Sensors and Actuators B 225 (2016) 453–462

Contents lists available at ScienceDirect

Sensors and Actuators B: Chemical

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

One-step fabrication of electrochemical biosensor based on

DNA-modified three-dimensional reduced graphene oxide and
chitosan nanocomposite for highly sensitive detection of Hg(II)
Zhihong Zhang a,b,∗ , Xiaoming Fu a , Kunzhen Li a , Ruixue Liu a , Donglai Peng a , Linghao He a ,
Minghua Wang b , Hongzhong Zhang b , Liming Zhou a,b,∗
a
Henan Provincial Key Laboratory of Surface and Interface Science, Zhengzhou University of Light Industry, No. 166, Science Avenue,
Zhengzhou 450001, China
b
Henan Collaborative Innovation Center of Environmental Pollution Control and Ecological Restoration, Zhengzhou University of Light Industry,
No. 166, Science Avenue, Zhengzhou 450001, China

a r t i c l e i n f o a b s t r a c t

Article history: A novel DNA-modified nanocomposite of three-dimensional reduced graphene oxide and chitosan parti-
Received 28 July 2015 cles (CS@3D-rGO@DNA) was prepared by one-step method and used as a new electrochemical biosensing
Received in revised form 7 November 2015 strategy to detect Hg2+ in drinking water. This biosensor was fabricated by the chemical coordination of
Accepted 19 November 2015
T-Hg2+ -T between the T-rich oligonucleotide strands contained in the as-prepared CS@3D-rGO@DNA
Available online 23 November 2015
nanocomposite and Hg2+ , thereby varying the electrochemical signals. The developed electrochemical
biosensor exhibited high sensitivity toward the determination of Hg2+ with a low detection limit of
Keywords:
0.016 nM, ranging from 0.1 nM to 10 nM. This novel biosensor also showed high selectivity, good stability,
DNA-templated nanocomposites
Detection of Hg2+
and repeatability. Hence, this proposed CS@3D-rGO@DNA-based electrochemical strategy is feasible and
Electrochemical biosensor may offer a simple, rapid, cost-effective, highly selective, and sensitive method for Hg2+ quantification.
chitosan © 2015 Elsevier B.V. All rights reserved.
Three-dimensional reduced graphene oxide

1. Introduction
Among the most prevalent toxic metal ions, Hg2+ is the most
stable modality of inorganic mercury [1]. Hg2+ is a highly toxic
heavy metal ion because of its accumulative characteristics and
toxic properties in the environment and biota [2]. A large amount
of mercury comes from the effluent of industrial manufacture, and
this element exists in water in the forms of organic and inor-
ganic mercury [3]. Long-term exposure to high levels of mercury
can lead to severe health problems, such as brain damage, kidney
failure, and other chronic diseases [4–6]. Hence, the sensitive and
specific detection of Hg2+ is important to control water pollution
and ensure safety of drinking water. Several methods have been
developed to detect mercury in environmental samples, such as
atomic adsorption spectroscopy [7], atomic emission spectroscopy
[8], atomic fluorescence spectroscopy [9], high-performance liquid
chromatography [10], inductively coupled plasma mass spec-
troscopy [11], and fluorescence analysis [12]. In accordance with
∗ Corresponding authors. Tel.: +86 37186609676; fax: +86 37186609676.
E-mail addresses: mainzhh@163.com (Z. Zhang), zlming1212@126.com
(L. Zhou).
the required limit of 1 ppb to 2 ppb in drinking water set by the
Italian and European Environmental Protection Agency, the com-
mon methods were limited from expanding applications because
of their complicated instrumentation or time-consuming sample
treatments.
Electrochemical methods are general and classic techniques in
analytical chemistry which demonstrate advantages of simplicity,
high sensitivity, good selectivity, and low cost. Electrochemical
approaches have been proven as effective tools to monitor mer-
cury [3]. The high specificity for the interaction of oligonucleotides
with metal ions prompts the oligonucleotides to be the current
tools for detecting metal ions. Adenine (A) and thymine (T) are
optimally suited to form complexes with Hg2+ because of their
high stability constant with Hg2+ [13]. Consequently, the for-
mation of T-Hg2+ -T coordination between Hg2+ and T-rich DNA
strands is often employed to test the Hg2+ in the environment
or water by using electrochemical approaches [14]. For example,
a metal–organic hybrid microarray formed by a nicking endonu-
clease assisted target-triggered strand release strategy via DNA
cyclic amplification was used to detect Hg2+ through the stable
T-Hg2+ -T linkage, yielding an ultra-low detection limit of 5 pM
[15]. Autonomously assembled hemin/G-quadruplex DNAzyme
nanowires were applied to amplify the signal for detecting Hg2+ as

http://dx.doi.org/10.1016/j.snb.2015.11.091
0925-4005/© 2015 Elsevier B.V. All rights reserved.
454 Z. Zhang et al. / Sensors and Actuators B 225 (2016) 453–462
a novel sensitive pseudobienzyme electrocatalytic DNA biosensor.
This method exhibited a high sensitivity for Hg2+ determination
with a detection limit of 2.5 pM [16]. Methylene blue and nick-
ing endonuclease were also applied to amplify the signal for Hg2+
detection [17]. As such, the complex fabrication of the sensing sys-
tem is typically used to detect heavy metal ions.
Chitosan, which is derived from the deacetylation of chitin, is
abundant, inexpensive, highly reactive, nontoxic, and multifunc-
tional [18]. Principally, chitosan contains large amounts of amino
( NH2 ) and hydroxyl ( -OH) groups, thereby exhibiting a high
adsorption capacity towards biomolecules. For several decades,
chitosan has been extensively investigated for adsorption of heavy
metal ions in water treatments because of its excellent quality,
multiple functional groups, and favorable cost [19,20]. However,
chitosan is only soluble in acidic solutions and difficult to dis-
perse in water [21]. Moreover, the non-solubility of chitosan in
water is a major limitation for its appropriate use in the removal
of heavy metal ions. Hence, chitosan is usually modified to expand
its application range. Chitosan/sulfhydryl-functionalized graphene
oxide composite was synthesized via covalent modification and
electrostatic self-assembly and used as an adsorbent material to
remove Cu(II), Pb(II), and Cd(II) in single- and multi-metal ion sys-
tems [22,23]. Furthermore, a new chitosan-based functional gel
containing multiwall carbon nanotube-poly(acrylic acid)-poly(4-
amino diphenyl amine) was fabricated to remove Cr(VI) [24].
Graphene, a monolayer of graphite, has recently elicited
considerable attention for its superior chemical stability, large
surface-to-volume ratio, tunable band gap, and good electronic,
mechanical, and thermal stabilities [25,26]. Given the strong pla-
nar stacking of sheets from the ␲–␲* interaction, graphene prefers
to self-assemble and form two-dimensional (2D) agglomerates
[27]. Compared with 2D graphene nanosheets, three-dimensional
graphene (3D-G) exhibits extraordinary properties, including
strong mechanical strength, large surface area, rapid transfer, and
mass transport kinetics [28,29]. Thus, 3D-G development is very
significant and offers potential applications in the field of biosen-
sors [30,31]. Previous research has shown that 3D-G comprising
various materials would be beneficial to construct 3D porous mate-
rials. For example, highly ordered polyaniline nanocone arrays
were synthesized on a 3D-G network via template-free electrode-
position method, yielding a specific capacitance of 751.3 F g−1 in
1 M HClO4 [32]. Si/porous reduced graphene oxide (rGO) compos-
ite fabricated by steam etching of Si/rGO aerogel was used as the
composite electrode, giving high specific capacity and good cycling
stability [33]. Flower-like NiCo2 O4 fabricated on 3D-G foam pro-
vides supercapacitors with specific capacitance of 1402 F g−1 [34].
Generally, chitosan combined with 3D-G exhibits high biocom-
patibility and is applied in environmental treatments [35], tissue
engineering [36], electrochemical biosensors [37], electrocatalysis
[38], and so on.
Although electrochemical DNA biosensors based on nucleic acid
hybridization or coordination chemistry offer great advantages,
such as specificity, selectivity, sensitivity, reusability, minia-
turization, and compatibility with micro-fabrication technology,
compared with existing traditional hybridization assays [39], such
biosensors are developed only after the immobilization of oligonu-
cleotide strands onto modified electrode surfaces, possibly leading
to long detection time and fabrication complexity. As such, the
present study is the first to apply a DNA-modified nanocomposite
of 3D-rGO and chitosan particles (CS) (CS@3D-rGO@DNA) to fabri-
cate an electrochemical biosensor for rapid and sensitive detection
of Hg2+ (Scheme 1). The analytical performance of the designed
electrochemical DNA biosensor was evaluated for the formation of
T-Hg2+ -T and direct adsorption of Hg2+ by using electrochemical
impedance spectroscopy (EIS). Overall, the unique hybrid nano-
structure of 3D-rGO offers good chemical performance and high
adsorption ability for heavy metal ions. Additionally, the porous
networks constructed by the nanocomposites can easily and rapidly
afford channels for DNA immobilization. The structure of CS, bene-
fiting from its functionality and adsorption ability, also guarantees
adsorption of heavy metal ions and DNA binding. On the basis of
systematic electrochemical testing, the CS@3D-rGO@DNA, in com-
parison with pure CS@3D-rGO nanocomposites, showed enhanced
sensitivity for detecting Hg2+ and significantly improved the detec-
tion limitation.
2. Experimental
2.1. Materials
Graphite powder, chitosan and hexadecyl trimethyl ammo-
nium bromide (CTAB) were purchased from Aladdin Reagent
Co. Ltd. (Shanghai, China). Sodium sulfate (Na2 SO4 ) and acetic
acid (CH3 COOH) were purchased from Tianjin Zhiyuan Chemical
Reagen Co. Ltd. (Tianjin, China). K3 [Fe(CN)6 ] and K4 [Fe(CN)6 ]·3H2 O
were obtained from Sinopharm Chemical Reagent Co., Ltd.
(Shanghai, China). DNA was obtained from Genesis Biotech Co. Ltd.
(Zhengzhou, China). All of the reagents were of analytical reagent
grade. All of the aqueous solutions were prepared with Milli-Q
water. The sequence of Hg2+ -targeted DNA was as following:
5 -CCC CCC CCC CCC TTC TTT CTT CCC CTT GTT TGT T-3 .
2.2. Solutions
Approximately 0.1 M phosphate buffer solution (PBS) was
prepared by mixing the stock solutions of 0.1 M NaH2 PO4
and 0.1 M Na2 HPO4 [v(Na2 HPO4 ):v(KH2 PO4 ) = 8:2]. Milli-Q water
(18.2 M cm−2 resistance) was used throughout the experiment.
An electrolyte solution was prepared immediately before use by
dissolving 1.65 g of K3 [Fe(CN)6 ] and 2.11 g of K4 [Fe(CN)6 ]·3H2 O in
1 L of PBS. Additionally, the Hg2+ -targeted DNA solution was used
as sensor to Hg2+ , and its stock solution (1 ␮M) in PBS was pre-
pared. Then PBS stock solution was then diluted to a concentration
of 500 nM. The Hg2+ stock solution (1 mM) was prepared by dis-
solving Hg(NO3 )2 with 0.5% HNO3 . Different concentrations (0.1,
0.5, 1.0, 5.0, and 10.0 nM) of Hg2+ were diluted using the Hg2+ stock
solution. Real water samples were collected from three sewage out-
falls along the Xushui River (Zhengzhou, China). All solutions were
prepared immediately before each experiment and stored at 4 ◦ C
until use.
2.3. Preparation of CS@3D-rGO and CS@3D-rGO@DNA
nanocomposites
The 3D-rGO used in the present work was prepared as described
in the previous work [40]. A brief description of the 3D-rGO used in
this study is presented in the Supporting Information. Up to 50 mg
of the as-prepared 3D-rGO was dispersed in 10 mL of Milli-Q water
to produce a homogeneous dispersion (5.0 mg mL−1 ). About 2.0 g
chitosan was dissolved in 100 mL of acetic acid (2%), followed by
stirring for 4 h until a homogeneous sticky liquid was formed. After-
ward, 100 mL of chitosan solution (20 mg mL−1 ) was added into
20 mL of the 3D-rGO dispersion and stirred for 0.5 h. Subsequently,
100 mg of CTAB and 0.5 g of Na2 SO4 were added into the above
mixture for further reaction. A precipitate was formed and washed
using Milli-Q water for three to five times. The precipitate was then
centrifuged and redispersed in water and ethanol to remove unde-
sirable particles, followed by drying in vacuum oven at 30 ◦ C for
24 h. The CS@3D-rGO@DNA nanocomposites were prepared using
the same procedure with 1 mL of DNA solution (500 nM). All resul-
tant nanocomposites were stored in a refrigerator at 4 ◦ C.
 Z. Zhang et al. / Sensors and Actuators B 225 (2016) 453–462
2.4. Characterizations
X-ray photoelectron spectroscopy (XPS) analysis was obtained
from an AXIS HIS 165 spectrometer (Kratos Analytical, Manches-
ter, UK) with a monochromatized Al KR x-ray source (1486.71 eV
photons). X-ray diffraction (XRD) analyses were performed with
D8 advance X-ray diffraction instrument (Germany) with a
scanning 2␪ angle of 10◦ to 80◦ . Fourier transform-infrared
(FT-IR) spectra of the samples were acquired from samples
in KBr pellets and using a Nicolet 5700 FT-IR instrument in
the range of 400 cm−1 to 4000 cm−1 . The surface morphology
was determined using a high-resolution transmission electron
microscope (HR-TEM, JEOL JEM-2100) with a field emission
gun of 200 kV. Field emission scanning electron microscopy
(FE-SEM) images were obtained using a JSM-6490LV SEM
(Japan).
2.5. Fabrication of electrochemical biosensors based on
CS@3D-rGO and CS@3D-rGO@DNA nanocomposites
Electrochemical measurements were performed on an electro-
chemical workstation (CHI 660B, CH Instruments). A conventional
three-electrode system was employed, which consisted of a mod-
ified Au electrode (with a diameter of 3 mm) as the working
electrode, an Ag/AgCl (saturated KCl) electrode as reference elec-
trode, and a platinum slide as counter electrode. EIS was performed
at room temperature. 5 mM Fe(CN)6 3−/4− with 1 M KCl and 140 mM
NaCl in PBS (pH 7.4) was employed as the electrolyte. Spectra were
recorded by applying a bias potential of 0.22 V versus Ag/AgCl (sat-
urated KCl) and 5 mV amplitude in the frequency range of 0.01 Hz
to 100 kHz. Each measurement was repeated for at least three
times.
For the electrochemical measurements, the prepared CS@3D-
rGO and CS@3D-rGO@DNA nanocomposites were first dispersed in
1 mg mL−1 ethanol. Up to 10 ␮L of different suspensions were then
dropped on each working electrode to form the electrochemical
sensors. After thorough washing with Milli-Q water, the work-
ing electrode based on CS@3D-rGO was immediately incubated
with 500 nM probe DNA solution. The other working electrode
based on CS@3D-rGO@DNA nanocomposite and the composite
electrode of CS@3D-rGO modified with DNA (CS@3D-rGO-DNA)
was successfully incubated with different concentrations of Hg2+
solution.
Scheme 1. Schematic diagram of Hg2+ detection using the developed electrochemical biosensor based on CS@3D-rGO@DNA nanocomposite.
455
3. Results and discussion
3.1. Sensor design
Two kinds of nanocomposites were used to detect Hg2+ ions
in the aqueous solution in the present work, i.e., CS@3D-rGO
and CS@3D-rGO@DNA. Three detection methods were fabricated,
including the direct adsorption of Hg2+ on the surface of CS@3D-
rGO (part I in Scheme 1) and detection of Hg2+ using the developed
electrochemical biosensor base on CS@3D-rGO immobilized with
DNA strands (part II in Scheme 1) and CS@3D-rGO@DNA (part III in
Scheme 1). Herein, DNA strands used is composed of 2 complemen-
tary G-C base pairs and 14 mismatched C bases if folded. 7 T-Hg2+ -T
base pairs will be formed and then cooperate to stabilize the duplex
in the presence of Hg2+ , leading to the construction of a duplex like
DNA scaffold. As mentioned above, the sensitive detection of Hg2+
using the developed nanocomposites was caused by three kinds of
interactions, namely the formation of surface complexes of Hg2+
ion with the amino group in chitosan, the electrostatic attraction
between the negatively charged surface of 3D-rGO, and the forma-
tion of the T-Hg2+ -T coordination between Hg2+ and DNA strands.
3.2. Chemical and crystal structures of CS@3D-rGO and
CS@3D-rGO@DNA nanocomposites
Fig. 1a shows the FT-IR spectra of CS@3D-rGO and @NCs@3D-
rGO @DNA nanocomposites. No substantial variation was observed
between them because of the tiny content of DNA (only 500 nM) in
the as-prepared nanocomposites. The characteristic peaks of the
two samples were associated with various oxygen functionalities,
including the broad peak of O H groups centered at 3410 cm−1 and
the C-O stretching peak at 1052 cm−1 [41]. The C C vibrations from
the graphene oxide domains was obtained at 1625 cm−1 [42]. The
peak at 2885 cm−1 to 2980 cm−1 was typical of C-H stretch vibra-
tion, whereas the peaks at 1545 and 1393 cm−1 were attributed
to amide groups [43]. The presence of amide can be ascribed to
the synergistic effect of hydrogen bonding between chitosan and
the oxygenated groups in GO, as well as the electrostatic interac-
tion between positively charged chitosan and the negative charge
on the 3D-GO network. The results were also confirmed by XPS
characterization.
To further study the structure of the products, Fig. 1b
presents the X-ray diffraction patterns of the CS@3D-rGO and
456 Z. Zhang et al. / Sensors and Actuators B 225 (2016) 453–462

(a) (b)

(i)
Transmitance / %

(ii)

(ii)

(i)

4000 3000 2000 1000 15 30 45 60 75

-1
W avenum ber / cm 2θ / °

Fig. 1. (a) FT-IR and (b) XRD spectra of (i) CS@3D-rGO and (ii) CS@3D-rGO@DNA nanocomposites.

(a) C 1s of CS@3D-rGO (b) N 1s of CS@3D-rGO

C-C
C-H O=N-C

C-N
+
C-N =N - N-H
C=O C-O
O=C-N
O=C-O

(c) C 1s of CS@3D-rGO@DNA (d) N 1s of CS@3D-rGO@ DNA

292 290 288 286 284 282 408 405 402 399 396 393
(e) P 2p of CS@3D-rGO@ DNA

138 135 132 129 126

Binding energy / eV
Fig. 2. C 1s, N 1s, and P 2p core-level XPS spectra of CS@3D-rGO and CS@3D-rGO@DNA nanocomposites.
 Z. Zhang et al. / Sensors and Actuators B 225 (2016) 453–462
CS@3D-rGO@DNA nanocomposites. For CS@3D-rGO, the main
diffractive region was observed in small peaks at 2␪ = 11◦ and
weak broad peak at 2␪ = 24◦ − 26.5◦ . Considering that chitosan is a
semi-crystalline polymer, its crystalline and amorphous structures
coexisted [44]. Notably, the incorporation of 3D-rGO significantly
affects the crystalline structure of chitosan, as supported by the
considerable variation of the characteristic peaks of neat chitosan.
In the presence of DNA, no substantial change in the XRD pattern
was observed (curve ii) in Fig. 1b, indicating that small amount of
DNA did not change the crystalline structure of chitosan. In addi-
tion, the peak of 2␪ = 26.5◦ indicates the existence of 3D-GO in the
nanocomposites [45].
XPS was employed to analyze the chemical composition of the
samples. The representative high-resolution XPS spectra of C 1s and
N 1s of CS@3D-rGO and C1s, N 1s, and P 2p of CS@3D-rGO@DNA are
shown in Fig. 2. The addition of DNA strands significantly reduced
the contents of C 1s and increased the content of N 1s and O 1s
(Table S1). The presence of the DNA strands was found in all spec-
tra as indicated by the high C 1s (Fig. 2c) and N 1s (Fig. 2d) peak
intensities together with the appearance of a new P 2p peak, which
was not present on the CS@3D-rGO surface. Moreover, the C 1s
coreline spectrum of CS@3D-rGO surface (Fig. 2a) was composed of
five components, in which the peaks at ∼284.84, ∼286.74, ∼288.34,
and ∼289.04 eV are attributed to C C/C H, C O, C O, and O C O
groups, respectively. All of these peaks resulted from the partly
reduced 3D-rGO [43] and chitosan [46]. The peak at ∼285.94 eV
caused by C–N groups was from chitosan [47]. However, the peak
at ∼288.34 is also attributed to O C N group, which is consistent
with the result of N 1s core line spectrum (Fig. 2b). As shown in
Fig. 2b, the N 1s core line spectra of CS@3D-rGO could be fitted
into three main parts, namely, ∼399.44, ∼400.44, and ∼401.94 eV,
which are due to C N/N H, O C N, and N+ groups, respec-
tively. In addition, the presence of a certain amount of O C N
groups in the sample indicated that the formation of covalent bonds
between the carboxyl groups of 3D-rGO and amino groups of chi-
tosan [48]. Comparing the CS@3D-rGO with CS@3D-rGO@DNA, the
peak at ∼289.04 eV (O C O) disappeared from the C 1s coreline
Fig. 3. Low- and high-resolution SEM images of (a, b) CS@3D-rGO and (c, d) CS@3D-rGO@DNA nanocomposites.
457
spectrum, indicating the chemical reaction with -NH2 groups in
DNA strands [49]. Meanwhile, two peaks were separated at ∼133.1
and ∼134.0 eV in the P 2p coreline spectra of CS@3D-rGO@DNA
(Fig. 2e). These peaks are assigned to P 2p3/2 and P 2p1/2 , respec-
tively.
3.3. Surface morphology of CS@3D-rGO and CS@3D-rGO@DNA
composites
Fig. 3 shows the SEM images of CS@3D-rGO and CS@3D-
rGO@DNA composites. As can be seen from Fig. 3a and b, the
NCs@3D-rGO presents a thick sheet-like structure, with smooth
surface and thin-layer wrinkled edge. In the magnified image
(Fig. 3b), the chitosan particles were homogeneously distributed
into the 3D-rGO network. The tight attachment indicated that 3D-
rGO could form bonds with chitosan, which is consistent with the
FT-IR and XPS analyses. The surface of the nanocomposites seems
to be rougher and became more wrinkled after adding DNA strands
(Fig. 3c,d). In the developed films, face-to-face adhering multilay-
ers were obtained because of the strong binding force between DNA
strands and chitosan, leading to shrinking of the nanocomposite 3D
network.
Typical TEM and HR-TEM images of the synthesized CS@3D-rGO
and CS@3D-rGO@DNA nanocomposites are shown in Fig. 4. The
TEM images of CS@3D-rGO showed a translucent thin wrinkled
film wrapped with aggregates of irregularly structured particles.
In the presence of DNA, chitosan particles form larger aggregates
(Fig. 4c), indicating the strong interaction of positively charged
amino groups of chitosan and negatively charged phosphate groups
in DNA strands. The addition of DNA in the reaction system had
improved the formation of chitosan particles.
3.4. Comparison of electrochemical performances of CS@3D-rGO
and CS@3D-rGO@DNA nanocomposites
The EIS were used to investigate changes in the electron trans-
fer process at the electrode-solution interface of the fabricated
458 Z. Zhang et al. / Sensors and Actuators B 225 (2016) 453–462

Fig. 4. TEM and HR-TEM images of (a, b) CS@3D-rGO and (c, d) CS@3D-rGO@DNA nanocomposites.

Fig. 5. EIS Nyquist plots of the (a) CS@3D-rGO, (b) CS@3D-rGO with immobilized DNA, and (c) CS@3D-rGO@DNA-modified Au electrodes and the coordinated electrode after
reaction with 100 nM of Hg2+ . (d) Variation in the Rct values for each stage of Hg2+ detection. Rct was measured using the developed immunosensors, in which CS@3D-rGO,
CS@3D-rGO-DNA, and CS@3D-rGO@DNA composite were used as the sensitive layers.

biosensor. In a typical EIS, the semicircle portion observed at high
frequencies corresponds to the charge-transfer limited process and
an increase of the diameter of the semicircle reflects an increase of
the interfacial charge-transfer resistance (Rct ). This circuit (inset
in Fig. 5a) included the ohmic resistance of the electrolyte (Rs ),
double-layer capacitance (CPE), Rct , and the Warburg impedance
(Zw ) resulting from the diffusion of the redox probe ions from the
bulk of the solution to the electrode surface [50]. The direct adsorp-
tion of Hg2+ onto the CS@3D-rGO and the CS@3D-rGO-DNA for
Hg2+ detection were investigated by EIS to evaluate the advantage
of using the CS@3D-rGO@DNA nanocomposite for Hg2+ detection
(Fig. 5a–c).
 Z. Zhang et al. / Sensors and Actuators B 225 (2016) 453–462
As shown in Fig. 5a, the bare gold electrode shows a very small
semicircle domain (Rct = 0.047 k), indicating a very fast charge-
transfer process. After coating of the CS@3D-rGO nanocomposite,
the Rct of the electrode increases to 0.180 k because of the rela-
tively weak conductivity of CS@3D-rGO. Subsequent adsorption of
Hg2+ also leads to an obvious increase in Rct (Rct = 0.365 k). This
result can be explained by the different binding behaviors among
Hg2+ and two components of the CS@3D-rGO nanocomposite (a2
in Scheme 1). As for CS, the formation of surface complexes of Hg2+
ion with the amino group in chitosan and the electrostatic attrac-
tion between the negatively charged surface of 3D-rGO and the
metal cations would take place, resulting in the Hg2+ adsorption
[51]. Additionally, the remained hydroxyl and carboxyl function
groups of 3D-rGO were deprotonated in PBS (pH = 7.4), further
leading to the binding of Hg2+ to negatively charged groups via
the electrostatic interaction [52]. When Hg2+ -targeted DNA was
immobilized onto the CS@3D-rGO nanocomposite (Fig. 5b, b1 in
Scheme 1), the Rct of the modified electrode increases to 0.362 k.
Two interactions, i.e., the electrostatic interaction between the
negative charged phosphate groups of the DNA strands and the
positive charged amino groups of chitosan nanoparticles [53] and
the strong ␲–␲ stacking interactions between DNA strands and the
3D-rGO [40], caused the closely binding of the DNA stands. It seems
that DNA strands would lie on the surface of the nanocomposite
[54]. The effective repulsion between the negatively charged DNA
strands and [Fe(CN)6 ]3−/4− leads to the increase of the Rct value of
the composite electrode [55]. After addition of Hg2+ into the sys-
tem, the Rct further increases significantly (Rct = 0.720 k) (b2 in
Scheme 1). Herein, Hg2+ ions would rather coordinate with the
T base to form the T-Hg2+ -T coordination than adsorb onto the
nanocomposite. Consequently, only small amounts of Hg2+ ions
were penetrated into the inner of the matrix and adsorbed. DNA
stands would undergo the change of the conformation to release
more bonded bases from the surface of the nanocomposite, which
also provides more free negative charged phosphate of the DNA
stands. Even the formation of the T-Hg2+ -T coordination can cause
the decrease of the Rct value [46], all of these effect is dominant and
contribute to the increase of the Rct value in this case. The same
result was observed in the previous work [40].
A similar trend was observed for Hg2+ detection using the devel-
oped electrochemical sensor based on CS@3D-rGO@DNA (Fig. 5c,
c2 in Scheme 1). The efficiency of the different electrochemical
sensors for detecting Hg2+ ions was investigated by plotting the
simulated values of Rct for each stage (Fig. 5d). Among the three
(a)
1.5
0 nM
0.1 nM
1.2 0.5 nM
1 nM
5 nM
0.9 10 nM
-Z''/kohm
0.6
0.3
0.0
0.0 0.6 1.2 1.8 2.4 3.0
Z'/kohm
Fig. 6. (a) EIS Nyquist plots for the detection of different concentrations of Hg2+ ions. The concentrations are of 0, 0.1, 0.5, 1.0, 5.0, and 10.0 nM. (b) The linear fit plots Rct
as function of the logarithm of Hg2+ concentration. Error bar represents the standard deviation of three parallel experiments.
459
tested samples, the CS@3D-rGO@DNA exhibited the highest affinity
to Hg2+ , resulting in the highest Rct of 0.412 k. The direct adsorp-
tion of Hg2+ on CS@3D-rGO shows the lowest Rct of 0.186 k.
By comparison, the CS@3D-rGO with immobilized DNA exhibited
a Rct of 0.357 k after Hg2+ coordinated with the ss-DNA; this
value is between that of the above two samples. As for the CS@3D-
rGO@DNA nanocomposite, DNA strands were immobilized on and
mixed with the CS@3D-rGO, leading to the uniform distribution. It
can cause Hg2+ adsorption and the T-Hg2+ -T coordination, simul-
taneously, resulting in much more Hg2+ ions determined out and
the high increase of the Rct value. Consequently, the as-prepared
CS@3D-rGO@DNA exhibits the highest detection efficiency toward
Hg2+ .
XPS characterization was used to evaluate the chemical com-
ponent of the CS@3D-rGO@DNA nanocomposite bonded with Hg2+
ions. The atomic percentages of the C 1s, N 1s, O1s, Hg 4f, and P 2p
of the sample were 64.01%, 2.48%, 27.52%, 5.68% and 0.31%, respec-
tively (Table S1). C 1s, N 1s, Hg 4f, and P 2p core-level XPS spectra
were shown in Fig S1. As for the C 1s and N 1score-level spec-
tra, the same characterized peaks were obtained. Doublet-peaks at
∼100.57 and ∼104.3 eV were observed in Hg 4f core-level spectrum,
contributing to the Hg 4f7/2 and Hg 4f5/2 , respectively. Because of
the binding of Hg2+ ions, the P 2p signal was demonstrated weaker
than that of the CS@3D-rGO@DNA nanocomposite.
3.5. Sensitivity of the developed electrochemical biosensor based
on CS@3D-rGO @DNA nanocomposites
The Nyquist plots of CS@3D-rGO@DNA-modified gold electrode
toward with different concentrations of Hg2+ from 0.1 nM to 10 nM
are shown in Fig. 6a. By the software, the parameters were cal-
culated and the dependence of Rct on the concentration of Hg2+
(0.1 to 10 nM) was shown in Fig. 6b. It is clear demonstrates that
Rct increases substantially when the concentration is below 1 nM.
Afterwards, the Rct reaches a plateau. This is somehow similar to
the Temknh adsorption isotherm model. The Rct value is linear
with logarithm of the Hg2+ concentration in the range of 0.1 nM
to 10 nM, as shown in the inset of Fig. 6b; the regression equation
is Rct (k) = 1.271 + 0.905 log CHg 2+ (unit of C is nM) and the corre-
lation coefficient R2 is 0.992). Each point is the mean value of three
measurements, and the error bars correspond to the standard devi-
ation. The detection limit was determined to 0.016 nM by using
3␴, where ␴ is the standard deviation of 11 parallel measure-
ments of the blank solution. The detection limit obtained using the
(b)
2.5
2.0
2.4
1.5
Rct / kohm
2.0
1.0 1.6
1.2
0.5
0.8
0.0 0.4
-1.0 -0.5 0.0 0.5 1.0
-0.5
3.6 4.2 0 2 4 6 8 10
2+
Hg concentration / nM
460 Z. Zhang et al. / Sensors and Actuators B 225 (2016) 453–462

Table 1 Table 2
Comparison of sensitivities of different methods. Recovery of real water samples spiked with different concentrations of Hg2+ as
determined by the prepared electrochemical biosensor.
Sensitive layer Detection Linear range LOD Ref.
technique (nM) (nM) Real samples Add. Found Recovery
(nM) (nM) (%)
Au-NPs–rGO Surface- 0.1–6000 0.1 [56]
enhanced Tap water 0.1 0.103 3.0
Raman 0.5 0.507 1.4
scattering 5.0 4.95 1.0
AuNPs Chemi- 0.1–10 0.05 [57] 10.0 10.29 2.9
luminescence River water 0.1 0.105 5.0
DNA-modified EIS 10–500 0.03 [58] 0.5 0.521 4.2
Au electrode 5.0 5.23 4.6
Ni(en)3 Ag2 I4 SWV 0.015–500 0.005 [15] 10.0 10.53 5.3
microarray
MWCNTs and DPV 0.1–20 0.03 [59]
Au-NPs The collected tap water samples were simply filtered, whereas the
Aptamer along QCM 0.5–100 0.24 [60] river water samples were filtered through 0.2 ␮m membranes to
with Au-NPs
This work EIS 0.1–10 0.016
remove impurities. Hg2+ stock solution at different concentrations
was added in the water samples, and the developed biosensor
was then used to determine the Hg2+ concentration. The analytical
results of Hg2+ determination in tap and river water samples are
shown in Table 2. The results determined for tap and river water
samples show good recovery values. All the measurements were
performed for five times. The results exhibited that the developed
electrochemical biosensor showed good selectivity and recovery.
Thus, the proposed strategy had potential for detecting Hg2+ in real
samples.

4. Conclusions

In this work, a facile co-reduction method was developed to

Fig. 7. Selectivity of Hg2+ detection in the presence of 10 nM Hg2+ and 100 nM of
other metal ions.
proposed new strategy is comparable with that reported so far
(Table 1) [56–58,15,59,60].
3.6. Selectivity, repeatability, and stability of the developed
biosensor
To evaluate the selectivity of the present sensing system, the
effects of 14 cations, including Ba2+ and Ca2+ , on the EIS mea-
surements were investigated. As shown in Fig. 7, the Rct value
was considerably or slightly increased after adsorption of differ-
ent metal ions. However, the Rct value is much higher for Hg2+
(10 nM) than for other types of metal ions even at a concentration
of 10 times that of Hg2+ (100 nM). The results indicated that this
new strategy has good selectivity for Hg2+ determination.
Repeatability of the results obtained using the developed elec-
trochemical sensor was evaluated by detecting Hg2+ at 1 and 5 nM
with five replicates. The relative standard deviations (RSDs) for both
concentrations were less than 5%, indicating a satisfactory repro-
ducibility of the proposed sensor. Repetitive EIS experiments of Rct
were conducted to determine the extent of stability. The results
indicated that after 10 continuous scan, the Rct of the developed
biosensor decreased by less than 3.9% (RSD = 1.6%).
3.7. Applications
The practical application of the fabricated electrochemical
biosensor based on CS@3D-rGO@DNA was evaluate by determining
the recovery of added Hg2+ in tap water and river water samples.
The tap water samples were collected from a local place and then
diluted with equal volume of 0.1 M PBS (pH 7.4). The river water
samples were collected from the Xushui River (Zhengzhou, China).
synthesize CS@3D-rGO nanocomposites in the presence of Hg2+ -
targeted DNA. Owing to the synergistic effect of 3D-rGO and
chitosan particles, the as-prepared CS@3D-rGO@DNA nanocom-
posite displays remarkably enhanced detection activity towards
Hg2+ in aqueous solution. The CS@3D-rGO@DNA-based electro-
chemical sensor demonstrates good sensitivity, wide linear range,
low detection limit, high selectivity, good repeatability, high sta-
bility, and prominent feasibility for real sample analysis. This study
not only introduces a new strategy for the one-step synthesis of
nanocomposites for electrochemical biosensors but also provides a
promising candidate material for the development of low-cost and
effective Hg2+ sensors.
Acknowledgement
This work was supported by Program for the National Natu-
ral Science Foundation of China (NSFC: Account No. 51173172 and
21441003).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.snb.2015.11.091.
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Biographies
Zhihong Zhang is a Professor in the Department of Materials and Chemical Engi-
neering Institute at Zheng Zhou University of Light Industry. She obtains her
undergraduate degree at the Zhengzhou University, China, and completed her doc-
toral work at Max-Plank Institute, German. After two years of postdoctoral work
at the national university of Singapore she joined Zheng Zhou University of Light
Industry in 2005, where she has since worked on plasma-polymerized polymer and
biosensors.
Xiaoming Fu is a graduate student at Zheng Zhou University of Light Industry. He
obtained his undergraduate degree from the Henan University of Urban Construc-
tion, China (2011). He mainly engaged in the preparation of plasma-polymerized
polymer and hydrogels.
Kunzhen Li is a graduate student at Zheng Zhou University of Light Industry. He
obtained his undergraduate degree in Chemistry from the Zheng Zhou University
of Light Industry, China (2015). He mainly engaged in the preparation of nanocom-
posites and the research of biosensors.
Ruixue Liu is a Professor in the Department of Materials and Chemical Engineer-
ing institute, Zhengzhou University of Light Industry. She obtained her Ph. D. from
Beijing University of Chemical Technology (2006). She mainly engaged in the prepa-
ration of plasma-polymerized polymer and hydrogels.
Donglai Peng is a lecturer in the department of polymer materials and engineering
in Zhengzhou University of Light Industry. She obtained her master degree in Chem-
istry from the Zheng Zhou University of Light Industry, China (2008). He mainly
engaged in the preparation of plasma-polymerized polymer and the research of
biosensors.
Linghao He is an Associate Professor in the department of polymer materials and
engineering, Zhengzhou University of Light Industry. She obtained her Ph. D. from
Zhengzhou University in 2011. Her research interests are in functional polymer
materials and in nanocomposites.
Minghua Wang is an associate professor in the department of polymer materials and
engineering, Zhengzhou University of Light Industry. He obtained his master degree
in Chemistry from the Zheng Zhou University of Light Industry, China (2008). Her
research interests are in environmental engineering and nanocomposites
Hongzhong Zhang is a Professor in the Department of Materials and Chemical
Engineering institute at Zheng Zhou University of Light Industry. He completed
his doctoral work at University of Science and Technology Beijing. His research is
interests in environmental engineering.
LiMing Zhou is a Professor in the Department of Materials and Chemical Engineering
institute at Zheng Zhou University of Light Industry. He completed his doctoral work
at Hebei University of Technology. His research is interests in Material science.