Article pubs.acs.org/ac
Increased Electrocatalyzed Performance through DendrimerEncapsulated Gold Nanoparticles and Carbon Nanotube-Assisted Multiple Bienzymatic Labels: Highly Sensitive Electrochemical Immunosensor for Protein Detection Bongjin Jeong,†,∥ Rashida Akter,‡,∥ Oc Hee Han,†,§ Choong Kyun Rhee,†,‡ and Md. Aminur Rahman*,† †
Graduate School of Analytical Science and Technology (GRAST) and ‡Department of Chemistry, Chungnam National University, Daejeon 305-764, South Korea § Analysis Research Division, Daegu Center, Korea Basic Science Institute, Daegu 702-701, South Korea ABSTRACT: A highly sensitive electrochemical carcinoembryonic antigen (CEA) immunosensor was fabricated by covalently immobilizing a monoclonal CEA antibody (anti-CEA, Ab1) and a mediator (thionine, Th) on a gold nanoparticle (AuNP)-encapsulated dendrimer (Den/AuNP). Multiwalled carbon nanotube (MWCNT)-supported secondary antibody (Ab2)-conjugated multiple bienzymes, glucose oxidase (GOx), and horseradish peroxidase (HRP) (Ab2/MWCNT/GOx/HRP) were used as electrochemical labels. The highly sensitive detection was achieved by the increased HRP-electrocatalyzed reduction of hydrogen peroxide, which was locally generated by the enzyme GOx. The immunosensor surface was characterized using electrochemical impedance spectroscopy, atomic force microscopy, and quartz crystal microbalance techniques. The Den/AuNP and Ab2/MWCNT/ GOx/HRP bioconjugates were characterized using high-resolution transmission electron microscopy, scanning electron microscopy, and X-ray photoelectron spectroscopy. Cyclic voltammetry and square wave voltammetry techniques were used to monitor the increased electrocatalyzed reduction of hydrogen peroxide by HRP. The linear dynamic range and the detection limit were determined to be 10.0 pg/mL to 50.0 ng/mL and 4.4 ± 0.1 pg/mL, respectively. The validity of the immunosensor response was tested in various CEA-spiked human serum samples, and the results were compared to those of an enzyme-linked immunosorbent assay method.
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Dendritic nanostructures such as dendrimers9−11 are synthetic highly branched polymers that show promise in several biomedical applications.12 The unique properties of dendrimers, such as structural homogeneity, integrity, controlled composition, and multiple functional surface terminal groups available for subsequent conjugation reactions expand their use as building blocks for nanostructures13−15 and as promising platforms for the fabrication of biosensors with increased sensitivity.16−20 However, as the conductivity of the dendrimer is low, a conducting nanomaterial such as gold nanoparticles (AuNPs) needs to be encapsulated into the interior of the dendrimer (Den/AuNPs)21 for accelerating the electron transfer process. Den/AuNP nanocomposites could be an excellent immunosensor platform because of the combination of physical and chemical properties of AuNPs and the surface reactivity of dendrimers for immobilizing a large amount of antibodies and mediators. The application of Den/AuNP as a sensor platform not only increases the amount of immobilized antibodies and mediator, but also accelerates the electron transfer process assisted by the encapsulated
he development of highly sensitive protein detection based on antigen−antibody binding plays a key role in the prognostic treatment of human diseases. Generally, protein biomarkers have been detected using conventional immunoassays.1 However, conventional immunoassays are complicated, time-consuming, tedious, expensive, labor-intensive, and not suitable for point-of-care applications. As an alternative to the conventional immunoassay procedures, the development of electrochemical immunosensors2,3 has shown great promise because of simple instrumentation, high sensitivity, fast response time, miniaturization, low cost, and point-of-care applications. Thus, it has received particular attention for the detection of proteins in clinical applications.4−7 However, improvement of sensitivity and minimization of nonspecific binding (NSB) are crucial for the successful clinical applications. Sensitivity can be improved by increasing the amount of immobilized antibodies and labels and using a suitable signal amplification strategy. On the other hand, the NSB effect can be minimized by using a negatively charged surface and an inert surface-passivating reagent.8 Dendritic nanostructures with negative surface charge can be used as nanocarriers for immobilizing an increasing amount of antibodies and labels and for minimizing the NSB event in highly sensitive immunosensor development. © 2013 American Chemical Society
Received: October 28, 2012 Accepted: January 4, 2013 Published: January 4, 2013 1784
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labels have been used for the amplified detection of biomolecules.29−31 However, antibody- and mediator-coimmobilized Den/AuNP (Den/AuNP/Ab/Th) as a capture probe and MWCNT-supported multiple bienzymatic label (MWCNT/GOx/HRP) as a detection probe has not yet been studied for the ultrasensitive detection of a cancer biomarker. Here, we report the DEN/AuNP- and multiple MWCNT/ GOx/HRP-based increased electrocatalyzed hydrogen peroxide reduction strategy for the highly sensitive detection of CEA. The Den/AuNPs and MWCNT/GOx/HRP have been characterized using transmission electron microscopy (TEM), scanning electron microscopy (SEM), and X-ray photoelectron microscopy (XPS). The immunosensor platform has been characterized using the atomic force microscopy (AFM), quartz crystal microbalance (QCM), and electrochemical impedance spectroscopy (EIS) techniques. Various experimental parameters have been optimized for CEA detection and selectivity, stability, and regeneration tests have been performed. The proposed CEA immunosensor has been applied in human serum samples and compared with a conventional enzymelinked immunosorbent assay (ELISA) method.
AuNPs for signal enhancement. There have been some reports on the application of Den/AuNP nanocomposites as sensor platforms in biosensor development.22,23 However, the sensitivities of those biosensors are not good and need to be increased. To further increase the detection sensitivity, nanomaterial-supported multiple labels can be used. Various nanomaterials such as magnetic nanoparticles,2 multiwalled carbon nanotubes (MWCNTs),5,6 silica nanoparticles,19 carbon spheres,24 etc. have been used as nanocarriers for loading multiple horseradish peroxidase (HRP) labels. However, MWCNT-assisted multiple bienzymatic glucose oxidase (GOx) and HRP labels (MWCNT/GOx/HRP) have not been reported yet. The combination of Den/AuNP as a sensor platform and MWCNT/GOx/HRP as multiple bienzymatic labels can be a promising amplification strategy for highly sensitive cancer biomarker detection. In the present work, a DEN/AuNP- and MWCNT/GOx/ HRP-based amplification strategy using enhanced amounts of anti-CEA and mediator, the electron promoting ability of DEN/AuNPs, and MWCNT-assisted multiple bienzymatic labels has been demonstrated for the development of a carcinoembryonic antigen (CEA) protein immunosensor. CEA has been associated with colorectal cancer and also identified as a biomarker for breast tumors and ovarian carcinoma.25 The normal physiological level of CEA is about 2.5−5.0 ng/mL26,27 and becomes elevated when inflammation or tumors arise.28 Thus, CEA detection plays an important role in clinical diagnostics. Figure 1 shows a schematic illustration of the proposed CEA immunosensor and its detection principle. CEA detection was based on the measurements of increased electrocatalytic reduction of GOx-generated hydrogen peroxide by the multiple HRP labels through the mediating ability of the loaded multiple thionine (Th). Previously, Th- and HRP-based
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EXPERIMENTAL SECTION Reagents. A third-generation (G3) poly(amidoamine) dendrimer (diameter ∼3.6 nm) with 32 surface-terminated succinamic acid groups [PAMAM(NHCOCH2CH2COOH)64], 2-mercaptoethanol, thionine, and sodium borohydride was obtained from Aldrich Co. Cysteamine, N-hydroxysuccinimide (NHS), 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide (EDC), β-glucose, sodium chloride, sodium citrate, glycine, hydrogen tetrachloroaurate (HAuCl4·H2O), hydrogen peroxide, sulfuric acid, nitric acid, glucose oxidase (GOx; Aspergillus niger), bovine serum albumin (produced in mouse), human serum sample, carcinoembryonic antigen, and monoclonal antiCEA antibody (produced in mouse) were obtained from Sigma Co. The MWCNTs were obtained from JEIO Co., Korea. The phosphate-buffered saline (PBS) solution was prepared by mixing 10 mM NaH2PO4 and 10 mM Na2HPO4 with 0.9% sodium chloride (NaCl). All other chemicals were of extrapure analytical grade and were used without further purification. All aqueous solutions were prepared with deionized distilled water obtained from a Milli-Q water-purifying system (18 MΩ cm). Instruments. Cyclic voltammograms and square wave voltammograms were recorded using a model 430B potentiostat/galvanostat (CH Instruments Inc., United States). In cyclic voltammetry (CV) and square wave voltammetry (SWV) experiments, Ag/AgCl (in saturated KCl) and a platinum (Pt) wire were used as reference and counter electrodes, respectively. SEM and TEM images were obtained using a scanning electron microscope (model JSM-7000F, JEOL, Japan) and high-resolution transmission electron microscope (JEM-2100, JEOL), respectively. XPS experiments were performed using a MultiLab 2000 (Thermo, United States). AFM images were taken by using XE-100 (Park Systems, Korea). Impedance spectra were recorded with a CHI 660D electrochemical workstation (CH Instruments Inc., United States). The frequency was scanned from 0.1 to 100 kHz at an open-circuit voltage with an ac voltage amplitude of 5 mV. QCM experiments were performed with a model 430B timeresolved electrochemical quartz crystal microbalance. An 8 MHz AT-cut quartz crystal (area 0.205 cm2) was used for the QCM experiments. The sensitivity factor was calculated
Figure 1. Schematic illustrations of the fabrication of the electrochemical CEA immunosensor and detection principle. 1785
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Figure 2. TEM image of (a) Den/AuNPs (the inset shows the high-magnification TEM image), (b) XPS analysis of (i) Den and (ii) Den/AuNPs, (c) SEM image of MWCNTs, and (d) SEM image of Ab2/MWCNT/GOx/HRP.
according to our previous report.5 Briefly, the mass change is related to the frequency change by the following relationship assuming that Δf is affected by only the mass change of the film:
of MWCNTs (hereafter designated as COOH−MWCNTs). Then, the COOH groups of MWCNTs were activated by treatment with 10 mM NHS/EDC for 6 h. Monoclonal antiCEA antibody Ab2 (5.0 μg/mL), GOx (1.0 mg/mL), and HRP (1.0 mg/mL) in 0.1 M PBS were added to the activated COOH−MWCNTs and stirred for 24 h at 4 °C. By this step, Ab2, GOx, and HRP were covalently bonded to the COOH− MWCNTs through the formation of the amide bond between the COOH groups of COOH−MWCNTs and NH2 groups of Ab2, GOx, and HRP. Fabrication of the Immunosensor Probe and the Sensing Principle. At first, Au/cysteamine (Cys) electrodes were prepared by dipping a Au electrode for 18 h in a 10 mM aqueous Cys solution. Prior to the modification, polycrystalline gold electrodes (3 mm in diameter) were polished with a 0.05 μm alumina/water slurry on a polishing cloth to a mirror finish, followed by sonication and rinsing with distilled water. Then the polished electrodes were rinsed with a fresh piranha solution (70% H2SO4, 30% H2O2). Caution: piranha solution reacts violently with most organic materials and must be handled with extreme care! This was followed by cycling the potential between +1.4 and −0.2 V at 100 mV/s 50 times in a 1.0 M H2SO4 solution. The Au/Cys electrode was then treated with mercaptoethanol for blocking the remaining empty sites of the Au electrode. The activated Den or Den/AuNP was chemically bonded to the Au/Cys through the formation of amide bonds between the NH2 groups of Cys and COOH groups of Den or Den/AuNP. The Au/Cys/Den- and Au/Cys/Den/AuNPmodified electrodes were rinsed with water and dried with nitrogen gas. Then monoclonal anti-CEA antibody Ab1 and Th were coimmobilized onto the Au/Cys/Den- and Au/Cys/Den/ AuNP-modified electrodes by incubating the electrodes in a 0.1 M PBS solution (pH 7.0) containing 0.1 mg/mL Ab1 and 1
Δm = −C Δf
where C is the sensitivity or conversion factor, Δf is the frequency change obtained from the QCM experiments, and Δm is the mass change calculated by using the area of the cathodic peak of voltammetric deposition of copper and Faraday’s law. From the slope of the Δm vs Δf plot, the sensitivity factor was calculated as 2.5 ng/Hz. Preparations of Den/AuNPs and Ab2/MWCNT/GOx/ HRP. The Den/AuNP was synthesized according to our previous report22 with a slight modification. Briefly, COOH group functionalized Den was reacted with an aqueous 0.1 M HAuCl4 solution for 1 h with stirring. By this step, Au ions were coordinated to the nitrogen ligand in the interior of the Den (Den−Au(III) ions). The Den/AuNP was obtained by reducing the Au(III) ions in the interior of Den with a 1.0 M NaBH4 solution for 20 min. The Den/AuNP was separated from the free Den and NaBH4 by centrifugation and was treated with mercaptoethanol. The COOH groups of the Den/ AuNP were then activated by treating the Den/AuNP with 10 mM NHS/EDC for 6 h. The activated Den/AuNP was separated from the free NHS/EDC by centrifugation. For preparing the Ab2/MWCNT/GOx/HRP bioconjugates, MWCNTs were first shortened through chemical oxidation by treating MWCNTs in a 3:1 (v/v) mixture of sulfuric and nitric acids at 60 °C for 12 h.5 The shortened MWCNTs were filtered and washed repeatedly with water until the pH reached 7.0. A shortening process removed metallic and carbonaceous impurities and generated carboxylic acid groups at the surfaces 1786
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mM Th for 24 h at 4 °C. After being washed three times with 0.1 M PBS, the Au/Cys/Den/Ab1/Th and Au/Cys/Den/ AuNP/Ab1/Th electrodes (immunosensors) were blocked by dipping them in 0.1% BSA solution for 2 h at 4 °C. After being washed three or four times with PBS and dried with N2, the blocked immunosensors were incubated with various concentrations of CEA antigen (Ag) at 4 °C. After the immunointeraction between Ab1 and Ag, the immunosensors were immersed into Ab2/MWCNT/GOx/HRP bioconjugates for 2 h for attaching the bioconjugates followed by washing three or four times with PBS to remove nonspecifically bound conjugates. The immunosensors were dipped in a 0.1 M PBS (pH 7.0) solution containing an optimized amount of glucose. Multiple HRP-catalyzed Th-mediated electroreduction of H2O2, which was locally generated by GOx, was then measured using the CV and SWV techniques.
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RESULTS AND DISCUSSION Characterization of the Den/AuNPs and Ab1/MWCNT/ GOx/HRP. The Den/AuNP was characterized using the fieldemission TEM and XPS techniques. Figure 2a shows an HRTEM image obtained for a Den/AuNP-modified surface. The inset of Figure 2a clearly shows that AuNPs were efficiently formed in the interior of the Den without any aggregation. The average particle size of AuNPs in the interior of the Den was determined to be 2.0 ± 0.6 nm. Figure 2b shows the XPS spectra obtained for (i) Den- and (ii) Den/ AuNP-modified surfaces. The survey spectrum obtained for the Den showed C1s, N1s, and O1s peaks. The two C1s peaks observed at 284.8 and 286.0 eV correspond to C−H, C−S, or C−C bonds and CO bonds, respectively. The N1s peak observed at 400.8 eV is due to the presence of NH groups. The O1s peaks observed at 532.3 and 533.2 eV are due to the presence of the surface carboxylic acid groups of the Den. The survey spectrum obtained for the Den/AuNPs showed two sharp peaks at 83.50 and 87.3 eV (inset of Figure 2b) along with other C1s, N1s, and O1s peaks. The peaks at 83.50 and 87.3 eV correspond to Au4f7 and Au4f5, respectively, which were not observed in the spectrum of only the Den. Furthermore, the N1s peak at 400.8 eV (for NH groups) shifted to a higher energy of 401.9 eV. These results clearly indicate that AuNPs were encapsulated in the interior of the Den. The Ab2/MWCNT/GOx/HRP bioconjugate was characterized using SEM experiments. Parts c and d of Figure 2 show the SEM images obtained for COOH−MWCNT and Ab2/ MWCNT/GOx/HRP surfaces, respectively. The diameter of the COOH−MWCNT before bioconjugation was 30 ± 5 nm, whereas it increased to 44 ± 3 nm after Ab2, HRP, and GOx bioconjugation. The 14 nm increase in diameter of the COOH−MWCNT after bioconjugation clearly indicates that Ab2, HRP, and GOx were attached to the COOH−MWCNT. Characterization of the Immunosensor Probe. The EIS, AFM, and QCM techniques were performed for the characterization of the immunosensor probe. The EIS32 technique provides information on the surface conductivity of various modification steps. Figure 3a shows the impedance changes observed for the Au/Cys-, Au/Cys/Den-, and Au/ Cys/Den/AuNP-modified electrodes recorded in a Fe(CN)63−/4− solution. The inset of Figure 3a shows a general equivalent circuit that can be used to model the impedance spectra of the present system. The equivalent circuit contains the solution resistance (Rs), the charge transfer resistance (Ret),
Figure 3. (a) EIS analyses of (i) Au/Cys, (ii) Au/Cys/Den, and (iii) Au/Cys/Den/AuNPs. The inset shows the general equivalent circuit. (b) AFM images obtained for (i) Au/Cys-, (ii) Au/Cys/Den-, and (iii) Au/Cys/Den/AuNP-modified surfaces. (c) QCM analyses of the frequency changes during (i) formation of the Cys monolayer, (ii) covalent attachment of Den/AuNPs on the Cys monolayer, (iii) covalent immobilization of a monoclonal anti-CEA antibody (Ab1), and (iv) interaction between anti-CEA and CEA.
the Warburg element (W), and the charge of the constant phase element (Qdl). The Ret value, which exhibits the charge transfer kinetics of the Fe(CN)63−/4− redox system can be determined by fitting the experimental data to the model circuit. The Ret can also be estimated from the diameter of the semicircle part at higher frequencies in the Nyquist plot. After the covalent attachment of the Den on the Au/Cys, the charge transfer resistance increased from 2.05 to 5.6 kΩ due to the low conductivity of the Den. However, the resistance decreased from 5.6 to 3.0 kΩ when Den/AuNP was covalently attached on the Au/Cys. This means that the encapsulated AuNPs in the interior of the Den increased the conductivity of the sensor surface and accelerated the electron transfer process of Fe(CN)63−/4−. AFM images (Figure 3b) obtained for the (ii) Au/Cys/Den- and (iii) Au/Cys/Den/AuNP-modified surfaces clearly show the homogeneous and supramolecular nanostructure of the Den and Den/AuNPs, respectively. Figure 3c shows the frequency−time responses obtained during the formation of a self-assembled monolayer (SAM) of Cys (i), covalent binding of Den/AuNPs (ii), and covalent immobilization of Ab1 (iii). During the formation of the Cys self-assembled monolayer, the frequency decreased gradually 1787
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and reached a steady state within 2 h, indicating that the monolayer formation was completed within 2 h. The average frequency change (Δf) was about 66 Hz, which corresponds to an average mass change (Δm) of 165 ng by using a sensitivity factor of 2.5.5 During the immobilization of the Den/AuNPs, the frequency decrease also attained a steady state within 2 h, and the Δf and Δm were determined to be 140 Hz and 350 ng, respectively. During the immobilization of Ab1 onto the Cys/ Den/AuNP electrode, the frequency response gradually decreased and reached a steady state. The average Δf was found to be about 92 Hz corresponding to a Δm of 230 ng. Assuming the the anti-CEA antibody has a similar molecular weight of anti-immunoglobulin G (IgG) antibodies (150 000), the surface coverage of Ab1 was calculated as 1.53 × 10−12 mol/ cm2. On the basis of the hydrodynamic diameter of the anti-IgG (∼10 nm), the theoretical surface coverage for a monolayer of anti-IgG was calculated as 4.15 × 10−13 mol/cm2. The experimental surface coverage obtained from the QCM experiment is about 4-fold higher than that of the theoretical value. The ∼4-fold enhancement of the Ab1 immobilization was due to the increased surface area and surface reactive groups of the dendrimer bound to the Au/Cys surface. From the experimental and theoretical surface coverage values, the surface coverage was determined to be 368%, indicating a greater Ab1 immobilization efficiency of the bound dendrimer. Additionally, the interaction between Ab1 and Ag was also studied using the QCM technique as shown in Figure 3a, curve iv. During the interaction between Ab1 and Ag, the Δf decreased to a steady state within 2 h, indicating the completion of Ab1−Ag interaction. The average Δf was 130.4 Hz, which corresponds to an average Δm of 326 ng. The surface coverage of Ag was calculated as 1.63 × 10−12 mol/cm2. The surface coverage of CEA was greater than that of anti-CEA, which might be due to the bivalent binding sites of the antibody. The QCM results on Δf and Δm changes confirmed the successful formation of a Cys SAM on Au, stable covalent attachment of Den/AuNPs on Au/Cys, and covalent immobilization of Ab1 on the Au/Cys/Den/AuNP electrode. The above EIS, AFM, and QCM results clearly indicate that the Den or Den/AuNPs was covalently attached on the Au/Cysmodified surface and anti-CEA antibody was covalently immobilized on the Au/Cys/Den- or Au/Cys/Den/AuNPmodified electrode. Electrochemical Characteristics of the CEA Immunosensor. Figure 4a shows the CV responses recorded for Au/ Cys/Den/Th-based (i, iii) and Au/Cys/Den/AuNP/Th-based (ii, iv) CEA immunosensors in a 0.1 M PBS solution without (i, ii) or with (iii, iv) glucose (1.0 mM). In the absence of glucose, the cyclic voltammogram for Au/Cys/Den/AuNPs/Th-based CEA immunosensors (ii) showed a well-defined redox peak at −0.15/−0.19 V, which was due to the mediated electron transfer of the heme group in HRP by Th. When the cyclic voltammogram was recorded for Au/Cys/Den/Th-based CEA immunosensors (i), the redox peak at −0.15/−0.19 V was very small, indicating that the encapsulated AuNPs in the interior of the Den promoted the electron transfer (ET) ability of the Thmediated heme ET process. The peak currents were directly proportional to the scan rate up to 0.4 V/s (Figure 4b), indicating that the electrode reactions were involved in the surface-confined process.33 The peak separation of the redox couple slightly increased with an increase in scan rate to 0.4 V/ s, indicating that the redox reaction was quasi-reversible. In the presence of glucose, the catalytic characteristics observed with a
Figure 4. (a) CV responses recorded for Au/Cys/Den/Th-based (i, iii) and Au/Cys/Den/AuNPs/Th-based (ii, iv) CEA immunosensors in a 0.1 M PBS solution without (i, ii) or with (iii, iv) 1.0 mM glucose, (b) redox peak currents vs scan rate dependence, and (c) SWV responses recorded for a Au/Cys/Den/AuNPs/Th-based CEA immunosensor in a 0.1 M PBS solution (i) and in a 1.0 mM glucose solution with (ii) Au/Cys/Den/Th- and (iii) Au/Cys/Den/AuNP/ Th-based CEA immunosensors.
sharp decrease of the oxidation current and dramatic increase of the reduction peak for both Au/Cys/Den/Th-based (iii) and Au/Cys/Den/AuNP/Th-based (iv) CEA immunosensors were due to the Th-mediated electrocatalytic reduction of enzymatically generated H2O2 by the enzyme GOx. The in situ generation of H2O2 by the immobilized GOx overcomes the diffusion limitation of the H2O2 by bringing it directly onto the detection probe34 and thus is expected to increase the sensitivity. The electrocatalytic reduction current observed for the Au/Cys/Den/AuNP/Th-based sensor was much higher than that observed for a Au/Cys/Den/Th-based CEA immunosensor, indicating that the encapsulated AuNPs in the interior of the Den enhanced the electrocatalytic performance by promoting the ET process. Figure 4c shows the square wave voltammograms recorded for a Au/Cys/Den/AuNP/Th-based CEA immunosensor in a 0.1 M PBS solution (i) and a PBS solution containing 1.0 mM glucose (ii, iii). The square wave voltammograms in a PBS solution showed a cathodic peak at −0.16 V for the reduction of the Th-mediated heme ET process. In the presence of glucose, the cathodic current increased through the electrocatalytic reduction of the in situ generated H2O2. However, the cathodic peak at −0.16 V was much higher for (iii) Au/Cys/ 1788
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higher than 7.5. The maximum reduction current was observed at a pH of 7.5. Thus, the optimum pH was chosen as 7.5. Detection of CEA with the Immunosensor. Under the optimized experimental conditions, the square wave (SW) electrocatalytic current responses were measured by varying the CEA concentration with the Au/Cys/Den/AuNP/Th-based sensor as shown in Figure 6a. Figure 6b shows the background-
Den/AuNP/Th- than (ii) Au/Cys/Den/Th-based sensors. Both the CV and SWV studies clearly revealed that the immunosensor response could be enhanced by using encapsulated AuNPs in the interior of the Den. To maximize the immunosensor response, various experimental parameters such as the Ab1 antibody dilution factor, Ab1−Ag interaction time, pH of the Ab1−Ag binding medium, etc. for the detection of CEA with Au/Cys/Den/AuNP/Thbased sensor were optimized. The effect of the Ab1 dilution factor on the electrocatalytic reduction of hydrogen peroxide was examined between 1:10000 and 1:100 (Figure 5a). The
Figure 6. (a) SWV responses recorded for Au/Cys/Den/AuNP/Thbased CEA immunosensors at (i) blank noise and at various CEA concentrations: (ii) 10 pg/mL, (iii) 50 pg/mL, (iv) 100 pg/mL, (v) 0.5 ng/mL, (vi) 1.0 ng/mL, (vii) 5.0 ng/mL, (viii) 10.0 ng/mL, (ix) 50.0 ng/mL, (x) 0.1 μg/mL, (xi) 0.5 μg/mL, and (xii) 1.0 μg/mL. (b) Background-subtracted SWV responses and (c) corresponding calibration plot.
Figure 5. Effects of (a) anti-CEA antibody dilution, (b) the interaction between the anti-CEA antibody and CEA, and (c) the pH of the CEA antigen solution during anti-CEA and CEA interaction.
electrocatalytic current response gradually increased from 1:10000 to 1: 2500 and rapidly increased from 1:2500 to 1:1000. Over a dilution factor of 1:1000, the current response did not significantly increase. Thus, the Ab1 antibody dilution factor was optimized to be 1:1000. The effect of the Ab1 antibody and Ag interaction time was investigated between 20 and 60 min (Figure 5b). The current response gradually increased from 20 to 30 min. Over 35 min of interaction time, the response steadily decreased. The maximum response was found at an interaction time of 35 min. Thus, the optimum interaction time was considered as 35 min. The effect of the pH of the Ab1−Ag binding medium was studied over the pH range of 5−9 (Figure 5c). The current response gradually increased from pH 5 to pH 7.5 and then rapidly decreased at pH values
subtracted SW responses, where the electrocatalytic reduction current responses of H2O2 were linearly proportional to the logarithm of the CEA concentration. Figure 6c shows the calibration plot constructed by plotting the current responses against the logarithm of the CEA concentration. The Au/Cys/ Den/AuNP/Th-based immunosensor exhibits a wide dynamic range between 10.0 pg/mL and 50.0 ng/mL. The reproducibility expressed in terms of the relative standard deviation (RSD) was about 6.3% (n = 5) at a CEA concentration of 1.0 ng/mL. The detection limit of CEA was determined to be 4.4 ± 0.1 pg/mL, which was based on three measurements of the standard deviation of the blank noise (95% confidence level, k = 3, n = 5). The observed dynamic range was much wider than 1789
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Table 1. Comparison of CEA Detection between the Proposed Immunosensor and ELISA Methods in CEA-Spiked Human Serum Samples CEA concn found (ng/mL) CEA concn added (ng/mL) 1 5 10 20
immunosensor 0.97 4.95 10.10 19.85
± ± ± ±
0.03 0.19 0.50 1.10
RSD (%)
recovery (%)
ELISA
immunosensor
ELISA
immunosensor
ELISA
± ± ± ±
3.1 3.8 5.0 5.5
6.8 3.0 7.8 5.5
97.0 99.0 101.0 99.20
103.0 99.0 101.0 99.3
1.03 4.95 10.25 20.5
0.07 0.15 0.8 1.2
After being washed with PBS, the immunosensor surface was successively incubated in a PBS solution containing 1.0 ng/mL CEA and Ab2/MWCNT/GOx/HRP bioconjugate solution and was stored in a dry condition at 4 °C. When the immunosensor was stored in dry conditions at 4 °C, the initial current response was not found to be significantly changed for a three week period. However, after three weeks, the immunosensor response gradually decreased due to the deactivation of the GOx and HRP in the bioconjugates. Real Sample Analysis. The proposed CEA immunosensor was applied in CEA-spiked human serum samples for the detection of CEA. The standard addition method was used to determine the CEA concentration. Table 1 shows the recovery results, which show an acceptable accuracy, with RSD values ranging between 3.1% and 5.5%. The CEA recovery was between 97.0% and 99.20%, which clearly indicated the potentiality of the proposed CEA immunosensor for CEA detection in real human serum samples. The validity of the immunosensor was evaluated by detecting the same CEAspiked samples using a conventional ELISA method. The results were presented in Table 1. Figure 7b shows a linear correlation plot between the proposed immunosensor and ELISA methods. Good agreement between immunosensor and ELISA methods was obtained, indicating the applicability of the proposed CEA immunosensor in real biological samples.
those of a nano-Au/multiwalled carbon nanotube/chitosan nanocomposite-based (0.3−2.5 and 2.5−20 ng/mL),35 HRP/ anti-CEA/nanogold/chitosan-based (0.5−25 ng/mL),36 and anti-CEA/glutathione//nanogold/poly(o-aminophenol)/Aubased (0.5−20 ng/mL)37 electrochemical immunosensors. The detection limit was also lower than those of HRP/anti-CEA/ nanogold/chitosan-based (0.22 ng/mL),36 anti-CEA/glutathione//nanogold/poly(o-aminophenol)/Au-based (0.1 ng/ mL),37 thionine-doped magnetic gold nanosphere-based (0.01 ng/mL),38 and anti-CEA/GA/GPMS/Fe3O4/SiO2/CPE-based (0.5 ng/mL)39 electrochemical immunosensors. Selectivity and Stability of Immunosensors. To assess the selectivity of the immunosensor, the SWV responses of the electrocatalytic reduction of H2O2 were measured for other common proteins such as prostate-specific antigen (PSA), human IgG, interleukin-6 (IL-6), human thrombin (TB), and human serum albumin (HSA). Figure 7a shows the current
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CONCLUSIONS A highly sensitive electrochemical immunosensor based on Den/AuNP as a sensor platform and MWCNT-supported multiple bienzymes (Ab2/MWCNT/GOx/HRP) as labels was developed for CEA biomarker detection. HRP-electrocatalyzed Th-mediated H2O2 reduction was monitored for the detection of CEA. The encapsulation of AuNPs in the interior of the Den increased the conductivity of the sensor probe and accelerated the electron transfer reaction and thus enhanced the immunosensor’s response. Also, the local generation of H2O2 by GOx overcame the diffusion limitation of the H2O2 by bringing it directly onto the detection probe and thus further increased the sensitivity. The linear dynamic range of the proposed immunosensor covered a 5-order wide concentration range in which CEA detection can be made without dilution. Also, the detection limit of the proposed CEA immunosensor was much lower than that of the conventional ELISA method. Importantly, the proposed immunosensor can be applied in biological samples and thus could be a valuable tool for clinical cancer diagnostics.
Figure 7. (a) SWV responses of Au/Cys/Den/AuNP/Th-based CEA immunosensors to other proteins and (b) the correlation response plot between the immunosensor and ELISA methods.
responses measured with the CEA immunosensor toward a 1.0 ng/mL concentration of various proteins. The proposed CEA immunosensor did not show any significant responses for other proteins, indicating the above proteins did not interfere in the CEA detection; i.e., it was very specific for CEA. The stability of the proposed CEA immunosensor was checked by measuring the response of 1.0 ng/mL CEA for one month. After each measurement, the immunosensor surface was regenerated by dipping it into a 0.2 M glycine− hydrochloric acid (Gly−HCl) solution (pH 2.8) for 5 min.
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AUTHOR INFORMATION
Corresponding Author
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[email protected]. 1790
dx.doi.org/10.1021/ac303142e | Anal. Chem. 2013, 85, 1784−1791
Analytical Chemistry
Article
Author Contributions ∥
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B.J. and R.A. contributed equally to this work.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This research was supported by the Research Fund of Chungnam National University in 2011 (Grant 2011-0716), the Basic Research Program for a regional university (20120742) funded by the National Research Foundation, Korean Government (MEST), and the 2012-University-Institute cooperation program funded by the National Research Foundation of the Korean Government (MEST).
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