Review Salivages 2510 Final Scanare Plagiat [622286]

1. Introduction
The success of a treatment, and even the difference between life and death, is often
related to early detection of the diseases. This desiderate is based in almost all situations on the
presence signaling or changes in concentrations for one or more analytes in biological fluid s.
These analytes are called markers or biomarkers based on their structure and the detection
environment i s very diverse, from blood to urine, saliva, tears or other body fluids. Due to the
benefits related to its ease sampling, manipulation and preservati on compared to blood, saliva is
increasingly used for dete ction and monitoring of biomarkers that accurate ly reflect normal and
abnormal states in humans. Thus, there is a great demand on developing new strategies,
procedures and methodologies for the fast and simple determination of different salivary
biomarkers even if they are found in small concen trations (in traces) . Another interest relies on
the compatibility with point -of-care (POC) applications, in order to improve clinical diagnosis
and treatment.
Biomarkers ( biological markers ) represent medical signs whi ch are accurately
measurable and reproducible , indicating the medical state of the patient [1]. A medical sign is
different from a medical symptom, as the last one can be identified by the patient itself. It is very
important that these signs should be the subject of accurate and reproducible measurem ent. The
World Health Organization (WHO) has given several definitions to a biomarker, such as “any
substance, structure, or process that can be measured in the body or its products and influence or
predict the incidence of outcome or disease” [1] or “a chemical, its metabolite, or the product of
an interaction between a chemical and some target molecule or cell that is measured in the
human body” [2]. Another definition of biomarkers stated by the WHO describes biomarkers as
"almost any measurement reflecting an i nteraction between a biological system and a potential
hazard, which may be chemical, physical or biological. The measured response may be
functional and physiological, biochemical at the cellular level, or a molecular interaction." [ 3].
This definition in cludes in the same category a substance, a structure, or a process that can be
measured in the body or its products and influence or predict the incidence and outcome of
disease as well as the monitoring of disease progression. Food and Drug Administration (FDA)
reports a biomarker as being ”a defined characteristic that is measured as an indicator of normal
biological processes, pathogenic processes or responses to an exposure or intervention, including

therapeutic interventions ” [4]. Thus, pulses , blood pressure, basic chemical compounds that are
the subject of com plex laboratory tests, are considered biomarkers [ 1].
Salivary fluid is a body fluid representing in fact a complex multianalyte matrix that
reflects hormonal, emotion al, immunological, metabolic and nutritional state of a person. Saliva
is the result of the exocrine secretion consisting mainly of water (about 99 %), electrolytes,
glucose, nitrogenous p roducts and various biocompounds. The main electrolytes are represen ted
by sodium, potassium, calcium, chloride, magnesium, bicarbonate and phosphate, while the
biocomponents are represented by enzymes, immunoglobulins, mucosal glycoproteins, albumin,
polypeptides, oligopeptides , antimicrobial factors , etc. All these compo nents are of great
importance for the health of the oral cavity and are responsible for the various functions
attributed to saliva. S alivary fluid includes the production of the salivary glands, the gingival
fold, mucous of the nasal cavity and pharynx, fo od scraps, cells from blood or epithel ial
desquamated ones and traces of chemical product s [5]. About 2000 proteins are present in human
saliva, and more than a quarter of these proteins are also found in human serum, which proves
once again the importance of saliva as a biological tool for disease diagnosis and treatment
monitoring [ 6-8]. An average daily pro duction of saliva for a healthy person ranges from 1 to 1.5
L (unstimulated and stimulated saliva ) [9, 10]. Saliva presence is critical for preserving and
maintaining the health of oral tissues and has been used as a source of non -invasive investigation
of metabolism and the elimination of many drugs.
There are three main conditions for technologies to be suit able for clinical applications:
(a) simple and inexpensive method for collecting biological samples with minimal discomfort for
patiens; (b) the existence of specific biomarkers as sociated with health or disease; (c) accurate,
portable and easy -to-use technology for disease diagnosis and health scr eening. Due to the low
levels of biomarkers in saliva , it becomes difficult to distinguish between background and target –
specific signal. Therefore, methods providing low limits of detection are required when
analyzing saliva. The vast m ajority of the meth ods traditionally used for salivary analysis are
applicable only at the laboratory level, most of these involving many st eps, such as sampling,
preconditioning, storage of the samples between sampling and testing, the actual testing, and
finally processing , analysis and reporting t he obtained results. Thus , the development of methods
and technolog ies in saliva ry diagnosis is important to allow the reliable detection of biomarkers
in this complex matrix. There are already several commercially available and r eliable devices for

protein assays but are all limited to laboratory tests, and thus shows limitations for POC
applications. Moreover, the instruments used for these tests are expensive, being accessible only
to large laboratories with high financial avail ability for maintenance and exploitation.
The main analytes that are currently tested from saliva, using commercially available
instruments are glucose, different hormones (cortisol, estriol, estradiol, estrone, estrogen,
progesterone, testosterone and dehydroepiandrosterone sulfate), human papilloma virus (HPV),
HIV antibody, different bacteria, interleukin 1 (IL-1) and interleukin 6 (IL-6) [11, 12 ]. However,
the increasing recognition of the correlation between the oral and overall health of humans had
led to the growing interest in using saliva for diagnostic purposes as well as for the discovery of
new biomarkers. Thus, the number of commercially available salivary diagnostic devices does
not correspond to the large number of possible applications and novel ones need to be developed.
Among techniques commonly used for the determination of salivary biomarkers we mention here
Liquid Chromatography –Mass Spectrometry (LC –MS) and UV –VIS Spectrophotometry for
glucose levels [ 13], Enzyme -Linked Immunosorbent Assay (ELISA), electrophoretic
immunoassay, Radioimmunoassay (RIA), mass spectrometry -based proteomics for protein
biomarkers, DNA based techniques for genetic biomarkers [ 8], microchips and micro fluidic
devices [ 14] and electrochemical biosensors . The last ones are simple and low cost technologies
that are important due to the simplicity of operation and instrumentation, easy integration in
compact analytical devices and suitability to multiplexed detection of a large plethora of
biomarkers [15-17]. Thus , the conductivity, selectivity and ability for immobilization of
biomolecules of electrochemical sensors are enhanced after the modification of the electrode ,
while the use of nanomaterials determine significant improvement s in sensitivity [12, 18].
This review focuses on the employment of several analytical techniques used for the
detection and quantification of salivary biomarkers with predictability in local or systemic
pathologies. The pros and cons will be presented highlighting the promising met hods for future
POC devices that used saliva as sources of diseases biomarkers.
2. The importance of saliva as real samples for biomarkers detection
Due to the recent advances in technology, saliva has become attractive for clinic al
applications and it is increasingly used for diagnosis and therapy monitoring , since it contains
several markers for toxicological and infectious diseases [ 19]. Moreover, the need for non –
invasive methods for biomarkers detection gained a significant im portance due to the increasing

numbers of pathologies which associate the biomarkers levels from different biological fluids,
with early screening and diagnosis. Sialometry and sialochemistry are more and more used to
monitor the general state of health, t o diagnose systemic illnesses, and as an indicator of risk for
diseases creating a close relation between oral and systemic health. It has been demonstrated that
saliva is in permanent exchange of substances with human serum thus both human fluids
contain ing the same biomarkers . Moreover, saliva is simple to collect and to processes, with
minimal invasiveness and low costs for collection . Salivary proteins as potential diagnostic
markers for various diseases such as breast cancer, ovarian cancer, Sjögren’s syndrome,
hepatocellular carcinoma, leukoplakia and oral cancer were of interest for many researchers [20].
Although there is a good correlation betw een the amount of some biomarker in saliva and serum ,
this is not completely clarified and understood why and how , thus a relationship between the
amount of these markers in saliva and the dise ases is more difficult to be establish. Moreover, the
therapeutic intervention based on the detection and quantification in saliva is therefore more
challenging com pared with other body fluids [ 21]. The advantages offered by saliva compared
with other biological samples are well known, but there are also several issues associated with its
use. The most important problems are related with its variable and heterogeneou s composi tion,
and its higher levels of mucins and proteolytic enzymes. Furthermore, the concentration of
biomarkers may be several orders of magnitude lower than in serum [ 15], the diurnal variations
in composition can lead to troubles in result interpret ation and its complex composition can
cause problems in sample preparations. However, the clinical utility of salivary diagnostics for
the assessment of systemic disease remains elusive even if in the last years a number of studies
showed the importance of fast screening non -invasive tools [22-25]. Despite of this , biomarkers
still can reflect the same expression profiles as found in blood and their detection may be
relevant for diagnosis.
Saliva may facilitate toxicological and biochemical investigation, being consi dered the
best research tool for scientific investigations on humans from the ethical point of view.
However, consents from the patients or voluntary participants have to be obtained prior to the
collection of samples and all local guidelines reg arding the work with human saliva have to be
followed before starting any study [ 26]. The most important applications of salivary analysis in
toxicology refers to the detection of drug dependence and alcohol abuse [ 27, 28 ], forensic
medicine [ 29] and the c orrel ation between the levels of drugs in saliva and blood [ 30, 31 ]. So far,

saliva has been successfully used for the diagnosis of several diseases, based on the qualitative
and/or quantitative determ ination of biomarkers. The envisaged pathologies are: local (oral
cancer [ 32]); endocrinological (diabetes or Cushing's syndrome [ 33, 34 ]); autoimmunological
(Sjörgen's sy ndrome, Hashimoto thyroiditis, celiac disease [ 35-37]); infectious (HIV, viral
hepatitis, malaria ([ 38, 39 ]); gastroesophageal reflux [ 40]; neurology, psychiatry (Al zheimer’s
disease [41]); systemic (breast, ovaries, pancreas or lung cancers [ 42-45]).
2.1. Methods used for saliva collection and manipulation
When working with real samp les, crucial steps are represented by the collection and
storage procedures . Saliva sampling involves mostly simple and noninvasive collection
procedures, easy depositing and transport. This is useful for people with problems in collecting
blood samples (eg. neonates, toddlers, hemophiliacs, patients with blood clotting disorders,
elderly and disabled people). Furthermore, the compliance of people who need periodical or
clinical monitoring increases, thus increasing the feasibility for monitoring their health
progression and treatment outcomes. Unlike blood, sali va samples have reduced risk of cross
contamination and very low exposure of personnel. However, it is important to standardize the
method of collection in order to obtain significant results. Saliva samples are collected in an easy
manner as whole saliva and as saliva collected from an individual salivary gland. Whole saliva
collection may be done personally, by the patient or volunteers, without the need for qualified
staff or specialists. Unstimulated whole saliva can be obtained by ‘passive drooling’ or ‘spitting’
directly into a sterile container (calibrated conical tube suitable for centrifugation ) [46, 47].
Stimulated saliva can be collected through mechanical stimulation by chewing inert materials
such as cotton or polystyrene Salivette® and/or gusta tory stimulation by using sugar -free sour
lemon drops [48]. The use of citric acid for the stimulation of salivary production influences the
pH of the collected samples, so this method is not suitable if the pH of the saliva should be
measured. Although, methods of salivary collection may have significant influence on the
precision and determination of salivary markers, there are no established uniform criteria for the
collection of human saliva. It is important to mention that the amount of analytes in unstimulated
and stimulated saliva may be different even if the material is not lost during the collection
protocol. This can be due to the change in flow rates or the different contributions of salivary
glands. Henc e the effect of the collection tools on the recovery of analytes should be studied
prior to any test on real saliva. Individual gland saliva can be obtained by glandular duct

cannulation (a thin tube inserted into a duct to obtain saliva) or by using speci fic collecting
devices to the ducts of interest (aspiration or drainage). These sampling methods are not widely
used because are slow and complex, and require a specialist for collecting the samples. It is very
important to remain steady in collection proc edures both across all patients and within all
collection visits for every individual. Among the listed collection methods, ‘ passive drool’ is
considered a standard of collection methods across different analytes and usually produces
enough samples for ana lysis [ 49]. During the collection of saliva samples, the harvesting
container should be immersed in ice -bath to prevent degradation of biologically important
salivary analytes [ 50]. To minimize the external and internal influences on the results of salivar y
investigation, saliva should be collected between 8 and 10 AM . Subjects inv olved should brush
teeth the night before the collection, avoid cigarette smoking, fo od ingestion or chewing gum at
least 1 hour before collection, sh ould not drink anything than pure water, and should not perform
any hygienic procedures insi de the oral cavity for two hours before sampling. Because of the
influence of many drugs on salivary secretion, patients should not take drugs at least 8 hours
before salivary collection [ 50]. Contamination of the collection pad must be avoided , by
avoiding the contact with skin, which presents numerous compounds and their transfer to the
saliva sample would generate erroneous results. Such an example is represented by lactate
detection in saliv a since it is found in large amounts on the skin and in sweat [ 51]. Nevertheless,
it is essential that devices used to collect oral fluid are checked to ensure reasonable stabilit y and
recovery of the analyte. T he most common applications are dealing with the workplace testi ng
for drugs of abuse, the roadsi de detection of illicit drugs and therapeutic drug monitoring after
detection in plasma or serum [ 52, 53 ].
2.2. Preservation and storage of saliva samples
After collection, saliva samples should be rapidl y processed or suitably stored, because
not all the components are stable at room temperature in this medium for long time. The first
category includes the inorganic compounds ( Mg, Ca, pho sphate), and the second one includes
hormones (cortisol and progeste rone) and proteins whose concentration has been found to be
stable over time only if the saliva samples have been stored at 5°C, −20°C and −80°C
respectively, immediately after sampling. In the case of proteins, their degradation is fast at room
temperature (about 30 minutes), thus, the use of protease inhibitors can help reduce this
phenomenon. Most of the target analytes in saliva can be prese rved if a chemical pretreatment

step is introduced before storage. For example, Nurkka et al. [ 53] have mixed the saliva sample
with an equal volume of 80% glycerol in H 2O and dipped into liquid nitrogen, thus succeeding
an instant freezing of the sample a nd inhibiting bacterial protease activity from degrading some
proteins in saliva. The concentration determined in this sample was about 50% higher than that
obtained in aliquots of the same saliva sample t hat were temporarily stored at 5°C even if the
samp le was treated with protease inhibitors. If necessary , the precipitation of the protein and the
extraction of the lipids to organic phase may be done, for example, by adding chloroform in to
the collecting tubes with saliva samples [ 54]. Care must be taken when pre -treatment is used,
because any added compound may produce interference and has to be investigated carefully [55 ].
The simple pretr eatment procedures (storage and transportation), or even more
complicate ones ( sample stabilization, lyses and signa l amplification ) can introduce uncertainties
into the saliva sample. For example, storing the saliva at -80oC provides long term stability of the
sample, but the thaw freeze cycle may damage the biomarkers inside the saliva. Usually, a
quality control should be carried out before measuring the saliva with traditional detection
methods [ 47]. Centrifugation of the salivary samples is included in the majority of the protocols
currently used for saliva pretreatment, when immediate testing is desired, as well as when the
medium or long term conservation of the real samples is desired. In this case, only the
deproteinized supernatant being further collected and store d for further analyse [55-58].
2.3. Testing human saliva – recovery tests
The recovery tests refer to the evaluation of the matrix effect on the performance of the
optimized analytical method used for the target analyte detection. Thus, direct detection is
applicable i n situation s when no pretreatment is required for the saliva before the measurement.
The collected samples can be directly used for measurements without any other interventions.
This is applicable only if the target analyte present high stabil ity at room temperature or the
analytical method is very fast. The direct detection is desirable because it removes the major
hindrance in the sample pretreatment [48, 55 , 57]. Recovery studies are usually carried out by
spiking collected saliva samples with different concentrations of target analyte, using standard
solution of known concentration [48, 59 ]. This is a type of quantitative analysis often used in
analytical chemistr y, so called standard addition method . This method is used in situations
where sample matrix also contributes to the analytical signal, a situation known as the matrix
effect, thus making it impossible to compare the analytical signal between sample and st andard

using the calibration curve obtained using standard probes [ 60]. The obtained recover ies should
be around 100% to assert that the chosen method is reliable for saliva detection.
3. Analytical and immunological methods used for biomarkers quantification
Methods used to detect biomarkers and metabolites from saliva include immunological
techniques, separation methods and lately , electrochemical methods through the use of
biosensors. Overviews of those methods are presented bellow. In most cases, biomarkers
analyz ed in saliva present a structure of peptides o r proteins. In these cases, the standard
analytical tool used for biomarker analysis is ELISA [20, 61-68]. However , recent studies have
shown that separation [69-77], or electrochemical [12, 78-84] methods can be useful alternatives
to the immunological assays used as ordinary analytical techniques.
3.1. Immunolog ical techniques
The immunological techniques that are capable of investigating tumoral and cellular
immune responses to oral organisms both in local oral tissues and fluids are enzyme -linked
immunosorbent assay (ELISA), which quantify specific antibody and cytokines in gingival
crevicular fluid , and saliva; flow cytometry used especially fo r the characterization of T cells
from peripheral blood and gingival tissues ; and immunohistologic al analysis of the
inflammatory cell infiltrate in gingival tissues [47]. Beside these , RIA and immunoblotting
techniques are other immunological methods useful for saliva analysis .
Modifications of these assays known as competitive inh ibition assays allow quantifying
the antigen (or antibody) in a mixture and determining the affinity of the antibody -antigen
interaction by using mathematical models. The key step, immobilization of the antigen of
interest, can be accomplished by direct adsorption to the assay plate or indirectly via a capture
antibody that has been attached to the plate. The antigen is then detected either di rectly (labeled
primary antibody) or indirectly (labeled secondary antibody). The most powerful ELISA assay
format is the sandwich assay. This t ype of capture assay is called “sandwich” because the analyte
to be measured is bound b etween two primary antibo dies: the capture antibody and the detection
antibody. The sandwich format is used because it is sensitive and robust. Competitive ELISA is a
strategy that is commonly used when the antigen is sma ll and has only one epitope ( antibody
binding site ). One variation of this method consists of labeling purified antigen instead of the
antibody. Unlabeled antigen from samples and the labeled antigen compete for binding to the

capture antibody. A decrease in signal from the purified antigen indicates the p resence of the
antigen in samples when compared to assay wells with labeled antigen alone. Antibodies are
directly labeled with alkaline phosphatase (AP) or horse radish peroxidase ( HRP ), this being
the most common ELISA detection strategy. HRP and AP subs trates typically produce a
colorimetric output that is read by a spect rophotometer. Detection can occur by fluores cently –
labeled antibodies, the assay being termed fluorescence -linked immunosorbent assay (FLISA).
Immunoblotting is usually performed in the form of Western blotting, which is reserved
to the detection of proteins and involves an electrophoresis separation step followed by
electroblotting of the separated proteins from the gel to a membrane and then probing with an
antibody. Detection of the antigen protein and antibody interaction is made in a similar way as in
RIA or ELISA depending on whether a radio -labeled or enzyme -coupled antibody is used.
Table 1 . Immunological methods and their advantages and disadvantages
Method Advantages Drawbacks
ELISA – useful for detecting and quantifying
substances such as peptides, proteins,
antibodies and hormones ;
– the detection strategy is based on a highly
specific antibody -antigen interaction . – many washing and incubation steps which
are time consuming ;
– false positive/ negative results alter the
reproducibility ;
– high cost due to use of antibodies ;
– allows the analysis of one protein per
plate ;
RIA – are capable of investigating t umoral and
cellular immune responses. – the use of a radio isotope require trained
personnel ;
– high cost of the technique .
Western blotting – enables the target protein to be identified in
a complex matrix ;
– produce qualitative/ semi -quantitative dat a
about the protein of interest ;
– used for direct and indirect detection ; – high cost of reagents ;
– expensive equipments ;
– need for skilled personnel ;
Immuno
fluorescence – suitable if the extract ion of the proteins
from the cells is not necessary . – use expensive flurophores ;
Flow cytometric
assays – enabling the detection of up to 100 different
analytes per assay;
– many protein analytes can be measured by
the multiplexed bead -based assay with a
single plate;
– extremely important for clinical studies
where sample volumes are limited;
– high accuracy due to the median
fluorescence;
– the readout is obtained of at least 50 –100
beads. – uses an expensive laser to excite t he
fluorescent labels ;
– cross -reactivity between antibodies;
– sensitivity may be compromised due to
the increase numbe r of beads;
– the performance in the m ultiplex assays
can be variable;
Abbreviations: ELISA – enzyme -linked immunosorbent assay , RIA – radioimmunological assay , HRP – horse radish
peroxidase , AP- alkaline phosphatase .

Fluorescent blotting is a newer technique and is growing in popularity as it affords the
potential to multiplex analysis . Whatever system is used in this technique , the intensity of the
signal should correlate with the abundance of the antigen on the membr ane.
3.2. Separation techniques
All types of separation methods, such as gas chromatography (GC) [71, 76 ] or LC [69,
70, 73 -75, 77 ], capillary electrophoresis, gel electrophoresis or electrochromatography [72, 75 –
77] have proved their val ue in the analysis of salivary biomarkers . The broad versatility, high
selectivity, efficiency and low time of analysis make them a good choice (Table 2) . An important
drawback of these method s is that the real sample needs a pretreatment which usually takes
longer than the anal ysis itself and, in the case of LC there is a high consumption of solvents.
Table 2. Separation methods and their advantages and disadvantages
Method Advantages Drawbacks
GC-MS – high sensitivity and selectivity ;
– relatively cheap ;
– low LOQs and LODs ;
– easy biomarker identification ;
– high efficiency . – not suitable for high molecular mass
proteins ;
– analytes must be volatile or easy to
derivatize in volatile compounds ;
– destructive method ;
– pretreatment can be long ;
LC-MS HPLC -MS – suitable for all types of
molecules;
– pretreatment is short. – high solvent consumption;
– relative ly expensive ;
– low ef ficiency ;
UPLC -MS – higher efficiency (compared to
HPLC);
– lower analysis time. – relatively expensive (compared to all
separation methods) ;
CE – suitable for all types of
molecules
– relatively cheap ;
– low analysis time;
– high efficiency . – ideal for ionized molecules ;
SDS-PAGE – dedicated to protei ns. – use of very toxic reagents ;
– gel-to-gel v ariation ;
CEC – suitable for all types of
molecules ;
– combines LC and CE principles . – relatively expensive ;
Abbreviations: GC -MS – gas chromatog raphy -mass spectrometry, LC -MS – liquid chromatography -mass
spectrometry, UPLC -MS – ultra -high performance liquid chromatography -mass spectrometry, CE – capillary
electrophoresis, SDS -PAGE – sodium dodecyl sulphate -polyacrylamyde gel electrophoresis, CEC – capillary
electrochromatography

3.3. Electrochemical methods

A large variety of electrochemical techniques are availab le for the detection of
biomarkers which are relevant from medical point of view in saliva. Thus, amperometry
measures the current produced by the oxidation or reduction of electroactive species in the
biochemical reaction, th e obtained current being related with the analyte concentration .
Potentiometry measures potential difference between a working electrode and a second reference
electrode under the condition of zero current flow . Voltammetric methods measure the current at
the surface of the working electrode as the potential is varie d, and electrochemical impedance
spectroscopy measures the dielectric properties of a medium as a function of frequency ( method
used for characterization of electrochemical systems and electrode functionalization ). More
sophisticated t echniques are square -wave voltammetry and differential pulse voltammetry ,
methods applied to minimize the influence of capacitative currents and for the improve ment of
the sensitivity and limit of detection for the measurements compa red with cyclic voltamme try
and linear sweep volt ametry . Despite all these advantages, electrochemical assays are relatively
less common in bi omedical and diagnostic aplications, since in many cases, the developed assays
and the optimized method s have not been tested with biological samples. This is mainly due to
the lack of validation related with t he difficulty of electrochemical measurement in real samples,
to poor specificity as there are many potenti al interferences . If the p roposed methods were
confirmed by v alidatio n, there would be more of these procedures used in diagnostic field.
Nowadays, electrochemical immunosensors with different sensing receptors and
transducers are consi dered promising tools in screening methods [ 78]. Specifically, over
conventional ELISA, the use of electrochemical immunosensors presents some advantages :
increased sensitivity; low detection limit s; low cost; simple design and ease of manipulation; low
consumption of expensive and/or toxic reagent. Electro chemical biosensors can be easy applied
in the dete ction of me tabolites for clinical, environmental and food analysis. Another advantage
of using biosensors is the possibility of sample process ing just after its sampling , without
separation steps , the response being obtained in minutes or even seconds. Another great
advantage is the specificity of the immobilized biocomponent and the selectivity , which make
the probe suitable for measurements in complex matrix , as are the biological fluids.
The electrochemical sensing devices present also several drawbaks due to the complexity
of the system if they are to be miniaturized. Typica lly, for the most of label -free electrochemical
sensor with a direct contact with liquid sample , many suplimen tary steps are necessary . The use

of nanomaterials in sensors determined enhanced sensitivity as well as increases the selectivity
of the electrochemical detection. Furthermore, t he development of various portable sensors :
microwire s, interdigitated, printed, stretchable and wearable electrodes , encouraged the uses of
electroanalytical methods in medical field [85]. Electrochemical sensors possesses great potential
for miniaturization technology, which includes improved accuracy, lower power and sample
consumption, and capab ility to be use in POC environments [ 86, 87 ].
3.4. Analytical parameters used to characterized analytical methods
Each analytical method is characterize d by its analytical parameters; the main ones being
needed for validation are mention by ICH [88] and are presented below .
Limit of detection (LOD) is the lowest amount of analyte in a sample which can be
detected b ut not necessarily quantified as an exact value. Limit of quantification (LOQ) is the
lowest amount of analyte in a sample which can be quantitatively determined with suitable
precision and accuracy being a parameter of quantitative assays for low levels of compoun ds in
samples . Linearity is the ability of an analytical procedure to obtain test results which are
direct ly proportional to the amount of analyte in the sample. Repeatability (intra-test accuracy) is
a measure of the exactness under the same working conditions . Specificity is the capacity of the
system to precisely gauge the analyte reaction in the vicinity of all potential specimen segments .
Range is the interval between the upper an d lower concentration of analyte in the sample for
whic h an analytical procedure has a suitable level of precision, accuracy and linearity. Precision
of a system is the event that it gives the right numerical response for the analyt e. Any technique
ought to have the capacity to figure out if the material being referred to comply with its detail for
instance, it ought to have the capacity to supply the accurate measure of substance present.
Accuracy is the measure of how close the information qualities are to one another for various
estimations under the same scientific conditions and is typically examined at three levels:
repeatability, transitional ex actness , and reproducibility . Reproducibility meas ures the accuracy
between labs, being considered in the institutionalization of a diagnostic methodolog y.
Robustness represents a measure of the system's ability to sta y unaffected by little varieties in
strategy parameters and provides an indication of its reliability during normal usage.
Even that the analytical techniques are well characterized false negative and false
positive results could appears especially when dealing with biological samples. In medicine are
known as false positive /negative diagnosis , while in statistical classification are false

positive /negative error [ 89]. False positive results refer to the situations in which the tests
performed generate positive results when they are actually negative. This situation is called
“false positive error”, term used in medical field and in software testing. False positive results
can cause serious problem s, especially if related to medical field (false positive diagnostic). In
statistics, a false positive is usually called a type I error ( the null hypothesis is incorrectly
rejected ). This creates a “false positive” for the research, leading the re searchers to believe that
their hypothesis is true, when in fact it isn’t. A related but opposite concept is represented by
false negative results . This refers to the situation in which the tests performed generate negative
results when they are actually pos itive. If a negative result is received erroneously and the null
hypothesis is not rejected (when, in fact it should be), this is known as a type II error. The terms
false positive/false negative and type I error /type II error are often used interchangeably, but
there are differences in detail and interpretation due to the differences between medical testing
and statistical hypothesis . False negative results can be found in different medical test ( for
alcoh ol or drugs , pregnancy , tuberculosis or Lyme disease), whi le an example of a false positive
is when a particular tes t designed to detect melanoma, tests positive for the disease, even though
the person does not have skin cancer. A good strategy w hich should be followed to avoid or at
least to minimize the possibility of getting inappropriate results is to double -check test results.
One could conclude that t he conventional strategies and methods used for the detection of
biomarkers in saliva are often the subject of some limitation. Aiming to overcome these
limitations like the need for skilled personal, the important cost of the equipments, high consume
of reagents, long analysis time etc., multiplexed assays and hyphenated techniques are
increasingly used. Even electrochemical methods offer interesting alte rnatives , due to the
advantages which results from their use, such as high sensitivity, selectivity, precision and
accuracy, low cost, small sample quantity needed, easiness in manipulation, simple
instrumentation, as well as the siutability for miniaturization and multiplexed detection.
Furthermore, coupling electrochemical transductors with different nanomaterials (e.g. carbon
nanotubes, graphene, metallic nanoparti cles or quantum dots ) greatly improve the sensing
performance while biomimetic elements (like aptamers or molecular imprinted polymers)
drastically increse the selectivity .
4. Pathologies diagnosed by using biomarkers from saliva

The compositio n of saliva can reflect the health of the local area (the oral cavity) and the
functioning of the entire organism, thus there have been identified different salivary biomarkers
with potential in prevention and/or diagnosis of different local and systemic pathologies. Several
pathologies could be diagnosed by using biomarkers and metabolites over expressed in saliva.
Thus, interleukins (IL -6, IL -8) [21, 83], L -phenylalanine and L -leucine [90] are reported in oral
cancer and oral squamous cell carcinoma (OSCC); ALT and P. gingivalis are associated with
periodontal disease [91]; lysophosphatidylcholine (18:1), lysophosphatidylcholine (22:6) and
monoacylglycerol (0:0/14:0/0:0) are related with breast cancer [75]; lactate is marker for cardiac
diseases [48, 51]; lactoferrin, β2-microglobulin 16 peptides and 27 mARN genes were used for
Sjörgen’s syndrome detection [72, 77]; 65 proteins were applied for diabetes mellitus, while
Alzheimer’s disease was tested using sphinganine -1-phosphate [70].
4.1. Local pathologies
Oral cancer is referred to a subgroup of head and neck malignancies affecting to lips,
tongue, salivary gl ands, oral cavity and other intraoral regions, being estimated by WHO as the
eight most frequent cancer worldwide. The majority of the oral cancers are located i n the oral
cavity, and 90% of these are OSCC [15, 20, 48, 79, 92, 93]. There is high mortality in oral
cancer, thus its early detection is challenging and may be done via the determination of several
biomarkers in biological fluids, the most common ones be ing IL -8, IL -6, IL -1β, IL -4, IL -10 [15,
20, 79, 92, 93 ], vascular endothelial growth factor (VEGF), human epidermal growth factor
receptor -2 (HER2), tissue polypeptide antigen (TPA) and epidermal growth factor receptor
(EGFR) [51, 94]. The use of saliva fo r the detection of oral cancer is envisaged by many
researchers but there is still a long way to using saliva as main matrix for early detection [87].
Some examples of immunological, separation and electrochemical methods used for detection of
biomarkers related with oral cancer are presented in Table 3 [20, 32, 51, 56, 90, 93, 95 -101].
Periodontal diseases represent chronic inflammatios of the periodontium caused by
persistent bacterial infectio n that results in tooth loss. Recent research studies have shown that
periodontal disease can act as a risk factor for cardiovascular and cerebrovascular diseases.
Several biomarkers were associated with these inflammation, soft tissue, and may be
determin ated in saliva (Table 3 ) [89, 102 -104].
4.2. Systemic pathologies

Knowledge about cancer and its biomarkers has increased during the last decades
providing great opportunities for improving the management of cancer patients by enhancing the
efficiency of detection an d efficacy of treatment. A broad range of biochemical compounds such
as nucleic acids, proteins, sugars, lipids, small metabolites, cytogenetic and cytokinetic
parameters as well as whole tumor cells found in the body fluid could be included (see examples
of cancer biomarkers and the methods used for their detection in Table 3 )[61, 62, 70 -72, 87 ].
Cardiac and cardiovascular diseases are the leading causes of death worldwide.
Inflammation is an important contributing factor for coronary heart diseases and atherosclerosis.
C-reactive protein (CRP) represent a strong risk factor for the development of cardiovascular
disease [105, 106]. There are a pletora of biomarkers for cardiac and cardiovascular diseases.
Among them, some of the most important and which can be detected from saliva are: myoglobin,
cardiac troponin I (cTnI), creatine phosphokinase MB (CK -MB), myeloperoxidase, brain
natriuretic peptide (NT -proBNP), exosomal miRNA, CRP, matrix metallo proteinase -8 (MMP -8),
MMP -9, tissue inhibitor of MMP -8 (TIMP -1), lactate THF -α, and other interleukins (Table 3 )
[48, 51 , 59, 107-121].
Sjörgen’s syndrome (SS) is an auto -immune disease which affect salivary and lachrymal
glands, characterized by dry mou th and dry eyes, and which could also develop to malignant
lymphoma. In this situation, early diagnostic and treatment of the disease is important, lactofer rin
and β2 -microglobulin being chosen as possible biomarkers for this disease [68, 69].
Diabetes mellitus (DM) is a metabolic disorder in which the concentration of glucose in
plasma is higher than 126 mg/mL, or in which blood glucose is above 200 mg/mL at any time of
day [121]. This abnormal values of glucose concentration are the consequences of abnormaliti es
in insulin secretion and action. There are three major types of DM that are preceded by pre –
diabetes, a metabolic condition wherein blood sugar rises to a level higher than that of the
normal range but lower than that of diabetes [122]. The causes that generate the development of
one or the other type of diabetes are different, and here monogenic diabetes syndromes (neonatal
diabetes and maturity -onset diabetes in young individuals), diseases of the exocrine pancreas
(cystic fibrosis), and drug – or chemi cal-induced diabetes (after the treatment for HIV/AIDS or
after organ transplantation) are included [123]. The development of any type of diabetes generate
a series of other healt problems such as heart and blood vessels diseases, eyes, kidneys, nerves,
teeth illnes or infections [ 124, 125 ]. Thus, it is very important to diagnose th ese diseasses in

early stages to prevent or delay long -term health complications. It is worthy to mention that in
the early stages all types of diabetes does not show any symptom s, thus fast and easy evaluation
of blood glucose levels remains the only way to prevent and slow the disease evolution. Periodic
harvesting of blood samples needed to analyze the glucose amount is an invazive and traumatic
procedure for most individuals, thus, the discovery of minimally invasive or noninvasive
procedures is a must. The use of saliva insteat of blood for diagnosis and treatment monitoring is
more and more intensively used but still challenging for researchers all over the world [ 6, 91 ,
125-127]. Many noninvasive technologies for continuous monitoring of glucose are currently
undergoing development [128], but none of them have reached till date the analytical
performance of the finger -prick testing [129-132].
Advanced glycation end -products (AGEs) are products obtained after glycation of protein
during the Maillard reaction. These analytes are involved in the development of long -term,
chronic complications such as: diabetes, diabetic neuropathy [133], atheroscl erosis,
hemodialysis -associated amyloidosis, oxidative stress and neurodegenerative diseases ( (e.g.
Parkinson’s and Alzheimer’s diseases ) [134], thus being intense vly detected (see Table 3 for
examples) 134-138]. It was demonstrated that elevated levels of uric acid in biological fluids
indicate a risk of type 2 diabetes [ 58, 130, 139, 140].
Cushing's syndrome, Addison's disease are two stress -related chronic diseases . Cortisol
is a stress b
iomarker and one of the most potent disease evoking hormones when its level is kept
continuously high , thus being a valuable possible biomarker for many diseases progression and
diagnosis [ 77, 141, 142 ]. The cortisol clinical evaluation has been performed with the aid of
ELISA, RIA [143], surface pl asmon resonance (SPR) [144] and electrochemical [145-147]
sensors (Table 3 ).
Alzheimer’s disease (AD) is a neuro -psychiatric disorder which involves dementia and total
memory loss. It has been proven that differences in saliva composition can appear even from
early stages of Alzheimer’s disease [67, 70 ], several examples of method used for possible
biomarkers detection being presented in T able 3 [28, 67, 70 ].
Table 3. Comparative presentation of the analytical parameters obtained with different analytical
methods for biomarkers involved in several pathologies
Pathology Method Analyte LOD Ref.

Oral cancer Amperometry IL-1β
IL-8
IL-8 mRNA 100 fg/mL
200 fg/mL
10 aM [101]
Fluorescence CEA 2 pg/mL [15]
ELISA IL-1β
IL-8 0.3 ng/mL
1 ng/mL [20]
Amperometry IL-8
IL-8 mRNA 72.4 pg/mL
0.21 nM [32]
Voltammetry IL-6 1 pg/mL: [99]
Amperometry IL-6 0.5 pg/mL [100]
ELISA IL-1β
IL-6
IL-8
GM-CSF 16.3 pg/mL
26.3 pg/mL
0.8 ng/mL
16.3 pg/mL [95]
NanoLC -MS/MS S100A8 – [48]
CE-TOF -MS 57 metabolites – [51]
Voltammetry CYFRA -21-1 80 pg/mL [56]
CYFRA -21-1 0.21 ng/mL [93]
CYFRA -21-1 0.12 ng/mL [98]
Voltammetry homocysteine
glutathione 0.9 μM
2 μM [79]
UPLC -ESI-MS L-phenylalanine
L-leucine – [90]
SISCAPA -MRM 19 targets 10-50 ng/mL [97]
Periodontal
disease ELISA neutrophils
osteoclast biomarkers – [103]
Amperometry HFA 16 pg/mL [104]
Brest cancer HILIC
RPLC -UPLC -ESI-MS LPO(18:1)
LPO(22:6)
MGO (0:0/14:0/0:0) – [70]
ESI-tandem MS proteins – [72]
ELISA IL-6
CRP 6.3 pg/mL
0.8 ng/mL [62]

Cardiac and
cardiovascular
diseases ELISA lysozyme
TIMP -1 1.2 pg/mL
1.2 ng/mL
[106] Luminex TNF -α
IL-1β,
IL-6
IL-8 1.2 pg/mL
0.5 pg/mL
0.5 pg/mL
0.5 pg/mL
IFMA MMP -8 0.08 ng/mL
Luminex TNF -α
IL-1β,
IL-6
IL-8 1.2 pg/mL
0.5 pg/mL
0.5 pg/mL
0.5 pg/mL [107]
ELISA lysozyme
TIMP -1 1.25 pg/mL
1.25 ng/mL
IFMA MMP -8 0.08 ng/mL
Luminex CRP
sICAM -1
ADIP 1.3 ng/mL
1.2 ng/mL
1.1 ng/mL [111]
AlphaLISA(R) immunoassay NT-proBNP 16 pg/mL [109]
Amperometry lactate 50 μM [59]
ECL 5 μM [115]
ELISA TNF -α 1 pg/mL [117]

8.2 pg/mL [118]
1.6 pg/mL [119]
Amperometry 1 pg/ml [57]
1 pg/mL [116]
Sjörgen’s
syndrome SELDI -TOF -MS
2D-DIGE 8 and 10 proteins – [68]
LC-MS/MS several proteins – [69]
Diabetes mellitus Amperometry glucose 0.1 mg/mL [12]
1 mg/mL [125]
0.1 mg/mL [126]
Colorimetry 1 mg/mL [127]
LC-MS/MS
Western immunoblotting
ELISA 65 proteins – [91]
Amperometry uric acid 1 μM [58]
Voltammetry 10 μM [140]
Cushing's
syndrome,
Addison's disease Chemiresistor
cortisol 10 pg/mL [77]
Voltammetry 10 pg/mL [141]
10 pg/mL [142]
0.76 nM [143]
EIS 1 pM [144]
Amperometry 0.1 ng/mL [145]
EIS bisphenolα 100 fM [147]
Alzheimer’s
disease UPLC -MS lactoferin – [28]
SDS-PAGE 7.43 μg/mL [67]
UPLC -MS SPG-1-P
ornithine
phenyllactic acid – [70]
ADIP – adiponectin; CRP – C-reactive protein; sICAM -1 – soluble intracellular adhesion molecule -1; NT-proBNP
myeloperoxidase, brain natriuretic peptide; SPG-1-P – sphinganine -1-phosphate AGEs -advanced glycation end
products ; CML – N-(carboxymethyl)lysine; TNF-α – Tumor Necrosis Factor ; IL- interleukin; IL-8 mRNA –
messenger RNA for IL -8 protein ; HFA – human fetuin A; LPO(18:1) and LPO(22:6) – lysophosphatidylcholine ;
MGO(0:0/14:0/0:0) – monoacylglycerol; LC -MS/MS – liquid chromatography with mass spectrometry; EIS –
electrochemical impedance spectroscopy; ECL – electrochemiluminescence; SISCAPA -MRM – peptide
immunoaffinity enrichment -coupled multiple reaction monitor ing-mass spectrometry; UPLC-MS – Ultra –
performance liquid chromatography -tandem mass spectrometry ; SDS-PAGE – sodium dodecyl sulfate –
polyacrylamide gel electrophoresis ; SELDI -TOF -MS – Surface -enhanced laser desorption/ionization time-of-flight
mass spectrometry; 2D-DIGE – 2-D Fluorescence Difference Gel Electrophoresis ; HILIC – Hydrophilic interaction
liquid chromatography ; RPLC -UPLC -ESI-MS – Reverse phase liquid chromatography coupled with ultra –
performance liquid chromatography -with electrospray tandem mass spectrometry ; CE-TOF -MS – capillary
electrophoresis time -of-flight mass spectrometry .
5. Future trends
Laboratory testing still remains the leading strategy in analytical evaluation of a large
number of samples involving those related with biochemistry, hematology, microbiology, and
anatomical pathology [148]. The financial limitations of healthcare budgets determined the urge
in finding solutions to avoid blockage of the health system in many cou ntries around the world.
The development of the primary care was considered the most suited solution for the world to
reduce costs. Poverty, chronic disease, infections lead to significant p roblems and adequate

diagnostic testing turns out to be difficult to accomplish. Consequently, there are more and more
initiatives to develop solid models based on POC systems and techno logy [ 130]. The necessity of
the development of POC technology, the potential of using saliva as medium, the selection of the
most appropriate biomarke rs for each disease, and the validation of the proposed methods for
their detection and quantification in saliva is very important. Some e xamples of such devices are
wearable and portable biosensors, smartphone technology, microfluidics and paper -based
technology. These futuristic technologies having as the ultimate goal the development of salivary
diagnostic s and reduc ing the hospital stays and costs related with this are common goals . The
number of POC testing devices has increased over the last decades and this development is likely
to continue, driven by changes in healthcare delivery in which are aimed le ss costly care a nd
closer to the patient’s home . POC technologies can be split into two categories: small handheld
devices and bench -top devices. The most important example of the first category is represented
by the glucose biosensors, followed by those used for cardiac markers and infectious pathogen s.

Figure 1 . (A) Image of the glucose biosensor on the polyethyleneterephthalate glycol (PETG)
mouthguard support . (B) Schematic image of the mouthguard biosensor . Reproduced with
permission from Elsevier and Copyright Clearance Centre from Ref. [130].
The second category is represented by the laboratory instruments and equipment such as
small haematology and immunology analysers. This field is in continues and rapid development
with many devices already designed, tested and prepared for the commercial m arket in the near
future. Only few examples of this kind of devices are listed below, all of them are related to the
continuous in situ detection of the target analyte in saliva. A “cavitas sensors” was developed
and applied for non -invasive monitoring of salivary glucose. The enzyme -based sensor was
screen -printed directly onto the surface of the mouth guard support and the measurement of
glucose amount in saliva was assured via the telemetry (see the schematic representation of the
glucose biosensor in Figure 1) [130].
The continuous monitoring of the salivary lactate was done with a m outhguard biosensors
that was fabricated by screen -printing three separate layers on a fexible PET substrate. An
Ag/AgCl conductiv e ink was first printed in order to form the reference electrode and the
contacts for all the electrodes. Graphite ink with Prussian blue was then used to print working
and auxiliary electrodes . An insulator layer was printed at the end using Dielectric paste .

Subsequently, the printed electrode system was attached to the mouthguard surface using a
double -sided adhesive (Figure 2 ) [59].

Figure 2. (A) Mouthguard biosensor, with the integrated printable 3 -electrode system . (B)
Illustration of the PB working electrode coated with the PPD -LOx l ayer in the mouthguard .
Reproduced with permission from Copyright Clearance Centre from Ref. [59].

6. Conclusion
In recent years, the possibility to assess the health or illness states, to monitor the disease
progression, or post -treatment therapeutic outcomes, through a non -invasive approach became
one of the most desirable goals for healthcare research. In this rev iew, we have discussed the
background of bioanalyte monitoring and introduced the recent development of analytical
techniques and technologies in saliv a analysis . Thus, saliva analysis represents a very interesting
biological fluid with high potential for biochemical, toxicological and immunological diagnostics
and health monitoring. Unlike blood sample, which is prone to clotting, saliva is much easier to
handle and its sampling is non -invazive , requir ing less pre -analysis manipulation.
Therefore, the salivary diagnostic using separation, immunologic and electrochemical
methods hold great potential for early -stage diagnostics without complicated and expensive
procedures. Almost all studies exemplified here proved a highly significant posi tive correlation
between the target analyte in saliva and serum in patients as well as in controls, and concluded
that salivary concentration can be used as useful matrix acquired from a non -invasive method .
Additionally, mouthguard wearable biosensors wit h telemetry system were developed for real –
time non -invasive saliva monitoring of several biomarkers such as: glucose, lactate and uric acid
to diagnose diabetes, heart failure, gout or other diseases.
Salivary diagnostic has developed into a sophisticated discipline, and serves as a
subdivision of the larger field of molecular diagnostics, now recognized as a central player in
biomedical, basic, and clinical research. Modern advances, mainly through standardization of
specimen collection using saliva colle ction devices, have made it easier for secure, effortless, and
non-invasive collection of samples. Further extensive research is required to make salivary
diagnostics a reality for different pathologies . Particularly, it can be envisaged that saliva protei n
profiling could be an attractive possibility to diagnose and monitor diseases in the near future.