A cathodic luminol-based electrochemiluminescence biosensor for detecting cholesterol using 3D-MoS2-PANI nanoflowers and Ag nanocubes for signal enhancement

Article abstract of DOI:10.1039/c5ra09638c

A sensitive cathodic luminol-based electrochemiluminescence (ECL) biosensor for detecting cholesterol was fabricated with three-dimensional MoS₂-polyaniline (3D-MoS₂-PANI) nanoflowers and Ag nanocubes (AgNCs) for signal enhancement. In this study, the synthesized 3D-MoS₂-PANI-AgNCs nanocomposites with a large surface area were used as a matrix for loading a high amount of cholesterol oxidase (ChOx). Subsequently, the loaded ChOx efficiently catalyzed the oxidation of cholesterol to produce H₂O₂in situ, which could promote the oxidation of luminol to generate a cathodic ECL signal. In addition, 3D-MoS₂-PANI-AgNCs nanocomposites accelerate the decomposition of H₂O₂ into reactive oxygen species (ROSs), which increase the ECL intensity. Due to the integration of the properties of 3D-MoS₂-PANI nanoflowers and AgNCs, the proposed cholesterol biosensor exhibits a wide linear response range from 3.3 nM to 0.45 mM with a low detection limit of 1.1 nM.

Full text of DOI:10.1039/c5ra09638c

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The illustration of the synthetic process of 3D-MoS -PANI-AgNCs nanocomposites and the
2
preparation of the ECL biosensor.

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DOI: 10.1039/C5RA09638C
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ARTICLE TYPE
A cathodic luminol-based electrochemiluminescence biosensor for
detecting cholesterol using 3D-MoS -PANI nanoflowers and Ag
nanocubes for signal enhancement
2
a
b
a
Xin Ou , Xingrong Tan , Xiaofang Liu , Hongmei Chen , Yu
a
a *a
Fan , Shihong Chen , Shaping Wei
*a
5
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
DOI: 10.1039/b000000x
A sensitive cathodic luminol-based electrochemiluminescence (ECL) biosensor for detecting cholesterol
was fabricated with three-dimensional MoS -polyaniline (3D-MoS -PANI) nanoflowers and Ag
2
2
1
0
nanocubes (AgNCs) for signal enhancement. In this work, the synthesized 3D-MoS -PANI-AgNCs
2
nanocomposites with large surface area were used as matrix for high loading of cholesterol oxidase
(ChOx). Subsequently, the loaded ChOx efficiently catalyzed cholesterol to in situ produce H O , which
2 2
could promote the oxidation of luminol to generate cathodic ECL signal. In addition, 3D-MoS -PANI-
2
AgNCs nanocomposites hasten the decomposition of H O2 into reactive oxygen species (ROSs),
2
1
5
amplifying the ECL intensity. Due to the integration of the virtues of 3D-MoS -PANI nanoflowers and
2
AgNCs, the proposed cholesterol biosensor performed a wide linear response range from 3.3 nM to 0.45
mM with a low detection limit of 1.1 nM.
Introduction
example, Jiang et al. reported an immunosensor based on
Luminol or its derivatives, as one of the most extensively
investigated and used luminophores for
cathodic luminol ECL for the detection of carcinoembryonic and
10
the immunosensor performed unity peak. Therefore, it is
2
2
3
3
4
4
0
5
0
5
0
5
5
5
6
6
7
7
0
5
0
5
0
5
electrochemiluminescence (ECL) system, has attracted much
attention for its excellent inherent properties, such as nontoxic,
low cost, low oxidation potential and high-emitting quantum
necessary to explore new cathodic luminol-based ECL sensor to
expand the ECL bioanalytical application.
Molybdenum disulfide (MoS ), with two-dimensionally
2
1,2
yield. As is well known, the ECL emission of luminol could be
(
2D) layered structures similar to graphite, was composed of
enhanced by reactive oxygen species (ROSs) such as hydroxyl
three stacked atomic layers (S-Mo-S) stacked together by weak
11,12
•
radical (OH ) and superoxide anion (O ). Meanwhile, ROSs
•
3
2
van der Waals interactions.
Indeed, with exceptional physical
could be generated from H O , which is exactly the product of
2
2
and chemical properties, MoS has aroused considerable attention
2
many biological active substrates with the catalysis of
4
corresponding enzymes. Thus, luminol-based ECL system has
13
in many fields, such as catalysis, sensors, and lithium
14
15
batteries. More importantly, Lin et al. discovered that MoS2
been widely applied in enzyme biosensing, here, H O could be
2
2
possessed an intrinsic peroxidase-like activity and can catalyze
•
16
the decomposition of H O into OH . Thus, MoS could be one
produced and further changed to ROSs, thus improving the
5
,6
2
2
2
luminol-based ECL emission. Especially, many luminol-based
sensors have been reported based on luminol’s anodic ECL and
the peak potential position was observed about at 0.5 V (vs.
of most ideal nanomaterials in luminol based ECL enzyme
biosensing. Additionally, MoS could be integrated with carbon-
2
based materials and metal or metal oxide nanoparticles to exhibit
7
,8
Ag/AgCl). However, there are many odd peaks to obstacle the
synergistic effects, such as excellent electrochemical behavior,
17
larger surface area and higher electron conductivity.
detection in this potential. Compared with luminol’s anodic ECL,
9
its cathodic ECL has been investigated relatively fewer. For
Noble-metal nanocrystals have attracted increasing attention
in recently years due to their remarkable physicochemical
18-20
a
Key Laboratory of Luminescence and Real-Time Analytical Chemistry
properties.
Particularly, Ag nanocrystals have been widely
(Southwest University), Ministry of Education, College of Chemistry and
Chemical Engineering, Southwest University, Chongqing 400715,
People’s Republic of China. E-mail: cshong@swu.edu.cn. (S.Chen);
shapingw@swu.edu.cn (S.Wei); Fax: +86-23-68253172; Tel: +86-23-
investigated for their intriguing properties and potential
21-22
applications in biomedicine, catalysis, imaging and sensing.
Importantly, Ag nanocrystals could catalyze H O to generate a
2
2
23
great amount of ROSs, improving ECL intensity of luminol. For
example, Lv et al. constructed a luminol ECL immunosensor for
the detection of Aflatoxin B1 based on a fact that Ag
nanoparticles could improve the ECL intensity of luminol, thus
6
8253172
b
Department of Endocrinology, 9 th People’s Hospital of Chongqing,
Chongqing 400700, China
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enhance the sensitivity and lower the detection limit of the
platinum wire was counter electrode and a modified glassy
carbon electrode (GCE) was working electrode. Raman spectra
22
immunosensor. Up to now, different shapes of Ag nanocrystals
have been synthesized, such as sphere, cubes, wires, bars, 60 were measured with Invia Reflex (Renishaw, UK). The
2
4-26
octahedrons and decahedrons.
Among them, Ag nanocubes
morphology of nanocomposites was analyzed by a scanning
electron microscopy (SEM, S-4800, Hitachi, Japan).
5
0
5
0
5
(AgNCs) have received extraordinary interest. For example,
Tharmaraj et al. constructed a silver nanocubes based sensor for
2+
detecting Hg
ions with rhodamine 6G as an external
Preparation of PANI and 3D-3D-MoS -PANI nanoflowers
2
27
spectroscopic probe.
65 Polyaniline (PANI) was prepared by chemical polymeration of
28
Based on above observations, in this work, a sensitive
cathodic luminol-based ECL biosensor was fabricated for
aniline according to the literature. 3D-MoS -PANI nanoflowers
2
1
1
2
2
were fabricated via a hydrothermal method. Briefly, 0.017 g
PANI was dissolved in 20 mL deionized water with sonication
for 20 min. Then, 0.12 g Na MoO ·2H O and 0.32 g thiourea
detecting cholesterol using three-dimensional MoS -polyaniline
2
(
3D-MoS -PANI) nanoflowers and AgNCs for signal
2
2
4
2
enhancement. Here, cholesterol, as an important component in 70 were added into the PANI suspension with stirring for 20 min.
cells and tissues of humans, plays a vital role in the construction
of steroid hormones, bile acids and fat soluble vitamins. Thus,
accurate and fast detection of cholesterol has attracted much
The obtained suspension was sealed in 50 mL Teflon-lined
autoclave and maintained at 200 ℃ for 24 h. After cooling to
room temperature, 3D-MoS -PANI was gathered by
2
interest. In this work, 3D-MoS -PANI nanoflowers and AgNCs
2
centrifugation and washed several times with ethanol and
were synthesized via a hydrothermal method. The obtained 3D- 75 deionized water, and then dried at 60 ℃. The preparation process
MoS -PANI-AgNCs nanocomposites with large surface area and
2
was pictured in scheme 1.
excellent biocompatibility were used as matrix for high loading
of cholesterol oxidase (ChOx). Then, the loaded ChOx efficiently
catalyzed cholesterol to in situ produce H O , which could
Preparation of AgNCs and 3D-MoS -PANI-AgNCs
2
2
2
nanocomposites
promote the oxidation of luminol to generate cathodic ECL
8
8
9
0
5
0
AgNCs were synthesized by a facile one-step hydrothermal
method. In brief, 0.16 g PVP was dispersed with 22 mL EG.
Next, 3 mL of AgNO (1 mM in EG) and 2.5 mL of FeCl (0.6
signal. In addition, 3D-MoS -PANI-AgNCs nanocomposites
2
could accelerate the decomposition of H O into ROSs,
2
2
3
3
enhancing the ECL intensity and leading to a highly sensitive
detection for cholesterol.
mM in EG) were successively added into the PVP solution. The
obtained mixture was then poured into a 50 mL Teflon-lined
autoclave followed by thermal treatment at 140 ℃ for 1 h.
AgNCs were collected by centrifugation and dispersed in 10 mL
Experimental
deionized water. To synthesize 3D-MoS -PANI-AgNCs, firstly, 1
2
Reagents and chemicals
mL AgNCs and 3 mL 3D-MoS -PANI (1 mg/mL) were mixed
2
30
35
40
45
Cholesterol oxidase (ChOx, EC 1.1.3.6, ≥50 units/mg),
6
under stirring overnight. Subsequently, 3D-MoS -PANI-AgNCs
2
cholesterol, Triton X-100, chitosan (Chi: MW ca. 1×10 , ＞85%
deacetylation) and AgNO were all purchased from sigma (St.
nanocomposites were separated by centrifugation, washing and
redispersion. The preparation process was pictured in scheme 1.
3
Louis, MO, USA). Na MoO ·2H O, aniline, ammonium
2
4
2
persulfate (APS), thiourea, poly vinylpyrrolidone (PVP-30000),
ethylene glycol (EG), FeCl3 were all obtained from KeLong
Chemical Reagent Co. (Chengdu, China). Human serum samples
were obtained from Ninth People’s Hospital (Chongqing, China).
Phosphate-buffered saline (PBS, containing 10% Triton X-100)
solutions with various pH were prepared using 0.050 M Na HPO4
2
and 0.050 M KH PO with 0.10 M KCl as supporting electrolyte.
2
4
PBS (0.050 M, pH 7.4) containing 10% (w/w) of Triton X-100
was used to prepare cholesterol stock solution in a heated water
bath at 65℃. Other chemicals used were of analytical grade and
were used as received. Deionized water was used all over the
experiments.
Apparatus
A CHI 600D electrochemical station (Shanghai CH Instruments
Co., China) was used to carry out electrochemical impedance
Scheme 1. The illustration of the synthetic process of 3D-MoS2-
PANI-AgNCs and the preparation of the ECL biosensor.
50
spectroscopy
(EIS)
experiments.
A
model
MPI-A
electrochemiluminescence analyzer (Xi’an Remax Electronic
Science & Technology Co. Ltd., China) was used to detect the
ECL signals. The voltage of the photomultiplier tube (PMT) was
set at 800 V and the potential scan was set as -0.3~0.3V
throughout the detection. All experiments were measured with a
95
Fabrication of 3D-MoS -PANI-AgNCs modified electrode
2
The fabrication process for the biosensor was pictured in Scheme
1. Prior to modification, the glassy carbon electrode (GCE, Φ=4
mm) was polished with 0.3 µm and 0.05 µm alumina slurry,
55
conventional three-electrode, in which a saturated calomel 100 respectively, and sonicated with ethanol and deionized water,
respectively. After dried in air, the pre-treated GCE was first cast
electrode (SCE) or Ag/AgCl (sat. KCl) was reference electrode, a
2
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with 15 µL 3D-MoS -PANI-AgNCs suspension and dried at the
2
1487
room temperature. Following that, 5 µL ChOx solutions were
a
incubated onto the electrode for 8 h. The resultant cholesterol
1164
biosensor (ChOx/3D-MoS -PANI-AgNCs/GCE) was stored at
2
1336
5
4 ℃ until further use.
Results and discussion
Characterization of different nanomaterials
379
405
b
The microstructures and morphologies of as-prepared
nanomaterials were characterized by SEM. As seen in the SEM
image of PANI (Fig. 1A), a typical aggregated fiber nanostructure
could be observed. Fig. 1B exhibits the SEM image of 3D-MoS2-
PANI nanoflowers. 3D hierarchical flower-like structures were
clearly observed, demonstrating the successful preparation of 3D-
1
1
2
2
3
0
5
0
5
0
2
50
500
750 1000 1250 1500
-1
Raman shift / cm
40
Fig. 2. Raman spectra of (a) PANI and (b) 3D-MoS -PANI.
2
MoS -PANI nanoflowers. For the AgNCs, the SEM image
2
displayed the cube-like shape with uniform particle size and
shape distribution (Fig. 1C). When AgNCs was combined with
ECL and EIS characterization of stepwise fabrication of the
electrode
45
3
D-MoS -PANI nanoflwers, the resultant SEM image showed the
2
ECL was performed to characterize the stepwise fabrication of
the electrode in PBS (pH 7.4) containing 0.25 mM luminol. As
depicted from the ECL dynamic curve (Fig. 3A) and ECL-
potential curve (Fig. 3B), a relatively low ECL intensity appeared
presence of many AgNCs on the 3D-MoS -PANI nanoflowers
2
(Fig. 1D), indicating that the 3D-MoS -PANI-AgCNs
2
nanocomposites were synthesized successfully.
Raman scattering was measured to study the chemical bonding
50
at bare GCE (curve a). When the 3D-MoS -PANI-AgNCs was
2
structure of PANI and 3D-MoS -PANI. Fig. 2 curve a showed the
2
cast on the GCE, an improved ECL intensity was observed (curve
Raman spectra of PANI, two Raman characteristic bands around
-
at 1487 cm and 1336 cm could be attributed to the C=N
1
-1
b), due to the excellent conductivity of 3D-MoS -PANI-AgNCs.
2
+
stretching vibration of the quinonoid ring and the C-N stretching
However, after ChOx was incubated onto the electrode, the ECL
intensity was decreased (curve c), ascribing to the obstacle of the
vibration of the benzenoid ring in the PANI, respectively. The
-
characteristic peak located around 1164 cm can be assigned to
1
55 non-conductive ChOx.
To gain a better understanding of assembly process of the
2
9,30
the C-H bending of the benzenoid ring.
Compared with the
electrode, EIS was conducted in PBS (pH 7.4) containing 5.0 mM
K [Fe(CN) ]/K [Fe(CN) ]. Fig. 3C exhibited the typical
Raman spectra of PANI, the 3D-MoS -PANI nanoflowers
2
3
6
4
6
exhibited two other Raman characteristic bands at 405 and 379
-
cm (Fig.2 curve b), corresponding to the A and E modes of
1
1
impedance spectra, which are presented in the form of the
60 Nyquist plot. The semicircle diameter at higher frequencies
1
g
2g
3
1,32
MoS nanostructure, respectively.
2
Above results indicated the
equals the electron-transfer resistance (R ). Compared with the
et
successful synthesis of 3D-MoS -PANI nanoflowers.
2
bare GCE (curve a), the 3D-MoS -PANI-AgNCs modified
2
electrode presented a decreased R (curve b), as the 3D-MoS -
et
2
PANI-AgNCs could promote the electron transfer. When ChOx
65
was cast onto the electrode, there was an obvious increase of Ret
(
curve c), indicating that the ChOx could hinder the electron
4−/3−
transfer of redox probe [Fe(CN)6]
.
3
5
450
300
150
0
b
c
A
a
Fig. 1. SEM images of (A) PANI, (B) AgNCs, (C) 3D-MoS2-
PANI and (D) 3D-MoS -PANI-AgNCs modified films.
2
1
2
3
4
5
Time / s
70
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experiments.
3
5
450
300
150
0
B
b
2000
1500
1000
A
c
a
500
-0.2
0.0
0.2
0
0
.0
0.1
0.2
0.3
0.4
Potential / V
luminol / mM
4
00
C
B
2
1
1
000
a
b
c
300
200
100
0
500
000
5
00
5
10
15
20
25
3D-MoS2-PANI-AgNCs / µL
200
400
600
800
Z' / ohm
C
Fig. 3. (A) ECL dynamic curve and (B) ECL-potential curve of
4800
5
0
5
0
5
0
(a) bare GCE, (b) 3D-MoS -PANI-AgNCs/GCE and (c)
2
ChOx/3D-MoS -PANI-AgNCs/GCE in 0.050 M PBS (pH 7.4)
2
3
200
containing 0.25 mM luminol, scan voltage: -0.3 V to 0.3 V, scan
rate: 200 mV/s. (C) EIS in PBS containing 5.0 mM
K [Fe(CN) ]/K [Fe(CN) ] (1:1) of above different modified
3
6
4
6
1600
0
1
1
2
2
3
electrodes.
Optimization of main experimental conditions
To achieve the excellent performance of the prepared luminol
based ECL biosensor for cholesterol detection, the concentration
of luminol was optimized. As pictured in Fig. 4A, the ECL
intensity showed a rapid increase with the increase of luminol
concentration from 0.050 to 0.25 mM. With the concentration up
to 0.25 mM, only a slight enhancement of ECL intensity was
observed. Therefore, 0.25 mM was selected as the optimal
concentration of luminol in subsequent work.
7
8
9
10
11
pH
4
4
5
5
0
5
0
5
Fig. 4. Effect of (A) luminol concentration, (B) the volume of
D-MoS -PANI-AgNCs suspension cast on the GCE on the ECL
3
2
response to 0.050 mM cholesterol in 0.050 M PBS (pH 7.4)
containing 0.25 mM luminol. Effect of pH (C) on the ECL
response of the biosensor to 0.050 mM cholesterol in 0.050 M
PBS containing 0.25 mM luminol. At the ChOx/3D-MoS -PANI-
2
The volume of 3D-MoS -PANI-AgNCs suspension was
2
AgNCs/GCE, scan voltage: -0.3 V to 0.3 V, scan rate: 200 mV/s.
investigated since it would affect the quantity of 3D-MoS -PANI-
2
AgNCs modified onto the electrode. The results indicated that the
The probable mechanism of the biosensor
ECL intensity enhanced with the augment of 3D-MoS -PANI-
2
As is well known, H O is an efficient coreactant for luminol
2
AgNCs volume from 5 to 15 µL and then reached a relatively
stable step after 15 µL (Fig. 4B). So the optimum volume of 3D-
2
ECL reactions. In the luminol-H O2 system, H O2 could be
2
2
catalyzed to generate ROSs. ROSs, as important intermediates of
luminol ECL reactions, could enhance ECL signal. To investigate
the ECL response of the modified electrode, first, the ECL
MoS -PANI-AgNCs was 15 µL.
2
The effect of pH on the ECL intensity was also surveyed over
the pH from 7.0 to 9.0 in PBS containing 0.25 mM luminol. As
evident from Fig. 4C, the maximum ECL intensity could be
obtained at pH 9.0. However, considering the normal
physiological pH of human body and the activity of ChOx, the
ECL detection was tested in PBS (pH 7.4) throughout the
response of 3D-MoS -PANI-Ag/GCE without ChOx and
2
ChOx/3D-MoS -PANI-Ag/GCE with ChOx were tested. As
2
presented in Fig. 5A, a relatively weak ECL intensity was
observed at 3D-MoS -PANI-Ag/GCE without ChOx (curve a),
2
4
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while the ECL intensity could be significantly enhanced at 35 AgNCs/GCE and (c) ChOx/3D-MoS -PANI-AgNCs/GCE.
2
ChOx/3D-MoS -PANI-AgNCs/GCE with ChOx (curve b). This
2
may be due to the fact that ChOx catalyzed cholesterol to in situ
produce H O , as a coreactant of luminol, which could promote
Performance of the biosensor
The performance of the biosensor for the detection of cholesterol
was explored under the optimized experimental conditions and
2
2
5
0
5
0
5
the oxidation of luminol to generate cathodic ECL signal.
Additionally, in order to investigate the role of MoS and AgCNs, 40 the results are exhibited in Fig. 6A. As expected, the cathodic
2
as control experiments, the ECL response of ChOx/3D-MoS2-
PANI/GCE without AgNCs and ChOx/PANI-AgNCs/GCE
ECL intensity increased proportionally with the concentration of
cholesterol in the range of 3.3 nM to 0.45 mM and the
corresponding calibration curve for cholesterol detection at -0.19
V is illustrated in the inset of Fig. 6B. The regression equation
without MoS were tested in 0.25 mM luminol. As observed, both
2
1
1
2
2
the ChOx/3D-MoS -PANI/GCE (Fig. 5B curve a) and
2
2
ChOx/PANI-AgNCs/GCE (Fig. 5B curve b) performed
a
45 was I (a.u.) = 503.67+23680 c (mM) (R =0.9970) and the
relatively low ECL intensity. However, as expected, a strong
detection limit was 1.1 nM (S/N=3). Compared with other
methods of cholesterol detection (Table 1), the proposed
biosensor in this work exhibited a lower detection limit down to
ECL intensity was obtained at the ChOx/3D-MoS -PANI-
2
AgCNs/GCE with both MoS and AgNCs, as shown in Fig. 5B
2
-
9
curve c. This phenomenon may be attributed to the fact that both
10 M. Additionally, the prepared biosensor also performed a
AgCNs and MoS could catalyze H O to generate ROSs, thus 50 high sensitivity and wide dynamic response range with five
2
2
2
amplifying the luminol-based ECL intensity. According to the
9
orders of magnitude. These results may be due to the following
,10
previous report and the experiment results,
the probable
facts. On one hand, 3D-MoS -PANI-AgNCs nanocomposites
2
mechanism is depicted as follows. First, ChOx modified on the
with large surface area and good biocompatibility could load
more ChOx on the electrodes. On the other hand, 3D-MoS2-
electrode catalyzed the oxidation of cholesterol to produce H O2
2
and cholest-4-en-3-one. Then, the MoS2 and AgCNs could 55 PANI-AgNCs
nanocomposites
could
accelerate
the
catalyze H O to generate ROSs, which could react with luminol decomposition of H O to produce ROSs, enhancing the ECL
2
2
2
2
to produce excited 3-aminophthalate dianion, thus leading to
improved ECL intensity. The mechanism for the cathodic ECL
reaction of luminol is presented in scheme 1.
intensity. Moreover, the cathodic luminol-base ECL has high
sensitivity.
12000
A
p
1
200
800
00
A
4000
3000
2000
1000
0
9000
6000
3000
0
b
a
g
a
4
0
-
0.2 0.0
0.2
a
-0.2
0.0
Potential / V
0.2
0.4
6
0
-
0.2
0.0
Potential / V
0.2
B
I (a.u.) =503.7+2.368E4 C (mM)
000 R2= 0.9972
1
2000
B
4
000
000
000
000
0
c
9
6
3
3
000
000
0
2
1
b
a
a
-
0.0
0.1
0.2
0.3
0.4
0.2
0.0
0.2
Cholesterol / mM
Potential / V
Fig. 5. (A) ECL response of (a) 3D-MoS -PANI-AgNCs/GCE
2
3
0
and (b) ChOx/3D-MoS -PANI-AgNCs/GCE to 0.15 mM
2
cholesterol in 0.050 mM PBS (pH 7.4) containing 0.25 mM
luminol. (B) ECL reponse of different modified electrodes to 0.15
mM cholesterol in 0.050 mM PBS (pH 7.4) containing 0.25 mM
luminol: (a) ChOx/3D-MoS -PANI/GCE, (b) ChOx/PANI-
2
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DOI: 10.1039/C5RA09638C
To evaluate the stability of the proposed biosensor, the biosensor
25 was measured under consecutive scanning in PBS containing
C
RSD=2.4%
0
.25 mM luminol. As seen in Fig. 6C, the ECL intensity did not
4
3
1
500
000
500
0
present obvious change after successive scanning for 12 cycles
and the relative standard deviation (RSD) was 2.4 %.
Simultaneously, the long-term storage stability was also studied.
3
0
The biosensor was stored at 4℃ and tested every three days in
PBS containing 0.25 mM luminol. In this way, the response of the
biosensor retained about 92.3 % of the initial response after a
storage period of 15 days. These results demonstrated that the
proposed biosensor exhibited a satisfactory stability.
35
The reproducibility of the proposed biosensor was
investigated as the following way. Five prepared biosensors were
tested with the same concentration of cholesterol (0.1 mM) in the
same conditions. Five biosensors performed similar ECL
responses and the RSD was 4.9 %, indicating an acceptable
0
20
40
Time / s
60
D
2400
1800
1200
40 reproducibility.
To assess the selectivity and specificity of the proposed
biosensor, several possible interferents were studied, such as
dopamine (DA), ascorbic acid (AA) and uric acid (UA). Fig. 6D
pictured the comparison of the biosensor toward different targets.
As expected, the biosensor performed negligible response to DA,
AA and UA, indicating a high specificity of the proposed
biosensor for cholesterol detection.
4
5
6
00
Table 2 Recoveries of cholesterol by the biosensor in human
0
Blank
UA
DA
AA MixtureCholesterol
5
0
serum samples
a
b
Found (mM)
Samples
Added (mM)
Recovery (%)
Fig. 6. (A) ECL response of the biosensor to (a) 0, (b)
.00000330, (c) 0.0000200, (d) 0.000220, (e) 0.000620, (f)
0.00262, (g) 0.0267, (h) 0.0567, (i) 0.0967, (j) 0.147, (k) 0.197,
l) 0.247, (m) 0.297, (n) 0.347, (o) 0.397, (p) 0.447 mM in 0.050
mM PBS (pH 7.4) containing 0.25 mM luminol. (B) Calibration
curve at different concentration of cholesterol. (C) The stability
of the biosensor to 0.18 mM cholesterol in 0.05 mM PBS (pH
7.4) containing 0.25 mM luminol. (D) The selectivity of the
biosensor against different interfering samples: (a) blank, (b) UA
0
5
0
5
1
2
3
4
0.00020
0.0040
0.040
0.00021±0.00001
0.0041±0.0003
0.039±0.003
0.21±0.01
105
103
97.5
105
(
1
0.20
(
0.3 mM), (c) DA (0.3 mM), (d) AA (0.3 mM), (e) a mixture
a
The human serum sample from 9 th People’s Hospital of Chongqing,
Chongqing
containing UA (0.3 mM), DA (0.3 mM), AA (0.3 mM) and
cholesterol (0.1 mM) and (f) cholesterol (0.1 mM).
b
average of three determination ± SD, n=3
1
5
5
Application in the real samples and recovery test
Table 1 Comparison of different methods for the determination of
cholesterol.
The analytical reliability and potential application of the proposed
biosensor was examined in human serum samples by a standard
additon method. A series of serum samples were diluted with
PBS. As seen from Table 2, the recoveries ranged between 97.5
and 105 %, indicating that the proposed biosensor was available
for detecting cholesterol in real biological samples.
Refe
renc
e
Determine
method
Detection
limit (mM)
Electrode materials
ChOx/CS-GR/GCE
Linear range (mM)
6
0
Differential pulse
voltammetry
/CT/GCE Amperometry
-3
5.0×10 -1.0
-4
7.2 ×10
33
34
-
3.0 ×10 -1.2×10
2
1
-3
ChOx/MnO
2
2.1×10
ChOx/ZnO-
CuO/ITO/glass
bioelectrode
Cyclic
voltammetry
-1
5.0×10 -1.2×10
1
―
35
Conclusions
ChOx/MWCNTs-GO-
Thi-Au/GCE
-4
1.5×10 -8.3×10
-1
-5
ECL
ECL
5.0×10
36
In conclusion, a sensitive cathodic luminol-based ECL biosensor
ChOx/3D-MoS
AgNCs/GCE
2
-PANI-
-6
3.3 ×10 -4.5 ×10
-1
-6
This 65 was fabricated with 3D-MoS -PANI nanoflowers and AgNCs for
work
2
1.1×10
ECL signal enhancement. The proposed biosensor exhibited a
high sensitivity and low detection limit for the determination of
2
0
CS-GR: chitosan-graphene MWCNTs: multiwalled carbon nanotubes, GO:
grapheme, Thi: thionine,Au: gold nanoparticles.
cholesterol due to the excellent properties of 3D-MoS -PANI
2
nanoflowers and AgNCs. Therefore, the integration of 3D-MoS2-
70 PANI nanoflowers and AgNCs would be the promising
Stability, reproducibility and interference determination
6
|
Journal Name, [year], [vol], 00–00
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DOI: 10.1039/C5RA09638C
2
2
7 V. Tharmaraj and K. Pitchumani, Nanoscale, 2011, 3, 1166.
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of China (21075100, 21275119), Ministry of Education of China
(708073), Science and Technology Commission of Beibei (2012-
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