DSpace at VNU: Gold-linked electrochemical immunoassay on single-walled carbon nanotube for highly sensitive detection of human chorionic gonadotropin hormone

Biosensors and Bioelectronics 42 (2013) 592–597 Contents lists available at SciVerse ScienceDirect Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios Gold-linked electrochemical immunoassay on single-walled carbon nanotube for highly sensitive detection of human chorionic gonadotropin hormone Nguyen Xuan Viet a,b, Miyuki Chikae a, Yoshiaki Ukita a, Kenzo Maehashi c, Kazuhiko Matsumoto c, Eiichi Tamiya d, Pham Hung Viet e, Yuzuru Takamura a,n a School of Materials Science, Japan Advanced Institute of Science and Technology (JAIST), 1-1 Asahidai, Nomi City, Ishikawa 923-1292, Japan Faculty of Chemistry, Hanoi University of Science, VNU, 19 Le Thanh Tong, Hoan Kiem District, Ha Noi, Vietnam 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 b c a r t i c l e i n f o a b s t r a c t Article history: Received 19 August 2012 Received in revised form 13 November 2012 Accepted 14 November 2012 Available online 23 November 2012 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 Â 30 mm2 in 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 2.0 mL for a group of 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 Keywords: Electrochemical immunoassay Gold nanoparticles Carbon nanotube electrode Sandwiched type Immunosensor hCG 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 sensitive 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), electrochemical (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 n Corresponding author E-mail address: yztakamura@jaist.ac.jp (Y Takamura) 0956-5663/$ - see front matter & 2012 Elsevier B.V All rights reserved http://dx.doi.org/10.1016/j.bios.2012.11.017 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 without affecting their sensitivity or limit of detection is highly desirable With advancements in the field of micro- and nanofabrication 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 technique 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 N Xuan Viet et al / Biosensors and Bioelectronics 42 (2013) 592–597 glassy 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 currents, 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 enhancement, blockage of undesired interference (co-existing proteins, nonspecific adsorption on the nanotube surfaces, etc.), and longterm 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 electrochemical immunoassay Experimental 2.1 Reagents Monoclonal anti-human a-subunit of follicle-stimulating hormone (Mab-FSH) with an affinity constant of 2.8 Â 10 À M À 1, and monoclonal anti-human chorionic gonadotropin (Mab-hCG) with an affinity constant of 4.9 Â 10 À M À 1, were purchased from Medix Biochemica (Kauniainen, Finland) The molecular weight of recombinant human chorionic gonadotropin (hCG) was determined 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, NaH2PO4 Á 2H2O, polyethylene glycol (PEG), KH2PO4 and 593 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 MO cm) 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 Electrochemical measurements were performed on an ALS/CH Instruments electrochemical analyzer, model 730C (Austin, Texas, USA) as shown in Fig 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 microelectrode as the working 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 (50 mg/mL in mM KH2PO4, pH 7.5) was mixed with 1.8 mL of 0.1% Au nanoparticle solution, and kept for 10 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 immobilization and blocking procedures, Au nanoparticle-conjugated Mab-hCGs (Au-Mab-hCGs) were collected by centrifugal operation (8000 g for 15 at 1C) Au-Mab-hCGs were suspended in 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 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 microelectrode as the reference electrode (RE) 594 N Xuan Viet et al / Biosensors and Bioelectronics 42 (2013) 592–597 in the preservation solution and the optical density was adjusted to an OD520 of 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 sputtering method A photoresist layer with a thickness of 15 mm (PMER photoresist) was subsequently spun over the chromium layer A disk-type pattern with a diameter of 180 mm 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 Then, a thermal SiO2 layer 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 successive steps, the SWCNT microelectrodes were washed by Milli-Q water for and then blown under N2 gas to dry Next, SWCNT microelectrodes were incubated in 30 mL dry DMF solution with 0.1 mM 1-pyrenebutanoic acid succinimidyl ester as a linker molecule for 30 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 400 mg/mL Mab-FSH in 10 mM phosphate-buffered saline (PBS, pH 7.4) by dropping 2.0 mL of Mab-FSH on each group of SWCNT microelectrodes Following this, the excess antibodies were rinsed with PBS To deactivate reactive groups and suppress nonspecific binding, 4.0 mL of 10 mM PBS solution containing 1% PEG–NH2 was added onto the resulting electrodes, and incubated for 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 electrochemical immunoassay (GLEIA) on SWCNT microelectrodes (SWCNT-GLEIA) is shown in Fig 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, 2.0 mL of the antigen solution was placed on a group of SWCNT microelectrodes for h at room temperature After rinsing with PBS, 2.0 mL of Au-Mab-hCG solution was applied onto the surface, and incubated for 30 at room temperature Finally, the SWCNT microelectrodes were rinsed carefully with PBS The direct redox reaction was performed in 0.1 M HCl solution (30 mL) covering the entire three-electrode zone at room temperature (as shown in actual photo of electrochemical Fig Scheme illustrating the principle of the gold-linked electrochemical immunoassay on SWCNT microelectrodes Fig SEM images of (a) SWCNTs immobilized with Mab-FSH and blocking agents, (b) Au nanoparticle-labeled antigen–antibody complexes immobilized on the surface of the SWCNT microelectrode with a hCG concentration of 1.0 ng/mL (10.0 mIU/mL) Scale bars ¼ 500 nm N Xuan Viet et al / Biosensors and Bioelectronics 42 (2013) 592–597 measurement in Fig 1) The pre-oxidation of Au nanoparticles was performed at a constant potential 1.2 V for 40 s, immediately 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) 595 2012), when some Au nanoparticles nonspecifically adsorbed onto the Si/SiO2 substrate 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 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 nanoparticlelabeled 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 microelectrode for GLEIA Because the surface of the SWCNT microelectrode was not totally covered with SWCNTs (Dumitrescu et al., 2008; Viet et al., Fig Cyclic voltammogram of Au-Mab-hCG immobilized on a SWCNT microelectrode at 50 mV/s in 0.1 M HCl solution The concentration of hCG was 100 ng/ml (1.0 Â 103 mIU/mL) Fig illustrates the cyclic voltammogram (CV) obtained from the Au-Mab-hCG-immobilized immunosensor after the antigen– antibody reaction (with 100 ng/mL hCG–1.0 Â 103 mIU/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 The positive shift of the gold reduction peak on SWCNT microelectrodes compared with SPCEs (from ỵ0.35 V (Idegami et al., 2008; Quinn et al., 2005) on SPCEs to around ỵ0.5 V on SWCNTs) in the CV curve illustrated that SWCNTs promote the reduction of Au ions better than 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 investigated 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 immunosensor with different concentrations of hCG (from 10.0 pg/mL to 2.0 Â 103 pg/mL–0.1 mIU/mL to 20.0 mIU/mL) in PBS containing 1% BSA at an applied potential of 1.20 V The reduction peaks Fig (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 2.0 Â 103 pg/mL (0.1 mIU/mL to 20.0 mIU/mL) in PBS containing 1% BSA 596 N Xuan Viet et al / Biosensors and Bioelectronics 42 (2013) 592–597 were observed at approximately ỵ0.52 V, nearly equal to the CV result of $ 0.5 V The peak current intensity increased in proportion to increasing hCG concentration The analytical range and sensitivity of the immunosensor were calculated by extracting the current intensity as a function of the hCG concentration from Fig 5a The results are shown in Fig 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 measurements 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 microelectrode 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 electrochemical 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 concentration 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 Â 103 pg/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 SWCNTmodified SPCEs are similar with those described above for the SWCNT microelectrode The LOD of GLEIA on SPCEs and SWCNTmodified 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 microelectrode with the ability to enhance electrochemical signals, reduce nonspecific binding, and effectively detect the signals directly from Au nanoparticles The performance of GLEIA on SWCNTmodified 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 SWCNTmodified 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 asgrown SWCNTs (Zhang et al., 2004) 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 containing 1% BSA and in male urine solution, correspondingly 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 at http://dx.doi.org/10.1016/j.bios.2012.11.017 References Bakker, E., 2004 Analytical Chemistry 76 (12), 3285–3298 Bard, 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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. .. solution to make different concentration For the detection of the antibody–antigen reaction, 2.0 mL of the antigen solution was placed on a group of SWCNT microelectrodes for h at room temperature... 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