Biosensors and Bioelectronics 90 (2017) 125–139

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Biosensors and Bioelectronics

journal homepage: www.elsevier.com/locate/bios

Recent advances in DNA-based electrochemical biosensors for heavy metal MARK

ion detection: A review
⁎
M.R. Saidura, A.R. Abdul Aziza, , W.J. Basirunb,c
a
Department of Chemical Engineering, Faculty of Engineering, University of Malaya, 50603 Kuala Lumpur, Malaysia
b
Department of Chemistry, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia
c
Institute of Nanotechnology and Catalysis (NANOCAT), University of Malaya, 50603 Kuala Lumpur, Malaysia

A R T I C L E I N F O A BS T RAC T

Keywords: The presence of heavy metal in food chains due to the rapid industrialization poses a serious threat on the
DNA environment. Therefore, detection and monitoring of heavy metals contamination are gaining more attention
Electrochemical biosensors nowadays. However, the current analytical methods (based on spectroscopy) for the detection of heavy metal
Heavy metal contamination are often very expensive, tedious and can only be handled by trained personnel. DNA biosensors,
T–Hg2+–T
which are based on electrochemical transduction, is a sensitive but inexpensive method of detection. The
DNAzyme
principles, sensitivity, selectivity and challenges of electrochemical biosensors are discussed in this review. This
G-quadruplex
review also highlights the major advances of DNA-based electrochemical biosensors for the detection of heavy
metal ions such as Hg2+, Ag+, Cu2+ and Pb2+.

1. Introduction
Elements with atomic weight more than 63.5 and a specific gravity
larger than 5g/cm−3 are classified as heavy metals. In small quantity,
heavy metals are co-factors and prosthetic groups of many enzymes,
and important for human health (Bagchi et al., 2000). However,
excessive intake of heavy metals through dietary ingestion and/or from
environmental contamination, poses a great threat to human and
environment (Järup, 2003; Martin and Griswold, 2009).
Essential biological molecules such as proteins and enzymes con-
tain nitrogen, oxygen or sulfur at their ligand sites, which leads to
greater possibility of heavy metal complexation. For example, they can
form a stable complex with the sulfur atom of methionine (–SCH) and
sulfhydryl group (–SH) of cysteine. Furthermore, a single heavy metal
ion can conciliate a crucial metal ion of similar size in the body's
metalloenzyme. In some dehydrogenating enzymes, Cd2+ can replace
Zn2+, a prelude to cadmium toxicity. This process interferes with the
enzymes’ and proteins’ normal functions, hindering metabolism and
delaying antioxidant protection, thus accumulating the reactive oxygen
species (Barnham et al., 2004; Stohs and Bagchi, 1995; Valko et al.,
2006). Reactive oxygen species irritate normal healthy cells and induce
toxicological and carcinogenic consequences mainly in the skin, bones
(Cr6+, Ni2+, Ag+, Cu2+, and Cd2+), kidney, liver (Cu2+, Hg2+, Pb2+, Ag+
and Cd2+), and the central nervous system (Pb2+, Hg2+, and As3+)
(Hellström et al., 2001; Hughes et al., 2011; Järup, 2003; Lidsky and
Schneider, 2003; Martin and Griswold, 2009; Panyala et al., 2008; Shi
et al., 2004; Smith and Steinmaus, 2009; Uriu-Adams and Keen, 2005).
The side-effects and health impact of heavy metals on human
beings have encouraged different organizations including the World
Health Organization (WHO) to set their safety limits (Griffiths et al.,
2012; Organization, 2004; Pescod, 1992). However, due to rapid
industrialization and urbanization, the awareness on the accepted daily
dose of heavy metals are overlooked in many parts of the world and
achieving the safety limits has become a challenge.
Many detection methods such as Atomic Absorption/ Emission
Spectroscopy (AAS/AES) (Pohl, 2009), Mass spectroscopy (MS)
(Flamini and Panighel, 2006), Inductively Coupled Plasma Mass
Spectrometry (ICP-MS) (Ashoka et al., 2009), Cold Vapor Atomic
Fluorescence Spectrometry (CV-AFS) (Aranda et al., 2009),
Ultraviolet–vis (UV) spectroscopy, X-Ray Fluorescence spectroscopy
(Lorber, 1986) and Potentiometric Methods (Alegret and Merkoçi,
2007) have been widely used to detect heavy metal ions at parts per
billion (ppb) and parts per trillion (ppt) levels. These methods are
extremely sensitive and selective but time-consuming. They require
tedious sample preparation, highly trained staff and cannot be used for
on-site detection due to the size of the analytical equipment (Jamali
et al., 2006). Therefore, the use of biological sensors or biosensors,
especially deoxyribonucleic acid (DNA) based biosensors appears to be
promising in heavy metal detection. This is because DNA and
DNAzymes are biodegradable, highly selective, easy to obtain using

⁎
Corresponding author.
E-mail address: azizraman@um.edu.my (A.R.A. Aziz).

http://dx.doi.org/10.1016/j.bios.2016.11.039
Received 10 September 2016; Received in revised form 3 November 2016; Accepted 15 November 2016
Available online 16 November 2016
0956-5663/ © 2016 Elsevier B.V. All rights reserved.
M.R. Saidur et al. Biosensors and Bioelectronics 90 (2017) 125–139

Fig. 1. (A) 1D 1H NMR spectrum of the T–Hg2+–T base pair: (i) in the absence of Hg2+. (ii) & (iii) in the presence of Hg2+(iv) Hg2+ and the duplex equilibrium system (Miyake et al.,
2006). (B) 1D 15N NMR spectrum of T–Hg2+–T: (b) the synthesized DNA oligomers with labeled thymidine indicated by color, and (c) 15N NMR spectrum of the duplex complex at a
molar ratio of 1:2 (Tanaka et al., 2007). (C) 1D 1H NMR spectrum of C–Ag+–C duplex formation: (i) spectrum of imino–proton region and (ii) spectrum for methyl proton region. Molar
ratio indicated at the left (Ono et al., 2008).

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in-vitro method and the analytical device is portable. Besides, electro-
chemical detection of certain analytes offers direct electronic signals
that overcome the use of expensive signal transforming instruments
(Drummond et al., 2003). It can be used on turbid or colored samples
because the sample components do not interfere with the electronic
signals (Ronkainen et al., 2010).
This review describes the sensing principles of DNA biosensors in
heavy metal detection with a focus on the recent developments of
sensitive and selective detection of different types of heavy metal ions
by electrochemical transduction.
2. Electrochemical DNA biosensor
According to the International Union of Pure and Applied
Chemistry (IUPAC), a biosensor is defined as “a self-contained
integrated device that is capable of providing specific quantitative or
semi-quantitative analytical information using a biological recognition
element (biochemical receptor), which is in direct spatial contact with a
transduction element” (Thévenot et al., 2001). A biosensor consists of
three segments (Liu et al., 2012a):
(1) The biological recognition elements (like enzyme, antibody, DNA
etc.).
(2) A signal transducer that transforms the detected signal into a
quantified, readable output.
(3) A signal processor to represent the transformed signal in an easy
and eco-friendly way.
The single stranded DNA (ss-DNA) molecules in DNA-biosensors
serve as the target recognition element and are known as the probe ss-
DNA. The principle is based on the hybridization of the probe ss-DNA
with complementary target ss-DNA or the disruption of the structural
integrity of probe ss-DNA by the analyte molecules. Both ways lead to
changes in the mass transport, light absorption, emission, or proton
concentration, which results in the generation of a signal. Then, the
signal is converted into a measurable response via an appropriate
transducer element such as optical, electrochemical or thermal ele-
ment, making the signal measurable as light, current, potential
(Sassolas et al., 2008).
An electrochemical DNA biosensor is a part of an electrochemical
cell (Drummond et al., 2003; Ronkainen et al., 2010). Like a conven-
tional three-electrode electrochemical cell, it consists of working,
reference and auxiliary electrodes. The working electrode is usually
Pt, Au or graphite modified with the probe ss-DNA molecules, while a
saturated calomel electrode (SCE) and platinum wire are the reference
and auxiliary electrodes, respectively (Labuda et al., 2010). It is
needless to acquire extra instrumentation for signal transduction, as
direct detection is accomplished by electrochemical techniques such as
amperometry, voltammetry, potentiometry, and electrochemical im-
pedance spectroscopy (EIS). Therefore, electrochemical transduction of
signals is a low cost, facile, and time-saving approach.
3. Principles of heavy metal ions detection in DNA biosensor
In heavy metal ion detection, the DNA probe is utilized as the metal
ion recognition element and based on the following principles:
• The selective binding of heavy metal ions with specific DNA bases
and formation of a stable DNA-duplex.
• Heavy metal ions facilitate cleavage of deoxyribozymes
(DNAzymes).
• The switching of a stable G-quadruplex structure of guanine-rich
probe DNA in the presence of heavy metal ions.
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Biosensors and Bioelectronics 90 (2017) 125–139
3.1. The selective binding of heavy metal ions with DNA bases and
formation of a stable DNA-duplex
Some heavy metal ions have selective affinity towards certain DNA
bases and form stable base pairs that are even stronger than the
Watson-crick base pair via coordination bond (Clever et al., 2007). Ono
and Togashi (2004) first reported the specific coordinating ability of
mercury ion (Hg2+) with two thymine (T) bases. They also proposed
that Hg2+ could form a stable T–Hg2+–T base pair in a DNA-duplex
using mismatches between the complementary sequences, which
thermally stabilize the DNA-duplex. They later proved the existence
of the T–Hg2+–T base pair structure using proton-1 (1H) and nitrogen-
15 (15N) nuclear magnetic resonance (NMR) spectroscopy (Miyake
et al., 2006; Tanaka et al., 2007). Fig. 1A shows the 1H NMR spectra of
the Hg2+ assisted duplex formation. Four peaks were observed between
10.4 and 11.1 ppm in the absence of Hg2+ due to the resonances of four
imino protons of the T–T mispairs, in amalgamation with those of the
Watson-crick base pairing (at 12.5–13.5 ppm). In the case of Hg2+
addition, the resonance of the imino protons in the T–T mispairs
decreased substantially.
According to Fig. 1B, the 1•3 duplex and 2•4 duplex clearly show
the splitting of the 15N resonance with a dipole-dipole coupling (J-
constant) almost similar to (2JNN). At the same time, the 15N NMR
spectrum of the 2•3 duplex was analyzed by using one labeled and one
unlabeled thymidine, but no splitting was observed. This confirmed
that the splitting of 15N NMR resonance spectrum for 1•3 and 2•4
duplexes was due to the J-constant (2JNN) and the T–Hg2+–T duplex.
Fig. 1C shows that the silver ion (Ag+) selectively binds with
cytosine (C) and forms a stable C–Ag+–C duplex, which is enough to
fully stabilize the duplex formation at pH 5 (Ono et al., 2008). The 1H
NMR spectrum of the imino proton region shows new peaks after
titration with Ag+, where the intensities of these peaks increase with
the concentration of Ag+, which confirms the duplex formation..
Therefore, the discovery of stable T–Hg2+–T and C–Ag+–C duplex
formations enables the design of selective DNA biosensors for heavy
metal ion detection. Therefore, T-rich or C-rich ss-DNA can be utilized
as a probe and complementary ss-DNA as a target. Hence, in the
presence of metal ions, they can form stable duplex structure and metal
ions can be detected by analyzing the hybridization event with different
transduction techniques (Li et al., 2016; Wen et al., 2010; Yan et al.,
2012).
3.2. Heavy metal ions facilitate cleavage of DNAzymes
DNAzymes are sequences that manifest catalytic properties such as
promoting DNA or ribonucleic acid (RNA) strands rupture (Fig. 2A and
B). After the discovery of ribozymes in 1980 s, it has been assumed that
DNA can act both as a catalyst and a genetic carrier. The first
DNAzymes was isolated in 1994 by an in-vitro selection method, which
was able to catalyze a trans-esterification reaction (Breaker and Joyce,
1994). Later on, many DNAzymes were successfully isolated.
Interestingly, most of the DNAzymes exhibit catalytic activity in the
presence of metal cofactors with high selectivity of the metal ions
(Wang et al., 2002). The first isolated DNAzyme is 400,000 times more
selective to lead ion (Pb2+) compared to other metal ions (Brown et al.,
2003; Lan et al., 2010). Different types of highly selective metal ion
dependent DNAzymes has been obtained using the in-vitro selection
process or systematic evolution of ligands by exponential amplification
(SELEX) (Hollenstein et al., 2008; Huang et al., 2015; Li et al., 2000;
Liu et al., 2007; Nelson et al., 2012).
The excellent metal selectivity enables the utilization of DNAzymes
as a biological recognition element in the design of improved DNA
biosensors for the detection of heavy metal ions. Fig. 2C shows the
typical reaction scheme of DNAzymes and the mechanism for heavy
metal ion detection. Here, in the absence of a selective metal ion, the
enzyme-oligonucleotide strand hybridizes with the substrate strand. A
M.R. Saidur et al. Biosensors and Bioelectronics 90 (2017) 125–139

Fig. 2. (A) Schematic illustration of DNAzymes. (B) 3D model of an 8-17E DNAzymes active site (Liu and Sen, 2008). (C) The principle of designing DNAzymes based biosensor for
heavy metal ions. (D) Hydrogen bonding between four Guanine bases and (E) a model of the G-quadruplex structure.

signal can be induced when the electrochemical redox reporter moves
closer to the surface, or when the labeled fluorophore and quencher
molecules come closer to each other. The metal ion then facilitates the
cleavage, which causes the melting temperature of the resulting two
strands to become lower than the ambient temperature and the DNA-
duplex is de-hybridized accordingly. The release of the reporter-
containing segment changes the signal and is related to the metal ions
concentration. Many heavy metal ion biosensors based on fluorescence,
colorimetric, and electrochemical measurements have been success-
fully fabricated (Lan and Lu, 2012; Li and Lu, 2000; Liu et al., 2009b;
Liu and Lu, 2003, 2007; Xiao et al., 2007).
3.3. The switching of a stable G-quadruplex structure of guanine-rich
probe DNA
Guanine (G) rich oligonucleotides and polynucleotides have a
strong tendency for self–association and aggregation to form a model
structure where the four strands are confined together by hydrogen

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bonding between the four-guanine groups (Fig. 2D). The polymorphic
four strand model structure shown in Fig. 2E is the G-quartet or G-
quadruplex (G4-DNA). In this four-fold helices, there is a central
channel which is a unique feature among the G-quadruplex structure
that distinguishes this particular DNA arrangement from other types of
DNA (Balagurumoorthy and Brahmachari, 1994; Howard and Miles,
1982).
It has been reported that some metal ions such as K+, Na+ and Pb2+
could influence the conformational switch of a G-rich DNA from
random coil to the G-quadruplex structure (Guschlbauer et al., 1990;
Smirnov and Shafer, 2000; Yang et al., 2010a). Some heavy metal ion
biosensors have been discovered based on the conformational switch-
ing by metal ions (Lin et al., 2011; Liu et al., 2009a; Zhan et al., 2013).
4. Heavy metal ion biosensors based on stable duplex
formation using DNA mismatches
When the T–T and C–C mismatches are present between two
complementary ss-DNA, Hg2+ and Ag+ can form a stable duplex even
stronger than the Watson-Crick base pair by co-ordination (Clever
et al., 2007; Xu et al., 2015). Therefore, with a T-rich or C-rich probe
ss-DNA and their immobilization on the electrode surface, it is possible
to form a duplex with complementary ss-DNA. Then, the surface-
confined DNA metal ions can be detected by different electrochemical
techniques, as summarized in Table 1.
4.1. T–T mismatches based biosensors for Hg2+ detection
4.1.1. T–Hg2+–T co-ordination based simple approach
Wu et al. (2010b) developed a simple T–Hg2+–T co-ordination
based biosensor for Hg2+ detection by immobilizing a thiolated
polythymine oligonucleotides (PTO) on a gold electrode via self-
assembly. Hg2+ captured by the T–Hg2+–T formation was initially
reduced at −0.2 Volt (V) on the PTO/Au modified electrode. The
oxidation signal of Hg2+ was analyzed with a detection limit of
60 picomoles (pM) using differential pulse voltammetry (DPV)
(−0.2 V~1.0 V). Although it is easy to fabricate direct electrochemical
redox signal based biosensor, it suffers from large background signals.
Therefore, the use of electrochemical redox indicator is recommended
in order to produce stronger electrochemical signals. For example, Niu
et al. (2011) developed a novel biosensor, where ferrocene (Fc) was
tagged on the ss-DNA as an indicator. This biosensor was able to detect
Hg2+ in the concentration range of 1 nanomole (nM) to 10 micromoles
(µM), with a detection limit of 0.6 nM based on the Fc signal. Later,
Zhang et al. (2013) used hexamine ruthenium complex [Ru(NH3)6]3+
where an increase of the peak current of [Ru (NH3)6]3+ was observed
after hybridization. Graphene oxide (GO) has a stronger interaction
with ss-DNA than ds-DNA (Varghese et al., 2009). Therefore, GO can
be used as an alternative electrochemical redox indicator. An electro-
chemical Hg2+ biosensor was developed by Park et al. (2012) using GO
as an active indicator.
4.1.2. Based on T–Hg2+–T co-ordination induce conformational
switching of probe DNA
It is well-known that target DNA molecule often induces conforma-

Table 1
Heavy metal ion detection biosensors based on stable duplex formation using DNA mismatches.

Strategy Analyte HM Electrode Electrochemical Electrochemical Limit of Linear range References

material technique indicator detection
(LOD)

Simple approach Hg2+ Gold DPV 60 pM 0.2–1 nM Wu et al. (2010b)

RGO/GCE CV, EIS, DPV [Ru(NH3)6]3+ 5 nM 8 nM−0.1 µm Zhang et al.
(2013)
SPGE CV Fc 0.6 nM 10–0.001 µm Niu et al. (2011)
Gold coated glass CV, EIS GO 1 nM 1 −300 nM Park et al. (2012)
electrode
Ag+ CPE and AuNPs/ DPV Ethyl green (EG) 0.104 nM/ 0.3–1 nM and 0.09 Ebrahimi et al.
CPE 0.0264 nM −1 nM (2015)
Fe3O4@3D-GO/ CV, EIS 2.0 pM 0.01–100 nM Yang et al. (2015)
gold
2+
Probe structural Hg Gold EIS [Fe(CN)6]3-/4- 100 pM 1 nM−1 mM Cao et al. (2009)
switch signal-on PGE DPV Fc 0.1 µm 0.1–2 µm Han et al. (2009)
Gold SWV Fc 2.5 nM 5 nM−1 µm Zhuang et al.
(2013)
Gold and SPGE SWV MB 0.1 nM 0.2 −100 nM /0.2 Tortolini et al.
−50 nM (2015a)
Gold DPV MB and Fc 0.08 nM 0.5 −5000 nM Xiong et al.
(2015)
+
Ag MHA/Gold DPV 1.3 nM 10–500 nM Yan et al. (2012)
NPG/GCE SWV AQDS 0.048 nM 0.1 nM−1 µm Zhou et al.
(2015)
Probe structural Hg2+ PGE DPV, CV, EIS Fc 0.5 nM 1 nM−2.0 µm Liu et al. (2009c)
switch signal-off PGE CV, DPV, EIS Fc 0.06 nM 0.1 nM- 5 µm Wu et al. (2010a)
Multi metallic (Au- CC RuHex 1 nM 6 −1066 nM Chen et al.
Ag) planar (2012)
electrode
Amplification based Hg2+ Gold CV [Ru(NH3)6]3+ 10 nM 10 nM−10 µm Miao et al.
(2009)
Gold SWV [Ru(NH3)6]3+ 0.5 nM 1 nM−0.1 µm Zhu et al.
(2009b)
3+
Gold DPV [Ru(NH3)6] 7.38 pM 0.05–2.5 nM Tang et al. (2012)
ITO coated glass DPV MB 0.2 nM 0.5 nM−20 µm Xuan et al.
chip (2013)
Ag+ Gold DPV MB 0.03 nM 0.1–120 nM Xu et al. (2013)

SPGE= Screen-printed gold electrode; PGE= Polycrystalline gold electrode. ITO= Indium tin oxide

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tional variations, or causes folding and unfolding of probe DNA during
hybridization (Hermann and Patel, 2000). This technique is a remark-
able general platform for the development of new DNA biosensors with
many attractive features (Liu et al., 2011). Recently, this approach has
attracted attention in the development of heavy metal ion biosensors.
4.1.2.1. Conformational switch signal-on biosensors. Cao et al.
(2009) developed a very sensitive and selective DNA biosensor using
EIS. Based on the co-ordination of Hg2+, the conformation of the poly-
T probes on the electrode changed from parallel linear to a hairpin
structure, increasing the surface crowding. This crowding initiates the
release of partial DNA molecules from the electrode surface, which
later influences the diffusion of the metal ions. The limit of detection
(LOD) of this biosensor was as low as 100 pM. However, was less
specific because of the non-specific adsorption and this increased the
possibility of false positive signals. Therefore, Han et al. (2009)
developed a signal-on strategy using a Fc-tagged DNA oligomer to
improve the specificity. A G–C base pair was added in the oligomer to
facilitate the intra-molecular folding. Thus, in the presence of Hg2+, the
T–Hg2+–T coordination triggered the probe oligomer to fold into a
hairpin-like structure, which pulled the Fc tags close to the electrode
surface and enhanced the electron transfer process (Fig. 3A). Then they
analyzed the Fc signal by DPV. Recently, Tortolini et al. (2015b) also
proposed a similar strategy using methylene blue (MB) as a redox tag
instead of Fc.
Zhuang et al. (2013) developed a sensitive and reusable signal-on
biosensor using Fc tag, but using a “compete for T–Hg2+–T coordina-
tion” approach in a specifically designed hairpin probe-DNA. The T–
Hg2+–T coordination opened the hairpin structure and led the Fc tags
closer to the electrode surface, enhancing the current signal generated
from the Fc-tag (Fig. 3B). Most recently, Xiong et al. (2015) has
reported an electrochemical biosensor based on the structural switch of
a T-rich probe DNA. Upon binding with the Hg2+, the steam loop probe
switched to a rigid ds-DNA. Interestingly, they used MB and Fc tags
respectively to indicate the steam loop and ds-DNA as shown in
Fig. 3C. Therefore, by examining the current ratio of MB to Fc, a
highly sensitive detection of about 0.08 nM Hg2+ was achieved.
4.1.2.2. Conformational switch signal-off biosensors. Liu et al.
(2009c) fabricated a facile signal-off biosensor for the detection of
Hg2+, where thiolated Fc-tagged poly-T oligonucleotides were
immobilized on the electrode surface via the self-assembly technique.
In the presence of Hg2+, a flexible single-strand poly-T oligonucleotide
probe formed a relatively rigid duplex with another poly-T probe via T–
Hg2+–T formation. Therefore, a flexible to rigid structure switching
puss out the Fc-tags from the electrode surface and decreased the redox
currents (Fig. 4A). This biosensor detected Hg2+ with high selectivity in
DPV, cyclic voltammetry (CV), and EIS measurements.
Wu et al. (2010a) developed a signal-off biosensor with higher
specificity. They started with the Hg2+ coordination, followed by the
substitution of the indicator. As shown in Fig. 4B, the competing
formation of T–Hg2+–T folds the mercury specific oligonucleotide
(MSO) probe into a hairpin-like structure, while the immediate release
of the Fc-tagged short ss-DNA decreases the redox current. It was
assumed that the decrease of non-specific adsorption increased the
sensitivity, (LOD 0.06 nM of Hg2+). Later on, Chen et al. (2012)
proposed another biosensor with the same strategy but using [Ru
(NH3)6]3+ as an indicator instead of Fc-tag via electrochemical
chronocoulometry (CC) technique (Fig. 4C).
4.1.3. Signal amplification based biosensors
Among all the strategies described above, only the probe-DNA
could incorporate the presence of Hg2+ and generate an electrochemi-
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Biosensors and Bioelectronics 90 (2017) 125–139
cal readout signal. However, the sensitivity was limited. Many
researchers have used nanomaterial-based signal amplification strate-
gies to increase the sensitivity (Aragay and Merkoçi, 2012; Gong et al.,
2013; Zhu et al., 2009a).
4.1.3.1. AuNPs-based signal amplification. Miao et al. (2009)
designed a facile signal amplification technique using AuNPs. They
first immobilized MSO onto the gold surface and at the same time
decorated the AuNPs with complementary ss-DNA. In the presence of
Hg2+, the AuNPs moved closer to the surface by forming a T–Hg2+–T
duplex and more electrochemical tags were bound to enhance the
signal. This work inspired Zhu et al. (2009b) to fabricate a highly
sensitive electrochemical biosensor using AuNPs. Fig. 5A, shows that
AuNPs were decorated with the MSO probes and additional linker
probes. The linker probes were complementary to the surface
immobilized capture probes. Therefore, hybridization between the
capture probe and the linker probe enabled AuNPs to move closer to
the gold electrode surface. Simultaneously the MSO probes could
capture a large number of Hg2+ through the T–Hg2+–T coordination.
This produced a highly amplified direct redox signal for the captured
Hg2+, around 3 orders of magnitude higher than other biosensors
fabricated from direct attachment of MSO on Au electrode without
using AuNPS.
Tang et al. (2012) designed an outstanding signal amplification
strategy based on the formation of a 3D DNA linked fishnet-sensing
platform using AuNPs as shown in Fig. 5B. In this strategy, the AuNPs
were functionalized with two types of thiolated probe-DNA (DNA1-
AuNPs and DNA2-AuNPs) and a capture probe with T–T mismatches
for the Hg2+ binding was used. The DNA1-AuNPs could combine with
the surface immobilized capture probe in the presence of Hg2+. At the
same time, the DNA2-AuNPs could bind with the DNA1-AuNPs. The
repetition of this type of conjugation could trigger a fishnet-like supra-
molecular structure. Therefore, when [Ru(NH3)6]3+ is used as the
electrochemical indicator, this fishnet structure adsorbs a large number
of indicators even at lower Hg2+ concentration. This biosensor could
detect Hg2+ concentration as low as 7.38 pM by analyzing the signal of
[Ru(NH3)6]3+ in DPV.
4.1.3.2. Exonuclease-III mediated signal amplification. Exonuclease-
III (Exo III) belongs to the 30–50 exonuclease family. It can digest 30-
end of ds-DNA without any particular recognition sequence (Xu et al.,
2012; Zuo et al., 2010). Exo-III facilitates the amplification as a
molecular beacon for the sensitive detection of several biomolecules
(Freeman et al., 2011; Liu et al., 2012b). Therefore, Exo-III mediated
signal amplification enables the fabrication of an electrochemical DNA
biosensor for the detection of Hg2+(Xuan et al., 2013). In this
immobilization-free electrochemical approach, the MB modified T-
rich ss-DNA folds into a hairpin structure upon coordination with
Hg2+. Subsequently, it acts as the substrate for EXO-III. When the
digestion reaction is completed, Hg2+ can be released from the duplex
for further coordination with another T-rich ss-DNA (Fig. 5C). The
cyclic shuffling of Hg2+ between the solution phase and the solid DNA
produces a highly sensitive biosensor.
4.2. C–C mismatches based electrochemical biosensor for Ag+
detection
Electrochemical detection of Ag+ ion by DNA biosensors is un-
common compared to detection of Hg2+. However, the application of
this technique for the detection of Ag+ is gaining interest with time.
4.2.1. C–Ag+–C coordination based simple approach
Ebrahimi et al. (2015) reported a simple C–Ag+–C co-ordination
M.R. Saidur et al. Biosensors and Bioelectronics 90 (2017) 125–139

Fig. 3. Probe structural switch signal-on biosensors. (A) Intramolecular folding of probe ss-DNA to a hairpin structure (Han et al., 2009). (B) Based on the competing conformation
among the probe hairpin and Hg2+ induced duplex (Zhuang et al., 2013). (C) Based on analysis of current ratio between MB and Fc (Xiong et al., 2015).

based biosensor for Ag+ detection by immobilization of 100% poly-C
oligonucleotide strands on both carbon paste electrode (CPE) and
AuNPs modified CPE. The Ag+ concentration was successfully deter-
mined in the real sample by voltammetry. The AuNPs modified
electrode achieved a better LOD compared to CPE. Yang et al. (2015)
designed another DNA biosensor for Ag+ through electrode modifica-
tion with Fe3O4@3D-GO and achieved an excellent limit of detection,
which was 2.0 pM of Ag+ in the range of 0.01–100 nM.
4.2.2. Based on C–Ag+–C coordination trigger structural switching of
probe DNA
Yan et al. (2012) developed a sensitive DNA biosensor based on the
C–Ag+–C induced structural switch neither with immobilization of the
probe DNA, nor labeling with any redox tag. Fig. 6A shows that a gold
electrode was modified with a monolayer of 16-mercaptohexadecanoic
acid (MHA) via self-assembly in the aqueous phase. The C-rich ss-DNA
was warped with multiwalled carbon nanotubes (MWCNTs) by π-π
interaction in the absence of Ag+. The electrode was isolated from the
electroactive indicator by MHA/SAM. In the presence of Ag+, the ss-
DNA formed a hairpin structure by C–Ag+–C. Thus, the MWCNT

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Fig. 4. (A) Associated with the intermolecular T–Hg2+–T duplex formation (Liu et al., 2009c). (B) Using the replacement of a complementary ss-DNA and investigation of the redox
current of Fc tags on ss-DNA (Wu et al., 2010a). and (C) Also based on the replacement of a complementary ss-DNA and analysis the chronocoulometric (CC) signal of RuHex (Chen
et al., 2012).
became free and attached on the electrode, leading to the electron
transfer process between the indicator and electrode. Therefore, the
sensor switched between “signal-off” to “signal-on” and was controlled
by the presence of Ag+ ion. Most recently, Zhou et al. (2015) proposed a
highly sensitive DNA biosensor based on Ag+ inducing structural
switch of probe-DNA. Upon immobilization of the probe ss-DNA (S1)
onto a nanoporous gold modified glassy carbon electrode (NPG/GCE),
it was hybridized with two types of ss-DNA (S2 and S3) (Fig. 6B). These
hybridizations initially formed a hairpin-like structure. However, in the
presence of Ag+ ions, the hairpin-like structure turned into a rigid
duplex like structure by forming the C–Ag+–C base pair which
subsequently increased the adsorption of electroactive signal inter-
calator disodium anthraquinone-2,6-disulfonate (AQDS). Higher elec-
tron conductivity of NPG-modified electrode enabled the detection of
0.048 nM of Ag+, with a wide linear range in square wave voltammetry
(SWV).
C–Ag+–C could also initiate the activity of the Exo-III like the T–
Hg –T duplex (Xuan et al., 2013). Therefore, a DNA biosensor with
2+
amplified signals was developed by Xu et al. (2013) by using EXO-III.
The signal of MB indicators could be amplified (Fig. 6C) with the
recycling of Ag+ from C–Ag+–C complex by the EXO-III activity.
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5. Heavy metal ion biosensors based on cleavage of
DNAzymes
Highly sensitive electrochemical biosensors have been developed
for the detection of heavy metal ions based on the excellent metal
selectivity of the DNAzymes, as summarized in Table 2. Xiao et al.
(2007) designed an electrochemical biosensor for the detection of Pb2+,
using an 8-17E lead dependent DNAzyme. Initially, they immobilized
the MB functionalized enzyme strand on a gold electrode. Then, the
DNAzyme was hybridized with the substrate strand that prohibited the
redox-active MB molecule from touching the electrode. However, in the
presence of Pb2+, it cleaved the substrate strand and made the enzyme
strand more flexible (Fig. 7A). Therefore, MB could move closer to the
electrode and facilitated the electron transfer process between the
electrode and MB. This electrochemical communication produced an
electrochemical signal, proportional to the Pb2+ concentration. This
biosensor was able to detect Pb2+ in soil samples and successfully
reached a detection limit of 300 nM. Similarly, Zhang et al. (2016)
reported higher sensitivity with Fc.
Shen et al. (2008) reported a signal amplification assay for the
detection of Pb2+ based on DNA-Au bio-barcode. Fig. 7B shows that a
large number of oligonucleotides strands called the bio-barcode DNA,
were functionalized with AuNPs. Concurrently, a Pb2+ dependent
M.R. Saidur et al. Biosensors and Bioelectronics 90 (2017) 125–139

Fig. 5. Schematic illustration of different signal amplification strategy: (A) Using AuNPs decorated by MSO probes and linking ss-DNA (Zhu et al., 2009b). (B) AuNPs mediated three-
dimensional fishnet electrochemical biosensor and amplified DPV curves (Tang et al., 2012). (C) Signal amplification associated with Exonuclease-III (Xuan et al., 2013).

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M.R. Saidur et al. Biosensors and Bioelectronics 90 (2017) 125–139

Fig. 6. Schematic presentation of (A) turn-off and turn-on strategy by controlled assembly of MWCNTs (Yan et al., 2012). (B) Surface fabrication and developed biosensor for Ag+
detection (Zhou et al., 2015). (C) Reshuffling of Ag+ by EXO-III activity and signal amplification (Xu et al., 2013).

Table 2
Heavy metal ion biosensors based on cleavage of DNAzymes.

Electrode Analyte HM Signal amplification Electrochemical Electrochemical Redox LOD Linear range Reference
material technique technique indicator

Gold Pb2+ DPV MB 300 nM 0.5–10 µm Xiao et al.

(2007)
Gold DPV Fc 0.25 nM 0.5 nM−5 µm Zhang et al.
(2016)
Gold AuNPs based bio barcode DPV [Ru(NH3)6]3+ 1 nM 5 nM−0.1 µm Shen et al.
(2008)
Gold AuNPs based CC [Ru(NH3)6]3+ 0.028 nM 0.1 nM−0.035 µm Yang et al.
amplification (2010b)
Magnetic Beads RCA amplification SWSV MB 7.8 pM 0.01 nM−1.0 µm Tang et al.
(2013)

Gold Cu2+ SWV Fc 20 nM 20 nM−600 µm Li et al. (2010)

AuNPs/Gold SWV Fc 0.1 pM 0.1 nM−10 µm Chen et al.
(2011)
GNCs EIS Fe(CN)64-/3- 0.0725 nM 0.1 nM−400 nM Hu et al. (2016)
Gold UO22+ HCR signal amplification SWV MB 20 pM 0.05 nM−4 nM Yun et al. (2016)

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M.R. Saidur et al. Biosensors and Bioelectronics 90 (2017) 125–139

Fig. 7. (A) A simple approach of DNAzyme based electrochemical detection of Pb2+(Xiao et al., 2007). (B) Schematic illustration of signal enhancing strategy using AuNPs (bio-barcode)
(Shen et al., 2008). (C) The principal of signal amplification by surface bound capture ss-DNA and AuNPs (Yang et al., 2010b). (D) Signal amplification by RCA for Pb2+ detection (Tang
et al., 2013).

DNAzyme was immobilized on a gold electrode and hybridized with the
substrate strand. Interestingly, the substrate strand was pre-designed
in such a way that it could also hybridize the DNA-Au bio-barcode
through an overhang. Therefore, a large amount of [Ru(NH3)6]3+ redox
mediator could be attached to the anionic phosphate in the DNA
backbone by simple electrostatic interaction. However, upon the
incubation with Pb2+, the catalytic reaction cleaved the substrate
strands into two pieces and detaches it from the catalytic strand. As
a result, an instant decrease in the amount of redox mediator on the
electrode surface and a sharp fall in electrochemical signals in DPV was
observed. In spite of sensitivity, it was a troublesome and time-
consuming method especially for field applications. Yang et al.
(2010b) constructed an extremely amplified biosensor for Pb2+ detec-
tion using AuNPs. The AuNPs was decorated with lead dependent
DNAzyme. Then, a capture ss-DNA was immobilized on the gold
electrode, as a complementary to the enzyme strand. When incubated
with the AuNPs modified DNAzyme (dsDNA-AuNPs) in Pb2+ solution,
it cleaved the substrate strand and the AuNPs modified enzyme strand
became free. Immediately, it was hybridized with the capture ss-DNA
on the gold electrode. This increased the adsorption of the redox
mediator on the DNA backbone and enhanced the electrochemical
signal (Fig. 7C). This signal amplification led to an outstanding
detection limit of 0.028 nM of Pb2+.
Recently, Tang et al. (2013) demonstrated a different signal
amplification strategy for the sensing of Pb2+ based on rolling circle
amplification (RCA) using cadmium-sulfide quantum dots (CdS-QD)
nanoparticles. As shown in Fig. 7D, upon the Pb2+ mediated cleavage,
the catalytic strand on the magnetic beads surface started to hybridize

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with the two ends of the RCA template and formed a circular RCA
template. In the presence of a polymerase, this RCA process produced
long ss-DNA with repeating units, which subsequently hybridized with
previously modified ss-DNA by CdS-QDs. Thereafter, a long DNA-
duplex with a repeating arrangement of QDs was produced, and
extremely low level of Pb2+(7.8 pM) could be detected. Even though
this method could detect extremely low level of concentration, the
application is limited due to the highly complicated procedures.
All the DNAzymes based biosensors described above are for the
detection of Pb2+, because biosensors for other heavy metal ions are
still very limited. Li et al. (2010) successfully designed a facile
DNAzyme based biosensor for the detection of copper (Cu2+) by
applying a Cu2+ dependent self-cleavage of DNAzyme composed of
87-nucleotides. Later, Chen et al. (2011) also proposed another
biosensor using a similar strategy with increased LOD. The large
surface area of AuNPs was able to immobilize a maximum number of
DNAzymes while decreasing the non-specific adsorption, thereby
increasing the sensitivity. In SWV, this sensor could reach a LOD of
0.1 pM, more sensitive than the previous one. A label-free biosensor for
Cu2+ detection was developed by Hu et al. (2016), where gold
nanoclusters (GNCs) were used as the immobilization platform for
DNAzymes and Fe(CN)64−/3− as the redox marker in EIS. Yun et al.
(2016) developed an ultrasensitive DNAzyme based biosensors for
uranium oxide ion (UO22+). Upon immobilization of a UO22+ specific
DNAzymes onto the gold electrode, two other hairpin DNA were used
in the UO22+ containing solution for the hybridization chain reaction
(HCR). The HCR amplification strategy produced a long nicked ds-
DNA, where a large number of MB could intercalate and produce a
highly sensitive electrochemical signal. In SWV, this biosensor could
detect concentration as low as 20 pM UO22+.
6. Heavy metal ion biosensors based on stable G-quadruplex
DNA formation
Lin et al. (2011) designed a label-free DNA biosensor for Pb2+
detection using [Fe(CN)6]4−/3− as a redox probe with the EIS method.
In the presence of Pb2+, the surface immobilized G-rich hairpin-like
DNA-probe opens its steam loop and switches to form a G4-DNA
structure, while the charge transfer resistance increases sharply. This
charge transfer resistance increases linearly with the of Pb2+ concen-
tration and this sensor could detect concentrations as low as 0.5 nM
Pb2+. Some smaller molecules have a selective binding affinity towards
G4-DNA over ss-DNA or ds-DNA through intercalation, such as crystal
violet (CV) (Kong et al., 2009). Therefore, Li et al. (2011) constructed
an electrochemical amperometric biosensor for the detection of Pb2+,
based on the specific interaction between G4-DNA and CV (Fig. 8A). A
linear correlation was found between the electrochemical response of
CV and the logarithm of Pb2+ concentration. The sensor could detect up
to 0.4 nM of Pb2+ within a dynamic range without the use of any
sophisticated equipment.
It has been reported that DNA G-quadruplex possesses peroxidase-
like activity when combined with hemin and is able to catalyze the
peroxidase reactions (Pelossof et al., 2010). The catalytic activity of
peroxidase-mimicking DNAzyme is also used in electrochemical bio-
sensors for the detection of heavy metal ions. For example, an
amperometric ‘signal-on’ sensing platform of Pb2+ was successfully
demonstrated by Li et al. (2013). The intercalation of hemin to the
Pb2+-conveyed G4-DNA initiates the catalytic activity of horseradish
peroxidase (HRP)-mimicking DNAzyme for the oxidative polymeriza-
tion of aniline (Fig. 8B). Therefore, the polyaniline (PANI) deposited on
the electrode can turn-on the electrochemical signal.
Zhang et al. (2015) also proposed a highly amplified sensing
platform for Pb2+ using hemin/G-quadruplex mediated HRP-mimick-
ing DNAzyme catalytic activity. The proposed biosensor could detect
Pb2+ concentration of as low as 32 pM through a complicated
procedure. After the cleavage, the substrate strand of a Pb2+ dependent
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Biosensors and Bioelectronics 90 (2017) 125–139
DNAzyme initiates the hybridization with two other auxiliary ss-DNA
strands, in an alternate way to produce a nicked double helix. With the
addition of hemin, the aptamer forms the hemin/G-quadruplex
DNAzyme, catalyzing the polymerization of aniline to form polyaniline
along with the ds-DNA, as shown in Fig. 8C.
7. Conclusion and future research
Heavy metal pollution is of great concern since it poses threat to the
public health and the environment. Electrochemical transduction
based DNA biosensors provide a convenient platform for early detec-
tion of heavy metal contamination. In this review, the principles of
heavy metal ion sensing by DNA biosensors were elaborated, with
particular emphasis on their selectivity and sensitivity. The recent
advancements in electrochemical DNA biosensors were also high-
lighted.
Literature shows that direct electrochemical redox signal based
electrochemical biosensors are easy to use. One of the challenges of
electrochemical biosensors is the background current which supressed
the electrical signals. However, the use of electrochemical redox
indicators/reporters could amplify the electrochemical signals. Some
excellent DNA biosensors have been developed for Hg2+, Ag+, Pb2+
using different electrochemical redox indicators/reporters. Gold is the
common choice as electrode material because it immobilizes the probe-
DNA through thiol-gold interaction. One option for achieving the
maximum hybridization efficiency and increasing detection limit in
DNA biosensor is to employ an upright configuration of probe ss-DNA.
However, the neutron reflectivity analysis confirms that the probe DNA
lies flat with multiple adsorption points on the gold surface as DNA
bases have a strong affinity for gold. Consequently, the hybridization
efficiency is low, usually about 10% or less (Levicky et al., 1998).
Obviously, the non-specific adsorption remains the major challenge
that limits broadspectrum detection. Thus, the fabrication of a working
electrode with improved specificity and sensitivity is one of the hall-
marks in the future. Different signal amplification strategies using
AuNPs, RCA and EXO-III activity could facilitate the transformation of
a trace amount of target metal ions into profuse DNA for subsequent
signal information. Despite the reported potentials these biosensors in
simple laboratory experiments, their suitability for complex environ-
mental samples needs to be verified.
The portability of the device is also an important criterion for
continuous monitoring of the samples. Thus, miniature electrodes are
desirable. However, for higher sensitivity and amplified signal, a
maximum number of probe-DNA must be immobilized on the elec-
trode surface. To this end, combining the interdigitated nanostructured
electrode with nanomaterials could address this problem. Recently,
graphene has attracted tremendous attention for the fabrication of
electrode surface. Graphene is an efficient material for the immobiliza-
tion of DNA probe without changing the stability and biological
activities of the DNA (Fang and Wang, 2013).
Since the current electrochemical DNA biosensors could only
selectively detect a single heavy metal ion, there is a need to develop
biosensors that could simultaneously detect multiple heavy metal ions.
Currently, most of the developed electrochemical DNA-based biosen-
sors are suitable for the detection of a limited number of heavy metal
ions such as Hg2+, Pb2+, Ag+, Cu2+, and UO22+. Future research should
focus on the design of new DNA sequences and DNAzymes for the
detection of other hazardous heavy metal ions such as cadmium (Cd2+),
chromium (Cr3+), iron (Fe2+ and Fe3+), arsenic (As6+) and nickel
(Ni2+). In the future, spectro-electrochemical measurements could be
further explored, as this technique potentially offers a new insight into
the mechanism of DNA biosensors by combining the electronic and
optical responses.
M.R. Saidur et al. Biosensors and Bioelectronics 90 (2017) 125–139

Fig. 8. Electrochemical detection of Pb2+ based on G-quadruplex DNA formation: (A) interaction of CV with Pb2+ induced G-quadruplex DNA as a signal producer (Li et al., 2011). (B)
HRP-mimicking DNAzyme catalyzed template guided deposition of polyaniline on the electrode (Li et al., 2013). (C) Hemin/G4-DNAzymes catalyze polymerization of aniline along ds-
DNA (Zhang et al., 2015).

Acknowledgements References

The authors are grateful to University of Malaya High Impact
Research Grant (UM.C/625/1/HIR/MOHE/ENG/37) from Ministry of
Higher Education Malaysia and PG149-2015B for financially support-
ing this work.
Alegret, S., Merkoçi, A., 2007. Electrochemical sensor analysis first ed.. Elsevier,
Amsterdam.
Aragay, G., Merkoçi, A., 2012. Nanomaterials application in electrochemical detection of
heavy metals. Electrochim. Acta 84, 49–61.
Aranda, P.R., Pacheco, P.H., Olsina, R.A., Martinez, L.D., Gil, R.A., 2009. Total and
inorganic mercury determination in biodiesel by emulsion sample introduction and
FI-CV-AFS after multivariate optimization. J. Anal. At. Spectrom. 24 (10),
1441–1445.

137
M.R. Saidur et al.
Ashoka, S., Peake, B.M., Bremner, G., Hageman, K.J., Reid, M.R., 2009. Comparison of
digestion methods for ICP-MS determination of trace elements in fish tissues. Anal.
Chim. Acta 653 (2), 191–199.
Bagchi, D., Bagchi, M., Stohs, S.J., Das, D.K., Ray, S.D., Kuszynski, C.A., Joshi, S.S.,
Pruess, H.G., 2000. Free radicals and grape seed proanthocyanidin extract:
importance in human health and disease prevention. Toxicol 148 (2), 187–197.
Balagurumoorthy, P., Brahmachari, S.K., 1994. Structure and stability of human
telomeric sequence. J. Biol. Chem. 269 (34), 21858–21869.
Barnham, K.J., Masters, C.L., Bush, A.I., 2004. Neurodegenerative diseases and oxidative
stress. Nat. Rev. Drug Discov. 3 (3), 205–214.
Breaker, R.R., Joyce, G.F., 1994. A DNA enzyme that cleaves RNA. Chem. Biol. 1 (4),
223–229.
Brown, A.K., Li, J., Pavot, C.M.-B., Lu, Y., 2003. A lead-dependent DNAzyme with a two-
step mechanism. Biochemistry 42 (23), 7152–7161.
Cao, R.G., Zhu, B., Li, J., Xu, D., 2009. Oligonucleotides-based biosensors with high
sensitivity and selectivity for mercury using electrochemical impedance
spectroscopy. Electrochem. Commun. 11 (9), 1815–1818.
Chen, C., Du, Y., Li, J., Yang, X., Wang, E., 2012. Fabrication of a sensor chip containing
Au and Ag electrodes and its application for sensitive Hg (II) determination using
chronocoulometry. Anal. Chim. Acta 738, 45–50.
Chen, Z., Li, L., Mu, X., Zhao, H., Guo, L., 2011. Electrochemical aptasensor for detection
of copper based on a reagentless signal-on architecture and amplification by gold
nanoparticles. Talanta 85 (1), 730–735.
Clever, G.H., Kaul, C., Carell, T., 2007. DNA–metal base pairs. Angew. Chem. Int. Ed. 46
(33), 6226–6236.
Drummond, T.G., Hill, M.G., Barton, J.K., 2003. Electrochemical DNA sensors. Nat.
Biotech. 21 (10), 1192–1199.
Ebrahimi, M., Raoof, J.B., Ojani, R., 2015. Novel electrochemical DNA hybridization
biosensors for selective determination of silver ions. Talanta 144, 619–626.
Fang, Y., Wang, E., 2013. Electrochemical biosensors on platforms of graphene. Chem.
Commun. 49 (83), 9526–9539.
Flamini, R., Panighel, A., 2006. Mass spectrometry in grape and wine chemistry. Part II:
the consumer protection. Mass Spectrom. Rev. 25 (5), 741–774.
Freeman, R., Liu, X., Willner, I., 2011. Amplified multiplexed analysis of DNA by the
exonuclease III-catalyzed regeneration of the target DNA in the presence of
functionalized semiconductor quantum dots. Nano Lett. 11 (10), 4456–4461.
Gong, J.-L., Sarkar, T., Badhulika, S., Mulchandani, A., 2013. Label-free chemiresistive
biosensor for mercury (II) based on single-walled carbon nanotubes and structure-
switching DNA. Appl. Phys. Lett. 102 (1), 013701.
Griffiths, C., Klemick, H., Massey, M., Moore, C., Newbold, S., Simpson, D., Walsh, P.,
Wheeler, W., 2012. US Environmental Protection Agency valuation of surface water
quality improvements. Rev. Environ. Econ. Policy 6 (1), 130–146.
Guschlbauer, W., Chantot, J.-F., Thiele, D., 1990. Four-stranded nucleic acid structures
25 years later: from guanosine gels to telomer DNA. J. Biomol. Struct. Dyn. 8 (3),
491–511.
Han, D., Kim, Y.R., Oh, J.W., Kim, T.H., Mahajan, R.K., Kim, J.S., Kim, H., 2009. A
regenerative electrochemical sensor based on oligonucleotide for the selective
determination of mercury(II). Analyst 134 (9), 1857–1862.
Hellström, L., Elinder, C.-G., Dahlberg, B., Lundberg, M., Järup, L., Persson, B., Axelson,
O., 2001. Cadmium exposure and end-stage renal disease. Am. J. Kidney Dis. 38 (5),
1001–1008.
Hermann, T., Patel, D.J., 2000. Adaptive recognition by nucleic acid aptamers. Science
287 (5454), 820–825.
Hollenstein, M., Hipolito, C., Lam, C., Dietrich, D., Perrin, D.M., 2008. A highly selective
DNAzyme sensor for mercuric ions. Angew. Chem. Int. Ed. 47 (23), 4346–4350.
Howard, F.B., Miles, H.T., 1982. Poly (inosinic acid) helixes: essential chelation of alkali
metal ions in the axial channel. Biochemistry 21 (26), 6736–6745.
Hu, W., Min, X., Li, X., Yang, S., Yi, L., Chai, L., 2016. DNAzyme catalytic beacons-based
a label-free biosensor for copper using electrochemical impedance spectroscopy. RSC
Adv. 6 (8), 6679–6685.
Huang, P.J.J., Vazin, M., Matuszek, Z., Liu, J.W., 2015. A new heavy lanthanide-
dependent DNAzyme displaying strong metal cooperativity and unrescuable
phosphorothioate effect. Nucl. Acids Res. 43 (1), 461–469.
Hughes, M.F., Beck, B.D., Chen, Y., Lewis, A.S., Thomas, D.J., 2011. Arsenic exposure
and toxicology: a historical perspective. Toxicol. Sci. 123 (2), 305–332.
Jamali, M.R., Assadi, Y., Shemirani, F., Hosseini, M.R.M., Kozani, R.R., Masteri-
Farahani, M., Salavati-Niasari, M., 2006. Synthesis of salicylaldehyde-modified
mesoporous silica and its application as a new sorbent for separation,
preconcentration and determination of uranium by inductively coupled plasma
atomic emission spectrometry. Anal. Chim. Acta 579 (1), 68–73.
Järup, L., 2003. Hazards of heavy metal contamination. Br. Med. Bull. 68 (1), 167–182.
Kong, D.M., Ma, Y.E., Wu, J., Shen, H.X., 2009. Discrimination of G‐quadruplexes from
duplex and single‐stranded DNAs with fluorescence and energy‐transfer fluorescence
spectra of crystal violet. Chem. -Eur. J. 15 (4), 901–909.
Labuda, J., Brett, A.M.O., Evtugyn, G., Fojta, M., Mascini, M., Ozsoz, M., Palchetti, I.,
Paleček, E., Wang, J., 2010. Electrochemical nucleic acid-based biosensors:
concepts, terms, and methodology (IUPAC Technical Report). Pure Appl. Chem. 82
(5), 1161–1187.
Lan, T., Furuya, K., Lu, Y., 2010. A highly selective lead sensor based on a classic lead
DNAzyme. Chem. Commun. 46 (22), 3896–3898.
Lan, T., Lu, Y., 2012. Metal ion-dependent DNAzymes and their applications as
biosensors. In: Sigel, A., Sigel, H., R.K.O (Eds.), Interplay between Metal Ions and
Nucleic Acids. Springer, Netherlands, 217–248, (Po Box 17, 3300 Aa Dordrecht).
Levicky, R., Herne, T.M., Tarlov, M.J., Satija, S.K., 1998. Using self-assembly to control
the structure of DNA monolayers on gold: a neutron reflectivity study. J. Am. Chem.
Soc. 120 (38), 9787–9792.
138
Biosensors and Bioelectronics 90 (2017) 125–139
Li, F., Feng, Y., Zhao, C., Tang, B., 2011. Crystal violet as a G-quadruplex-selective probe
for sensitive amperometric sensing of lead. Chem. Commun. 47 (43), 11909–11911.
Li, F., Yang, L., Chen, M., Qian, Y., Tang, B., 2013. A novel and versatile sensing platform
based on HRP-mimicking DNAzyme-catalyzed template-guided deposition of
polyaniline. Biosens. Bioelectron. 41 (1), 903–906.
Li, J., Lu, Y., 2000. A highly sensitive and selective catalytic DNA biosensor for lead ions.
J. Am. Chem. Soc. 122 (42), 10466–10467.
Li, J., Zheng, W., Kwon, A.H., Lu, Y., 2000. In vitro selection and characterization of a
highly efficient Zn (II)-dependent RNA-cleaving deoxyribozyme. Nucl. Acids Res. 28
(2), 481–488.
Li, L., Luo, L., Mu, X., Sun, T., Guo, L., 2010. A reagentless signal-on architecture for
electronic, real-time copper sensors based on self-cleavage of DNAzymes. Anal.
Methods 2 (6), 627–630.
Li, L., Wen, Y., Xu, L., Xu, Q., Song, S., Zuo, X., Yan, J., Zhang, W., Liu, G., 2016.
Development of Mercury (II) Ion biosensors based on Mercury-specific
oligonucleotide probes. Biosens. Bioelectron. 75, 433–445.
Lidsky, T.I., Schneider, J.S., 2003. Lead neurotoxicity in children: basic mechanisms and
clinical correlates. Brain 126 (1), 5–19.
Lin, Z., Chen, Y., Li, X., Fang, W., 2011. Pb2+ induced DNA conformational switch from
hairpin to G-quadruplex: electrochemical detection of Pb2+. Analyst 136 (11),
2367–2372.
Liu, A., Wang, K., Weng, S., Lei, Y., Lin, L., Chen, W., Lin, X., Chen, Y., 2012a.
Development of electrochemical DNA biosensors. Trends Anal. Chem. 37, 101–111.
Liu, C.-W., Huang, C.-C., Chang, H.-T., 2009a. Highly selective DNA-based sensor for
lead (II) and mercury (II) ions. Anal. Chem. 81 (6), 2383–2387.
Liu, G., Wan, Y., Zou, Z.Y., Ren, S.Z., Fan, C.H., 2011. Deoxyribonucleic acid molecular
design for electrochemical biosensors. Fenxi Huaxue Chin. J. Anal. Chem. 39 (7),
953–962.
Liu, J., Brown, A.K., Meng, X., Cropek, D.M., Istok, J.D., Watson, D.B., Lu, Y., 2007. A
catalytic beacon sensor for uranium with parts-per-trillion sensitivity and millionfold
selectivity. Proc. Natl. Acad. Sci. USA 104 (7), 2056–2061.
Liu, J., Cao, Z., Lu, Y., 2009b. Functional nucleic acid sensors. Chem. Rev. 109 (5),
1948–1998.
Liu, J., Lu, Y., 2003. A colorimetric lead biosensor using DNAzyme-directed assembly of
gold nanoparticles. J. Am. Chem. Soc. 125 (22), 6642–6643.
Liu, J., Lu, Y., 2007. A DNAzyme catalytic beacon sensor for paramagnetic Cu2+ ions in
aqueous solution with high sensitivity and selectivity. J. Am. Chem. Soc. 129 (32),
9838–9839.
Liu, S.J., Nie, H.G., Jiang, J.H., Shen, G.L., Yu, R.Q., 2009c. Electrochemical sensor for
mercury(II) based on conformational switch mediated by interstrand cooperative
coordination. Anal. Chem. 81 (14), 5724–5730.
Liu, X., Aizen, R., Freeman, R., Yehezkeli, O., Willner, I., 2012b. Multiplexed aptasensors
and amplified DNA sensors using functionalized graphene oxide: application for
logic gate operations. ACS Nano 6 (4), 3553–3563.
Liu, Y., Sen, D., 2008. A contact photo-cross-linking investigation of the active site of the
8-17 deoxyribozyme. J. Mol. Bio 381 (4), 845–859.
Lorber, K.E., 1986. Monitoring of heavy metals by energy dispersive X-ray fluorescence
spectrometry. Waste Manag. Res. 4 (1), 3–13.
Martin, S., Griswold, W., 2009. Human health effects of heavy metals. Environ. Sci.
Technol. Br. Cit. 15, 1–6.
Miao, P., Liu, L., Li, Y., Li, G., 2009. A novel electrochemical method to detect mercury
(II) ions. Electrochem. Commun. 11 (10), 1904–1907.
Miyake, Y., Togashi, H., Tashiro, M., Yamaguchi, H., Oda, S., Kudo, M., Tanaka, Y.,
Kondo, Y., Sawa, R., Fujimoto, T., 2006. MercuryII-mediated formation of thymine-
HgII-thymine base pairs in DNA duplexes. J. Am. Chem. Soc. 128 (7), 2172–2173.
Nelson, K.E., Ihms, H.E., Mazumdar, D., Bruesehoff, P.J., Lu, Y., 2012. The importance
of peripheral sequences in determining the metal selectivity of an in vitro‐selected
Co2+‐dependent DNAzyme. ChemBioChem 13 (3), 381–391.
Niu, X., Ding, Y., Chen, C., Zhao, H., Lan, M., 2011,a. A novel electrochemical biosensor
for Hg2+ determination based on Hg2+-induced DNA hybridization. Sens. Actuators
B: Chem. 158 (1), 383–387.
Ono, A., Cao, S., Togashi, H., Tashiro, M., Fujimoto, T., Machinami, T., Oda, S., Miyake,
Y., Okamoto, I., Tanaka, Y., 2008. Specific interactions between silver (I) ions and
cytosine–cytosine pairs in DNA duplexes. Chem. Commun. 39, 4825–4827.
Ono, A., Togashi, H., 2004. Highly selective oligonucleotide‐based sensor for mercury (II)
in aqueous solutions. Angew. Chem. Int. Ed. 43 (33), 4300–4302.
Organization, W.H., 2004. Guidelines for drinking-water quality: recommendations.
World Health Organization, (WHO),Geneva.
Panyala, N.R., Peña-Méndez, E.M., Havel, J., 2008. Silver or silver nanoparticles: a
hazardous threat to the environment and human health. J. Appl. Biomed. 6 (3),
117–129.
Park, H., Hwang, S.J., Kim, K., 2012. An electrochemical detection of Hg2+ ion using
graphene oxide as an electrochemically active indicator. Electrochem. Commun. 24
(1), 100–103.
Pelossof, G., Tel-Vered, R., Elbaz, J., Willner, I., 2010. Amplified biosensing using the
horseradish peroxidase-mimicking DNAzyme as an electrocatalyst. Anal. Chem. 82
(11), 4396–4402.
Pescod, M.B., 1992. Wastewater treatment and use in agricultureBull. 47. FAO, Rome,
125.
Pohl, P., 2009. Determination of metal content in honey by atomic absorption and
emission spectrometries. Trends Anal. Chem. 28 (1), 117–128.
Ronkainen, N.J., Halsall, H.B., Heineman, W.R., 2010. Electrochemical biosensors.
Chem. Soc. Rev. 39 (5), 1747–1763.
Sassolas, A., Leca-Bouvier, B.D., Blum, L.J., 2008. DNA biosensors and microarrays.
Chem. Rev. 108 (1), 109–139.
Shen, L., Chen, Z., Li, Y., He, S., Xie, S., Xu, X., Liang, Z., Meng, X., Li, Q., Zhu, Z., 2008.
M.R. Saidur et al.
Electrochemical DNAzyme sensor for lead based on amplification of DNA−Au Bio-
Bar codes. Anal. Chem. 80 (16), 6323–6328.
Shi, H., Hudson, L.G., Liu, K.J., 2004. Oxidative stress and apoptosis in metal ion-
induced carcinogenesis. Free Rad. Bio. Med 37 (5), 582–593.
Smirnov, I., Shafer, R.H., 2000. Lead is unusually effective in sequence-specific folding of
DNA. J. Mol. Bio. 296 (1), 1–5.
Smith, A.H., Steinmaus, C.M., 2009. Health effects of arsenic and chromium in drinking
water: recent human findings. Annu. Rev. Public Health 30, 107.
Stohs, S., Bagchi, D., 1995. Oxidative mechanisms in the toxicity of metal ions. Free Rad.
Bio. Med 18 (2), 321–336.
Tanaka, Y., Oda, S., Yamaguchi, H., Kondo, Y., Kojima, C., Ono, A., 2007. 15N–15N J-
coupling across Hg (II): direct observation of Hg (II)-mediated TT base pairs in a
DNA duplex. J. Am. Chem. Soc. 129 (2), 244–245.
Tang, S., Tong, P., Li, H., Tang, J., Zhang, L., 2013. Ultrasensitive electrochemical
detection of Pb2+ based on rolling circle amplification and quantum dots tagging.
Biosens. Bioelectron. 42 (1), 608–611.
Tang, X., Liu, H., Zou, B., Tian, D., Huang, H., 2012. A fishnet electrochemical Hg2+
sensing strategy based on gold nanoparticle-bioconjugate and thymine-Hg 2+-
thymine coordination chemistry. Analyst 137 (2), 309–311.
Thévenot, D.R., Toth, K., Durst, R.A., Wilson, G.S., 2001. Electrochemical biosensors:
recommended definitions and classification. Biosens. Bioelectron. 16 (1), 121–131.
Tortolini, C., Bollella, P., Antonelli, M.L., Antiochia, R., Mazzei, F., Favero, G., 2015a.
DNA-based biosensors for Hg2+ determination by polythymine-methylene blue
modified electrodes. Biosens. Bioelectron. 67, 524–531.
Tortolini, C., Bollella, P., Antonelli, M.L., Antiochia, R., Mazzei, F., Favero, G., 2015b.
DNA-based biosensors for Hg2+ determination by polythymine-methylene blue
modified electrodes. Biosens. Bioelectron. 67, 524–531.
Uriu-Adams, J.Y., Keen, C.L., 2005. Copper, oxidative stress, and human health. Mol.
Asp. Med. 26 (4), 268–298.
Valko, M., Rhodes, C., Moncol, J., Izakovic, M., Mazur, M., 2006. Free radicals, metals
and antioxidants in oxidative stress-induced cancer. Chem. Biol. Inter. 160 (1),
1–40.
Varghese, N., Mogera, U., Govindaraj, A., Das, A., Maiti, P.K., Sood, A.K., Rao, C.N.R.,
2009. Binding of DNA nucleobases and nucleosides with graphene. ChemPhysChem
10 (1), 206–210.
Wang, W., Billen, L.P., Li, Y., 2002. Sequence diversity, metal specificity, and catalytic
proficiency of metal-dependent phosphorylating DNA enzymes. Chem. Biol. 9 (4),
507–517.
Wen, Y., Xing, F., He, S., Song, S., Wang, L., Long, Y., Li, D., Fan, C., 2010. A graphene-
based fluorescent nanoprobe for silver (I) ions detection by using graphene oxide and
a silver-specific oligonucleotide. Chem. Commun. 46 (15), 2596–2598.
Wu, D., Zhang, Q., Chu, X., Wang, H., Shen, G., Yu, R., 2010a. Ultrasensitive
electrochemical sensor for mercury(II) based on target-induced structure-switching
DNA. Biosens. Bioelectron. 25 (5), 1025–1031.
Wu, J., Li, L., Shen, B., Cheng, G., He, P., Fang, Y., 2010b. Polythymine oligonucleotide-
modified gold electrode for voltammetric determination of mercury(II) in aqueous
solution. Electroanalysis 22 (4), 479–482.
Xiao, Y., Rowe, A.A., Plaxco, K.W., 2007. Electrochemical detection of parts-per-billion
lead via an electrode-bound DNAzyme assembly. J. Am. Chem. Soc. 129 (2),
262–263.
Xiong, E., Wu, L., Zhou, J., Yu, P., Zhang, X., Chen, J., 2015. A ratiometric
electrochemical biosensor for sensitive detection of Hg2+ based on thymine-Hg2+-
thymine structure. Anal. Chim. Acta 853 (1), 242–248.
Xu, E., Lv, Y., Liu, J., Gu, X., Zhang, S., 2015. An electrochemical study based on
thymine-Hg2+-thymine DNA base pair mediated charge transfer processes. RSC Adv.
139
Biosensors and Bioelectronics 90 (2017) 125–139
5 (61), 49819–49823.
Xu, G., Wang, G., He, X., Zhu, Y., Chen, L., Zhang, X., 2013. An ultrasensitive
electrochemical method for detection of Ag+ based on cyclic amplification of
exonuclease-III activity on cytosine-Ag+-cytosine. Analyst 138 (22), 6900–6906.
Xu, Q., Cao, A., Zhang, L.-f., Zhang, C.-y., 2012. Rapid and label-free monitoring of
exonuclease III-assisted target recycling amplification. Anal. Chem. 84 (24),
10845–10851.
Xuan, F., Luo, X., Hsing, I.-M., 2013. Conformation-dependent exonuclease-III activity
mediated by metal ions reshuffling on thymine-rich DNA-duplexes for an
ultrasensitive electrochemical method for Hg2+ detection. Anal. Chem. 85 (9),
4586–4593.
Yan, G., Wang, Y., He, X., Wang, K., Su, J., Chen, Z., Qing, Z., 2012. A highly sensitive
electrochemical assay for silver ion detection based on un-labeled C-rich ssDNA
probe and controlled assembly of MWCNTs. Talanta 94, 178–183.
Yang, X., Li, T., Li, B., Wang, E., 2010a. Potassium-sensitive G-quadruplex DNA for
sensitive visible potassium detection. Analyst 135 (1), 71–75.
Yang, X., Xu, J., Tang, X., Liu, H., Tian, D., 2010b. A novel electrochemical DNAzyme
sensor for the amplified detection of Pb2+ ions. Chem. Commun. 46 (18),
3107–3109.
Yang, Y., Kang, M., Fang, S., Wang, M., He, L., Feng, X., Zhao, J., Zhang, Z., Zhang, H.,
2015. A feasible C-rich DNA electrochemical biosensor based on Fe3O4@3D-GO for
sensitive and selective detection of Ag+. J. Alloy. Compd. 652, 225–233.
Yun, W., Jiang, J., Cai, D., Wang, X., Sang, G., Liao, J., Lu, T., Yan, K., 2016.
Ultrasensitive electrochemical detection of UO22+ based on DNAzyme and
isothermal enzyme-free amplification. RSC Adv. 6 (5), 3960–3966.
Zhan, S., Wu, Y., Liu, L., Xing, H., He, L., Zhan, X., Luo, Y., Zhou, P., 2013. A simple
fluorescent assay for lead (II) detection based on lead (II)-stabilized G-quadruplex
formation. RSC Adv. 3 (38), 16962–16966.
Zhang, B., Chen, J., Liu, B., Tang, D., 2015. Amplified electrochemical sensing of lead ion
based on DNA-mediated self-assembly-catalyzed polymerization. Biosens.
Bioelectron. 69, 230–234.
Zhang, Y., Xiao, S., Li, H., Liu, H., Pang, P., Wang, H., Wu, Z., Yang, W., 2016. A Pb2+-ion
electrochemical biosensor based on single-stranded DNAzyme catalytic beacon.
Sens. Actuators B: Chem. 222, 1083–1089.
Zhang, Y., Zhao, H., Wu, Z., Xue, Y., Zhang, X., He, Y., Li, X., Yuan, Z., 2013. A novel
graphene-DNA biosensor for selective detection of mercury ions. Biosens.
Bioelectron. 48, 180–187.
Zhou, Y., Tang, L., Zeng, G., Zhu, J., Dong, H., Zhang, Y., Xie, X., Wang, J., Deng, Y.,
2015. A novel biosensor for silver (I) ion detection based on nanoporous gold and
duplex-like DNA scaffolds with anionic intercalator. RSC Adv. 5 (85), 69738–69744.
Zhu, Z., Su, Y., Li, J., Li, D., Zhang, J., Song, S., Zhao, Y., Li, G., Fan, C., 2009a. Highly
sensitive electrochemical sensor for mercury (II) ions by using a mercury-specific
oligonucleotide probe and gold nanoparticle-based amplification. Anal. Chem. 81
(18), 7660–7666.
Zhu, Z., Su, Y., Li, J., Li, D., Zhang, J., Song, S., Zhao, Y., Li, G., Fan, C., 2009b. Highly
sensitive electrochemical sensor for mercury(II) ions by using a mercury-specific
oligonucleotide probe and gold nanoparticle-based amplification. Anal. Chem. 81
(18), 7660–7666.
Zhuang, J., Fu, L., Tang, D., Xu, M., Chen, G., Yang, H., 2013. Target-induced structure-
switching DNA hairpins for sensitive electrochemical monitoring of mercury (II).
Biosens. Bioelectron. 39 (1), 315–319.
Zuo, X., Xia, F., Xiao, Y., Plaxco, K.W., 2010. Sensitive and selective amplified
fluorescence DNA detection based on exonuclease III-aided target recycling. J. Am.
Chem. Soc. 132 (6), 1816–1818.