Aiomt 68

REVIEW
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Advances in Electrochemical Biosensors Based on

Nanomaterials for Protein Biomarker Detection in Saliva
Tao Dong,* Nuno Miguel Matos Pires,* Zhaochu Yang, and Zhuangde Jiang
1. Introduction
The focus on precise medicine enhances the need for timely diagnosis and
frequent monitoring of chronic diseases. Moreover, the recent pandemic of Proteins are a class of macromolecules pre-
severe acute respiratory syndrome coronavirus 2 poses a great demand for sented on the surface of infectious viruses
and bacteria and are involved in key biolog-
rapid detection and surveillance of viral infections. The detection of protein
ical processes regulating the status of dis-
biomarkers and antigens in the saliva allows rapid identification of diseases or eases. Differently expressed proteins can be
disease changes in scenarios where and when the test response at the point the target of drugs and are often used as
of care is mandated. While traditional methods of protein testing fail to biomarkers for detection and diagnosis.[1,2]
provide the desired fast results, electrochemical biosensors based on Hence, the early identification and quantifi-
cation of proteins in body fluids are vital
nanomaterials hold perfect characteristics for the detection of biomarkers in
to the control of infectious and chronic dis-
point-of-care settings. The recent advances in electrochemical sensors for eases.
salivary protein detection are critically reviewed in this work, with emphasis Body fluids including blood, urine, in-
on the role of nanomaterials to boost the biosensor analytical performance terstitial fluid, and saliva are commonly
and increase the reliability of the test in human saliva samples. Furthermore, targeted in the measurement of protein
this work identifies the critical factors for further modernization of the biomarkers.[3] Saliva in particular is prefer-
able for testing in point-of-care (POC) set-
nanomaterial-based electrochemical sensors, envisaging the development
tings where rapid diagnosis is demanded.[4]
and implementation of next-generation sample-in-answer-out systems. Saliva can be easily and repetitively collected
in sufficient volumes for detection, involv-
ing a low risk of infection and the procedure
is quite patient-compliant. Therefore, a powerful method of rapid
N. M. Matos Pires, Z. Yang, Z. Jiang
Chongqing Key Laboratory of Micro-Nano Systems and Intelligent disease tracing and detection can be developed by targeting saliva
Transduction as the diagnostic fluid and analyzing it with a sensor with simple
Collaborative Innovation Center on Micro-Nano Transduction and operation and high analytical performance.
Intelligent Eco-Internet of Things The global pandemic of severe acute respiratory syndrome
Chongqing Key Laboratory of Colleges and Universities on Micro-Nano
Systems Technology and Smart Transducing coronavirus 2 (SARS-CoV-2) has stimulated the rapid develop-
National Research Base of Intelligent Manufacturing Service ment of saliva analysis tools. During the last 2 years, a great
Chongqing Technology and Business University number of saliva-based sensor technologies for SARS-CoV-2-
Nan’an District, Chongqing 400067, China related proteins, namely viral antigens or SARS-CoV-2 antibod-
E-mail: [email protected]
ies, have been reported.[5–8] The positive test for SARS-CoV-2-
T. Dong
related proteins rapidly identifies the infected individuals in sit-
Department of Microsystems- IMS
Faculty of Technology uations where and when the RT-PCR test is not available. The
Natural Sciences and Maritime Sciences clinical value of saliva-based protein sensors is extended to other
University of South-Eastern Norway-USN infectious disease diagnostics (i.e., rapid tests for malaria),[9] per-
P.O. Box 235, Kongsberg 3603, Norway sonalized medicine (i.e., routine tests for chronic lung disease or
E-mail: [email protected]
heart disease),[10,11] and other medical conditions affecting mil-
Z. Jiang
State Key Laboratory for Manufacturing Systems Engineering lions of people (i.e., on-site tests for physiological stress detection,
International Joint Laboratory for Micro/Nano Manufacturing and early-stage detection of cancer, etc.).[12–14]
Measurement Technology Protein detection methods applied to saliva have been bene-
Xi’an Jiaotong University fiting from the standardization of specimen collection through
Xi’an 710049, China
the use of modern non-invasive devices.[15] However, several
The ORCID identification number(s) for the author(s) of this article challenges persist with saliva-based protein detection. In the
can be found under https://doi.org/10.1002/advs.202205429 first place, the protein biomarkers are present at significantly
© 2022 The Authors. Advanced Science published by Wiley-VCH GmbH. low concentrations in saliva, which poses a challenge to the
This is an open access article under the terms of the Creative Commons resolution and detection limits of current analysis tools. Second,
Attribution License, which permits use, distribution and reproduction in the target proteins may vary in concentration by several orders
any medium, provided the original work is properly cited.
of magnitude, adding to the difficulty in terms of detection
DOI: 10.1002/advs.202205429
Adv. Sci. 2023, 10, 2205429 2205429 (1 of 28) © 2022 The Authors. Advanced Science published by Wiley-VCH GmbH
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range. Finally, the specific detection of salivary protein can be tein signatures.[23] Molecular “barcodes” can be constructed us-
hampered by interfering molecules and ions present in saliva.[16] ing SERS and the combination with advanced machine learning
The exploitation of nanomaterials in conjugation with advanced techniques for further signal processing enhances the detection
sensor architectures may constitute a pathway to mitigate those accuracy.[24] Alterations in immunoglobulin and other proteins
challenges. were identified by attenuated total reflection-Fourier transform
infrared spectroscopy which exhibited a significant power of dis-
crimination between SARS-CoV-2 infected patients and healthy
1.1. Overview of Salivary Protein Detection individuals.[25] The technique can also generate diagnostic fin-
gerprints from saliva samples in combination with multivariate
Analyses of protein biomarkers in saliva are typically conducted analysis.
by the enzyme-linked immunosorbent assay (ELISA).[17] The The confinement of the immunological methods, LC-MS/MS,
common procedure involves the immobilization of the protein and spectroscopic-based techniques to the clinical chemistry lab-
on the ELISA microtiter plate either by direct adsorption or by oratory hinders, despite their reliability, the use of these tech-
indirect immobilization using a capture antibody pre-coated on niques for rapid diagnosis of diseases or rapid feedback on results
the plate. The detection is executed by loading an enzyme-labeled of disease treatment. The advantage of saliva-based diagnostics is
antibody whose interaction with a chemical substrate produces a centered on the sample’s easy accessibility, which is ideal for POC
colorimetric or a luminescent signal. Western blotting is another settings. The use of complex instrumentation and laborious op-
immunological-based method more commonly used in confir- erations of the assay prevent the test from providing the detection
mation studies of candidate salivary biomarkers.[18] Gel elec- result immediately. The problem has motivated the development
trophoresis is employed in this technique with the separated pro- of protein biosensors[26] which can provide detection results in
teins visualized on PVDF membranes using specific antibodies minutes and are amenable to analyzing the biofluid sample in
coupled with either radio-conjugates or enzyme labels. settings outside a clinical laboratory. Various biosensors have
Screening of the saliva proteome with high throughput has been studied for detecting protein biomarkers in saliva, includ-
been performed by liquid chromatography/tandem mass spec- ing electronic sensors,[27] electrochemical sensors,[17] fluorescent
trometry (LC-MS/MS). Hsiao et al. for instance have exploited sensors,[28] interferometer sensors,[6] plasmonic sensors,[29] ab-
multiplexed LC-MS/MS assays to screen hundreds of peptides sorbance sensors,[30] and quartz crystal microbalance sensors.[31]
representing sets of protein biomarkers.[19] The study has char- However, many of the reported technologies do not exhibit suf-
acterized variabilities in 90 proteins among samples collected ficient analytical performance and easy operation in the field.
from the same individual and samples collected from different Satisfying the growing demand for saliva-based biosensors with
individuals. For the analysis of up to a few hundred proteins, the characteristics of low cost, ultra-high sensitivity, and fast
the use of gel electrophoresis coupled to MS is a common pro- test cycles from sampling to analyte detection remains an im-
cedure; however, the need for analyzing low-abundance salivary portant challenge nowadays.[32] The electrochemical sensor may
proteins (as in the case of interleukins for instance) calls for offer the best compromise between low cost and high analyti-
advanced MS techniques such as matrix-assisted laser desorp- cal performance among the existing sensor platforms. The elec-
tion/ionization time-of-flight (MALDI-TOF/TOF) and linear ion trochemical sensor is acknowledged to be sensitive, fast, with
trap. These techniques would ensure high resolution for salivary low detection limits, easily integrated, and amenable to minia-
protein identification.[20] By utilizing an assay similar to that re- turization at reasonable costs.[16,33–36] These characteristics make
ported by Hsiao et al., Kipping et al. have screened surrogate pep- the electrochemical assay suitable for perfect POC devices. Nev-
tides derived from SARS-CoV-2 nucleocapsid protein.[21] Despite ertheless, when challenged with the saliva sample, the sensor
the broad versatility and rapid analysis time (within a few min- shall exhibit superior selectivity to the target due to the com-
utes), the technique requires intensive protocols for sample pre- plex composition of the sample, as well as superior sensitiv-
treatment and target labeling before the analysis. ity and detection limit as the salivary protein biomarkers are
Raman spectroscopy is an alternative tool for protein profil- commonly present in the sample at low concentration com-
ing with much simpler sample preparation compared with LC- pared with blood for instance. Nanomaterials and related com-
MS/MS. The technique creates fingerprints of proteins based on posites are often selected as the technology solution to enhance
the phenomenon of Raman scattering which originates from a the analytical merits of the electrochemical sensor while retain-
frequency shift in the radiation of a laser upon to interaction of ing its costs and miniaturization advantages. Various classes
light with proteins. Typically, the Raman signal needs to be en- of nanomaterials have been used to modify the electrode sur-
hanced by the use of metallic nanostructures exploiting chemical faces, including Au nanoparticles,[37] carbon nanotubes,[38] mag-
enhancement or amplification via surface plasmon resonance. netic nanoparticles,[39] exfoliated graphene,[40] reduced graphene
Surface-enhanced Raman spectroscopy (SERS) has been evalu- oxide,[41] metal oxide nanoparticles,[42] metal-based thin-films,[43]
ated for profiling various salivary cytokines as potential biomark- and organic-based thin-films.[44]
ers of asthma.[22] Gold (Au) nanorods were employed as signal This review intends to present the latest advances in electro-
enhancers, and the method has shown good accuracy for the chemical sensing for protein assays in saliva, focusing on the
early identification of bronchial inflammation in asthmatic pa- routes to be explored for the deployment of nanomaterial-based
tients. Silver nanoparticles were used in another study revealing electrochemical sensors in this field. Current and future sensor
the potential of SERS to decipher the diagnosis of lung cancer platforms incorporating nanomaterials or their associated com-
patients compared to the control group by analyzing salivary pro- posites are outlined in this paper.
Table 1. Summary of clinically relevant protein biomarkers in saliva.
Protein biomarker Physiological rangea) Disease Ref.
Interleukin-1𝛽 161.51c) –1312.75d) (pg mL−1 ) Periodontitis [45]

Interleukin-6 10c) –>30d) (pg mL−1 ) Periodontitis [46]
0.6c) –43.6d) (pg mL−1 ) Oral squamous cell carcinoma [47]
Interleukin-8 210.10c) –1718.61d) (pg mL−1 ) Oral squamous cell carcinoma [48]
Interleukin-2 2.07c) –3.06d) (U mL−1 ) Mucositis in individuals with acute [49]
lymphoblastic leukemia
Vascular endothelial growth factor—VEGF 280c) –4321d) (pg mL−1 ) Oropharyngeal cancer [50]
Tumor necrosis alpha—TNF-𝛼 8.60c) –27.75d) (pg mL−1 ) Oral squamous cell carcinoma [51]
2.15c) –12.92d) (pg mL−1 ) Periodontitis [52]
Human epidermal growth factor receptor 2—HER2 9.93e) –146.70f) (pg mL−1 ) Breast cancer [53]
Triosephosphate isomerase—TPI1 >800c) –<400d) (U mL−1 ) Gastric cancer [54]
Matrix metalloproteinase-8—MMP-8 190.91c) –348.26d) (ng mL−1 ) Periodontitis [55]
Matrix metalloproteinase-9—MMP-9 145.87c) –231.02d) (ng mL−1 ) Sjögren´s syndrome [56]
C-reactive protein <1c) –>80d) (ng mL−1 ) Cardiovascular disease [57]
Secretory immunoglobulin A—sIgA 72.83c) –103.11d) (mg L−1 ) Human immunodeficiency virus infections [58]
Immunoglobulin G 12.4c) –27.0d) (mg per mg of Human papillomavirus infections [59]
total protein concentration)
Cardiac troponin T 8.9c) –45.8d) (pg mL−1 ) Myocardial infarction [60]
Galectin-3 282.0c) –601.3d) (ng mL−1 ) b) Heart failure [61]
N-terminal proB-type natriuretic peptide—NT-proBNP <16c) –76.8d) (pg mL−1 ) b) Heart failure [62]
𝛼-amylase <50–100c) –>150d) (U mL−1 ) Stress [63]
Procalcinotin 0.09c) –0.50d) (ng mL−1 ) b) Lung inflammation [64]
Beta amyloid 42—A𝛽42 21.1c) –51.7d) (pg mL−1 ) Alzheimer’s disease [65]
DJ-1 <4e) –>5f) (ng mL−1 ) Parkinson’s disease [66]
SARS-CoV-2 nucleocapsid protein (N-protein) <1c) –100d) (pg mL−1 ) SARS-CoV-2 infections [67]
SARS-CoV-2 IgG 7.0c) –25.5d) (μg mL−1 ) SARS-CoV-2 infections [68]
SARS-CoV-2 IgA 43c) –201d) (AU-arbitrary units) SARS-CoV-2 infections [69]
a) b) c) d) e) f)
Mean values; Median; Control condition; Disease condition; Stage I of disease; Advanced stage of disease.
1.2. Disease-Marker Proteins in Saliva mation in chronic lung disease patients.[57,64] Nevertheless, CRP
is presented in saliva at concentration values at least one thou-
Salivary proteins have been associated with various diseases, in- sand times lower than CRP measured in the blood, making its
cluding localized and systemic diseases. Table 1 lists examples of detection in saliva challenging. Troponins are another class of
salivary proteins targeted as biomarkers of diseases. proteins involved in systemic events. The cardiac troponin I and
Active transport and passive diffusion either transcellular T (cTnI and cTnT, respectively) when targeted by high-sensitivity
or paracellular are normally the biological pathways for the assays have an important role in the early and accurate identifica-
biomarkers to enter saliva.[70] The antibodies IgA and IgG form tion of acute coronary syndrome and myocardial infarction.[60,114]
a first-line immune barrier in the oral cavity against intruding These cardiac biomarkers have been detected at a few picograms
pathogens. IgA appears in saliva via active transport from secre- per milliliter (pg mL−1 ) concentrations in saliva from healthy sub-
tory cells. IgG is believed to enter saliva by passive means through jects and tens to hundreds of pg mL−1 in samples from diagnosed
the gingival crevicular fluid.[2] Variations of these two antibodies patients. In addition, the N-terminal pro-brain natriuretic pep-
are indicative of viral exposure in saliva tests.[58,68,69] Particularly, tide (NT-proBNP), which is the gold standard biomarker for heart
in cases of SARS-CoV-2 infections, the levels of IgG were found failure monitoring, has also been identified in saliva samples al-
in good correlation with the levels in serum.[68] The findings de- though at concentrations (minimum detection of 1 pg mL−1 ) dif-
noted the potential of salivary IgG for monitoring the immune ficult to be accurately analyzed by current biosensors.[72]
response to systemic infections. Cytokines are largely produced in the oral cavity and there-
C-reactive protein (CRP) is a marker of systemic inflammation fore are often associated with oral pathologies. Elevations of
whose levels measured in saliva correlate well to blood levels.[71] interleukins (IL)-1𝛽, IL-6, TNF-𝛼, and matrix metalloproteinases
This protein is acknowledged to be an important risk marker of (MMPs) were identified in periodontitis and hold great potential
cardiovascular disease. Recent studies have shown the value of as biomarkers of diagnosis for this disease.[45,46,52,55] IL-6, IL-8,
salivary CRP as a confirmatory biomarker of acute myocardial and TNF-𝛼 have exhibited an area under the curve (AUC) greater
infarction as well as a predictive biomarker of acute lung inflam- than 0.8 for the diagnosis of oral squamous cell carcinoma
compared with controls.[73] Other reports have suggested a cor- electrode response typically by variations in voltage, current, or
relation between cytokines and systemic diseases. For instance, impedance. Antibodies are specific recognition elements often
elevations of salivary MMP-9 were statistically significant in pri- used in saliva-based biosensors. The antibody can modify the
mary Sjögren´s syndrome, a systemic autoimmune disorder.[56] surface of the nanomaterial on the electrode surface, and the
Levels of salivary IL-8 were found to have increased significantly variation of the signal is induced either by the complex protein-
in patients with bowel diseases and with muscle and joint antibody directly[81] or enhanced by a secondary antibody la-
diseases.[74] IL-19 has been implicated in systemic inflammatory beled with an enzyme catalyzing electron donor reactions.[82]
disorders and has been associated with asthma severity being a Oligonucleotide (or peptide) aptamers are also common recogni-
potential biomarker for therapy response.[75] tion probes for protein biomarkers. Aptamers benefit from sim-
Besides oral cancer, salivary proteins have also been impli- ple chemical synthesis and high stability, are easily tuned in size
cated in lung cancer, breast cancer, and gastric cancer. A panel and conformation, and possess a less complex chemical structure
of three proteins, haptoglobin, zinc-a-2-glycoprotein, and calpro- compared with antibodies.[83,84]
tectin, has exhibited an AUC of 0.9 for the detection of lung Nanobodies are an emerging class of bio-receptors with con-
cancer in comparison with negative controls.[76] Clinical sensi- siderable interest in electrochemical protein biosensors.[85,86]
tivity and specificity of nearly 90% were achieved with this pro- Nanobodies are variable domains of antibodies, with smaller
teomic biomarker panel. In other work, the proteins cystatin B, sizes and higher affinity to targets compared with conventional
triosephosphate isomerase, and deleted in malignant brain tu- antibodies. Besides the high binding specificity and high stabil-
mors 1 protein were found to differentiate gastric cancer patients ity, the nanobody possesses an improved solubility and its lower
from controls with a detection accuracy of 0.93.[54] These reports dimensions enable the detection below the Debye lengths.[87]
have also elucidated the need to measure multiple biomarkers “Artificial antibodies” or molecularly imprinted polymers (MIPs)
in parallel for one diagnosis. Proteomics using mass spectrom- are also reported in recent electrochemical biosensors. MIPs use
etry is typically exploited for the discovery of biomarker panels, the target proteins as templates for synthesizing rigid tridimen-
whereas ELISA or Luminex assays are used to confirm the levels sional (3D) polymer structures around the binding sites of the
of candidate protein biomarkers in the biofluid.[53,66,76] protein.[14] Following the extraction of the template, the target
The following sections of this review mention other protein protein is detected by rebinding to the MIP containing empty
biomarkers in connection to targets of newly developed saliva- binding sites. MIPs may comprise conducting polymer struc-
based protein biosensors. Trends in the area of salivary protein tures and may form electroactive films on the top of the electrodes
biomarkers encompass the discovery of new marker panels with for the transduction of the signal.[88]
enhanced detection accuracy and further validation of the identi- Specific detection of salivary protein has also been conducted
fied biomarkers in large-scale clinical trials.[67,77] with no use of biomolecular probes. Cascade enzymatic reactions
involving the hydrolysis of the target and subsequent activation of
redox probes have been proposed as one strategy for the specific
1.3. Electrochemical Sensing Method detection of 𝛼-amylase.[89] Moreover, functional chemical groups
expressed on the nanomaterial surface can be used for the recog-
Due to the unique characteristics of the electrochemical sensor nition of free terminals of the target protein in the presence of
for POC settings, this type of sensor has been widely used in the activating agents. For the activation, the immobilization yields of
sensitive and specific detection of salivary biomarkers.[34–44,78] In carbodiimide hydrochloride/N-hydroxy succinimide (EDC/NHS)
addition to its promising analytical performance, inherent com- chemistry for instance may allow a strong linkage between the
pactness, low cost, and non-complicated operation, the miniatur- functionalized nanomaterial and the target.[6]
ized electrochemical sensor may allow direct detection in saliva Biomolecular probes or functional chemical groups are mainly
with minimal or no addition of extra electrolytes. Compton´s used to modify the working electrode of the sensor. Alternatively,
group is a pioneer in electrochemical analyses of saliva sam- or in addition, the biomolecular probe or the activating chemi-
ples by exploiting the relatively strong ionic strength of saliva cal groups may functionalize nanostructures in the solution as
(50–100 mM).[79,80] In this case, molecules with redox proper- signal enhancers to the electrochemical sensor.[39,90]
ties may undertake direct oxidation or reduction on the miniatur-
ized electrode and be measurable by the sensor. However, most
proteins identified as chronic or infectious disease biomarkers 1.3.2. Sensing Technique
are electrochemically inert. To realize their detection, the redox
probes are used as either label conjugates of biorecognition ele- The electrochemical sensor provides sensitive and fast signal
ments or as a reagent in the solution and subsequently combined transduction of the protein-receptor binding events. Various tech-
with electrode surfaces modified with functional materials.[11,32] niques of electrochemical sensing have been employed in pro-
The roadmap concept of electrochemical detection for protein tein detection from saliva samples, including potentiometry, am-
biomarkers in the saliva is depicted in Figure 1. perometry, differential pulse voltammetry (DPV), square wave
voltammetry (SWV), electrochemical impedance spectroscopy
(EIS), and electrochemical capacitance.
1.3.1. Biorecognition Element Amperometric sensors measure the current response between
a working electrode (WE) and counter-electrode (CE) caused
In the electrochemical immunoassay, the binding of the pro- by redox reactions triggered by the recognition of the target
tein marker with the respective antibody induces changes in the analyte.[82] The amperometric response is often measured as a
Figure 1. Illustration of electrochemical detection of protein biomarkers in saliva by the nanomaterial-based biosensor.
function of applied potential (either fixed or swept) or as a func- measured at the end of a forward square-wave pulse and the value
tion of time upon applying a voltage pulse (chronoamperom- at the end of the returning square pulse.
etry). In potentiometric sensors, the electrochemical potential Electrolyte-gated transistors (EGTs) are increasingly popular in
measured between the WE and reference electrode (RE) varies sensitive protein detection.[96] EGTs have a similar structure and
upon immobilization of the analyte onto the WE surface.[91] The operation mode to the conventional MOSFETs—metal-oxide-
measurement is conducted with no current present. Impedimet- semiconductor field-effect transistors (FETs). While the MOS-
ric sensing is related to the changes in conductance and capaci- FET uses a dielectric material for the gate, the EGT uses an elec-
tance at the interface of the WE to the electrolyte. In this case, trolyte as the gate dielectric. In EGT the sensor response is con-
the specific recognition of the analyte cause variations in the trolled by the movement of ions in the electrolyte which occurs
interfacial impedance.[92,93] EIS is a representative impedimet- following the application of a gate voltage. Depending on the per-
ric technique involving the measurement of changes in either meability of the EGT channel to the ions of the electrolyte, the
charge transfer resistance or electrode interface capacitance due transistor acts as an “electrical double-layer transistor” (EDLT)
to protein-receptor binding. In EIS electrode currents are mea- or as an “electrochemical transistor” (ECT).[97,98] The former is
sured following the application of an AC signal of varied fre- the impermeable one in which the gate controls the channel cur-
quency. The impedimetric measurement can also be done with rent via a mechanism of capacitive field effect at the channel-
constant frequency probing variations of dielectric properties at electrolyte interface. In the “electrochemical transistor” the chan-
the electrode–electrolyte interface.[94] In voltammetric biosen- nel is redox-active and events of doping/dedoping occur upon
sors, the detection of analytes is normally reflected as variations injection of ions from the electrolyte. For sensing purposes, the
of peak current as a function of applied potentials.[37,95] The tech- EGT is compatible with both amperometric and potentiometric
nique of DPV probes electron transfer from and to electrodes us- signal transduction.[99]
ing small pulses whose potential is increased on a linear ramp. Photoelectrochemical (PEC) sensors are also an advanced
The current of detection is measured as the difference between generation of electrochemical sensors. The PEC sensor has the
values at two time points, before the application of the pulse and unique characteristic that the excitation signal (potential) is of a
at the end of it. Similar to DPV, SWV obtains the detection (peak) different energy form (optical) than that of the detection signal
signal by determining the difference between the current value (electrical). This endows the sensor with enhanced sensitivity
and low background noise.[83,90] In PEC sensing the photocat- conductivity. TNF-𝛼 was detected in artificial saliva samples by
alytic properties of semiconductor electrodes are exploited to a CA immunoassay employing an Au electrode with surface
obtain a current or voltage response, and this response is either modification by magnetic particles.[129] TNF-𝛼-capture antibody
enhanced or hindered by the assay involving the binding of the was coated on the magnetic particle surface. As a result, the LOD
target protein to the electrode surface.[100] of the sub-micron rough Au electrode has improved to 0.3 pg
To maximize the performance of each sensing technique, re- mL−1 , corresponding to one order of magnitude lower than the
searchers have made use of the high catalytic efficiency, the large planar Au electrode.[34]
reaction area, and the great biocompatibility of nanomaterials or Screen-printed electrodes (SPEs) are a low-cost alternative to
their related nanocomposites.[101] The merits of nanomaterials the Au electrodes for protein assays exhibiting no loss of analyt-
equip the electrochemical sensor with superior sensitivity, low ical performance. Carbon-based SPEs were used in amperomet-
detection limit, high biosensor stability, and fast response. ric assays for salivary interferon-gamma (IFN-𝛾).[130] This SPE-
This review reports and discusses the latest developments based sensor has also handled a “sandwich-type” immunoassay,
of electrochemical sensors in saliva-based detection targeting in which the IFN-𝛾-capture antibody modified with the surface of
disease-signaling protein biomarkers. The synergy of electro- the SPE and a secondary antibody labeled with HRP completed
chemical techniques, nanomaterials, and specific recognition the assay (see Figure 2A). The amperometric response was ob-
probes is highlighted. Other reviews, namely the recent works tained through HRP/hydroquinone/H2 O2 redox cycling on the
of Kaya et al.[102] and Mostafa et al.,[103] have shed light on elec- WE. The LOD of this SPE-based assay was around 1 pg mL−1
trochemical sensors for biomarkers of various cancers. In these while the detection range was from a few pg mL−1 to 2000 pg
works, the focus is scattered over various biofluid types and an- mL−1 . Of remark, the assay has handled undiluted samples of hu-
alytes, including transcriptomic and metabolic markers. Mani et man saliva by utilizing WE surface blockage with bovine serum
al.[78] discussed the progress of electrochemical sensors with a albumin (BSA). Screen printing can also be applied to make
dispersed focus on drugs, toxins, proteins, and pathogens. Cam- low-cost Au electrodes and turn them more amenable to use in
puzano et al.[104] reviewed affinity-based saliva biosensors before resource-poor settings. An Au-based SPE was fabricated for CA
2017. The present review highlights the advances in electrochem- immunoassays analyzing a periodontal disease protein.[106] Con-
ical protein biosensors within the latest 5 years. trary to the amperometric sensors above-reviewed, this SPE was
coupled with aptamers used as the biorecognition elements. A
first “capturing” aptamer has coated the SPE surface and a sec-
2. Electrochemical Sensors for Salivary Protein ond “detecting” aptamer labeled with HRP recognized the target
Biomarkers protein and completed the assay. The redox reaction of TMB cat-
alyzed by the HRP label was exploited for the CA measurements.
The following section is divided according to the types of elec-
The assay times were still over 1 h.
trochemical sensors that recently emerged in the literature. The
Besides modifying the surface of the Au electrode with
discussion of the recent developments in each type of electro-
micro- or nanoparticles, the Au electrode can be patterned with
chemical sensor includes a critical overview of the strengths and
nanoscale features by adding a second Au layer via electrodepo-
pitfalls of each type. Representative works of each category of sen-
sition. The nanostructured Au electrode has exhibited good elec-
sor accompanied by main performance metrics are described in
tronic properties and it was used in a new concept of ampero-
Table 2.
metric tethered sensors.[107,131] The tethered sensor consisted of
immobilizing a biomolecular complex made of a thiolated DNA
2.1. Amperometric Sensors linker and a detection antibody atop the nanostructured Au elec-
trode. A redox reporter (ferrocene) was tethered to the DNA linker
Amperometry is one of the primary forms of biosensing since the and mediated the detection via its redox reaction on the electrode
demonstration of the amperometric measurement of glucose by surface. The tethered sensor has detected SARS-CoV-2 spike (S)
Leland C. Clark.[128] For protein analysis in saliva, amperometric protein with a LOD as low as 1 pg mL−1 . The assay is shown in
sensing is commonly conducted using functionalized Au elec- Figure 2B. By applying a positive potential (+0.5 V), the biomolec-
trodes. Salivary TNF-𝛼 has been detected by an Au WE function- ular complex is brought into contact with the electrode surface oc-
alized with a TNF-𝛼-capture antibody.[34] The amperometric re- curring the oxidation of ferrocene. Using the CA measurement, it
sponse was obtained from the redox reaction of tetramethylben- can be observed a slower decay in the current response due to the
zidine (TMB) on the Au electrode catalyzed by the horseradish hydrodynamic drag force (Fd ) that balances the force induced by
peroxidase (HRP). The HRP enzyme is commonly utilized as a la- the electric field (Fe ). The lower the concentration of protein the
bel for a secondary antibody that recognizes the protein target. In faster the current decays. Each analyte measurement took only
this work,[34] chronoamperometry (CA) was employed to record 5 min, and tests were conducted with clinical saliva samples re-
the differences in the detection signal among the tested TNF-𝛼 vealing minimal interference from the sample matrix on the am-
samples ranging from 1 to 30 pg mL−1 in concentration. A limit perometric response.
of detection (LOD) of 1 pg mL−1 protein was reported. Although In summary, planar and nanostructured Au electrodes and
the CA measurement was executed in only 5 s, the total assay SPEs are still predominant in amperometric sensors targeting
time exceeded 1 h. protein detection. These sensors typically utilize HRP as a label
The Au electrode can be modified with micron or nano-sized of detection antibody and exploit its catalytic activity to redox
particles to increase the surface area and enhance electrode reagents in solution. This type of assay exhibits potential for
Table 2. Overview of recently developed electrochemical sensors for disease-signaling protein biomarkers in saliva.
Biosensor type Sensor material Recognition Target Detection range Limit of Comment Ref.
element detection
Adv. Sci. 2023, 10, 2205429

Amperometric/ Gold WE Antibody TNF-𝛼 1–30 pg mL−1 1 pg mL−1 Detection in 5 s after incubation with HRP-labelled secondary [34]
chronoamperometric antibody and following the addition of TMB substrate.
Amperometric/ Screen-printed Antibody SARS-CoV-2 S1 0.5–5 ng mL−1 0.15 ng mL−1 Incubation with a secondary antibody and addition of a third [105]
chronoamperometric carbon electrode protein HRP-labelled antibody. Detection with a portable analyzer.
www.advancedsciencenews.com
Amperometric/ Screen-printed gold Aptamer ODAM 0–15 nM 1 nM Recognition by a primary aptamer immobilized on electrode and [106]
chronoamperometric electrode detection by HRP-linked secondary aptamer. Results were
displayed on a smartphone.
Amperometric/ Nanostructured gold Antibody on SARS-CoV-2 S1 — 1 pg mL−1 Reagent-free sensor with ferrocene attached to a DNA linker. [107]
chronoamperometric coating a DNA protein Probe binding with protein causes hydrodynamic drag on the
linker sensor affecting the kinetics of the sensor response.
Potentiometric Nano-rough gold film Molecularly SARS-CoV-2 >102 –106 pg mL−1 100 pg mL−1 Target proteins used as template molecules fitted conformally [108]
imprinted and MERS S on the concave nanostructures of a gold film.
substrate proteins
Impedimetric/EIS Nanostructured Y2 O3 Antibody CYFRA-21-1 0.01–50 ng mL−1 0.01 ng mL−1 Y2 O3 nanoparticles enhanced electrode biocompatibility, charge [109]
coating transfer efficiency, and surface-to-volume ratio.
Impedimetric/EIS Organic Antibody IL-1𝛽 0.01–3 pg mL−1 3 fg mL−1 Organic nano-coating enhanced binding sites for antibody [110]
polythiophene- immobilization and improved charge transfer efficiency of
based coating electrodes.
Impedimetric/EIS MWCNT-AuNP Antibody DJ-1 4.7–4700 fg mL−1 0.5 fg mL−1 Nanocomposite improved the catalytic activity. MWCNT-AuNP [111]
2205429 (7 of 28)
nanocomposite facilitates electron transfer between antibody and electrode.
Impedimetric/EIS Gold WE Aptamer SARS-CoV-2 S1 4–44 000 fM 1 fM Dimeric DNA aptamer immobilized on a thiolated gold [112]
protein electrode with superior affinity to S proteins. Detection under
10 min.
Impedimetric/EIS Screen-printed gold Molecularly SARS-CoV-2 2–40 pg mL−1 0.7 pg mL−1 Immobilization of target protein in a MIP film hindered diffusion [113]
electrode with imprinted RBD of the redox probe on the electrode surface. Nano-porosities
nano-porosities polymer enhanced active surface area. Detection in 20 min.
Impedimetric/EC Al/Si/SiO2 /Si3 N4 Antibody TNF-𝛼 1–30 pg mL−1 1 pg mL−1 Measurement of EC capacitance changes by protein-antibody [94]
capacitance binding complexes on a high dielectric material (film, 100-nm
thick).
Voltammetric/DPV ZnO-rGO Antibody IL-8 10−4 –5 ng mL−1 ≈50 pg mL−1 Nanocomposite film exhibited quasi-reversible electrochemical [114]
nanocomposite characteristics in the cyclic voltammogram for Zobell´s
solution. Tests in undiluted saliva samples.
Voltammetric/DPV Nitrogen-doped rGO Aptamer cTnI 1–105 pg mL−1 1 pg mL−1 Decreased peak current by the complex DNA aptamer-cTnI. rGO [115]
provided a large surface area and its functionalization by
py-PEG allowed optimizing bioreceptor density.
(Continued)
© 2022 The Authors. Advanced Science published by Wiley-VCH GmbH

Table 2. (Continued).
Biosensor type Sensor material Recognition Target Detection range Limit of Comment Ref.
element detection
Voltammetric/DPV MWCNTs Antibody IL-1𝛽 10–1200 pg mL−1 5.2 pg mL−1 Azide-functionalized MWCNTs bound to ethynylated primary [116]
antibody by electro-click chemistry. Secondary antibody
labeled with AP-Strep label for enzymatic redox.
Adv. Sci. 2023, 10, 2205429

Voltammetric/DPV AuNPs coupled to Peptide SARS-CoV-2 — 0.35 ag mL−1 Both AuNPs and magnetic particles were modified with ACE2 [117]
magnetic particles sequence RBD peptide to specifically bind RBD. Detection by reduction of
[AuCl4 ]− to Au after the addition of HCl solution.
www.advancedsciencenews.com
Voltammetric/DPV Bi2 WO6 /Bi2 S3 Antibody SARS-CoV-2 N 0.01–1 pg mL−1 3 fg mL−1 Bi2 WO6 /Bi2 S3 was used as the sensor platform coated by [118]
heterostructure protein primary antibody. Signal enhancement by g-C3 N4 /Au/WO3
composite as a label of secondary antibody.
Voltammetric/SWV Au nanowires Antibody CRP 8–140 fg mL−1 4 fg mL−1 Arrays of nanowires improved electron transfer efficiency for [119]
[Fe(CN)6 ]3-/4− , and enhanced the surface area and the
biocompatibility to adsorbed biomolecules.
Voltammetric/SWV Nanoporous anodic Aptamer SARS-CoV-2 2.5–40 ng mL−1 0.8 ng mL−1 Nanoporous membrane coated with AuNPs and functionalized [120]
aluminum oxide on RBD with DNA aptamer. The aptamer-RBD complex hindered
LEGE access of an electrode to a redox probe.
Electrolyte gated Carbon nanofibers Antibody Nesfatin-1 10–106 fM 10 fM A FET channel was made of multi-pore carbon nanofibers. [121]
transistor/EDLT Analyte binding to the channel-immobilized antibody led to
variation in channel charge transport.
Electrolyte gated rGO Aptamer HPV-16 E7 — 100 pg mL−1 rGO prepared onto silanized interdigitated electrodes. Change [122]
transistor/EDLT protein of conformation of an RNA aptamer by binding with protein
altered current output. Limit of detection in a buffer.
2205429 (8 of 28)
Electrolyte gated Poly(3- Antibody CRP — ≈13 molecules An organic semiconductor is used as the FET channel. Gate [123]
transistor/ECT hexylthiophene-2,5- per 100 μL functionalized with antibody. Detected shifts in the transfer
diyl) I–V curves due to analyte binding to the respective antibody.
Electrolyte gated PEDOT:PSS Spike SARS-CoV-2 10–108 fM 10 fM The surface potential of the FET gate varied with the specific [124]
transistor/ECT protein, IgG protein binding of IgG on the gate and led to shifts in the I–V transfer
His Tag curves. Assay time of 5 min including incubation.
Pd NPs/g-C3 N4 - Antibody SARS-CoV-2 S1 1–106 fg mL−1 1 fg mL−1 The binding of S1 proteins to antibodies decreased the efficiency [125]
Photoelectrochemistry S/SrTiO3 protein of photon-to-current conversion in PdNPs/g-C3 N4 -S/SrTiO3
nanocomposite composite in contact with an electrolyte.
Ti3 C2 Tx /NiWO4 Antibody Prostate- 1.2–0.18 × 1012 fg 0.15 fg mL−1 NiWO4 nanoparticles and Ti3 C2 Tx sheets formed a [126]
Photoelectrochemistry nanocomposite specific mL−1 heterostructure with fast interfacial charge transfer kinetics.
antigen Detection is conducted by inhibition of photocurrent.
CdS QDs/g-C3 N4 Aptamer SARS-CoV-2 0.5–32 nM 0.12 nM Immobilization of RBD protein by a DNA aptamer caused [127]
Photoelectrochemistry nanocomposite RBD hindrance to mass transport of redox probe to the
nanocomposite surface, reducing photocurrent response.
Abbreviations: HRP—Horseradish peroxidase; TMB—Tetramethylbenzidine; ODAM—Human odontogenic ameloblast-associated protein; MERS—Middle-East respiratory syndrome coronavirus; EIS—Electrochemical
impedance spectroscopy; Y2 O3 —Yttrium oxide; MWCNT-AuNP—Multiwalled carbon nanotube-gold nanoparticle; EC—Electrochemical; RBD—Receptor-binding domain; Al—Aluminum; SiO2 —Silicon dioxide; Si3 N4 sili-
con nitride; ZnO—Zinc oxide; rGO—Reduced graphene oxide; cTnI—Cardiac troponin I; AP-Strep—Alkaline phosphatase-streptavidin conjugate; ACE2—Human angiotensin-converting enzyme 2; Bi2 WO6 /Bi2 S3 —Bismuth
tungstate/bismuth sulfide composite; g-C3 N4 /Au/WO3 —Graphitic carbon nitride sheet decorated with AuNPs and tungsten trioxide composite; LEGE—Laser engraved graphene electrode; EDLT—Electrical double-layer
transistor; HPV—Human papillomavirus; ECT—Electrochemical transistor; PEDOT:PSS—Poly(3,4-ethylenedioxythiophene)−poly(styrenesulfonate); Pd NPs/g-C3 N4 -S/SrTiO3 —Palladium nanoparticles/sulfur-doped carbon
nitride/strontium titanate; CdS QDs—Cadmium sulfide quantum dots.
© 2022 The Authors. Advanced Science published by Wiley-VCH GmbH

Figure 2. Amperometric sensors for detection of salivary protein biomarkers. A) Carbon-electrode-based immunoassay for IFN-𝛾 coupled to chronoam-
perometry. Reproduced with permission.[130] Copyright 2020, Elsevier. B) Reagent-free electrode-tethered immunosensor. Inset shows different current
decay due to different protein biomarker concentrations. Reproduced with permission.[107] Copyright 2021, American Society of Chemistry. Abbreviations:
EDC/NHSS—1-ethyl-3-(3-dimethylamino-propyl) carbodiimide/N-hydroxy sulfosuccinimide; HQ—hydroquinone; HRP-Strep—horseradish peroxidase-
labeled streptavidin.
analyzing minimally processed saliva samples by coating the 2.1.1. Pros and Cons of Amperometric Sensors
electrode surface with BSA; on the other side, this coating can
minimize the electroactive area to some extent. The advent of The advantages of amperometric sensors are 1) the simplicity of
tethered sensors with surface-linked redox reporters creates the sensor with easy integration on-chip and potentially low cost,
an opportunity to further simplify the amperometric sensor, 2) the fast signal transduction and rapid assaying with tethered
enabling a faster test besides making it non-reagent based. sensors, and 3) the possibility of detecting protein markers
Incubation with the saliva sample becomes the single step of in minimally prepared saliva samples. The disadvantages of
the assay, thereby facilitating the realization of a fully automated this electrochemical technique include: 1) The LODs are still
sensor. reported in the pg mL−1 level; 2) the activity of enzyme-based
Figure 3. Potentiometric sensor for saliva-based protein detection exploring a nanostructured working electrode. a) Nano-rough gold (Au) surface on a
silicon substrate. b) Imprinting of template molecules (hereby viral particles; the process is the same as spike proteins alone) on the thiol-Au surface. c)
Removal of template molecules forming an imprinted thiol layer. d) Potentiometric sensing of the analyte. e) Potentiometric response when the analyte
is added (V0 , starting baseline voltage). f) Detection of target protein biomarker. Reproduced with permission.[108] Copyright 2022, American Chemical
Society.
amperometric sensors is affected by variations in pH and tem- polishing grade of the silicon substrate surface that defines the
perature. This can be of concern upon rapid introduction of an roughness of Au. The sensor was demonstrated for saliva sam-
unprepared biological specimen; 3) the assay times are relatively ples and exhibited a detection time shorter than 5 min. Its analyti-
long with the introduction and incubation of labeled detection cal performance in saliva has so far been shown for viral particles,
antibody which follows the analyte diffusion and incubation on namely Zika virus or Dengue virus,[132] and protein markers such
the electrode surface. as SARS-CoV-2 S proteins[108] and cancer embryonic antigen.[133]
Based on carbon ink electrodes and utilizing no biomolecular
probes, another approach of the potentiometric sensor was re-
2.2. Potentiometric Sensors alized for 𝛼-amylase, a common disease marker analyzed from
human saliva.[134] The carbon electrodes were part of reagent
In saliva, the potentiometric sensor is widely exploited for the de- strips containing two separate channels under different pH
tection of inorganic indicators such as pH or ions such as salivary (one under alkaline pH and the other under neutral pH). The
thiocyanate.[78] For protein analysis, the developments of poten- target 𝛼-amylase can hydrolyze starch into maltose;[89] subse-
tiometric sensors have been focusing on exploiting the readout quently, under the alkaline solution, the generated maltose re-
properties of nanostructured electrodes. Potentiometric sensors duces Fe(CN)6 ]3− to [Fe(CN)6 ]4− . The reaction does not occur in
incorporating a nanostructured Au coating on silicon and a MIP the neutral condition. Therefore, the difference in the ion ratio
recognition element were proposed for the detection of various between two parallel channels can be explored for obtaining an
salivary biomarkers.[108,132] Hereby, the target protein is used as electrical potential difference between two electrodes, thereby in-
the template for a 3D imprinting technique applied to an Au coat- directly detecting 𝛼-amylase. This potentiometric sensor can pro-
ing with nano-roughness. The template molecules are adsorbed vide the result in 2 min and measures the target at the physiolog-
onto the concave areas of the Au coating, and thiols are crystal- ically relevant concentrations.
lized around the template by reacting with Au and forming the In conclusion, the lower utilization of the potentiometric sen-
binding sites of the sensor. After the removal of the template, sor for protein analysis compared to other electrochemical tech-
the imprinted thiol layer binds specifically to the target proteins, niques such as the amperometric sensor or impedimetric sensor
and the binding complexes cause variation in the open-circuit is explained by the difficulty of adopting potentiometric measure-
potential of the sensor. This type of potentiometric sensor is ments in affinity-based sensing.[91] Despite this issue, the combi-
shown in Figure 3. The performance of detection is controlled by nation of MIPs and nanostructured Au electrodes offers potential
the roughness of the Au coating whose concave structures shall for a class of potentiometric sensors with utility in biomarker de-
match the size of the bio-target. Smaller molecule sizes would tection cases demanding fast responses. The concept is suitable
demand a smoother Au surface at the nanoscale, and it is the for mass production.
2.2.1. Pros and Cons of Potentiometric Sensors ciencies, and enhance biocompatibility for the immobilization of
biomolecular probes, in addition to providing an increased sur-
The strengths of the potentiometric sensor are 1) the detection in face area. A carbon-based SPE nanostructured with gold nanopar-
a few min, 2) the simplicity of the sensor, and 3) the use with no ticles (AuNPs) was demonstrated for the detection of SARS-CoV-
sample treatment. The main drawbacks include 1) low detection 2 nucleocapsid (N) protein in tenfold diluted saliva samples.[136]
sensitivity, 2) limited linear response, 3) susceptibility to varia- The sensor was constructed by electrodeposition of the AuNPs
tions of pH and temperature, and requires frequent calibrations, on the SPE forming a nanostructured film, followed by surface
and 4) high requirement for a stable and accurate reference elec- modification with streptavidin used as a linker to the attachment
trode. of a biotinylated antibody. A solution of [Fe(CN)6 ]3−/4− was added
to the nanostructured film electrode to obtain the EIS signal
following a similar procedure as with the aforementioned ITO-
2.3. Impedimetric Sensors based impedimetric sensors. The nanostructured Au impedimet-
ric sensor exhibited a LOD of few pg mL−1 and good reproducibil-
Impedance sensing in saliva has been realized using unstruc- ity thanks to the large catalytic area and highly oriented linking
tured and nanostructured electrodes made of metal oxides, car- of the detection antibody. In another work,[113] an impedimetric
bon, and Au. Moreover, it is denoted an increasing use of sensor was formed by an Au-based SPE with nano-sized porosi-
graphitic nanomaterials in electrochemical impedance sensing. ties (peak to valley height of 30.6 nm). The sensor detected the
Indium tin oxide (ITO) is a widely used material in impedance receptor-binding domain (RBD) of the SARS-CoV-2 S protein
sensors despite its limitations in achieving a prominent charge with a MIP film formed on the porosity valleys of the nanostruc-
transfer efficiency.[104,110] The ITO electrode has the advantages tured electrode. While the nanoporosities enabled an electrode
of being low-cost and providing a surface highly compatible with surface with low background resistance to charge transfer, the in-
a variety of chemical methods for the immobilization of biorecog- teraction of the target RBD with the specific binding sites on the
nition probes.[93,135] Unstructured ITO is often functionalized MIP increased substantially Ret of the electrode in the presence of
with self-assembled layers (SAMs) for the attachment of anti- [Fe(CN)6 ]3−/4− . The effect was proportional to the concentration
bodies specific to the target protein. After incubation of the pro- of protein. Of remark, this nanostructured impedimetric sensor
tein (typically taking more than 30 min), the [Fe(CN)6 ]3−/4− re- measured RBD in twofold diluted saliva samples with an analysis
dox probe is added to the electrode surface to obtain the EIS time of 20 min.
signal which varies with the interaction between the protein Carbon nanotubes and graphene are among the most promis-
and the immobilized antibodies. This approach was used to de- ing graphitic nanomaterials for EIS sensors due to their effi-
tect IL-1𝛽 on ITO electrodes modified with carboxyl-activated 6- cient electron transfer, high catalytic activity, and low interfa-
phosphohexanoic acid as the SAM.[135] The sensor monitored the cial resistance.[102,137] A graphene ink formulation made of ex-
resistance of the electrode to electron transfer (Ret ) upon redox re- foliated graphene nanosheets has been used for preparing sen-
actions of [Fe(CN)6 ]3−/4− on the electrode surface. Diffusion and sitive EIS sensors. These sensors have targeted SARS-CoV-2 S1
kinetics of [Fe(CN)6 ]3−/4− were hindered on the electrode surface protein and RBD in artificial saliva exhibiting a detection range
due to increasing concentrations of IL-1𝛽. This is a typical assay of 1–1000 ng mL−1 and LODs of ≈20 pg mL−1 and ≈110 pg
methodology to achieve protein detection in EIS biosensors. The mL−1 for RBD and S1, respectively.[138] An overview of the con-
EIS detection on the unstructured ITO electrode exhibited a LOD cept is shown in Figure 4A. The approach has benefited from
of 7.5 fg mL−1 and a detection range of 0.025 to 3 fg mL−1 from a facile modification of the graphene film electrode by antibody
centrifuged and 20-fold diluted saliva samples.[135] and surface blocking agent. The high-throughput and inexpen-
Conductive composite materials are a solution with great po- sive printing of the electrodes ($3.39 per unit) are also advantages
tential to further improve the analytical characteristics of the un- of these graphene-printed sensors. Nevertheless, [Fe(CN)6 ]3−/4−
structured ITO electrode for impedance detection. A composite is still used to generate the EIS response. Analysis time has ex-
electrode made of ITO and a polythiophene derivative conjugated ceeded 30 min. Multi-walled carbon nanotubes (MWCNTs) alone
polymer has been proposed to enhance the charge transfer effi- or as part of composite electrodes are also promising materials
ciency of the electrode while providing more binding sites for for sensitive impedance detection. A nanocomposite electrode
the immobilized of the antibody.[110] The synergy of these two made of MWCNTs and AuNPs was prepared to detect DJ-1 pro-
effects led to a twofold decreased LOD for IL-1𝛽 detection com- tein as an important biomarker of Parkinson´s disease and ox-
pared with the aforementioned SAM-ITO electrode. The incu- idative stress.[111] Antibodies specific to DJ-1 were immobilized
bation time and total assay duration were not changed with the on the surface of MWCNTs (Figure 4B), and the composite in-
use of the composite electrode. In another work,[93] a twofold re- terface MWCNTs-AuNPs ensure high charge transfer character-
duced LOD for detecting salivary interleukins was achieved by istics. The interaction of the target protein with the immobi-
modifying the surface of ITO with a composite film made of car- lized antibodies created variations of both Ret and electrochemi-
bon black, poly(glycidyl methacrylate), and polyvinylidene fluo- cal capacitance of the electrodes in the presence of [Fe(CN)6 ]3−/4− .
ride. Hereby, the interleukin IL-8 was detected in 50-fold diluted This nanocomposite-based EIS sensor achieved a remarkably low
saliva samples, and the incubation times were still longer than LOD (0.5 fg mL−1 ); nevertheless, a 106 -fold dilution of saliva was
30 min. necessary to execute detection in clinical samples with minimal
Another class of impedimetric sensors involves the nanostruc- signal interferences.
turing of SPEs. Nanostructured SPEs can be taken as an alterna- In conclusion, low-cost electrodes such as ITO and SPEs are
tive to ITO composite electrodes to improve charge transfer effi- coated with nanostructured materials to decrease the LODs of
Figure 4. Impedimetric sensors for detection of salivary protein biomarkers. A) Illustration of a graphene-based EIS sensor with the steps: a) Aerosol
jet printing of graphene ink with dispersed nanosheets. b) Immobilization of antibodies. c) Blocking of unmodified graphene areas against non-specific
adsorption. d) Sampling method proposed for the saliva test with the sensor. e) Incubation of the sample containing either S1 protein or RBD). f) Nyquist
plot with and with no analyte. g) Charge transfer resistance response (ΔRct ) due to RBD concentrations in saliva. Reproduced with permission.[138]
Copyright 2022, IOP Publishing. B) Illustration of a nanocomposite impedimetric sensor made of ITO electrode immobilized with MWCNTs and AuNPs.
Reproduced with permission.[111] Copyright 2021, Elsevier. Abbreviation: 11-AUT—1-amino-1-undecanethiol.
impedance sensors which can reach fg mL−1 . The basis of signal strategy was used in another DPV sensor using AuNPs bound to
amplification is to maximize electron transfer efficiency between human angiotensin-converting enzyme 2 (ACE2) peptide which
the electrode and electrolyte, and hence to reduce the background acted as the bio-receptor to SARS-CoV-2 RBD.[117] A remarkably
Ret before surface-binding of the protein. Among the reports an- low LOD of 0.35 ag mL−1 was achieved with this AuNP-ACE2 sen-
alyzed, the detection range has seldom surpassed four orders of sor when an extra bio-recognition probe made of ACE2 labeled
magnitude either with uncoated metal or carbon electrodes or with magnetic particles was added to the assay.
with those coated with nanomaterials. A detection range of five AuNPs have acted as modifiers of voltammetric sensors to en-
orders of magnitude was achieved by coating a dimeric DNA ap- hance their conductivity and confer larger catalytic areas (Figure
tamer on a thiolated Au electrode.[112] This work may reinforce 5A). For a DPV sensor detecting SARS-CoV-2 S1 protein in saliva,
further the fact that the design of the biosensor needs to consider AuNPs were used to modify the surface of fluorine-doped tin ox-
the synergy of electrode materials and properties of biorecog- ide electrodes.[140] The nanoparticles guaranteed a high peak cur-
nition elements. The analysis times with impedimetric sensors rent response in the presence of [Fe(CN)6 ]3−/4− , which has de-
are typically in the order of tens of min and are regulated by creased with the formation of protein-antibody complexes atop
the diffusion and incubation of the analyte on the electrode sur- the AuNPs. For an SWV sensor detecting SARS-CoV-2 RBD,
face. The steric hindrance created by the protein/bio-recognition AuNPs have modified the surface of a composite electrode made
probe complexes on the surface reactivity of a redox probe is the of nanoporous aluminum oxide membranes and graphene.[120]
basic sensing mechanism in impedimetric sensors. This mech- The nanoparticles were expressed atop the composite electrode
anism often waives the use of a labeled secondary antibody sim- and were bound to thiolated aptamers via Au–S bonds. The SWV
plifying the assay. Saliva dilution is still the common procedure was maximum with no presence of analyte indicating the high
for preparing the sample for impedance sensing.[110,136] diffusivity of the nanoporous sensor to a redox probe. By bind-
ing of target RBD with the surface-immobilized aptamer, mass
transfer of the redox probe was hindered on the electrode sur-
2.3.1. Pros and Cons of Impedimetric Sensors face, leading to a decreased Faradaic current. Besides offering
a large surface area for aptamer binding, AuNPs have also en-
Pros of impedimetric sensors are 1) the compatibility of the sens- hanced charge transfer through this nanoporous electrode. The
ing technique to use of different bio-receptors including antibod- SWV response was obtained from 1:4 diluted saliva samples and
ies, aptamers, and MIPs, 2) the possibility of developing sensitive the analysis time surpassed 20 min.
biosensors from low-cost electrode surfaces, 3) the wide avail- The utilization of AuNPs to confer nanostructures on voltam-
ability of EIS electrode designs comprising various types of com- metric sensors is a common procedure; nevertheless, Au in form
posite materials. The major cons concerning protein detection in of other nano-scale architectures can also be exploited to achieve
saliva encompass: 1) The need for introducing a redox probe in notable charge transfer efficiencies and enhance peak currents.
solution to execute the detection; the reagent-based assay incre- Au in form of nanowire arrays was synthesized on top of poly-
ments one step in the operation of the sensor following incuba- mer substrates for the detection of salivary CRP at a limit of a
tion with the analyte, 2) the need for more complex data analy- few fg mL−1 .[119] The nanowires provided a truly enlarged surface
sis compared to amperometric or impedimetric sensors involv- for antibody immobilization and facilitated the redox cycling of
ing data fitting to equivalent circuits, 3) the limitation of sensing [Fe(CN)6 ]3−/4− , being responsible for the high catalytic activity of
mechanisms to steric hindrance which may restrain the develop- the voltammetric sensor (Figure 5B). Of note, the SWV response
ment of strategies to enhance detection sensitivity. of this sensor can be optimized for various bio-targets by reg-
ulating the size and density of nanowires using a nanoimprint
lithography process. This SWV sensor achieved three orders of
2.4. Voltammetric Sensors magnitude as the detection range for CRP measured in tenfold
diluted saliva samples.
DPV and SWV are the most representative voltammetry-type Graphitic nanomaterials have emerged as alternatives to Au
techniques for protein detection in saliva. Both techniques pro- nanostructures for sensitive voltammetric sensors. Besides ex-
duce well-defined peak currents in rapid assays, exhibit high hibiting naturally high catalytic activity and in the meanwhile
signal-to-noise ratios, and require no complex signal processing good biocompatibility, the graphitic nanomaterials benefit from
such as fitting to equivalent circuits as in the case of EIS. facile manipulation of their surface and lattice composition, tun-
AuNPs have been intensively exploited in DPV[117,139] and ing physicochemical properties, and creating a new generation of
SWV[119,120] sensors due to their high catalytic properties for re- voltammetric sensors with outstanding peak current responses.
dox reactions. For analysis of protein biomarkers in saliva, AuNPs Graphene oxide (GO) with or without atomic and surface mod-
have majorly been used in two sensing formats, either as labels ifications has been designed for a variety of DPV and SWV sen-
of biorecognition probes[117] or as modifiers of the metal elec- sors. GO with modification can be used as a coating for common
trode or composite electrode surfaces.[120] A DPV sensor for sIgA SPEs and glassy carbon electrodes (GCEs). A series of SWV sen-
was realized by labeling secondary (detection) antibodies with sors with GO-coated SPEs and GCEs were developed for SARS-
AuNPs. A peak current response was obtained from the electro- CoV S1 proteins using antibodies as the biorecognition element.
chemical reduction of the complex [AuCl4 ]− to Au in the presence After the addition of an electrolyte, the S1 proteins were quanti-
of diluted acid, and the response was proportional to the amount fied in the range of attograms to femtograms per mL from saliva
of sIgA bound to the AuNP-labeled antibody. The LOD of this samples pretreated with a lysis buffer.[141] This GO-based SWV
DPV assay was in the order of a few ng mL−1 .[139] A similar assay sensor demonstrated a diagnostic accuracy of over 90% in posi-
Figure 5. Voltammetric sensors based on Au nanostructures. A) DPV sensor for the detection of SARS-CoV-2 S proteins exploiting the catalytic ef-
fect and large surface area of AuNPs. Inset (f) demonstrates decreasing in DPV response due to increasing protein concentrations. Reproduced with
permission.[140] Copyright 2021, Elsevier. B) SWV sensor with Au nanowires for the detection of salivary CRP. Insets of the bottom side of (B) show the
SWV response due to increasing CRP concentrations (from “a” to “o”) and the respective calibration curve based on peak currents. Reproduced with
permission.[119] Copyright 2019, Elsevier. Abbreviation: MPA—3-mercaptopropionic acid.
tive virus-infected samples which contrasted with less than 67% On the other side, there was observed no gain in the detection
achieved with a commercial antigen test kit. range which shifted to lower protein concentrations. Especially
Atomic modification of GO has also been exercised to tune the for voltammetric sensors based on graphitic nanomaterials, the
electrocatalytic properties of this nanomaterial. GO can be trans- biocompatibility of nanomaterial surfaces to various methods of
formed into a porous material and doped with different elements electrode passivation against non-specific biomolecule binding,
to enhance the electroactive area of GO-coated electrodes. Using such as the surface modification with poly(ethylene glycol) or
this route of GO modification, a nitrogen-doped GO coating on casein, has enabled the analysis of undiluted saliva samples. In
a GCE electrode was reported for the detection of cTnI in undi- summary, the analysis times with state-of-art voltammetric sen-
luted saliva samples.[115] The porous structure of GO enabled a sors are not inferior to 30 min and are mainly affected by the
large surface area for the immobilization of a DNA aptamer and mass transport phenomenon of the analyte toward the electrode
guaranteed high DPV peak currents. The interactions of cTnI and surface and by the use of a secondary biorecognition probe if a
the aptamer were measured at cTnI concentrations spanning six “sandwich-type” immunoassay would be necessary for the target
orders of magnitude, which indicated the good signal-to-noise ra- analyte.[116]
tio of the modified GO sensor. Minimal biological interferences
in the test of undiluted samples were ensured by a poly(ethylene
glycol) coating which was highly compatible with the nitrogen- 2.4.1. Pros and Cons of Voltammetric Sensors
doped GO.
Moreover, GO can be used to stabilize metal oxide nanopar- The pros of the voltammetric sensors for protein detection in
ticles which also hold promising electrocatalytic properties for saliva are 1) the wide availability of electrocatalytic nanomateri-
voltammetric sensors. Nanocomposites of GO with zinc oxide als for DPV and SWV electrodes, 2) the flexibility of assay for-
(ZnO) nanoparticles were prepared for DPV sensors targeting mats either using single bio-receptors or combining multiple bio-
salivary IL-8.[114] Chemical functionalization of the nanocompos- receptors in one assay, thereby widening the possibility of tar-
ite surface with ethanolamine ensured reproducible measure- geting many types of proteins, 3) the readout of the voltammet-
ments of IL-8 in undiluted saliva samples. GO has also stabi- ric signals requires less processing compared to impedimetric
lized yttria-doped zirconia nanoparticles exploited as a sensing sensors, and 4) the possibility of analyzing minimally processed
platform to detect salivary CYFRA-21-1.[142] These GO-stabilized saliva samples. The disadvantages of voltammetric sensors are
DPV sensors exhibited LODs for protein detection in the order related to 1) the dependence on the redox activity of an externally
of pg mL−1 . added probe and subsequent dependence on its diffusion and re-
Besides GO, other graphitic nanomaterials have emerged in action kinetics, 2) the reduced charge transfer efficiency in the
the literature for voltammetric sensing. MWCNTs with surface electrode caused by poly(ethylene glycol) or casein-based surface
modification with 11-azide-3,6,9-trioxaun-decan-1-amine have passivation, and 3) the influence of orientation and polarity of tar-
significantly improved the peak current response of carbon-based get protein molecules on the charge transfer between the redox
SPEs.[116] This azide-MWCNTs-based sensor has detected IL-1𝛽 probe and the electrode.[140]
in minimally processed saliva samples. For the assay, a primary
antibody was immobilized on MWCNTs via electro-click chem-
istry, and a secondary antibody labeled with alkaline phosphatase- 2.5. Electrolyte-Gated Field-Effect Transistor Sensors
streptavidin was loaded on the electrode after incubation of IL-
1𝛽. Alkaline phosphatase catalyzed the conversion of 1-naphthyl EGTs are FETs in which an electrolyte acts as a gate insulator di-
phosphate to 1-naphthol leading to DPV currents (Figure 6A). electric in contact with the conducting channel. In this setup, the
The azide-MWCNTs formed a perfect surface for passivation with ions in the electrolyte are displaced in opposite charges at the in-
1% casein which facilitated measurements in undiluted saliva terface channel/gate when an electrical field is applied. In EGTs,
samples. The possibility of tuning the conductivity and catalytic nanomaterials are commonly applied to the FET-conducting
activity of graphitic carbon foils was exploited in DPV-based channel exploiting their superior charge transport characteris-
sensing.[143] It is known that the physicochemical properties of tics.
graphitic nanomaterials can be tuned by exfoliation and/or sur- The EDLT is the type of EGT in which the binding of the
face activation with specific functional groups. By using a strategy biorecognition element with the target protein causes variation of
of partial exfoliation and surface activation with carboxyl groups, EGT-channel charge density. Multi-pore carbon nanofibers were
modified graphitic carbon foils have exhibited a high sensitivity synthesized for an EDLT conducting channel used for detecting
to variations in the diffusion and kinetics of [Fe(CN)6 ]3−/4− reac- nesfatin-1, a biomarker of epilepsy.[144] The sensor is shown in
tion (Figure 6B). This sensitivity was exploited for the detection of Figure 7A. The carbon nanofibers were impermeable to ions, and
SARS-CoV-2 S proteins recognized by surface-immobilized anti- the operation of the sensor was based on the formation of an
bodies. The LOD was in the order of tens of pg mL−1 while the ultra-thin electrical double layer at the electrode/electrolyte in-
detection was conducted in artificial saliva. terfaces. Artificial saliva was used as the electrolyte.[121] At fixed
In conclusion, Au nanostructures and doped or surface- gate voltage and fixed source-drain voltage, the source-drain cur-
modified GO have extensively been used in voltammetric sen- rent (Isd ) decreased with the formation of protein (nesfatin-1)
sors. Lowering the detection limit of DPV and SWV sensors and antibody (anti-nesfatin-1) immune complexes onto the sur-
from the pg mL−1 level to a few fg mL−1 has been made possi- face of the carbon nanofibers. These immune complexes in-
ble by engineering nanostructures on Au surfaces or by form- duced changes in the charge transport properties of the carbon
ing heterostructures of two or more nanomaterials (see Table 2). nanofibers channel, decreasing the hopping rate of charges. The
Figure 6. Voltammetric sensors based on graphitic nanomaterials for the detection of salivary interleukin IL-1𝛽 and SARS-CoV-2 S proteins. A) Screen-
printed electrode modified with MWCNTs. Deposition of MWCNTs was followed by adding CuSO4 containing anti-IL-1𝛽 IgG. DPV was measured using
1-naphthylphosphate (1-NPP) in presence of an alkaline phosphatase label. Reproduced with permission.[116] Copyright 2020, Elsevier. B) Biosensor with
oxidized graphitic carbon foil (OGCF) and functionalized with antibody against S1 protein. Inset shows the DPV signals from detecting various protein
concentrations causing a decrease in peak current. Reproduced with permission.[143] Copyright 2022, Elsevier. Abbreviation: EDA—ethylenediamine.
Figure 7. Electrolyte-gated field effect transistors (EGTs). A) Detection of Nesfatin-1 in saliva by an electrical double layer transistor (EDLT) comprised of
multi-pore carbon nanofibers deposited on interdigitated electrodes and functionalized by antibody. Reproduced with permission.[121] Copyright 2021,
Royal Society of Chemistry. B) A graphene-based EDLT for protein detection with resolution in the attomolar level. In this sensor, the conformation of a
flexible single-stranded DNA (ssDNA) cantilever is adjusted according to ion-gate voltage (Vlg ), bringing an aptamer probe bound to the analyte near
the EDLT channel. Detection occurs within the Debye length. Reproduced with permission.[146] Copyright 2022, Springer Nature. C) An organic electro-
chemical transistor for detection of salivary IgG with biorecognition occurring through the gate modified with antigen. Reproduced with permission.[124]
Copyright 2021.
performance of this EDLT benefited from the porous structure of nel was modified with an RNA aptamer.[122] The change of con-
carbon nanofibers which increased the surface area for antibody formation of the aptamer upon binding with the target protein
binding, thereby detecting channel charge density variations with enabled an increased Isd response under a positive gate voltage.
high sensitivity. Proteins were detected in the range of femtomo- Before binding with the protein, the negatively charged RNA
lar (fM) concentration as the limit with this EDLT. aptamer was folded in the vicinity of the FET channel, which
Graphitic nanomaterials are also specially designed for EDLTs counteracted the applied electric field leading to negligible alter-
to achieve conducting channels ultra-sensitive to variations of ation in Isd . The graphene monolayer is another nanomaterial
charge in the vicinity of the channel. GO is one of the most re- with a high potential for ultra-sensitive EDLTs. Figure 7B shows
ported graphitic nanomaterials for EDLTs. For the detection of an EDLT with a graphene monolayer channel coupled to an ad-
human papillomavirus-16 E7 protein in saliva, a GO EDLT chan- vanced biomolecular structure for protein recognition (named
DNA cantilevers). The DNA cantilever controls the induction of unprocessed saliva samples. In conclusion, the OECTs provide
electric charges on the graphene monolayer which is known to a fast sample-to-result time (<15 min) in clinical samples with
be very sensitive to electrical potential variations.[145] The target little or no sample processing, and their ultra-high sensitivity is
protein binds to an aptamer as part of the biological cantilever accompanied by the potential for device miniaturization.[86,124]
linked to a flexible single-stranded DNA (ssDNA). The aptamer- Overall, nanomaterials are the essential components of the
protein complex is detected under a negative potential across the EGTs whose sensing performance demands the sensitive mea-
electrolyte gate (Vlg < 0 in Figure 7B). At such potential, the neg- surement of tiny changes in the FET channel conductivity.
atively charged ssDNA moves downward bringing the aptamer- Among the graphitic materials reported for ion-impermeable
protein complex within the Debye length in which the variation channel EGTs, the graphene monolayer may exhibit the highest
in the electrical potential of the graphene channel is maximum. potential. Coupling the graphene monolayer conducting channel
By the measurement of Isd which decreased with increasing pro- with flexible single-stranded oligonucleotide probes allows tak-
tein concentrations before signal saturation, this EDLT detected ing advantage of the ultra-sensitivity of graphene to variations of
thrombin in the range of 0.5 aM to 0.25 pM.[146] In conclusion, charge in the vicinity of the material. For ion-permeable EGTs,
the graphitic nanomaterial-based EDLT exhibits ultra-high sensi- the progress in the synthesis of new conjugated polymers may
tivity to protein detection in unprocessed saliva samples, on the deliver novel ion-permeable materials enabling further improve-
condition that the biorecognition probe-protein complexes are ment of detection limits and lowering the power to operate the
measured within the Debye length. FET. The operation of the EGTs concerning electrolyte gate volt-
The ECT is another modality of EGT with the merit of ultra- ages is generally dependent upon the charge mobilities of the
high sensitivity to salivary biomarkers´ detection within the De- conducting channel materials, the intrinsic charges of the target
bye length. Organic semiconductors (OSCs) are a class of nano- protein, and the ionic compositions of the electrolyte.
materials mostly employed as the ECT channel due to their
high permeability to ions. The ECTs based on OSCs (or OECTs)
are commonly characterized by their high transconductance, 2.5.1. Pros and Cons of Electrolyte-Gated Field-Effect Transistor
low working voltage, and excellent stability in contact with the Sensors
electrolyte.[98] When arranged as a FET channel the OSC with-
stands volumetric doping/dedoping upon injection of ions via The major advantages of the EGTs for protein sensing in saliva
the gate electrode. This unique feature makes the OECT ultra- are 1) the extremely high signal amplification in the conduct-
sensitive to minute binding events at the gate surface. With the ing channel due to the ultra-high conduction gain in graphitic
immobilization of the target protein on the gate, large modula- nanomaterials (used in EDLT) and the volumetric coupling be-
tion of Isd occurs due to the intrinsic charges of the protein or tween ionic and electronic charges in the ECT channel, leading
protein-induced charge redistribution.[147] to ultra-low detection limits, 2) the great biocompatibility of EGT
Poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonate) (PE- nanomaterials, 3) the sensitive and accurate detection in unpro-
DOT:PSS) is a hole-conductive OSC widely utilized in OECTs. cessed saliva samples taking advantage of sensing within the De-
Upon application of a positive Vlg , cations can be injected into bye length, 4) the high potential of the EGT to be coupled with
the PEDOT:PSS channel, compensating for the depleted holes. integrated circuit designs, further miniaturizing the biosensor.
A representative OECT with a PEDOT:PSS channel for salivary EGTs also involve some disadvantages that need to be consid-
protein detection is depicted in Figure 7C. The sensor detected ered in the design stage of the biosensor, namely: 1) The depen-
SARS-CoV-2 IgG on an Au gate electrode coated with S1 protein dence upon intrinsic charges of the target analytes which are de-
(hereby used as the biorecognition element). This OECT has ex- fined by the respective isoelectric points in saliva. Any variation
emplified the role of the intrinsic charges of the protein to induce of protein charges between samples would significantly affect the
a varied response of the OECT channel. The positively charged reproducibility of detection; 2) less availability of nanomaterials
IgG bound to the S1 protein at the gate switched the Vlg to more compared to other electrochemical sensors, which limits design
negative potentials. The detection was demonstrated for eight or- flexibility; 3) the possibility of Isd signal saturation for protein
ders of magnitude of IgG concentrations while the LOD was in concentrations exceeding the picomolar level.[146]
the order of a few fM.[124] As the IgG-S1 protein complex has ex-
ceeded the Debye length in the unprocessed saliva sample, dilu-
tion was necessary to increase the Debye barrier. 2.6. Photoelectrochemical Sensors
Other conjugated polymers have been proposed to replace PE-
DOT:PSS in the OECT channel. A conductive polymer named The PEC sensor is relatively new in the field of saliva-based
p(g0T2-g6T2) was introduced in an OECT for the detection of diagnostics.[100,148] Generally, PEC-based protein detection in-
SARS-CoV-2 RBD.[86] The new polymer allowed the ECT to be volves the functionalization of a semiconductor electrode with
operated at even lower gate voltages than that in the PEDOT:PSS bio-receptors whose binding with the target protein induces
OECT, and with no cost in transconductance efficiency. Of re- variations of the sensor photocurrent response. Upon illumina-
mark, in addition to the new OECT channel, RBD was detected tion, the semiconductor or semiconductor composite catalyzes
by nanobodies immobilized on the gate electrode. Due to their the redox reaction of charge-scavenging species at the elec-
lower dimensions compared to antibodies, the nanobodies en- trode/electrolyte interface thereby generating the photocurrent
sured the detection of the protein marker under the Debye barrier response. TiO2 , ZnO, and other wide bandgap semiconductors
with no need for saliva sample dilution. The p(g0T2-g6T2) OECT were commonly used in PEC biosensors because of their PEC
has achieved a single-molecule sensitivity (in the range of zM) in stability. However, their low response in the visible light range
Figure 8. Photoelectrochemical sensors incorporating mixed-dimensional nanomaterials. A) A photoelectrochemical sensor for prostate-specific antigen
targeted in saliva. The sensor is made of oxidized Ti3 C2 Tx nanosheets with adsorbed NiWO4 nanoparticles. The heterostructure is used to modify
the surface of a glassy carbon electrode-GCE. Reproduced with permission.[126] Copyright 2021, Elsevier. B) A photoelectrochemical sensor to detect
S proteins based on the heterostructure of a 2D metal-organic framework (Yb-TCPP nanosheets) covered with AuNPs. Photocurrent response was
enhanced via plasmon-induced resonance energy transfer-PRET. Reproduced with permission.[151] Copyright 2021, American Chemistry Society.
has prompted a shift in the research toward semiconductors or protein concentrations and it is an extensive strategy for any other
semiconductor composites exhibiting a lower bandgap. protein marker. A major merit of this g-C3 N4 -based composite
Graphitic carbon nitride (g-C3 N4 ) is a rising star nanomate- sensor was the achievement of a wide detection range covering
rial in the field of PEC biosensing.[149] It has a bandgap of 2.7 eV fg mL−1 to ng mL−1 (six orders of magnitude).
and allies great photosensitivity with excellent chemical stabil- Another composite of g-C3 N4 with CdS quantum dots was de-
ity. Due to its graphene-like nature, g-C3 N4 possesses tunable veloped for the detection of RBD protein.[127] The mechanism
electronic properties. Nevertheless, to achieve sufficiently high of interfacial charge transfer occurred similarly as in the afore-
charge transfer efficiency, g-C3 N4 is normally associated with mentioned g-C3 N4 /SrTiO3 /PD sensor. The g-C3 N4 /CdS compos-
other semiconductors and metals forming PEC nanocomposites. ite surface was functionalized with a DNA aptamer and, in the
A nanocomposite made of g-C3 N4 , a perovskite (SrTiO3 ), and pal- presence of RBD, the bound biomolecular complex formed a bar-
ladium (PD) nanoparticles were proposed for the sensitive detec- rier to the redox activity of ascorbic acid. One of the merits of the
tion of S1 protein in artificial saliva.[125] The principle of detection g-C3 N4 /CdS PEC sensor was the good accuracy of detection as
involved the specific binding of S1 with an antibody coated on recovery rates exceeded 95% for saliva samples spiked with RBD.
the composite surface which created steric hindrance to a redox Another graphite-like nanomaterial with a 2D structure,
probe (ascorbic acid) in solution. Upon illumination of the com- namely Ti3 C2 Tx nanosheets, was synthesized for a PEC sensor
posite by visible light, and with no presence of S1 protein, photo- targeting the prostate-specific antigen (PSA) cancer biomarker in
generated holes in the semiconductor composite were scavenged saliva (Figure 8A). For the electrode fabrication, Ti3 C2 Tx was sta-
by ascorbic acid. In the presence of the S1 protein, the immune bilized by partial oxidation in the presence of NiWO4 nanopar-
complex hindered the diffusion and kinetics of the redox probe at ticles coating the nanosheets. Besides exhibiting high stability,
the PEC electrode/electrolyte interface, resulting in a decreased the Ti3 C2 Tx -based PEC sensor has achieved protein detection
photocurrent response. The effect is proportional to increasing covering fg mL−1 to sub-mg mL−1 concentrations (eleven orders
of magnitude) exploiting steric hindrance of PSA/antibody com- insulating compounds[148] have not been much explored in saliva
plexes on the redox activity of ascorbic acid. The ultra-wide range biosensing.
of detection was attributed to the efficient charge transfer be-
tween Ti3 C2 Tx nanosheets and the supporting GCE and between
2.6.1. Pros and Cons of Photoelectrochemical Sensors
Ti3 C2 Tx and the electrolyte and also due to reduced charge re-
combination in the oxidized Ti3 C2 Tx . The LOD of this graphitic
The main pros of PEC sensors are 1) wide detection range for
nanomaterial-based sensor was below 1 fg mL−1 .
protein detection, 2) low LOD below fg mL−1 which can cover
Composites based on 2D metal–organic frameworks (MOFs)
the physiological concentration limit of most protein markers in
are another emerging class of materials for highly sensitive and
saliva, 3) fast signal generation from redox reactions at the elec-
stable PEC sensors.[150,151] In the nanosheet form, a 2D MOF
trode/electrolyte interface, 4) variety of sensor designs with mul-
named Yb-TCPP was developed for PEC sensing of S1 proteins
tiple electrode architectures and suitability to various biorecogni-
(Figure 8B). The Yb-TCPP semiconductor nanosheets displayed
tion elements. The major pitfalls are: 1) The complexity of PEC
high electron-hole separation (quantum) efficiency and good
electrodes that is necessary to achieve the desired photocatalytic
electrode charge transfer properties, especially in combination
efficiency; 2) the PEC stability is affected by the pH and ionic
with AuNPs. The Schottky junction formed by the 2D Yb-TCPP
strength of the electrolyte; 3) the addition of electroactive reagents
and AuNPs enhanced the photoelectric conversion in the PEC
for the sensing mechanism which limits automation of the assay;
electrode due to the phenomenon of plasmon-induced resonance
and 4) the need for processing the saliva sample before testing
energy transfer (PRET). PRET is considered one of the state-of-
which increments assay steps and hinders rapid detection.
art mechanisms for signal enhancement in PEC sensors.[100] To
maximize the enhancing effect of PRET, the 2D MOF was de-
signed with an absorption spectrum overlapped with the sur- 2.7. Comparison of Electrochemical Sensors for Salivary Protein
face plasmon resonance spectrum of AuNPs. A DNA aptamer Detection
was coated on the surface of AuNPs for the specific detection
of S1 protein; the aptamer/protein complex formed a barrier to The amperometric, potentiometric, impedimetric, and voltam-
charge transfer between the composite electrode and a Na2 SO4 metric sensors are classical electrochemical biosensors for sali-
electrolyte containing a redox probe. This work[151] exemplified vary protein measurement. All of these sensors take advantage of
the amplification effect of AuNPs in PEC sensors not only for the signal enhancement with the incorporation of nanomaterials and
photocurrent signal but also for the surface binding area. most of them can perform satisfactorily in minimally processed
In general, the analysis times for PEC sensors exceed 30– saliva samples by implementing nanomaterial surface modifi-
40 min and are majorly limited by the incubation times of the cation with blocking agents against non-specific adsorption of
target proteins. To handle the saliva samples, the PEC working interfering compounds (which may include large glycoproteins
electrode is commonly passivated with a BSA solution after sur- or other untargeted biomolecules naturally occurring in saliva).
face immobilization with biorecognition probes.[125,151] Although However, the amperometric and potentiometric sensors namely
being pre-treated with BSA, the majority of PEC sensors reviewed those based on enzymes for signal generation are more suscep-
have not analyzed unprocessed samples. The PEC detection is tible to variations in pH and temperature compared to the im-
still demonstrated for saliva in its diluted form or pre-filtered pedimetric or voltammetric analogs. On the other hand, in label-
by porous membranes.[126,127] The electrolyte solution contain- free impedimetric and voltammetric utilizing steric hindrance as
ing the redox probe is commonly loaded on the PEC sensor after the sensing mechanism, the orientation and charge of the target
the incubation step with the analyte present in the saliva sample. protein molecules are relevant factors that can interfere with the
Nevertheless, the good electrolytic properties of saliva biofluid[80] detection signals. For these cases, there are practical solutions
open the possibility of adding the redox probe (i.e., ascorbic acid) to minimize these interferences with the electric control of bio-
into the saliva sample which reduces the number of incubation receptor surface immobilization[153] and the standardization of
steps. background ionic composition of saliva with an associated cost
In conclusion, despite the intensive research on g-C3 N4 -based in adding a step of sample pre-treatment.[154] With the minimiza-
composites, Ti3 C2 Tx as part of a new family of 2D metal car- tion of interferences, the label-free impedimetric and voltammet-
bides has shown the highest promise among the nanomateri- ric sensor can perform more accurately and precisely than the
als reported for PEC sensing of salivary proteins. The reason enzyme-based amperometric or potentiometric sensor.
for the higher catalytic activity of this metal carbide compared The mode of detection and the automation of assay are also fac-
to g-C3 N4 might be related to the exposed Ti sites on the metal tors of differentiation among the above-mentioned sensors. The
carbide nanosheets which endow stronger redox reactivity than continuous mode of detection for protein biomarkers in situ is
that of carbon-based materials.[152] Higher reactivity might justify needed in various clinical situations, including but not limited to
the wide detection range achieved by Ti3 C2 Tx . Nonetheless, due patient safety against infections or organ failure by measuring in-
to the low cost of g-C3 N4 , research on g-C3 N4 -based composites flammatory biomarkers.[155] Potentiometric sensors and tethered
shall continue with the goal of realizing new PEC electrodes with amperometric sensors with near real-time response and incor-
improved detection range and sensitivity. Moreover, future works porating reversible binding bio-receptors[107,156] have the perfect
may exploit the use of saliva as the electrolyte medium and com- characteristics for analyte sensing in continuous mode. Further-
pare PEC sensing in saliva to that in standard electrolytes. It is more, by analysis of existing assay protocols, the tethered sensor
also observed that other PEC sensing mechanisms, besides PRET with an embedded redox reporter offers the best potential for as-
and steric hindrance, namely in situ generation of electroactive or say simplification and subsequent automation even compared to
recent label-free impedimetric and voltammetric sensors that still the other side, conjugated polymers are the optimal choice for
require the addition of redox reagents. ECT sensors, which is justified by their record-high transcon-
Despite the merit of rapid responses of potentiometric and am- ductance and volumetric balance between ionic and electronic
perometric sensors, their limits of detection are seldom to be charges.[44,99] Nevertheless, although the single-nanomaterial-
lower than the pg mL−1 level and their detection range is situ- based sensor may perform satisfactorily, a substantial number
ated above 1 pg mL−1 . There are cases of salivary biomarkers, of works indicated the need to modify the nanomaterial with
for instance, interleukin-6 or SARS-CoV-2 N protein, whose low- doping elements[115] and/or form composites with another one
est concentration with clinical relevance was detected below pg or more nanomaterials.[148] Especially, the design of heterostruc-
mL−1 .[47,67] Other biomarkers including but not limited to NT- tures or nanocomposites of two or more nanomaterials is cru-
proBNP and TNF-𝛼 have their lowest concentration close to 1 pg cial in impedimetric, voltammetric, and PEC sensors to max-
mL−1 , and the variability in the biomarker determinations among imize charge transfer efficiencies at the electrode/electrolyte
subjects and between clinical studies needs to be considered. interfaces.[111,120,125]
Therefore, achieving biosensor resolutions and detection ranges The role of electrode materials is not dissociated from the
covering levels below pg mL−1 would ensure a “safe zone” for the biorecognition probes. Nanobodies and aptamer/peptide probes
analysis of salivary biomarkers. The detection of salivary proteins with fold/unfold states are currently exploited to ensure ultra-
in the sub-pg mL−1 and fg mL−1 concentration has been made sensitive detection within the Debye length. This is particularly
possible with nanomaterial-based impedimetric and voltammet- relevant for the test of saliva or other biofluids in which the De-
ric sensors (Table 2). bye length may recede to less than 1 nm.[87,147] The Debye length
EGTs and PEC sensors are emerging classes of electrochemi- in an electrochemical sensor defines how far the electrostatic ef-
cal sensors with a great prospect for continuous innovations in fect from a protein-receptor complex on the response of a nano-
this field. To date, among the types of electrochemical sensors material electrode persists. The bio-receptors are also associated
hereby reviewed, the EGTs may give the best compromise be- with electroactive labels. By labeling a secondary receptor, poly-
tween low LODs, simplicity of assay formats, rapid detection, dopamine for instance can act as an effective electron donor in
and amenability for testing unprocessed saliva samples. On the an electrochemical sensor to further enhance charge transfer ki-
other side, the PEC sensor would find great applicability in clin- netics at the electrode/electrolyte interface, or to sweep holes at
ical cases, in which is necessary to analyze biomarkers across a the interface to further reduce undesired recombination in elec-
wide range of concentrations. The PEC sensor may potentially trodes made of semiconductors.[158] On the other hand, the pri-
achieve accurate protein measurements from fg mL−1 to near mg mary receptor bound on the nanomaterial surface can also immo-
mL−1 .[126] This feature relates to the high signal-to-noise ratio of bilize enzymes that catalyze reactions generating charge donors
the sensor due to the physical separation of light input from the in situ.[159] In this case, the specific binding of these receptors to
electrical output. Determinations across a wide detection range target proteins ceases this electroactive species generation.
with the same sensing principle can enable the analysis of multi-
ple biomarkers or different disease conditions from one sensing
platform. 3.2. Incorporating Signal Amplification Techniques
Significant efforts have been made to endow the working elec-

3. Current Trends and Future Perspectives trodes with protein detection sensitivities and LODs at the
In conformity with the recent research progresses in saliva-based femtomolar or even some at the zeptomolar (single-molecule)
electrochemical sensors being reviewed in Section 2, the follow- levels.[86,157] To obtain these levels of sensitivity and LOD, strate-
ing discussion focuses on trends and key factors for the advance- gies of signal amplification have been adopted in the design of
ment of electrochemical biosensors based on nanomaterials. Im- the nanomaterial-based electrodes. The enhancement of the elec-
proving the detection sensitivity and the detection limit, reducing tron and mass transfer processes at the electrode/electrolyte in-
the analysis time with minimal sample handling, and reaching terfaces are the desired effects with the use of signal amplification
the automation and miniaturization of the biosensor are com- methods in electrochemical sensors.
mon goals for researchers and engineers to achieve in clinically The charge transfer efficiencies are controlled by the com-
useful POC devices. position, surface modification, and dimensionality of the elec-
trode materials. Several materials such as carbon nanotubes
and graphene oxide, for instance, improve the electrical con-
3.1. Modifying Electrodes with Nanomaterials nection between the reaction sites and the electrode. Element
doping is a flexible strategy to tune electron energy band struc-
Nanostructured Au, carbon nanostructures, graphitic nanoma- tures of the employed nanomaterials.[160] Moreover, the forma-
terials (GO, MWCNTs, g-C3 N4 ), and conjugated polymers are tion of nanocomposites aims at reducing the pitfalls and maxi-
among the rising nanomaterials used for modifying Au and mizing the strengths of individual electrode materials.[118] Sur-
SPEs or MEMS interdigitated electrodes and thereby achieving face modification with metal and metal oxide nanoparticles not
the desired performance. Certain types of electrochemical sen- only enhances the conductivity of the electrode but also increases
sors have been predominantly adopting a specific class of nano- the reaction sites. The rising focus on 2D layered materials,
material. For instance, graphitic nanomaterials are widely used with thickness reduction from bulk to single-layer, allows ex-
in DPV sensors due to their easily tunable electronic proper- ploiting the quantum confinement effect to tune the electronic
ties and fast response to induced conductivity changes.[95,116] On properties.[145,161] Moreover, at the single-layer or few multi-layer
forms, the nanomaterial may exhibit surface defects or lattice va- chemical signal. Another electric-field manipulated sensor was
cancies which can further improve the reactivity and conductivity produced by developing ssDNA cantilevers bound to rigid ds-
of the electrode.[161,162] DNA structures.[146] In these cases, the one-step detection in-
Besides the exploitation of nanomaterials, the response of the volved no addition of reagents which promotes the autonomous
electrode can be magnified by electroactive reagents in solu- operation of the biosensor devices. Foldable biomolecular sys-
tion. One approach is to exploit mediator shuttles[163] which en- tems independent from the applied electric field also hold po-
hance the kinetics of the redox reactions on the electrode surface, tential in electrochemical sensors with one-step and reagent-less
thereby lowering the electrochemical potential. Another strategy detection features. One of the extremities of DNA/RNA aptamer
is the design of two or more redox couples acting in synergy to can be conjugated with a redox molecule (commonly methylene
continuously generate charge species and increase charge accu- blue) while the other extremity is bound to the nanomaterial elec-
mulation on the electrode.[130] Nevertheless, the use of extra electrode surface. Folding/unfolding of the aptamer by recognition
troactive agents in the electrolyte comes with the cost of increas- of the target protein can alter the distance of the redox molecule
ing the complexity and duration of the assay. from the sensor surface, and this event occurs with minimal ef-
In electrochemical analysis and sensing, the mass transfer effi- fect from the electrical potential.[165] The same phenomenon can
ciencies of reactants or coexisting ions have an impact on the elec- be exploited by the use of reversibly folded single-domain pro-
trochemical response. Enhancing mass transfer on the electrode tein receptors with chemical groups conjugated to a redox-active
surface would make the electrode more sensitive to variations of label.[166] Overall, the above-discussed one-step assays normally
redox kinetics and signal intensity at the time of introducing the retain their performance merits in full biofluid samples.[131,165]
target protein. Nanoporous structures, for instance by pattern-
ing Au in form of nanowires[119] or nano-concave structures,[133]
can reduce the distance of the interaction between the redox 3.4. Handling Full Saliva Sample
probe and active sites on the electrode, leading to a gain in re-
action efficiency. Furthermore, engineering 2D materials with Reducing the handling of the saliva-based sensor after sam-
surface atom defects can tune the surface wettability of the elec- ple collection is a vision in POC settings. Detection in the full
trode affecting the mass transfer (adsorption/desorption) of the biofluid sample accelerates the assay, facilitates sensor automa-
analytes.[162] tion, and promotes the user-friendliness of the device. Saliva con-
The nanoconfinement effect observed in nanochannels can tains a significant amount of large glycoproteins, mucus, and
also alter the mass transfer processes for electrochemical sens- debris which can be removed by centrifugation. Filters are also
ing. For protein detection in saliva, nanoporous membranes with used to remove assay-interfering particles and large molecules
two open ends[120] were introduced to increase the diffusion rate following protocols with or without centrifugation.[126] Neverthe-
of a redox probe on the supporting electrode. Subsequently, the less, the dilution of the sample is a widely practiced method for
binding of biomolecules onto the nanoporous membrane was ef- minimizing interfering substances; moreover, it introduces ben-
fective in reducing access of the electrode to the redox probe, thus efits for electrochemical sensing such as the regulation of Debye
altering the signal with high sensitivity. Moreover, the technique length.[96] Apart from the fact that dilution brings an additional
of “confined thin liquid layer”[164] involving for instance the con- step to the assay, it decreases the concentration of biomarkers
finement of the electrode/electrolyte system between two planes from their original levels. This is critical for the detection of pro-
can significantly accelerate mass transfer via incrementing the teins in saliva at very low concentrations. The modification of the
collision frequency of electroactive species with the electrode sur- nanomaterial electrode surface has therefore been investigated
face. for realizing practical and effective devices.
Coating the sensor surface with negatively charged DNA se-
quences has been demonstrated to minimize the binding of un-
3.3. Realizing Assays with One-Step Detection targeted biomolecules in saliva.[112,167] Moreover, a more univer-
sal procedure is the modification of the electrode surface with
Decreasing the number of assay steps and subsequently decreas- SAMs and especially BSA. Decreased electron transfer at the
ing incubation times is essential for rapid protein testing at the electrode/electrolyte interface can be encountered in electrodes
point of care. Numerous electrochemical sensors exploit single coated with traditional anti-fouling molecules.[168] The electrical
antibodies or single aptamers bound to target protein to cre- response and sensitivity of the sensor may largely be preserved
ate steric hindrance to the diffusion and redox activity of probe in devices in which the anti-fouling molecules form an interlaced
species on the electrode surface.[83,109,114,143] Although the analy- composite with conductive nanomaterials on the electrode sur-
sis times of these sensors are majorly controlled by the step of face. A porous 3D composite can be formed by cross-linking BSA
sample incubation, their operation typically involves adding an with glutaraldehyde and interlacing it with Au nanowires.[169] The
electrolyte reagent containing the redox probe or charge scav- amperometric response preserved nearly 90% of its original sig-
enger. One-step detection would perfectly be realized with the nal after one month of exposure to unprocessed biofluid.
conjugation of the redox probe to the biorecognition element. Reversible functionalization of electrode surfaces with im-
Ferrocene was bound to a negatively charged double-stranded munomagnetic particles (IMPs) is another strategy for handling
DNA (dsDNA) which served as an electrode surface linker to a the full saliva sample.[82,170] IMPs capture the target protein in the
regular antibody.[131] The variation of the applied potential to this sample and subsequently bring it to the vicinity of the electrode
sensor allowed the manipulation of the distance between the re- surface by the application of a magnetic field. Electrochemical
dox probe and electrode surface thereby regulating the electro- detection occurs with the aid of enzymes labeling a detection an-
tibody. This approach permits the reuse of the electrode although out systems. For “sample-in”, the microfluidic platform can be
the long-term operation may likely be hindered by the unspecific integrated into a higher fluidic structure in which a user-friendly
adhesion of biomolecules. The sensor architecture may also aid saliva aid collector can be inserted and the sample loaded.[180]
the anti-fouling behavior of the sensor regardless of the type of For “answer-out”, a portable and miniaturized potentiostat can
surface modification applied. In the case of PEC sensors, for in- be idealized with the results wirelessly sent to a personal smart-
stance, the utilization of photo-cathodes for electrochemical sig- phone or tablet (Figure 7C).
nal transduction reduces the interference of reductive agents (i.e.,
dopamine, uric acid, etc.) that are oxidized in the opposite elec-
trode (anode).[171] 3.6. Achieving Multiplexed Detection
The accurate and precise diagnosis of many infectious and

3.5. Developing Sample-in-Answer-out Systems chronic diseases may demand the analysis of multiple pro-
teins simultaneously.[26,73,76,181] Optical transduction using either
Microfluidics is desirable not only for providing controlled trans- fluorescence or absorbance is predominantly exploited by re-
port of biofluid and reagents over the electrochemical sensor, searchers to realize multiplexed detection in saliva.[33,172] A con-
ensuring accuracy and reproducibility of the signal but also cept of L-O-Disc coupled with a bead-based sandwich immunoas-
for realizing the automation of the assay. A range of microflu- say has simultaneously detected three cardiac biomarkers, CRP,
idic devices has been introduced during the last decades for cTnI, and NT-proBNP from one sample of saliva.[30] Carboxyl-
saliva handling and downstream biomarker analysis. The early functionalized microspheres encoded with different combina-
microsystems were derived from the need for miniaturization tions of fluorescent dyes have been effective in detecting ten
and automation of ELISA plate protocols. To this end, mi- salivary proteins.[33] These fluorescence-encoded microspheres
croarray well chips were realized by silicon photolithography or were run in PDMS/glass microchips and performed a parallel
poly(dimethylsiloxane) (PDMS) soft lithography and coupled to analysis of low-abundance proteins (namely interleukins) at the
flow cell apparatuses.[28,33,172] Microporous agarose beads were pg mL−1 range from saliva obtained from patients. Moreover,
immobilized on the microwells to enlarge surface areas and re- the concept of digital ELISA has recently emerged as a promis-
duce assay times.[28,172] Other concepts include the lab-on-disc ing technique for analyzing multiple salivary proteins simultane-
(L-O-Disc) and the passive-flow polymer microchip (P-F-Chip). ously and at femtomolar or below concentration levels.[182] Com-
L-O-Disc employs centrifugal pumping force to transfer sam- bined with microfluidic flow cytometry, digital ELISA with dye-
ples and reagents between multiple zones of the microfluidic encoded beads may detect hundreds of proteins from sample vol-
chip intended for different steps of the assay.[30] The L-O-Disc umes of a few μL.
was essentially created to improve reaction kinetics in sandwich Multiplexed measurements of salivary proteins by electro-
immunoassays. P-F-Chip is based on capillary flow assays with chemical sensors have been reported although their throughput
on-chip dried reagents. A representative example of a P-F-Chip capacity has generally been inferior to that exhibited by current
integrates microchannels for sample loading, capillary pumps optical sensing platforms. Practically, many multiplex electro-
for fluid waste removal, dried detection antibodies, time delay chemical assays are still confined to a series of diluted concentra-
microchannels for controlling incubation times, and circular- tions of the same protein biomarker rather than analyzing differ-
shaped reaction chambers coated with capture antibodies for the ent biomarkers in one platform. Amperometric sensors arranged
bio-detection.[173] The concept can reduce the time and improve in arrays of Au electrodes have been sought as a solution;[175,183]
the accuracy of sandwich-type amperometric assays.[174] however, these multiplexed amperometric sensors were confined
Great focus is increasingly spent on the integration of the elec- to measurements of salivary IL-8. Another amperometric sens-
trochemical sensor into paper-based microfluidic platforms. Two ing platform using dual SPEs functionalized with double-walled
approaches have been adopted in this regard: the first involves the carbon nanotubes has simultaneously detected IL-1𝛽 and TNF-
attachment of SPEs with channels and reaction areas arranged on 𝛼.[184] Other electrochemical techniques have also been exploited
the paper substrate which can be regular filter paper or nitrocel- for multiplexed electrochemical measurements. DPV was cou-
lulose strip;[175,176] the second deals with the impregnation of the pled with an eight-sensor array made of carbon SPEs; neverthe-
paper substrate with electrode materials which can be performed less, its analytical demonstration was limited to the analysis of
by screen printing for electrodes in the millimeter dimension various concentrations of the same analyte (RBD protein).[117] A
range or by inkjet printing for analytical strips in the microm- multiplexed fluidic-impedimetric sensor was formed by combin-
eter range.[177,178] Laser scribing can also be exploited to realize ing nanostructured Au electrodes and MIPs;[185] hereby, the an-
electrodes made of carbon in the paper substrate.[178] The second alytical performance was only shown for SARS-CoV-2 spike pro-
approach may offer unlimited prospects regarding the creation tein despite the promises of parallel biomarker analysis.
of rapid and disposable electrochemical sensors; it is associated There is still room for advancing electrochemical sensors with
with very low production costs and simplifies the integration of high-throughput capability for multiple salivary biomarkers. The
sensing and microfluidic parts. The capacity of the porous struc- recent progress of one-step assays, the realization of nanocom-
ture of the paper to incorporate both the conductive materials and posites with anti-fouling properties, and the viability of low-
preloaded reagents makes a perfect platform for one-step operat- cost production of nanomaterial-based electrodes[186,187] and in-
ing sensors.[179] tegrated microfluidic platforms[188,189] may in synergy contribute
Minimal user intervention in the analysis of saliva samples at to the further development of multiplex electrochemical sensing
the point of care motivates the realization of sample-in-answer- in complex biofluid samples. Apart from the engineering devel-
opments, continuous discovery of new salivary biomarkers will The support from RFF Vestfold og Telemark (project nos. 328480 and
itself stimulate the emergence of novel multiplexed sensor tech- 341831) and Oslofjordfondet (project. no. 285575) is also acknowledged.
nologies and in the meantime widen the screening and diagnos-
tic scope of saliva. Followingly, rigorous population studies will
be demanded not only to confirm the clinical usefulness of the Conflict of Interest
biomarkers but also to validate the responses of the sensors in
clinical and non-clinical situations. In the future, multiplexed The authors declare no conflict of interest.
electrochemical detection in saliva may likely be in connection
with medical big data and artificial intelligence to maximize the
usage of biomarker data in the context of POC settings. Keywords
biosensors, electrochemical sensor materials, nanomaterials, point-of-
4. Conclusion care systems, protein biomarkers
The recent SARS-CoV-2 pandemic, along with urges for stricter Received: September 19, 2022
monitoring of various chronic diseases and other infections, Revised: November 20, 2022
have elucidated the need for progressing saliva diagnostics to Published online: December 30, 2022
clinicians and engineers. The detection of protein markers and
antigens in saliva offers unlimited opportunities to conduct fast
screening or fast diagnosis that can accelerate proper medica-
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Tao Dong is a Full Professor at the Department of Microsystems, Faculty of Technology, Natural Sci-
ences and Maritime Sciences, University of South-Eastern Norway. He received a Ph.D. degree in Me-
chanical Engineering from Nanjing University of Science and Technology, China, in 2003 and obtained
the Post-Doctoral Diploma in 2005. He is the Chair Professor at Chongqing Technology and Business
University and Nanjing University of Science and Technology, China. His research interests include
lab-on-chip devices, micro-nanofluidics, biosensors, transducers, etc.
Nuno Miguel Matos Pires received an integrated M.S. degree in Biomedical Engineering at the Univer-
sity of Minho, in 2011. He obtained a Ph.D. degree in Applied Micro- and Nano-Systems Technology
at Buskerud and Vestfold University College (now University of South-Eastern Norway), in 2014. He
is currently working as an Associate Professor at Chongqing Technology and Business University. His
research interests are micro-nanosensors, lab-on-chips, and analytical devices.
Zhaochu Yang is a Full Professor at the Chongqing Key Laboratory of Micro-Nano Systems and Intel-
ligent Transduction, Chongqing Technology and Business University, Chongqing, China. He received
a Ph.D. degree in Microsystems Technology from the University of Oslo, Norway, in 2015, and a Doc-
toral degree in Science Engineering in Power Engineering and Engineering Physics from Xi’an Jiaotong
University, China, in 2009, respectively. His research interests include microfluidics, biosensors, and
heat mass transport in microsystems.
Zhuangde Jiang is a Professor at the School of Mechanical Engineering, Xi’an Jiaotong University,
China, an Academician of the Chinese Academy of Engineering, Honorary Professor of the University
of Birmingham, UK. He is also an Honorary Chair Professor at Chongqing Technology and Business
University, China. He has been engaged in the research of micro-nano-manufacturing, microelec-
tromechanical systems (MEMS), ultraprecision machining technology, sensing technology, and in-
strumentation, for decades. His research interests also cover quantum sensing, biological detection
technology, and biomedical instruments.

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