Biosensors and Bioelectronics 113 (2018) 142–147

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Biosensors and Bioelectronics

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

Novel electrochemical sensing platform for ultrasensitive detection of T

cardiac troponin I based on aptamer-MoS2 nanoconjugates
⁎ ⁎⁎
Xiujuan Qiaob, Kunxia Lib, Jinqiong Xub, Ni Chenga, Qinglin Shenga,b, , Wei Caoa, Tianli Yuea, ,
⁎⁎
Jianbin Zhengb,
a
College of Food Science and Engineering, Northwest University, Xi’an, Shaanxi 710069, China
b
College of Chemistry & Materials Science/Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of Ministry of Education/Shaanxi Provincial Key
Laboratory of Electroanalytical Chemistry, Northwest University, Xi’an, Shaanxi 710069, China

A R T I C LE I N FO A B S T R A C T

Keywords: Cardiac troponin I (cTnI) is a specific and sensitive biomarker for the early diagnosis of acute myocardial in-
Electrochemical aptasensor farction and for the subsequent clinical treatments. In this work, novel electrochemical sensing platform for
Nanoconjugates sensing of cTnI based on aptamer-MoS2 nanoconjugates was proposed. For comparison, core-shell Au@SiO2@Au
MoS2 nanoparticles were also used for sensing of cTnI. The sensing schemes and electrochemical responses of the
Au@SiO2@Au
proposed sensors were investigated by electrochemical impedance spectroscopy (EIS) in 5.0 mM K3[Fe(CN)6]/
Cardiac troponin I
K4[Fe(CN)6] (1:1) solution containing 0.1 M KCl, respectively. Results showed that the aptamer-Au@SiO2@Au
based aptasensor shows a linear rage of 10 pM-10.0 μM with the detection limits of 1.23 pM For the aptamer-
MoS2 nanosheets based aptasensor, the linear range for cTnI detection was from 10 pM to 1.0 μM with a lower
detection limit of 0.95 pM Meanwhile, both the sensors were successfully applied for detection of cTnI in human
blood samples. The two kinds of aptsensors have been successfully used for detecting of cTnI in human blood
serums. Moreover, no negligible signal changes could be observed in the presence of non-targets of CK-MB and
Myo, suggesting the good potential for clinic diagnosis.

1. Introduction
Cardiovascular disease (CVDs) is a severe public health problem in
the world, especially acute myocardial infarction (AMI) that is a clinical
disease and would cause serious damage to health (Jian et al., 2017).
Therefore, the prevention and control of cardiovascular disease are
necessary for health care systems in the whole society. Moreover, it is
highly required to build an equipment to detect CVDs timely and ac-
curately. Among various biomarkers, Cardiac troponin I (cTnI) is re-
garded as the new “gold standard” for CVDs diagnosis by the European
Society of Cardiology (Sheng et al., 2017). Moreover, the concentration
of cTnI would provide the basis for the doctor to judge the size of the
infarction area.
Currently, various previous reported methods, such as enzyme-
linked immunosorbent assay (ELISA) (Kallempudi and Gurbuz, 2011;
De Antonio et al., 2013; Lequin, 2005), electrochemiluminescence im-
munoassay (ECLIA) (Shen et al., 2011; Giannitsis et al., 2010), chemi-
luminescent immunoassay (CLIA) (Cho et al., 2009), fluorescence im-
munoassay (FIA) (Hayes et al., 2009), and gold-labeled immunoassay
(Wu et al., 2012a, 2012b) were used for the determination of AMI.
However, most of these methods take a long experimental period, need
a sophisticated experimental technique, and result in low sensitivity. At
present, the most common method for troponin detection is ELISA that
develops rapidly and is widely used. But, ELISA still exhibits several
disadvantages: (a) the detection process requires optical instruments
and involves many washing steps; (b) antibody used in the experiment
can be easily inactivated. These would limit its wide applications. Ap-
tamers, which was the single-stranded DNA or RNA molecules, have the
same property to antigen and can instead of the function of antibody.
According to the previous reported work (Jo et al., 2015), the affinities
of aptamer with cTnI is characterized by the Kd value. The Kd value of
aptamer is 270 pM, which was lower than that of the antibody with Kd
of 20.8 nM. What's more, aptamers can be generated from an in vitro
selection process known as systematic evolution of ligands by ex-
ponential enrichment (SELEX) (Robertson and Joyce, 1990; Tuerk and
Gold, 1990; Ellington and Szostak, 1990). In principle, compared with
antibodies, aptamers have good stability during long-term storage,
versatility in modification, low toxicity, chemical synthesis at low cost,

⁎
Corresponding author at: College of Food Science and Engineering, Northwest University, Xi’an, Shaanxi 710069, China.
⁎⁎
Corresponding authors.
E-mail addresses: qlsheng@nwu.edu.cn (Q. Sheng), yuetl305@nwsuaf.edu.cn (T. Yue), zhengjb@nwu.edu.cn (J. Zheng).

https://doi.org/10.1016/j.bios.2018.05.003
Received 21 February 2018; Received in revised form 21 April 2018; Accepted 3 May 2018
Available online 04 May 2018
0956-5663/ © 2018 Elsevier B.V. All rights reserved.
X. Qiao et al.
and they can be renatured and denatured many times without losing
their binding ability with targets (Elshafey et al., 2015). Therefore,
aptamers have become increasingly important tools to instruct various
biosensors, especially for developing rapid, sensitive, and low-cost
electrochemical sensors for the detection of cTnI.
To obtain a better performance, some traditional nanomaterials
(like: noble metal nanomaterials, carbon nanomaterials) have been
introduced to improve the sensitivity of electrochemical sensors due to
their large specific surface area, rich bonding sites, and fast electron
transfer rates (Zhang et al., 2017; Fu et al., 2017; Saleh et al., 2018,
2017a, b). Recent years, molybdenum disulfide (MoS2) is a 2D layered
nanomaterial that has attracted an increasing number of scientists’ in-
terests because of its unique properties and graphene-like structure
(Huang et al., 2013). MoS2 is a semiconducting, which has an inter-
esting indirect-to-direct bandgap transition (from 1.2 to 1.9 eV), de-
pending on its thickness (Radisavljevic et al., 2011; Eda et al., 2011).
This phenomenon may offer a valuable solution to overcome the
shortages of graphene. So, the increasing potential uses of MoS2 is going
to develop in sensing combined with fluorescence, electrochemistry,
and surface-enhanced Raman scattering techniques (Wu et al., 2012a,
2012b; Su et al., 2014; Balcioglu et al., 2014；Al-Shalalfeh et al., 2017;
Saleh et al., 2017a, b; Al-Shalalfeh et al., 2016a, b). Similar to gra-
phene, MoS2 possesses high fluorescence quenching efficiency. There-
fore, MoS2 has been used as a nanoprobe for the homogeneous detec-
tion of biomolecules. As previous works reported, MoS2 nanosheets
demonstrated the ability of adsorbing single-stranded DNA (ssDNA)
spontaneously via the van der Waals force (Zhu et al., 2013; Ge et al.,
2014). Furthermore, MoS2 nanosheets are also efficient dye quenchers,
which always be employed to develop fluorescent sensors for the de-
tection of small molecules or DNA by using aptamers as the recognition
units. Thus, it is expected that the MoS2 nanosheets can be used for the
construction of electrochemical sensors for highly sensitive detection of
cTnI.
In this paper, a novel electrochemical aptasensor based on the
conjugation of aptamer with the layer-structured MoS2 nanosheets to
detect cTnI was proposed. For comparison, the core-shell structured
Au@SiO2@Au was also used for the construction of electrochemical
sensor. Not only can we give a comparison of the two sensing schemes,
but also the sensing performances of the two sensors were evaluated. In
addition, the application of the two biosensors for detecting cTnI in
human plasma samples was conducted.
2. Experimental
2.1. Reagents
All oligonucleotides are synthesized by Sangon Biotech. Co., Ltd.
(Shanghai, China). The sequences used were as follows:
For Au@SiO2 @Au
5′-SH-(C 6 )-CGTGCAGTACGCCAACCTTTCTCATGCGCTGCCCCTC
TTA-MB-3′
For MoS2
5′-CGTGCAGTACGCCAACCTTTCTCATGCGCTGCCCCTCTTA-3′
Tris-HCl, TCEP and cTnI are also purchased from Sangon Biotech.
Co., Ltd. (Shanghai, China). Gold (III) tetrachloride trihydrate
(HAuCl4·3H2O, ≥99%), tetraethyl orthosilicate (TEOS), and 2-pro-
panol, (3-aminopropyl) triethoxysilane (APTES) were purchased from
Aldrich.
All other reagents are of analytical grade and used without further
purification. All solutions were prepared with ultrapure water.
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Biosensors and Bioelectronics 113 (2018) 142–147
2.2. Apparatus
The morphologies of the Au@SiO2@Au nanocomposite and MoS2
nanosheets were observed using a scanning electron microscope (SEM,
JEOL, Japan) and a transmission electron microscope (TEM, Hitachi H-
7500). Square wave voltammetry (SWV), and electrochemical im-
pedance spectroscopy (EIS) were carried out with CHI 660 electro-
chemical workstation (Shanghai CH Instrument Co. Ltd., China). A
conventional three-electrode cell was used in this work. The modified
glassy carbon electrodes (GCE, geometric area=0.071 cm2) were used
as the working electrodes, and a platinum wire and an Ag/AgCl (3 M
KCl) electrode were used as the counter and reference electrode, re-
spectively.
2.3. Preparation of Au@SiO2 @Au and MoS2 nanosheets
2.3.1. Preparation of Au@SiO2 @Au nanomaterials
We could use the sol-gel process directly for coating the surfaces of
these gold nanoparticles with conformal shells of amorphous silica. In
this process, the formation of silica coatings involved from hydrolysis of
tetraethyl orthosilicate (TEOS) to generate silica sols, followed by nu-
cleation and condensation of these sols onto the surfaces of gold na-
noparticles. Ammonia could be used as a catalyst to speed up the hy-
drolysis of TEOS. In a typical procedure, 4 mL gold colloids (10 nm in
diameter, 4.5*1010 particles/mL) (as reported (Sheng et al., 2018)) was
added to 20 mL 2-propanol in 100 mL beaker under continuous stirring.
Then, 0.5 mL ammonia solution (30 wt%) and 500 μL TEOS (0.01 M)
were consecutively added to the reaction mixture. The reaction pro-
ceeds for about 1 h at room temperature with continuous stirring. The
product could be well-dispersed in water without adding any surfactant
due to the presence of negative charges on the surfaces of silica shells.
The Au@SiO2 nanomaterials was obtained after washing by centrifu-
ging at 4000 rpm. To get the Au@SiO2@Au nanocomposites, Au@SiO2
nanomaterials obtained was suspended in methanol (10 mL), followed
by adding APTES (150 μL) under stirring overnight. The function of
APTES here is to provide positive valence on Au@SiO2 nanomaterials.
Finally, the APTES-functionalized Au@SiO2 with positive valence was
then dispersed in citrate-capped AuNPs collosol with negative valence
and stirred 12 h. So the Au@SiO2 @Au nanocomposites were synthe-
sized through electrostatic attraction. The product was centrifuged,
washed and dry in the air.
2.3.2. Preparation of monolayer MoS2 nanosheets
MoS2 powder (typical particle size 10 µm, 0.3 g) was mixed with a
10 mL, 1.6 M n-butyllithium solution applied to intercalate into MoS2
powder at room temperature for 2 days. MoS2 has a structure like
sandwich. When organic groups are introduced into the layers, two
dimensional nanocomposites can be formed. A group of alkali metal
ions such as Li+ or transitional elements such as Fe+ can be inserted
between layers. When ions are inserted, they form nano-laminated
compounds. The reaction is as follows:
C4H9Li + MoS2 = LiMoS2 + C4H9 (1)
+
Then, deoxidized water was used to exfoliate the Li intercalated
MoS2 by washing. And the suspension was sonicated for 1 h to facilitate
the exfoliation process. The generated LiMoS2 can be abscissed in the
ionospheric reagent (such as water). The LiMoS2 after abscission is
negatively charged on the surface. So, it can be become a stable single
layer of molybdenum disulfide suspension due to the electrostatic re-
pulsion. The LiOH and other soluble impurities were removed from
MoS2 nanosheets by centrifuging twice. Finally, the product was stored
at 4 °C for further application.
X. Qiao et al.
2.4. Preparation of the modified electrodes
The construction of nanocomposites modified GCE was performed
as follows: 1 mg Au@SiO2 @Au/MoS2 was dissolved in 1 mL of 0.5%
chitosan liquid to obtain homogeneous suspension by sonicating for
15 min. Prior to modification, bare GCE (diameter of 3 mm) was po-
lished by 0.3 mm and 0.05 mm of alumina powder respectively. Then
sonicating in ethanol and deionized water (volume ratio = 1:1) is al-
lowed for 15 min to remove adsorbed substances. Finally, a 6 μL aliquot
of the suspension was pipetted onto GCE and dried at room tempera-
ture.
After drying in the ambient air, in order to immobilization of sul-
fydryl modified aptamer on Au@SiO2 @Au/GCE, the Au@SiO2@Au/
GCE was incubated in a 0.5 μM aptamer mixture (containing 1.0 μM
sulfydryl modified aptamer, 10 mM Tris-HCl, and 0.1 mM TCEP, pH
7.4) for 12 h at room temperature. Then, the aptamer nanoconjugates
modified electrodes were washed by ultrapure water and dried using a
mild nitrogen stream. Finally, the aptamer nanoconjugates modified
electrodes was incubated in a series of concentrations of cTnI for further
detection.
For the immobilization of non sulfydryl modified aptamer on MoS2/
GCE, the process of immobilization is almost same as immobilization
steps of sulfydryl modified aptamer on Au@SiO2@Au/GCE except
0.5 μM aptamer mixture (containing 1.0 μM non sulfydryl modified
aptamer, 10 mM Tris-HCl, and 0.1 mM TCEP, pH 7.4).
3. Results and discussion
3.1. Characterization of Au@SiO2@Au nanomaterials and MoS2
nanosheets
The morphologies of the as-prepared core-shell Au@SiO2@Au and
MoS2 nanosheets were first characterized by SEM and TEM. Fig. 1A
shows the SEM and TEM images of Au@SiO2 nanoparticles with uni-
form sphere structure of about 240 nm in diameter. The as-prepared
Au@SiO2 nanoparticles is global and smooth. The inset of Fig. 1A
provide an evidence that the Au@SiO2 nanoparticles show a clear core-
shell structure, which illustrates the success of synthesis of basic na-
nomaterial for further modification. The process of preparation is due
to the hydrolysis of tetraethyl orthosilicate (TEOS) to generate silica
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Biosensors and Bioelectronics 113 (2018) 142–147
sols, followed by nucleation and condensation of these sols onto the
surfaces of gold nanoparticles. After the growth of Au NPs on Au@SiO2
nanoparticles, a core-shell structure of Au@SiO2@Au was successfully
obtained (Fig. 1B). Unlike the smooth surface of Au@SiO2 nano-
particles, a core-shell structure of Au@SiO2 @Au surface is totally
rough and we can clearly see that Au NPs can be uniformly distributed
on the surface of Au@SiO2 nanoparticles (Inset of Fig. 1B). In this
process, the APTES-functionalized Au@SiO2 with positive valence
combine with citrate-capped AuNPs with negative valence through
electrostatic attraction. The preparation of target nanomaterials need
more steps is as same as the former paper reported (Saleh et al., 2016).
Fig. 1C and D illustrate the images of TEM images of the as-prepared
MoS2 nanosheets. From the images, it is clearly seen that a monolayer
structure of the MoS2 nanosheet was successfully prepared. MoS2 na-
nosheets is exfoliated form MoS2 powder through sonication. A good
MoS2 unilaminar nanosheet can be observed. The size of MoS2 na-
nosheets ranges between 200 and 400 nm. The image indicates well-
dispersed MoS2 nanosheets with a uniform shape. The uniform dis-
persion in shapes is similar as the reported papers (Al-Shalalfeh et al.,
2016a, b).
3.2. The scheme of detection of cTnI based on Au@SiO2@Au
nanomaterials and MoS2 nanosheets
There are many ways for the construction of electrochemical apta-
sensors. The first common type is assembling of hairpin ssDNA labeled
with signal molecules on various nanomaterials by Au-S bond. In this
work, we have introduced two kinds of nanomaterials with different
nanostructures to build different types of aptasensors. The core-shell
Au@SiO2@Au was used for assembling of the hairpin ssDNA labeled
with methylene blue (Mb). The sensing scheme for cTnI based on the
Au@SiO2@Au is showed in Scheme 1A. When the target was added into
the system, a structure changes would results in the leave of the signal
of Mb away from the electrode surface, thus resulting a signal decrease
of the sensing process. Scheme 1B demonstrate the scheme of the sensor
for label-free detection of cTnI based on MoS2-aptasensor. The me-
chanism of selectivity of the proposed aptasensor was based on the
high-affinity of aptamer towards cTnI. At the beginning, the MoS2 na-
nosheets was immobilized on the electrode surface. Then, the MoS2
nanosheets modified electrode was incubated in a 0.5 μM aptamer
Fig. 1. A) The SEM image of the Au@SiO2
nanoparticles. Inset of Fig. 1 A is the TEM
image of Au@SiO2 nanoparticles. B) The SEM
image of the core-shell Au@SiO2 @Au nano-
particles. Inset of Fig. 1 B is the TEM image of
the core-shell Au@SiO2@Au nanomaterials. C,
D) The TEM images of MoS2 nanosheets. Inset
of Fig. 1 C is the TEM image of the one of single
MoS2 nanosheet.
X. Qiao et al.
Scheme 1. A) The scheme of detection of cTnI by the traditional aptasensor based on Au@SiO2@Au. B) The scheme of detection of cTnI by the aptasensor based on
MoS2 nanosheets.
mixture to build an aptasensor. In the absence of cTnI, the aptamer
probe was mainly in the unfolded and flexible state and lay on MoS2
nanosheets naturally. After adding cTnI into the sensing system, the
aptamer adopted a rigid and definite tertiary structure due to the spe-
cific binding with the target cTnI. The affinity of this rigid aptamer
structure with the MoS2 was weaker than that of aptamer with cTnI, so
the aptamer probes would released from the MoS2 nanosheets surface.
Based on the impedance changes of the sensing process, the detection of
cTnI was realized.
3.3. Characterization of the fabrication of the aptasensors
SWV and EIS are useful techniques for characterizing the properties
of modified electrode. The self-assembly procedures can be well traced
by electrochemical impedance spectroscopy. The EIS characterization
of the Au@SiO2@Au-based and MoS2-based aptasensor was recorded in
Fig. 2. The potentials changed with every modified step, which proved
the process of the aptasensor preparation. EIS measurement was per-
formed in 5.0 mM K3[Fe(CN)6]/K4[Fe(CN)6] (1:1) solution containing
0.1 M KCl. The equipment was set at 5 mV of alternative voltage am-
plitude, 200 mV of the applied potential and the voltage frequencies
ranged from 0. 01 Hz to 100 kHz. As shown in Fig. 2A, the bare GCE
145
Biosensors and Bioelectronics 113 (2018) 142–147
showed a small semicircle diameter (111.10 Ω, red line). When Au@
SiO2 @Au was immobilized on the GCE, the semicircle diameter be-
came smaller than that of bare GCE (70.09 Ω, atrovirens line), in-
dicating the electron transfer from [Fe(CN)6]3-/4- to the electrode sur-
face was enhanced due to the good conductivity of Au@SiO2 @Au.
After assembling of aptamer, the semicircle diameter of aptamer-Au@
SiO2 @Au/GCE (251.62 Ω, blue line) was bigger than nanomaterial
based GCE, implying that the electron transfer is hindered by aptamer
with poor conductivity. After incubation of the electrode in cTnI target
solution, the semicircle diameter increased dramatically (402.03 Ω,
black line), which was attributed to the presence of protein blocking the
electron transfer. As for the MoS2 nanosheets-based based system, the
semicircle diameter of MoS2 (240.1 Ω, black line) is bigger than the
semicircle diameter of GCE because the relative poor conductivity of
MoS2 that hinder the electron transfer from [Fe(CN)6]3-/4- to the elec-
trode. The semicircle diameter changed when stabilizes the aptamer on
MoS2 nanosheets (950.2 Ω, atrovirens line). After the incubation of
electrode with the cTnI target, the semicircle diameter became smaller
than before (560.5 Ω, blue line), which can be ascribed to the release of
aptamer from MoS2 nanosheets (Fig. 2B).
Fig. 2. A) Nyquist plots of GCE (red
line), Au@SiO2@Au/GCE (atrovirens
line), aptamer-Au@SiO2 @Au/GCE
(blue line), cTnI&aptamer-Au@
SiO2@Au/GCE (black line). B) Nyquist
plots of GCE (red line), MoS2/GCE
(black line), aptamer-MoS2/GCE (atro-
virens line), cTnI&aptamer-MoS2/GCE
(blue line) reacted in 5.0 mM K3[Fe
(CN)6]/K4[Fe(CN)6] solution con-
taining 0.1 M KCl at 5 mV of alternative
voltage amplitude, 200 mV of the ap-
plied potential and the voltage fre-
quencies ranged from 0. 01 Hz to
100 kHz.
X. Qiao et al.
3.4. Electrochemical detection of cTnI
After immobilization of Mb functionalized aptamer with the hairpin
structure on the Au@SiO2 @Au devices through Au-S bond, the tradi-
tional biosensor based on Au@SiO2@Au is studied. The electrochemical
signals of Mb were studied by SWV in the potential ranging from 0.7 to
− 0.5 V under the conditions of a step potential of 5 mV, an amplitude
of 20 mV and a frequency of 25 Hz. Tris hydroxymethyl aminomethane
hydrochloride (Tris-HCl) buffer (pH 7.4, 10 mM) was employed as the
supporting electrolyte. Electrolyte solutions were kept under a nitrogen
atmosphere during electrochemical measurements through deox-
ygenating with nitrogen bubbling for at least 30 min Fig. 3A shows the
signal decrease in SWV as a function of cTnI concentration (10.0 pM to
10.0 μM, lines b–h). The light blue line (line a) show the former signal
of Mb without cTnI. After adding different concentrations of cTnI into
the detection system, the Mb-aptasensor following react with the in-
creasing concentration of cTnI, leading to the shape of aptamer changed
and faraway from electrode surface, which will make the signal of Mb
to reduce. As expected, the electrochemical responses decrease (ΔI˭Ino
target - Itarget) of Mb was linear with the increasing cTnI concentration
from 10.0 pM to 10.0 μM with the linear correlation coefficient of
0.9995 (Fig. 3B). The detection limit is determined to be through the
3 N/SS/N=3 rule with 1.23 pM The detection limit of this biosensor for
cTnI is comparable and shows some improvements over other nano-
material-based approaches in Table S1 (Support Information). The de-
tection limit of this proposed aptasensor is 3 or 4 orders of magnitude
lower than the literature values, respectively (Wu et al., 2017;
Periyakaruppan et al., 2013).
EIS technique is applied for detecting cTnI by the novel biosensor
based on MoS2 nanosheets at 5 mV of alternative voltage amplitude,
200 mV of the applied potential and the voltage frequencies ranged
from 0.01 Hz to 100 kHz. EIS measurement was performed in 5.0 mM
K3[Fe(CN)6]/K4[Fe(CN)6] (1:1) solution containing 0.1 M KCl. After
assembling of aptamer on the MoS2/GCE, the aptamer was mainly in
the unfolded and flexible state and lay on MoS2 nanosheets naturally.
The results of EIS for aptasensor based on MoS2 can be saved, which is
showed in Fig. 4A (dark blue line, 1069 Ω). After that, as expected, in
the presence of cTnI in the sensing system, the aptamer adopted a rigid
and definite tertiary structure due to the specific binding with the target
cTnI. The affinity of this rigid aptamer structure with the MoS2 was very
weak so that aptamer with cTnI can be released from the nanosheets
surface and the final impedance signal was recorded. So, the semicircle
diameter of aptamer-MoS2/GCE binding with cTnI decreases with the
increasing of concentrations of cTnI from 10.0 pM to 1.0 μM, which
confirms that the aptamer molecules released successfully from the
MoS2 nanosheets after reacting with target. Moreover, the native
polyacrylamide gel electrophoresis image of aptamer sequence and
cTnI before and after incubation were also proved that the aptamer
146
Biosensors and Bioelectronics 113 (2018) 142–147
Fig. 3. A) SWV of the aptasensor based
on Au@SiO2@Au incubated in the rage
of 10.0 pM to 10.0 μM cTnI (from a to
h: 0, 10.0 pM, 100.0 pM, 1.0 nM,
10.0 nM, 0.1 μM, 1.0 μM, 10.0 μM) in
the potential ranging from 0.7 to
− 0.5 V under the conditions of a step
potential of 5 mV, an amplitude of
20 mV and a frequency of 25 Hz. B) The
linear between the response changes of
Mb (ΔI˭Ino target ─ Itarget) and the con-
centration of cTnI.
molecules released successfully from the MoS2 nanosheets (Fig. S1 in
Supporting Information). Therefore, the polyacrylamide gel electro-
phoresis results match well with the EIS results, suggesting a reliable
sensing function as designed. At the relatively high concentration, in-
tercalation of cTnI into the aptamer approaches a saturated-status.
Assuming the interaction process meets the requirements of the Lang-
muir isotherm (Atkins, 2001; Szymánska et al., 2007; Li et al., 2011),
the binding constant (i.e., equilibrium constant, Kb) can be expressed
as:
K a c = [R et (Ci )–R et (C0)]/ R et (C0) = ΔR et / R et (C0) (2)
where Ret(C0) and Ret(Ci) indicate charge transfer resistance without
cTnI and with desired concentration of cTnI, respectively. By plotting of
ΔRet/Ret° as a function of the concentration of cTnI, a straight line was
obtained. The linear regression equation is: ΔRet/Ret° = 0.095 log C
+ 0.614 with correlation coefficient of 0.9997 (Fig. 4B). The aptasensor
based on MoS2 nanosheets show the detection limit of 0.95 pM The
detection limit of this biosensor for cTnI is also compared in Table S1
(Support Information). The detection limit of the aptasensor based on
MoS2 nanosheets really shows a better performance than other bio-
sensors compared in Table S1. However, the linear range of the apta-
sensor based on MoS2 nanosheets is narrower than that of the apta-
sensor based on Au@SiO2@Au. The possible reason may be due to the
conductivity of MoS2 is lower than Au@SiO2@Au's conductivity. The
above results suggest that the layer-structure possess better sensing
performances than the core-shell structure of nanomaterials.
3.5. Selectivity of the proposed aptasensors in real samples
In order to evaluate the practical application capability and relia-
bility of the two types of proposed sensing system for the detection of
cTnI in 10% human serum. The human serum samples were gotten from
healthy man provided by the Hospital of Northwest University. In order
to exclude the adsorption of sample constituents at the electrode surface
which will suppress the ferricyanide signal, pretreatments were con-
ducted. Firstly, The human serum samples were concentrated by pre-
cipitation at room temperature with an equal volume of saturated
(NH4)2SO4, gentle mixing on a magnetic stirrer for 30 min, cen-
trifugation at 22 °C for 15 min. Secondly, the serum samples were 40-
fold diluted with Tris-HCl buffer, which was then used as the assay
medium. Finally, due to the generated LiMoS2 can be abscised in the
ionospheric reagent (such as water), the single layer MoS2 after ab-
scission is negatively charged on the surface, which can exclude the
adsorption of blood sample constituents like albumin with negative
charge at the electrode surface through electrostatic repulsion. The
selectivity of these two kinds of aptasensors was also evaluated by
testing several other molecules [creatin kinase (CK-MB) and myoglobin
(Myo)] seemed as biomarkers of AMI some years before in the same
X. Qiao et al.
experimental conditions. The experimental results were shown in Fig.
S2. Results reveal that the aptasensor can be applied for based on Au@
SiO2 @Au detection of cTnI is successful with increase in the con-
centration of cTnI in the range from 0.01 μM to 10.0 μM in human
plasma. Interestingly, there were almost no signal changes when adding
CK-MB (10.0 μM) and Myo (10.0 μM) into the human plasma at the
same experiment condition. The result demonstrates that our approach
based on Au@SiO2@Au nanoparticles could be a promising convenient
strategy for cTnI detection in real samples. Furthermore, we have de-
monstrated that troponin can also be detected by the proposed apta-
sensor based on MoS2 nanosheets in human plasma. As shown in Fig.
S2B, an outstanding enhancement of signal responses in the presence of
the different concentration of target cTnI was observed. However,
negligible signal changes could be observed after adding non-targets of
CK-MB (1.0 μM) and Myo (1.0 μM). The two results obviously showed
sufficient selectivity for detecting cTnI and provide a means for ultra-
sensitive detection of cTnI in practical samples.
4. Conclusions
In this work, an aptamer-MoS2 nanosheets nanoconjugates was
utilized for electrochemical sensing of cTnI. For comparison, core-shell
Au@SiO2 @Au nanoparticles was also used for sensing of cTnI. These
two kinds of biosensors are with totally different scheme of detection of
target. Upon the addition of cTnI, the electrochemical signal changed
with the switching of aptamer structure and keep away from nanoma-
terials in the traditional biosensor based on Au@SiO2@Au nano-
particles. Whereas, the presence of cTnI can combine with aptamer laid
on the MoS2 and adopt a rigid and definite tertiary structure that cause
a very weak affinity with the MoS2 so that aptamer with cTnI can be
released from the nanosheets surface, so that it will decrease the re-
sistance value. The aptamer-Au@SiO2@Au based aptasensor shows a
linear rage of 10 pM-10.0 μM with the detection limits of 1.23 pM For
the aptamer-MoS2 nanosheets based aptasensor, the linear range for
cTnI detection was from 10 pM to 1.0 μM with a lower detection limit of
0.95 pM These two kinds of biosensors also have high selectivity against
other molecules in the blood, which illustrates that it has great potential
for practical applications.
Acknowledgments
The authors gratefully acknowledge the financial support of this
project by the National Science Fund of China National Natural Science
Foundation of China (21575113, 21775120), the State Key Laboratory
of Analytical Chemistry for Life Science (SKLACLS1811), the Natural
Science Fund of Shaanxi Province in China Natural Science Foundation
of Shaanxi Province in China (2017JM2036), and the Scientific
Research Foundation of Shaanxi Provincial Key Laboratory (15JS100
147
Biosensors and Bioelectronics 113 (2018) 142–147
Fig. 4. A) Nyquist plots of the apta-
sensor based on MoS2 incubated with
10.0 pM to 1.0 μM cTnI (from a to g: 0,
10.0 pM, 100.0 pM, 1.0 nM, 10.0 nM,
0.1 μM, 1.0 μM) at 5 mV of alternative
voltage amplitude, 200 mV of the ap-
plied potential and the voltage fre-
quencies ranged from 0. 01 Hz to
100 kHz. B) The linear between ΔRet/
Ret0 and concentration of cTnI in the
rage of 10.0 pM to 1.0 μM with RSD of
3.84% (n = 5).
and 16JS099).
Appendix A. Supplementary material
Supplementary data associated with this article can be found in the
online version at http://dx.doi.org/10.1016/j.bios.2018.05.003.
References
Al-Shalalfeh, M.M., Onawole, A.T., Saleh, T.A., Al-Saadi, A.A., 2017. Mat. Sci. Eng. C 76,
356–364.
Al-Shalalfeh, M.M., Saleh, T.A., Al-Saadi, A.A., 2016a. RSC Adv. 6, 75282–75292.
Al-Shalalfeh, M.M., Sohail, M., Saleh, T.A., Aziz, M.A., 2016b. Aust. J. Chem. 69, 1314–1320.
Atkins, P.W., 2001. Physical Chemistry. PWN, Warsaw, pp. 827–829.
Balcioglu, M., Rana, M., Robertson, N., Yigit, M.V., 2014. ACS Appl. Mater. Interfaces 6,
12100–12110.
Cho, I.H., Paek, E.H., Kim, Y.K., Kim, J.H., Paek, S.H., 2009. Anal. Chim. Acta 632, 247–255.
De Antonio, M., Lupón, J., Galán, A., Vila, J., Zamora, E., Urrutia, A., Díez, C., Altimir, S.,
Bayes-Genis, A., 2013. Clin. Chem. Acta 426, 18–24.
Eda, G., Yamaguchi, H., Voiry, D., Fujita, T., Chen, M., Chhowalla, M., 2011. Nano. Lett. 11,
5111–5116.
Ellington, A.D., Szostak, J.W., 1990. Nature 346, 818–822.
Elshafey, R., Siaj, M., Zourob, M., 2015. Biosens. Bioelectron. 68, 295–302.
Fu, Y.Y., Sheng, Q.L., Zheng, J.B., 2017. Anal. Methods 9, 2812–2820.
Ge, J., Ou, E.C., Yu, R.Q., Chu, X., 2014. J. Mater. Chem. B 2, 625–628.
Giannitsis, E., Kurz, K., Hallermayer, K., Jarausch, J., Jaffe, A.S., Katus, H.A., 2010. Clin. Chem.
56, 254–261.
Hayes, M.A., Petkus, M.M., Garcia, A.A., Taylor, T., Mahanti, P., 2009. Analyst 134, 533–541.
Huang, X., Zeng, Z.Y., Zhang, H., 2013. Chem. Soc. Rev. 42, 1934–1946.
Jian, Y., Lin, D., Tan, X.R., 2017. World J. Clin. Cases 5, 349.
Jo, H., Gu, H., Jeon, W., Youn, H., Her, J., Kim, S.K., Lee, J., Shin, J.H., Ban, C., 2015. Anal.
Chem. 87, 9869–9875.
Kallempudi, S.S., Gurbuz, Y., 2011. Sens. Actuators B Chem. 160, 891–898.
Lequin, R.M., 2005. Clin. Chem. 51, 2415–2418.
Li, L.D., Zhao, H.T., Chen, Z.B., Mu, X.J., Guo, L., 2011. Sens. Actuators B: Chem. 157, 189–194.
Periyakaruppan, A., Gandhiraman, R.P., Meyyappan, M., Koehne, J.E., 2013. Anal. Chem. 85,
3858–3863.
Radisavljevic, B., Radenovic, A., Brivio, J., Giacometti, V., Kis, A., 2011. Nat. Nanotechnol. 6,
147–150.
Robertson, D.L., Joyce, G.F., 1990. Nature 344, 467–468.
Saleh, T.A., Al-Shalalfeh, M.M., Al-Saadi, A.A., 2016. Sci. Rep. 6, 32185.
Saleh, T.A., Al-Shalalfeh, M.M., Al-Saadi, A.A., 2017a. Mater. Res. Bull. 91, 173–178.
Saleh, T.A., Al-Shalalfeh, M.M., Al-Saadi, A.A., 2018. Sens. Actuators B: Chem. 254, 1110–1117.
Saleh, T.A., Al-Shalalfeh, M.M., Onawole, A.T., Al-Saadi, A.A., 2017b. Vib. Spectrosc. 90,
96–103.
Shen, W., Tian, D.Y., Cui, H., Yang, D., Bian, Z.P., 2011. Biosens. Bioelectron. 27, 18–24.
Sheng, Q.L., Qiao, X.J., Zheng, J.B., 2018. Electroanalysis 30, 137–145.
Sheng, Q.L., Qiao, X.J., Zhou, M., Zheng, J.B., 2017. Microchim. Acta 184, 1573–1585.
Su, S., Zhang, C., Yuwen, L.H., Chao, J., Zuo, X.L., Liu, X.F., Song, C.Y., Fan, C.H., Wang, L.H.,
2014. ACS Appl. Mater. Interfaces 6, 18735–18741.
Szymánska, I., Radecka, H., Radecki, J., Kaliszan, R., 2007. Biosens. Bioelectron. 22,
1955–1960.
Tuerk, C., Gold, L., 1990. Science 249, 505–510.
Wu, Q.Q., Li, Y.X., Xian, H.Y., Xu, C.D., Wang, L., Chen, Z.B., 2012a. Nanotechnology 24,
025501.
Wu, Q., Sun, Y., Zhang, D., Li, S., Zhang, Y., Ma, P.Y., Yang, Y., Wang, X.H., Song, D.Q., 2017.
Biosens. Bioelectron. 96, 288–293.
Wu, S.X., Zeng, Z.Y., He, Q.Y., Wang, Z.J., Wang, S.J., Du, Y.P., Yin, Z.Y., Sun, X.P., Chen, W.,
Zhang, H., 2012b. Small 8, 2264–2270.
Zhang, S., Fu, Y.Y., Sheng, Q.L., Zheng, J.B., 2017. New J. Chem. 41, 13076–13084.
Zhu, C.F., Zeng, Z.Y., Li, H., Li, F., Fan, C.H., Zhang, H., 2013. J. Am. Chem. Soc. 135,
5998–6001.