Microchimica Acta (2019) 186: 465

https://doi.org/10.1007/s00604-019-3588-1

REVIEW ARTICLE

Electrochemical sensors and biosensors based on the use

of polyaniline and its nanocomposites: a review on recent advances
Nahid Shoaie 1 & Maryam Daneshpour 2 & Mostafa Azimzadeh 3,4,5 & Sara Mahshid 6 & Seyyed Mehdi Khoshfetrat 7,8 &
Fatemeh Jahanpeyma 1 & Alieh Gholaminejad 1 & Kobra Omidfar 7,8 & Mehdi Foruzandeh 1

Received: 15 February 2019 / Accepted: 6 June 2019 / Published online: 24 June 2019
# Springer-Verlag GmbH Austria, part of Springer Nature 2019

Abstract
Polyaniline and its composites with nanoparticles have been widely used in electrochemical sensor and biosensors due to their
attractive properties and the option of tuning them by proper choice of materials. The review (with 191 references) describes the
progress made in the recent years in polyaniline-based biosensors and their applications in clinical sensing, food quality control,
and environmental monitoring. A first section summarizes the features of using polyaniline in biosensing systems. A subsequent
section covers sensors for clinical applications (with subsections on the detection of cancer cells and bacteria, and sensing of
glucose, uric acid, and cholesterol). Further sections discuss sensors for use in the food industry (such as for sulfite, phenolic
compounds, acrylamide), and in environmental monitoring (mainly pesticides and heavy metal ions). A concluding section
summarizes the current state, highlights some of the challenges currently compromising performance in biosensors and nano-
biosensors, and discusses potential future directions.

Keywords Sensing system . Nanoparticles . Clinical detection . Food quality control . Environmental monitoring . Drugs . Cancer
cells . Bacteria . Glucose . Uric acid . Cholesterol . Sulfite . Phenolic compounds . Acrylamide . Pesticides . Heavy metal ions

Introduction biomolecules in a short time has attracted wide interest in

There are different strategies for biological analytes monitor-
ing. The conventional analytical methods have some disad-
vantages such as time-consuming, requiring initial preparation
steps, well-trained personnel, and expensive materials and in-
struments [1, 2]. Therefore, studies in biosensors designing
field for quantitative and accurate determination of
recent years [3, 4]. According to the International Union of
Pure and Applied Chemistry (IUPAC) definition, a biosensor
is “a device that uses specific biochemical reactions mediated
by isolated enzymes, immunosystems, tissues, organelles or
whole cells to detect chemical compounds usually by electri-
cal, thermal or optical signals”[5]. A typical biosensor is com-
posed of a biorecognition element and a transducer and

* Kobra Omidfar
omidfar@tums.ac.ir
* Mehdi Foruzandeh
foroz@modares.ac.ir
1
Department of Biotechnology, Tarbiat Modares University of
Medical Science, P.O. Box 14115-111, Tehran, Iran
2
Biotechnology Department, School of Advanced Technologies in
Medicine, Shahid Beheshti University of Medical Sciences,
Tehran P.O. Box: 1985717443, Iran
3 8
Medical Nanotechnology & Tissue Engineering Research Center,
Yazd Reproductive Sciences Institute, Shahid Sadoughi University of
Medical Sciences, PO Box: 89195-999, Yazd, Iran
4
Stem Cell Biology Research Center, Yazd Reproductive Sciences
Institute, Shahid Sadoughi University of Medical Sciences, Yazd P.O.
Box: 89195-999, Iran
5
Department of Advanced Medical Sciences and Technologies,
School of Paramedicine, Shahid Sadoughi University of Medical
Sciences, Yazd, Iran
6
Department of Bioengineering, McGill University, Montreal,
Quebec P.O. Box: H3A 0E9, Canada
7
Biosensor Research Center, Endocrinology and Metabolism
Molecular-Cellular Sciences Institute, Tehran University of Medical
Science, Tehran P.O. Box:1411713137, Iran
Endocrinology and Metabolism Research Center, Endocrinology and
Metabolism Research Institute, Tehran University of Medical
Sciences, Tehran, Iran
465 Page 2 of 29
biosensors are classified based on their transduction type
which are mostly electrochemical [6–9].
The need to improve the sensitivity of the electrochemical
biosensors leads to the use of novel strategies such as appli-
cation of conductive polymers (CP), Nanoparticles (NPs), and
also CP/NPs composites [10–14]. Scientists have used variety
of conductive polymers to fabricate biosensors with high se-
lectivity, sensitivity, and low limit of detection (LOD). These
polymers have attracted considerable attention as an appropri-
ate supporting matrix in immobilization of biological ele-
ments. Furthermore, they often have employed as sensing
elements in biosensors and subsequently in improving the
sensitivity of biosensing systems [10, 14–16]. The CPs are
divided into polyenes and polyaromatics such as
polyacetylene, polyaniline (PANI), polypyrrole (Ppy),
polythiophene, poly (p-phenylene), poly (phenylene vinylene)
[17]. Among them, PANI has excellent and controllable
chemical and electrical features. It can be applied as a trans-
ducer which is also a suitable material for loading various
biomolecules [10, 18, 19]. In addition to polymers, different
types of nanomaterials have been used in biosensors. The
nanoparticles provide an extensive surface area for the immo-
bilization of bio-components while simultaneously enhancing
the conductivity [7, 20, 21]. In addition, the CP/NPs compos-
ites noticeably enhance the sensitivity and detection limits
down to even single molecules recognition [12, 22, 23].
This review describes recent advances in the use of
polyaniline and nanomaterials in electrochemical biosensing
systems. Furthermore, we will also offer an authoritative re-
view on the recently developed Polyaniline-based electro-
chemical nanobiosensors that can be deployed in clinical, en-
vironmental and food analysis applications.
Polyaniline applications in nanosensors
Polyaniline (PANI) in electrochemical nanosensors are often
applied along with nanomaterials to enhance the sensitivity.
PANI is a member of the semi-flexible CP family that was
discovered over 180 years ago [24, 25]. The use of PANI in
biosensor construction has been frequently reported due to its
unique properties, such as unique electrical, optical, electro-
optical, photoelectric, high conductivity, structural flexibility,
low cost, easy synthesis, good electrochemical activity, chem-
ical, and considerable stability [18, 26–28]. All these features
are associated with the chemical structure of PANI (Fig. 1)
[29]. PANI contains “n” reduced (benzenoid diamine) and
“m” oxidized (quinoid diamine) repeating units which the ox-
idation state of PANI relates to the rate of “m” [18].
PANI has four basic states including the leucoemeraldine
base (LEB), emeraldine base (EB), per nigraniline base
(PNB), and emeraldine salt (ES). The LEB, EB, and PNB
are different redox forms of PANI having m:n ratio as 0:1,
Microchim Acta (2019) 186: 465
1:1, and 1:0, respectively [29, 30]. The ES is the only
conducting state among the various forms of PANI that can
be achieved in acidic solutions (pH <3.0) [26, 31]. The
emeraldine base is deprotonated form and transition between
ES and EB that are controlled by pH. This form of PANI can
be easily obtained by HCl-doping that displays good environ-
mental and thermal stability [17]. PANI is synthesized using
chemical and electrochemical methods [32]. The chemical
method includes exposing the aniline monomers to an oxidiz-
ing or reducing agent; whereas electrochemical synthesis can
be performed by anodic and cathodic polarization in proper
electrolytes [33].
PANI is the first CP that was commercialized. Nowadays,
numerous studies have reported the use of this polymer in
different applications ranging from batteries to chemical sen-
sors and biosensors [34]. Researchers continuously have been
applying PANI in designing and constructing biosensors be-
cause of its unique features, such as its function as a good
mediator for electron transfer in redox or enzymatic reactions.
This phenomenon is possibly due to the inherent
electroactivity of PANI. In addition, PANI can be employed
as an appropriate matrix for immobilization of biomolecules
[18, 35]. PANI has obtained reputation in the biosensor field,
partially due to its high conductivity, redox reversibility, long-
term environmental stability, high solution process ability, and
easy synthetic procedures with a controllable thickness on the
sensor electrode. There are different nanostructures of PANI
including nanospheres, nanowires, nanorods, and nanotubes
that can increase PANI-characteristics through making the
high interfacial area between PANI and its environment [18,
36–38].
With advancement of nanotechnology, different types of
nanomaterials and nanocomposites have been extensively
used in medicine (e.g. diagnostics, imaging, therapeutics)
and industry (e.g. Cosmetics, Clothing) and have offered
promising results in these fields [39–41]. Nanomaterials does
have appropriate physical and chemical features such as size
scale, high surface-to-volume ratio, good electrocatalytic
properties, and chemical stability [42, 43]. Use of
nanomaterials in nanobiosensor fabrication can lead to obtain
excellent platform for the immobilization of great amount of
bio-component units [12]. Nanomaterials can introduce an
extended surface area for immobilization of biorecognition
elements in a biosensor and improve analytical performance
of biosensing systems via enhancement of sensitivity and re-
duction of detection limit [38]. Furthermore, nanomaterials
can be employed as carriers to load signal markers or directly
as signal reporters in the detection of analytes. These materials
accelerate electron transfer when being used for modifying the
surface of electrode [39, 44]. There are different types of
nanomaterials including gold and silver nanoparticles, quan-
tum dots, magnetic nanoparticles, carbon nanostructures,
graphene [12, 21, 45, 46]. In the next parts, we are going to
Microchim Acta (2019) 186: 465
Fig. 1 a 3D structure of
polyaniline. The carbon atoms
(cyan balls), nitrogen atoms (red
balls), hydrogen atoms (small
yellow balls), and clouds
(molecular orbitals). (b)
Polyaniline 2D structure.
Reprinted with the permission
from Dhand et al. 2011 [18]
review the application of PANI and nanomaterials combina-
tions in electrochemical nanosensors for different
applications.
Polyaniline-based nanobiosensors in clinical
applications
Cancer and biomarker detection
Cancer is the first leading cause of death in developed coun-
tries and the second leading cause of death in developing
countries. This disease has more than 200 diverse forms and
can affect over 60 human organs [47, 48]. The early detection
of cancer reduces the mortality and is decisive for its success-
ful treatment [49–51]. Therefore, the need for new diagnostic
tools appears to be evident [52]. Hui et al. fabricated a sensi-
tive electrochemical cytosensor by self-assembly of folate on
PANI- nanofibers (NF)/AuNP nanocomposite for detecting
cancer cells. As the Fig. 2 demonstrates, the glassy carbon
electrode (GCE) was first modified with a PANI-NF. Then
AuNPs, glutathione (GSH) and Folic acid (FA) were added
onto PANI-NF modified electrode. Using bovine serum albu-
min (BSA) the surface of GCE/PANI-NF/AuNP/ GSH/FA
was blocked. Due to the overexpression of folate receptors
on the surface of many tumor types, a folic acid receptor-
overexpressing human cervical carcinoma HeLa cell was used
as a model in this study. The detection mechanism of the
proposed cytosensor is based on interaction folate
immobilized on PANI-NF/AuNP surface with folate receptor
on cancer cell membranes. By electrochemical impedance
technique, the linear detection range and the detection limit
were obtained 1.0 × 104 to 6.4 × 106 cells. mL-1 and 2000
cells. mL-1, respectively. The stability cytosensor was also
Page 3 of 29 465
evaluated. After four weeks, the electrochemical response of
cytosensor had no visible change in relation to its initial sen-
sitivity to cells that indicated the significant long-term stability
of the biosensor. In addition, the cytosensor showed good
repeatability in cell number determination. The relative stan-
dard deviation was calculated at 1.9% The above results con-
firmed that this cytosensor has high sensitivity, excellent re-
peatability, appropriate stability, and ease of regeneration and
hence it can as an ideal candidate for application in detection
of cancer cells [53].
Prostate cancer (PCa) with approximately 1.1 million cases
and 307,000 related deaths in 2012 is one of the major chal-
lenging health problems in all around the world [54]. This
cancer is the fourth common cancer in the world. Therefore,
early and accurate detection of PCa plays an important role in
reducing mortality risk. Prostate-specific antigen (PSA) is the
most effective biomarker for the clinical detection of PCa that
is produced by the prostate [55]. In compare with a healthy
person, the level of PSA is increased in patients. There are
some conventional methods for the recognition of PSA such
as enzyme-linked immunosorbent assay (ELISA), chemilumi-
nescence immunoassay (CLIA), chemiluminescent micropar-
ticle immunoassay (CMIA) and radioimmunoassay (RIA)
[55]. Recently, Vural et al. designed an Immunosensor in or-
der to quantification of PSA in raw samples. In this research,
by using Anti-PSA (Ab1) and (HRP) labeled anti-PSA (HRP-
Ab2) a disposable amperometric sandwich-type immunoassay
was designed on PANI/AuNP/peptide nanotube (PNT)-modi-
fied pencil graphite electrode (PGE). The immunosensor pre-
sented a low detection limit (0.68 ng. mL-1) , a wide linear
range (1-100 ng. mL-1), high reproducibility with RSD 5.6%
and good stability. The function of the biosensor was also
compared with ELISA as a control method and similar results
were achieved with both analysis. In addition, the function of
465 Page 4 of 29
Fig. 2 Schematic of fabricated
sensitive electrochemical
cytosensor using self-assembly of
folate on PANI-NF/AuNP nano-
composite for detecting HeLa
cells as a model. Reprinted with
the permission from Hui et al.
[53]
biosensor for assaying PSA in real samples was evaluated.
The chronoamperometric results presented good correlation
with commercially available ELISA method. The maximum
RSD and maximum relative error were calculated 14.7% and
10.9%, respectively. Also, in comparison with ELISA meth-
od, this immunoassay is done in a shorter time ( 65 min) and
with lower cost- effective [55]. In another study by Tezerjani
et al. an amperometric DNA biosensor based on PANI/
graphene oxide (GO) nanocomposite-modified GCE has been
developed for PCa detection. Using cyclic voltammetry (CV)
and electrochemical impedance spectroscopy (EIS) tech-
niques, the hybridization of complementary strands at the
PANI/GO-modified electrode surface were studied. A wide
range from 1.0×10-16 M to 1.0×10-8 M along with a limit of
detection of 3.3×10-17 M have been reported for this nano-
composite platform [56]. Furthermore, Dey et al. have used
Polyaniline nanowires/AuNP-modified Au electrode for fab-
ricating a mediator-free immunosensor for PSA detection.
After the immobilization of anti-PSA on the modified Au
electrode, the PSA was added as the analyte. The different
concentrations of PSA were assayed by differential pulse volt-
ammetry (DPV) mode. The proposed immunosensor reached
a detection limit of 0.6 pg. mL-1 (lower than ELISA) in a linear
range between 1 pg. mL-1 and 100 ng. mL-1 and, a sensitivity
of 1.4 mAM-1 (higher than ELISA). In fact, the results showed
that use of AuNP–PANI nanocomposite provides an effective
surface area for the immobilization of anti-PSA and increases
electron transport [57].
Based on the World Health Organization (WHO), Breast
cancer is the most commonly occurring cancer in women
(https://www.who.int/cancer/prevention/diagnosis-screening/
breast-cancer/en/). Although the mortality rate of this cancer
Microchim Acta (2019) 186: 465
has been declining over the last 10 years, it is assessed that
627,000 women died from breast cancer that is nearly 15% of
all cancer deaths among women in 2018. For improving breast
cancer outcomes and survival, early detection is significant
(https://www.who.int/cancer/prevention/diagnosis-screening/
breast-cancer/en/). Recently, Salahandish et al. fabricated a
label-free electrochemical nanosensor in order to quantify hu-
man epidermal growth factor receptor 2 (HER2) as a breast
cancer biomarker in human serum samples. A nanocomposite
with three layers including nitrogen-doped graphene (NFG),
silver nanoparticles (AgNPs), and PANI was prepared onto
the fluorine doped tin oxide (FTO) electrode. Then, anti-
HER2 antibodies were captured on the NFG/AgNPs/PANI-
modified FTO electrode surface. After immersing the biosen-
sor in culture medium with spiked breast cancer cells, detec-
tion of SK-BR3 was done by field emission scanning electron
microscope (FESEM) and DPV electrochemical method. A
dynamic range of 10-5×106 cells. mL-1 and a detection limit
of 2 cells. mL-1 with a fast response time of 30 min have been
reported for the detection of SK-BR3 breast cancer cell [58].
Ovarian cancer (OC) is the seventh most commonly diag-
nosed cancer among women and the eighth most common
cause of cancer-related deaths in the world [59, 60].
Carcinoma Antigen 125 (CA125) is known as an appropriate
and specific biomarker associated with epithelial OC.
Therefore, accurate identification of CA125 can play an im-
portant role in diagnosis and therapy of ovarian cancer [61].
The ELISA, radiometric immunoassay, sandwich immunoas-
say, and fluoroimmunoassay are current techniques that are
being used for detecting of CA125. In 2017, Zheng et al.
fabricated a label-free voltammetric immunosensor based on
a signal multi-amplification strategy by employing gold
Microchim Acta (2019) 186: 465
nanoparticle-Prussian blue-platinum nanoparticle-polyaniline
(AuNP-PB-PtNP-PANI) hydrogel nanocomposite for highly
sensitive detection of CA 125. The PANI hydrogel was firstly
produced with the excellent porous 3D structure that it can
maintain plenty of water molecules. In the next step, for en-
hancing the electrical conductivity, the surface area and pro-
vide supporting points for PB catalytic growth, the PtNPs
were deposited on the PANI hydrogel-modified GCE surface.
Then, AuNPs were deposited onto the surface of electrode.
Finally, anti-CA125 was immobilized on AuNP-PB-PtNP-
PANI hydrogel/GCE. Using such signal multi-amplification
strategy, this immunosensor achieved a linear range of 0.01 -
5000 U. mL-1 and detection limit 4.4 mU. mL-1 and sensitivity
119.76 μA·U.mL-1·cm-2 [61].
Hepatocellular carcinoma (HCC) with a global incidence
of over 600,000 cases per year is one of the most common
cancers in the world [62]. Alpha-fetoprotein (AFP) is a highly
sensitive and specific biomarker for detecting hepatocellular
carcinoma [63]. It is a type of glycoprotein with a molecular
weight of approximately 70 kDa, derived from embryonic
endoderm tissue cells [62, 63]. The concentration in healthy
human serum is less than 20 ng mL-1, but enhances signifi-
cantly in patient serum with liver cancer [63]. There are dif-
ferent methods for assaying level of AFP in human serum
such as CLIA, ELISA, surface plasmon resonance, fluores-
cent immunoassay, and electrochemical immunoassay [63].
Liu et al. using three-dimensional macroporous conducting
polymer PANI fabricated an electrochemical immunosensor
in order to quantification of AFP as cancer liver biomarker
(Fig. 3). The electroactive surface area of the 3D macroporous
PANI- modified electrode is 3.4 times larger than of the planar
Fig. 3 Schematic diagram of the
detection of AFP using the
developed electrochemical
immunosensor based on 3D-
macroporous PANI. Reprinted
with the permission from Liu
et al. 2018 [63]
Page 5 of 29 465
PANI- modified electrode. Therefore, allows the immobiliza-
tion of many AFP antibodies. In this sensor, the electrochem-
ical signal was generated by the inherent redox signal from
PANI, without the addition of other redox reagents. Under
optimum conditions, the porous PANI-based immunosensor
showed a wide linear range from 0.01 to 1000 pg. mL-1 and a
detection limit of 3.7 fg. mL-1 [63]. Also, a hybrid structure
composed of electrochemically synthesized PANI nanowires,
AuNPs, and polyethylene glycols (PEG) has been used for
AFP immunosensor manufacture by Hui et al. The GCE sur-
face was modified by PANI nanowire and AuNPs. Then, the
modified- AuNPs/PANI electrodes were immersed in 4-armed
PEG-amine. By forming a highly non-fouling cross-linked
hydrated polymeric network via the interaction AuNPs and
PEG -NH2groups, the PEG was immobilized onto the surface
of AuNP/PANI. After adding AFP sample, the final detection
was assayed by DPV analysis based on the redox reaction of
PANI as the sensing signal on the GCE surface [64].
Chronic myeloid leukemia (CML) is a malignancy of he-
matopoietic cells that extremely effects on patients bone mar-
row, peripheral blood and lymphatic system [65]. In more than
90% of patients with CML, there is a reciprocal translocation
of the long arms of chromosomes 9 and 22, t (9;22) [66]. This
aberrant chromosome is named Philadelphia chromosome
[66]. Some traditional techniques that were used for detection
CML includes immunophenotyping, cytochemistry test, bone
marrow examination, and bone marrow minimal residual dis-
ease examination [65]. In 2018, Wang et al. constructed a
novel electrochemical biosensor based on signal amplified
strategy for sensitive detection of K562 cell. They used the
multiple layer cadmium quantum dots (CdS QDs)
465 Page 6 of 29
functionalized polystyrene microspheres (PS) and GO PANI
composite as the signal probe and sensor platform, respective-
ly. K562 cell is a type of most uncontrolled and destructive
human CML cell line. The biosensor showed a wider linear
range from 10 to 1.0 × 107 cells. mL-1 with a lower detection
limit of 3 cells. mL-1 (S/N = 3) [67].
Carcinoembryonic antigen (CEA) is a cell surface glyco-
protein that is produced by gastrointestinal tract cells during
embryonic development [41]. The CEA level is minimal in the
bloodstream of healthy adults while a significant level in-
crease has been seen in some cancers, especially colon and
rectal cancer [41]. The CEA level is reported ∼2.5 μg.L-1 in
serum from patients with colorectal carcinoma that is higher
than healthy individuals [41]. Therefore, the CEA can be used
as a tumor biomarker and a prognostic indicator in clinical
tests [41]. A hybrid structure composed of PANI nanowire
and hyaluronic acid has been used for non-fouling electro-
chemical immunosensor construction by Wang and his co-
worker. This nanocomposite was functionalized by a tumor
biomarker CEA-antibodies and exposed to CEA (the target).
The DPV electrochemical technique was applied for evalua-
tion the antifouling performance of PANI/ hyaluronic acid
surface and acts as the analytical information. The current of
immunoelectrode displayed a detection limit of0.0075 pg.
mL-1 and a linear response of 0.01 to 10,000 pg. mL−1. The
biosensing system function was also assessed with the con-
centrations of CEA in different serum samples. The recovery
rates were obtained between 101.6 to 104.2% and the RSD
varied from 4.86 to 6.08%. The high recovery and low RSD
percentages demonstrate the high functionality of the
immunosensor for direct detection of the CEA in plasma. It
can be concluded that coating an electrode with hyaluronic
acid can largely decrease non-specific adsorption of proteins
on the electrode surface. Therefore, the composite of
hyaluronic acid with conductive polymers such as PANI is a
suitable platform for the construction of other non-fouling
electrochemical biosensors [68].
The chondroitin sulphate proteoglycan4 (CSPG4) is a
membrane-bound tumor antigen that is expressed in differen-
tiated tumor cells and also in tumor-initiating cells or cancer
stem cells [69]. It has been reported that CSPG4 is expressed
in more than 85% of surgically removed melanoma lesions
[69]. In contrast, there is limited distribution of this antigen in
normal tissues. Therefore, CSPG4 is an ideal target for immu-
notherapy in patients with advanced melanoma [69]. The
ELISA, reverse transcription-PCR, flow cytometry, and the
immunohistochemical (IHC) are common methods that are
used to detect CSPG4 in melanoma cells and tissues [69].
These methods are disadvantages such as expensive fluores-
cent labeling, time-consuming, tedious liquid handling, large
sample consumption, and require specialist equipment and
well-trained personnel [69]. In 2016, Fu et al. developed an
impedimetric immunosensor for fast, low-cost, and label-free
Microchim Acta (2019) 186: 465
detection of CSPG4. At the first, PANI@graphene oxide film
was codeposited on the ITO electrode. Using glutaraldehyde-
mediated cross-linking, the CSPG4 antibody mAbD2.8.5 was
covalently immobilized onto PANI@GO-modified ITO elec-
trode. The CSPG4-positive cell lines M14/CSPG4 and MV3
were employed. In compare with ELISA, immunosensor was
acted with higher point-of-care potential, lower reagent con-
sumption (5 μl), without labeled antibodies, and shorter assay
time (˜ 2h). The function of the impedimetric immunosensor in
assaying real sample was validated by evaluating CSPG4 mol-
ecules secreted in cell culture medium and cell lysate. The
results indicated that the sensor has a high potential to quickly
measure of CPSG4. It can be concluded that this low-cost,
simple, and label-free electrochemical biosensor can be a suit-
able alternative to ELISA and flow cytometry techniques [69].
Neutrophil gelatinase-associated lipocalin(NGAL) is a
25 kDa protein of the lipocalin family that produced by in-
jured nephron epithelia and then released into blood and urine
[70]. Therefore, it is identified as one of the most promising
novel biomarkers for the detection of renal epithelial injury
[70, 71]. By using a nano-hybrid structure composed of
graphene and PANI, in 2017, Yukird et al. designed a novel
label-free electrochemical immunosensor in order to NGAL
detection. The screen-printed carbon electrode (SPCE) was
first modified using electrospraying of graphene/PANI
nanodroplets on the working electrode. The NGAL antibodies
were then captured onto graphene/PANI-modified SPCE by
covalent bonding between the NH2 groups of PANI and
COOH group of NGAL antibodies. For NGAL assay, a solu-
tion of recombinant NGAL was dropped onto graphene/
PANI/NGAL antibodies-modified electrode surface. Under
optimal conditions, a wide dynamic range from 50 to 500
ng.mL-1 , high sensitivity along with a limit of detection of
21.1 ng.mL-1 and high specificity toward NGAL recognition
from small samples (10 μL) have been reported for this bio-
sensing platform. The immunosensor function was tested in
human urine as a real sample; the results correspond well with
the amounts obtained from the ELISA method. In compare
with the conventional ELISA method, the biosensor needs
shorter analysis time (˜30 min/sample), lower sample and op-
erating cost. Using graphene/PANI as the platform leads to a
dramatic enhance (58-fold) in the oxidation current upon the
binding between NGAL and its antibody [72].
Circulating miRNA is a suitable biomarker for the diagno-
sis and assessment of cancer and non-cancer patients. Many
studies have shown that MicroRNA-16 (miR-16) family
members have an abnormal expression in different types of
tumors. MicroRNAs are suitable biomarkers in diagnosis can-
cer and can distinguish cancer patients from non-cancer peo-
ple [73]. Many studies have shown that MicroRNA-16 (miR-
16) have an abnormal expression in different types of cancers.
Saberi and colleagues constructed a biosensor based on PANI
and AuNPs as a sensor platform and alkaline phosphatase
Microchim Acta (2019) 186: 465
enzyme as a label. For this purpose, Saberi and colleagues
simultaneously employed PANI and AuNPs as a sensor plat-
form and alkaline phosphatase enzyme as a label. This
nanogenosensor showed a linear response over a concentra-
tion range between 0.2-10 nM for the quantification of
microRNA-16 with a limit of detection of 0.1 nM [31].
There are 13 different let-7 in humans which has been
shown to be down-regulated or up-regulated in various can-
cers. Therefore, they have been proposed as diagnostic tools in
serum/plasma [74]. Peng et al. proposed a highly sensitive
electrochemical biosensor for direct miRNA expression pro-
filing. In this biosensing system, the capture probes were
immobilized onto the Au electrode surface. Due to the inter-
action of the capture probe with a ruthenium oxide nanoparti-
cle (RuO2 NP)-tagged target miRNA, the polymerization of
aniline was catalyzed in the presence of H2O2. The hybridized
miRNA strands act as templates for the PANI polymerization.
The genosensor was examined by analyzing the expression
levels of three representative miRNAs of the let-7 family.
The lowest amount of total RNA required for miRNA recog-
nition was obtained to be approximately 1.0 ng. The function
of biosensor compared with northern blot method and accept-
able results were reached [75, 76].
Bacterial species and infectious diseases
In the last century, diagnosis and treatment of bacterial infec-
tions have remained important global health problem that en-
hance the rate of morbidity and mortality, and are also respon-
sible for the increase of the costs and hospitalization times of
patients. Therefore, the rapid diagnosis of pathogenic bacteria
and timely treatment plays a significant role in the clinical and
general health field [77–80].
Tuberculosis (TB) is an infectious disease caused by the
bacillus Mycobacterium tuberculosis (MTB). After human
immunodeficiency virus infection (HIV), TB is the second
cause of deaths by an infectious disease [81, 82]. According
to the World Health Organization (WHO), in 2014 approxi-
mately 9.6 million cases of TB were reported worldwide [83].
It has been expected that the global number of TB cases is
growing at a rate of 2% per year. Accurate and timely diagno-
sis is an important factor in TB infection control [82]. Due to
the low growth rate and fastidious nature, the recognition of
this pathogen has been generally difficult. The sputum smear
microscopy is a standard method that widely used for diag-
nosing of TB [81, 82]. In this method, the bacteria are exam-
ined in sputum samples by a microscope, which associates
with low detection accuracy and demand for skilled
personnel[82]. This method is unable to detection half of the
positive TB infections [81]. In addition, the optimum time for
TMB culture ranges from 4 to 6 weeks, therefore, the culture
can't provide early and rapid diagnosis of TB[83]. To avoid
the limitations of these methods, various diagnostic methods
Page 7 of 29 465
for TB including polymerase chain reaction (PCR) ,immune
assays and southern hybridization are developed [81, 83].
These diagnostic methods have high specificity and sensitivity
but have some limitations such as expensive, time-consuming,
usage of hazardous materials, and needs well-trained person-
nel [81, 83]. By using point-of-care (POC) diagnostic with
100% accuracy can annually be prevented of death 625
000 TB patients [82]. In recent years, researchers have de-
signed various biosensing platforms for accurate, sensitive,
and rapid determination of TB with cost-effective [81, 84,
85]. In 2014, Liu et al. fabricated an electrochemical
nanogenosensor by employing reduced graphene oxide
(rGO)/AuNP-modified electrode as sensing platform and
AuNP/PANI as tracer label for sensitive detection of MTB
bacteria. The rGO/AuNPs nanocomposite was assembled on
a GCE providing a large surface area and facilitating electron
transfer as an excellent biocompatible matrix for immobiliza-
tion DNA capture probe sequence. The target DNA and the
Au/PANI nanocomposite-labelled DNA detector probe were
added onto rGO/AuNPs/GCE modified electrode surface and
the final signal was recorded via the excellent electrochemical
activity of Au/PANI nanocomposites (Fig. 4a) [84, 86]. Chen
et al. have also designed a sandwich-type of DNA biosensor
for sensing the specific IS6110 DNA fragment of MTB that
has been confirmed to be a key molecular marker for the
diagnosis of TB. The capture probe was immobilized onto
the surface of the Au electrode via amino-Au affinity.
Subsequently, the target DNA was added onto the Au elec-
trode and the PANI-rGO/AuNPs composite was used as a
redox detector probe for the generation of the voltammetric
signal due to appropriate electrochemical activity. The authors
believed that PANI/rGO/AuNP nano hybrid enhanced the
electron transfer rate and increased the sensitivity of the bio-
sensor. In fact, incorporating PANI with rGO provided many
advantages including increasing the immobilization of PANI
that improves the electrochemical signals and also create a
good platform for other modifications. This is probably be-
cause of having the numerous amino group of PANI.
Moreover, high conductivity and good adsorption properties
of AuNPs improved the electrical characteristics and tightly
immobilize detector probes via strong Au-S bond. This
genosensor presented a linear range of 0.1 pM -10 nM and a
limit of detection of 50 fM (Fig. 4b) [83].
In addition, Mohd Azmi et al. fabricated a sandwich elec-
trochemical immunonanosensor using ESAT-6-like protein
esxB (CFP10) as a protein biomarker in order to early quan-
tification of MTB in raw samples. The CFP10 protein that also
known as secreted antigenic protein MTSA-10 or 10 kDa cul-
ture filtrate antigen CFP-10 is a 10 kDa secreted antigen from
MTB. The immunoassay was designed based on a two-step
strategy including graphene/PANI-modified screen printed
gold electrode (SPGE) and a sandwich immunoassay format.
Therefore, the capture anti-CFP10 antibodies (CapAb) were
465 Page 8 of 29
Fig. 4 Examples of using PANI/AuNP in Mycobacterium tuberculosis
biosensor construction. (a) Schematic demonstration of a nanobiosensor
fabricated using rGO and AuNPs for electrode modification and Au-
PANI as the electrochemical label. Reprinted with the permission from
immobilized onto GP/PANI-modified SPGE . After adding
sample, the target was sandwiched between the CapAb and
primary anti-CFP10 antibodies that were labeled with iron
oxide-gold magnetic nanoparticles (Fe 3 O 4 -Au MNPs).
Finally, the electrochemical measurement was performed via
DPV mode. The sandwich type immunosensor was capable to
detect the CFP10antigen with a linear range from 20 to 100
ng.mL-1 (R2 = 0.99) and a low detection limit of 15 ng.mL-1
within 3 h. The blood test-free method that detects TB disease
by detection of CFP10 biomarker in sputum can provide wide
potential applications in clinical analysis [87].
In this type of biosensors that are based on antibody/
antigen interaction, the binding capacity of antibodies affected
by the pH and temperature conditions and this is one of the
most important drawbacks in designing immunesensors [88].
Neisseria gonorrhea bacteria is the cause of gonorrhea,
which is the second most prevalent bacterial sexually trans-
mitted disease [89]. If this infection is not diagnosed and treat-
ed properly, it result in pelvic inflammatory disease (PID),
infertility and ectopic pregnancy in women, urethritis and epi-
didymitis in men; and proctitis, pharyngitis, disseminated
Microchim Acta (2019) 186: 465
Liu et al. 2014 [84]. (b) Schematic of the sandwich-type of DNA biosen-
sor based on PANI-rGO/AuNPs composite. Reprinted with the permis-
sion from Chen et al. 2017 [83]
gonococcal infection in both males and females [76]. Singh
et al. used PANI/CNTs as the biosensing platform for detec-
tion of Neisseria gonorrhea infection. The amino-labeled
Neisseria gonorrhea probe was covalently immobilized onto
PANI/CNT composite on platform onto indium tin oxide
(ITO) electrode surface by glutaraldehyde. Clinical patient
samples were analyzed by fabricated biosensor and gonorrhea
was detected by employing methylene blue (MB) as the redox
indicator in a response time of 60 s. This bioelectrode exhib-
ited a linear range from 10-6 to 10-17 M and limit of detection
of 1.2×10-17 M [76]. Additionally, in 2012, Singh et al. pre-
sented a bacterial electrochemical biosensor based on
PANI/nFe3O4/CNT nanocomposite for rapid and accurate di-
agnosis of gonococcal infection. They applied biotin/avidin
interaction for immobilization of the Neisseria gonorrhea spe-
cific biotinylated nucleic acid probe onto PANI–nFe3O4–
CNT/ITO nano electrode and methylene blue as electroactive
indicator. The presence of nFe3O4 not only increases the
electroactive surface area which can improve the immobiliza-
tion of DNA but also enhances the stability and electron trans-
fer kinetics between medium and the electrode. Their result
Microchim Acta (2019) 186: 465
showed that this biosensor able to detect low concentrations
(1×10−19 M). In fact, using the PANI-nFe3O4-CNT as the
platform can increase specificity and sensitivity in biosensing
(Fig. 5a) [90].
Spain et al. reported a biosensor for detecting low concen-
trations of Staphylococcus aureus DNA. As the authors de-
scribed, AuNPs were synthesized on the PANI nanofibers
(NF) and the PANI-NF/AuNP nanocomposite was obtained
and deposited on gold electrodes. The nanocomposite provid-
ed a high surface area, good conductivity, and great porosity
that was ideal for biosensing analysis [91].
E s c h er i c h i a co l i (E . c o l i ) is on e o f m e m b e r s
Enterobacteriaceae family that usually found in the intestines
of humans and animals [35, 92]. Most strains of this bacterium
are not harmful; however, some strains have the pathogenic
Fig. 5 a Schematic of bacterial electrochemical biosensor based on
PANI/nFe3O4/CNT nanocomposite for rapid and accurate diagnosis of
gonococcal infection. Reprinted with the permission from Singh et al.
2012[90] b Schematic of DNA electrochemical biosensor for
Page 9 of 29 465
potential and can lead to a wide range of infections from mild
to life-threatening conditions [93]. It routinely used as an in-
dicator to monitor fecal contamination of water [93, 94].
Shoaie et al. applied sandwich strategy assay to design a
DNA electrochemical biosensor for specific and rapid detec-
tion of E. coli. This biosensor was fabricated on a SPCE mod-
ified with PANI and AuNPs as biosensing platform. The bio-
tinylated capture probe was immobilized onto the
PANI/AuNPs/avidin-modified SPCE surface using biotin/
avidin interaction. The hybridization solution including
E.coli DNA derived from single-step bacterial lysis and dig-
labeled detector probe was dropped on the electrode surface.
The CV electrochemical signal, after adding the anti-dig
horseradish peroxidase (HRP) enzyme and TMB/H2O2 solu-
tion was generated via enzymatic reaction and electron-
quantification E.coli. With the combination of suitable properties PANI,
AuNPs and, HRP enzyme activity. Reprinted with the permission from
Shoaie et al. 2018 [35]
465 Page 10 of 29
producer on the SPCE surface. The suggested biosensor was
able to detect 4×106 to 4 colony-forming units per milliliter
CFU.mL-1) of E. coli in less than 70 min without DNA am-
plification. The sensitivity was obtained 353 μA. CFU-1. Cm-2
(Fig. 5b) [35].
In 2019, Mo et al. designed a sensitive and regenerative
sandwich electrochemical immunoassay in order to quantifi-
cation of E. coli O157: H7 sample. The immonunosensor
fabricated using PANI/AuNp modified-SPCE as a platform
and rGO/ neutral red (NR) / gold@platinum nanoparticles
(Au@Pt) composite as the non-enzyme signal label. At the
first PANI monomers were electrodeposited on the bare
SPCE surface via potentiostatic method. Then, AuNPs were
dropped onto PANI- modified SPCE and linked to PANI film
by Au-N bond. The anti-E. coli O157:H7 monoclonal anti-
body (Ab1) was added onto SPCE-PANI/AuNP platform. The
Ab1s were automatic immobilized on PANI/AuNP-modified
electrode surface through amino and AuNPs interaction. In the
next step, E.coli, Ab2, and rGO-NR-Au@Pt were placed on
the electrode and the final signal was measured with the CV
electrochemical method. The immonunosensor presented a
wide linear range ( 8.9 ×103 to 8.9 ×109 CFU.mL-1) and limit
of detection 2.84 ×103 CFU.mL-1. Therefore, this biosensing
system is ideal for E.coli detection and also it is readily adapt-
able for the assay of other bacterial species[95].
The main limitation of using the offered electrochemical
biosensors based on screen-printed electrodes is need of an
analysis non-portable device such as potentiostat for obtaining
the final response. Thus, the design and construction of a
portable read-out device may considerably increase the appli-
cation potential of these sensing systems[35].
Glucose
Diabetes mellitus is the most common endocrine disorder that
can be characterized by symptoms of chronic hyperglycemia
[96–98]. The prevalence of diabetes has increased worldwide
and it is defined as the most challenging health problem in the
twenty-first century [96, 99]. A two-fold increase in the num-
ber of people with diabetes was reported between 1980 and
2008 [100]. The controlling of blood glucose is an integral
part of medical diagnostics and the best way for the manage-
ment of diabetes [101]. The most commercially successful
tools for glucose monitoring are enzyme-based glucose bio-
sensors [42]. The initial development of the biosensor for
measuring glucose level was offered in 1962 by Clark and
Lyons. Usually, glucose assay is done based on its interaction
with one of three enzymes, glucose oxidase (GOx), hexoki-
nase or glucose-1-dehydrogenase (GDH). However, glucose
oxidase (GOx) is the most famous used enzyme in designing
and constructing glucose biosensors because it has a relatively
higher selectivity for glucose [97, 102].
Microchim Acta (2019) 186: 465
Tamer et al. by using PANI/gold nanorod (GNR) compos-
ite fabricated an electrochemical enzymatic nanobiosensor for
the recognition of glucose. Glucose oxidase (GOx) enzyme
was immobilized on modified PANI/GNR electrode with the
covalent bonding via glutaraldehyde. This composite provid-
ed an appropriate matrix for enzyme immobilization due to the
large specific surface area and higher electroactivity. Their
results showed a calibration curve with a linear range from
17.6 μM to 1 mM, the sensitivity of 13.8 μA mM-1. Cm-2, and
LOD of 5.8 μM [103]. Zhai et al. used the benefits of both
conducting hydrogel and the nanoparticle catalyst for devel-
oping a highly sensitive glucose nanosensor based on the
platinum (Pt) NP/PANI hydrogel. The PANI hydrogel porous
structure provided a welcoming platform for immobilization
PtNPs and GOx enzyme and diffusion of water-soluble mol-
ecules, which efficiently face-lifted the catalysis of glucose
oxidation. The PtNPs was used as the active catalyst for the
electro-oxidation of hydrogen peroxide (H2O2) which was
generated during the performance of the enzymatic reaction.
Finally, the high-conducting PtNP/PANI hydrogel
heterostructures assembled the transferred charges from these
electrochemical processes and enhanced the sensitivity of the
glucose sensor. The designed glucose nanobiosensor repre-
sented a good high sensitivity of 96.1 μA mM-1 .cm-2, with
an average response time of 3 s, a wide linear range from 0.01
to 8 mM, and a low detection limit of 0.7 μM [102]. In 2012,
Nguyen et al. represented a glucose electrochemical biosensor
with the help of the Graphene/Fe3O4 NPs/PANI composite.
The authors reported the synthesis of graphene sheets with the
thickness of 5 nm on copper stripes by thermal chemical vapor
deposition (CVD) method. The graphene films were then
transferred to the layer-by-layer PANI/Fe3O4 NPs/GOx
preformed interdigitated array (IDA). The graphene patterned
sensor has shown an excellent and linear amperometric re-
sponse to glucose within the concentration range from 2.9 to
23 mM and a sensitivity of 47 μA mM-1 .cm-2 [104].
In 2017, Xu et al. designed a new non-enzymatic electro-
chemical biosensor for detecting glucose using flexible
AuNPs/PANI/carbon cloth (CC) integrated electrode. The
flexible CC was used as a supporting electrode that its elec-
trochemical properties is close to the bare GCE but exhibit a
smaller charge transfer resistance (Rct) owing to its porous
structure and good electrical conductivity. The PANI film
was formed on the flexible CC electrode surface as a dispers-
ing and binding agent. Then, the AuNPs with good catalytic
activity were electrodeposited on the PANI/CC- modified
electrode surface that resulted in a good performance to glu-
cose detection with a wide linear range from 10.26 μM to10
mM. Also, this flexible glucose electrochemical biosensor
showed sensitivity and detection limit of 150 μA mM-1
.cm-2 and 3.08 μM, respectively. Furthermore, proposed bio-
sensor had an appropriate accuracy for detecting glucose in
real serum samples [105]. In another study by Wu et al. a
Microchim Acta (2019) 186: 465
highly sensitive dual mode electrochemical platform has been
fabricated based on plasma polyaniline (pPANI)-modified tin
oxide and 3D reduced graphene oxide (SnO2@3D-rGO)
nanocomposite for detection of glucose. The gold electrode
was first modified with SnO2@3D-rGO nanocomposite that
this nanostructure was made by simultaneously reducing 3D-
GO and converting SnCl4 into SnO2, followed by pPANI
modification. After adding glucose onto GOx-SnO2@3D-
rGO@pPANI-modified Au electrode, the amperometric
current–time responses have been recorded. A low detection
limit of 0.047 ng. mL−1 (0.26 nM) (S/N = 3) within the wide
concentration range of 0.1 ng. mL−1 to 5 μg. mL−1 have been
reported for this biosensing platform [106].
Moreover, in 2018, Esmaeeli and his co-workers simulta-
neously employed polyaniline nanofiber (PANI-NFs) and
copper oxide (CuO) onto fluorine doped tin oxide (FTO) elec-
trode for designing a non-enzymatic glucose sensor (Fig. 6).
This biosensor presented the chronoamperometry (CA) linear
range between 0.25 μM to 4.6 mM and the detection limit of
0.24 μM (S/N = 3). The selectivity in the quantification of
glucose was assayed with some important species such as
sodium chloride, ascorbic acid, uric acid, dopamine and, urea
and no considerable interfering effect was observed. The re-
sult indicates the proposed non-enzymatic electrochemical
biosensor can be a promising candidate for glucose sensing
[107]. In the same year, Wang et al. designed a bifunctional
electrochemical sensor for glucose electro-catalysis and rutin
Fig. 6 Schematic illustration of the stepwise non-enzymatic chronoamperometry biosensor construction process onto (CuO/PANI-NF/FTO) electrode
for sensing glucose. Reprinted with the permission from Esmaeeli et al. 2018 [107]
Page 11 of 29 465
oxidation onto NiCo2S4/rGO@PANI-modified GCE. Rutin is
one of the most abundant bioactive flavonoids extensively
found in fruits, buckwheat, vegetables, and medicinal herbs
[108]. For preparing NiCo2S4/rGO@PANI nanocomposite,
they used of the two-step hydrothermal treatment and calcina-
tion, coupled with in situ polymerization process. This bifunc-
tional sensor exhibited excellent electrocatalytic activity in
assaying glucose with a linear range from1 to 7000 μM along
with a limit of detection of 0.14 μM (S/N=3) and sensitivity of
4.924 μA·μM-1·cm-2. For rutin, the linear range, detection
limit and sensitivity have been reported 0.01-200 μM,
0.007 μM (S/N=3) and 75.50 μA·μM-1·cm-2, respectively.
The obtained results towards glucose and rutin in real samples
show that electrochemical sensor based on NiCo 2 S 4 /
rGO@PANI nanocomposite is a suitable candidate for the
improvement of biosensor devices [108].
Uric acid
Uric acid (2,6,8-trihydroxypurine) is the main end product
of purine metabolism that is present in blood and urine of
humans, birds, and other animals. In human, produced uric
acid is excreted by kidneys and intestinal tract without
further metabolism. Serum concentration of uric acid is
controlled by the balance of production and excretion.
The abnormal uric acid level is a strong indicator for rou-
tine clinical diagnosis some of the disorders such as gout,
465 Page 12 of 29
hypertension, insulin resistance, arthritis, neurological dis-
eases, hyperuricemia or Lesch-Nyan syndrome, cardiovas-
cular diseases, and renal insufficiency. Therefore, an accu-
rate determination uric acid in the different biological fluids
is clinically valuable [109–113]. In 2010, Bhambi et al.
described a sensitive amperometric uric acid biosensor. In
an electrochemical cell system, they electropolymerized an-
iline monomers and multi-walled carbon nanotubes
(MWCNT) onto the ITO coated glass plate by
chronoamperometry technique. Then Bacillus uricase en-
zyme was covalently immobilized onto MWCNT/PANI
composite. This enzymatic sensor was used for evaluating
serum samples from healthy individuals and patients suffer-
ing from hyperureicemia, gout, and other nephropathies.
The reported nanobiosensor demonstrated a very quick re-
sponse time of 8 sec with a linear range from 0.005 to 0.6
mM, and also low detection limit of 0.005 mM [114].
Rawal et al. constructed an amperometric enzymatic
biosensor for recognition uric acid in clinical samples
by employing the Prussian blue nanoparticles (PBNPs),
MWCNT and PANI on the surface of the Au electrode.
In order to prepare c-MWCNT/PANI/Au electrode, the
PANI modified electrode was immersed into the c-
MWCNT solution. The PBNPs were electrodeposited on-
to the c-MWCNT/PANI/Au electrode. Uricase enzyme
was covalently immobilized onto the c-MWCNT/PANI/
PBNPs hybrid nanocomposites modified Au electrode by
chitosan (CH) and glutaraldehyde. This enzymatic bio-
sensor assayed uric acid in real serum samples and ex-
hibited optimum response within 4 s with a linear work-
ing range from 0.005 to 0.8 mM and a detection limit of
5 μM. The sensor maintained 63% of its initial activity
after a period of 7 months [110].
Devi et al. designed a uric acid biosensor based on iron
oxide nanoparticles (Fe 3 O 4 )/chitosan graft (CHIT-g)/
(PANI) composite film on the surface of the Pt electrode.
Under an alkaline condition, the Fe3O4-NPs were made
by the co-precipitation of Fe (II) and Fe (III). Then,
Fe3O4-NPs were added into CHIT solution and a very
viscous solution of CHIT with homogenously dispersed
Fe3O4-NPs was achieved. In the next step, a solution in-
cluding monomers aniline, Fe 3 O 4 -NPs/ CHIT and
Hydrochloric acid (HCl) was prepared in a glass cell.
Nanocomposite film of Fe 3 O 4 -NPs/CHIT-g-PANI was
electrodeposited on the Pt electrode by CV mode.
Uricase enzyme was immobilized onto the Fe3O4-NPs/
CHIT-g-PANI/ modified Pt electrode surface via glutaral-
dehyde coupling. The nano-biosensor checked with serum
of apparently healthy persons and its result equaled well
with a standard enzymatic colorimetric method (r = 0.98).
This sensor demonstrated a linear range from 0.1 to
800 μM with the sensitivity of 0.44 mA. mM-1.cm-2 and
a detection limit of 0.1 μM (S/N = 3) [111].
Microchim Acta (2019) 186: 465
Cholesterol
Cholesterol is a biochemical metabolite with significant
natural functions, found in all cells of the body.
Cholesterol and its ester act as a precursor for the synthe-
sis of different bio-component such as hormones, vitamin
D, and bile acids [115, 116]. The assessment of cholester-
ol level in blood is an important parameter in the diagno-
sis and prevention of a large number of clinical disorders
such as arteriosclerosis, hypertension, cerebral thrombo-
sis, myocardial infarction and many heart-related disor-
ders [116–118]. Colorimetric and spectrophotometric ana-
lytical methods have been used for determination of blood
cholesterol level. These techniques are complex and ex-
pensive since a high volume of the enzyme is needed in
each assay [118]. In 2014, Srivastava et al. fabricated a
biosensor with high sensitivity and accuracy for determi-
nation of cholesterol by using CH and PANI–Au nano-
composite. The PANI/Au nanocomposite was chemically
synthesized and employed along with CH as a matrix for
immobilization of cholesterol oxidase (ChOx) enzyme on
the modified electrode surface. This enzymatic biosensor
represented linearity in a wide range of 50–500 mg. dL-1
with a detection limit of 37.89 mg. dL-1 and a sensitivity
of 0.86 mA. Mg -1. dL-2. It can be concluded that the
excellent immobilization and enzyme–substrate reactions
are a benefit of this matrix over other matrixes applied for
cholesterol sensors [119]. In another study, Dhand et al.
designed a nanobiosensor based on PANI/MWCNT nano-
composite film for quantification of cholesterol. Using the
electrophoretic technique, a thin film of PANI–MWCNT
composite was deposited on the ITO-coated glass plate.
The ChOx enzyme was covalently immobilized onto the
PANI–MWCNT composite via EDC/NHS chemistry. The
results indicated that ChOx/PA NI–MWCNT/ITO
bioelectrode can detect cholesterol in the concentration
range of 1.29 to 12.93 mM with sensitivity and response
time of 6800 nA mM -1 and 10 s, respectively [120].
Furthermore, in 2012, Deepshikha et al. developed a nov-
el amperometric cholesterol nanobiosensor by employing
a bienzyme system including ChOx and horseradish per-
oxidase (HRP). The AuNPs decorated graphene-
nanostructured polyaniline nanocomposite (NSPANI/
AuNP/GR) was chemically synthesized and electrochem-
ically deposited onto ITO electrode. ChOx and ChOx/
HRP were fixed onto NSPANI/AuNP/GR/ITO modified
electrodes by glutaraldehyde as a cross-linker. The
C h O x / N S PA N I - A u N P - G R / I T O a n d C h O x - H R P /
NSPANI-AuNP-GR/ITO nanobioelectrodes were used to
analyze the real samples via amperometric and photomet-
ric studies. In a comparison view, the bienzyme
nanobioelectrodes showed better performance in terms of
sensitivity, detection limit, and response time [121].
Microchim Acta (2019) 186: 465
Drug
In clinical practice, therapeutic drug monitoring (TDM) is
essential for evaluating pharmaceutical drugs with dosage lim-
itations or toxicity issues [122]. In fact, TDM is a routinely
practiced clinical laboratory technique that aids the monitor-
ing of patients undergoing treatment with these drugs and
measurements the concentration of specific narrow therapeu-
tic drugs at designated intervals in a patient's biofluid (urine
and blood) [122, 123]. Therefore, it provides access to the
pharmacokinetics of drugs and consequently a clear clinical
judgment of dosage or in medical intervention if the situation
becomes urgent [122, 123]. In centralized laboratories, TDM
is usually done by immunoassay analyzers and mass spec-
trometers that these instruments can be run only by trained
personnel [124]. The time required for the preparation, sam-
ples analysis, and data processing, and high cost are draw-
backs of these methods that severely affect the TDM applica-
tion in medical practices [124]. The sensors as a new genera-
tion of analyzer tools can pave the way for the TDM of many
commonly administered drugs and also provide safe and op-
timal dosing across a board range of pharmaceuticals [122,
124].
Calycosin is a typical isoflavonoid that has the potential
pharmaceutical properties such as the prevention and treat-
ment of tumors, diabetic cognitive impairment, and the reduc-
tion of inflammation, stroke, and cardiovascular disease [125,
126]. In 2018, Cai et al. designed a novel electrochemical
method by PANI /graphene quantum dots nanocomposite for
calycosin analysis. PANI functionalized graphene quantum
dots (PAGD) was prepared by situ polymerized, then, The
PAGD nanocomposite was added onto bare GCE surface.
Subsequently, calycosin different concentrations were pre-
pared in PBS and enriched on the functionalized sensor by
open circuit potential for 180 s. The final detection was per-
formed by DPV measurements. A wide detection range of
1.1×10 -5 to 3.52×10 -4 mol.L and a detection limit of
9.8×10-6 mol.L (S/N = 3) have been reported for this electro-
chemical sensor. In addition, when the sensor was used for
calycosin analysis in the extracts of Radix Astragali and Radix
Hedysari, the acceptable recovery (91.22% to 105.85%) was
obtained. According to the obtained results, the sensor has
acceptable reproducibility, long-term stability, good selectivi-
ty, and sensitivity and can offer a new way for the detection of
calycosin [126].
5-Fluorouracil (5-FU) is one of the most important drugs
that widely used in combination with other drugs for treating
different types of cancers such as breast, stomach, pancreatic,
colorectal, and cervical cancer [127, 128]. High-performance
liquid chromatography (HPLC), liquid chromatography-mass
spectrometry/mass spectrometry (LC-MS/MS), capillary elec-
trophoresis, gas chromatography-mass (GC–MS) are methods
which employed for recognition of anti-cancer 5-FU drug
Page 13 of 29 465
[128]. These techniques have disadvantages containing diffi-
cult procedure and complicated pre-treatment and expensive
instruments, therefore, researchers have paid more attention to
biosensing systems due to the possibility of miniaturization,
easy preparation of sample and rapid recognition [128]. In
2018, Modarres Zahed et al. have used silver nanoparticles
decorated PANI nanocomposite- modified pencil graphite
electrode in order to fabricate a highly efficient electrochem-
ical sensor for the determination of anti-cancer drug 5-FU.
Silver nanoparticles-polyaniline nanotube
(AgNPs@PANINT) nanocomposite was first synthesized by
a simple one-step method. Then, pencil graphite rods were
immersed in AgNPs@PANINTs solution hence nanocompos-
ites physically adsorbed to the surface of pencil graphite elec-
trode. Final electrochemical signal was obtained via DPV as
electrochemical mode and Britton-Robinson (B-R) buffer (pH
8.0) as supporting electrolyte. The electrochemical behavior
of the 5-FU on bare and AgNPs@PANINT -modified elec-
trode indicated that nanocomposite has a notable catalytic ac-
tivity towards the oxidation of 5-FU. This phenomenon might
be associated with the synergistic electrocatalytic effect of
AgNPs and PANINTs, higher conductivity and larger surface
area. The sensor reached a detection limit of 0.06 μM in linear
range between 1.0 to 300.0 μM. Also, the sensor exhibited
excellent selectivity in the presence of two anticancer drugs
including oxaliplatine and irinotecan. It is believed that by
taking advantage of unique properties of PANINTs and
AgNPs, the proposed biosensor can facilitate the analysis of
5-FU in the serum sample [128].
In the same year, Mostafavi and her coworkers constructed
a novel diclofenac (DCF) molecularly imprinted electrochem-
ical sensor using PANI/reduced graphene oxide nano-
composite [129]. DCF is a member of non-steroidal anti-in-
flammatory drugs that extensively applied as anti-rheumatic,
anti-inflammatory, analgesic and antipyretic [129, 130]. They
applied Molecular Imprinting Technology (MIT) for modifi-
cation carbon-based electrode which is a technique to design
artificial receptors called Molecularly Imprinted Polymers
(MIPs) and is formed of selective sites in a polymer matrix
with the memory of an analyte (template molecule) [129,
131]. DCF-imprinted polymer was synthesized by employing
PANI/ triphenylamine/ reduced graphene oxide nanocompos-
ite. The determination of DCF was done via CV analysis and
the detection limit of 1.1 mg.mL-1 was evaluated in the linear
concentration range from 5 to 80 mg.mL-1. The relative stan-
dard deviations of the inter- and intra-day analysis were also
calculated by this sensing method were found to be as 2.43%
and 2.47%, respectively. To assay the specificity of this meth-
od for DCF detection, the binary solutions DCF/glucose,
DCF/ascorbic acid, and DCF/urea were prepared. These com-
binations were chosen due to glucose and ascorbic acid exist
in blood, and urea is routinely found in urine. The results
showed that DCF molecularly imprinted electrochemical
465 Page 14 of 29
sensor can be successfully employed for DCF detection in
pharmaceutical and urine samples [129]. The simultaneous
application of PANI, reduced graphene oxide, and
triphenylamine as cross-linker is associated with some bene-
fits. The simultaneous application of PANI, reduced graphene
oxide, and triphenylamine as cross-linker is associated with
some benefits. Use of triphenylamine provides the possible
formation of three dimension polymers, the presence of re-
duced graphene oxide with PANI enhance electro-catalytic
activities and make the fast electron transfer process possible
[129].
In MIPs technique for completely remove the template
from MIPs are often needed harsh conditions and the kinetics
are slow for the removal and rebinding of the template, that
these properties are disadvantages of this technique [88, 132].
In order to evaluate the dose of Bezafibrate (BZF) drug in
pharmaceutical formulations and real samples (blood and
urine), Rajpurohit et al. fabricated an electrochemical sensor
by applying carbon nanofibers (CNF) /PANI/Pt NPs compos-
ite onto the carbon paste electrode (CPE) surface [133]. BZF
(2-[4-[2-[(4-chlorobenzoyl) amino] ethyl] phenoxy]-2-
methylpropanoic acid) is a fibric acid derivative that widely
used as lipid regulating agent in the patients with high choles-
terol and triglyceride [133]. After preparing CNF/PANI/Pt/
CPE, the determination of BZF was performed via DPV
mode. Using this nanocomposite platform, a detection limit
of 2.46 nM and a linear range of 0.025 mM to 100 mM were
obtained. Due to the modified electrode demonstrated an ex-
cellent recovery in the synthetic samples, the sensor is a prom-
ising candidate for the quantitative determination of BZF in
clinical and pharmaceutical analyses [133].
Quercetin (3,3',4’,5,7-pentahydroxyflavon) is the most
abundant flavonoid molecule in the plants with many biolog-
ical and pharmacological activities, which may be beneficial
to human health [134, 135]. The appropriate level of quercetin
is an important parameter in clinical medicine, biochemistry,
natural pharmaceutical chemistry, and nutrition science [136].
A high quercetin intake may associate with side effects such as
headache, kidney cancer and stomach upset. In 2018,
Ponnaiah et al. used a binary nanocomposite composed of a
copper tungstate (CuWO4) and PANI in order to designing an
electrochemical sensor for voltammetric determination of
quercetin [136]. The main drawback related to using WO3
metal oxide for electrochemical sensing is poor stability dur-
ing sensing process, the use of CuWO4 nanocomposite over-
comes this problem and provide a platform with long-term
stability, high surface area, suitable electrocatalytic activity,
and reusability, which can enhance the kinetics of electron
transfer reactions [136]. At room temperature, the nano-
hybrid structure (CuWO4@PANI) was synthesized by chem-
ical oxidative single step polymerization on the surface of a
GCE. Then, different concentrations of quercetin were
dropped onto CuWO4@PANI-modified GCE surface and
Microchim Acta (2019) 186: 465
the sensor performance was evaluated. When the concentra-
tion of quercetin is enhanced (0.001–0.500 μM), the peak
current increases linearly. The value of limit of detection and
sensitivity of the CuWO4@PANI/GCE were calculated to
be1.2 nM and 1.6025 μA μM-1 cm-2, respectively. The results
confirm that the CuWO4@PANI/GCE nanostructure can very
well be used as a most promising sensing technique for quer-
cetin detection. Also, the voltammetric sensor function was
validated using analysis of natural samples such as green
tea, grape juice, apple juice, honey, pear and onion juice and
blood and urine samples of pregnant woman and adult man
[136] (Fig. 7).
Dexamethasone (DXN) is a synthetic hormone which de-
rivative of the glucocorticoid hydrocortisone group [139,
161]. In many medical applications, it is regularly used as an
anti-allergic, anti-inflammatory, and immunosuppressive
agent [161]. In addition, it is employed as a growth promoting
agent to enhance body mass. This drug has been added to the
list of the banned drug because of its misuse as a doping agent
in sports [161]. Also, it can lead to decreasing fertility and
ovarian function such as chronic ovulation and polycystic
ovarian syndrome [161]. In fact, it can be presumed as a hu-
man reproductive toxicant [161]. Therefore, accurate monitor-
ing of DXN concentration is very important and development
of a rapid and sensitive detection method for the DXN identi-
fication is required [139]. Recently, a novel sensitive and se-
lective electrochemical sensor based on a Fe3O4/PANI–CuII
microsphere modified carbon ionic liquid electrode (CILE)
has introduced for the determination of dexamethasone by
Fatahi and her coworkers. Using the solvothermal method,
magnetite particles were synthesized. In the next step, the
Fe3O4/PANI microspheres were produced by in situ chemical
oxidative polymerization of aniline. The Cu was loaded on
Fe3O4/PANI microspheres and subsequently Fe3O4/PANI–
CuII composite synthesized. The Fe3O4/ PANI–CuII micro-
spheres can remarkably accelerate the electron transfer rate
and provide an excellent synergistic electrochemical activity
for the dexamethasone oxidation. The CILE was fabricated by
mixing paraffin oil, solid I(EMIMPF6) and graphite powder
and then modified Fe3O4/PANI–CuII composite. For prepar-
ing CILE that widely used in the field of electrochemical
sensing, an ionic liquid is applied as both binder and modifier
in carbon paste electrode. Finally, the dexamethasone concen-
tration was evaluated by Fe3O4/PANI–CuII/ CILE sensing
platform and DPV electrochemical mode. The oxidation peak
current is linear with the concentration of dexamethasone in
the range of 0.05 to 30 mM with a detection limit of 3.0 nM
[139].
Nifedipine is a 1,4-dihydropyridine-based calcium channel
blocker that is mainly used in in the treatment of various
cardiovascular diseases such as hypertensive crisis, angina
pectoris, and chronic hypertension [140, 162]. The high-
dose consumption of this drug is toxic and may lead to
Microchim Acta (2019) 186: 465
Fig. 7 a Schematic representation of Synthesis of CuWO4@PANI
nanocomposite and the possible mechanism of oxidation and reduction
reaction of the quercetin. b Schematic representation of the quercetin
pounding heartbeats, dizziness, nausea, and vomiting [140].
Wang et al. developed a highly selective electrochemical sen-
sor based on layer-by-layer assembly films from PANI and
MWCNTs for nifedipine detection. The ITO electrode surface
was modified using nano- hybrid uniformly distributed film
composed of PANINPs and carboxylic acid functionalized
MWCNTs (MWNTs-COOH) via a layer-by-layer method.
The PANINPs and MWNTs-COOH was successfully assem-
bled by using electrostatic interactions that appropriate conju-
gation between PANINPs and MWNTs-COOH associated
with notable electrochemical performance of the produced
film. Moreover, MWNT-COOH/PANINP-modified ITO elec-
trode as sensing platform displayed synergistic effect to the
nifedipine electrochemical oxidation. The calibration curve
showed that the peak current was increased linearly with the
increase of NIF concentrations. Using such MWNT-COOH/
PANINP-modified ITO electrode as platform, this nanosensor
achieved a detection limit of 1,0×10–6 mol.L and a linear
range of 1.10×10–6 to1.10 ×10–4 mol.L [140].
Cefixime (CEF) is a semi-synthetic antibiotic and the third
generation of cephalosporin that is extensively used for the
treatment of a number of bacterial infections by preventing
the cell wall formation in bacteria as well as their growth
and proliferation [138, 163]. The spectrophotometry, fluorom-
etry, capillary electrophoresis, ultra-performance liquid
chromatography-tandem mass spectrometer (UPLC–MS/
Page 15 of 29 465
assay in different samples using CuWO4@PANI/GCE as a sensing
platform. Reprinted with the permission from Ponnaiah and
Periakaruppan 2018 [136]
MS),chemiluminescence, fluorescence, and electrochemistry
are techniques that used for antibiotics detection in biological
or aqueous samples [138]. In these methods, several steps
requisite for purification and preconcentration of antibiotics
such as solid phase microextraction (SPME), solid- phase ex-
traction (SPE), and liquid-liquid phase extraction (LLE).
Dehghani et.al. have recently introduced a novel electrochem-
ical sensor by using gold nanowires/graphene oxide/
electropolymerized molecular imprinted polymer in order to
determination of CEF. The molecularly imprinted electro-
chemical sensor displayed good sensitivity and selectivity in
quantification of CEF and obtained a linear concentration
range from 20.0 to 950.0 nM with a detection limit of 7.1
nM. The recovery percentages of CEF assay in the real sam-
ples (i.e. serum and urine) were measured between 97.8% and
102.9%. In fact, with the combination of suitable properties
PANI, graphene oxide, gold nanowires and the simultaneous
use of techniques MIP and electrochemical, this sensor exhib-
ited advantages such as simplicity of operation, high sensitiv-
ity, the decrease required sample amount, low cost, and short
response time [138].
Azithromycin (AZT) is a macrolide antibiotic that is ap-
plied to treat or prevent bacterial infections in humans such as
respiratory, urogenital, ear, pneumonia, bronchitis, sinusitis,
and typhoid [14]. Excessive use of AZT leads to the resistance
of bacteria strains to this antibiotic [14]. Jafari and his
465 Page 16 of 29
coworker designed a selective and sensitive electrochemical
nanosensor based on an aniline MIP film and gold nano ur-
chins (GNU) /graphene oxide-modified GCE for assaying
AZT antibiotic [14]. At the first, the GCE surface was modi-
fied by graphene oxide and GNU, all the steps of the GCE
modification were characterized by CV, EIS, SEM. For
forming AZT-aniline MIP film, in the presence of AZT as a
template, aniline monomers were electropolymerized on
GNU /graphene oxide-modified GCE by CV mode. Due to
the solubility of AZT in aqueous mediums, AZT molecules
can be extracted from polymer matrix hence pores known as
molecular imprints, were produced at the surface. The sensor
was dipped into AZT solutions with different concentrations
and final electrochemical signals were reached by DPV elec-
trochemical technique. The analytical performance of the sen-
sor was obtained in the linear range of 0.3 nM to920.0 nM
with the detection of limit 0.1 nM for AZT. The proposed
sensor was used for AZT detection in human serum. The
relative standard deviation for eight repetitive measurements
was found to be 2.5% that confirms that it may be an appro-
priate technique for the trace recognition of AZT in real sam-
ples. In compare with the regular spherical gold nanoparticles,
the gold nanourchines create higher surface area. This proper-
ty along with high conductivity of gold nanomaterials and also
graphene oxide can be a way to increase the sensitivity of the
offered biosensor. It can be concluded that the modification of
GCE surface by the MIP/GNU/ graphene oxide porous com-
posite via aniline electropolymerization enhance the electron
transfer, analytical signal, and sensitivity of the sensing plat-
form to AZT antibiotic [14]. Table 1 exhabits a comparative
study on the polyaniline- based nanobiosensors and their ap-
plications in the cilinical field.
Polyaniline-based nanosensors in the food
industry
Sulfite
Sulfites are inorganic salts generally used as a preservative,
antibacterial, antioxidant or reducing agent in the food and
pharmaceutical industries [164]. The accurate determination
of sulfite amount is very important because the intake extra
amount of sulfite can causes harmful side effects on human
health, such as asthma, hepatic steatosis and may enhance
transaminase activity in serum [165]. In 2014, Yang et al.
constructed an amperometric nanosensor for determination
of sulfite in real samples. This sensor was designed based on
PANI-coated copper hexacyanoferrate modified GCE
(PANI/CuHCF/GCE) that displayed higher electrocatalytic
activity and suitable stability toward the oxidation of sulfite
with detection limit 0.6 μmol·L-1 [141].
Microchim Acta (2019) 186: 465
Phenolic compounds
There are polyphenols and their compounds in different types
of plants as the result of secondary metabolisms production.
Due to abundant antioxidants properties, polyphenols can be
useful against a variety of diseases, particularly cardiovascular
disease and cancer [143, 144]. Furthermore, polyphenols have
antimicrobial property and can inhibit the reproduction and
growth of many microorganisms, such as bacteria, fungi,
yeasts, and viruses. In the food industry, the polyphenolic
components can be applied in food processing to increase
the shelf-life of foods [166]. Rawal et al. fabricated an enzy-
matic nanobiosensor for determination polyphenol using sil-
ver nanoparticles (AgNPs), MWCNT, and PANI. The AgNPs/
MWCNT/PANI composite was constructed through electro-
deposition of MWCNTs, aniline, and AgNPs on the Au elec-
trode surface. The purified laccase from Ganoderma sp. was
immobilized on the surface of AgNPs/MWCNT/PANI/Au
electrode via a covalent coupling. By using this sensor, total
phenolic content in tea, alcoholic beverages, and pharmaceu-
tical formulations was quantified. The presented polyphenol
biosensor was shown linear range, response time, and detec-
tion limit 0.1-500 μM, 6 s, and 0.1 μM, respectively [143]. In
2012, Chawla et al. designed an improved electrochemical
nanobiosensor for detection of phenolic compounds in fruit
juices. Laccase enzyme was covalently immobilized onto
nickel nanoparticles (NiNPs) MWCNT/PANI composite that
electrodeposited on the Au electrode surface. The results in-
dicated that the biosensor has presented an excellent sensitive
platform with a wide linear range 0.1-10 μM (lower concen-
tration range) and 10-500 μM (higher concentration range)
and a limit of detection of 0.05 mM [144].
Enzyme-based biosensing devices have been extensively
used in quantitative analysis of a variety of substrates over a
wide range of applications [167]. Enzymes have catalytic ac-
tivity and specific binding capacity; also, they can act fast [88,
(http://ocw.utm.my/file.php/74/CHAPTER_2_OCW.pdf)].
Therefore, the application of enzymes leads to improving the
sensitivity of biosensing systems. However there are some
disadvantages in using enzyme for construction biosensor
such as tend to lose activity after a relatively short period of
time (unless stored under appropriate conditions), the limited
stability, the dependency of activities on various factors such
as ionic strength, pH, temperature, and chemical inhibition,
the high cost of source and extracting, isolating, purifying
processes [(http://ocw.utm.my/file.php/74/CHAPTER_2_
OCW.pdf), 168].
Acrylamide
Acrylamide (AA) has strong neurotoxicity and genotoxicity
properties, and shows sever carcinogenicity potential in ani-
mals and human [145, 169]. This compound can induce point
Microchim Acta (2019) 186: 465 Page 17 of 29 465

Table 1 Summary of recent electrochemical nanobiosensor based on polyaniline and their applications in medicine

Clinical Biomarker Composition Electrochemical Detection limit References

applications method

Cancer PSA PANI/AuNP/ PNT Amp 0.68 ng. mL-1 [55]

PCa DNA PANI/ GO Amp 3.3×10-11μM [56]
PSA PANI nanowires/AuNP DPV 0,0006 ng. mL-1 [57]
CA125 AuNP-PB-PtNP/PANI SWV 4.4 mU. mL-1 [61]
HER2 NFG/AgNPs/PANI DPV 2 cells. mL-1 [58]
-1
AFP 3D- macroporous PANI DPV 0,0037 ng. mL [63]
AFP PEG/AuNP/PANI nanowires DPV 7×10-6 ng. mL-1 [64]
K562 cell CdS QDs/ PS as signal probe, GO/ PANI as platform ASV 3 cells. mL-1 [67]
Bacteria E. coli PANI/ AuNP CV 4 CFU.mL-1 [35]
E. coli O157: PANI/AuNp as platform and rGO-NR-Au@Pt as signal CV 2.84 ×103 [95]
H7 label CFU.mL-1.
N. gonorrhoeae PANI-nFe3O4-CNT DPV 1×10-13 μM [90]
M. tuberculosis rGO/AuNP as sensing platform and AuNP/PANI as DPV - [84]
tracer label
M. tuberculosis PANI-rGO/AuNP as redox detector probe DPV 5×10-8 μM [83]
M. tuberculosis graphene/PANI as a platform and Fe3O4-Au as label DPV 15 ng.mL-1 [87]
Glucose Glucose AuNP/PANI/ CC CV 3.08 μM [105]
Glucose GOx-SnO2@3D- Amp 0,0026 μM [106]
Glucose rGO@pPANI CA 0.24 μM [107]
Glucose CuO/PANI-NF Amp 0.14 μM [108]
Glucose NiCo2S4/rGO@PANI Amp 0.38 μM [137]
Uric acid Uric Acid c-MWCNT/PANI Amp 5 μM [110]
Uric Acid Fe3O4/ CHIT-g/ PANI Amp 0.1 μM [111]
Cholesterol Cholesterol NSPANI/AuNP/GR Amp - [121]
Drug Cholesterol PANI–Au–CH CV 378×103 ng.mL-1 [119]
Calycosin PANI/GQD DPV 0,98 μM [126]
5-Fluorouracil AgNP@PANINT DPV 0.06 μM [128]
Diclofenac PANI/rGO CV 11×105 ng.mL-1 [129]
Cefixime PANI/GO/AuNW DPV 0.007 μM [138]
Bezafibrate CNF/PANI/PtNP DPV 0.00246 μM [133]
Azithromycin PANI/GO/GNU DPV 0.0001 μM
Quercetin PANI/CuWO4 DPV 0.0012 μM [136]
Dexamethasone Fe3O4/PANI–CuII DPV 0.003 μM [139]
Nifedipine PANI/MWCNT CV 1 μM [140]

PSA, Prostate-specific antigen, PANI, polyaniline, AuNP, gold nanoparticle, PNT, peptide nanotube, Amp, amperometry, PCa, prostate cancer, GO,
graphene oxide, DPV, differential pulse voltammetry, CA125, carcinoma antigen 125, PB-PtNP, Prussian Blue platinum nanoparticles, SWV, square
wave voltammetry, HER2, human epidermal growth factor receptor 2, NFG, nitrogen-doped graphene, AgNPs, silver nanoparticles, AFP, Alpha-
fetoprotein, 3D,Three-dimensional, PEG, polyethylene glycols, CdS QDs, cadmium quantum dots, PS, polystyrene microspheres, ASV: anodic stripping
voltammetry, E.coli: Escherichia coli, CV: cyclic voltammetry, CFU, colony forming unit, rGO, reduced graphene oxide, NR: neutral red, Au@Pt:
gold@platinum nanoparticles, N. gonorrhea: Neisseria gonorrhea, Fe3O4, iron oxide nanoparticles, CNT, carbon nanotubes, CC, carbon cloth,
M. tuberculosis: Mycobacterium tuberculosis, pPANI, plasma polyaniline, CuO, copper oxide, PANI-NF, polyaniline nanofiber, CA:
chronoamperometry, cMWCNT, carboxylated multi walled carbon nanotubes, CHIT-g, chitosan graft, NSPANI: nanostructured polyaniline, GR,
graphene, CH, chitosan, GQD, graphene quantum dot, PANINT, polyaniline nanotube, AuNW, gold nanowires, CNF, carbon nanofibers, PtNP, platinum
nanoparticle, GNU, gold nano urchins, CuWO4,copper tungstate, CuII, copper, MWCNT, multi wall carbon nanotube

mutations, act as a Michael acceptor, and form adducts with
thiol, hydroxyl, and amino groups in DNA, that leads to DNA
damage [170]. Another consequence of human exposure to
AA is the formation of acrylamide/hemoglobin adduct
[171]. Despite of all these harmful effects, different levels of
this toxic compound are present in various food products
[170]. The mechanism leading to the formation of AA is
called Maillard reaction between reducing sugars (e.g. glucose
or reactive carbonyls) and proteins/amino acids (mainly aspar-
agine) [169, 172]. In fact, AA frequently generates in high-
465 Page 18 of 29
starch foods (potato crisps, cookies, and breads) during
cooking at high temperatures [169]. In 2013, Batra et al. de-
scribed a high sensitive electrochemical acrylamide biosensor
based on covalent immobilization of hemoglobin (Hb) onto
MWCNT/CuNPs/PANI composite on pencil graphite elec-
trode surface. The offered nanosensor has relied on the reac-
tion of Hb with acrylamide and generation of Hb-acrylamide
adducts that changes the electroactivity of Hb. There was an
inverse relationship between Hb-acrylamide adduct concen-
tration and the current peak. The biosensor showed a wide
linear range of 5 nM to 75 mM. The reported sensitivity,
response time and detection limit for this biosensor were
72.5 μA.nM-1.cm-2, low down of 2 s and 0.2 nM, respectively.
The results of this study showed that the simultaneous use of
unique features of metallic nanoparticles along with CNTs and
PANI played an important role in improving the analytical
performance of the biosensor (Fig. 8) [145].
Polyaniline-based nanosensors
for environmental health
Pesticides
The wide use of pesticides in agricultural is the cause of their
abundant existence in the environment, mainly in water and
soil. Chlorpyrifos (O, O-diethyl-O- (3, 5, 6-trichloro-2-pyri-
dyl)-phosphorothioate) is one of the most general
Fig. 8 Schematic representation
of the chemical reaction involved
in the fabrication of Hb/
cMWCNT/CuNP/PANI/PG
electrode. Reprinted with the
permission from Batra et al. 2013
[145]. The recent electrochemical
nanosensors based on polyaniline
and their applications in food
industry were mentioned in
Table 2
Microchim Acta (2019) 186: 465
organophosphate pesticides used in agriculture for control
pests and improve production. However, high toxicity level
of these compounds has raised public concern for food safety
and human health [146]. In 2013, Sun et al. fabricated a stable,
high sensitive, and low-cost immunosensor based on
AuNP/PANI/MWCNT/CH and GCE for detecting chlorpyri-
fos. The chemically-synthesized PANI/MWCNT/CH nano-
composite and AuNPs were adsorbed onto the electrode.
AuNPs were used as the linker and firmly adsorbed anti-
chlorpyrifos due to its good biocompatibility, large specific
surface area, and high surface free energy. In addition, the high
surface area and conductivity of MWCNTs increase the redox
properties of conducting polymers and facilitate electron
transfer between the redox center and the electrode surface.
The proposed immunosensor was used for evaluating real
samples including cabbage, pakchoi, lettuce, and leek that
were spiked by different concentrations of chlorpyrifos. The
results showed linear range from 0.1 to 40 × 10-6 mg.mL-1 and
from 40 × 10−6 mg.mL-1 to 500 × 10-6 mg.mL-1, and detection
limit of 0.06 × 10-6 mg.mL-1 (Fig. 9) [146].
Organophosphate compounds interfere with the appropri-
ate functioning of enzyme acetylcholinesterase (AChE) and
affect the central nervous system of human (CNS) [173].
Cesarino et al. employed PANI and MWCNTs to prepare a
sensitive electrochemical acetylcholinesterase (AChE) nano-
biosensor for recognition carbamate pesticides in fruit and
vegetables (apple, broccoli and cabbage). PANI/MWCNT
core–shell film was electrochemically formed on the GCE
Microchim Acta (2019) 186: 465 Page 19 of 29 465

Table 2 Summary of recent electrochemical nanosensor based on polyaniline and their applications in food industry

Food applications Biomarker Composition Electrochemical method Detection limit References

Sulfite Sulfite PANI/CuHCF Amp 0.6 μM [141]

Sulfite AuNP/CHIT/cMWCNT/PANI Amp 0.5 μM [142]
Phenolic compounds Polyphenol AgNP/MWCNT/PANI Amp 0.1 μM [143]
Polyphenol NiNP/ MWCNT/PANI Amp 50 μM [144]
Acrylamide Acrylamide MWCNT/CuNP/PANI DPV 0.0002 μM [145]

PANI, polyaniline, CuHCF, copper hexacyanoferrate, Amp, amperometry, AuNP, gold nanoparticle, CHIT, chitosan, cMWCNT, carboxylated multi-
walled carbon nanotubes, AgNP, silver nanoparticles, MWCNT: carboxylated multi-walled carbon nanotubes, NiNP, nickel nanoparticles, CuNP, copper
nanoparticles, DPV, differential pulse voltammetry

surface via CV method. Acetylcholinesterase enzyme was
then fixed onto PANI/MWCNT/GCE. The amount of
methomyl and carbaryl in liquefied apple, broccoli and cab-
bage samples was finally examined by chronoamperometry.
The detection limit of this biosensor for carbaryl and
methomyl were obtained 1.4 and 0.95 μmol. L-1, respectively.
The achieved results were in complete agreement with those
from the HPLC technique [174].
Malathion (S-bis (etoxycarbonyl) ethyl O,O-dimetyl
phosphorodithioate) is a broad spectrum, non-systemic insecti-
cide that belonging to the organophosphorus (OP) class
pesticide[175]. The toxicity of this component differs from
other organophosphorus species. It has a relatively low toxicity
to mammals and birds, but it has high toxicity to aquatic organ-
isms and bees [175, 176]. There are various methods for de-
tecting and monitoring amount of malathion such as
colorimetric, high performance liquid chromatography
(HPLC), Fourier transform–Raman (FT–Raman) spectrometry,
gas chromatography-mass spectrometry (GC-MS), and capil-
lary electrophoresis. However, the above-mentioned tech-
niques are sensitive, reliable and efficient, but they have limi-
tations such as requiring expensive and bulky instrumentation,
complex sample preparation and pretreatment processes, well-
trained personnel and, high volumes of toxic reagents and long
analysis time [175, 177]. Therefore, most researches focused
on developing the electrochemical biosensors for fast recogni-
tion malathion employing acetylcholinesterase (AChE) en-
zyme, the function of this type of biosensors is based on the
irreversible inactivation of enzymatic activity [175].
In 2018, He et al. designed a novel electrochemical biosen-
sor by anchoring enzyme (AChE) onto the core-shell nano-
structured composite of hollow carbon spheres covered with

Fig. 9 Schematic illustration of the stepwise immunosensor fabrication process based on AuNPs/PANI/MWCNTs/ CH and glassy carbon (GC) electrode
for detecting chlorpyrifos. Reprinted with the permission from Sun et al. 2013 [146]
465 Page 20 of 29
needle-like polyaniline (HCS@PANI) for rapid recognition of
malathion. After synthesizing HCS@PANI, the (AChE) en-
zyme was absorbed onto this nanocomposite. The electro-
chemical catalysis of AChE/HCS@PANI-based biosensor to-
wards acetylthiocholine chloride (ATCl) was inhibited in the
presence of malathion, that this phenomenon was applied to
distinguish malathion. Using such a nanocomposite platform
and AChE, this nano-electrochemical biosensor achieved a
low detection limit of0.16 pg·mL-1 and a linear range of 1.0
ng·mL-1 to 10 μg·mL-1. It can be concluded the use of nano-
composite HCS@PANI only demonstrations high specific
surface area and chemical functionality but also improves
electrochemical activity. In addition, the HCS@PANI nano-
composite can act as a suitable layer for the AChE immobili-
zation and catalysis of ATCl has a strong potential application
in the high-sensitive detection of pesticide [147].
Fenitrothion, [O, O-dimethyl O-(3-methyl-4-nitrophenyl)
phosphorothioate] is another (OP) pesticide that broadly used
in pre-harvest treatment in a variety of crops, horticulture, and
forestry[148, 178]. Moreover, it is also employed against
some diseases such as malaria [148]. The degradation of this
pesticide is associated with producing some of the secondary
metabolites such as fenitrooxon and 3-methyl-4-nitrophenol.
These compounds are toxic and partially water-soluble [148].
Khan et al. constructed a sensitive and selective electro-
chemical sensor on PANI/AgNP/MWCNT- modified GCE
for examining and quantifying fenitrothion in surfactant media.
The sensor based on PANI/AgNP/MWCNT composite re-
vealed a linear range from 0.1 to 1.5 μg. mL-1 and 0.1 to5.67
μg. mL-1 with low detection limits of 0.000265 μg. mL-1 and
0.000193 by CV and adsorptive differential pulse stripping
voltammetry (AdDPSV) methods, respectively [148].
Nitrophenols (NPs) are a class of nitro compounds that wide-
ly used in the production of explosives, pesticides, dyes, and
pharmaceuticals [149, 179]. Typically, Nitrophenol derivatives
are formed via photochemical reactions in the presence of nitro-
gen oxides [150]. These compounds are widely distributed in
nature such as in the soil and water environment, during their
production and application [150, 179]. Due to the characteristics
of strong bio-refractory and their toxic effect on humans, ani-
mals, aquatics, and plants, the quantification and determination
of NPs have attracted significant interest [150, 179].
In 2017, Zhang et al. developed an electrochemical sensor
by decorating of silver nanowire-polyaniline (AgNW/PANI)
composite onto GCE for 4- NP detection. This sensing meth-
od can detect 4- NP with the concentration range from 0.6 to
32 μM and the limit of detection of 52 nM (S/N = 3). The
sensor was applied to detect the 4-NP in real water samples.
The results indicated that designed sensor is effective for4-NP
determination in the real water samples [149].
p-Nitrophenol ( p- NP) is one of the member of nitrophenol
family that is easily adsorbed through the skin and mucous
membranes and has mutagenic, carcinogenic effect on
Microchim Acta (2019) 186: 465
mammals cells[150]. Using PANI/GO nanocomposite as plat-
form and ammonium persulfate as the initiator, a molecularly
imprinted polymer (MIP) electrochemical sensor was de-
signed for quantitative detection of p- NP by Saadati et al.
Under the optimized experimental conditions, sensor showed
linearly concentration range 6 ×10 −5 –14 ×10 −5 mol.L-1 with
a limit of detection 2 ×10 −5 mol.L-1 (correlation coefficient of
0.991). Moreover, this nonosensor presented good selectivity
for p-NP in the presence of related phenol compounds such as
4-bromophenol, 2,4- dinitrophenol, 2-nitrophenol and 4-
chlorophenol [150].
Acetamiprid belongs to the class of neonicotinoid pesti-
cides [151, 180]. It is one of the synthetic derivatives of nic-
otine that acts as a neuro-active agent and nicotine
acetylcoline receptor antagonist which causes paralysis and
death [151, 180]. In order to assaying of acetamiprid, Rapini
et al. fabricated a portable and sensitive electrochemical
aptasensor with employing PANI film and AuNPs onto graph-
ite screen-printed electrode (SPGE) surface. PANI/AuNP
nanocomposite firstly was electrodeposited on the electrode
surface by CV method. Then, thiol-tethered DNA aptamers
were immobilized onto SPGE surface. After the addition of
the different acetamiprid solutions containing a fixed amount
of biotin-labeled complementary oligonucleotide sequence,
streptavidin conjugated to alkaline phosphatase was dropped
onto the electrode prior to electrochemical analysis. The final
detection was done by DPV measurements, which was based
on enzymatic reaction performance and the hydrolysis reac-
tion of 1-naphthyl phosphate to 1-naphthol on the SPGE sur-
face. When the acetamiprid concentration was enhanced, a
decline of the signal was showed, making the aptamer sensor
act as signal off sensor. In this offered sensor, a dose-response
plot was obtained between 0.25 and 2.0 mM acetamiprid con-
centration range. Under optimal condition, the detection limit
of sensor was calculated to be 0.086 mM [151].
Despite aptamer-based biosensors have high sensitivity
and request to a smaller volume of the sample for function,
the lack of high-quality aptamers for important targets is one
of the most limitations of aptamer-based biosensors[181].
Atrazine (ATZ) (2-chloro-4-ethylamino-6-isopropylamino-
s-triazine) is another pesticide in agriculture that widely
employed for control weeds in the production of corn and
other agricultural crops as well as in non-cropland situations
[153, 154, 182]. Atrazine displays its phytotoxic effects via
binding to quinine-binding proteins, suppressing electron
transport, destruction of the chloroplast, inhibition of tissue
respiration, decreasing the carbohydrate amount, changes in
the enzyme activity, and blocking photosynthesis of the weeds
[153, 182]. Due to its high stability, degradation with a low
rate, and good solubility, atrazine is generally found in soil,
water, and agriculture products [183, 184]. Recently, it was
reported that this herbicide may have harmful effects on the
environment, the aquatic organisms, and even the human
Microchim Acta (2019) 186: 465
health [153]. In 2010, Hayes and his coworkers presented that
ATZ is a strong endocrine disruptor and can convert male
frogs into female frogs at relatively low concentration (0.1
ppb) [153, 185]. Also, ATZ has dangerous effects on human’s
reproductive system, central nervous system, endocrine sys-
tem, and the immune system and causes different types of
cancers, birth defects, stoppage of hormone acts, and poor
semen quality in men [153, 182, 184]. Therefore, the devel-
opment of fast and high sensitive assaying systems is essential
for monitoring the presence and quantity of ATZ and preven-
tion of toxic risks [182]. In 2015 Van Chuc et al. developed a
label-free electrochemical immunosensor on PANI/ graphene-
modified Pt electrode for ATZ detection. The PANI monomers
were electrodeposited onto Pt working electrode by CV mode.
The graphene film was then prepared by CVD method and
transferred to PANI / Pt working electrode by polymethyl
methacrylate (PMMA) as sacrificial layer. The anti-ATZ (α-
ATZ) was immobilized onto PANI/ graphene film using glu-
taraldehyde as a cross linker. After adding ATZ sample, the
electrochemical determination was done via square wave volt-
ammetry (SWV) technique. This immunosensor achieved a
detection limit of (˜43 ×10-12 g.L-1) and a linear range of
10-11 to 10-7 mol.L-1. The layer-by-layer assembling improves
the electrochemical performance of ATZ sensor via more con-
trol on the nanostructured films behaviors [153]. Also, Deep
et al. proposed a PANI based immuno nanosensor for highly
sensitive impedimetric recognizing of ATZ. Styrene sulphonic
acid (SSA) doped PANI was chemically synthesized. In order
to obtain the functional -NH2 terminal, the SSA-PANI struc-
ture was treated with 3-(aminopropyl) triethoxysilane
(APTES). The functional nanopolymer was electrodeposited
on the surface of SPCE. Then, the SSA/PANI- modified SPCE
was incubated with anti-ATZ antibody and the pesticide
immuno nanosensor was formed. This sensing method exhib-
ited a wide dynamic range from 0.01 to 50 ng.mL-1 along with
a limit of detection of 0.01 ng.mL-1. This work proposed that
PANI alone can be used for designing immunosensing plat-
form and specific and highly sensitive detection of ATZ [154].
Heavy metals
Heavy metals are important environmental pollutants with
toxic effects on soil microorganism through the change in
the diversity, population size, and biological activities of the
microbial communities. Heavy metals are absorbed by the
plants and subsequently accumulated in the food chain, which
is a risk factor for human health [186]. Mercury (Hg), lead
(Pb), arsenic (As), cadmium (Cd), nickel (Ni), copper (Cu),
chromium (Cr), zinc (Zn), silver (Ag), and selenium (Se) are
among the most frequent pollutant heavy metals [187].
Different analytical methods, such as atomic absorption spec-
trometry, inductively coupled plasma-optical mass spectrom-
etry (ICP-MS), capillary electrophoresis, and X-ray
Page 21 of 29 465
fluorescence spectrometry has been used for the recognition
of heavy metal ions [155, 188]. However, these techniques are
time-consuming and need complex and expensive equipment
and well-trained personnel to perform the operational proce-
dures [188]. Yang et al. fabricated an electrochemical nano-
biosensor based on 3D-rGO/PANI composite as the sensitive
layer for detecting Hg2+ in aqueous solution. The T-rich DNA
strands were immobilized onto electrode surface by amino-
group-rich 3D-rGO/PANI that preferred to bind with Hg2+
and formed T-Hg2+-T coordination. This sensor exhibited high
sensitivity, selectivity and repeatability for Hg2+ in a concen-
tration range from 0.1 nM to 100 nM with LOD of 0.035 nM.
The authors claimed that rGO/PANI composite improved the
specific surface area, electrical conductivity, and electrochem-
ical performance of the presented biosensor (Fig. 10a) [155].
In another research by Tang et al., an ultrasensitive and
reusable electrochemical DNA biosensor has been developed
onto GCE for Hg2+ detection using mesoporous carbon
(OMC)/self-doped polyaniline nanofiber (SPAN)/AuNP com-
posite as a platform and DNA anionic intercalated as signal
indicator. As the Figure 10B demonstrates, the GCE surface
was first modified with SPAN nanofibers and OMC by layer-
by-layer self-assembly. Then, AuNPs were electrodeposited on
the SPAN/OMC- modified electrode surface. The capture
DNA probes were immobilized on the surface of modified
electrode via the stable gold- sulfide band (Au-S). For remov-
ing non-specific DNA adsorption, the modified-GCE was treat-
ment by 6-mercapto-1-hexanol (MCH). In the presence of
Hg2+ ions, the ssDNA probes were folded into hairpin structure
by thymine- thymine (T-T) mismatch with Hg2+. Finally, the
AQDS was used as the electroactive signal indicator. Dou to
Hg2+ was easily separated from the T-Hg2+-T complexes, the
biosensor was immersed into cysteine solution for the forma-
tion bond between Hg2+and cysteine. This bond is more con-
stant than T-Hg2+-T structure and causes to recovery of free-
state single-stranded oligonucleotides from hairpin structure. A
relatively wide dynamic range from 10 fM to 1 μM along with
a limit of detection of 0.6 fM (S/N = 3) have been reported for
this DNA electrochemical sensor [156] (Fig. 10b).
In electrochemical biosensors based on DNA, the buffered
solution may highly interfere in the reaction, this is a draw-
back of electrochemical DNA biosensors [189].
In 2014, Dong et al. prepared the GNPs/PANI/GR ter-
nary nanocomposite on GCE surface to construct a sensi-
tive biosensor for determination of Pb 2+ . The sensor
reached a linear range of 0.5 to 10 nM and LOD of 0.1
nM. Compared to the GNP/GCE, the PANI/GCE, and the
GR/GCE, the ternary nanocomposite showed higher sen-
sitivity for the recognition of trace Pb2+ in aqueous solu-
tion. The result indicated that ternary nanocomposite can
be used for heavy metal analysis due to its effective char-
acteristics, such as good electric conductivity and appar-
ent synergistic effect for the metal ion detection [188]. In
465 Page 22 of 29
Fig. 10 Examples of PANI/nanoparticles based biosensor for sensitive
detection of Hg2+. (A) Schematic diagram of the detection of Hg2+ ions
using the developed electrochemical DNA biosensor based on 3D-
rGO@PANI nanocomposite. Reprinted with the permission from Yang
et al. 2015 [155]. (B) Schematic illustration of a DNA nanobiosensor for
2017, Barrosa et al. employed of AuNPs, emeraldine salt
polyaniline (PANI-ES) and sodium montmorillonite clay
mineral (Na+-MMT) for detecting of Cd2+, Pb2+ and Cu2+
ions simultaneously. The sensor achieved low detection
limits of 0.001, 0, 009 and 0,02 μg. L-1 for Cd2+, Pb2+
and Cu2+ ions, respectively [158].
In 2018, Kong et al. fabricated an easy-to-use electrochem-
ical sensor using core-shell ferroferric oxide@polyaniline
(Fe3O4@PANI) nanoparticles as a sensitive platform for the
quantitative detection of Pb2+ and Cd2+ ions. Under the opti-
mal experimental conditions, the linear ranges from 0.1 to 104
and 1.0 to 0.9×104 nmol.L-1 with low detection limits of 0.03
and 0.3 nmol.L-1 were obtained for detecting Pb2+ and Cd2+
ions, respectively [159]. In the same year, Deshmukh and et al.
developed an electrochemical sensor based on ethylenedi-
aminetetraacetic acid (EDTA) -modified PANI/SWNT nano-
composite onto stainless steel electrode (SSE) for detecting
Cu2+ ions concentration. EDTA is an agent that has chelation
ability. Therefore, it can provide advanced selectivity and sen-
sitivity towards metal ions. Interaction of Cu2+ with EDTA-
PANI/SWNT-modified SSE was studied by electrochemical
DPV technique. The sensor exhibited a broad linear range of
2 mM to 4 μM and limit of detection of 1.4 μM [160]. The
Microchim Acta (2019) 186: 465
Hg2+ ions quantification by using mesoporous carbon (OMC)/self-doped
polyaniline nanofiber (SPAN)/AuNP composite as a platform and DNA
anionic intercalator as signal indicator. Reprinted with the permission
from Tang et al. 2017 [156]
nanosensors based on polyaniline and their applications to
enviromental assays were displayed in Table 3.
Conclusion
Applications of PANI polymer in biosensors and nanobiosen-
sors have been reviewed in this work. Recent advancements in
this field were listed and evaluated carefully and both advan-
tages and drawbacks were discussed for sensing devices in
various fields such as clinical diagnosis, food industry, envi-
ronmental monitoring. In fact, these biosensors are promising
alternatives for routine analysis methods due to their diverse
and flexible structure. PANI is a CP that attracted significant
attention as a suitable substrate for immobilization biological-
components in fabrication biosensors. Furthermore, it acts as
an effective mediator for electron transfer in redox or enzy-
matic reactions. Numerous studies have indicated that the
combination of nanomaterials and PANI in a biosensing con-
struct can increase the surface area, conductivity, and stability
of the sensing platform. Such devices have been showing
great potentials for obtaining specific and sensitive detection
of various analytes valuable in medicine, food industry, and
Microchim Acta (2019) 186: 465 Page 23 of 29 465

Table 3 Summary of recent electrochemical nanosensor based on polyaniline and their applications to environmental assays

Applications Biomarker Composition Electrochemical method Detection limit References

Pesticides Chlorpyrifos AuNP/PANI/MWCNT/CH CV 6 × 10 -2 μg.L-1 [146]

Malathion HCS@PANI CV 16×10 -5 μg.L-1 [147]
Fenitrothion PANI/AgNP/MWCNT CV and AdDPSV 0.000265 and 0, 193 μg.L-1 [148]
4-NP AgNW/PANI DPV 0.052 μM [149]
p- NP PANI/GO CV 20 μM [150]
Acetamiprid PANI /AuNP DPV 86 μM [151]
Profenofos PANI/AuNP DPV 0.27 μM [152]
˜43 ×10 μg.L
-6 -1
ATZ PANI/ graphene SWV [153]
ATZ SSA/PANI EIS 10 μg. L
-2 -1
[154]
Heavy metals Hg 3D-rGO/PANI EIS 35 μM [155]
Hg SPAN/OMC/AuNP DPV 6×10 -10 μM [156]
As AuNP/PANI CV and ASV 0.4 μg. L-1 [157]
Cd, Pb ,Cu PANI-ES/Na+-MMT/AuNP DPV 0.001 μg. L-1 for Cd , 0, 009 μg. L-1 [158]
for Pb and 0,02 μg. L-1 for Cu
Pb and Cd Fe3O4@PANI DPV 3 × 10 -5 μM for Pb and 3 × 10 -4 μM for Cd [159]
Cu PANI/SWNT DPV 1.4 μM [160]

PANI, polyaniline, AuNP, gold nanoparticle, cMWCNT, multi-walled carbon nanotubes, CH, chitosan, CV, cyclic voltammetry, HCS@PANI, hollow
carbon spheres covered with needle-like polyaniline, AgNP, silver nanoparticles, 4-NP, 4-Nitrophenol, AgNW, silver nanowire, DPV, differential pulse
voltammetry, ATZ, Atrazine, SWV, square wave voltammetry, SSA, Styrene sulphonic acid, Hg, mercury, 3D-rGO, Three-dimensional-reduced
graphene oxide, EIS, electrochemical impedance spectroscopy, SPAN, self-doped polyaniline nanofiber, OMC, mesoporous carbon, As, arsenic,
ASV, Anodic stripping voltammetry, Cd, cadmium, Pb, lead, Cu, copper, PANI-ES, emeraldine salt polyaniline, Na+ -MMT, sodium montmorillonite
clay mineral, Fe3O4: ferroferric oxide, SWNT, single walled carbon nanotubes.

environmental monitoring. In this way, the main challenge is
to choose a suitable nanoparticle and maybe other polymers to
make the nanocomposite with PANI. Another challenge is to
mix them as it should be to get the best results in nanobiosen-
sors, especially with cost-effective and fast methods. Same to
most of the electrochemical systems, the PANI-based electro-
chemical biosensors does have some minor problems such as
difficult mass production for commercialization and also rel-
atively low repeatability of the fabricating process compared
to other commercialized methods. In addition, low stability of
the biological components (DNA, Antibody, Aptamer, etc.) in
different environment and temperatures, as well as need of
portability can be a hard step for their commercialization. In
future, it is expected to see more novel nanoparticles and
nanostructures to be combined with PANI for application in
nanobiosensors to enhance the sensitivity.
Compliance with ethical standards The author(s) declare that
they have no competing interests.
References
1. Šefčovičová J, Tkac J (2015) Application of nanomaterials in
microbial-cell biosensor constructions. Chemical Papers 69:42–53
2. Paniel N, Baudart J (2013) Colorimetric and electrochemical
genosensors for the detection of Escherichia coli DNA without
amplification in seawater. Talanta 115:133–142
3. Farka Zk, Juřík Ts, Kovář D, Trnková Le, Skládal P (2017)
Nanoparticle-based immunochemical biosensors and assays: re-
cent advances and challenges. Chem Rev 117:9973–10042
4. Kissinger PT (2005) Biosensors—a perspective. Biosens
Bioelectron 20:2512–2516
5. Nagel B, Dellweg H, Gierasch L (1992) Glossary for chemists of
terms used in biotechnology (IUPAC Recommendations 1992).
Pure Appl Chem 64:143–168
6. Topkaya SN, Azimzadeh M (2016) Biosensors of in vitro detec-
tion of cancer and bacterial cells. In: Nanobiosensors for
Personalized and Onsite Biomedical Diagnosis. Institution of
Engineering and Technology, pp 73–94
7. Topkaya SN, Azimzadeh M, Ozsoz M (2016) Electrochemical
biosensors for cancer biomarkers detection: Recent advances and
challenges. Electroanalysis:n/a–n/a
8. Mahshid SS, Camiré S, Ricci F, Vallée-Bélisle A (2015) A
highly selective electrochemical dna-based sensor that em-
ploys steric hindrance effects to detect proteins directly in
whole blood. J Am Chem Soc 137:15596–15599
9. Omidfar K, Darzianiazizi M, Ahmadi A, Daneshpour M, Shirazi
H (2015) A high sensitive electrochemical nanoimmunosensor
based on Fe3O4/TMC/Au nanocomposite and PT-modified elec-
trode for the detection of cancer biomarker epidermal growth fac-
tor receptor. Sens Actuators B 220:1311–1319
10. Aydemir N, Malmström J, Travas-Sejdic J (2016) Conducting
polymer based electrochemical biosensors. Phys Chem Chem
Phys 18:8264–8277
11. Gerard M, Chaubey A, Malhotra B (2002) Application of
conducting polymers to biosensors. Biosens Bioelectron 17:
345–359
12. Holzinger M, Le Goff A, Cosnier S (2014) Nanomaterials for
biosensing applications: a review. Front Chem 2:63
465 Page 24 of 29
13. Daneshpour M, moradi LS, Izadi P, Omidfar K (2016)
Femtomolar level detection of RASSF1A tumor suppressor gene
methylation by electrochemical nano-genosensor based on
Fe3O4/TMC/Au nanocomposite and PT-modified electrode.
Biosens Bioelectron 77:1095–1103
14. Jafari S, Dehghani M, Nasirizadeh N, Azimzadeh M (2018) An
azithromycin electrochemical sensor based on an aniline MIP film
electropolymerized on a gold nano urchins/graphene oxide mod-
ified glassy carbon electrode. J Electroanal Chem 829:27–34
15. Park CS, Lee C, Kwon OS (2016) Conducting polymer based
nanobiosensors. Polymers 8:249
16. Barsan MM, Ghica ME, Brett CM (2015) Electrochemical sensors
and biosensors based on redox polymer/carbon nanotube modified
electrodes: a review. Anal Chim Acta 881:1–23
17. Arshak K, Velusamy V, Korostynska O, Oliwa-Stasiak K, Adley C
(2009) Conducting polymers and their applications to biosensors:
emphasizing on foodborne pathogen detection. IEEE Sensors
Journal 9:1942–1951
18. Dhand C, Das M, Datta M, Malhotra B (2011) Recent advances in
polyaniline based biosensors. Biosens Bioelectron 26:2811–2821
19. Chowdhury AD, Gangopadhyay R, De A (2014) Highly sensitive
electrochemical biosensor for glucose, DNA and protein using
gold-polyaniline nanocomposites as a common matrix. Sens
Actuators B 190:348–356
20. Spain E, Keyes TE, Forster RJ (2013) Vapour phase polymerised
polyaniline–gold nanoparticle composites for DNA detection. J
Electroanal Chem 711:38–44
21. Azimzadeh M, Nasirizadeh N, Rahaie M, Naderi-Manesh H
(2017) Early detection of Alzheimer's disease using a biosensor
based on electrochemically-reduced graphene oxide and gold
nanowires for the quantification of serum microRNA-137. RSC
Adv 7:55709–55719
22. Shrivastava S, Jadon N, Jain R (2016) Next-generation polymer
nanocomposite-based electrochemical sensors and biosensors: A
review. TrAC Trends Anal Chem 82:55–67
23. Shoaie N, Forouzandeh M, Omidfar K (2018) Highly Sensitive
Electrochemical Biosensor Based on Polyaniline and Gold
Nanoparticles for DNA Detection. IEEE Sensors Journal 18:
1835–1843
24. Ćirić-Marjanović G (2013) Recent advances in polyaniline re-
search: Polymerization mechanisms, structural aspects, properties
and applications. Synth Met 177:1–47
25. Hun X, Li Y, Wang S, Li Y, Zhao J, Zhang H, Luo X (2018)
Photoelectrochemical platform for cancer cell glutathione detec-
tion based on polyaniline and nanoMoS2 composites modified
gold electrode. Biosens Bioelectron 112:93–99
26. Xu Q, Leng J, Li H-b, Lu G-j, Wang Y, Hu X-Y (2010) The
preparation of polyaniline/gold nanocomposites by self-assembly
and their electrochemical applications. React Funct Polym 70:
663–668
27. Chang H, Yuan Y, Shi N, Guan Y (2007) Electrochemical DNA
biosensor based on conducting polyaniline nanotube array. Anal
Chem 79:5111–5115
28. Kang Z, Jiao K, Xu X, Peng R, Jiao S, Hu Z (2017) Graphene
oxide-supported carbon nanofiber-like network derived from
polyaniline: A novel composite for enhanced glucose oxidase
bioelectrode performance. Biosens Bioelectron 96:367–372
29. Dhand C, Dwivedi N, Mishra S, Solanki P, Mayandi V, Beuerman
R, Ramarishna S, Lakshminarayanan R, Malhotra B (2015)
Polyaniline-based biosensors. Nanobiosensors in Disease
Diagnosis 4:25–46
30. Mallick K, Witcomb MJ, Scurrell MS (2006) Gold in polyaniline:
recent trends. Gold Bulletin 39:166–174
31. Saberi RS, Shahrokhian S, Marrazza G (2013) Amplified electro-
chemical DNA sensor based on polyaniline film and gold nano-
particles. Electroanalysis 25:1373–1380
Microchim Acta (2019) 186: 465
32. Arora K, Prabhakar N, Chand S, Malhotra B (2007) Ultrasensitive
DNA hybridization biosensor based on polyaniline. Biosens
Bioelectron 23:613–620
33. Mickova I, Prusi A, Grčev T, Arsov L (2006) Electrochemical
polymerization of aniline in presence of TiO2 nanoparticles.
Maced J Chem Chem Eng 25:45–50
34. Tahir ZM, Alocilja EC, Grooms DL (2005) Polyaniline synthesis
and its biosensor application. Biosens Bioelectron 20:1690–1695
35. Shoaie N, Forouzandeh M, Omidfar K (2018) Voltammetric de-
termination of the Escherichia coli DNA using a screen-printed
carbon electrode modified with polyaniline and gold nanoparti-
cles. Microchimica Acta 185:217
36. Sen T, Mishra S, Shimpi NG (2016) Synthesis and sensing appli-
cations of polyaniline nanocomposites: a review. RSC Adv 6:
42196–42222
37. Wang J, Li Y, Li C, Zeng X, Tang W, Chen X (2017) A
voltammetric study on the interaction between isoproterenol and
cardiomyocyte DNA by using a glassy carbon electrode modified
with carbon nanotubes, polyaniline and gold nanoparticles.
Microchim. Acta 184:2999–3006
38. Zhang X, Lai G, Yu A, Zhang H (2013) A glassy carbon electrode
modified with a polyaniline doped with silicotungstic acid and
carbon nanotubes for the sensitive amperometric determination
of ascorbic acid. Microchim. Acta 180:437–443
39. Syedmoradi L, Daneshpour M, Alvandipour M, Gomez FA,
Hajghassem H, Omidfar K (2017) Point of care testing: The im-
pact of nanotechnology. Biosens Bioelectron 87:373–387
40. Mahshid SS, Mahshid S, Dolati A, Ghorbani M, Yang L, Luo S,
Cai Q (2011) Template-based electrodeposition of Pt/Ni nano-
wires and its catalytic activity towards glucose oxidation.
Electrochim Acta 58:551–555
41. Hasanzadeh M, Shadjou N (2017) Advanced nanomaterials for
use in electrochemical and optical immunoassays of
carcinoembryonic antigen. A review. Microchim. Acta 184:389–
414
42. Lai J, Yi Y, Zhu P, Shen J, Wu K, Zhang L, Liu J (2016)
Polyaniline-based glucose biosensor: A review. J Electroanal
Chem 782:138–153
43. Yazdanparast S, Benvidi A, Banaei M, Nikukar H, Tezerjani MD,
Azimzadeh M (2018) Dual-aptamer based electrochemical sand-
wich biosensor for MCF-7 human breast cancer cells using silver
nanoparticle labels and a poly(glutamic acid)/MWNT nanocom-
posite. Mikrochim Acta 185:405
44. Azimzadeh M, Rahaie M, Nasirizadeh N, Ashtari K, Naderi-
Manesh H (2016) An electrochemical nanobiosensor for plasma
miRNA-155, based on graphene oxide and gold nanorod, for early
detection of breast cancer. Biosens Bioelectron 77:99–106
45. Mahshid S, Li C, Mahshid SS, Askari M, Dolati A, Yang L, Luo S,
Cai Q (2011) Sensitive determination of dopamine in the presence
of uric acid and ascorbic acid using TiO2 nanotubes modified with
Pd, Pt and Au nanoparticles. Analyst 136:2322–2329
46. Ghazizadeh E, Naseri Z, Jaafari MR, Forozandeh-Moghadam M,
Hosseinkhani S (2018) A fires novel report of exosomal electro-
chemical sensor for sensing micro RNAs by using multi covalent
attachment p19 with high sensitivity. Biosens Bioelectron 113:74–81
47. Chandra P (2015) Electrochemical nanobiosensors for cancer di-
agnosis. J Anal Bioanal Tech 6:1000e119
48. Siegel RL, Miller KD, Jemal A (2017) Cancer statistics, 2017. CA
Cancer J Clin 67:7–30
49. Wu L, Qu X (2015) Cancer biomarker detection: recent achieve-
ments and challenges. Chem Soc Rev 44:2963–2997
50. Keshavarz M, Behpour M, Rafiee-pour H-A (2015) Recent trends
in electrochemical microRNA biosensors for early detection of
cancer. RSC Adv 5:35651–35660
Microchim Acta (2019) 186: 465
51. Smith RA, Cokkinides V, Eyre HJ (2006) American Cancer
Society guidelines for the early detection of cancer, 2006. CA
Cancer J Clin 56:11–25
52. Selvolini G, Marrazza G (2017) MIP-based sensors: Promising
new tools for cancer biomarker determination. Sensors 17:718
53. Hui W, Tian W, Yan-Xia Y, Zhang Y-X, Pei-Hui Y, Huai-Hong C,
Ji-Ye C (2012) Construction of an electrochemical cytosensor
based on polyaniline nanofiber/gold nanoparticle interface and
application to detection of cancer cells. Chin J Anal Chem 40:
184–190
54. Damborska D, Bertok T, Dosekova E, Holazova A, Lorencova L,
Kasak P, Tkac J (2017) Nanomaterial-based biosensors for detec-
tion of prostate specific antigen. Microchimica Acta 184:3049–
3067
55. Vural T, Yaman YT, Ozturk S, Abaci S, Denkbas EB (2018)
Electrochemical immunoassay for detection of prostate specific
antigen based on peptide nanotube-gold nanoparticle-polyaniline
immobilized pencil graphite electrode. J Colloid Interface Sci 510:
318–326
56. Tezerjani MD, Benvidi A, Rezaeinasab M, Jahanbani S,
Moshtaghioun SM, Youssefi M, Zarrini K (2016) An
impedimeric biosensor based on a composite of graphene nano-
sheets and polyaniline as a suitable platform for prostate cancer
sensing. Anal Methods 8:7507–7515
57. Dey A, Kaushik A, Arya SK, Bhansali S (2012) Mediator free
highly sensitive polyaniline–gold hybrid nanocomposite based
immunosensor for prostate-specific antigen (PSA) detection. J
Mater Chem 22:14763–14772
58. Salahandish R, Ghaffarinejad A, Naghib SM, Majidzadeh-A K,
Zargartalebi H, Sanati-Nezhad A (2018) Nano-biosensor for high-
ly sensitive detection of HER2 positive breast cancer. Biosens
Bioelectron 117:104–111
59. Webb PM, Jordan SJ (2017) Epidemiology of epithelial ovarian
cancer. Best Pract Res Clin Obstet Gynaecol 41:3–14
60. Reid BM, Permuth JB, Sellers TA (2017) Epidemiology of ovar-
ian cancer: a review. Cancer Biol Med 14:9
61. Zheng Y, Wang H, Ma Z (2017) A nanocomposite containing
Prussian Blue, platinum nanoparticles and polyaniline for multi-
amplification of the signal of voltammetric immunosensors: high-
ly sensitive detection of carcinoma antigen 125. Microchimica
Acta 184:4269–4277
62. Wang X, Wang Q (2018) Alpha-Fetoprotein and hepatocellular
carcinoma immunity. Can J Gastroenterol Hepatol 2018 Article
ID 9049252:8
63. Liu S, Ma Y, Cui M, Luo X (2018) Enhanced electrochemical
biosensing of alpha-fetoprotein based on three-dimensional
macroporous conducting polymer polyaniline. Sens Actuators B
255:2568–2574
64. Hui N, Sun X, Song Z, Niu S, Luo X (2016) Gold nanoparticles
and polyethylene glycols functionalized conducting polyaniline
nanowires for ultrasensitive and low fouling immunosensing of
alpha-fetoprotein. Biosens Bioelectron 86:143–149
65. Yu T, Zhang H, Huang Z, Luo Z, Huang N, Ding S, Feng W
(2017) A Simple Electrochemical Aptamer Cytosensor for
Direct Detection of Chronic Myelogenous Leukemia K562
Cells. Electroanalysis 29:828–834
66. Alves PU, Vinhas R, Fernandes AR, Birol SZ, Trabzon L,
Bernacka-Wojcik I, Igreja R, Lopes P, Baptista PV, Águas H
(2018) Multifunctional microfluidic chip for optical nanoprobe
based RNA detection–application to Chronic Myeloid
Leukemia. Sci Rep 8:381
67. Wang J, Wang X, Tang H, Gao Z, He S, Li J, Han S (2018)
Ultrasensitive electrochemical detection of tumor cells based on
multiple layer CdS quantum dots-functionalized polystyrene mi-
crospheres and graphene oxide–polyaniline composite. Biosens
Bioelectron 100:1–7
Page 25 of 29 465
68. Wang J, Hui N (2018) A nonfouling voltammetric immunosensor
for the carcinoembryonic antigen based on the use of polyaniline
nanowires wrapped with hyaluronic acid. Microchimica Acta 185:
329
69. Fu J, Shi Z, Li M, Wang Y, Yu L (2016) Label-free detection of
chondroitin sulphate proteoglycan 4 by a polyaniline/graphene
nanocomposite functionalized impedimetric immunosensor.
Journal of Nanomaterials 2016:2
70. Singer E, Markó L, Paragas N, Barasch J, Dragun D, Müller DN,
Budde K, Schmidt-Ott KM (2013) Neutrophil gelatinase-
associated lipocalin: pathophysiology and clinical applications.
Acta physiologica 207:663–672
71. Devarajan P (2010) Neutrophil gelatinase-associated lipocalin: A
troponin-like biomarker for human acute kidney injury.
Nephrology 15:419–428
72. Yukird J, Wongtangprasert T, Rangkupan R, Chailapakul O,
Pisitkun T, Rodthongkum N (2017) Label-free immunosensor
based on graphene/polyaniline nanocomposite for neutrophil
gelatinase-associated lipocalin detection. Biosens Bioelectron
87:249–255
73. Cui J (2015) MiR-16 family as potential diagnostic biomarkers for
cancer: a systematic review and meta-analysis. Int J Clin Exp Med
8:1703
74. Larrea E, Sole C, Manterola L, Goicoechea I, Armesto M, Arestin
M, Caffarel M, Araujo A, Araiz M, Fernandez-Mercado M (2016)
New concepts in cancer biomarkers: circulating miRNAs in liquid
biopsies. Int J Mol Sci 17:627
75. Peng Y, Yi G, Gao Z (2010) A highly sensitive microRNA bio-
sensor based on ruthenium oxide nanoparticle-initiated polymeri-
zation of aniline. Chem Commun 46:9131–9133
76. Singh R, Dhand C, Sumana G, Verma R, Sood S, Gupta RK,
Malhotra BD (2010) Polyaniline/carbon nanotubes platform for
sexually transmitted disease detection. J Mol Recognit 23:472–
479
77. Sin ML, Mach KE, Wong PK, Liao JC (2014) Advances and
challenges in biosensor-based diagnosis of infectious diseases.
Expert Rev Mol Diagn 14:225–244
78. Lazcka O, Del Campo FJ, Munoz FX (2007) Pathogen detection:
A perspective of traditional methods and biosensors. Biosens
Bioelectron 22:1205–1217
79. Zourob M, Elwary S, Turner AP (2008) Principles of bacterial
detection: biosensors, recognition receptors and microsystems.
Springer Science & Business Media
80. Hashemi E, Forouzandeh M (2018) Designing a new biosensor
“DNA ELISA” to detect Escherichia coli using genomic DNA and
comparison of this method to PCR-ELISA. J Enzyme Inhib Med
Chem 33:722–725
81. Torati SR, Reddy V, Yoon SS, Kim C (2016) Electrochemical
biosensor for Mycobacterium tuberculosis DNA detection based
on gold nanotubes array electrode platform. Biosens Bioelectron
78:483–488
82. Hiraiwa M, Kim J-H, Lee H-B, Inoue S, Becker AL, Weigel KM,
Cangelosi GA, Lee K-H, Chung J-H (2015) Amperometric
immunosensor for rapid detection of Mycobacterium tuberculosis.
J Micromech Microeng 25:055013
83. Chen Y, Li Y, Yang Y, Wu F, Cao J, Bai L (2017) A polyaniline-
reduced graphene oxide nanocomposite as a redox nanoprobe in a
voltammetric DNA biosensor for Mycobacterium tuberculosis.
Microchimica Acta 184:1801–1808
84. Liu C, Jiang D, Xiang G, Liu L, Liu F, Pu X (2014) An electro-
chemical DNA biosensor for the detection of Mycobacterium tu-
berculosis, based on signal amplification of graphene and a gold
nanoparticle–polyaniline nanocomposite. Analyst 139:5460–
5465
465 Page 26 of 29
85. Zhou L, He X, He D, Wang K, Qin D (2011) Biosensing technol-
ogies for Mycobacterium tuberculosis detection: status and new
developments. Clin Dev Immunol 2011 Article ID 193963:8
86. Rodovalho V, Alves L, Castro A, Madurro J, Brito-Madurro A,
Santos A (2015) Biosensors Applied to Diagnosis of Infectious
Diseases–An Update. Austin J Biosens & Bioelectron 1:1015
87. Mohd Azmi U, Yusof N, Kusnin N, Abdullah J, Suraiya S, Ong P,
Ahmad Raston N, Abd Rahman S, Mohamad Fathil M (2018)
Sandwich electrochemical immunosensor for early detection of
tuberculosis based on graphene/polyaniline-modified screen-
printed gold electrode. Sensors 18:3926
88. Kumar H, Neelam R (2016) Enzyme-based electrochemical bio-
sensors for food safety: A review. Nanobiosens Dis Diagn 5:29–
39
89. Hailemariam M, Abebe T, Mihret A, Lambiyo T (2013)
Prevalence of Neisseria gonorrhea and their antimicrobial suscep-
tibility patterns among symptomatic women attending gynecolo-
gy outpatient department in Hawassa Referral Hospital. Ethiop J
Health Sci 23:10–18
90. Singh R, Verma R, Sumana G, Srivastava AK, Sood S, Gupta RK,
Malhotra B (2012) Nanobiocomposite platform based on
polyaniline-iron oxide-carbon nanotubes for bacterial detection.
Bioelectrochemistry 86:30–37
91. Spain E, Kojima R, Kaner RB, Wallace GG, O’Grady J, Lacey K,
Barry T, Keyes TE, Forster RJ (2011) High sensitivity DNA de-
tection using gold nanoparticle functionalised polyaniline
nanofibres. Biosens Bioelectron 26:2613–2618
92. Ariffin EY, Lee YH, Futra D, Tan LL, Karim NHA, Ibrahim NNN,
Ahmad A (2018) An ultrasensitive hollow-silica-based biosensor
for pathogenic Escherichia coli DNA detection. Anal Bioanal
Chem 410:2363–2375
93. Kellner K, Ettenauer J, Zuser K, Posnicek T, Brandl M (2016) An
automated, robotic biosensor for the electrochemical detection of
E. coli in water. Procedia Engineering 168:594–597
94. Wildeboer D, Amirat L, Price RG, Abuknesha RA (2010) Rapid
detection of Escherichia coli in water using a hand-held fluores-
cence detector. Water Res 44:2621–2628
95. Mo X, Wu Z, Huang J, Zhao G, Dou W (2019) A sensitive and
regenerative electrochemical immunosensor for quantitative de-
tection of Escherichia coli O157: H7 based on stable polyaniline
coated screen-printed carbon electrode and rGO-NR-Au@ Pt.
Anal Methods 11:1475–1482
96. Zimmet P, Alberti KG, Magliano DJ, Bennett PH (2016) Diabetes
mellitus statistics on prevalence and mortality: facts and fallacies.
Nat Rev Endocrinol 12:616–622
97. Yoo E-H, Lee S-Y (2010) Glucose biosensors: an overview of use
in clinical practice. Sensors 10:4558–4576
98. Salek-Maghsoudi A, Vakhshiteh F, Torabi R, Hassani S, Ganjali
MR, Norouzi P, Hosseini M, Abdollahi M (2018) Recent advances
in biosensor technology in assessment of early diabetes bio-
markers. Biosens Bioelectron 99:122–135
99. Tracey ML, Gilmartin M, O’Neill K, Fitzgerald AP, McHugh SM,
Buckley CM, Canavan RJ, Kearney PM (2016) Epidemiology of
diabetes and complications among adults in the Republic of
Ireland 1998-2015: a systematic review and meta-analysis. BMC
Public Health 16:132
100. Danaei G, Finucane MM, Lu Y, Singh GM, Cowan MJ, Paciorek
CJ, Lin JK, Farzadfar F, Khang Y-H, Stevens GA (2011) National,
regional, and global trends in fasting plasma glucose and diabetes
prevalence since 1980: systematic analysis of health examination
surveys and epidemiological studies with 370 country-years and 2·
7 million participants. The Lancet 378:31–40
Microchim Acta (2019) 186: 465
101. Thakur B, Amarnath CA, Sawant SN (2014) Pectin coated
polyaniline nanoparticles for an amperometric glucose biosensor.
RSC Adv 4:40917–40923
102. Zhai D, Liu B, Shi Y, Pan L, Wang Y, Li W, Zhang R, Yu G (2013)
Highly sensitive glucose sensor based on Pt nanoparticle/
polyaniline hydrogel heterostructures. ACS Nano 7:3540–3546
103. U. Tamer, A.İ. Seçkin, E. Temur, H. Torul, Fabrication of biosen-
sor based on polyaniline/gold nanorod composite, International
Journal of Electrochemistry, 2011 (2011) Article ID 869742, 7
pages.
104. Nguyen HB, Ngo TTT, Nguyen NT, Dang TTH, Do PQ, Nguyen
XN, Nguyen XP, Phan HK, Phan NM (2012) Graphene patterned
polyaniline-based biosensor for glucose detection. Adv Nat Sci
Nanosci Nanotechnol 3:025011
105. Xu M, Song Y, Ye Y, Gong C, Shen Y, Wang L, Wang L (2017) A
novel flexible electrochemical glucose sensor based on gold
nanoparticles/polyaniline arrays/carbon cloth electrode. Sens
Actuators B 252:1187–1193
106. Wu S, Su F, Dong X, Ma C, Pang L, Peng D, Wang M, He L,
Zhang Z (2017) Development of glucose biosensors based on
plasma polymerization-assisted nanocomposites of polyaniline,
tin oxide, and three-dimensional reduced graphene oxide. Appl
Surf Sci 401:262–270
107. Esmaeeli A, Ghaffarinejad A, Zahedi A, Vahidi O (2018) Copper
oxide-polyaniline nanofiber modified fluorine doped tin oxide
(FTO) electrode as non-enzymatic glucose sensor. Sens
Actuators B 266:294–301
108. Wang Y, Zhong J, Ding F, Zhao Q, Zhang Z, Liu X, Liu Y, Rao H,
Zou P, Wang X (2018) A bifunctional NiCo 2 S 4/reduced
graphene oxide@ polyaniline nanocomposite as a highly-
efficient electrode for glucose and rutin detection. New J Chem
42:9398–9409
109. Chauhan N, Pundir CS (2011) An amperometric uric acid biosen-
sor based on multiwalled carbon nanotube–gold nanoparticle
composite. Anal Biochem 413:97–103
110. Rawal R, Chawla S, Chauhan N, Dahiya T, Pundir C (2012)
Construction of amperometric uric acid biosensor based on uricase
immobilized on PBNPs/cMWCNT/PANI/Au composite. Int J
Biol Macromol 50:112–118
111. Devi R, Pundir C (2014) Construction and application of an am-
perometric uric acid biosensor based on covalent immobilization
of uricase on iron oxide nanoparticles/chitosan-g-polyaniline com-
posite film electrodeposited on Pt electrode. Sens Actuators B 193:
608–615
112. Erden PE, Kılıç E (2013) A review of enzymatic uric acid biosen-
sors based on amperometric detection. Talanta 107:312–323
113. El Kurdi R, Patra D (2018) Gold nanoparticles functionalized with
Pluronic are viable optical probes for the determination of uric
acid. Microchimica Acta 185:185
114. Bhambi M, Sumana G, Malhotra B, Pundir C (2010) An
amperomertic uric acid biosensor based on immobilization of
uricase onto polyaniline-multiwalled carbon nanotube composite
film. Artif Cells Blood Substit Biotechnol 38:178–185
115. Xu L, Zhang M, Hou Y, Huang W, Yao C, Wu Q (2015) An Au
nanocomposite based biosensor for determination of cholesterol.
Anal Methods 7:3480–3485
116. Muhammet SM, Çete S, Arslan F, Yaşar A (2009) Amperometric
cholesterol biosensors based on the electropolymerization of pyr-
role and aniline in sulphuric acid for the determination of choles-
terol in serum. Artif Cells Blood Substit Biotechnol 37:273–278
117. Singh K, Chauhan R, Solanki PR, Basu T (2013) Development of
Impedimetric Biosensor for Total Cholesterol Estimation Based on
Microchim Acta (2019) 186: 465
Polypyrrole and Platinum Nanoparticle Multi Layer
Nanocomposite. J Org Chem 3:262
118. Khan R, Solanki PR, Kaushik A, Singh S, Ahmad S, Malhotra B
(2009) Cholesterol biosensor based on electrochemically prepared
polyaniline conducting polymer film in presence of a nonionic
surfactant. J. Polym. Res. 16:363–373
119. Srivastava M, Srivastava S, Nirala N, Prakash R (2014) A
chitosan-based polyaniline–Au nanocomposite biosensor for de-
termination of cholesterol. Anal Methods 6:817–824
120. Dhand C, Arya SK, Datta M, Malhotra B (2008) Polyaniline–
carbon nanotube composite film for cholesterol biosensor. Anal
Biochem 383:194–199
121. Deepshikha R, Chauhan PR, Solanki TB (2012) Fabrication of
bienzymatic cholesterol biosensor based on gold nanoparticles
decorated graphene-nanostructured polyaniline nanocomposite.
Int. J. Biomed. Nanosci. Nanotechnol 2:251–275
122. McKeating KS, Aubé A, Masson J-F (2016) Biosensors and nano-
biosensors for therapeutic drug and response monitoring. Analyst
141:429–449
123. Chawla PK, Udwadia ZF, Soman R, Mahashur AA, Amale RA,
Dherai AJ, Lokhande RV, Naik PR, Ashavaid TF (2016)
Importance of therapeutic drug monitoring of rifampicin. J
Assoc Physicians India 64:68–72
124. Meneghello A, Tartaggia S, Alvau MD, Polo F, Toffoli G (2018)
Biosensing technologies for therapeutic drug monitoring. Curr
Med Chem 25:4354–4377
125. Gao J, Liu ZJ, Chen T, Zhao D (2014) Pharmaceutical properties
of calycosin, the major bioactive isoflavonoid in the dry root ex-
tract of Radix astragali. Pharm Biol 52:1217–1222
126. Cai J, Sun B, Gou X, Gou Y, Li W, Hu F (2018) A novel way for
analysis of calycosin via polyaniline functionalized graphene
quantum dots fabricated electrochemical sensor. J Electroanal
Chem 816:123–131
127. Prasad BB, Srivastava S, Tiwari K, Sharma PS (2009)
Development of uracil and 5-fluorouracil sensors based on mo-
lecularly imprinted polymer-modified hanging mercury drop
electrode (S & M 0769). Sensor Mater 21:291
128. Zahed FM, Hatamluyi B, Lorestani F, Es’haghi Z (2018) Silver
nanoparticles decorated polyaniline nanocomposite based electro-
chemical sensor for the determination of anticancer drug 5-fluoro-
uracil. J Pharm Biomed Anal 161:12–19
129. Mostafavi M, Yaftian MR, Piri F, Shayani-Jam H (2018) A new
diclofenac molecularly imprinted electrochemical sensor based
upon a polyaniline/reduced graphene oxide nano-composite.
Biosens Bioelectron 122:160–167
130. Wong A, Marestoni LD, Sotomayor MD (2014) Monitoring of
diclofenac with biomimetic sensor in batch and FIA systems. J
Braz Chem Soc 25:1283–1291
131. Chen L, Xu S, Li J (2011) Recent advances in molecular imprint-
ing technology: current status, challenges and highlighted appli-
cations. Chem Soc Rev 40:2922–2942
132. Adumitrăchioaie A, Tertiș M, Cernat A, Săndulescu R, Cristea C
(2018) Electrochemical methods based on molecularly imprinted
polymers for drug detection. A review. Int J Electrochem Sci 13:
2556–2576
133. Rajpurohit AS, Punde NS, Rawool CR, Srivastava AK (2018)
Application of carbon paste electrode modified with carbon
nanofibres/polyaniline/platinum nanoparticles as an electrochem-
ical sensor for the determination of bezafibrate. Electroanalysis
30:571–582
134. Rezazadeh F, Mohamadi M, Afzali D, Shamspur T, Mostafavi A
(2015) A quercetin biosensor based on chitosan-entrapped carbon
Page 27 of 29 465
nanotube paste electrode coated with DNA. J AOAC Int 98:1375–
1381
135. Jiang W, Yang S, Sun X, Lu W, Jiang D, Xu L, Xu H, Gao B, Ma
M, Cao F (2018) Quercetin-coated Fe 3 O 4 nanoparticle sensors
based on low-field NMR for determination and removal of Pb 2+
and Cu 2+ in biological samples. Anal Methods 10:2494–2502
136. Ponnaiah SK, Periakaruppan P (2018) A glassy carbon electrode
modified with a copper tungstate and polyaniline nanocomposite
for voltammetric determination of quercetin. Microchim Acta 185:
524
137. Yu Z, Li H, Zhang X, Liu N, Tan W, Zhang X, Zhang L (2016)
Facile synthesis of NiCo2O4@ Polyaniline core–shell nanocom-
posite for sensitive determination of glucose. Biosens Bioelectron
75:161–165
138. Dehghani M, Nasirizadeh N, Yazdanshenas ME (2019)
Determination of cefixime using a novel electrochemical sensor
produced with gold nanowires/graphene oxide/
electropolymerized molecular imprinted polymer. Mater Sci Eng
C 96:654–660
139. Fatahi A, Malakooti R, Shahlaei M (2017) Electrocatalytic oxida-
tion and determination of dexamethasone at an Fe 3 O 4/PANI–Cu
II microsphere modified carbon ionic liquid electrode. RSC Adv
7:11322–11330
140. Wang Q, Zhao R, Wang S, Guo H, Li J, Zhou H, Wang X, Wu X,
Wang Y, Chen W (2016) A highly selective electrochemical sen-
sor for nifedipine based on layer-by-layer assembly films from
polyaniline and multiwalled carbon nanotube. J Appl Polym Sci:
133
141. Yang Y, Yan Y, Chen X, Zhai W, Xu Y, Liu Y (2014) Investigation
of a polyaniline-coated copper hexacyanoferrate modified glassy
carbon electrode as a sulfite sensor. Electrocatalysis 5:344–353
142. Rawal R, Chawla S, Dahiya T, Pundir CS (2011) Development of
an amperometric sulfite biosensor based on a gold nanoparticles/
chitosan/multiwalled carbon nanotubes/polyaniline-modified gold
electrode. Anal Bioanal Chem 401:2599–2608
143. Rawal R, Chawla S, Pundir C (2011) Polyphenol biosensor based
on laccase immobilized onto silver nanoparticles/multiwalled car-
bon nanotube/polyaniline gold electrode. Anal Biochem 419:196–
204
144. Chawla S, Rawal R, Sharma S, Pundir CS (2012) An amperomet-
ric biosensor based on laccase immobilized onto nickel
nanoparticles/carboxylated multiwalled carbon nanotubes/
polyaniline modified gold electrode for determination of phenolic
content in fruit juices. Biochem Eng J 68:76–84
145. Batra B, Lata S, Sharma M, Pundir C (2013) An acrylamide bio-
sensor based on immobilization of hemoglobin onto multiwalled
carbon nanotube/copper nanoparticles/polyaniline hybrid film.
Anal Biochem 433:210–217
146. Sun X, Qiao L, Wang X (2013) A novel immunosensor based on
Au nanoparticles and polyaniline/multiwall carbon nanotubes/
chitosan nanocomposite film functionalized interface. Nano-
Micro Letters 5:191–201
147. He L, Cui B, Liu J, Song Y, Wang M, Peng D, Zhang Z (2018)
Novel electrochemical biosensor based on core-shell nanostruc-
tured composite of hollow carbon spheres and polyaniline for
sensitively detecting malathion. Sens Actuators B 258:813–821
148. Khan I, Pandit UJ, Wankar S, Das R, Limaye SN (2017)
Fabrication of electrochemical nanosensor based on polyaniline
film-coated AgNP-MWCNT-modified GCE and its application
for trace analysis of fenitrothion. Ionics 23:1293–1308
149. Zhang C, Govindaraju S, Giribabu K, Huh YS, Yun K (2017)
AgNWs-PANI nanocomposite based electrochemical sensor for
detection of 4-nitrophenol. Sens Actuators B 252:616–623
465 Page 28 of 29
150. Saadati F, Ghahramani F, Shayani-jam H, Piri F, Yaftian MR
(2018) Synthesis and characterization of nanostructure molecular-
ly imprinted polyaniline/graphene oxide composite as highly se-
lective electrochemical sensor for detection of p-nitrophenol. J
Taiwan Inst Chem Eng 86:213–221
151. Rapini R, Cincinelli A, Marrazza G (2016) Acetamiprid
multidetection by disposable electrochemical DNA aptasensor.
Talanta 161:15–21
152. Selvolini G, Băjan I, Hosu O, Cristea C, Săndulescu R, Marrazza
G (2018) DNA-based sensor for the detection of an organophos-
phorus pesticide: Profenofos. Sensors 18:2035
153. Van Chuc N, Binh NH, Thanh CT, Van Tu N, Le Huy N, Dzung
NT, Minh PN, Thu VT, Dai Lam T (2016) Electrochemical
immunosensor for detection of atrazine based on polyaniline/
graphene. J. Mater. Sci. Technol 32:539–544
154. Deep A, Saraf M, Bharadwaj SK, Sharma AL (2014) Styrene
sulphonic acid doped polyaniline based immunosensor for highly
sensitive impedimetric sensing of atrazine. Electrochim Acta 146:
301–306
155. Yang Y, Kang M, Fang S, Wang M, He L, Zhao J, Zhang H, Zhang
Z (2015) Electrochemical biosensor based on three-dimensional
reduced graphene oxide and polyaniline nanocomposite for selec-
tive detection of mercury ions. Sens Actuators B 214:63–69
156. Tang L, Xie X, Zhou Y, Zeng G, Tang J, Wu Y, Long B, Peng B,
Zhu J (2017) A reusable electrochemical biosensor for highly
sensitive detection of mercury ions with an anionic intercalator
supported on ordered mesoporous carbon/self-doped polyaniline
nanofibers platform. Biochem Eng J 117:7–14
157. Chowdhury AN, Ferdousi S, Islam MM, Okajima T, Ohsaka T
(2007) Arsenic detection by nanogold/conducting-polymer-modi-
fied glassy carbon electrodes. J Appl Polym Sci 104:1306–1311
158. de Barros A, Constantino CJL, da Cruz NC, Bortoleto JRR,
Ferreira M (2017) High performance of electrochemical sensors
based on LbL films of gold nanoparticles, polyaniline and sodium
montmorillonite clay mineral for simultaneous detection of metal
ions. Electrochim Acta 235:700–708
159. Kong Y, Wu T, Wu D, Zhang Y, Wang Y, Du B, Wei Q (2018) An
electrochemical sensor based on Fe 3 O 4@ PANI nanocompos-
ites for sensitive detection of Pb 2+ and Cd 2+. Anal Methods 10:
4784–4792
160. Deshmukh MA, Patil HK, Bodkhe GA, Yasuzawa M, Koinkar P,
Ramanaviciene A, Shirsat MD, Ramanavicius A (2018) EDTA-
modified PANI/SWNTs nanocomposite for differential pulse volt-
ammetry based determination of Cu (II) ions. Sens Actuators B
260:331–338
161. Mehennaoui S, Poorahong S, Jimenez GC, Siaj M (2019)
Selection of high affinity aptamer-ligand for dexamethasone and
its electrochemical biosensor. Sci Rep 9:6600
162. Mantas A, Mihranyan A (2019) Immediate-release nifedipine bi-
nary dry powder mixtures with nanocellulose featuring enhanced
solubility and dissolution rate. Pharmaceutics 11:37
163. Fakhri A, Shahidi S, Agarwal S, Gupta VK (2016) Electrocatalytic
oxidation behavior of cefixime antibiotic at bimetallic Pt-W nano-
particle-decorated multi-walled carbon nanotubes modified glassy
Carbon electrode and its determination. Int. J. Electrochem. Sci
11:1530–1540
164. Pundir CS, Rawal R (2013) Determination of sulfite with empha-
sis on biosensing methods: a review. Anal Bioanal Chem 405:
3049–3062
165. Bahmani B, Moztarzadeh F, Hossini M, Rabiee M, Tahriri M,
Rezvannia M, Alizadeh M (2009) A sulfite biosensor fabricated
by immobilization of sulfite oxidase on aluminum electrode mod-
ified with electropolymerized conducting film (polyaniline).
Asian J. Chem 21:923
Microchim Acta (2019) 186: 465
166. Ozcan T, Akpinar-Bayizit A, Yilmaz-Ersan L, Delikanli B (2014)
Phenolics in human health. International Journal of chemical
Engineering and applications 5:393
167. Nguyen H, Lee S, Lee U, Fermin C, Kim M (2019) Immobilized
enzymes in biosensor applications. Materials 12:121
168. Hasan A, Nurunnabi M, Morshed M, Paul A, Polini A, Kuila T, Al
Hariri M, Lee Y-K, Jaffa AA (2014) Recent advances in applica-
tion of biosensors in tissue engineering. Biomed Res Int 2014
Article ID 307519:18
169. Huang S, Lu S, Huang C, Sheng J, Zhang L, Su W, Xiao Q (2016)
An electrochemical biosensor based on single-stranded DNA
modified gold electrode for acrylamide determination. Sens
Actuators B 224:22–30
170. Hu Q, Xu X, Fu Y, Li Y (2015) Rapid methods for detecting
acrylamide in thermally processed foods: A review. Food
Control 56:135–146
171. Stobiecka A, Radecka H, Radecki J (2007) Novel voltammetric
biosensor for determining acrylamide in food samples. Biosens
Bioelectron 22:2165–2170
172. Batra B, Lata S, Pundir C (2013) Construction of an improved
amperometric acrylamide biosensor based on hemoglobin
immobilized onto carboxylated multi-walled carbon nanotubes/
iron oxide nanoparticles/chitosan composite film. Bioprocess
Biosyst Eng 36:1591–1599
173. Dhull V, Gahlaut A, Dilbaghi N, Hooda V (2013)
Acetylcholinesterase biosensors for electrochemical detection of
organophosphorus compounds: A review. Biochem Res Int
2013) Article ID 731501:18
174. Cesarino I, Moraes FC, Lanza MR, Machado SA (2012)
Electrochemical detection of carbamate pesticides in fruit and
vegetables with a biosensor based on acetylcholinesterase
immobilised on a composite of polyaniline–carbon nanotubes.
Food Chem 135:873–879
175. Bolat G, Abaci S (2018) Non-Enzymatic Electrochemical Sensing
of Malathion Pesticide in Tomato and Apple Samples Based on
Gold Nanoparticles-Chitosan-Ionic Liquid Hybrid
Nanocomposite. Sensors 18:773
176. Bala R, Swami A, Tabujew I, Peneva K, Wangoo N, Sharma RK
(2018) Ultra-sensitive detection of malathion using quantum dots-
polymer based fluorescence aptasensor. Biosens Bioelectron 104:
45–49
177. Guler M, Turkoglu V, Kivrak A (2016) Electrochemical detection
of malathion pesticide using acetylcholinesterase biosensor based
on glassy carbon electrode modified with conducting polymer
film. Environ Sci Pollut Res 23:12343–12351
178. Tefera M, Admassie S, Tessema M, Mehretie S (2015)
Electrochemical Sensor for Determination of Fenitrothion at
Multi-wall Carbon Nanotubes Modified Glassy Carbon
Electrode. Anal Bioanal Chem Res 2:139–150
179. Rao H, Guo W, Hou H, Wang H, Yin B, Xue Z, Zhao G (2017)
Electroanalytical investigation of p-nitrophenol with dual
electroactive groups on a reduced graphene oxide modified glassy
carbon electrode. Int J Electrochem Sci 12:1052–1063
180. Rapini R, Marrazza G (2015) DNA technology for small mole-
cules sensing: a new approach for Acetamiprid detection, AISEM
Annual Conference, 2015 XVIII. IEEE:1–4
181. Zhou W, Huang P-JJ, Ding J, Liu J (2014) Aptamer-based biosen-
sors for biomedical diagnostics. Analyst 139:2627–2640
182. Norouzi P, Larijani B, Ganjali M, Faridbod F (2012)
Admittometric electrochemical determination of atrazine by
nano-composite immune-biosensor using FFT-square wave volt-
ammetry. Int J Electrochem Sci 7:10414–10426
183. Švorc Ľ, Rievaj M, Bustin D (2013) Green electrochemical sensor
for environmental monitoring of pesticides: Determination of at-
razine in river waters using a boron-doped diamond electrode.
Sens Actuators B 181:294–300
Microchim Acta (2019) 186: 465
184. Liu X, Li W-J, Li L, Yang Y, Mao L-G, Peng Z (2014) A label-free
electrochemical immunosensor based on gold nanoparticles for
direct detection of atrazine. Sens Actuators B 191:408–414
185. Hayes TB, Khoury V, Narayan A, Nazir M, Park A, Brown T,
Adame L, Chan E, Buchholz D, Stueve T (2010) Atrazine induces
complete feminization and chemical castration in male African
clawed frogs (Xenopus laevis). Proc Natl Acad Sci 107:4612–
4617
186. Jiwan S (2011) Effects of heavy metals on soil, plants, human
health and aquatic life. IJRCE 1:15–21
187. Singh R, Gautam N, Mishra A, Gupta R (2011) Heavy metals and
living systems: an overview. Indian J Pharm 43:246
Page 29 of 29 465
188. Dong Y, Zhou Y, Ding Y, Chu X, Wang C (2014) Sensitive detec-
tion of Pb (II) at gold nanoparticle/polyaniline/graphene modified
electrode using differential pulse anodic stripping voltammetry.
Anal Methods 6:9367–9374
189. Kumar SaA (2008) Recent advances in DNA biosensor. Sensors
& Transducers 92:122–133
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