Gold-linked electrochemical immunoassay on single-walled carbonnanotube for highly sensitive detection of human chorionic gonadotropin hormone Nguyen Xuan Vieta,b, Miyuki Chikaea, Yoshia
Trang 1Gold-linked electrochemical immunoassay on single-walled carbon
nanotube for highly sensitive detection of human chorionic
gonadotropin hormone
Nguyen Xuan Vieta,b, Miyuki Chikaea, Yoshiaki Ukitaa, Kenzo Maehashic,
a
School of Materials Science, Japan Advanced Institute of Science and Technology (JAIST), 1-1 Asahidai, Nomi City, Ishikawa 923-1292, Japan
b
Faculty of Chemistry, Hanoi University of Science, VNU, 19 Le Thanh Tong, Hoan Kiem District, Ha Noi, Vietnam
c
The Institute of Scientific and Industrial Research, Osaka University, 8-1 Mihogaoka, Ibaraki, Osaka 567-0047, Japan
d Department of Applied Physics, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan
e
Research Center for Environmental Technology and Sustainable Development (CETASD), Hanoi University of Science, VNU, 334 Nguyen Trai, Thanh Xuan
District, Ha Noi, Vietnam
a r t i c l e i n f o
Article history:
Received 19 August 2012
Received in revised form
13 November 2012
Accepted 14 November 2012
Available online 23 November 2012
Keywords:
Electrochemical immunoassay
Gold nanoparticles
Carbon nanotube electrode
Sandwiched type
Immunosensor
hCG
a b s t r a c t
A new sensitive gold-linked electrochemical immunoassay (GLEIA) for the detection of the pregnancy marker human chorionic gonadotropin (hCG) has been developed using the direct electrochemical detection of Au nanoparticles We utilized single-walled carbon nanotube (SWCNT) microelectrodes; 24 SWCNT microelectrodes were arrayed on a single Si substrate 25 30 mm2in size, for the development
of a new GLEIA (SWCNT-GLEIA) This SWCNT-GLEIA provided convenient and cost-effective tests with the required antibody and antigen sample volumes as small as 2.0mL for a group of 4 SWCNT microelectrodes In addition, this assay also exhibited properties of high sensitivity and selectivity benefitting from the intrinsic extraordinary features of SWCNTs Using scanning electron microscopy,
we also observed Au nanoparticle-labeled antigen–antibody complexes immobilized on the surface of the SWCNT microelectrodes The concentration of the pregnancy marker (hCG) showed a linear relationship with the current intensity obtained from differential pulse voltammetry measurements with a limit of detection (LOD) of 2.4 pg/mL (0.024 mIU/mL) hCG This LOD is 15 times more sensitive than a previous GLEIA, which used screen-printed carbon electrodes
&2012 Elsevier B.V All rights reserved
1 Introduction
An immunosensor, a type of biosensor, can be defined as a
compact analytical device incorporating antibodies or antigens or
their fragments, either integrated within or intimately associated
with a physicochemical transducer Immunosensors provide
sen-sitive and selective tools for determining the presence of proteins
on the basis of a specific reaction between an antibody and
antigen (Veetil and Ye, 2007) Immunosensors can help in directly
monitoring a molecular recognition event on the surface of a chip
A large number of immunosensors have been developed using
different transducers that exploit changes in mass (Janshoff et al.,
2000;Ward and Buttry, 1990), heat (Luong et al., 1988),
electro-chemical (Dzantiev et al., 1996; Shah and Wilkins, 2003), or
optical properties (Brecht and Gauglitz, 1995; Haes and Van
Duyne, 2002;Morgan et al., 1996) Most of the reagents employed
in immunosensor, such as antibodies, enzymes, and fluorescence labels are very expensive, and additionally, analytes such as blood from a neonate or spinal fluid are precious commodities (Veetil and Ye, 2007) Hence, miniaturization of diagnostic devices with-out affecting their sensitivity or limit of detection is highly desirable With advancements in the field of micro- and nano-fabrication and lab-on-chip concepts, novel high-throughput immunosensors that offer decreased analysis time and ease of automation, integration, and portability are being explored Among the various immunosensor developed, electrochemical immunosensor have become the predominant analytical techni-que for the quantitative detection of biomolecules due to their simplicity, high sensitivity, low cost, fast analysis and ease of miniaturization (Bakker, 2004;Privett et al., 2010;Skla´dal, 1997) Moreover, sandwich-type electrochemical immunosensors have gained much attention because of their high specificity and sensitivity (Campbell et al., 2001; Chen et al., 2006; Idegami
et al., 2008)
It is well-known that, in conventional single-walled carbon nanotube (SWCNT)-modified electrodes, such as SWCNT-modified
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n
Corresponding author.
E-mail address: yztakamura@jaist.ac.jp (Y Takamura).
Trang 2glassy carbon electrodes (Luo et al., 2001;Wang et al., 2001,2002),
screen-printed carbon electrodes (SPCEs) (Lin et al., 2004; Sha
et al., 2006), and platinum electrodes (Okuno et al., 2007a,2007b;
Tsujita et al., 2009,2008), the electrochemical signals come from
both the SWCNTs and the supporting electrodes (carbon or
platinum, etc.) because the supporting electrodes are also exposed
to the electrolyte solutions In most of these cases, SWCNTs exhibit
greatly enhanced electrochemical signals, so that the contribution
of the supporting electrodes is negligible However, in some special
cases, such as when measuring electro-double layer charge
cur-rents, or in cases where the reactions are specifically enhanced on
plane supporting electrodes, this becomes a problem In addition,
the nonspecific adsorption of protein on nanotubes is not desirable,
especially when using actual biological fluid samples that contain
many co-existing proteins (Nedelkov and Nelson, 2001;Tombelli
et al., 2005; Wang, 2002) More sophisticated sensors, therefore,
are needed to address issues such as target recognition
enhance-ment, blockage of undesired interference (co-existing proteins,
nonspecific adsorption on the nanotube surfaces, etc.), and
long-term storage Nonspecific binding directly affects the selectivity
and sensitivity of devices
In this paper, we describe a sandwich-type electrochemical
immunoassay for highly sensitive and selective detection of the
biomarker molecule hCG, which is used as a model of detection
A SWCNT microelectrode (Viet et al., 2012) was used in this
electrochemical immunoassay instead of conventional electrodes
such as glassy carbon electrodes or SPCEs Au nanoparticles were
used to label the antibody immunocomplex in this
electrochemi-cal immunoassay
2 Experimental
2.1 Reagents
Monoclonal anti-humana-subunit of follicle-stimulating
hor-mone (Mab-FSH) with an affinity constant of 2.8 109M1, and
monoclonal anti-human chorionic gonadotropin (Mab-hCG) with
an affinity constant of 4.9 109M1, were purchased from
Medix Biochemica (Kauniainen, Finland) The molecular weight
of recombinant human chorionic gonadotropin (hCG) was
deter-mined as 57.1 kDa using sodium dodecyl sulfate polyacrylamide
gel electrophoresis (SDS-PAGE), and its potency was measured as
10,000 IU/mg (Rohto Pharmaceutical Co., Ltd., Osaka, Japan)
A colloidal solution of Au nanoparticles of diameter 40 nm was
purchased from British Biocell International Ltd., (Cardiff, UK)
HCl, NaH2PO42H2O, polyethylene glycol (PEG), KH2PO4 and
dimethylformamide (DMF) were purchased from Wako Pure Chemical Industries (Osaka, Japan) Sodium azide (NaN3) was purchased from Nakarai Tesque (Kyoto, Japan) 1-pyrenebutanoic acid succinimidyl ester was purchased from Life Technologies Corporation (Carlsbad, CA, USA) Bovine serum albumin (BSA) was purchased from Sigma-Aldrich, (St Louis, MO, USA) Polyethylene glycol amine with a molecular weight of 5000 Da was purchased from SUNBRIGHT (NOF Corporation, Tokyo, Japan) Male urine solution was purchased from Lee Biosolution, Inc Other reagents were of analytical grade, and all solutions were prepared and diluted using ultra-pure water (18.2 MOcm) from the Milli-Q system (Millipore, Billerica, MA, USA)
2.2 Instrument Scanning electron microscopy (SEM) images were obtained using Hitachi S-4100 with accelerating voltage 20 kV Electroche-mical measurements were performed on an ALS/CH Instruments electrochemical analyzer, model 730C (Austin, Texas, USA) as shown inFig 1, in which a 3-electrode system was used with a Pt wire as the counter, an AgCl/Ag micro-electrode as the reference (Microelectrodes Inc., Bedford, NH, USA), and a SWCNT micro-electrode as the working micro-electrode
2.3 Sandwiched immunosensor procedure a) Preparation of Au nanoparticle-labeled hCG antibody (Au-Mab-hCG)
The preparation of Au-Mab-hCGs was performed by a similar method as previously reported by our group (Idegami et al.,
2008;Nagatani et al., 2006;Tanaka et al., 2006) with a slight modification Briefly, an aliquot (200 microliter) of Mab-hCG solution (50mg/mL in 5 mM KH2PO4, pH 7.5) was mixed with 1.8 mL of 0.1% Au nanoparticle solution, and kept for 10 min at room temperature Then, 100 microliter of 1% PEG in 50 mM
KH2PO4 solution (pH 7.5) and 200 microliter of 10% BSA in
50 mM KH2PO4 solution (pH 9.0) were added to block the uncoated surfaces of the Au nanoparticles After the immobi-lization and blocking procedures, Au nanoparticle-conjugated Mab-hCGs (Au-Mab-hCGs) were collected by centrifugal operation (8000 g for 15 min at 4 1C) Au-Mab-hCGs were suspended in 2 mL of the preservation solution (1% BSA, 0.05% PEG 20,000, 0.1% NaN3, and 150 mM NaCl in 20 mM Tris-HCl buffer, pH 8.2), and collected again in the same manner For the stock solution, Au-Mab-hCGs were suspended
Fig 1 Electrochemical measurement set-up with a SWCNT microelectrode as the working electrode (WE), a Pt wire as the counter electrode (CE), and an Ag/AgCl
Trang 3in the preservation solution and the optical density was
adjusted to an OD520of 6
b) Fabrication of immunosensor
An array of 24 SWCNT microelectrodes on a single Si substrate
was made following a procedure that has been previously
described and characterized in detail (Viet et al., 2012) Briefly,
an SWCNT network was synthesized by catalyst chemical
vapor deposition using ethanol as the carbon source on the
Si substrate Next, a chromium layer (200 nm) was thermally
evaporated onto the SWCNT network using the plasma
sput-tering method A photoresist layer with a thickness of 15mm
(PMER photoresist) was subsequently spun over the chromium
layer A disk-type pattern with a diameter of 180mm was
formed inside the Pt contacts by exposing them to 458-nm
helium light for 30 s and then developing in PG-7 solution The
exposed chromium layer was removed by chromium etchant
solution in 2 min Then, a thermal SiO2layer of 250 nm was
sputtered onto the exposed SWCNT network by the plasma
sputtering method Finally, the residual disk-type pattern of
the photoresist layers and chromium layers was cleaned using
remover and chromium etchant solution, respectively Note
that between 2 successive steps, the SWCNT microelectrodes
were washed by Milli-Q water for 1 min and then blown under
N2gas to dry
Next, SWCNT microelectrodes were incubated in 30mL dry
DMF solution with 0.1 mM 1-pyrenebutanoic acid
succinimi-dyl ester as a linker molecule for 30 min at room temperature,
followed by rinsing with DMF solvent to remove the unbound
molecules from the SWCNTs (Chen et al., 2001;Okuno et al.,
2007a) In order to covalently immobilize Mab-FSH on the SWCNTs, SWCNT microelectrodes were exposed overnight to
400mg/mL Mab-FSH in 10 mM phosphate-buffered saline (PBS, pH 7.4) by dropping 2.0mL of Mab-FSH on each group
of SWCNT microelectrodes Following this, the excess anti-bodies were rinsed with PBS To deactivate reactive groups and suppress nonspecific binding, 4.0mL of 10 mM PBS solution containing 1% PEG–NH2was added onto the resulting electro-des, and incubated for 1 h at room temperature The array was then rinsed with PBS
c) Sandwich-type immunoreaction and electrochemical measurement
A scheme illustrating the principle of the gold-linked electro-chemical immunoassay (GLEIA) on SWCNT microelectrodes (SWCNT-GLEIA) is shown inFig 2 Different concentrations of the hCG antigen solution were made by diluting the stock solution in PBS containing 1% BSA for detection In case of detection of hCG in biological fluid, stock solution of hCG was spiked in male urine solution to make different concentration For the detection of the antibody–antigen reaction, 2.0mL of the antigen solution was placed on a group of 4 SWCNT microelectrodes for 1 h at room temperature After rinsing with PBS, 2.0mL of Au-Mab-hCG solution was applied onto the surface, and incubated for 30 min at room temperature Finally, the SWCNT microelectrodes were rinsed carefully with PBS
The direct redox reaction was performed in 0.1 M HCl solution (30mL) covering the entire three-electrode zone at room temperature (as shown in actual photo of electrochemical
Fig 2 Scheme illustrating the principle of the gold-linked electrochemical immunoassay on SWCNT microelectrodes.
Fig 3 SEM images of (a) SWCNTs immobilized with Mab-FSH and blocking agents, (b) Au nanoparticle-labeled antigen–antibody complexes immobilized on the surface of
Trang 4measurement inFig 1) The pre-oxidation of Au nanoparticles
was performed at a constant potential 1.2 V for 40 s,
immedi-ately followed by DPV, while scanning the potential range
from 1.0 V to 0.0 V with a step potential of 4.0 mV, pulse
amplitude of 50 mV, and a pulse period of 0.2 s The potentials
were recorded against the Ag/AgCl microelectrode as the
reference (Idegami et al., 2008)
3 Results and discussion
3.1 SEM images of GLEIA using SWCNT microelectrodes
Fig 3a shows the SEM image of the SWCNT network inside the
SWCNT microelectrode after immobilizing with Mab-FSH and
blocking agent Fig 3b is the SEM image of Au
nanoparticle-labeled immunocomplexes immobilized on the surface of the
SWCNTs (see white arrows in Fig 3b) Au nanoparticles were
distributed on the surface of the SWCNT network with a hCG
concentration of 1.0 ng/mL (10.0 mIU/mL) This shows that the
antigen, hCG, was successfully detected using the SWCNT
micro-electrode for GLEIA
Because the surface of the SWCNT microelectrode was not
totally covered with SWCNTs (Dumitrescu et al., 2008;Viet et al.,
2012), when some Au nanoparticles nonspecifically adsorbed onto the Si/SiO2substrate and did not contact the SWCNTs, they were not able to generate electrochemical signals This should improve the effect of depressing the background signal, resulting
in a lower limit of detection This is the advantage of using a SWCNT network over other CNT-modified electrodes
3.2 Electrochemical operation of GLEIA using SWCNT microelectrode
Fig 4illustrates the cyclic voltammogram (CV) obtained from the Au-Mab-hCG-immobilized immunosensor after the antigen– antibody reaction (with 100 ng/mL hCG–1.0 103mIU/mL) in the potential range from 0.0 to 1.4 V vs Ag/AgCl in 0.1 M HCl solution The reduction peak of Au ions could be observed at a potential of around þ0.5 V, corresponding with reaction (1) in Fig 4 The positive shift of the gold reduction peak on SWCNT microelec-trodes compared with SPCEs (from þ0.35 V (Idegami et al., 2008;
Quinn et al., 2005) on SPCEs to around þ0.5 V on SWCNTs) in the
CV curve illustrated that SWCNTs promote the reduction of Au ions better than do SPCEs; one reason is the difference in the environment of the reference electrode In SPCEs, the reference electrode is immersed directly in 0.1 M HCl solution and has a potential of 0.2881 V compared with a normal hydrogen electrode (NHE) On the other hand, in the case of SWCNT electrodes, the reference electrode is immersed in 3.0 M KCl solution, thus it has
a potential of 0.21 V vs the NHE (Bard 2001)
In the operation of the GLEIA, the reduction peak current of DPV was used for the detection of Au nanoparticles in 0.1 M HCl solution This process involves the oxidation of Au nanoparticles into Au ions before the Au ions are reduced on the electrode surface to obtain a good electrochemical signal (Idegami et al.,
2008) The effect of the pre-oxidation potential on the current densities of the DPV reduction peak of the Au ion was investi-gated The pre-oxidation potentials were measured at 1.20, 1.50, and 1.70 V vs Ag/AgCl with a pre-oxidation time of 40 s in the presence of 250 pg/mL (2.5 mIU/mL) hCG, shown in Fig s1 of supplemental document A rapid decrease in the reduction peak current intensity was observed with increasing pre-oxidation potential This indicates that the loss of Au ions occurs more easily at high pre-oxidation potential than at lower pre-oxidation potentials Therefore, in this electrochemical measurement, 1.20 V was the optimum pre-oxidation potential Fig 5a shows DPV curves obtained from the Au-Mab-hCG-immobilized immu-nosensor with different concentrations of hCG (from 10.0 pg/mL
to 2.0 103pg/mL–0.1 mIU/mL to 20.0 mIU/mL) in PBS contain-ing 1% BSA at an applied potential of 1.20 V The reduction peaks
Fig 4 Cyclic voltammogram of Au-Mab-hCG immobilized on a SWCNT
micro-electrode at 50 mV/s in 0.1 M HCl solution The concentration of hCG was 100 ng/ml
(1.0 10 3
mIU/mL).
Fig 5 (a) Differential pulse voltammograms of the Au-Mab-hCG on SWCNT microelectrodes in 0.1 M HCl solution (b) Normalized calibration curves in GLEIA using SWCNT microelectrodes (curve I and II), SWCNT-modified SPCEs (curve III), and SPCEs (curve IV) as the platform The concentration of hCG ranged from 10.0 pg/mL to
3
Trang 5were observed at approximately þ0.52 V, nearly equal to the CV
result of 0.5 V The peak current intensity increased in
propor-tion to increasing hCG concentrapropor-tion
The analytical range and sensitivity of the immunosensor were
calculated by extracting the current intensity as a function of the
hCG concentration fromFig 5a The results are shown inFig 5b
(curve I) The reduction peak current intensity of Au ions
depended linearly on the hCG concentration in this concentration
range, and the correlation coefficient (R2) of the linear fitting
curve for this relationship was 0.9906 Under the above measured
conditions, an LOD of 2.4 pg/mL (0.024 mIU/mL) for hCG was
calculated as 3SD (where SD is the standard deviation of 5
mea-surements of blank samples) This value is 15 times lower than
the previous work of our lab using SPCEs (Idegami et al., 2008) as
platform for this immunosensor
The LOD of this immunosensor increases to 53 pg/mL
(0.53 mIU/mL) in the male urine solution (curve II in Fig 5b)
This value of LOD is around 20 times higher than that measuring
in PBS containing 1% BSA For a comparison, we also conducted
the GLEIA using planar SPCE for same urine sample, and the result
got the LOD of 1.85 ng/mL (18.5 mIU/mL) (Fig s2), which is 51
times higher than LOD in PBS containing 1% BSA using planar
SPCE The LOD of SWCNT microelectrode increases 20 times in
urine sample and this is considered to be caused by the deviation
of signal due to non-specific binding of various bio-substances in
urine sample The value of 20 is still less than the value of 51 for
the increase in SPCE This fact indicates that our SWCNT
micro-electrode has better suppression property of non-specific binding
and better selectivity not only in PBS with 1% BSA but also in urine
sample than conventional planar SPCE These values for SWCNT
microelectrode were obtained using the same described condition
above, and may be improved more by further optimization
This sandwich-type immunosensor using Au nanoparticles as
label has several advantages over the use of enzyme as the label
In the case of enzyme-based detection systems, the electrode
surface is covered with the immune-complexes and blocking
agents; these biomolecules remain on the surface during
electro-chemical measurement, and may disturb the performance of the
electrode In our method, the pre-oxidation of Au nanoparticles at
a high potential and the denaturation of the biomolecules in
highly acidic conditions were carried out simultaneously Thus,
the detachment of possible blocking molecules from the surface
provided a large electroactive area for oxidized Au ions to be
reduced again efficiently during the DPV scan Additionally, the
loss of oxidized Au ions by diffusion was avoided because of the
negative charge of the chelated compounds with the high
con-centration of chloride ions in the acidic electrolyte The constant
application of highly positive voltage rapidly attracted negatively
charged Au chelates and promoted their electrodeposition on the
carbon surface (Idegami et al., 2008)
3.3 Sensitivity of GLEIA using SWCNT microelectrodes, GLEIA on
SWCNT-modified SPCEs, and SPCEs
Fig 5b shows the normalized calibration curves of GLEIA on a
platform of SWCNT microelectrodes (curve I) (this study), SPCEs
(curve IV) (Idegami et al., 2008), and SWCNT-modified SPCEs
(curve III), with hCG concentrations ranging from 10.0 pg/mL to
2.0 103pg/mL (0.1 mIU/mL to 20.0 mIU/mL) These normalized
curves determine the current density on each type of electrode
used for GLEIA The procedure for GLEIA on SPCEs and
SWCNT-modified SPCEs are similar with those described above for the
SWCNT microelectrode The LOD of GLEIA on SPCEs and
SWCNT-modified SPCEs were 36 pg/mL (0.36 mIU/mL) and 13 pg/mL
(0.13 mIU/mL), correspondingly These results show that GLEIA
using the SWCNT microelectrode has the highest sensitivity
The high sensitivity of this SWCNT-GLEIA was attributed to the combination of the high performance of our SWCNT microelec-trode with the ability to enhance electrochemical signals, reduce nonspecific binding, and effectively detect the signals directly from Au nanoparticles The performance of GLEIA on SWCNT-modified SPCEs was better in comparison with GLEIA on SPCEs This comes from the enhancement of SPCE performance due to the presence of SWCNTs However, the performance of SWCNT-modified SPCEs was lower than that of SWCNT microelectrodes because the SWCNTs using for modifying SPCEs underwent acid treatment (Gooding et al., 2003), which leads to shortening, more sidewall defects, and lower electrical conductivity than with as-grown SWCNTs (Zhang et al., 2004)
4 Conclusion
A new sensitive gold-linked electrochemical immunoassay for the detection of the pregnancy marker, hCG, has been successfully developed based on the sandwich-type immunosensor This SWCNT-GLEIA, based on microelectrodes that use an SWCNT network directly grown on Si, exhibited the highest sensitivity compared with those of GLEIAs conducted using SPCEs and SWCNT-modified SPCEs This SWCNT-GLEIA also showed good selectivity when detecting hCG in male urine solution The LOD of SWCNT-GLEIA got the values of 2.4 pg/mL (0.024 mIU/mL) and
53 pg/mL (0.53 mIU/mL) hCG, when it was spiked in PBS contain-ing 1% BSA and in male urine solution, correspondcontain-ingly
Acknowledgments This work was partially supported by a Grant-in-Aid for Scientific Research on Priority Areas (No 19054011) and the Cooperative Research Program of ‘‘Network Joint Research Center for Materials and Devices’’ from the Ministry of Education, Culture, Sports, Science and Technology of Japan
Appendix A Supporting information Supplementary data associated with this article can be found in the online version athttp://dx.doi.org/10.1016/j.bios.2012.11.017
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