C7LC00955K 104. Khamina K A microRNA next-generation-sequencing discovery assay (miND) for genome-scale analysis and absolute quantitation of circulating microRNA biomarkers Int. J. Mol. Sci. 2022 23 1226 10.3390/ijms23031226 35163149 105. Sander LE Hepatic acute-phase proteins control innate immune responses during infection by promoting myeloid-derived suppressor cell function J. Exp. Med. 2010 207 1453 1464 10.1084/jem.20091474 20530204 106. Jain S Gautam V Naseem S Acute-phase proteins: as diagnostic tool J. Pharm. Bioallied. Sci. 2011 3 118 127 10.4103/0975-7406.76489 21430962 107. Byrne ML Acute phase protein and cytokine levels in serum and saliva: a comparison of detectable levels and correlations in a depressed and healthy adolescent sample Brain Behav. Immun. 2013 34 164 175 10.1016/j.bbi.2013.08.010 23999491 108. Gulhar, R., Ashraf, M. A. & Jialal, I. Physiology, acute phase reactants, in StatPearls. StatPearls Publishing Copyright © 2022, (StatPearls Publishing LLC., Treasure Island (FL), 2022). 109. Devaraj S Singh U Jialal I The evolving role of C-reactive protein in atherothrombosis Clin. Chem. 2009 55 229 238 10.1373/clinchem.2008.108886 19095731 110. Megson E C-reactive protein in gingival crevicular fluid may be indicative of systemic inflammation J. Clin. Periodontol. 2010 37 797 804 10.1111/j.1600-051X.2010.01603.x 20618548 111. Pay JB Shaw AM Towards salivary C-reactive protein as a viable biomarker of systemic inflammation Clin. Biochem. 2019 68 1 8 10.1016/j.clinbiochem.2019.04.006 30995442 112. Ouellet-Morin I Validation of a high-sensitivity assay for C-reactive protein in human saliva Brain Behav. Immun. 2011 25 640 646 10.1016/j.bbi.2010.12.020 21236331 113. Cho, Y. R. & Oh, Y. I. Comparative analysis of C-reactive protein levels in the saliva and serum of dogs with various diseases. Animals 10, 1042 (2020). 114. Szabo YZ Slavish DC Measuring salivary markers of inflammation in health research: a review of methodological considerations and best practices Psychoneuroendocrinology 2021 124 105069 10.1016/j.psyneuen.2020.105069 33316694 115. Jacobs R Host biomarkers detected in saliva show promise as markers for the diagnosis of pulmonary tuberculosis disease and monitoring of the response to tuberculosis treatment Cytokine 2016 81 50 56 10.1016/j.cyto.2016.02.004 26878648 116. Helmi ZR Sabri RA Hameed BH Assessment of oral health status, leptin, and inflammatory markers in serum and saliva of patients with polycystic ovarian syndrome in reference to metabolic syndrome Mustansiriya Med. J. 2016 15 11 19 117. Sali V Dynamics of salivary adenosine deaminase, haptoglobin, and cortisol in lipopolysaccharide-challenged growing pigs Front. Vet. Sci. 2021 8 698628 10.3389/fvets.2021.698628 34722692 118. Bozovic D Racic M Ivkovic N Salivary cortisol levels as a biological marker of stress reaction Med. Arch. 2013 67 374 377 10.5455/medarh.2013.67.374-377 24601177 119. Arafah BM Measurement of salivary cortisol concentration in the assessment of adrenal function in critically ill subjects: a surrogate marker of the circulating free cortisol J. Clin. Endocrinol. Metab. 2007 92 2965 2971 10.1210/jc.2007-0181 17535998 120. Palmier, N. R. & Leme, A. F. P. Salivary alpha-1-antitrypsin and macrophage migration inhibitory factor may be potential prognostic biomarkers for oncologic treatment-induced severe oral mucositis. Support Care Cancer 29, 2939–2946 (2021). 121. Slavish DC Salivary markers of inflammation in response to acute stress Brain Behav. Immun. 2015 44 253 269 10.1016/j.bbi.2014.08.008 25205395 122. Fernandez-Botran R Correlations among inflammatory markers in plasma, saliva and oral mucosal transudate in post-menopausal women with past intimate partner violence Brain Behav. Immun. 2011 25 314 321 10.1016/j.bbi.2010.09.023 20888902 123. Izawa S The diurnal patterns of salivary interleukin-6 and C-reactive protein in healthy young adults Brain Behav. Immun. 2013 27 38 41 10.1016/j.bbi.2012.07.001 22796263 124. Riis JL Salivary cytokines in healthy adolescent girls: Intercorrelations, stability, and associations with serum cytokines, age, and pubertal stage Dev. Psychobiol. 2014 56 797 811 10.1002/dev.21149 23868603 125. Riis JL Salivary cytokines as a minimally-invasive measure of immune functioning in young children: correlates of individual differences and sensitivity to laboratory stress Dev. Psychobiol. 2015 57 153 167 10.1002/dev.21271 25604242 126. Aleksandra Nielsen A Saliva Interleukin-6 in patients with inflammatory bowel disease Scand. J. Gastroenterol. 2005 40 1444 1448 10.1080/00365520510023774 16316893 127. Äyräväinen, L. et al. Inflammatory biomarkers in saliva and serum of patients with rheumatoid arthritis with respect to periodontal status. Ann. Med. 50, 333–344 (2018). 128. Rathnayake N Salivary biomarkers for detection of systemic diseases PLoS ONE 2013 8 e61356 10.1371/journal.pone.0061356 23637817 129. Kosaka T Salivary inflammatory cytokines may be novel markers of carotid atherosclerosis in a Japanese general population: the Suita study Atherosclerosis 2014 237 123 128 10.1016/j.atherosclerosis.2014.08.046 25238219 130. Rapado-González Ó Human salivary microRNAs in Cancer J. Cancer 2018 9 638 649 10.7150/jca.21180 29556321 131. Park NJ Salivary microRNA: discovery, characterization, and clinical utility for oral cancer detection Clin. Cancer Res. 2009 15 5473 5477 10.1158/1078-0432.CCR-09-0736 19706812 132. Michael A Exosomes from human saliva as a source of microRNA biomarkers Oral. Dis. 2010 16 34 38 10.1111/j.1601-0825.2009.01604.x 19627513 133. Dang J MicroRNA-137 promoter methylation in oral lichen planus and oral squamous cell carcinoma J. Oral. Pathol. Med. 2013 42 315 321 10.1111/jop.12012 23121285 134. Jiang J Real-time expression profiling of microRNA precursors in human cancer cell lines Nucleic Acids Res. 2005 33 5394 5403 10.1093/nar/gki863 16192569 135. Tandon M Deep sequencing of short RNAs reveals novel microRNAs in minor salivary glands of patients with Sjögren’s syndrome Oral. Dis. 2012 18 127 131 10.1111/j.1601-0825.2011.01849.x 21895886 136. Nijakowski K Surdacka A Salivary biomarkers for diagnosis of inflammatory bowel diseases: a systematic review Int. J. Mol. Sci. 2020 21 7477 10.3390/ijms21207477 33050496 137. Setti G Salivary microRNA for diagnosis of cancer and systemic diseases: a systematic review Int. J. Mol. Sci. 2020 21 907 10.3390/ijms21030907 32019170 138. Sommakia S Baker OJ Regulation of inflammation by lipid mediators in oral diseases Oral. Dis. 2017 23 576 597 10.1111/odi.12544 27426637 139. Singh S Evaluation of serum and salivary lipid profile: a correlative study J. Oral. Maxillofac. Pathol. 2014 18 4 8 10.4103/0973-029X.131881 24959029 140. Hatanaka, K. et al. Enzymatic measurement of short-chain fatty acids and application in periodontal disease diagnosis. PLoS ONE 17, e0268671 (2022). 141. Kawase T Simultaneous determination of 7 short-chain fatty acids in human saliva by high-sensitivity gas chromatography-mass spectrometry CHROMATOGRAPHY 2020 41 63 71 10.15583/jpchrom.2019.025 142. Labat C Inflammatory mediators in saliva associated with arterial stiffness and subclinical atherosclerosis J. Hypertens. 2013 31 2251 2258 10.1097/HJH.0b013e328363dccc 23868086 143. Gaber F Increased levels of cysteinyl-leukotrienes in saliva, induced sputum, urine and blood from patients with aspirin-intolerant asthma Thorax 2008 63 1076 1082 10.1136/thx.2008.101196 18757457 144. Syndergaard B Salivary biomarkers associated with gingivitis and response to therapy J. Periodontol. 2014 85 e295 e303 10.1902/jop.2014.130696 24502627 145. Gümüş P Evaluation of the gingival inflammation in pregnancy and postpartum via 25-hydroxy-vitamin D3, prostaglandin E2 and TNF-α levels in saliva Arch. Oral. Biol. 2016 63 1 6 10.1016/j.archoralbio.2015.11.018 26658365 146. Sánchez GA Salivary IL-1β and PGE2 as biomarkers of periodontal status, before and after periodontal treatment J. Clin. Periodontol. 2013 40 1112 1117 10.1111/jcpe.12164 24118119 147. Sánchez GA Relationship between salivary leukotriene B4 levels and salivary mucin or alveolar bone resorption, in subjects with periodontal health and disease J. Periodontal Res. 2013 48 810 814 10.1111/jre.12070 23488687 148. Slomiany BL Lipid composition and viscosity of parotid saliva in Sjögren syndrome in man Arch. Oral. Biol. 1986 31 699 702 10.1016/0003-9969(86)90100-7 3477215 149. Tishler M Salivary eicosanoid concentration in patients with Sjögren’s syndrome Ann. Rheum. Dis. 1996 55 202 204 10.1136/ard.55.3.202 8712887 150. Fineide F Characterization of Lipids in saliva, tears and minor salivary glands of Sjögren’s Syndrome patients using an HPLC/MS-based approach Int. J. Mol. Sci 2021 22 8997 10.3390/ijms22168997 34445702 151. Longo M TM6SF2/PNPLA3/MBOAT7 loss-of-function genetic variants impact on NAFLD development and progression both in patients and in in vitro models Cell Mol. Gastroenterol. Hepatol. 2022 13 759 788 10.1016/j.jcmgh.2021.11.007 34823063 152. Maciejczyk M Zalewska A Gerreth AK Salivary redox biomarkers in selected neurodegenerative diseases J. Clin. Med 2020 9 497 10.3390/jcm9020497 32059422 153. Bullon P Newman HN Battino M Obesity, diabetes mellitus, atherosclerosis and chronic periodontitis: a shared pathology via oxidative stress and mitochondrial dysfunction? Periodontol 2000 2014 64 139 153 10.1111/j.1600-0757.2012.00455.x 24320961 154. Maciejczyk, M. & Nesterowicz, M. Oxidation, glycation, and carbamylation of salivary biomolecules in healthy children, adults, and the elderly: can saliva be used in the assessment of aging? J. Inflamm. Res. 15, 2051–2073 (2022). 155. Šteňová, E., Bakošová M. & Lauková, L. Biological anti-TNF-α therapy and markers of oxidative and carbonyl stress in patients with rheumatoid arthritis. Oxid. Med. Cell Longev. 2021, 5575479 (2021). 156. Tóthová L Salivary markers of oxidative stress in oral diseases Front. Cell Infect. Microbiol 2015 5 73 10.3389/fcimb.2015.00073 26539412 157. Vona, R. & Gambardella, L. Biomarkers of oxidative stress in metabolic syndrome and associated diseases. Oxid. Med. Cell Longev. 2019, 8267234 (2019). 158. Maciejczyk, M. et al. Salivary redox biomarkers in insulin resistance: preclinical studies in an animal model. Oxid. Med. Cell Longev. 2021, 3734252 (2021). 159. Zalewska A Dysfunction of salivary glands, disturbances in salivary antioxidants and increased oxidative damage in saliva of overweight and obese adolescents J. Clin. Med 2020 9 548 10.3390/jcm9020548 32079369 160. Fejfer K Oxidative modification of biomolecules in the nonstimulated and stimulated saliva of patients with morbid obesity treated with bariatric surgery Biomed. Res. Int. 2017 2017 4923769 10.1155/2017/4923769 29457027 161. Smriti K Salivary glucose as a diagnostic marker for diabetes mellitus J. Diabetes Sci. Technol. 2016 10 991 992 10.1177/1932296816637619 26961976 162. Maciejczyk M Taranta-Janusz K Wasilewska A Kossakowska A Zalewska A A case-control study of salivary redox homeostasis in hypertensive children. Can Salivary Uric Acid be a Marker of Hypertension? J. Clin. Med. 2020 9 837 10.3390/jcm9030837 32204502 163. Maciejczyk M Szulimowska J Taranta-Janusz K Wasilewska A Zalewska A Salivary gland dysfunction, protein glycooxidation and nitrosative stress in children with chronic kidney disease J. Clin. Med. 2020 9 1285 10.3390/jcm9051285 32365532 164. Ghimenti, S. & Lomonaco, T. Salivary lactate and 8-isoprostaglandin F(2α) as potential non-invasive biomarkers for monitoring heart failure: a pilot study. Sci. Rep. 10, 7441 (2020) 165. Abdul Rehman S Role of salivary biomarkers in detection of cardiovascular diseases (CVD) Proteomes 2017 5 21 10.3390/proteomes5030021 28783097 166. Galindez JM Salivary heme oxygenase-1: a potential biomarker for central neurodegeneration J. Cent. Nerv. Syst. Dis. 2021 13 11795735211029114 10.1177/11795735211029114 34290541 167. Gornitsky M Altered levels of salivary 8-oxo-7-hydrodeoxyguanosine in breast cancer JDR Clin. Trans. Res. 2016 1 171 177 30931794 168. Porto-Mascarenhas EC Salivary biomarkers in the diagnosis of breast cancer: a review Crit. Rev. Oncol. Hematol. 2017 110 62 73 10.1016/j.critrevonc.2016.12.009 28109406 169. Guentsch A Lipid peroxidation and antioxidant activity in saliva of periodontitis patients: effect of smoking and periodontal treatment Clin. Oral. Investig. 2008 12 345 352 10.1007/s00784-008-0202-z 18509684 170. Wenk MR The emerging field of lipidomics Nat. Rev. Drug Discov. 2005 4 594 610 10.1038/nrd1776 16052242 171. Wolfram RM Salivary isoprostanes indicate increased oxidation injury in periodontitis with additional tobacco abuse Biofactors 2006 28 21 31 10.1002/biof.5520280103 17264390 172. Morrow JD Increase in circulating products of lipid peroxidation (F2-isoprostanes) in smokers. Smoking as a cause of oxidative damage N. Engl. J. Med. 1995 332 1198 1203 10.1056/NEJM199505043321804 7700313 173. Huang Y Mass spectrometry-based metabolomic profiling identifies alterations in salivary redox status and fatty acid metabolism in response to inflammation and oxidative stress in periodontal disease Free Radic. Biol. Med. 2014 70 223 232 10.1016/j.freeradbiomed.2014.02.024 24607715 174. Buduneli N Fatty acid profiles in smokers with chronic periodontitis J. Dent. Res. 2011 90 47 52 10.1177/0022034510380695 21041552 175. Kułak-Bejda A Waszkiewicz N Bejda G Zalewska A Maciejczyk M Diagnostic value of salivary markers in neuropsychiatric disorders Disease Markers 2019 2019 4360612 10.1155/2019/4360612 31191750 176. Nguyen TT Salivary oxidative stress biomarkers in chronic periodontitis and acute coronary syndrome Clin. Oral. Investig. 2017 21 2345 2353 10.1007/s00784-016-2029-3 27987039 177. Ciui B Cavitas electrochemical sensor toward detection of N-epsilon (carboxymethyl)lysine in oral cavity Sens. Actuators B: Chem. 2019 281 399 407 10.1016/j.snb.2018.10.096 178. Arakawa, T. et al. A wearable cellulose acetate-coated mouthguard biosensor for in vivo salivary glucose measurement. Anal. Chem. 92, 12201–12207 (2020). 179. Tseng, P. et al. Functional, RF-trilayer sensors for tooth-mounted, wireless monitoring of the oral cavity and food consumption. Adv. Mater. 30, e1703257 (2018).