Journal of Electroanalytical Chemistry 878 (2020) 114596

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Journal of Electroanalytical Chemistry

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Review

Recent advances and challenges in electrochemical biosensors for emerging

and re-emerging infectious diseases
Shalini Menon, Manna Rachel Mathew, Sonia Sam, K. Keerthi, K. Girish Kumar
⁎
Department of Applied Chemistry, Cochin University of Science and Technology, Kochi 682022, Kerala, India

A R T I C L E I N F O A B S T R A C T

Article history: The rise of emerging infectious diseases (EIDs) as well as the increase in spread of existing infections is threatening
Received 16 June 2020 global economies and human lives, with several countries still fighting repeated onslaught of a few of these epidemics.
Received in revised form 1 August 2020 The catastrophic impact a pandemic has on humans and economy should serve as a reminder to be better prepared to
Accepted 19 August 2020
the advent of known and unknown pathogens in the future. The goal of having a set of initiatives and procedures to
Available online 25 August 2020
tackle them is the need of the hour.
Keywords:
Rapid detection and point-of-care (POC) analysis of pathogens causing these diseases is not only a problem entailing
Voltammetry the scientific community but also raises challenges in tailoring appropriate treatment strategies to the healthcare sec-
Impedance tor. Among the various methods used to detect pathogens, Electrochemical Biosensor Technology is at the forefront in
Amperometry the development of POC devices. Electrochemical Biosensors stand in good stead due to their rapid response, high sen-
Potentiometry sitivity and selectivity and ease of miniaturization to name a few advantages.
Corona Virus This review explores the innovations in electrochemical biosensing based on the various electroanalytical techniques
COVID-19 including voltammetry, impedance, amperometry and potentiometry and discusses their potential in diagnosis of
emerging and re-emerging infectious diseases (Re-EIDs), which are potential pandemic threats.
© 2020 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
2. Review of recent electrochemical biosensors for EIDs and Re-EIDs based on various electrochemical sensing techniques . . . . . . . . . . . . . . . . . 3
2.1. Voltammetric biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
2.2. Impedance biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
2.3. Amperometric biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
2.4. Potentiometric biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
3. Conclusions and future prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

1. Introduction R&D models do not account for the application of enhanced disease detec-
tion/ prevention kits to epidemics that are intermittent or unpredictable,
The inherent risk of EID's is that the introduction of a pathogen into a especially when they occur in countries with minimal investment in
community drastically decreases the percentage of healthy individuals in healthcare infrastructure [1]. When faced with the challenge of a novel
a society which would leave an ireversible social and economic stain. The pathogen, the situation becomes even graver. The international community
number of potential pathogens worldwide is on the rise, while the research has recognized the need for innovation to improve our capacity to respond
and development (R&D) resources are minimal [1]. Currently, medical to emerging threats and the need to plan for potential epidemic outbreaks

⁎ Corresponding author.
E-mail address: giri@[Link]. (K.G. Kumar).

[Link]
1572-6657/© 2020 Elsevier B.V. All rights reserved.
S. Menon et al. Journal of Electroanalytical Chemistry 878 (2020) 114596

with an emerging R&D paradigm. The need for R&D in this area to be pri- strategies dependent on cell culture. Although they are more sensitive, pre-
oritized cannot be stressed enough, through which R&D could also be cise and reliable in detection of microorganisms with reduced diagnosis
instrumental in planning the response to an outbreak. time (1–4 h), they do have certain shortcomings in comparison with cell
In its 2007 study, the World Health Organization cautioned that infec- culture [7]. Problems that restrict the application of molecular-related ap-
tious diseases are emerging at an alarming rate [2]. There have been proaches to routine diagnosis includes false positive and false negative re-
about 40 infectious diseases reported since the 1970s, including Middle sults and lack of uniformity in molecular testing [8]. Moreover there are
East respiratory syndrome (MERS), severe acute respiratory syndrome possibilities for wrongly interpreting and differentiating between a disease
(SARS), ebola, chikungunya, swine flu, avian flu, Zika and most recently and an infection as the existence of nucleic acid does not necessarily indi-
Novel Coronavirus Disease (COVID-19) [2]. The potential for these diseases cate the presence of viable species [8].
to spread rapidly and have catastrophic global impact has high probability Most of the technologies dicusssed are out of reach to majority of the
due to the increasing international travel, population explosion in develop- world's population since they are complex, centralized and need skilled
ing/ under-developed countries and ever increasing proximity with wild technicians to operate. The need for portability, cost reduction and ease
animals. of use is thus largely appreciable, particularly in the case of neglected
Re-EIDs are those caused by pathogens that are raising health concerns diseases.
for a significant proportion of the population after being dormant for a Biosensing technique is a promising diagnostic technology which has
while. Schistosomiasis is re-emerging in Egypt; Ebola hemorrhagic fever gained popularity in recent decades due to its numerous benefits [9]. Biosen-
is making a come back in West Africa and Legionellosis had been reported sors have aided in revolutionizing the treatment of various health problems
in Philadelphia [3]. Tuberculosis has made a come back too as the pathogen since its inception, five decades ago [9]. Their effectiveness in clinical man-
has developed tolerance to the antibiotics used for treatment (either by ge- agement, and characteristics like specificity, rapidity and responsiveness are
netic exchange or mutation). The prevalence of the pathogen has been the considered key in initiating early diagnosis and therapy [9]. The advance-
reason for the long-term usage of antibiotics (both within an organism and ments made in emerging techniques like nanotechnology and microfluidics,
throughout the population) [3]. Malaria has also shown to become drug- coupled with the identification of biomarkers would boost the efficiency of
resistant, while the mosquito host has also developed tolerance to pesti- healthcare sector. Transducer integration in biomaterials has allowed for
cides [3]. Whooping cough (pertussis) and Diphtheria have also shown to the development of interfaces capable of producing signals - aptly discussed
be prevalent in communities [3]. as biosensors [10]. Biosensors, which are bio-electromechanical systems,
Identification, monitoring and treatment of diseases are the primary ob- can be classified based on transducer form, label and general configuration
jectives of all public health programs [4]. Effective methods of identifica- [10]. Biosensors are designed to suit specific functionalities and affinities.
tion are paramount in preventing or mitigating the spread of a virus Recognition of analytes can often contribute to a transition in three-
before the consequences make an impact to the society. Hence, the global dimensional structure that is further transduced as a signal [10].
market for diagnoses of infectious diseases is projected to increase substan- Numerous electrochemical biosensors have been proposed for identifi-
tially in the coming years as per a report obtained from various sources cation of various diseases, citing features such as low cost, sensitivity, selec-
which includes expert interviews, secondary literature, market and market tivity and rapid response, in recent years [11–16]. Electrochemical
analysis (Fig. 1) [5]. biosensors, a subsidary of biological sensors, comprise of a biological sens-
Healthcare facilities usually utilize cell culture systems that require a ing system and an electrochemical transducer. These devices are focused
complex of cell separation processes from their normal (in vivo) setting primarily on detecting actual or future changes related to interactions hap-
and subsequent growth in an artificially (in vitro) created environment pening at the interface of the sensor sample matrix. The recognition factor
with different nutrients and antibiotics [6]. Thereby pathogen recognition (antibodies, enzymes, tissues, DNA/RNA or other biomolecules) interacts
is visually determined based on the observed distinct growth patterns [6]. selectively with the target analyte, resulting in the production of an electri-
However, cell culture is increasingly losing its role and its relative impor- cal signal, which is transmitted to the signal processor through the trans-
tance in the diagnosis of human diseases as this technique requires exper- ducer [12]. The signal is further amplified and noise is separated out
tises and trained personnel, sophisticated instruments and is also time before relevant information is retrieved.
consuming [7]. The need of the hour is the clinical diagnosis for early Electrochemical biosensing techniques will be compared to the most
and successful detection and treatment. Consequently, molecular-related widely used conventional methods (Cell culture systems, immunofluores-
approaches, including nucleic acid sequencing and polymerase chain reac- cence (IF) method and molecular approach) for the diagnosis of infectious
tion (PCR) have taken center stage as methods of diagnosis to substitute diseases. Table 1 summarizes the advantages and disadvantages of electro-
chemical biosensors over these popular conventional methods.
Electrochemical biosensors are being developed and manufactured in
large scale nowadays owing to their increasing demand in environmental,
agricultural, clinical and industrial research sectors [22]. One such success
story is of an electrochemical biosensor, a Glucometer, used to monitor glu-
cose levels in diabetic patients. Electrochemical biosensors are categorized
based on parameter measured such as: Current (Amperometric/
Voltammetric biosensors), Impedance (Impedance biosensors) or Potential
(Potentiometric biosensors) [12].
A few articles relevant to this subject have been previously released
which includes an outline of the basic concepts of sensing, case studies
and difficulties in designing point-of-care sensors for detection in a clinical
environment. A comprehensive review on electrochemical biosensor-based
pathogen detection has been done by Ellen and Blake [23]. Although elec-
trochemical biosensors are broadly reviewed in the article, latest articles on
the detection of pathogens causing EID's and Re-EID's have not been inves-
tigated in detail. A summary of recent advances in electrochemical sensors
for virus detection and usage of these sensors to track the climate, health,
and food have been presented in a recent review by Tugba et al. [24].
Fig. 1. Represents the infectious disease diagnostic market (2017–2022). This review focusses on viral infections only and the biosensors presented

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S. Menon et al. Journal of Electroanalytical Chemistry 878 (2020) 114596

Table 1
Advantages and disadvantages of electrochemical techniques over widely used conventional methods.
Method Advantages Disadvantages References

Cell culture systems Isolate wide variety of viruses (including mixed cultures & Technical expertise required to read cytopathic effect; Long [6,17]
unanticipated agents); Highly sensitive over rapid antigen tests; incubation period for most viruses; Needs an in-house
Antiviral susceptibility testing, epidemiologic studies and procurement and maintenance of a variety of cell culture forms
serotyping possible
IF assays Usually exhibits good sensitivity and excellent specificity Not as sensitive as cell cultures; Not useful for all viruses; [17,18]
Requires trained experienced hands in reading results; poor
adenovirus sensitivity
Molecular approach Excellent specificity and sensitivity; Quick turnaround using PCR FDA-cleared kits and approved protocols not commonly [19–21]
(Nucleic acid detection) in real time; Suitable for viruses which cannot be cultivated in available for most viruses; In-house technical skills needed to
conventional cell cultures establish and standardize the methods; expensive
instrumentation; Highly specific probes and primers (may skip
mutated virus); Detects only the sought viruses and may miss
mixed infections and unexpected agents in most cases; Most
assays available only at research labs
Electrochemical biosensors Rapid response, cost-effective, robust, easy to miniaturize, Sensitive to sample matrix effects; [11–16]
excellent detection limits, requires less sample volume, has the Not as sensitive as conventional methods;
ability to be used in turbid biological fluids with optically Lower shelf life
absorbing and fluorescent molecules.
Conversion to a sensor device significantly reduces cost of
analysis, saves time & enable regions with limited resources to
perform healthcare diagnostics without the need of trained
professionals

are divided based on the biorecognition element as antibody-based, nucleic 2. Review of recent electrochemical biosensors for EIDs and Re-EIDs
acid-based, aptamer-based and antigen-based electrochemical biosensors. based on various electrochemical sensing techniques
A mini-review published by Ojla and Keith in 2019 presents a detailed
overview of the latest developments in the design and production of elec- 2.1. Voltammetric biosensors
trochemical sensing techniques for pathogenic bacteria detection over the
last three years, with a special emphasis on three main clinically important Scientific community has been indebted to voltammetric biosensors
pathogens, P. aeruginosa, S. Aurores and E. coli [7]. The advancement in as it is able to provide the information of a biological system by
point-of-care bacterial pathogen detection, comprehensive characteristics converting it into an electronic signal. They belong to a class of electroan-
of bio-recognition components, immobilisation strategies and the funda- alytical sensing methodologies, where the current generated is monitored
mental concepts of optical-based biosensors used in bacterial pathogen de- between the working electrode and a counter electrode upon sweeping
tection was evaluated in a study published in 2018 by Jean and colleagues potential between working electrode and a reference electrode [30]. In
[25]. In fact, they illustrate the respective advantages and disadvantages of voltammetry, the heterogeneous electron transfer takes place at the
each methodology. In 2018, reviews on the Applications and Perspectives electrode-electrolyte interface, where electroactive species reaches by
of Biosensors for Diagnostics in Infectious Diseases [26] and Recent ad- mass transport from the bulk of the solution. The measured current is
vances in graphene-based biosensor technology with applications in life sci- the resultant of oxidation/reduction processes of the electroactive spe-
ences [27] were published. The advantages of Gene Specific DNA Sensors cies, which takes place at the surface of the working electrode. Depending
for Diagnosis of Pathogenic Infections were presented in a review by Manali upon the applied potential waveform, voltammetric techniques are classi-
et al. [10]. The mini review discusses numerous transducer dependent sen- fied as linear weep voltammetry (LSV), cyclic voltammetry (CV), square
sors and their role in acute and chronic disease diagnosis. Point-of-Care wave voltammetry (SWV) and differential pulse voltammetry (DPV)
Testing for Infectious Diseases- Past, Present, and Future was discussed in [31]. In voltammetric biosensors, the process of bio-recognition between
a mini review by Thomas and co-workers in 2017 [28]. Through this the recognition layer and the analyte brought to the current response ei-
minireview, they analyzed the POC research environment, its roots, details ther by redox processes of the analyte or via labelling. The current re-
of the variety of existing implementations, and potential technology needs. sponses are usually observed as a peak, which corresponds to the
Ting-Yen-Wei presented a review on Synthetic Biology-Based Point-of-Care concentration of the electroactive species [32] (Fig. 2). In order to over-
Diagnostics for Infectious Disease in 2016 which outlined the barriers to come certain limitations of the bare electrodes, researchers have been fo-
treatment of infectious diseases and addressed two new alternatives: bio- cusing on developing modified layers on the surface of the electrodes.
sensors and engineered virus diagnostic systems [29]. They concluded These modifications include an extensive class of materials such as
that such methods focused on synthetic biology would resolve and over- nanomaterials, polymer films, metal complexes etc. and such transducers
come diagnostic obstacles in infectious diseases. In the same year Bobby are generally named as chemically modified electrodes. Even though ad-
et al. discussed the role of biosensors in the detection of EIDs [4].This vantages like excellent sensitivity and less analysis time makes
gives a description of the various forms of biosensor systems used to iden- voltammetric sensors superior, the challenges suffered from the lack of
tify EIDs, and explains some of the techniques behind them in terms of selectivity (in comparison to conventional methods) and demand for
transduction and the concepts of bioreceptors. the targets to be redox active in the given potential range [33] make
This presented overview offers a detailed description of recent develop- the systems complicated than other electroanalytical techniques.
ments in designing and improving electrochemical sensing techniques for The detection of toxins and pathogens of infectious diseases have re-
the determination of pathogens over the last five years, with a special em- ceived much attention, as its diagnosis is essential for human health care.
phasis on EIDs and Re-EIDs. This review covers some of the important arti- Various toxins and pathogens have been detected via voltammetric biosens-
cles that have been published on the detection of EIDs and Re-EIDs and has ing approach, where simple to complex sensing platforms including specific
been structured into four main sections: voltammetric biosensors, imped- nanostructures, nanomaterials [34], electropolymers etc. have been used.
ance biosensors, amperometric biosensors, and potentiometric biosensors. In addition to the modification layer, biomolecules like aptamers
Furthermore, the respective advantages and limitations of each technique [35–39], DNAs [40,41], proteins or antibodies (depending on the mode
are demonstrated. of biorecognition) are also immobilized for the selective recognition.

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S. Menon et al. Journal of Electroanalytical Chemistry 878 (2020) 114596

the probe by depositing graphene modified hybrid nanocomposite films of

chitosan-iron oxide on fluorine tin oxide (FTO) with DNA aptamer se-
quence specific to MPT64 immobilized and found that the detection limit
is extended in the range of 0.9 fg/mL in 0.05 M PBS (pH 7.0). The stability
and the recoveries obtained by spike-in studies have shown the ability of
the aptaelectrode for the diagnosis of tuberculosis at early stage. Recently,
the electrochemical detection of chikungunya virus was first reported by
Singhal et al., where they developed a voltammetric biosensor based on
molybdenum disulphide nanosheets modified screen-printed gold elec-
trode [45]. The detection approach adopted is based on the ability of meth-
ylene blue (MB) molecules to interact with the guanine bases in single and
double stranded DNA via Vander Waals interaction. Since MB molecules
are heterocyclic aromatic compound and alkaline conditions are more
suited, 0.1 M PBS of pH 7.8 was chosen for all studies. The genosensor ex-
hibited a wide linear range from 0.1 nM to 100 μM with 3.4 nM as the limit
of detection. In addition to that, Ilkhani and Farhad demonstrated a DNA
biosensor for the detection of ebola virus, having determination possible
in the range 10 nM −75 nM with a limit of detection 4.7 nM [46]. They
used gold screen-printed electrode for the detection and prior to detection,
the probe DNA was immobilized on its surface. During the detection step,
biotinylated target DNA strand gets hybridized with the probe DNA and
gets interacted with streptavidin-alkaline phosphatase enzyme. The analyt-
ical signal is acquired by the enzymatic conversion of 4-Aminophenyl phos-
phate to 4-Aminophenol, which was then detected by DPV. The nanomolar
Fig. 2. Schematic representation of functioning of a voltammetric biosensor.
detection of influenza genes was reported by Subak et al. by using a simple
carbon transducer without any further modifications [47]. The label-free
The mechanism behind the immobilization of recognition elements DNA biosensor allowed detection limit as low as 21 nM for Influenza B se-
clearly depends on the physical and chemical properties of the transducer quences. The electrochemical oxidation of guanine before and after probe
and the environment in which the system operates. Although, physical in- and target DNA hybridization was evaluated using DPV in 0.5 M acetate
teractions such as hydrophobic or electrostatic are simple, it causes nega- buffer solution (ABS-pH 4.8). The rapid, direct and simple detection of
tive impact on the analytical performance, stability and reproducibility of the genosensor made it to be superior among other DNA biosensors and
the sensor [33]. Another effective mode of immobilization is the covalent can be easily adapted for the development of POC analysis. The detection
tethering of receptor surface to transducer exposed carboxyl or amine of Zika virus (ZIKV) sometimes becomes a major problem because of the
units. Among this, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide cross-responses between zika and dengue viruses. A DNA based biosensor
(EDC) coupling is a popular method for receptor immobilization, wherein for the detection of ZIKV was revealed by Alves et al. using poly(3-amino-
a covalent bridging occurs between amine groups and carboxyl groups 4-hydroxybenzoic acid) modified pencil carbon graphite electrode, which
via the amine nucleophilic attack on a generated active ester intermediate was superior over immunosensors in terms of low cost and its simple mod-
[33]. Affinity binding approaches like receptor biotinylation and its in- ification procedure [48]. They have validated the bioassay in human serum
duced high affinity for pre-immobilised streptavidin, is another more func- samples. The possibility for picomolar detection (25.4 pM) and its ability to
tional and stable potential mode for receptor immobilization [33]. differentiate ZIKV from dengue virus made the bioassay a promising sensor
Since all biological processes are dependent on pH, it is important to put for POC applications.
more concern on the pH of the buffer, where the studies are executed. The Yet another DNA based sensor for dengue virus was proposed by
pH of the blood must be maintained at 7.4 and hence researchers have done Tripathy et al. in 2016, where they developed a label free sensing platform
most of the studies in this physiological pH. using Manganese (III) oxide nanofiber modified Glassy carbon electrode
DNA biosensing approaches have been commonly proposed in the field which allowed a zeptomolar electrochemical detection of dengue virus
of biosensors for EIDs. For instance, a DNA based sensor was reported for (120 × 10−21 M) using DPV in PBS pH 7.4 [49]. The sensing mechanism
the selective detection of dengue virus serotype 3 (DENV-3), which causes was based on DNA hybridization and its efficiency was investigated in
the most predominant vector-borne disease in the world, dengue fever. blood serum samples. Their proposed sensor was superior to other labelled
Oliveira et al. designed a DNA based biosensor for the detection of dengue and label free sensors for the detection of dengue viruses in respect to sen-
virus with pencil graphite electrode modified with a probe specific to sitivity and detection without any complex labelling and sophisticated
DENV-3 [42]. The electrochemical analysis was carried out using DPV and instrumentation.
the sensor was able to achieve the detection limit as low as 3.09 nM in In addition to the above-mentioned biosensor, zeptomolar detection
20 mM Tris-HCl buffer (pH 7.0). Later, a genosensor for the detection of oli- was also achieved for the detection of bacterial pathogens. Mobed et al.
gonucleotide sequences of avian influenza virus (AIV) type H5N1 was re- in 2019 designed the ultrasensitive DNA biosensor for the detection of bac-
ported by Kurzątkowska et al., where they proposed a novel mechanism for terial pathogen Legionella pneumonia, which causes severe form of pneu-
the generation of electrochemical signal based on an ion-barrier “switch monia in humans (Fig. 3) [50]. The fabrication was done by assembling a
on” system and achieved picomolar detection [43]. Two types of genosensors gold nano architecture (AuNPs) on the surface of poly (dopamine-β-Cyclo-
were reported incorporating (dipyrromethene)2Cu(II) and (dipyrromethene) dextrin) modified gold electrode. Further immobilization and hybridization
2Co(II) complexes on gold electrode which is covalently attached to a 20mer of probe and target DNA leads to the generation of voltammetric signal
probe via redox-active monolayers. The (dipyrromethene)2Cu(II) incorpo- using toluidine blue redox indicator. The ultrasensitive bioassay permits
rated genosensor was able to perform in the dynamic linear range of 1 pM linear range and low limit of quantification as 1 ZM to 1 μM and 1 ZM, re-
−10 pM with a detection limit of 1.3 pM, whereas using (dipyrromethene) spectively, in Tris-HCl buffer (pH 7.4).
2Co(II) complex the detection limit was extended to 1.2 pM at buffer condi- Researches have also been done for developing voltammetric biosensors
tions of 0.9 M NaCl and 0.09 M sodium citrate (pH 7.0). An aptamer based capable for simultaneous determination, as it needs less analysis time, cost
biosensor was reported for the detection of Mycobacterium tuberculosis spe- effective detection and comparatively lower sample size. Yan et al. reported
cific antigen MPT64 by Thakur et al. in the year 2017 [31]. They fabricated a novel biosensor for the simultaneous electrochemical detection of two

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S. Menon et al. Journal of Electroanalytical Chemistry 878 (2020) 114596

Fig. 3. Schematic representation of DNA biosensor for the detection of legionella pneumonia using AuNPs/poly (dopamine-β-Cyclodextrin) modified gold electrode.
Reprinted from Int. J. Biol. Macromol., 128, Mobed et al., DNA-based bioassay of legionella pneumonia pathogen using gold nanostructure: A new platform for diagnosis
of legionellosis, 692–699, Copyright (2019), with permission from Elsevier, License number 4850890192113.

DNA targets related to human immune deficiency virus (HIV) and tubercu- CoV-2 or COVID-19 outbreak has emerged as a global pandemic and
losis (TB) in 2015 [51]. The bioassay is based on quantum dots-polymer resulting as a serious public health issue all over the world. Mahari et al.
nanotracers on the surface of glassy carbon electrode and the detection fabricated a biosensor device for COVID-19 detection in spiked saliva sam-
was made by measuring square wave voltammetric signals from metal ples using nCOVID-19 antibody (nCOVID-19Ab) immobilized screen
ions (Pb or Cd). The excellent signal amplification capacity of the polymer printed electrode (eCovSens) and compared it with a potentiostat based
nanotracers caused the assay to detect target DNAs as low as 0.2 f. and ex- sensor fabricated using FTO electrode modified with gold nanoparticles
hibited dynamic concentration range from 0.5 f. to 500 pM. and immobilized with nCovid-19Ab [55]. Both the sensing strategies
An important immunosensing based bioassay for the detection of achieved a femtomolar detection and the eCovSens device offers as a stable
MERS-CoV, a highly pathogenic virus was first reported by Layqah et al. and rapid diagnostic tool for POC applications.
[52]. The novel competitive immunosensor was fabricated by electrodepos- The rapid and sensitive POC detection has become the need of the hour.
iting AuNPs on carbon disposable array electrodes and immobilized with From the conventional methods of detection of infectious diseases through
MERS-CoV antigen – 725 Spike protein S1. The single step detection was RT-PCR (Reverse transcription polymerase chain reaction), which involves
achieved by measuring the redox peak current of 5 mM ferro/ferri cyanide time consuming and cumbersome procedures, the voltammetric biosensors,
redox system in 0.1 M PBS (pH 7.4). The sensitive and selective detection of be it DNA or immuno based sensors, stands out as it possess some unique
MERS-CoV enable linear response in the concentration range 0.001 ng/mL advantages. Nevertheless, a study of recent literatures clearly reveals that
to 100 ng/mL and detection limit as low as 1.0 pg/mL, which is lower than DNA sensors are more exploited than immuno based sensors and has
the reported ELISA (enzyme linked immunosorbent assay) approach. They emerged as an effective alternate method for the conventional techniques.
have also mentioned that the proposed sensing strategy can be extended in Some limitations like limited shelf life, temperature sensitivity and thereby
future for the multiplex detection of different CoVs. Similarly, for ZIKV denaturation of antibodies makes immunosensors a less preferred tech-
detection, Faria et al. reported an electrochemical immunosensor, where nique for on-site clinical diagnosis [44]. Labeling in DNA sensors also
bioassay is based on arranging ZnO nanostructures immobilized with often lead to change in the properties of the macromolecules, which results
ZIKV-NS1 antibody on printed circuit board [53]. The analytical responses in loss of bioactivity and affinity to the target [42,56]. Currently, the re-
were evaluated using CV in 10 mmolL−1 K4 [Fe(CN)6] and 0.5 molL−1 searchers are focusing on developing label free DNA sensing, where the in-
NaNO3 solution as mediator and the bioassay permits a rapid detection in herent electrochemical properties of DNA like electro-oxidation of purines,
the concentration range 0.1 ng/mL −100 ng/mL and limit of detection as are relied for signal generation. These label free DNA sensors have many ad-
low as1.00 pg/mL. vantages such as rapidness in detection, simplicity in fabrication, and least
Though a good number of researches are going on in the field of pre sample requirements. Though voltammetric immunosensors have been
voltammetric biosensors, the platform for miniaturization to a porta- designed to portable device for onsite monitoring, many of the label-free
ble device is necessary for POC applications. In an approach to develop DNA sensors reported are also promising approaches for the development
an immuno based voltammetric sensor device for the detection of of POC diagnostic applications.
cholera toxin, Archibald et al. in 2015 designed a vertically oriented,
nanocoaxial electrodes in array format [54]. The linear range was ob- 2.2. Impedance biosensors
tained as 10 ng/mL - 1 μg/mL and the limit of detection was found to
be 2 ng/mL, which is comparable to the optical ELISA approach. The -Electrochemists have been familiar with the Electrochemical Imped-
fabricated sensor array proved to be an excellent platform for diagno- ance Spectroscopic (EIS) technique for over a century [57]. The fact that
sis of infectious cholera toxin in the POC scenario. Recently, SARS- EIS can be used effectively for the label free detection makes it a powerful

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S. Menon et al. Journal of Electroanalytical Chemistry 878 (2020) 114596

tool in biosensing applications [4]. The occurrence of a bio-recognition pro- In an approach by Tamayo and co-workers, self-assembled monolayer
cess is always followed by some changes in various physical and chemical of biotin modified 19 base-oligonucleotide probe on a gold electrode was
properties at the electrode electrolyte interface, and EIS technique makes used to detect complementary proviral sequences of HIV-1 [68]. Surface
use of the changes in charge transfer resistance (Rct) or interfacial capaci- Enhanced Raman Spectroscopy (SERS) confirmed the direct linking of
tance to mark the biochemical changes occurring at the sensor surface biotin- DNA to the gold electrode. EIS was used to monitor the hybridiza-
[4,57]. tion of proviral sequences with the oligonucleotide probe where Rct value
Unlike other electrochemical methods such as cyclic voltammetry showed an increase upon hybridisation and is explained to be due to an in-
which involves large amplitude perturbations, small amplitude perturba- crease in blocking effect at the electrode surface. Using Rct value as a mea-
tion in EIS makes it a non-destructive technique [58]. In spite of being an sure of the blocking capacity of the monolayer to the electron transfer
ideal method for understanding dynamics of biochemical reactions, EIS reactions of (Fe(CN)4− 3−
6 / Fe(CN)6 ) probe, the surface coverage of the
technique suffers from several challenges. One among them is the sensitiv- biotin-DNA monolayer on the gold electrode was calculated to be 89%.
ity of the so developed label free biosensor [4,58]. Studies reveal that sen- Yet another impedance DNA biosensor for HIV-1 gene was proposed by
sitivity of these sensors is lower compared to the biosensors that utilize Gong et al. using graphene-nafion composite film modified glassy carbon
labels. Though labelling can enhance selectivity and sensitivity, it con- electrode as the sensing platform [69]. The sensor responded to the HIV-1
sumes extra time, involves difficult sample handling and is also expensive gene over a concentration range of 1.0 × 10 −13 M - 1.0 × 10 −10 M
[59]. Another major challenge to be addressed is whether the technique and achieved a detection limit of 2.3 × 10−14 M. The single stranded cap-
works good in real samples such as blood serum, where there is a significant ture probe DNA was adsorbed onto the graphene-nafion modified electrode
amount of non-target molecules. [60]. Now a great deal of effort has been surface via п-п* stacking interactions and this led to an increased electron
done by researchers in an attempt to improve the sensitivity by modifying transfer resistance value of the (Fe(CN)4− 3−
6 / Fe(CN)6 ) redox couple. The
electrodes with nanocomposites, conducting polymers, metal nanoparticles authors reported that in the presence of HIV-1 target DNA, the probe
etc. [61,62]. DNA gets hybridized, forming the double stranded helix DNA (dsDNA)
In simpler terms impedance can be regarded as a resistance to the flow and this strong and effective binding forces the dsDNA to leave the elec-
of current in an electrical circuit. Basically, information from impedance trode surface, which resulted in a decreased electron transfer resistance of
measurement consists of resistive and capacitive parts based on which the redox couple. In a recent work done by Ravina et al., the authors dem-
there are faradaic and non-faradaic sensors [59]. In faradaic biosensors onstrate a genosensor for early detection of swine flu (H1N1) infection in
the electrode containing the bioreceptor is immersed in a solution of an human [70]. The sensor was fabricated by immobilizing amino labelled sin-
electrochemical redox probe and electron transfer resistance of the elec- gle stranded DNA specific to the glycoprotein hemagglutinin (HA) found on
trode is measured whereas in the case of non-faradaic sensors it does not re- the surface of H1N1 virus onto the cysteine modified screen printed gold
quire a redox probe because it measures the interfacial capacitance (ie, the electrode (Fig. 5). The hybridization of the single stranded complementary
charge storage of the system when voltage is applied). Impedance can be DNA (ss-cDNA) of H1N1 to the probe DNA was carried out in Tris-EDTA
represented as Nyquist (for faradaic process) and Bode plots (for capacitive buffer (10 mM Tris, 1 mM EDTA, pH 8.0) and was confirmed by EIS.
or non-faradaic process) [63]. This section focuses on impedance biosen- They also studied the specificity of the biosensor towards H1N1 in presence
sors based on faradaic process. Literatures reveal that most of the studies of human DNA and ssDNA of other infectious pathogens. More importantly
use (Fe(CN)4− 3−
6 / Fe(CN)6 ) as the redox probe for monitoring the charge they validated the biosensor with samples from H1N1 infected patients.
transfer resistance (Rct) before and after the bio-recognition event. Other Most recently, a label free, impedance DNA biosensor was developed by
redox probes used include [Ru(NH3)6]3+/2+ and ferrocene (Fc+/Fc) [64]. Antonio et al. for the detection of synthetic antibody sequences of Zika virus
Currently active research is progressing on impedance biosensors for in- [71]. The thiol-probe DNA was immobilized on the gold working electrode
fectious diseases. When the bioreceptor immobilized on the electrode sur- (exploiting the strong affinity of sulphur towards gold) of the planar three
face captures the target analyte, there occurs a change in the Rct value of contact disposable electrode fabricated on a polyethylene terephthalate
the redox probe, which can be linked to the concentration of the target an- (PET) substrate. The sensor was able to directly detect the Zika virus se-
alyte. EIS technique has been found very much successful in monitoring hy- quences selectively over a concentration range of 25 nM −340 nM with a
bridization of probe DNA with the target DNA in DNA based biosensors detection limit of 25 nM.
[65–67]. A general schematic illustration of the fabrication of an imped- In addition to DNA biosensors, significant research has been dedicated
ance DNA biosensor is given in Fig. 4. to the field of impedance immunosensors for infectious diseases [72].

Fig. 4. General schematic illustration of the fabrication of an impedance DNA biosensor.

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S. Menon et al. Journal of Electroanalytical Chemistry 878 (2020) 114596

Fig. 5. Schematic representation of the fabrication of gene specific impedance biosensor for determination of swine flu (H1N1) in human. Reprinted from Int. J. Biol.
Macromol., 130, Ravina et al., Hemagglutinin gene based biosensor for early detection of swine flu (H1N1) infection in human, 720–726, Copyright (2019), with
permission from Elsevier, License number 4850890410830.

Kaushik et al. reports a more rapid, selective and sensitive micro enough potential to be developed into a POC diagnostic device for elim-
immunosensor for Zika virus protein [73]. The immunosensor was devel- inating malaria. The impedance sensor demonstrated the use of an in-
oped using interdigitated gold array micro electrode (IDE-Au). The elec- terdigitated electrode sensor surface for immobilizing the capture
trode was functionalized with self-assembled monolayer of dithiobis probe anti- fpHRP2 monoclonal antibodies. They validated the sensor
(succinimidyl propionate) (DTSP). The Zika virus specific envelop protein in human saliva samples for ensuring applicability in real world and it
antibody were then immobilized onto this modified electrode via electro- showed sensitivity as low as 25 pg/mL for the detection of fpHRP2.
static interactions. The response of the immunosensor towards different Nidzworski et al. [77] developed a novel biosensor for the specific de-
concentrations of Zika virus protein was analyzed by EIS technique in tection of M1 proteins, the universal biomarker of influenza disease, at ul-
5 mM PBS (pH = 7.4) containing 5 mM Fe(II)/Fe(III) and it allowed the de- tralow concentrations. Boron doped diamond (BDD) electrode, due to its
tection in the range of 10 pM to 1 nM with a detection limit of 10 pM. The rapid electrochemical response, low background current and broad poten-
authors compared the developed sensor and it showed better performance tial window, was used as the sensing surface. The anti-M1antibodies were
with those reported in literature. However the presented sensor was not immobilized onto the surface of the BDD electrode functionalized with 4-
tested in real samples and it questions its real life applicability. Similarly, aminobenzoic acid via self assembly. In order to avoid nonspecific binding
a label free impedance immunosensor was fabricated by Darwish and co- during analysis, bovine serum albumin (BSA) molecules were capped onto
workers for the direct detection of Non-structural protein (NS1) biomarker the open sites of the modified BDD electrode. The response of the electrode
for dengue virus [72]. They modified the indium tin oxide (ITO) electrode after incubating in M1 proteins was followed by EIS analysis and the sensor
surface with antifouling agents derived from aryl-diazonium cations, which presented lower detection time of less than 5 min. Furthermore, this rapid,
was followed by the electrodeposition of gold nanoparticles and its label free biosensor showed a limit of detection of 1 fg/mL and exhibited
functionalization with 1,4-phenylenediamine. The anti-NS1 IgG antibodies high specificity towards strains of influenza virus in presence of bacteria
where then immobilized onto the modified electrode surface. The and yeast.
immunosensor responded to the NS1 antigen over a wide range from Recently, Chowdhury and Park reported another promising work [78].
5 ng/mL-4000 ng/mL. They evaluated reproducibility, selectivity and sta- They developed an ultrasensitive impedance immunosensor for hepatitis E
bility of the sensor and also assessed it's cross-reaction with malaria infected virus (HEV) detection. Notable significance is that the sensor was able to at-
human sera. tain sensitivity comparable to that achieved by real time quantitative re-
Although there have been considerable research works reported in the verse transcription polymerase chain reaction. The fabrication of the
area of biosensors for infectious diseases, the ultimate aim is miniaturiza- immunosensor consisted of modifying the glassy carbon electrode with
tion to provide POC diagnosis [74]. Sepulveda et al. designed, constructed graphene quantum dots and gold nanoparticles enclosed polyaniline nano-
and tried bio-microsystems based on printed circuit board platforms con- wires (GQDs@AuNP-PAni) onto which the anti-hepatitis E antibody was
taining independent electro-immunosensors for early secretary antigen easily loaded. The AuNP-PAni increases electron transfer process and also
target-6 (ESAT-6) produced by Mycobacterium tuberculosis [75]. They fabri- provides high surface area. They ensured the sensitive detection of HEV
cated three bio-microsystems with each microsystem containing 40 electro- by applying an external pulse at the time of loading virus. The sensor exhib-
immunosensors. The so developed electro-immunosensor consisted of ited good linear range of concentration from 1 fg/mL to 10 pg/mL with a
polyclonal antibodies immobilized onto the gold nano layer surface by detection limit of 0.8 fg/mL.
self-assembly. As a non-destructive technique, EIS was used to monitor all Infectious diseases have emerged as a major health problem worldwide
the stages involved and it enabled rapid detection of the disease. Indeed and electrochemical biosensors have evolved as an attractive tool as it en-
the work done by Soraya et al. [76] reported the development of an ultra- ables rapid, sensitive and selective detection. For the past few years, there
sensitive and label free sensor for histidine-rich protein 2 produced by the has been considerable progress in research works in the area of impedance
malaria parasite Plasmodium falciparum (fpHRP2) and the sensor showed DNA biosensors and immunosensors for several EIDs and Re-EIDs.

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S. Menon et al. Journal of Electroanalytical Chemistry 878 (2020) 114596

Possibility of label-free detection and thus reduction of assay complexity

thereby favours miniaturization and this differentiates impedance tech-
nique from other existing methods. Compared to the fabrication of
immunosensors, which use antibodies, DNA biosensors show several ad-
vantages. High purity DNAs can be synthesized and can be easily modified
with functional groups such as thiols or biotin so that they can be easily cap-
tured onto the electrode surface. Moreover, they are highly stable and can
be regenerated from the double stranded DNA, formed after hybridization
with target DNA of the sample, by thermally melting it [79]. On the con-
trary, the sensitivity of DNA biosensors was found to be lesser compared
to impedance immunosensors. This may be attributed to the repulsion
between the redox probe Fe(CN)4− 3−
6 / Fe(CN)6 and the negatively charged
sugar phosphate backbone of DNA, which may significantly affect the
resulting EIS signals. Moreover, the extent of interaction of the target mol-
ecule with the immobilized probe, which in turn determines the perfor-
mance of the biosensor, depends on various factors such as pH and ionic
strength of the reaction medium, temperature, duration of probe immobili-
zation etc. Optimization of these parameters is of utmost importance while
developing a biosensor. Instead of merely acting as a platform for sensing,
the structure and configuration of the electrode plays a key role in the per-
formance of a sensor and interdigitated microelectrodes is found to be more
advantageous over conventional electrodes in miniaturization. Although
several advances have been made in impedance biosensors for infectious
diseases, a number of hurdles still exist and further studies need to be
done before these sensors can find real-time applicability in future. Fig. 6. Schematic representation of an amperometric biosensor.

2.3. Amperometric biosensors remarkable stability inherent to nucleic acids [91]. Recently, Chen et al. de-
veloped a dual probe amperometric biosensor which can distinguish hepa-
Literature clearly reveals that the era of electrochemical biosensors was titis B virus genotypes, B and C through temperature control [92]. B and C
established on amperometric and potentiometric transducer platforms type capture DNA probes were simultaneously immobilized on BSA based
[80,81]. An amperometric glucose biosensor itself was the stepping stone probe carrier and the current produced by an enzyme attached to the target
towards the development of cost effective, reliable, rapid and handy POC DNA (horseradish peroxidase (HRP)) mediated reduction of H2O2 was used
diagnostic devices, which then created a revolution in medical diagnosis for detection. The BSA monolayer is capable of enhancing spatial position-
[82]. The current-time response of electro-oxidation/reduction of an ing and controlling the inter probe and probe-target interactions in a better
electroactive species, at an optimal potential is monitored in amperometry way [92]. Detection of B and C genotypes of hepatitis using this single sens-
[83]. The current generated will be proportional to the concentration of the ing platform is possible due to the difference in the temperature of hybrid-
electroactive species and the excellent selectivity in detection offered by ization exhibited by these genotypes with their respective capture single
this potentiostatic technique made it the widely used one in chemical sen- stranded DNAs in phosphate buffer of pH 7.4. The sensor is applicable in
sor development [84]. In addition, wide concentration range of detection real biological samples with a limit of detection 1.12 pM (B type) and
and low-cost instrumentation of amperometric technique has made it a pre- 1.64 pM (C type). A different strategy involving an enzyme was used by
ferred one for sensor developers [85]. Minimization of charging current Li and co-workers for the development of a DNA based biosensor for HIV
(current needed to apply potential) which affects the detection limit is gene [93]. This DNA hybridisation based label free electrochemical sensor,
also an advantage of amperometry [32]. Indeed, the blend of selectivity involves a series of processes and two hair pin probes HP1 and HP2 and only
provided by amperometry and the specificity in binding offered by the later one is immobilized on the gold working electrode through self as-
biorecognition elements have led to the successful development of numer- sembled monolayers. The enzyme exonuclease (III) induces selective diges-
ous highly sensitive and reliable biosensors so far [86]. In amperometric tion of duplex DNA formed by the hybridization of target ssDNA with HP1.
biosensors, specific bioreceptor-target binding produces amperometric sig- A help DNA formed as the digestion product release a c-myc template DNA
nal either directly or indirectly. Charge transfer from electron rich label at- from HP2 and the guanine nanowire formed from the c-myc provides am-
tached to the target molecule can produce direct signals. On the other hand, perometric detection of HIV gene upto concentration of 3.60 pM (Fig. 7).
indirect signalling is possible through redox processes catalysed by enzyme All the solutions were prepared and studies were done in phosphate buffer
labels on the target molecule. Natural polymers like glycans also act as a (pH 7.4) and TECEP buffer (pH 6.5) to maintain physiological pH. Possibil-
bioreceptors that selectively react with certain proteins to give indirect am- ity of target DNA recycling and the good accuracy exhibited in complex bi-
perometric signals. Conducting polymers and nanomaterials have been also ological samples by this biosensor clearly indicates its potential in real
incorporated in these biosensors to enhance the immobilization of the rec- sample analysis [93].
ognition element and thereby stability and sensitivity [87,88]. Simplicity in Recently, another DNA hybridization based amperometric sensor was
design of an amperometric detector paves possibilities for miniaturisation reported by Bouhemadou and co-workers, for dengue virus serotype-2
of these biosensors as mentioned earlier [89]. However, signal reduction [94]. In this sensor, Cu2CdSnS4 alloy nanostructure based silver coated in-
due to interference from sample matrix exists as a challenge of amperomet- terdigitated electrode allows more stable immobilization of the probe DNA.
ric enzyme based sensors [90]. Fig. 6 is a general schematic representation The carboxyl terminated probe DNA was covalently linked to the electrode
of an amperometric biosensor. using (3-Aminopropyl)triethoxysilane and its hybridization with dengue
For the last few years, very staunch research has been progressing in viral DNA alter the electrical property of the electrode. It is shown that
amperometric DNA based, enzyme based and immunosensors for clinical there exists an inverse relationship between the amperometric signal and
analysis. A number of biosensors for the selective detection of biomarkers concentration of target DNA in the range 100 f. to 10 nM at a working volt-
of some EIDs and Re-EIDs have become a part of the above. It is quite inter- age 1.5 V. Inspite of the rapidness and low power consumption claimed for
esting that DNA based sensors are now in the forefront than conventional this biosensor, lack of any mention about the medium of studies or applica-
enzyme and immunosensors for EID detection, which might be due to the tion in real samples may affect the reliability of the sensor. In addition,

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S. Menon et al. Journal of Electroanalytical Chemistry 878 (2020) 114596

Fig. 7. Schematic representation of DNA based biosensor for HIV. Reprinted from Sensors Actuators, B Chem., 238, Huang et al., Sensitive detection of HIV gene by coupling
exonuclease III-assisted target recycling and guanine nanowire amplification, 1017–1023, Copyright (2017), with permission from Elsevier, License number
4850890539754.

Dong et al. developed a very sensitive amperometic sensor which specifi- It is shown that detection of Mycobacterium tuberculosis cells upto a concen-
cally detect AIV type H7N9 using a very similar strategy [95]. Here they tration of 100 CFU/mL is possible with the immunoassay.
have utilized a tetrahedral DNA nanostructure containing the capture Long assay time, low target DNA levels in biological fluids (need for pre-
DNA, immobilized on a gold electrode via Au-S bond to enhance the molec- vious amplification of sequences) still remains as the main obstacle for
ular recognition (DNA hybridisation), and thereby sensitivity to H7N9 virus miniaturisation of DNA biosensors, in spite of the ease of synthesis, high
gene could reach 100 fM. Moreover, signal amplification was done through chemical stability and reusability of DNA sequences [99]. The coupling of
oxidation of 3,3′ 5,5′–tetramethylbenzidine dihydrochloride (TMB) simple glycan bioreceptor with amperometry rather than the usual imped-
catalysed by HRP linked to the target DNA. The practical utility of this ance technique has opened facile possibilities for POC settings. It can be ex-
assay is clear from its successful application in throat- swab samples con- pected to make it more specific through the incorporation of different
taining the virus. It is notable that a good reduction in assay times glycan chains. Likewise, the integration of microtip and amperometry in
(~ 1 h) can be seen in all these biosensors, when long time consumption the immunoassay for tuberculosis widens the possibilities for on-site analy-
of DNA based sensors exist as a drawback in POC analysis. sis of infectious diseases. However, in the current situation of the emer-
The possibilities of glycans (carbohydrate based polymers) were also gence of deadly mutated pathogens, it is expected that the research in the
employed for the detection of ID biomarkers recently. A glycan based am- near future will be focussing on the excellent specificity offered by
perometric biosensor was reported for the specific and sensitive detection genosensing for rapid bedside testing.
of influenza viruses, by Cui et al. [96]. The glycoprotein present on the in-
fluenza viral surface can selectively release galactose from glycans. The 2.4. Potentiometric biosensors
strategy of indirect detection of virus through the detection of released
galactose amperometrically via glucose strips bearing dehydrogenase was The working principle behind potentiometric sensors is that, the po-
used in this assay. Successful application in human nasal swab samples tential difference between working and reference electrodes varies di-
and very less assay time (15 min) are the salient features of this sensor. It rectly with the concentration of the analyte under study, at zero current
is clear that the rapidity and simplicity in design of this sensor enables to flow [100]. Ion selective, gas sensitive electrodes and Field Effect Transis-
present it in a POC device form. In fact, the detection via glycan based sen- tors (FET) are the usually employed transducers in potentiometric sen-
sors are limited to glycan-binding proteins and viruses [97]. sors, which are selected, based on the nature of the species under study.
Furthermore, Hiraiwa et al. developed an immunosensor for Mycobacte- This tuning of the working electrode can enhance the selectivity and sen-
rium tuberculosis in human sputum using a microtip microtip with perfor- sitivity [101] and which in combination with other key features of poten-
mance comparable to PCR [98]. The amperometric signals were obtained tiometric technique such as non-invasion, cost effectiveness and
by the capture of Mycobacterium tuberculosis cells spiked in sputum on sensitivity have led to the development of many biosensors important
anti-Mycobacterium tuberculosis functionalized microtip followed by binding in biomedical analysis [102,103]. Since fabrication of biosensors to
of fluorescein labelled antibodies over the former. Charge transfer from POC devices have been the prime goal among researchers, FET based po-
electron rich fluorescein label to the electrode results in the amperometric tentiometric sensors have gained very much attention recently, which
signal, which is further, amplified through a duplicated coffee ring effect. might be due to FET's built-in potential for miniaturisation [104]. Ease

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S. Menon et al. Journal of Electroanalytical Chemistry 878 (2020) 114596

detect both human H1N1 and avian H5N1 influenza viruses in nasal
mucus [109]. Rapid detection in the wide concentration range 100.5 to
108.5 TCID 50/mL was made possible by functionalizing the gate termi-
nals with two different sialic acid containing glycans that recognize
human and avian viruses respectively. Feasibility of the assay was also
successfully demonstrated by connecting it to a smartphone. All the mea-
surements were carried out at physiological pH (pH 7.4). The remarkable
stability and sensitivity exhibited by glycan-immobilized FET biosensor
over an antibody-immobilized (antibodies are the widely used recogni-
tion elements with FET) [110] one is also discussed. In addition,
Goswami and co-workers developed an aptamer based extended gate
Fig. 8. Schematic representation of Field effect transistor based biosensor. FET biosensor for malaria biomarker, Plasmodium falciparum glutamate
dehydrogenase (Pfgd) [105]. The extended gate was used for increasing
the sensitivity and biocompatibility if the FET. Anti-Pfgd aptamer was
of fabrication, rapid response and highly sensitive label-free detection by immobilized on an interdigitated gold microelectrode, which was at-
FET has also amplified the interest of researchers for this transducer plat- tached to gate terminal of the FET. The net charge produced on the elec-
form [105]. FET is a semiconductor (transducer) based potentiometric trode surface due to aptamer-Pfgd binding led to detection of Pfgd in
device containing three terminals- source, drain and gate. In biologically human serum samples in the linear range 100 f. to 10 nM with a limit
sensitive field effect transistors, gate terminal (dielectric material) is of 48.6 pM within a minimal response time (~ 5 s). The developed device
modified with target specific bioreceptors. The surface-charge changes has a very good potential for POC settings as well (Fig. 9).
on the gate terminal through the binding of charged biomolecules like Even though the excellent sensitivity and specificity offered by
proteins, DNA, RNA etc. can alter the gate voltage and thereby the charge immunosensors have been utilized in bioassays, last two assays discussed
transport properties of FET channel [106]. In fact, defects like impurities above have presented viable alternatives (glycans, aptamers) for antibodies
of the semiconductor exist as a limitation of FET based sensors [107]. A in FET based biosensing. Glycan based sensors developed so far are really
typical schematic representation of Field effect transistor based biosensor rays of hope for the effective control of the pandemic influenza viruses at
is given in Fig. 8. its source itself. But the sensing activity of glycan based assays are limited
Three efficient FET based biosensors were developed recently for in- to glycan binding viruses, as discussed earlier. Since aptamers have already
fectious diseases. The most interesting and important one in the present been proved as effective bio-recognition elements for the detection of many
scenario is the FET based immunosensor for the very pandemic COVID- infectious diseases, its combination with the potential FET transducer can
19 causative virus developed by Seo et al. [108] Graphene based FET lead to the development of accurate, rapid and portable analysis devices
functionalized with highly immunogenic SARS- COV-2 spike protein spe- for infectious diseases in future.
cific antibodies act as the early detection platform for the Corona virus Table 2 provides a detailed summary of the literature discussed in the
upto 1 fg/mL. The antibodies were immobilized on the graphene surface four different sub-sections.
and the protein binding induced response of the FET is monitored for the
SARS-COV-2 detection using PBS of pH 7.4 as the electrolyte. The suc-
cessful and rapid real time application of the immunosensor in human na- 3. Conclusions and future prospects
sopharyngeal swab specimens, SARS-COV-2 cultured cells and clinical
samples make it very significant in this crucial situation. In addition, Shortfalls in epidemic management, disease diagnosis & healthcare fa-
the assay responds specifically to SARS CoV-2 in presence of MERS cilities have had devastating impact on humans in recent memory, be it
CoV. Hideshima et al. reported a glycan based FET biosensor which can in terms of life or the economic burden on nations. Throughout this

Fig. 9. Schematic representation of aptamer based FET biosensor for malaria biomarker. Reprinted from Biosens. Bioelectron. 123, Singh et al., Development of an aptamer-
based field effect transistor biosensor for quantitative detection of Plasmodium falciparum glutamate dehydrogenase in serum samples, 30–35, Copyright (2019), with
permission from Elsevier, License number 4850890651665.

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S. Menon et al. Journal of Electroanalytical Chemistry 878 (2020) 114596

Table 2
A summary of selected biosensors referenced in this review for the determination of EIDs and Re-EIDs.
Sl. Transducer Target Biosensor format Linear range Detection Limit Reference
No.

1. Differential Pulse Dengue Virus Serotype 3 DNA sensor – 3.09 nM [42]

Voltammetry
2. Square Wave Avian Influenza DNA sensor 1 - 10 pM 1.39 pM [43]
Voltammetry
5
3. Differential Pulse Mycobacterium Tuberculosis DNA aptasensor 1.00 - 1 × 10 fg/mL 0.9 fg/mL [44]
Voltammetry
4. Cyclic Voltammetry Chikungunya Virus DNA sensor 0.1 nM - 100 μM 3.4 nM [45]
5. Differential Pulse Ebola Virus DNA sensor 10-75 nM 4.7 nM [46]
Voltammetry
6. Differential Pulse Influenza genes DNA sensor – 21 nM [34]
Voltammetry
7. Square Wave Zika Virus DNA sensor 84 pM -1.41 nM 25.4 pM [48]
Voltammetry
8. Differential Pulse Dengue Virus DNA sensor 1 ZM-1μM 120 ZM [49]
Voltammetry
9. Square Wave Legionella pneumophilia DNA sensor 1 ZM - 1 μM 1 ZM [50]
Voltammetry
10. Square Wave DNA related to Human Immuno Deficiency Virus and DNA sensor 0.5 f. - 500 pM 0.2 fM [51]
Voltammetry Tuberculosis
11. Square Wave Middle East Respiratory Syndrome Corona Virus Immunosensor 0.001 - 100 ng/mL 1.0 pg/mL [52]
Voltammetry
12. Cyclic Voltammetry Zika Virus Immunosensor 0.1 - 100 ng/mL 1.00 pg/ mL [53]
13. Square Wave Cholera toxin Immunosensor 10mng/mL - 1 μg/mL 1 ng/mL [54]
Voltammetry
14 Differential Pulse Severe Acute Respiratory Syndrome Corona virus Immunosensor 1 f. -1 μM 90 fM [55]
voltammetry
15. Impedance Human immune deficiency virus -1 DNA biosensor – – [68]
16. Impedance Human immune deficiency virus -1 DNA biosensor 1.0 × 10 -13 M - 1.0 × 2.3 × 10-14 M [69]
10 -10 M
17. Impedance H1N1 DNA biosensor – 6.6 ×10-4 [70]
ng/ul
18. Impedance Zika virus DNA biosensor 25-340 nM 25 nM [71]
19. Impedance Zika virus Immunosensor 10 pM-1 nM 10 pM [73]
20. Impedance Dengue virus Immunosensor 5- 4000 ng/mL [72]
21. Impedance Mycobacterium Tuberculosis Immunosensor [75]
22. Impedance Plasmodium Falciparum Histidine rich peotein 2 Immunosensor 25 pg/mL [76]
23. Impedance Influenza virus Immunosensor 1fg/mL [77]
24. Impedance Hepatitis E Immunosensor 1 fg/mL - 10 pg/mL 0.8 fg/mL [78]
virus
25. Amperometry B/C genotyping of hepatitis B virus DNA based sensor 100 - 800 f. (B type) 1.12 f. (B type) [92]
100 – 800 f. (C type)

1.64 fM
(C type)
26. Amperometry Human immune deficiency virus DNA based sensor 10 fM- 100 nM 3.60 fM [93]
27. Amperometry Dengue virus DNA based sensor _ 17 nM [94]
28. Amperometry Avian influenza virus type H7N9 DNA based sensor 1.0 f. – 2.5 nM 0.75 fM [95]
29. Amperometry Influenza viruses Glycan based _ _ [96]
30. Amperometry Mycobacterium Tuberculosis Immuno sensor 102-105 CFU/mL 102 CFU/mL [98]
31. Potentiometry Plasmodium falciparum glutamate dehydrogenase Aptamer based FET 100 f. – 10 nM 48.6 nM [105]
(malaria biomarker) biosensor
32. Potentiometry Corona virus Immuno sensor _ Culture medium-1.6 [108]
pfu/mL
Clinical samples- 242
copies/ mL
33. Potentiometry Influenza virus Glycan based FET 100.5 - 108.5 TCID 50/mL 100.5 TCID 50/mL [109]
biosensor

study, we have highlighted the applications of electrochemical biosensor The electrochemical biosensors discussed have been identified as proto-
systems in detecting EIDs and Re-EIDs in an attempt to tackle this menace. types and have been evaluated under laboratory conditions only thus far.
The research discussed in this review highlight the ongoing paradigm The implementation was restricted to a minimum number of actual samples
shift in health monitoring and disease diagnosis sector, via increased appli- that were inadequate to establish a satisfactory validation. Optimizing the
cation of electrochemical biosensing diagnostic systems. Despite promising stability, storage, and logistics of electrochemical biosensors, as well as
research and advancements made towards electrochemical biosensors in maintaining their optimum functionality in diverse samples that are barely
recent years, availability of commercially viable devices in the real-world treated or diluted, are challenges to be addressed prior to developing mar-
setting is far from a reality. ketable products.
As discussed in the review, every modality does have pros and cons, but Additional work into microfluidic techniques & microfabrication, cost-
they can all facilitate in disease diagnosis at a POC level with immaculate effective materials and electronics are required to promote the manufacture
planning. The glucometer is an example of the scope of electrochemical bio- of smaller, compact and cheaper sensors. The miniaturization of electro-
sensors, with sound research, meticulous planning and clinical execution chemical cells and deployment of more autonomous handheld readers are
the future of POC detection would be along similar lines. also manadated to improve the efficiency of POC systems, ensuring these

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S. Menon et al. Journal of Electroanalytical Chemistry 878 (2020) 114596

would also tick the guidelines required for large scale production such as [19] Z. Danielle Alves Gomes, PCR and Infectious Diseases, Perspectives on Polymerase
Chain Reaction, IntechOpen, 2019.
complete integration, automation, large-scale miniaturization, flexibility [20] W.J. Shieh, Advanced pathology techniques for detecting emerging infectious disease
and cost reduction. pathogens, Advanced Techniques in Diagnostic Microbiology, Springer, Cham 2018,
In addition, there is an imminent need to deepen collaboration between pp. 543–561.
[21] S. Powell, C. Zachary, The impact of molecular approaches to infectious disease diag-
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