In living organisms under physiological conditions, there is a preservation of the sub-cellular redox balance, also called “redox homeostasis”. Disruption of redox homeostasis, due to an unbalance between prooxidant and antioxidant reactions, results in oxidative stress and in altered redox signalling [1,2]. In turn, this contributes to aging [3] and to the onset and progression of many disorders, including neurodegenerative diseases [4], cardiovascular diseases/atherosclerosis [5], metabolic diseases such as diabetes [6], oral diseases as periodontitis [7], and cancers [8]. Indeed, mounting evidence has proven that oxidative stress and chronic inflammation are strictly linked in the etiopathogenesis of these disorders [[9], [10], [11]].

The inflammatory scenario is characterized by an increase in oxidation. Inflammation may be triggered by oxidative stress and increased levels of reactive oxygen species (ROS), frequently through the activation of transcription factors and expression of pro-inflammatory genes. On the other hand, inflammation causes immune cells to secrete various cytokines and chemokines acting in the amplification of the immune cell recruitment and activity at the site of injury; this determines an enhanced ROS generation, that in turn causes oxidative stress, further tissue damage, and inflammation [12,13]. Accordingly, reducing oxidative stress through antioxidants treatment has been considered as a potentially effective anti-inflammatory therapeutic strategy [14].

Inside the cells, oxidants (free radicals or non-radical species) attack nucleic acids, proteins, carbohydrates, and mainly unsaturated lipids, especially polyunsaturated fatty acids (PUFAs) [11,15,16], forming lipid hydroperoxides and, as by-products, highly bio-reactive carbonyl species. These include 4-hydroxy-2-nonenal (4-HNE), propanal, hexanal, and malondialdehyde (MDA) [11,15,16], all of which occur via both chemical and enzyme-catalyzed reactions. Antioxidant defence mechanisms can address physiological or low levels of LipoPeroxidation by-Products (LPb-Ps), however chronic development of abnormally large amounts of end-products from lipid oxidation overwhelms cell repair, accelerating aging and facilitating the occurrence of some pathological conditions [[3], [4], [5], [6], [7], [8],[17], [18], [19], [20]]. In particular, MDA is produced by enzymatic and non-enzymatic processes, linked to the decomposition of polyunsaturated fatty acids, particularly linoleic acid and arachidonic acid, present mainly in phospholipids [21]. It is also produced during arachidonic acid metabolism [21]. Upon its synthesis, MDA can react with specific groups on biomolecules, DNA and proteins principally, forming MDA adducts. It has been reported as the most mutagenic molecule among the ROS by-products, thus playing a role in the cancerogenesis process [16,21]. At controlled doses, MDA may also act as a signalling messenger and a regulator of gene expression, both as free MDA or as MDA adducts [[21], [22], [23]]. In conclusion, the overall picture suggests that LPb-Ps can be widely used as effective biomarkers of oxidative stress for an early diagnosis and a valuable and efficacious corrective intervention.

Few technologies for detecting LPb-P concentrations in biological fluids have been developed in recent years. Unfortunately, these analytical procedures require a complex and time-consuming multi-step sample preparation, and thus they do not appear useful for a routine diagnostic use [20]. Among LPb-Ps, MDA represents a valuable biomarker with predictive utility in clinical practice. Since aldehydes are released in biological fluids (blood, saliva, gingival crevicular and cerebral fluids, urine) when cells are harmed by lipid peroxidation, serum or plasma levels of MDA have been used as an indirect diagnostic indicator of oxidative stress in several acute and chronic conditions and diseases. These include cardiovascular disorders such as myocardial infarction and reperfusion hypoxia, atherosclerosis, nephropathy, chronic obstructive pulmonary disease, diabetes, Alzheimer disease [[24], [25], [26], [27], [28], [29], [30]], and different types of cancer. Increased levels of free MDA have also been found in the plasma of amyotrophic lateral sclerosis patients, women with preeclampasia [30], or in the plasma and breath condensates from asthmatic subjects [27]. Very recently, plasma MDA content has been evaluated as a factor associated with a negative prognosis and exitus of the multiorgan dysfunction syndrome in critically ill patients [28]. Finally, detection of MDA in oral fluids may contribute to estimating the degree of oxidative stress-related tissue damages and, thus, to the accurate diagnosis and follow-up of several oral diseases, such as oral squamous cell carcinoma [29] and chronic inflammatory conditions such as periodontitis.

Periodontitis is considered the most frequent cause of tooth loss in adults in industrialized countries [31]. It is a dysbiotic bacterial biofilm-initiated, chronic, inflammatory, multifactorial disease, causing the progressive resorption of tooth-supporting tissues and, finally, tooth loss, with potential significant consequences for masticatory function, aesthetics, and systemic and psychological health. The imbalance of the ROS and antioxidant systems may contribute to functional and structural periodontal tissue changes that favors the occurrence of periodontitis [32].

The main strategy to make a diagnosis of periodontitis is the detection of clinical periodontal parameters such as bleeding on probing, probing depth, and clinical attachment level, as well as the radiographic evidence of alveolar bone loss. This clinical approach, strongly affected by the operator's skill and experience, does not allow for early diagnosis and therapy since periodontitis is usually detected after the biological damage has already occurred. Modern dentistry may today rely on adjunctive practical and non-invasive diagnostic aids through the assessment of inflammatory and oxidative stress biomarkers in oral fluid specimens (saliva and gingival crevicular fluid, GCF). This strategy implies that periodontitis may be detected early, at the onset, when the periodontal damage is in progress. The levels of oxidative stress parameters in saliva, such as MDA, can reflect the activity and severity of periodontitis [32].

According to recent meta-analyses [33,34], patients with periodontitis have significantly elevated MDA levels in both their GCF and saliva. The increased MDA level in GCF does not only reflect the elevated periodontal tissue oxidative damage but also suggests an overlayed status of inflammation. In fact, the elevated level of MDA in GCF could be derived from superoxide anion production during the interaction between periodontal pathogens or their metabolites with neutrophils within periodontal tissues. This might be associated with an increased leakage of GCF in the saliva of periodontitis patients [34] and suggests that the more severe the inflammation status of the periodontal tissue, the higher the level of released MDA would be. On these bases, salivary levels of MDA might potentially be utilized as an indicator of an oxidative stress-dependent periodontal injury [[33], [34], [35], [36]]. More precisely, MDA, along with immune response host products, such as the inflammatory interleukins 1β and 6 (IL-1β and IL-6), and factors related to extracellular tissue degradation, such as the matrix metalloproteinase-8 (MMP-8), are considered the most reliable biomarkers of periodontitis. It has been shown a strong correlation between the onset or progression of periodontitis and the presence in the GCF and, through this, in the saliva, of these inflammation-associated biomolecules. Detecting MDA in oral fluids, in combination with MMP-8 and IL-6, may be most effective to diagnose periodontitis and to monitor individual therapy response, especially in subjects who, according to evidence-based medicine, are more susceptible to developing periodontitis, and particularly to develop severe (Stage 3 and Stage 4) and/or rapidly progressive (Grade C) periodontitis, less responsive to standard prevention/treatment, and presumably more predisposed to have periodontitis-impacting systemic diseases.

On the other hand, so far, a definite physiological MDA concentration range in body fluids, particularly in oral fluids such as saliva, has not been established. This is probably due to the presence of many factors that affect its levels, including age, smoking habits, body mass index, daily diet, and many lifestyle behaviours. Moreover, differences in the basal MDA levels might be due to the specific methods for quantification. Despite that, putative reference MDA values have been determined. They are reported as 1.25 μM mean value (0.36–2.80 μM) in plasma of adult subjects, increasing to 2.54 (2.19–3.63) μM in elderly [37], or 6.7 μM (SD 0.46) total MDA concentration, as determined by Mendonça et al. [38], as MDA quantity per gram of carnitine in urine (0.07–0.12 mg/g) [39], and as mean salivary MDA levels of 0.42 ± 0.08 μM in periodontally healthy subjects [40]. The same authors also reported a value of 0.80 ± 0.09 μM MDA concentrations in saliva of patients with chronic periodontitis [40]. These data should however be considered with caution, and well-designed large-scale studies, with adequately matched controls and correct distribution across the clinical disease stages are still strongly required to definitely determine the precise MDA concentration ranges in the different stages of the diseases associated to oxidative stress and in the prognosis of inflammation-associated conditions. A major difficulty to be overcome regards the analytical method that must be applied to detect MDA (as free compound or as adducts).

Several tests, mainly based on thiobarbituric acid-reactive substance (TBARS) or dinitrophenyl hydrazine (DNPH) condensation, have been developed and widely used to detect MDA and oxidative stress. However, they are not specific for MDA determination since the presence of other aldehydes represents a strong confounding factor [41]. Particularly, both TBARS- and DNPH-based strategies might be affected by methodological inaccuracies due to the derivatization process that is used to increase sensitivity [42]. Particularly, the biological sample processing is carried out at high temperatures (∼95 °C) and acidic conditions. This can generate a further matrix oxidation with overestimation of the results. An improvement in the specificity of TBARS- and DNPH-based methods can be obtained by means of techniques such as HPLC (high pressure liquid chromatography) coupled with UV-absorbance or fluorimetric detection or by GC-MS (gas-chromatography-mass-spectrometry) [38,[43], [44], [45], [46]]. Although these methodologies might be automated, their use for routinely clinical analyses appear unlikely. More recently, ELISA assays have been introduced for conjugated MDA [47]. These methods present several advantages, particularly in terms of sample preparation and specificity; however, they are time-consuming and require dedicated laboratory equipment.

In the last years, the need for point-of-care diagnostics has accelerated the development of straightforward, portable devices that are suited for any scope and may be used to assess the systemic redox balance in clinical settings, at the patient side. On our knowledge, few assay kits are commercially suitable for point-of-care MDA detection, primarily developed as test strips for monitoring oxidative stress degree by the semi-quantitative detection of MDA amount. These tests have been developed as colorimetric and no precise measurement of MDA concentrations are achieved. Other commercially available kits can be more precise, but in a linear range from 1 to 100 μM, reached only by using plate readers and high temperatures for incubations. However, as described above, these conditions can alter the MDA amount in the biological specimens during the in vitro handling.

The objective of our study is to exploit surface plasmon resonance (SPR) technique to develop a portable plasmonic-based plastic optical fiber (POF) biosensing chip for highly sensitive detection of MDA (free and conjugated) in biological fluids. The obtained POF biosensor relies on the high specificity of antigen recognition due to an anti-MDA antibody self-assembled monolayer (SAM) on a gold surface, while the monitoring is achieved by a simple experimental setup based on two components: a white light source and a spectrometer [48]. The experimental results reported here will be useful for developing biosensing approaches based on this kind of PoC-Ts for monitoring oral diseases like periodontitis, oral cancers, or systemic oxidative-stress associated pathologies.