Biosensors and Bioelectronics 54 (2014) 199206

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Biosensors and Bioelectronics

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

A cost-effective sandwich electrochemiluminescence immunosensor

for ultrasensitive detection of HIV-1 antibody using magnetic
molecularly imprinted polymers as capture probes
Jing Zhou a, Ning Gan a,n, Tianhua Li a, Futao Hu b, Xing Li a, Lihong Wang c, Lei Zheng d
a
State Key Laboratory Base of Novel Functional Materials and Preparation Science, Faculty of Materials Science and Chemical Engineering, Ningbo University,
Ningbo 315211, PR China
b
Faculty of Marine, Ningbo University, Ningbo 315211, PR China
c
Faculty of Science, Ningbo University, Ningbo 315211, PR China
d
Nanfang Hospital, Southern Medical University, Guangzhou 510515, PR China

art ic l e i nf o

a b s t r a c t

Article history:
Received 17 September 2013
Accepted 24 October 2013
Available online 9 November 2013

In this report, a rapid and cost-effective sandwich electrochemiluminescence (ECL) immunosensor was
constructed for the ultrasensitive detection of human immunodeciency virus type 1 antibody (anti-HIV-1)
using magnetic molecularly imprinted polymers (MMIPs) as capture probes by combining surface and
epitope imprinting techniques and antigen conjugated with horseradish peroxidase (HRP-HIV-1) as
labels. First, 3-aminobenzeneboronic acid (APBA) was used as the functional monomer and cross-linking
reagent, which was polymerized on the surface of silicate-coated magnetic iron oxide nanoparticles
(Fe3O4@SiO2 NPs) in the presence of human immunoglobulin G (HIgG), as the template exhibiting the
same Fc region but different Fab region to anti-HIV-1 after the addition of the initiator, ammonium
persulfate. This process resulted in grafting a hydrophilic molecularly imprinted polymer (MIP) lm on
the Fe3O4@SiO2 NPs. Thus, MMIPs, which could be reused after eluting the template, were used to
recognize and enrich ultra-trace levels of anti-HIV-1. Subsequently, a novel sandwich ECL immunosensor
was formed through the immunoreaction between MMIPs conjugated with varied concentrations of
anti-HIV-1 and HRP-HIV-1. By the catalysis of HRP immobilized onto HRP-HIV-1 on the ECL system of
Luminol-H2O2, a linear response range of the anti-HIV-1 dilution ratio (standard positive serum) was
achieved from 1:20,000 to 1:50, with a detection limit of 1:60,000 (S/N 3). The developed method
provides a low-cost, simple, and sensitive way for the early diagnosis of HIV infected patients.
& 2013 Elsevier B.V. All rights reserved.

Keywords:
Sandwich electrochemiluminescence
immunosensor
Surface imprinting
Epitope imprinting
Human immunodeciency virus type
1 antibody
Horseradish peroxidase
Luminol

1. Introduction
Acquired immune deciency syndrome (AIDS) is caused by
infection with human immunodeciency virus (HIV), and is the
end-stage of disease (Weiss, 1993). HIV is divided into two types,
that is, HIV-1 and HIV-2, of which HIV-1 is more common and
extensively studied (Wang et al., 2013; Jangam et al., 2013;
Esseghaier et al., 2013; Ruslinda et al., 2013). To date, no drug or
vaccine has become available to cure or stop the onset of disease
(Garber et al., 2004; McMichael, 2006; Levy, 2009; Ross et al., 2010).
The early diagnosis and monitoring of HIV-1 infection in patients
is of great importance for the initiation and the evaluation of
antiviral therapies (Cohen et al., 2011).
HIV-1 antibody (anti-HIV-1) is generated after HIV-1 infection
(Ly et al., 2001). However, the window of opportunity to detect
anti-HIV-1 is generally 48 weeks (Weiss, 1993), which causes great

Corresponding author. Tel./fax.: 86 574 87609987.

E-mail address: ganning@nbu.edu.cn (N. Gan).

0956-5663/$ - see front matter & 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.bios.2013.10.044

difculty in the early diagnosis of HIV infection. Therefore, various

methods have been developed for the early detection of antiHIV-1 and reduce the diagnostic window, hoping to nd the
potential opportunity of a cure in the early stages of infection
(Ly et al., 2001).
At present, these methods mainly include enzyme-link immunosorbent assay (ELISA) (Louie et al., 2006; Yeom et al., 2006),
enzyme immunoassay (EIA) (Barbe et al., 1994; Galli et al., 1996),
and Western Immunoblot (WB) analysis (Dodd and Fang, 1990).
Although these methods offer a highly accurate approach in the
testing of anti-HIV-1, their low sensitivity makes the diagnostic
window lag behind. In this case, there would be no opportunity to
cure this disease after anti-HIV-1 was detected (Cohen et al., 2008).
Thus these methods are not suitable for the reduction in the
diagnostic window of opportunity. To address this problem and
obtain a rapid diagnosis, developing new methods for the detection of anti-HIV-1 to improve sensitivity is a key step.
Recently, electrochemiluminescence (ECL) has received a great
deal of interest in many elds due to its unique advantages, such
as an innately high sensitivity, simple instrumentation and low

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cost, including environmental pollutant determination, pharmaceutical analysis, and immunoassays (Richter, 2004; Zhang et al.,
2008; Jia et al., 2009; Hull et al., 2009; Wu et al., 2010; Zhao and
Zhou, 2012; Carvajal et al., 2012; Dai et al., 2012). Even so, it
commonly needs the use of magnetic beads to label the antibody
or antigen as the capture probes for commercial ECL immunoassays (ECLIA). This makes the labeling steps cumbersome when
preparing such probes. Moreover, the prepared probes are difcult
to preserve and easily inactivated, which directly affects the
sensitivity of ECL detection and increases the testing costs.
With the development of molecular imprinting technology,
molecularly imprinted polymers (MIPs) have been assessed as
articially synthesized receptors and widely used in sensors,
catalysis reactions, and separations, in which MIPs act as substitutes for antibodies or enzymes due to their unique binding
characteristics (in terms of both afnity and specicity), high
chemical and physical stability, easy availability and low cost
(Haupt and Mosbach, 2000; Ye and Mosbach, 2008). However,
imprinting of large structures, such as proteins and other biomacromolecules, remains a challenge, and is attributable to their
restricted mobility within highly cross-linked polymer networks,
and the poor efciency in rebinding. Meanwhile, the molecular
size, conformational exibility and sensitivity to denaturation of
proteins would also make them difcult for imprinting (Lu et al.,
2012a). Therefore, some approaches involving surface imprinting
(Nematollahzadeh et al., 2011) and epitope imprinting (Tai et al.,
2010) have been developed to help solve these issues. Recently,
Lu et al. reported a new magnetic surface imprinting technique
based on Fe3O4@SiO2 NPs as the core (Lu et al., 2012b). The MIPs
were coated on Fe3O4@SiO2 NPs, and the resulting polymer was
magnetically susceptible and thus easily separated by external
magnetic elds after completing both adsorption and recognition.
Moreover, a mass of recognition sites were situated at the surface
of the coreshell material, which made the templates rebind faster
and removed easily due to easy accessibility and low mass transfer
resistance to the target molecules. Furthermore, the MMIPs also
display a high adsorption ability and excellent adsorption selectivity. These advantages of MMIPs make this approach attractive
and broadly applicable to biological enrichment, separation and
biosensors (Kan et al., 2010). On the other hand, epitope imprinting has been an important area of research since it was rst
demonstrated by Minoura et al. for peptide recognition (Rachkov
and Minour, 2000). In this process, a fragment exposed on the
epitope of the target macromolecule is used as a template. The
resultant MIP recognizes not only the template but also the whole
macromolecule. For example, Shea et al. constructed macromolecular receptors for proteins using the epitope-peptides of cytochrome
C and bovine serum albumin as templates (Nishino et al., 2006).
Recently, Lu et al. developed a biomimetic sensor for the detection
of HIV-1 related protein (glycoprotein 41, gp41) based on an
epitope imprinting technique (Lu et al., 2012a). Compared with
traditional protein imprinting approaches, epitope imprinting has
several advantages (Ge and Turner, 2008). Firstly, more specic
and stronger interactions with a fragment or small part of the
macromolecule can reduce non-specic binding and improve
afnity. Secondly, the polymer can recognize both the template
and the entire protein, and the operational procedures are easier
to complete. Thirdly, short peptides as epitopes for imprinting are
cost-effective. However, little work has been reported thus far on
combining surface imprinting with epitope imprinting for detection of proteins in human samples.
Based on the above-mentioned factors, a novel sandwich ECL
immunosensor for the ultratracible detection of anti-HIV-1 was
designed using MMIPs as an alternative to HIV-1 antigens as
capture probes by combining surface and epitope imprinting
techniques and using HRP-HIV-1 as labels. The whole process for

the construction of the immunosensor is shown in Scheme 1.

Since HIgG bears the same Fc region, and a different Fab region to
anti-HIV-1, it can be used as the dummy template for MMIPs in
place of anti-HIV-1. Moreover, it is much cheaper, more available
than anti-HIV-1 and the resulting MMIPs can be reused after
eluting the template, all of which make the immunoassay costeffective. Furthermore, the obtained MMIPs also simplied the
fabrication process of the immunosensor by the use of external
magnetic elds. By the catalysis of HRP immobilized onto HRPHIV-1 and detected by the ECL system of Luminol-H2O2, a linear
response range of anti-HIV-1 dilution ratios (standard positive
serum) was achieved from 1:20,000 through 1:50 with a detection
limit of 1:60,000 (S/N 3). This method associated the recognition
property of MMIPs towards a target molecule with the high
specicity of an antibody-antigen reaction, which provided an
improved specicity when compared with MMIPs alone. In addition, the features of this improved method including its low cost,
simplicity, and ultrasensitivity, would pave the way for a new
approach in clinical immunoassay.

2. Experiments
2.1. Chemicals and reagents
HIV ELISA Kits were from Rongsheng Biological Pharmaceutical
Co. Ltd. (Shanghai, China). Elecsys HIV Kits were from Roche
Diagnostics GmbH., Germany. Human immunoglobulin G (HIgG)
was from Beijing Biosynthesis Biotechnology Co. Ltd. Tetraethyl
Orthosilicate (TEOS), 3-aminobenzeneboronic acid (APBA) was
from J&K chemical Co. Ltd. Luminol was from Sigma-Aldrich
(St. Louis, MO, USA). Bovine serum albumin (BSA, 9699%) and Triton
X-100 (TX-100) were bought from sinophram chemical reagent Co.
Ltd. (Shanghai, China). Other chemicals were analytically pure and
used as received. A 0.01 M stock solution of luminol was prepared
by dissolving luminol in 0.1 M NaOH solution and was kept at 4 1C.
Working solutions of luminol were obtained by diluting the stock
solution. 0.1 M phosphate buffer solutions (PBS, pH 8.0) containing
0.1 M KCl and NaCl was used as the electrolyte. Washing buffer
solution consisted of a PBS (pH 7.4) with 0.05% (v/v) Tween 20
(PBST). Eluting solution contained 1% sodium dodecyl sulfate
(SDS). Deionized water was used throughout the experiments.
2.2. Instrumentation
ECL experiments were carried out using a MPI-B model
electrochemiluminescence analyzer (Xi'an Remax Electronic
Science&Technology Co. Ltd., Xi'an, China) with the voltage of
the photomutiplier tube (PMT) set at 600V. A three-electrode
system used consisted of a screen-printed carbon working electrode (SPCE, DropSens Corporation, Spain), a carbon auxiliary
electrode and an Ag reference electrode. The developed assay
was validated using a commercialized electrochemiluminescence
immunoassay (ECLIA) on the fully automated immuno-analyzer
Elecys 2010 (Roche). A eld emission-scanning electron microscope (FE-SEM, SU-70, Hitachi, Japan), and transmission electron
microscope and high resolution transmission electron microscope
(TEM and HRTEM, Tecnai G20, Philip) were employed to characterize the surface morphology and size of the nanoparticles.
Atomic force microscopic (AFM) images were obtained by a
Nanoscope IIIa multimode atomic force microscope (Veeco Instruments, USA). The XRD characterization was performed using X-ray
diffraction (Bruker, D8 Focus) with Cu K radiation at room
temperature. Thermogravimetric analysis (TGA) was carried out
by a ZRY-2P thermal analyzer (Shanghai Balance Instrument

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201

Scheme 1. Describing the systematic process for the synthesis of the immunosensor.

Company, China). Magnetic properties were obtained with a

LakeShore 7307 (Lakeshore Cryotronic) VSM at 300 K.
2.3. Preparation of MMIPs and the ECL immunosensor
MMIPs and magnetic nonmolecular imprinted polymers (MNIPs)
were prepared according to a slightly modied report (Li et al., 2009).
Firstly, an aqueous suspension of superparamagnetic magnetite
nanoparticles were prepared by the controlled chemical coprecipitation reaction. FeCl2  4H2O (3.44 g) and FeCl3  6H2O (9.44 g) were
respectively dissolved under a N2 atmosphere in deaerated deionized
water (160 mL) with vigorous mechanical stirring (800 rpm). A
nitrogen gas environment was maintained in the vessel during the
reaction to prevent critical oxidation. When the solution was preheated to 80 1C, ammonium hydroxide (20 mL) was added to achieve
alkaline conditions. After 30 min, black superparamagnetic MNPs

were obtained by sedimentation with the help of an external

permanent magnet and the supernatant was decanted. The MNPs
were washed with deionized water and absolute ethanol several
times (150 mL each time) to remove unreacted chemicals until a
transparent ferrouid was obtained. Then, the superparamagnetic
MNPs were coated with silica by using a solgel method. Superparamagnetic MNPs (0.120 g) was redispersed in 2-propanol
(240 mL) and deionized water (18 mL) by sonication for approximately 15 min. Then, under continuous mechanical stirring
(800 rpm), ammonium hydroxide (21 mL) and TEOS (4 mL) were
consecutively added to the reaction mixture. The reaction proceeded
at room temperature for 14 h under continuous mechanical stirring.
The resultant product was obtained by magnetic separation with
the help of an external permanent magnet and was thoroughly
washed with deionized water and absolute ethanol. Lastly, for the
preparation of human IgG-imprinted polymers (HIgG-MMIPs), HIgG

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J. Zhou et al. / Biosensors and Bioelectronics 54 (2014) 199206

(0.01 g) was dissolved in sodium phosphate buffer (5 mL, pH 8.0)

containing APBA (100 mM), and the mixture was incubated at
room temperature for 1 h. After adding silica-coated MNPs
(0.04 g), the solution was then incubated for 2 h at room temperature. Prior to use, the silica-coated MNPs were subjected to
extensive deionized water and absolute ethanol, and washed
thoroughly. Subsequently, a 100 mM aqueous solution of ammonium persulfate (6.5 mL) as initiator was slowly added dropwise to
the above solution for about 20 min and the polymerization
process was executed at room temperature. After 14 h, HIgGMMIPs were obtained. Finally, the HIgG-MMIPs were eluted with
1% SDS to remove the entrapped HIgG. The MMIPs were then
equilibrated with the buffer used in the polymerization process.
MNIPs were prepared using the same procedure but without HIgG.
The procedures are shown in Scheme 1A and B.
The ECL immunosensor was constructed as Scheme 1B: 10 L
10 mg mL  1 MMIPs and 20 L different dilution ratio of anti-HIV1 solution (1:20,000 to 1:10) was rstly mixed, adsorbing and
stirring for 60 min at 25 1C. Then 50 L 2.5% BSA was added to
prevent the nonspecic adsorption. After 1 h, the mixture was
separated by magnet and washed twice with water, dispersing
into 40 L HRP-HIV-1 conjugates and incubating at 37 1C for
30 min. Finally, the sandwich-type immunocomplexes were isolated by magnet, washed with PBST three times, dispersed in
20 L of PBS (pH 8.0) and stored at 4 1C for ECL tests.
2.4. ECL detection
As shown in Scheme 1C, 10 L aforementioned immunocomplexes was dropped on the SPCE with a magnet pasted in advance,
then ECL detection could be performed by applying a double-step
potential (10 s pulse period, 1 s pulse time, 0.3 V pulse potential
and 0.15 V initial pulse potential) to the working electrode at a
scan rate of 100 mV s  1 after adding 5 L 2  10  4 M luminol
solution and 10 L 5  10  3 M H2O2 solution. The resulting ECL
signals from different concentration of anti-HIV-1 solution were
detected by the PMT below. Finally, standard curves were obtained
according to the linear relationship between the logarithm of the
ECL intensity IECL (IECL I  IO, here I and IO are the ECL
intensities of ECL immunosensor prepared with and without
anti-HIV-1, respectively) and the logarithm of the dilution ratio
of anti-HIV-1 in the range of 1:20,000 to 1:50.
2.5. ECL adsorption measurement
To investigate the adsorption dynamics of MMIPs and MNIPs,
10 L 10 mg mL  1 MMIPs and MNIPs were separately added into
30 L anti-HIV-1 solution with a dilution ratio of 1:10, then the
optimal adsorbed time was obtained through measuring the
changes in ECL intensity, ECL of ECL immunosensor prepared
at different time intervals. To investigate the adsorption equilibrium of MMIPs, 10 L 10 mg mL  1 MMIPs was equilibrated with
varied initial different dilution ratio of anti-HIV-1 solution
(1:10,0001:10). After 60 min, the polymers were separated by
magnet and the saturated adsorption capacity Q0 on MMIPs was
assessed by ECL at adsorption equilibrium.
3. Results and discussion
3.1. Characterization of synthesized nanoparticles
The SEM and TEM images of Fe3O4, Fe3O4@SiO2 and MMIPs
nanocomposites are shown in Fig. 1. The diameters of the
uncoated Fe3O4 nanoparticles were in the range of 1018 nm
(Fig. 1A and D), while the mean diameter of the Fe3O4@SiO2

nanoparticles increased to approximately 100 nm with a narrow

size distribution (Fig. 1B and E). The latter size was determined by
the TEOS concentration in the reaction mixture. It was also shown
that the Fe3O4 nanoparticles were fully coated by the silica
(Fig. 1E). After the imprinted polymer further covered the Fe3O4@SiO2 nanoparticles, the surface of the nanoparticles became less
smooth, suggesting the successful formation of imprinted layers
(Fig. 1C and F). The HRTEM was used to further conrm the
formation of the protein-templated imprinted sites. It could be
seen from Fig. 1G that there existed many cavities on the surface of
MMIPs. AFM was also used to characterize the surface morphology
of the imprinted layers before (Fig. 1H) and after (Fig. 1I) the
elution of the HIgG templates. Each scan represents a 200 nm 
200 nm lateral area and the vertical scale is 2.8 nm per division.
Compared with Fig. 1H, I showed many dark points whose lateral
areas were about (612) nm  (815) nm and depths were in the
range of 0.51.5 nm, indicating the formation of the cavities on the
imprinted layers.
Fig. 1J showed XRD patterns of Fe3O4 (a), Fe3O4@SiO2 (b) and
MMIPs (c). It was observed that the six diffraction peaks of Fe3O4
(2 30.11, 35.51, 43.11, 53.41, 57.01, 62.61) appeared in the 2
range of 20801. After Fe3O4 was coated by SiO2 NPs, the peak
intensity decreased and a new parabolic peak attributed to
amorphous SiO2 NPs appeared at approximately 2 231. Finally,
from curve c, it was observed that the peak intensity of MMIPs was
slightly reduced when compared with that of Fe3O4@SiO2 NPs,
which revealed the formation of a thin imprinted lm. However,
the peak positions of Fe3O4, Fe3O4@SiO2 and MMIPs were
unchanged, demonstrating that their crystalline structure was
essentially maintained.
Fig. S1 (please refer to the Supporting information) essentially
compared the TGA curves of Fe3O4@SiO2 and MMIPs, which
illustrated that the weight loss of curve a, and curve b below
130 1C (about 9%) was due to volatilization of the solvent or water.
The vast and rapid weight loss of curve b between 300 1C and
620 1C (about 30%) originated from the imprinted polymer on the
surface of Fe3O4@SiO2. Therefore, these observations indicated the
existence of an imprinted polymer.
VSM was employed to study the magnetic properties of the
synthesized nanocomposites: Fe3O4 (a), Fe3O4@SiO2 (b), and
MMIPs (c). The magnetic hysteresis loops of the samples dried at
300 K are illustrated in Fig. 2. The saturation magnetization of
MMIPs was reduced to 8.572 emu g  1 as compared with bulk Fe3O4
and Fe3O4@SiO2 (saturation magnetization are 58.921 emu g  1
and 10.388 emu g  1, respectively). However, the saturation magnetization of MMIPs retained sufcient magnetic responsiveness
to satisfy the need of the magnetic separation (Fig. 2 (inset)).
When an external magnetic eld was applied, the dark particles
were attracted to the wall of the vial in a very short time (about
20 s) and the dispersion became transparent and clear. After
removing the external magnetic eld, a brownish black homogeneous dispersion appeared. The results demonstrated the superparamagnetism property of MMIPs.
3.2. Characterization of the ECL immunosensor
As shown in Fig. 3, no signicant difference in the ECL signal
could be found in MNIPs (curve a) and MMIPs (curve b) for the ECL
immunosensor when there was no anti-HIV-1 in sample. However,
when there existed anti-HIV-1 in sample, the ECL intensities from
MNIPs (curve c) and MMIPs (curve d) immunosensor were both
enhanced because the immobilization of HRP on them could
catalyze the ECL reaction of the luminol-H2O2 system. Additionally, the signal originating from MMIPs was stronger as compared
with that of MNIPs, which was ascribed to a higher specic
adsorption capacity of anti-HIV-1 on MMIPs.

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203

Fig. 1. The SEM images of Fe3O4 (A), Fe3O4@SiO2 (B) and MMIPs (C); TEM images of Fe3O4 (D), Fe3O4@SiO2 (E) and MMIPs (F); HRTEM images of MMIPs (G); AFM images
of the imprinted layers before (H) and after (I) the elution of the HIgG templates; (J) XRD patterns of Fe3O4 (a), Fe3O4@SiO2 (b) and MMIPs (c).

3.3. Optimization of the experimental conditions

In order to enhance the detection sensitivity of the sandwich
immunosensor and reduce background signals, we optimized some
conditions including the amount of MMIPs, and the pH of the

electrolyte, which was achieved by changing the examined conditions while the others were xed.
The effect of the amount of MMIPs used to construct the
sandwich immunosensor on the sensitivity of the ECL signal was
investigated from 20 to 200 g (Fig. S2, Supporting information).

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J. Zhou et al. / Biosensors and Bioelectronics 54 (2014) 199206

An increase in the ECL signal was observed up to 100 g.

Whereas, the increasing amount of MMIPs lowered the sensitivity
for ECL detection, which was probably due to an excess of MMIPs
blocking the mass transformation, and minimizing the electric
conductivity. For this reason, an immobilized MMIPs, at an amount
of 100 g was chosen for further work. Since the luminol-H2O2
system in base solution could lead to a strong ECL background
signal, 2  10  4 M was selected as the concentration of luminol
solution in this study. By contrast, to ensure sufcient signal
amplication of HRP from the signal tags HRP-HIV-1 and preservation of HRP activity, 5  10  3 M H2O2 solution and pH 8.0 PBS
as the electrolyte were thus adopted in this paper (Fig. S3, Supporting
information).
3.4. Imprinting effect

Fig. 2. The hysteresis loops of Fe3O4 (a), Fe3O4@SiO2 (b), and MMIPs (c). The insert
shows the separation and redispersion process of a solution of MMIPs in the
presence (left) and absence (right) of an external magnetic eld.

Fig. 3. ECL curves of a mixture included 5 L 2  10  4 M luminol solution, 10 L

5  10  3 M H2O2 solution and 10 L ECL immunosensor prepared by MNIPs and
HRP-HIV-1 without (a) and with (c) anti-HIV-1 at a dilution ratio of 1:50;
additionally, 5 L 2  10  4 M luminol solution, 10 L 5  10  3 M H2O2 solution
and 10 L ECL immunosensor prepared by MMIPs and HRP-HIV-1 without (b) and
with (d) anti-HIV-1 at a dilution ratio of 1:50.

The adsorption kinetics (Fig. 4a) and isotherm curves (Fig. 4b)
of MMIPs and MNIPs are shown herein. It could be seen that
MMIPs achieved a much higher ECL and a faster adsorption rate
than MNIPs during the rst 60 min and until adsorption equilibrium was achieved (Fig. 4a). However, it should be noticed MNIPs
reached adsorption equilibrium much faster (40 min) than MMIPs
(60 min), which might be attributed to the higher adsorption
capacity of MMIPs and the need of more adsorption time. But
the purpose of this paper is to develop an ultrasensitive ECL
immunosensor and reduce the diagnostic window between the
time of HIV-1 infection and laboratory diagnosis. Thus improving
the detection sensitivity and reducing the detection limit is the
key to this work. To achieve the goal, what is more important is
the higher adsorption of MMIPs, which contributes to the enrichment of ultratrace analytes. As presented in Fig. 4b, the ECL
increased as the initial dilution ratio of anti-HIV-1 increased at
dilution ratios below 1:50, and reached a plateau at a dilution ratio
of 1:50. Moreover, the MMIPs exhibited a higher ECL change for
anti-HIV-1 than MNIPs did at the same concentration. This
indicated specic adsorption of MMIPs and the non-specic
binding of MNIPs within the range of the experimental concentrations. By tting the experimental data with the isothermic
adsorption curves, the saturated adsorption capacity Q0 of MMIPs
was approximately 0.006 C0 g  1 (where C0 was the original concentration of anti-HIV-1 for standard positive serum).
3.5. Analytical performance
The ECL signals that were responsive to the changing dilution
ratio of anti-HIV-1 (aj) are shown here (Fig. 5). It could be seen
that the ECL peak intensity increased gradually with increasing

Fig. 4. (a) Adsorption kinetics of MMIPs and MNIPs; (b) Adsorption isothermic curves of MMIPs and MNIPs.

J. Zhou et al. / Biosensors and Bioelectronics 54 (2014) 199206

Fig. 5. ECL proles of immunosensor detection of dilution ratios of anti-HIV-1:

(a) 0, (b) 1:20,000, (c) 1:10,000, (d) 1:8000, (e) 1:5000, (f) 1:2000, (g) 1:500,
(h) 1:200, (i) 1:100, and (j) 1:50. Inset: calibration curve.

concentrations of anti-HIV-1 (bj). The standard calibration curve

for anti-HIV-1 detection is shown in the inset. Under optimal
conditions, the logarithm of the ECL intensity IECL (IECL I  IO,
here I and IO are the ECL intensities of ECL immunosensor prepared
with and without anti-HIV-1, respectively) was proportional to the
logarithm of the dilution ratio of anti-HIV-1 in the range of
1:20,000 to 1:50 (R2 0.9927), and the detection limit was
1:60,000 (S/N 3), which was much lower than that found for
ELISA (1:300) (Saifuddin et al., 1995). This result indicated that the
emergence of MMIPs as a candidate for biological recognition
elements and signal amplication would offer new perspectives
on the improvement of current immunoassays.
3.6. Specicity, reproducibility and feasibility of this proposed
immunosensor
To study the specicity of this proposed immunosensor, MMIPs
were binded respectively with anti-HIV-1 standard negative serum
(SNS), 20 ng mL  1 carcinoembryonic antigen (CEA), 10 ng mL  1
carbohydrate antigen 19-9 (CA19-9), 1 mg mL  1 BSA, 1 g mL  1
HIgG, 100 ng mL  1 alpha fetoprotein (AFP), anti-HIV-1 with a dilution ratio of 1:2000 and a mixture containing all the above
molecules/proteins, blocked, then incubated with HRP-HIV-1. The
subsequent detection was followed by using the current immunosensor procedure (Fig. S4, Supporting information). The ECL response
obtained from MMIPs binding with anti-HIV-1 and incubated with
HRP-HIV-1 was much stronger than that obtained from MMIPs
binding with SNS, CEA, CA19-9, BSA, HIgG and AFP, and incubated
with HRP-HIV-1, which was similar to that obtained with MMIPs
binding with a mixture containing all of the above molecules/
proteins and incubated with HRP-HIV-1. These results indicated that
this immunosensor had adequate specicity for the diagnosis of
anti-HIV-1.
The reproducibility of the developed immunosensor was
investigated in the online assay for anti-HIV-1 with a dilution
ratio of 1:500. High reproducibility of the ECL response (relative
standard deviation (RSD) of 4.52%) was obtained for 5 repetitive
detections of anti-HIV-1 with a dilution ratio of 1:500. The
electrode-to-electrode reproducibility was also examined between
six independent SPCE electrodes in the above solutions, and the
corresponding RSD was calculated to be 3.89%. In addition, the
available times of MMIPs eluting from the sandwich immunosensor were studied by repetitive fabrication of the immunosensor
and detection of the ECL signal. The observation informed us that

205

there was no signicant difference in ECL signals observed for ten

measurements, which might be explained by the excellent stability of MMIPs.
To monitor the feasibility of the developed immunosensor in
clinical analyses, three blank serum samples from different
patients were diluted and analyzed. Recovery experiments were
carried out by the standard addition method in these serum
samples. The results showed that the recovery ranged from
84.15% to 104.50%, and that the corresponding RSD ranged from
4.28% to 8.31% (see Supporting information, Table S1), which
revealed that the feasibility of the developed immunoassay could
be preliminarily applied to the detection of ultratrace levels of
anti-HIV-1 in clinical human serum samples for routine clinical
diagnosis. In addition, to further validate the proposed method,
forty-three human serum samples from Nanfang Hospital (China)
were tested with the proposed method and a commercialized
ECLIA kit (Roche), which were consistent with each other (25
samples were positive and the rest were negative). The efciency
value of 100% could be calculated using the following
equation (Shukla et al., 2009): Efciency (TPTN)  100/Total
(TP, True Positive; TP 25; TN, True Negative; TN 18; Total 43).
Such result demonstrated that the novel method exhibited good
assay performance comparable to the commercialized ECLIA kit
and was potentially applicable in detecting anti-HIV-1.

4. Conclusions
We have successfully designed a rapid and cost-effective
sandwich ECL immunosensor for the ultrasensitive detection of
anti-HIV-1 based on the combination of surface imprinting and
epitope imprinting techniques. We have also studied its effectiveness for detecting human serum specimens by ECLIA. The resulting
imprinted lm showed a high afnity and enrichment capacity for
anti-HIV-1 at ultra low concentration levels. Additionally, ECL
measurements revealed that the developed sandwich ECL immunosensor had a stronger capacity of resisting disturbance than
MMIPs alone, owing to the use of the high specicity of an
antibody-antigen reaction, which overcame the interference of
the possible template molecules. The capture probes (MMIPs)
could also be recycled, and thus greatly reduced the experimental
cost. Therefore, the obtained immunosensor was applied successfully for monitoring anti-HIV-1 in human serum samples. The
simplicity, cost-effectiveness and high sensitivity of the developed
method proposes a merger of imprinting techniques and immunoassay for biomolecular analysis.

Acknowledgments
This work was supported by the National Natural Science
Foundation of China (No. 30901367), the Natural Science Foundation
of Zhejiang (LY13C200017, LY12C20004, and LY12B01005), the
Science and Technology Project of Zhejiang (2012C23101 and
2011C23126), the Natural Science Foundation of Ningbo City
(2011B82014 and 2013A610241, and K.C. Wong Magna Fund in
Ningbo University.

Appendix A. Supporting information

Supplementary data associated with this article can be found in
the online version at http://dx.doi.org/10.1016/j.bios.2013.10.044.

206

J. Zhou et al. / Biosensors and Bioelectronics 54 (2014) 199206

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