A sandwich-like electrochemiluminescent biosensor for the detection of concanavalin A based on a C60-reduced graphene oxide nanocomposite and glucose oxidase functionalized hollow gold nanospheres

Article abstract of DOI:10.1039/c4ra08274e

A sensitive sandwich-like electrochemiluminescent (ECL) biosensor was designed for the detection of concanavalin A (ConA) using a C₆₀-reduced graphene oxide (C₆₀-rGO) nanocomposite as a platform and glucose oxidase (GOX) decorated hollow gold nanospheres (HGNSs) as a label. First, C₆₀-rGO with a large surface area was prepared for combining with phenoxy-derivatized dextran, which served as the recognition element for interacting with ConA by biospecific carbohydrate-protein (lectin) interactions. Then, GOX decorated HGNSs (GOX-HGNSs) were linked to the electrode surface through the biospecific interaction between the intrinsic carbohydrate residues of GOX and ConA. These localized GOX and HGNSs amplified the ECL signal of luminol intensely, which was achieved by the efficient catalysis of the GOX towards the oxidation of glucose to in situ generate an improved amount of hydrogen peroxide (H₂O₂) as a coreactant, and the excellent catalysis of HGNSs towards the ECL reaction of luminol-H₂O₂. The prepared biosensor exhibited a sensitive response for the determination of ConA, ranging from 0.10 to 100 ng mL^-1 with a detection limit down to 30 pg mL^-1 (signal to noise = 3). With excellent stability, sensitivity, selectivity and simplicity, the prepared biosensor showed great prospects in lectin sensing or carbohydrate sensing. This journal is

Full text of DOI:10.1039/c4ra08274e

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A sandwich-like electrochemiluminescent
biosensor for the detection of concanavalin A
Cite this: RSC Adv., 2014, 4, 48465
based on a C –reduced graphene oxide
6
0
nanocomposite and glucose oxidase functionalized
hollow gold nanospheres
Juanjuan Zhang, Yuan Ruo, Shihong Chen,* Xia Zhong and Xiaoping Wu
A sensitive sandwich-like electrochemiluminescent (ECL) biosensor was designed for the detection of
concanavalin A (ConA) using a C60–reduced graphene oxide (C60–rGO) nanocomposite as a platform
and glucose oxidase (GOX) decorated hollow gold nanospheres (HGNSs) as a label. First, C60–rGO with a
large surface area was prepared for combining with phenoxy-derivatized dextran, which served as the
recognition element for interacting with ConA by biospecific carbohydrate–protein (lectin) interactions.
Then, GOX decorated HGNSs (GOX-HGNSs) were linked to the electrode surface through the
biospecific interaction between the intrinsic carbohydrate residues of GOX and ConA. These localized
GOX and HGNSs amplified the ECL signal of luminol intensely, which was achieved by the efficient
catalysis of the GOX towards the oxidation of glucose to in situ generate an improved amount of
hydrogen peroxide (H ) as a coreactant, and the excellent catalysis of HGNSs towards the ECL
2
O
2
Received 7th August 2014
Accepted 16th September 2014
reaction of luminol–H O . The prepared biosensor exhibited a sensitive response for the determination of
2
2
ꢀ1
ꢀ1
ConA, ranging from 0.10 to 100 ng mL with a detection limit down to 30 pg mL (signal to noise ¼ 3).
With excellent stability, sensitivity, selectivity and simplicity, the prepared biosensor showed great
prospects in lectin sensing or carbohydrate sensing.
DOI: 10.1039/c4ra08274e
www.rsc.org/advances
What's more, the sensitivity needed to be further improved.
These challenges drive us to explore a new detection technique
Introduction
Concanavalin A (ConA) is a plant lectin isolated from the jack combined with novel signal amplication strategy to realize the
1
bean. As a protein, it has an affinity for mannose, glucose and highly sensitive determination of ConA.
some glycoproteins, such as horseradish peroxidase and glucose
Electrochemiluminescence is a new detection technique
oxidase. The investigation of ConA is signicant because ConA developed in recent years. It has become a powerful analytical
2
is a powerful probe to search for cell-surface sugar chains when tool owing to its advantages such as sensitivity, rapidity,
3
19
ConA interacts with the glycoprotein on the cell surface.
simplicity, controllability, and low background noise. Among
Besides, ConA affects mitogenic effect and lymphocyte trans- various electrochemiluminescent (ECL) systems, luminol–H
2
O
2
4
formation and is applicable to the analysis of malignant tumor system integrates inexpensive reagent consumption, low
5
cells. Furthermore, ConA was oen chosen as a lectin model for oxidation potential and high emission yields, thus arousing
6
–8
20
further study of carbohydrate–protein interactions.
Up to now, the main detection techniques for ConA are catalyze the oxidation of glucose to in situ generate H
electrochemical method including differential pulse voltam- served as a coreactant of luminol to enhance ECL signal of
some concern. As well-known, glucose oxidase (GOX) could
which
O
2 2
9
–11
12,6
metry
optic method including uorescence spectroscopy,
and electrochemical impedance spectroscopy, and luminol. This amplication strategy will overcome the shortage
1
3,14
2 2
UV- of instability of H O directly as coreactant, improving the
15,16
17,18
21
visible spectroscopy,
and surface plasmon resonance.
sensitivity of the biosensor. Additionally, it is demonstrated
Generally, these methods suffered from tedious procedures for that some noble metal nanoparticles especially gold nano-
preparing derivatives of carbohydrate for binding with ConA. particles exhibit excellent catalytic performance to directly
22
2 2
enhance the ECL signal of luminol–H O system. For example,
Cui's research group reported ECL biosensors based on luminol
functionalized gold nanoparticles for the detection of cardiac
troponin I and thrombin with satisfying performance. Since
Education Ministry Key Laboratory on Luminescence and Real-Time Analysis, College
of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715,
People's Republic of China. E-mail: cshong@swu.edu.cn; Fax: +86-23-68253172; Tel:
23
24
the size and shape would affect the properties of materials,
+
86-23-68252277
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different nanostructured-gold materials have been synthesized A (ConA, from CanaValia ensiformis jack bean), 1,2-epoxy-3-phe-
2
2–24
25
for various applications, such as, nanoparticles,
nanorods,
noxypropane, and phytohaemagglutinin were purchased from
26
27
nanotubes, hollow nanospheres. Among them, hollow gold Sigma Chemical Co. (St. Louis, MO, USA). Hydrogen tetra-
nanospheres (HGNSs) have attracted considerable interest due chloroaurate (HAuCl $4H O) was obtained from Shanghai Chem-
4
2
to their large surface area, good biocompatibility and excellent ical Reagent Co., (Shanghai, China). Alpha fetoprotein and
conductivity. Particularly, the tunable interior and exterior carbohydrate antigen 15-3 were received from Biocell Co.
structure of HGNSs allows more molecules to adsorb on the (Zhengzhou, China). Poly(ethylenimine) (PEI, 50%) was purchased
28
inside and outside surface of the walls.
from Fluka (Switzerland). Graphene oxide (GO) was purchased from
Graphene, as a one-atom-thick planar sheet comprising an Nanking Xianfeng Nano Co. (Nanking, China). Phosphate-buffered
2
2+
sp -bonded carbon structure, has emerged as a rapidly rising saline (PBS) solutions containing 0.10 M KCl, 0.10 mM Ca and
29
2+
star in the eld of nanomaterial science. Fullerene (C ), as a 0.10 mM Mg were prepared with 0.10 M H PO , adjusted pH with
6
0
3
4
zero-dimensional carbon material, has attracted enormous concentrated NaOH solution. Ferricyanide solutions containing
interest recently owing to its excellent photo/physical behavior, 5.0 mM K Fe(CN) and 5.0 mM K Fe(CN) were used as a redox
charge transport, and efficient charge separation. The nano- reporter. A stock ConA solution (1.0 mg mL ) was prepared in PBS
3
6
4
6
ꢀ1
30
2+
2+
composite of C60 and graphene could combine the merit of C60 (0.075 M, pH 7.4) containing 0.10 mM Ca and 0.10 mM Mg then
ꢁ
2+
2+
and graphene, thus showing new properties. For example, this stored at 4 C. Herein, Ca and Mn were necessary to activate
6
nanocomposite with three-dimensional structure would ConA conformation. A stock solution of luminol (0.010 M) was
increase the efficient surface area of graphene. Despite each of prepared by dissolving luminol in 0.10 M NaOH solution and was
ꢁ
them had been applied in several elds, ranging from sensors kept at 4 C when not in use. Phenoxy-derivatized dextran (PDN) was
and photovoltaic cells to nanostructure devices for advanced synthesized according to a previous method in ref. 33, and its
29,31
electronic applications,
the report about the nanocomposite molecule structure is shown in Scheme 1, inset (B). All other
of C60 and graphene is rare. The possible reasons may be as chemicals were of analytical grade and were used as received.
follows. C60 is easily soluble in toluene but difficultly soluble in Deionized water was used throughout this study.
water. However, graphene is usually water-soluble aer func-
tionalized by hydrophilic groups such as amido and oxygen
containing groups. This difference makes their homogeneous
Apparatus
Cyclic voltammetry (CV) was performed with a CHI 600D elec-
trochemical work station (Shanghai CH Instruments Co.,
China). ECL emission was measured using a model MPI-A
electrochemiluminescence analyzer (Xi'an Remax Electronic
Science & Technology Co. Ltd., China), equipped with a pho-
tomultiplier tube with its voltage set at 800 V. The potential was
co-interaction difficult. Luckily, the water-soluble C60 could be
achieved by long-time ultrasonic of the mixture of C –toluene
dispersion and water, which makes the homogeneous co-
interaction of C60 and graphene possible.
6
0
32
In this work, we synthesized a nanocomposite of C60 and
reduced graphene oxide (C60–rGO) as matrix for immobilizing
biomolecules. Through sandwich scheme, a sensitive ECL
biosensor for ConA was designed based on C –rGO, HGNSs
ꢀ1
set from 0.2 to 0.8 V at a scan rate of 0.3 V s in the detection
process. The conventional three-electrode system with a modi-
6
0

ed glassy carbon electrode (GCE) as working electrode, a
and the advantages of in situ generating coreactant. Concretely,
using poly(ethylenimine) as linking reagent, C –rGO was
saturated calomel electrode (SCE) or Ag/AgCl (saturated KCl) as
reference electrode, and a platinum wire as counter electrode
6
0
synthesized and cast on the electrode surface for interacting
with phenoxy-derivatized dextran (PDN) through p–p stacking.
Next, ConA was modied on the electrode surface by biospecic
interaction with PDN. Then, the GOX-HGNSs were linked to the
electrode surface via sandwich biospecic reactions. These
localized GOX and HGNSs would greatly amplify the ECL signal
of luminol due to the facts that GOX could catalyze the oxida-
tion of glucose to in situ generate improved amount of H O as
2
2
coreactant and HGNSs could enhance the ECL signal of lumi-
nol–H system. Therefore, as the immobilization amount of
2 2
O
ConA increased, the amount of GOX-HGNSs increased, and the
ECL signal enhanced. This sandwich scheme not only inte-
grated virtues of C60–rGO and HGNSs, but also utilized a novel
signal amplication strategy with in situ generating coreactant,
thus achieving the highly sensitive detection of ConA.
Experimental
Reagents
Scheme 1 The schematic representation of the stepwise fabrication
process of the biosensor. Inset (A) is the synthesis process of C60–rGO.
Inset (B) is the structure of PDN. Inset (C) represents the biospecific
Glucose oxidase (GOX), bovine serum albumin (BSA, 96–99%),
luminol (5-amino-2, 3-dihydro-1,4-phthalazinedione), concanavalin interaction. Inset (D) is the TEM image of HGNSs.
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was used in the detection process. Absorption spectra were
recorded by a UV-2450 UV-vis spectrophotometer from Shi-
madzu Corp. (Japan). The morphologies of HGNSs were
analyzed by scanning electron microscopy (SEM, Hitachi, S-
4800, Japan) and transmission electron microscope (TEM,
TECNAI 10, Philips Fei Co., Hillsboro, OR). The topographs of
different modied lms were investigated with atomic force
microscopy (AFM, dimension ICON, USA). The fourier-trans-
form infrared spectroscopy (FT-IR) was recorded on a Nexus 670
FT-IR spectrophotometer (Nicolet Instruments) using a KBr
pellets. All of the above experiments were carried out at room
temperature.
Scheme 2 The mechanism of lumimol ECL emission.
HGNSs preparation
HGNSs were prepared according to the ref. 27 with minor electrode was immersed into PDN solution. Aerwards, 20 mL of
modication. Firstly, 200 mL 0.10 M sodium citrate solution was 0.5% BSA solutions was cast onto the electrode for 30 min to
added into 50 mL double distilled water with rapid magnetic block the non-specic binding sites. Then, the resultant elec-
stirring under the nitrogen (N
2
) atmosphere. Then, 200 mL of a trode was immersed into ConA solution for 1 h to achieve the
(0.50 M) immobilization of ConA on the electrode surface through the
freshly prepared NaBH (1.0 M) and 50 mL CoCl
4
2
solution were added, respectively. When the color of the solu- biospecic interaction between ConA and the sugar chains of
tion changed to gray, 150 mL 1% HAuCl solution was drop- PDN, as shown in Scheme 1, inset (C). Finally, GOX-HGNSs were
4
wisely added for six times, each time 25 mL. The color of the linked onto the modied electrode. The modied electrode was
solution changed from dark brown to deep blue and then cleaned with PBS (pH 7.4) to remove the unstably adsorbed
reacted for 30 min under rapid stirring. The resultant mixture species aer every modication step. The schematic diagram of
was separated and washed by centrifugation with double the fabrication of the biosensor is depicted in Scheme 1.
distilled water to get HGNSs.
Mechanism of the ECL emission and detection principle of
the biosensor
Preparation of C60–rGO nanocomposite
First, amido functionalized reduced graphene oxide (rGO) was
34
According to the report, the oxidation of luminol is believed to
prepared according to a previously described method. 5.0 mL
ꢀ
ꢀ
1
involve the oxygen-related radicals (for example, OHc, O c , and
2
dispersion of GO sheets (1.0 mg mL ) and 0.50 mL PEI (3%)
ꢁ
other radical derivatives) which were generated from H
2
O
2
in
were mixed and heated under reux in an oil bath at 135 C for
35–38
luminol–H O system.
These active oxygen-related radicals
2
2
about 3 h. The resultant PEI–rGO composite was centrifuged
and washed using double distilled water to remove redundant
PEI, and then redispersed in 2.0 mL water. Second, water-
react with luminol monoanion that is from the deprotonation of
luminol to produce 3-aminophthalate anions which emit light,
as shown in Scheme 2. In the present study, H O is generated
ꢀ
1
2
2
soluble C60 was prepared by adding 5.0 mL 1 mg L
60–toluene solution to 5.0 mL ultrapure water. With the aid of
from the oxidation of glucose catalyzed by GOX. In the presence
of enough amount of glucose in detection cell, the concentra-
tion of H O increases with the amount of GOX. Therefore, the
2 2
more target ConA are immobilized, the more GOX is assembled
through biospecic interaction, and the stronger ECL signal is
generated. The intensity of the ECL signal is directly propor-
tional to the concentration of ConA. So ConA can be detected
quantitatively using the prepared biosensor.
C
ultrasonic agitation, toluene in the solution would completely
volatilize, and C60 would be transferred to water phase. Aer-
wards, the yellow product was collected through centrifugation
and washed three times with distilled water. Finally, resultant
water-soluble C60 was mixed with PEI–rGO dispersion, and then
keep stirring at room temperature over a night. C60 will interact
with rGO by the amido on the surface of PEI–rGO to form
30
C
60–rGO nanocomposite. Aer centrifuged to remove the
unadsorbed C60, the C60–rGO nanocomposite was obtained and
characterized by UV-vis absorption spectrum and FT-IR. The
Results and discussion
synthesis process of C60–rGO is illustrated in Scheme 1, Characterization of C60–rGO nanocomposite
inset (A).
Fig. 1(A) shows the UV-vis absorption spectra of rGO, C60 and
60–rGO nanocomposite. For C60 aqueous solution (curve (a)),
three strong optical absorption peaks belonged to the dipole-
C
Fabrication of the ECL biosensor
The glassy carbon electrode (GCE, F ¼ 4 mm) was carefully allowed transitions in pristine C60 were observed at 211, 264,
polished with 0.3 mm and 0.05 mm alumina to obtain mirror-like and 341 nm, respectively. In the spectrum of rGO (curve (b)), the
surface, and then ultrasonically washed with ethanol and water, peak at 265 nm was ascribed to the p / p* transition of C]C.
respectively. Subsequently, 8 mL dispersion of C60–rGO was cast Curve (c) is the spectrum of C60–rGO nanocomposite. As
on the pretreated GCE and allowed to dry in air. Next, the observed, the three strong optical absorption peaks of C60 red-
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(
(
Fig. 2(A) (curve (b))). For the FT-IR spectroscopy of C60–rGO
ꢀ1
Fig. 2(A) (curve (c))), the peak at 1558 cm disappeared and the
ꢀ1
peak at 1631 cm decreased, conforming the fact that C
interacted with rGO via amido to successfully achieve C –rGO
6
0
30
6
0
nanocomposite. Aer C60–rGO interacted with PDN, compared
with the spectrum of C60–rGO (Fig. 2(A) (curve (c))), a new peak
ꢀ
1
was observed at 1046 cm
which was attributed to the
stretching vibration of C–O–C in dextran (Fig. 2(A) (curve (d))).
With the further modication of BSA, ConA and GOX-HGNSs,
the bending vibration of N–H band in amino acid residues of
ꢀ1
ꢀ1
proteins at 1640 cm and 1550 cm appeared again at the
spectrum of GOX-HGNSs/ConA/BSA/PDN/C –rGO composite
6
0
(Fig. 2(A) (curve (e))). Above results conrmed the change in
functional groups involved in each reaction step and showed
the successful preparation of the biosensor.
Cyclic voltammetry (CV) is a useful tool to evaluate the
assembly process of the modied electrodes. The cyclic vol-
tammograms (CVs) of different modied electrodes using
4
ꢀ/3ꢀ
Fe(CN)6
as an electroactive probe are presented in Fig. 2(B).
As seen from Fig. 2(B), a couple of quasi-reversible redox peaks
of the probe was observed at the bare electrode (curve (a)). When
the electrode was modied with C60–rGO, an increase in the
peak current was observed (curve (b)) owing to that C60–rGO
could accelerate the electron transfer rate between the electrode
Fig. 1 (A) The UV-vis absorption spectra of (a) C60, (b) rGO, and (c)
C
60–rGO nanocomposite. SEM images of (B) C60, (C) rGO, (D) C60
–
rGO, and (E) HGNSs. (F) is an enlarged SEM image of HGNSs.
4
ꢀ/3ꢀ
and Fe(CN)
6
, and meanwhile increase the interface area.
Aer consecutively modied with PDN (curve (c)), BSA (curve
shied to 222, 268, 345 nm, respectively, indicating that C60
interacted with rGO.
The surface morphologies of rGO, C60 and C60–rGO nano-
composite were investigated using SEM. Compared with the
SEM image of sole C60 (Fig. 1(B)) or rGO (Fig. 1(C)), the SEM
image of C –rGO (Fig. 1(D)) showed many heaves or sunkens,
6
0
indicating that C –rGO possessed a larger surface area than C
60
6
0
or rGO for achieving higher biomolecules loading. Fig. 1(E) and
F) display the SEM images of HGNSs. As seen from the enlarged
(
SEM image of HGNSs (Fig. 1(F)), a relatively rough surface
morphology was clearly noticed, indicating a large surface area
of HGNSs. Also, a darker centre was noticed, which may be
attributed to the non-conductivity of hollow structure. The
conguration of HGNSs was further conrmed by TEM char-
acterization and the TEM image is shown in Scheme 1, inset (D).
As seen from SEM and TEM images of HGNSs, some very small
gold particles were observed on the surface of HGNSs. They are
maybe the byproduct of the shell growth, namely, some small
particles that do not grow into the shell, but break off early in
27,39
the process.
Characterization of the fabrication process of the biosensor
The change in functional groups involved in each reaction step
was conrmed by FT-IR technique since it is considered as an
important tool for characterizing functional groups. There are
ꢀ
1
Fig. 2 (A) The FT-IR spectra of (a) C60, (b) rGO, (c) C60–rGO, (d)
60–rGO/PDN, and (e) C60–rGO/PDN/BSA/ConA/GOX-HGNSs. (B)
CVs of (a) bare GCE, (b) C60–rGO/GCE, (c) PDN/C60–rGO/GCE, (d)
BSA/PDN/C60–rGO/GCE, (e) ConA/BSA/PDN/C60–rGO/GCE, and (f)
four characteristic bands at 524, 573, 1179 and 1425 cm
Fig. 2(A) (curve (a))), which consistently matched the typical
four dipole-allowed of C . The PEI reduced graphene oxide
C
(
6
0
ꢀ
1
ꢀ1
showed characteristic peaks at about 1631 cm and 1558 cm
,
3 6
GOX-HGNSs/ConA/BSA/PDN/C60–rGO/GCE in 5.0 mM K [Fe(CN) ]/
ꢀ1
which were ascribed to the bending vibration of N–H band
4 6
K [Fe(CN) ] (1 : 1). Scan rate: 50 mV s .
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d)), and ConA (curve (e)), the current response successively
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(
declined, which was caused by their non-conductivity. When
the electrode was incubated in the dispersion of GOX decorated
HGNSs (curve (f)), an obvious increase in peak currents was
noticed at corresponding CVs, which was ascribed to good
conductivity of HGNSs. The CV studies proved that the
biosensor has been successfully prepared.
The surface topographies of differently modied lms were
noticed with AFM and the results are shown in Fig. 3. In
Fig. 3(A), a large quantity of C60 nanoparticles were homoge-
nously distributed on the rGO nanosheets. Aer modied with
PDN, the surfaces became blurry, which may be attributed to
PDN coating on the C60–rGO surface by p–p stacking, seen from
Fig. 3(B). When blocked by BSA and further interacted with
ConA protein by biospecic interaction (Fig. 3(C)), the AFM
image of the resulting lm exhibited a smoothing effect as
compared with that of C60–rGO/PDN lm, which might be due
to BSA and ConA molecules lling the interstitial places
between C60 nanoparticles, suggesting that the ConA and BSA
were successively immobilized. Aer GOX labelled HGNSs were
assembled by biospecic interaction between ConA and GOX,
some obvious nanoparticles emerged again, suggesting
GOX-HGNSs were successfully modied (Fig. 3(D)).
Fig. 4 The effects of (A) pH and (B) glucose concentrations on the ECL
response of the biosensor.
prohibited forming effective sandwich conguration, thus
leading to a signicant decrease in ECL response. Whereas,
high pH led to the denaturation of ConA. At room temperature,
the ECL signals were recorded at the prepared biosensor
(
GOX-HGNSs/ConA/BSA/PDN/C60–rGO/GCE) in an ECL detector
ꢀ4
cell containing different pH PBS with 2.0 ꢂ 10 M luminol and
0
.030 M glucose. The intensity of ECL signal at the biosensor
ꢀ1
incubated without and with 10 ng mL ConA was denoted as Io
and I , respectively. I /I was dened as the standard of optimal
t
t o
value. The corresponding results are shown in Fig. 4(A). As seen,
pH 7.4 is optimal for further experiments.
The concentration of glucose needs to be investigated since
glucose, as the substrate of enzyme catalytic reaction, could be
oxidized to gluconic acid and meanwhile produce H O , the
2 2
coreactant of luminol for enhancing ECL signal. The effect of
glucose concentration on ECL response was investigated in
Optimization of analytical conditions
In order to achieve the maximum sensitivity for ConA, experi-
mental parameters including pH and glucose concentration
which affect the performance of the biosensor had been
discussed.
The pH affects the luminol's ECL signal for the following
reasons. (1) Luminol emitted strong chemiluminescent/ECL
signal in alkaline condition. (2) The catalytic activity of GOX was
affected by pH condition. (3) At low pH, ConA released the metal
ions, and consequentially presented in an inactive form, which
ꢀ4
0
.075 M PBS (pH 7.4) containing 2.0 ꢂ 10 M luminol at the
ꢀ1
biosensor incubated with 10 ng mL ConA, and the corre-
sponding results are shown in Fig. 4(B). As seen, a strong signal
was obtained with 0.030 M glucose. Thus, 0.030 M was chosen
in the further experiments.
Fig. 5 (A) The ECL response of (a) GOX-HGNSs/BSA/PDN/C60–rGO/
GCE and (b) GOX-HGNSs/ConA/BSA/PDN/C60–rGO/GCE. (B) ECL
responses of the biosensor to different concentrations of ConA. (C)
Calibration curve for ConA assay. (D) The stability of the biosensor
ꢀ1
Fig. 3 AFM images of differently modified films: (A) C60–rGO, (B) PDN/ incubated with 50 ng mL ConA under consecutive cyclic potential
C
60–rGO, (C) ConA/BSA/PDN/C60–rGO, and (D) GOX-HGNSs/ConA/ scans for 13 cycles. All working buffers were 0.10 M PBS (pH 7.4)
ꢀ4
BSA/PDN/C60–rGO.
containing 2.0 ꢂ 10 M luminol and 0.030 M glucose.
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Table 1 Analytical performance of different detection approaches for the determination of ConA
Linear range
Detection limit
ꢀ
1
ꢀ1
Immobilization method
Detection approach
(mg mL
)
(mg mL
)
Reference
AuNp/SPCE/Sugar-MPS/ConA
Electrochemical impedance
spectroscopy
2.23–40
0.8
6
Glucose assembled onto multi-wall carbon nanotube-
polyaniline
Capture ConA with glycosylated aniline polymer
Differential pulse voltammetry
0.00034–0.95
0.3–0.15
0–30
0.00010
0.012
0.12
11
12
13
15
Electrochemical impedance
spectroscopy
Fluorescence
Trityl-derivatized mannose immobilized on a polystyrene
microplate
Glycolipid molecules self-assembled onto gold
nanoparticles
UV-vis spectrum
2.5–7.5
0.01
GO/DexP/ConA/Dex-AuNPs modied gold chips
GOX-HGNSs/ConA/BSA/PDN/C60–rGO/GCE
Surface plasmon resonance
Electrochemiluminescence
1.0–20.0
0.00010–0.10
0.39
0.000030
18
This work
Quantitative analysis of ConA
Second, through a sandwich protocol, the localized GOX and
HGNSs were assembled onto the electrode which could
generate coreactant in situ for ECL signal enhancement.
Fig. 5(A) compared the ECL response of GOX-HGNSs/BSA/PDN/
C –rGO/GCE and GOX-HGNSs/ConA/BSA/PDN/C –rGO/GCE.
6
0
60
Compared with the control electrode GOX-HGNSs/BSA/PDN/
C –rGO/GCE without ConA (curve (a)), the prepared biosensor Reproducibility, stability, and selectivity of ECL biosensor
6
0
GOX-HGNSs/ConA/BSA/PDN/C60–rGO/GCE with ConA pre-
sented a greatly enhanced ECL signal (curve (b)). This is due to
the localized GOX and HGNSs which amplied the luminol ECL
signal. The quantitative analysis of ConA was performed at
GOX-HGNSs/ConA/BSA/PDN/C60–rGO/GCE under optimal
experimental conditions and the results are shown in Fig. 5(B).
As seen, with an increase in the incubated ConA concentration,
an increase in ECL signal was noticed. The calibration curve
The reproducibility of the biosensor was estimated by testing
the 1 ng mL ConA with ve modied electrodes in the same
batch. The relative standard deviation (RSD) was found to be
ꢀ1
4.6%, indicating an acceptable reproducibility.
The stability of the biosensor was investigated by testing its
ECL response in PBS under consecutive cyclic potential scans
for 13 cycles. The curve of ECL signal (vs. time) is shown in
Fig. 5(D) (RSD ¼ 1.0%).
(
Fig. 5(C)) of ECL intensity versus ConA concentration was linear
To further study the selectivity of the biosensor, different
ꢀ
1
from 0.10 to 100 ng mL , with a low detection limit of 30 pg
ꢀ1
interfering agents such as alpha fetoprotein (50 ng mL ), BSA
ꢀ1
mL (signal to noise ratio ¼ 3). The regression equation was I
ꢀ1
(
0.5%, w/w), carbohydrate antigen 15-3 (0.1 U mL ) and phy-
ꢀ1
(
a.u.) ¼ 18.0c (ng mL ) + 144.7 (where I was the ECL intensity
ꢀ1
tohaemagglutinin (100 ng mL ) were investigated. The corre-
sponding results are shown in Fig. 6. As seen, the change in ECL
signal caused by these interfering substances was neglectable
compared with that of blank solution. However, the ECL signal
and c was the concentration of ConA) with a correlation coeffi-
cient of 0.994. The details of a comparison between our method
and previous methods are provided in Table 1. Compared to the
results of most previous reports, our prepared biosensor
exhibited a high sensitivity and a low detection limit, which was
due to the following reasons. First, the C60–rGO as the sup-
porting nanomaterials enabled low background ECL signal.
ꢀ1
obviously enhanced for 1 ng mL ConA compared with the
blank solution. This result indicated a good selectivity of the
prepared biosensor towards ConA.
Conclusions
In summary, a novel sandwich ECL biosensor for the sensitive
detection of ConA was developed using C –rGO as a substrate
6
0
for the immobilization of PDN and GOX-HGNSs as amplica-
tion reagent. The amplication of the luminol ECL signal was
achieved as follows. (1) HGNSs own large surface area, and good
biocompatibility, which is propitious to immobilize more GOX
for efficient catalysis towards glucose to in situ generate H
2 2
O .
(2) Due to excellent conductivity and large surface area, and
excellent electroactivity to luminol oxidization, the HGNSs
could amplify the ECL signal of luminol. Additionally, C –rGO
6
0
ꢀ1
with large surface area as a substrate for immobilizing PDN
presented low background signal. Therefore, the prepared
biosensor presented high sensitivity towards the determination
Fig. 6 The ECL response at the biosensor for (a) 1 ng mL ConA, (b)
blank solution, (c) carbohydrate antigen 15-3, (d) phytohaemag-
glutinin, (e) alpha fetoprotein, and (f) BSA.
48470 | RSC Adv., 2014, 4, 48465–48471
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of ConA. Furthermore, a satisfying stability, selectivity and 14 L. Zou, H. L. Pang, P. H. Chan, Z. S. Huang, L. Q. Gu and
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Acknowledgements
This work was nancially supported by the NNSF of China 18 C. F. Huang, G. H. Yao, R. P. Liang and J. D. Qiu, Biosens.
21075100, 21275119, 21105081), Ministry of Education of
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