Glucose oxidase-encapsulated nanogold hollow microspheres as labels based on a sensitive electroluminescent immunoassay

Article abstract of DOI:10.1039/c4ra08896d

We herein report the synthesis of glucose oxidase-encapsulated nanogold hollow microspheres (GOx-GHS), which show excellent catalytic performance for the reduction reaction of glucose, and the nanogold on the GOx-GHS provided the nanoparticles with good biocompatibility. In addition, the electrochemiluminescence (ECL) property of luminol was investigated and a type of luminol nano-composite (AuNPs-EGN-luminol) was prepared. The as-prepared nano-composite was found to have good ECL property of the luminol, as well as good electroconductibility. The prepared GOx-GHS and AuNPs-EGN-luminol were applied in a sandwich-type ECL immunosensor for cancer antigen 125 (CA125). In the immunosensor, AuNPs-EGN-luminol acted as a solid support for CA125 primary antibody, and GOx-GHS was employed as the supporter of CA 125 secondary antibody. With the addition of glucose, the hydrogen peroxide was prepared and the ECL property of the AuNPs-EGN-luminol was enhanced. The proposed immunosensor enabled CA125 concentrations to be determined in the range of 1 pg mL^-1 to 100 ng mL^-1, with a detection limit of 0.38 pg mL^-1.

Full text of DOI:10.1039/c4ra08896d

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PAPER
Glucose oxidase-encapsulated nanogold hollow
microspheres as labels based on a sensitive
electroluminescent immunoassay
Cite this: RSC Adv., 2014, 4, 52796
Xiu Wang,a Zhaoquan Bian,a Chengchao Chu,a Xiaoxiao Zheng,a Shenguang Ge,b
a
a
c
*
*
Jinghua Yu, Mei Yan and Xianrang Song
We herein report the synthesis of glucose oxidase-encapsulated nanogold hollow microspheres
(GOx–GHS), which show excellent catalytic performance for the reduction reaction of glucose, and the
nanogold on the GOx–GHS provided the nanoparticles with good biocompatibility. In addition, the
electrochemiluminescence (ECL) property of luminol was investigated and a type of luminol nano-
composite (AuNPs–EGN–luminol) was prepared. The as-prepared nano-composite was found to have
good ECL property of the luminol, as well as good electroconductibility. The prepared GOx–GHS and
AuNPs–EGN–luminol were applied in a sandwich-type ECL immunosensor for cancer antigen 125
(CA125). In the immunosensor, AuNPs–EGN–luminol acted as a solid support for CA125 primary
antibody, and GOx–GHS was employed as the supporter of CA 125 secondary antibody. With the
addition of glucose, the hydrogen peroxide was prepared and the ECL property of the AuNPs–EGN–
luminol was enhanced. The proposed immunosensor enabled CA125 concentrations to be determined in
Received 19th August 2014
Accepted 1st October 2014
DOI: 10.1039/c4ra08896d
www.rsc.org/advances
the range of 1 pg mLꢀ1 to 100 ng mLꢀ1, with a detection limit of 0.38 pg mLꢀ1
.
strong ECL at positive potentials.12 We have demonstrated that
the utilization of cost-effective luminol–hydrogen peroxide
system could overcome the disadvantages of conventional ECL
signal probes by simplifying analytical procedure and short-
ening analysis time. In addition, the previous works indicate
that the ECL behavior of luminol is strongly dependent on the
materials and surface status of the electrodes involved in the
ECL experiments. The luminol ECL on a platinum or gold NP-
modied electrode has been reported, and the luminol ECL
process could be accelerated due to the catalytic property of the
Pt or AuNPs.13,14
Recently, gold nanoparticles (AuNPs) have attracted consid-
erable attention as one of the most popular and promising
conducting materials because of its large specic surface area,
good stability and interesting electrical and optical properties15,
and biocompatibility.16,17 More importantly, AuNPs have
provided a good pathway of electron transfer, and they have
enhanced the immobilized amount of biomolecules.18 In addi-
tion, considerable attention has been paid to exfoliated gra-
phene (EGN) due to its fascinating two-dimensional structure,
unusual electrochemical properties, and large accessible
surface area.19,20 Because of the large surface area of EGN and
related derivatives it can also be used as an excellent carrier for
loading more active probes for binding biomolecules, offering a
signicant amplication on the electrochemical sensing
signals.21 Herein, the AuNPs are immobilized on the surface of
EGN through electrostatic incorporation using poly-
(diallyldimethylammonium chloride) (PDDA), fabricating the
1. Introduction
To date, many inspection methods, including uorescence
immunoassay,1,2 radioimmunoassay,3 enzyme-linked immuno-
sorbent assay (ELISA),4 mass spectrometric immunoassay,5,6
electrophoretic immunoassay,7 and chemiluminescence
immunoassay, have been used for the detection of cancer
antigen 125 (CA125) in clinical serum samples. Among various
detection methods, electrochemiluminescence (ECL) that
represents a fusion between electrochemical and chemilumi-
nescent methods has become an important and powerful tool in
analytical and clinical applications because of its simplicity, low
cost, and high sensitivity.8,9 Moreover, ECL has several advan-
tages over other analytical approaches because it does not
require a light source, which results in a simple instrumenta-
tion and low- or zero-background signal. In view of the afore-
mentioned superior characteristics, ECL has been paid
considerable attention during the past several decades.10
ECL detection with luminol has attracted much attention in
bioanalytical and analytical chemistry.11 In addition, the lumi-
nol–hydrogen peroxide system has been well-known to produce
aKey Laboratory of Chemical Sensing & Analysis in Universities of Shandong, School of
Chemistry and Chemical Engineering, University of Jinan, Jinan 250022, P. R. China.
E-mail: chm_yanm@126.com; Fax: +86-531-88959988; Tel: +86-531-82767161
bShandong Provincial Key Laboratory of Preparation and Measurement of Building
Materials, University of Jinan, Jinan 250022, China
cCancer Research Center, Shandong Tumor Hospital, Jinan 250012, P. R. China
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gold nanoparticles–exfoliated graphene (AuNPs–EGN) compos- 760D electrochemical workstation (Shanghai CH Instruments,
ites. The hydroxyl of EGN can connect with luminol through China). Electrochemical impedance spectroscopy (EIS) was
N,N0-carbonyl diimidazole (CDI).
carried out on an IM6x electrochemical station (Zahner,
Recently, ECL-based detection has played a signicant role at Germany). Transmission electron microscopy (TEM) images
the forefront of the bioanalysis area because of its ultrahigh were obtained using a Hitachi H-800 microscope (Japan). The
sensitivity and selectivity. Furthermore, the ECL strategy can morphologies of AuNPs–EGN and GOx–GHS were characterized
decrease the detection limit to the single-molecular level. with scanning electronic microscopy (SEM) on a QUANTA FEG
Because enzymes exhibit outstanding specicity, selectivity, and 250 thermal eld-emission scanning electron microscope (FEI
catalytic activity, these merits have led to their widespread Co., USA). UV-vis experiments were performed with a UV-3900
applications in research, medicine, and industry.22–24 To date, spectrophotometer (Hitachi, Japan). X-ray diffraction (XRD)
enzymes have been widely applied as recognition and signaling patterns were obtained using a D8 Advance (Bruker) X-ray
ꢀ
elements for the detection of some specic molecular analy- diffractometer with Cu KR radiation (l ¼ 1.5418 A). All the
tes.25,26 Glucose oxidase (GOx) can catalyze the oxidation of experiments were carried out with a conventional three-
glucose to gluconolactone along with the generation of electrode system with a modied glassy carbon electrode
hydrogen peroxide (H2O2).27 By quantifying the amount of H2O2 (GCE, 3 mm in diameter) as the working electrode (WE), a
generated, one can indirectly quantify the amount of GOx. Here, platinum counter electrode (CE) and an Ag/AgCl (sat. KCl)
we observed that H2O2 can efficiently increase the ECL of the reference electrode (RE).
intensity of luminol. Therefore, an ECL immunosensor based
on the GOx–GHS system is available.
2.3. Preparation of PDDA–EGN
A signal-type ECL sandwich immunosensor for the detection
of CA125 has been prepared. In the immunosensor, the AuNPs–
EGN–luminol acted to be the solid support for CA125 primary
antibody (Ab1), and CA125 secondary antibody (Ab2) was incu-
bated on the surface of the GOx–GHS nanoparticles. Thus, we
obtained the ECL intensities, and thus the amount of CA125
could be ascertained.
First, a modied Hummers method28,29 was applied to synthe-
size the graphene oxide (GO) from natural graphite powder.
Then, the as-prepared GO was exfoliated by ultrasonication for
more than 2 h, and the obtained dispersion was then subjected
to 10 min of centrifugation at 10 000 r$minꢀ to get a black-
brown supernatant (0.5 mg mLꢀ1). Finally, the homoge-
neously exfoliated GO (EGO) aqueous dispersion was reduced
into PDDA–EGN.
In a typical procedure, to a three-necked ask containing 100
mL EGO solution (0.50%), 500 mL PDDA (20%) solution was
added and stirred for 30 min. Next, 500 mL hydrazine hydrate
(80%) was added and subjected to stirring for 24 h at 90 ꢁC.
Finally, excess of PDDA was removed by centrifugation, and the
composite was rinsed twice with water, and the black PDDA–
EGN was then readily redispersed in water by mild ultra-
sonication, forming a black suspension with a nal concentra-
2. Experiments
2.1. Reagents
All the chemicals and solvents used were of analytical grade or
of the highest purity available. CA125, Ab1 and Ab2, and bovine
serum albumin (BSA, 96–99%) were obtained from Shanghai
Linc-Bio Science Co. Ltd (Shanghai, China). Luminol, dimethyl
sulfoxide (DMSO), CDI, poly(diallyldimethylammonium chlo-
ride) (PDDA, 20%, w/w in water, Mw 200 000–350 000) and bis(2-
ethylhexyl) sodium sulfosuccinate (AOT) were obtained from
Sigma-Aldrich (St. Louis, Mo., USA). HAuCl4$4H2O, hydrazine
hydrate (80%), GOx (100–250 units per mg) and sodium citrate
were purchased from Shanghai Chemical Reagent Company
(Shanghai, China).
All solutions were prepared using Milli-Q water (Millipore) as
a solvent. Phosphate buffered solutions (PBS, pH 7.4) were
prepared using 0.01 mol Lꢀ1 KH2PO4 and 0.01 mol Lꢀ1
Na2HPO4. PBS (0.01 mol Lꢀ1, pH 7.4), containing 1% glucose,
was used as the electrolyte. The CA125 was stored at 4 ꢁC, and its
standard solution was daily prepared with PBS solution when
required. The clinical serum samples were provided by Shan-
dong Tumor Hospital.
tion of 1.0 mg mLꢀ1
.
2.4. Composition of AuNPs–EGN–luminol hybrids
AuNPs with a diameter of ꢂ13 nm were produced by reducing
gold chloride tetrahydrate with citric acid at 96 ꢁC for 15 min.
AuNPs should be dispersed by ultrasonication prior to use. In
a typical expeꢀri1ment of self-assembly, 100 mL of PDDA–EGN
(1.00 mg mL ) was added into 5 mL of AuNPs solution
(0.10 mg mLꢀ1) with stirring and ultrasonic processing for
10 min to form a homogeneous mixture. The mixture was then
kept undisturbed under ambient conditions for 12 h. Finally,
the precipitate was collected by centrifugation with water for
several times. The as-prepared AuNPs–EGN hybrids were
redispersed in 1 mL of water, and the nal concentration
obtained was 0.10 mg mLꢀ1
Preparation of AuNPs–EGN–luminol: rst, the AuNPs–EGN
.
2.2. Apparatus
The ECL measurements were carried out using a MPI-E multi- hybrids were dried under vacuum at 60 ꢁC for 12 h, and the
functional electrochemical and chemiluminescent analytical composite was dipped in 5 mL of CDI solution (1%) at room
system (Xi'an Remex Analytical Instrument Ltd, Co.) with the temperature for 4 h. Subsequently, the precipitate was collected
voltage of the photomultiplier tube (PMT) set at 800 V. Cyclic by centrifugation. The mixture was then dispersed into 5 mL of
voltammetric measurements (CVs) were performed with a CHI luminol solution (1.00 ꢃ 10ꢀ4 mol Lꢀ1) and kept undisturbed
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under ambient conditions for 4 h. Finally, the precipitate was incubation for 5 h, the residual Ab2 was removed by centri-
collected by centrifugation with water for several times. The as- fugation and washed several times with 0.1 mol Lꢀ1 PBS.
prepared AuNPs–EGN–luminol was redispersed in 1 mL of Subsequently, the GOx–GHS composite-labeled Ab2 was
distilled water.
redispersed in 2 mL of 1% BSA solution for 2 h to block the
excess of amino groups and nonspecic binding sites of the
GOx–GHS composite-labeled Ab2. Aer being centrifuged and
washed with PBS, the resultant GOx–GHS composite-labeled
Ab2 was dispersed in 0.01 mol Lꢀ1 of pH 7.4 PBS to a nal
2.5. Preparation of Ab1–AuNPs–EGN–luminol
To generate AuNPs–EGN–luminol composite immunological
labels, 1 mL of the above AuNPs–EGN–luminol suspension
was mixed with 1 mL of Ab1 solution (20 mg mLꢀ1 of Ab1 in
0.01 mol Lꢀ1 PBS) (pH 7.4). Aer incubation for 4 h, the
residual antibody was removed by centrifugation and washing
with PBS (0.1 mol Lꢀ1) for several times. Subsequently, excess
of antibodies were washed with PBS (pH 7.4) and incubated in
1% BSA for 1 h to block non-specic binding sites. The
resultant Ab1–AuNPs–EGN–luminol composite was dispersed
with PBS (pH 7.4) to a nal volume of 2 mL and stored at 4 ꢁC
for subsequent usage.
ꢁ
volume of 2 mL and stored at 4 C for subsequent usage.
2.7. Fabrication of the sandwich-type
electrochemiluminescence immunosensor
The entire process for constructing the modied electrode is
schematically shown in Scheme 1. A GCE electrode with a 3
mm diameter was carefully polished with 1.0, 0.3 and 0.05
mm alumina powder on ne abrasive paper and ultrasonically
washed with water. First, 5 mL Ab1–AuNPs–EGN–luminol
hybrids were coated on the working electrodes and dried at
room temperature for 30 min. The electrode was then incu-
bated with various concentrations of CA125 solution for 30
min. Subsequently, the prepared GOx–GHS composite-
labeled Ab2 was dropped onto the electrode surface and
incubated for another 30 min. Finally, the electrode was
washed with PBS (pH 7.4), and the immunosensor was
prepared.
2.6. Preparation of GOx–GHS-labeled Ab2
First, the preparation of GOx–GHS composite: the GOx–GHS
were synthesized according to the literature30 with a little
modication. Two solutions were initially prepared as
follows: solution A, including 200 mL of 0.05 mol Lꢀ1 CaCl2,
100 mL of 1.50 ꢃ 10ꢀ4 mol Lꢀ1 GOx aqueous solution, and
15 mL of 0.10 mol Lꢀ1 AOT isooctane suspension, and solu-
tion B, containing 200 mL of 0.05 mol Lꢀ1 Na2CO3, 100 mL of
1.50 ꢃ 10ꢀ4 mol Lꢀ1 GOx aqueous solution, and 15 mL of
0.10 mol Lꢀ1 AOT isooctane suspension. Aer adequately
stirring the GOx, solution B was added dropwise into solution
A with violent stirring until the translucent GOx-doped CaCO3
colloids were formed. Following that 100 mL of 5.00% HAuCl4
solution was added dropwise into the reverse micellar solu-
tion. Aer stirring the mixture further for 2 h at room
temperature, 100 mL of 0.05 mol Lꢀ1 aqueous hydrazine
hydrate was injected into the prepared suspension. With the
aid of NH2OH, the Au3+ ions coating the CaCO3 surface were
reduced to metallic Au0. With the progressive addition of
hydrazine hydrate to reverse micelles, the solution acquired a
fawn-to-red color due to the formation of gold colloids. Aer
stirring for 1 h at room temperature, 3 mL of absolute ethanol
was added and stirred for 10 min, which resulted in the
complete breakdown of reverse micelles with the formation of
two immiscible layers of aqueous ethanol and isooctane. The
ethanol was carefully removed using a separating funnel. The
particles thus obtained were washed four times with isooc-
tane and centrifuged to remove any residual AOT. The pel-
leted particles were then dispersed in 10 mL of water by
vigorous stirring for 30 min, and the dispersed system was
dialysed against distilled water for 2 h using a 12 kDa cut-off
dialysis bag. The hollow microspheres of nanogold particles
were nally formed by the addition of 0.001 mol Lꢀ1 HCl into
the nanogold coated GOx-doped-CaCO3 suspension. Thus,
GOx–GHS was prepared.
2.8. ECL detection of CA125 with the immunosensor
ECL detection of CA125 with the immunosensor ECL
measurements was done at room temperature. The modied
electrode was dipped in the 0.01 mol Lꢀ1 PBS containing 1%
glucose, and aer a reaction of 10 min, the ECL intensity was
measured. The potential swept from 0 to 0.6 V with a scan rate
of 50 mV sꢀ1 with a photomultiplier tube voltage of ꢀ800 V. The
ECL signals related to the CA125 concentrations could then be
measured.
Second, to generate GOx–GHS composite immunological
labels, 1 mL of the aforementioned GOx–GHS suspension was
mixed with 1 mL of Ab2 solution (20 mg mLꢀ1, pH 7.4). Aer Scheme 1 Fabrication process of the electrochemical immunosensor.
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molecules were adsorbed on one GHS. Furthermore, it also
provided a larger area for the immobilization of the external Ab2.
When appropriately applied in bioanalysis, the GOx–GHS
provided a signicant improvement in analytical sensitivity. The
SEM and magnied SEM images of GOx–GHS are shown in Fig. 2A
and B, and the TEM image of GOx–GHS is shown in Fig. 2C, which
displayed good dispersion with a mean diameter of ꢂ50 nm.
In addition, to the binding energy differences, we also
observed the geometrical differences between the considered
systems, as measured by 2q. As shown in the XRD of GOx–GHS
(Fig. 3B), for the systems of GOx–GHS, the curve exhibited two
diffraction peaks at 38.2ꢁ and 44.6ꢁ, which can be indexed to
diffraction from the (111) and (220) of the fcc structure of
metallic Au, respectively. To further monitor the formation of
GOx–GHS, the UV-vis absorption spectrum of the GOx–GHS was
also investigated. The GOx (curve d on Fig. 3A) showed
absorption peaks at 377 and 453 nm. When GOx–GHS were
prepared (curve e on Fig. 3A), the peaks at 379 and 445 nm were
mainly ascribed to the GOx molecules, whereas the peak at
522 nm was attributed to the AuNPs. Thus, we could conclude
that GOx molecules and colloidal gold could be doped into the
synthesized nanocomposites via the reverse micellar method.
3. Results and discussion
3.1. Characterization of AuNPs–EGN–luminol composite
SEM was used to characterize the prepared EGN (Fig. 1A), which
have a large surface area for loading more AuNPs. In addition,
the SEM revealed the structure of AuNPs–EGN. As shown in
Fig. 1C and D, AuNPs with a diameter of ꢂ13 nm (Fig. 1B) were
dispersed on the surface of EGN, and some of them are covered
with EGN. In addition, AuNPs could also be found inside the
cavity of EGN, which implied that the AuNPs were dispersed in a
three-dimensional structure along with EGN, and no aggrega-
tion of EGN sheets or free AuNPs was observed, which suggested
that the AuNPs almost absorbed in to the EGN. The unique
structure of the prepared composite couple be might benet the
electrochemical or ECL applications.
To monitor the formation of AuNPs–EGN–luminol, we
studied the UV-vis absorption spectra of the luminol, AuNPs,
and AuNPs–EGN–luminol, respectively (Fig. 3A). The pure
luminol showed absorption peaks at 301 and 348 nm (curve b),
whereas there was a peak at 523 nm for the pure AuNPs (curve
a). When luminol and AuNPs were encapsulated into the EGN,
three absorption peaks were simultaneously observed (curve c).
The peaks at 301 and 349 nm were mainly ascribed to the
luminol, whereas the peak at 522 nm was attributed to the
AuNPs. Compared with those obtained with pure luminol
solution and pure AuNPs, the slight deviation between
absorption wave numbers was due to the interaction between
AuNPs–EGN and the luminol solution. Thus, we could conclude
that the AuNPs–EGN–luminol composite were prepared.
3.3. Possible mechanisms for ECL production
The mechanisms for ECL production of the fabricated immu-
nosensor were studied and inferred as follows: rst, glucose
reduced the avin adenine dinucleotide (FAD) of GOx to
reduced FAD (FADH2), simultaneously forming gluconic acid.
Second, FADH2 obtained was reoxidized by oxygen dissolved in
the solution, and as a result H2O2 was produced. Third, under
the weak alkaline condition, the H2O2 molecules were mostly
converted to HOOꢀ. Fourth, the luminol on the surfaces of the
EGN–AuNPs nanoparticles were excited by electron (eꢀ), form-
ing the excited state of luminol (luminol*). Fih, HOOꢀ could
transfer the energy of luminol* (a), generating a stable-state
luminol. Finally, the remaining luminol* (b) could emit light
(hn). The possible mechanism of ECL can be described by the
following equations:
3.2. Characterization of GOx–GHS composite
In this study, the GHS provided a large area for the encapsula-
tion of the inner GOx, and hundreds to thousands of GOx
GOx (FAD) + 2 glucose / 2 gluconic acid + GOx (FADH2)
(1)
GOx (FADH2) + O2 / GOx (FADH2) + H2O2
H2O2 + OHꢀ / HOOꢀ + H2O
(2)
(3)
(4)
(5)
(6)
Luminol + eꢀ / luminol*
Luminol* (a) + 2HOOꢀ / luminol + 2OHꢀ + O2
Luminol* (b) / luminol + hn
3.4. ECL and EIS behaviors of the immunosensor
To investigate the technique of the AuNPs–EGN–luminol
composites for ECL analysis, we designed two experiments. One
Fig. 1 (A) SEM image of EGN, (B) TEM image of AuNPs, (C) SEM image
of AuNPs–EGN, (D) magnified SEM image of AuNPs–EGN.
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Fig. 2 SEM (A) and magnified SEM (B) image of GOx–GHS, (C) TEM image of GOx–GHS.
glucose (curve a on Fig. 5A), the intense ECL signal for the as-
prepared electrode originated from the synergistic effect of
the H2O2, which resulted from the GOx-mediated electro-
oxidation of glucose in the presence of oxygen. On the whole,
the multiple enzymatic turnovers signicantly amplied the
generated ECL photons.
EIS is an effective method to monitor the changes of
interfacial properties, which allow understanding the chem-
ical transformation and processes associated with the
conductive electrode surface.31–33 In EIS, the diameter of the
Fig. 3 (A) UV-vis absorption spectra of (a) AuNPs, (b) luminol, (c)
AuNPs–EGN–luminol composite, (d) GOx, and (e) GOx–GHS semicircle at higher frequencies corresponds to the electron-
composite. (B) XRD of AuNPs-EGN.
transfer resistance (Ret); a change in the value of Ret was
associated with the blocking behavior of the modication
processes in the GCE, which was reected in the EIS as a
experiment was designed without the EGN for the sensing
platform, the other without the AuNPs for the signal label. In
Fig. 4A, we compared the ECL intensity of AuNPs–luminol
composite-labeled Ab1 and AuNPs–EGN–luminol composite-
labeled Ab1. As can be seen, AuNPs–EGN–luminol composites
could increase the ECL, which could benet the immobilization
analysis. In the Fig. 4B, we compared the ECL intensity of EGN–
luminol composite-labeled Ab1 and AuNPs–EGN–luminol
composite-labeled Ab1. The AuNPs–EGN–luminol composite-
modied GCE reveals excellent ECL performance compared to
EGN–luminol composites.
To verify the enzymatic amplication of the immuno-
sensor, the ECL behavior of the GCE/Ab1–AuNPs–EGN/CA125/
GOx–GHS–Ab2 electrode was studied by adding 1% glucose
into the aforementioned PBS solution, and the ECL signal of
luminol showed a sharp increase (curve b in Fig. 5A).
Compared with the ECL behavior without the addition of the
change in the diameter of the semicircle at high frequencies.
The EIS of the electrodes were performed in a background
solution of 5.0 mmol Lꢀ1 K3Fe(CN)6 containing 0.1 mol Lꢀ1
KCl, and the frequency range was at 100 mHz to 10 kHz with a
signal amplitude of 5 mV. Fig. 5B shows the EIS of the GCE at
different stages. The EIS of bare GCE revealed a relatively small
semicircle domain (curve a). When Ab1–AuNPs–EGN–luminol
was modied on the GCE surface, a much lower resistance was
obtained (curve b), implying that the AuNPs–EGN–luminol is
an excellent electrical conducting material and accelerates the
electron transfer. Furthermore, CA125 could resist all the
electron-transfer kinetics of the redox probe at the ber
surface, resulting in the increase of resistance (curve c), which
veried the immobilization of these substances. Finally, when
GOx–GHS–Ab2 interacted with CA125 (curve d), the resistance
decreased, indicating that the synthesized GOx–GHS
possessed high conductivity and good electron transfer effi-
ciency, although the protein adsorption layer acted as a barrier
to the interfacial electron transfer.
3.5. Optimization of experimental conditions
To achieve an optimal ECL signal, the pH value of the substrate
solution was an important factor for the ECL intensity.
According to the experimental results in Fig. 5C, the ECL
intensity of the immunosensor is obviously affected at different
pH, which may be a result of the chemical properties of the
immunosensor varying with the changes of acidity. Obviously,
PBS (0.01 mol Lꢀ1, pH 7.8) can be selected as the detection
solution. Fig. 5D shows that the ECL intensity is responsive to
different potentials (outlined in Fig. 5D). It can be observed that
the optimal scanning potential is 0–0.6 V.
Fig. 4 (A) The immunosensor performance with (a) and without (b)
EGN. (B) The immunosensor performance with (a) and without (b)
AuNPs.
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Fig. 5 (A) GOx–GHS–Ab2/CA125/Ab1/AuNPs–EGN/GC electrode (without (curve a) and with (curve b) 1% glucose) in 0.1 mol Lꢀ1 PBS solution
(pH 7.4). (B) EIS of bare GCE (a), GCE/Ab1–AuNPs–EGN–luminol (b), GCE/Ab1–AuNPs–EGN–luminol/CA125 (c), GCE/Ab1–AuNPs–EGN–
luminol/CA125/GOx–GHS–Ab2 (d) in 0.01 mol Lꢀ1 PBS (2.5 mmol Lꢀ1 Fe(CN)64ꢀ/3ꢀ + 0.1 mol Lꢀ1 KCl, pH 7.4). The frequency range is between
0.01 and 100 000 Hz with signal amplitude of 5 mV. (C) ECL profiles of the immunosensor at various pH of PBS containing 1% glucose. (D) The
potential range (V): (a) 0–0.5, (b) 0–0.55, (c) 0–0.6, and (d) 0–0.65. The CA125 concentration was 5 ng mLꢀ1. Scan rate: 100 mV sꢀ1. The voltage
of the photomultiplier tube was set at 800 V.
Compared with other methods,38–40 the as-prepared immuno-
3.6. Analytical performance
sensor has a relatively large linear range and low detection limit.
The results demonstrated that the proposed method has huge
application potential in the eld of cancer diagnosis.
Under the optimal conditions, the proposed immunosensor
was applied to detect the different concentrations of CA125. As
shown in Fig. 6B and C, the ECL intensity of the immunosensor
increased with the increasing CA125 concentrations, and it
exhibited a good linear relationship with the logarithm of
CA125 concentration from 0.001 to 100 ng mLꢀ1. The linear
3.7. Specicity, stability, and reproducibility of the
immunosensor
regression equation can be given as IECL
¼
1345.62 +
410.63 log cCA125 (cCA125/ng mLꢀ1), with a correlation coefficient
of 0.9921. The detection limit value for CA125 was determined
at 0.38 pg mLꢀ1, estimated at a signal-to-noise ratio of three
criterion, which suggested a satisfactory detection limit and
linear range.
To further characterize the specicity of the immunosensor,
5 ng mLꢀ1 CA125, containing 200 ng mLꢀ1 human serum
albumin (HSA), 200 ng mLꢀ1 alpha fetoprotein (AFP), and
200 ng mLꢀ1 prostate protein antigen (PSA) was, analysed by the
immunosensor, and the results are shown in Fig. 6A. The rela-
tive deviations (RD) were 1.86%, 2.08% and 1.79%. These
results indicated that the relative standard deviations are all
Table 1 shows the linear range and detection limit of
immunosensors along with those of previous reports.34–37
Fig. 6 (A) Specificity of the immunosensor. (B) ECL profiles of the immunosensor in the absence (a–g) of different concentrations of CA125 in
PBS solution containing 1% glucose at pH of 7.4. CA125 concentration (ng mLꢀ1): (a) 0.001, (b) 0.01, (c) 0.1, (d) 1, (e) 5, (f) 10, and (g) 100 (the
voltage of the photomultiplier tube was set at 800 V). (C) The calibration curve of the immunosensor.
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Table 1 Comparison of analytical properties with other immunoassay biosensing systems
Detection methods
Linear range
Detection limit
Ref.
Immunosensor
mPADs
ELISA
0–50 pg mLꢀ1
1.0 pg mLꢀ1
0.001 ng mLꢀ1
0.16 ng mLꢀ1
0.38 pg mLꢀ1
34
35
36
0.001–100 ng mLꢀ1
0.825–165 ng mLꢀ1
0.001–100 ng mLꢀ1
Electrochemiluminescence
This work
Table 2 Comparison of CA125 determinations on human serum samples by ECL immunosensor and ELISA
Immunosensor concentrations (ng mLꢀ1 ELISA concentrations (ng mLꢀ1
)
)
Sample
Added
Found
Recoveries (%)
Added
Found
Recoveries (%)
Relative deviation (%)
1
2
3
4
0.5
1.0
2.0
3.0
0.507
0.983
2.022
3.044
101.4
98.3
101.1
101.5
0.5
1.0
2.0
3.0
0.512
0.976
2.036
3.031
102.4
97.6
101.8
101.0
ꢀ0.99
0.712
0.69
ꢀ0.49
less than 3.00%, which suggested that the specicity of the as- focused on the determination of the target antigen molecules, it
prepared immunosensor was acceptable. can be easily extended to the detection of other antigens or
The stability of this immunosensor was tested by carrying biocompounds. Moreover, the potential of this method for
out 10 continuous cyclic scans in 0.1 mol Lꢀ1 PBS (pH 7.4). In application is its simple and efficient diagnostic strategy for
addition, when the immunosensor was dried and stored at 4 ꢁC, immunoassays. Importantly, this approach does not require
the ECL response retained 94.12% of the initial response aer a sophisticated fabrication, and it is well suited for high-
storage period of 30 days. The slow decrease in response may be throughput biomedical sensing and application in both clin-
related to the gradual deactivation of the immobilized antibody ical and biodefense areas.
incorporated in the composite.
The reproducibility played a vital role in the performance of
the prepared immunosensor. When the ECL response for CA125
Acknowledgements
of six fabricated immunosensors was tested, the result showed a
relative standard deviation of 3.12%, giving an acceptable
fabrication reproducibility of the immunosensors.
This work was nancially supported by National Natural
Science Foundation of China (51273084, 21277058), Natural
Science Foundation of Shandong Province, China
(ZR2012BZ002), and Technology Development Plan of Shan-
dong Province, China (Grant no. 2012GGB011813).
3.8. Application of ECL immunosensor in human serum
samples
References
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method. It clearly indicates that there is no signicant differ-
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developed immunosensor could be satisfactorily applied to the
clinical determination of CA125 levels in human serum.
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