Highly Specific and Ultrasensitive Graphene-Enhanced

Feb 12, 2013 - Hyojeong Han , Jae-Chul Pyun , Hyein Yoo , Hong Seog Seo , Byung Hwa Jung , Young Sook Yoo , Kyoungja Woo , and Min-Jung Kang...
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Highly Specific and Ultrasensitive Graphene-Enhanced Electrochemical Detection of Low-Abundance Tumor Cells Using Silica Nanoparticles Coated with Antibody-Conjugated Quantum Dots Yafeng Wu,† Peng Xue,† Yuejun Kang,*,† and Kam M. Hui*,‡ †

School of Chemical and Biomedical Engineering, Nanyang Technological University, 70 Nanyang Drive, Singapore 637457, Singapore ‡ Division of Cellular and Molecular Research, National Cancer Centre, 11 Hospital Drive, Singapore 169610, Singapore S Supporting Information *

ABSTRACT: A dual signal amplification immunosensing strategy that offers high sensitivity and specificity for the detection of low-abundance tumor cells was designed. High sensitivity was achieved by using graphene to modify the immunosensor surface to accelerate electron transfer and quantum dot (QD)-coated silica nanoparticles as tracing tags. High specificity was further obtained by the simultaneous measurement of two disease-specific biomarkers on the cell surface using different QD-coated silica nanoparticle tracers. The immunosensor was constructed by covalently immobilized capture antibodies on a chitosan/electrochemically reduced graphene oxide film-modified glass carbon electrode. Cells were captured with a sandwich-type immunoreaction and the different QD-coated silica nanoparticle tracers were captured on the surface of the cells. Each biorecognition event yields a distinct voltammetric peak, which position and size reflects the corresponding identity and amount of the respective antigen. This strategy was vividly demonstrated by the simultaneous immunoassay of EpCAM and GPC3 antigens on the surface of the human liver cancer cell line Hep3B using anti-EpCAM-CdTeand anti-GPC3-ZnSe-coated silica nanoparticle tracers. The two tracers gave comparable sensitivity, and the immunosensor exhibited high sensitivity and specificity with excellent stability, reproducibility, and accuracy, indicating its wide range of potential applications in clinical and molecular diagnostics.

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characteristics of CTCs, including polymerase chain reaction (PCR)-based analysis, cytometric analysis, and cell-enrichment steps to boost sensitivity.9 Although each of these methods represents significant technological advances and provides a basis from which to anticipate ongoing technological developments, none of these current approaches constitute the optimal platform for CTC isolation.10 Moreover, with few exceptions, almost all these methods are assays relating to the enumeration of CTC and remain limited by the inability to elute the captured cells for further molecular analysis. However, it is the molecular characterization of CTCs that offers confirmation and potential insights into the metastasis process. The goal is, therefore, to develop alternative quantitative, sensitive, and specific detection methods for CTCs that enable the elution of the bound tumor cells. Great efforts have been made toward the signal amplification to significantly enhance the sensitivity for biomarker detection, including the employment of new redox-active probes, the

etastasis is the primary cause of mortality in most cancer patients and is initiated by cancer cells that are shed and transported through the circulation from the primary tumor to distant organs. It has been estimated, using an experimental rat model system, that as many as 3−4 × 106 mammary carcinoma cells can be shed into the circulation from a gram primary tumor each day,1,2 though few of these ever form metastases. It holds, nevertheless, that the number of metastases formed is generally proportional to the number of tumor cells shed into circulation,3 and it therefore has been suggested that circulating tumor cells (CTC) can be used as a noninvasive measure to monitor treatment efficacy and disease progression or recurrence.4−8 However, even if one assumes that there is no clearance of the tumor cells shed into circulation, one would have a maximum estimation of 200 tumor cells mL−1 of blood for an average individual with approximately 5 L of blood. This concentration equates to about 0.004% of all white cells in the blood and, hence, poses key technical challenges for the detection and quantification of these low-abundance CTCs due to the high sensitivity and specificity needed for these methodologies. Currently employed strategies rely on the physical properties, expression of biomarkers, or the functional © 2013 American Chemical Society

Received: November 23, 2012 Accepted: February 12, 2013 Published: February 12, 2013 3166

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integration of enzyme-assisted signal amplification processes, and the incorporation of nanomaterials to increase the upload of electrochemical tags, etc.11−15 The latter approach is particularly effective by introducing multiple redox species per binding event. Silica nanoparticles (SiO2) are the most commonly used nanoparticles in electrochemical immunosensors, due to its unique features of monodispersed size, uniform structure, good biocompatibility, and easy functionalization.16,17 QD-based electrochemical bioassay has received particular attention because of inherent miniaturization, low detection limits, low cost, and low power requirement.18,19 The high fluorescence quantum yield (QY) and high optical stability of QDs in comparison with other alternatives, such as organic fluorescent molecules and phosphorescent dyes, also make them highly promising nanomaterials for photovoltaic cells,20,21 biosensors,22,23 and light-emitting diodes.24,25 Chen et al. has recently described the use of CdTe QDcoated SiO2 as a signal amplification method to detect the latent membrane protein 1 (LMP-1).26 Anti-LMP-1/QDcoated SiO2 nanoparticles were used as a label, which were introduced to the electrode surface through two immunoreactions. A detection limit of 1 pg mL−1 was achieved, and the signal was amplified 2.8 times compared with the traditional immunoassay. We have also proposed an electrochemical immunosensor based on CdSe and PbS QD-coated SiO2, as dual labels for IgG and CEA determination.27 These labels were introduced to the surface of gold substrates through a subsequent sandwich immunoreaction, which allowed the simultaneous detection of IgG and CEA. The lowest detectable concentration achieved was 3 pg mL−1 for IgG and 5 pg mL−1 for CEA, yielding a higher sensitivity than most of the traditional sandwich immunoassay.28 In this paper, for the first time, we reported the combination of quantum dot-coated silica nanoparticles with grapheneaccelerated electron transfer to develop a dual signal amplification strategy, producing an ultrasensitive and highly specific electrochemical and fluorescent protocol for the simultaneous measurement of two different types of protein biomarkers on the surface of tumor cells. CdTe- and ZnSecoated silica nanoparticles, having uniform size distribution and good stability, could easily serve as tracing tags to label antiEpCAM and anti-GPC3 (Figure 1A). The graphene was immobilized on an immunosensor surface using chitosan and electrochemically reduced to accelerate the electron transfer. The amino group on the chitosan film enabled the covalent binding of the capture antibody (Figure 1B). After a sandwichtype immunoreaction, the two tracers were coimmobilized on the cell surface. Additionally, the binding between antibodies and tumor cells is noncovalent and thus offers the potential to elute the bound tumor cells. The present design enabled detection of at least 10 cells mL−1 and exhibited good stability, precision, and accuracy, demonstrating its wide range of potential applications for clinical and molecular diagnostics.

Figure 1. Pictorial representation of (A) the preparation of Si/Zn/ anti-GPC3, Si/Cd1/anti-EpCAM, and Si/Cd2/anti-GPC3 and (B) the fluorescent and electrochemical detection procedures of circulating tumor cells.

received. The phosphate buffer solution (PBS) was prepared by mixing NaH2PO4 and Na2HPO4. Doubly distilled water was used throughout the study. Apparatus. Square-wave voltammetric (SWV) stripping measurements were performed with a CHI 660C electrochemical workstation (CH Instrument Company, Shanghai, China). A conventional three-electrode system consisting of a modified bismuth film-modified glassy carbon electrode (BFE), a platinum wire, and a saturated calomel electrode (SCE) were used as the working, auxiliary, and reference electrodes in the electrochemical measurements, respectively. The morphology of the bare silica nanoparticles, CdTe QDcoated silica nanoparticles (Si/Cd), and ZnSe QD-coated silica (Si/Zn) nanoparticles were analyzed with a transmission electron microscope (TEM, S-2400N, HITACHI, Japan). X-ray photoelectron spectroscopy (XPS) was performed with ESCALAB 250 Multitechnique Surface Analysis system (Thermo Electron Company). Phase contrast and fluorescent images were acquired by Nikon inverted microscopy (ECLIPSE TE-2000U, Nikon, Japan) equipped with a video camera (DS-U1, Nikon, Japan). Cell Culture. Hep3B, MCF-7, PP5 cells were cultured in RPMI 1640 medium (GIBCO) supplemented with 10% fetal calf serum (FCS, Sigma), penicillin (100 μg mL−1), and streptomycin (100 μg mL−1) at 37 °C in a humidified atmosphere containing 5% CO2. Cells in the exponential growth stage were harvested by centrifugation at 1000 rpm for 5 min, washed thrice with sterile 0.01 M PBS, pH 7.4, and the

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EXPERIMENTAL SECTION Reagents. Anti-EpCAM antibody and anti-GPC3 were obtained from Meridian Life Science Inc. (Memphis, TN). 1Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), (3-aminopropyl)-triethoxysilane (APTS), acetic acid (HAc), sodium acetate (NaAc), bismuth nitrate pentahydrate, and bovine serum albumin (BSA) were purchased from Sigma-Aldrich (St. Louis, MO). All other chemicals were of analytical grade and were used as 3167

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sonicating a 0.05 wt % aqueous dispersion for 30 min. Unexfoliated graphite oxide was removed by a 5 min ultrafiltration at 2000 rcf. The glassy carbon electrode (GCE) with a 3 mm diameter was polished to a mirror using 1.0, 0.3, and 0.05 μm alumina slurry, followed by rinsing thoroughly with deionized water. After successive sonication in 1:1 nitric acid, acetone, and deionized water, the electrode was rinsed with water and allowed to dry at room temperature. Five microliters of 0.5 mg mL−1 GO solution was dropped on the pretreated GCE, which was dried in air. Then, 3 μL of 0.05% chitosan solution was dropped on the GO film and dried in air. After electrochemical reduction of GO at −1.0 V in pH 7.4 PBS, the modified electrode was washed with water and incubated with 5 μL of 2.5% glutaraldehyde (in 50 mM PBS, pH 7.4) for 2 h, followed by washing with water. Five microliters of 0.2 mg mL−1 anti-EpCAM was then dropped onto the surface and incubated sequentially at room temperature for 60 min at 4 °C overnight in a 100% moisturesaturated environment. Subsequently, excess antibody was removed with washing buffer. Finally, 5 μL of BSA blocking solution was dropped on the electrode surface and incubated for 60 min at room temperature to block possible remaining active sites against nonspecific adsorption. The anti-EpCAM/ chitosan/electrochemically reduced GO/GCE electrodes were thus obtained. The anti-EpCAM/chitosan/GCE was similarly prepared and used for a comparative experiment. Sandwich Immunoassay with Ab-Coupled Nanobioprobe. A sandwich immunoassay using Si/Cd1/anti-EpCAM and Si/ Zn/anti-GPC3 as labels was shown in Figure 1B. The antiEpCAM/chitosan/electrochemically reduced GO/GCE electrode was incubated in 1 mL of Hep3B cell suspension at 37 °C for 30 min to capture cells with the first immunoreaction. After being washed thoroughly with PBS, the cells attached-GCE electrode was exposed to 0.4 mL of Si/Cd1/anti-EpCAM and Si/Zn/anti-GPC3 suspension at 37 °C for 50 min to introduce the labels onto the GCE electrode through the second immunoreaction. After that, it was thoroughly rinsed with PBS containing 0.05% Tween to remove immunological labels due to the physical adsorption to complete the sandwich immunoreactions. SWV Analysis. Fabrication of Bismuth Film Modified Glassy Carbon Electrode (BFE). The pretreated GCE electrode was immersed in a bismuth nitrate solution in acetate with a final pH of 2.0 and Bi(III) ion concentration of 1.25 mg mL−1 and applied potential of −1.2 V (vs SCE) for 120 s. SWV Analysis. The QDs remaining on the cell surface were dissolved by 30 μL of HNO3 solution (0.1 mol L−1). The solution containing the dissolved metal ions was then transferred into 3 mL of acetate buffer (0.1 mol L−1) at pH 4.5. The amount and identity of the dissolved metal ions were determined by stripping voltammetry. The analytical procedure involved three 120 s electrodeposition at −1.2 V (with 10 s stirring between each accumulation period). After that, it was stripped by scanning from −1.2 to −0.6 V, using SWV measurements with 4 mV potential steps, 25 Hz frequency, and 25 mV amplitude. Fluorescence Analysis. The anti-EpCAM-modified GCE electrode was incubated in 1 mL of Hep3B solution at 37 °C for 30 min to capture the cells. After being washed thoroughly with PBS, the cells-attached GCE electrode was exposed to 0.4 mL of Si/Cd1/anti-EpCAM and Si/Cd2/anti-GPC3 suspension at 37 °C for 30 min to introduce the labels onto the GCE electrode surface. After that, it was thoroughly rinsed with PBS

cell pellet was resuspended in 10 mM PBS, pH 7.4. The cell number was determined using a Petroff−Hausser cell counter. Preparation of the Antibody (Ab)-Coupled Nanobioprobe. Preparation of SiO2 Nanoparticles, CdTe, and ZnSe. Synthesis of monodispersed SiO2 nanoparticles was carried out according to the reported seed-growth methods.29,30 CdTe1 (λ=550 nm), CdTe2 (λ=620 nm), and ZnSe were synthesized according to a previously reported procedure.31−34 The solution with the QDs was further purified by ultrafiltration.35 Preparation of QD-Coated Silica Nanoparticles. For preparation of CdTe1 QD-coated silica nanoparticles (Si/ Cd1), CdTe2 QD-coated silica nanoparticles (Si/Cd2), and ZnSe-coated silica (Si/Zn) nanoparticles, 0.02 g silica nanospheres were first dispersed in 2 mL ethanol and treated with 0.4 mL APTS. After stirring for 6 h, the suspension was centrifuged and washed with ethanol; the washing was repeated four times. After that, the amino-functionalized silica nanoparticles were harvested. They were then redispersed in a mixture of 2 mL of CdTe1 QDs, CdTe2 QDs, or ZnSe QDs and 1 mL EDC (20 mg mL−1). The suspension was stirred at 4 °C for 12 h. Unbound QDs were removed by successive centrifugation and washed several times with water. Finally, the as-prepared Si/Cd1, Si/Cd2, and Si/Zn nanoparticles obtained were dispersed in water to a final volume of 1 mL. Preparation of Ab-Coupled Nanobioprobe. To generate QD-coated silica nanosphere immunological labels, 1 mL of Si/ Cd1 suspension was mixed with 1 mL of anti-EpCAM (25 μg mL−1 in 0.01 M PBS, pH 7.4); 1 mL of Si/Cd2 suspension was mixed with 1 mL of anti-GPC3 solution (25 μg mL−1 in 0.01 M PBS, pH 7.4) and 1 mL of Si/Zn suspension was mixed with 1 mL of anti-GPC3 solution (25 μg mL−1 in 0.01 M PBS, pH 7.4). Subsequently, 100 μL of freshly prepared EDC (20 mg mL−1 in 0.01 M PBS, pH 7.4) and 100 μL of NHS (10 mg mL−1 in 0.01 M PBS, pH 7.4) were added. After incubation at room temperature for 2 h, free antibodies were removed by centrifugation and the pellet was washed with 0.01 M PBS several times to yield the antibody-modified Si/Cd1 nanoparticles (Si/Cd1/anti-EpCAM), Si/Cd2 nanoparticles (Si/ Cd2/anti-GPC3), or Si/Zn nanoparticles (Si/Zn/anti-GPC3). Finally, the Si/Cd1/anti-EpCAM, Si/Cd2/anti-GPC3, and Si/ Zn/anti-GPC3 nanoparticles were resuspended individually in 5 mL of 1% BSA solution for 2 h to block the excess amino group and nonspecific binding sites of the immunological labels. After being centrifuged and washed with PBS, the resultant Si/Cd1/anti-EpCAM, Si/Cd2/anti-GPC3, and Si/ Zn/anti-GPC3 nanoparticles were resuspended individually with 0.01 M of pH 7.4 PBS to a final volume of 5 mL and stored at 4 °C for later use. The fabrication of the immunological labels was illustrated in Figure 1A. For control experiments, Cd/anti-EpCAM or Zn/anti-GPC3 were prepared by incubating the mixture of 1 mL of CdTe QDs solution and 200 μL of anti-EpCAM or 1 mL of ZnSe QDs solution and 200 μL of anti-GPC3 solution in the presence of 300 μL of 2 mg mL−1 EDC solution at room temperature for 2 h. The free nonconjugated QDs were removed by ultrafiltration using a 50000 MW filter. Fabrication of Biosensors for SWV Analysis. Preparation of Antibody-Modified GCE Electrode. GO (graphene oxide) was synthesized from graphite by the modified Hummers method.36,37 The as-synthesized graphite oxide was suspended in water and subjected to dialysis for one week to remove residual salts. After drying at 50 °C overnight, aspurified graphite oxide was exfoliated into GO by ultra3168

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Figure 2. (A) Electrochemical impedance spectroscopy of (a) chitosan/GCE, (b) chitosan/GO/GCE, and (c) chitosan/electrochemically reduced GO/GCE in 0.1 M KCl containing 5 mM Fe(CN)62−/3−. (B) Records of anodic stripping voltammograms after the anti-EpCAM/chitosan/ electrochemically reduced GO/GCE was incubated in 1 × 106 cells mL−1 Hep3B cell solution for 30 min, and then in (a) Si/Cd/anti-EpCAM and Si/Zn/anti-GPC3 solution or in (b) CdTe QD-labeled anti-EpCAM and ZnSe QD-labeled anti-GPC3 for 50 min. Records of anodic stripping voltammograms after the anti-EpCAM/chitosan/GCE were incubated in 1 × 106 cells mL−1 Hep3B cell solution for 30 min, and then in (c) Si/Cd/ anti-EpCAM and Si/Zn/anti-GPC3 solution for 50 min. Records of anodic stripping voltammograms after the anti-EpCAM/chitosan/ electrochemically reduced GO/GCE was incubated in (d) PBS solution without a cell or (e) PP5 cell solution. Records of anodic stripping voltammograms after the anti-EpCAM/chitosan/electrochemically reduced GO/GCE was incubated in 1 × 106 cells mL−1 MCF-7 cell solution for 30 min, and then in (f) Si/Cd/anti-EpCAM and Si/Zn/anti-GPC3 solution for 50 min.

nanospheres. After further coupling with the antibodies, the XPS spectra showed two decreased Cd3d (Figure S2A, curve b, Supporting Information) or Zn2p (Figure S2B, curve b, Supporting Information) and one enhanced N1s line (Figure S2A, curve b, and Figure S2C of the Supporting Information) at the same binding energy with QD-coated silica nanospheres. This was due to the coating of the antibody on the surface of the QDs. Characterization of the Immunosensor. Graphene, especially electrochemically reduced GO, exhibits high conductivity and mediates electron transfer at its edge planes. It has been widely used in the biosensor to improve the detection sensitivity.39−41 Our present strategy employed reduced GO to construct a sensitive immunosensor for cell detection. After GO was doped in chitosan, the resulting chitosan/GO/GCE showed much lower electron transfer resistance, Ret (Figure 2A, curves a and b). Upon the electrochemical reduction of GO, the Ret further decreased (Figure 2A, curve c). The antibodies-modified GCE was used to capture Hep3B cells from the cell suspension through the first immunoreaction. Due to the presence of EpCAM and GPC3 on the cell surface, Si/Cd/anti-EpCAM and Si/Zn/antiGPC3 labels were introduced onto the cell surface through the secondary binding event. After the captured CdTe and ZnSe dissolved to generate Cd2+ and Zn2+ from the GCE electrode in a 0.1 M HNO3 solution, the captured CdTe and ZnSe QDs on the GCE electrode could be detected by a BFE. Figure 2B shows the SWV curves of different electrodes with the Hep3B cell of 1 × 106 cells mL−1. A well-defined peak for the oxidation of Cd and Zn was observed from the immunosensor at around −0.75 V and −1.15 V (Figure 2B, curves a and b). However, the oxidation current of 14.76 μA by Si/Cd/anti-EpCAM and 8.88 μA by Si/Zn/anti-GPC3 labels (Figure 2B, curve a) was 2 times and 2.7 times larger than the 7.11 μA by Cd/antiEpCAM and 3.26 μA by Zn/anti-GPC3 labels (Figure 2B, curve b), respectively, showing signal amplification by silica nanoparticles. The signal amplification was due to the increase of the CdTe or ZnSe QDs loading in per immunological events. To verify the signal amplification of graphene, anti-EpCAM was directly coupled to the GCE through chitosan. Although anti-

containing 0.05% Tween to remove immunological labels due to physical adsorption and observed under a fluorescent microscope.

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RESULTS AND DISCUSSION Preparation of the Ab-Coupled Nanobioprobe. Silica nanoparticles with good monodispersion and similar surface morphology were synthesized according to the previously reported seed-growth method.38 The TEM images showed that the as-prepared silica nanoparticles had a chemically clean and homogenized structure, with a diameter of 100 ± 3.0 nm (Figure S1A of the Supporting Information). Coating of QDs onto the surface of silica nanospheres was achieved through acylamide binding in the presence of EDC as the activator. In this case, APTS was first coupled to the hydroxyl group on the surface of silica nanospheres to yield an amino-terminated selfassembled monolayer. Subsequently, the carboxylic groups located on the surface of CdTe or ZnSe QDs reacted with amino distal points, which formed QD-coated silica nanoparticles. The coating of CdTe (Figure S1B of the Supporting Information) or ZnSe (Figure S1C of the Supporting Information) QDs on silica nanospheres was confirmed by TEM images, demonstrating the deposition of QDs on the surface of silica nanospheres with a uniform distribution. The other carboxyl groups located on the QD surface can be further coupled to the amino groups of antibodies through acrylamidebonding in the presence of EDC and NHS as activating reagents. Figure S2 of the Supporting Information showed the XPS spectra of the QD-coated silica nanoparticles before and after antibody coating. The XPS spectrum of the CdTe QDfunctionalized silica nanoparticles exhibits the binding energy of the electrons for Cd3d at 405.3 and 412.1 eV (Figure S2A, curve a, of the Supporting Information). The XPS spectrum of the ZnSe QDs functionalized silica nanoparticles exhibits the binding energy of the electrons for Zn2p at 1022 and 1048 eV (Figure S2B, curve a, of the Supporting Information). A weaker N1s line was exhibited at 400.0 eV for the N−H group in the APTS (Figure S2A, curve a, and Figure S2C of the Supporting Information). This confirmed that CdTe QDs or ZnSe QDs have been successfully coated on the surface of silica 3169

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Figure 3. (A) SWV of Si/Cd1/anti-EpCAM-Hep3B-anti-EpCAM/chitosan/electrochemically reduced GO/GCE at Hep3B cell concentration of 5, 10, 30, 60, 80, 100, 500, 1000, 1 × 104, 5 × 104, 1 × 105, 5 × 105, and 1 × 106 cells mL−1. (B) Plot of peak current obtained by dissolved Si/Cd1/ anti-EpCAM-Hep3B-anti-EpCAM/chitosan/electrochemically reduced GO/GCE vs Hep3B cell concentration in incubation solution. Inset in (B): linear regression plot.

Figure 4. (A) SWV of Si/Zn/anti-GPC3-Hep3B-anti-EpCAM/chitosan/electrochemically reduced GO/GCE at Hep3B cell concentration of 10, 30, 60, 80, 100, 500, 1000, 1 × 104, 5 × 104, 1 × 105, 5 × 105, and 1 × 106 cells mL−1. (B) Plot of peak current obtained by dissolved Si/Zn/anti-GPC3Hep3B-anti-EpCAM/chitosan/electrochemically reduced GO/GCE vs Hep3B cell concentration in an incubation solution. Inset in (B): linear regression plot.

(EpCAM and GPC3 positive). Additionally, the wide flat baseline (e.g., between the Cd and Zn peaks or before Cd) indicates the possibility of measuring additional protein biomarkers simultaneously on the cell surface. Optimization of the Conditions for Immunoassay. Kinetically, the number of cells captured from the solution depends on the incubation time before it reaches thermodynamic equilibrium. By incubating antibodies modified GCE in the cell-containing solution for different period of time, the oxidation current of Cd was observed to reach a maximum value at 30 min, so the incubation time was chosen to be 30 min (Figure S3A of the Supporting Information). The amount of Si/Cd/anti-EpCAM and Si/Zn/anti-GPC3 labels immobilized on the cell surface was associated with the incubation time between labels and cells. With an increasing incubation time, the oxidation current of Cd increased and trended to a constant value after an incubation time of 50 min (Figure S3B of the Supporting Information), which showed a saturated binding between the labels and the cells. Therefore, the incubation time of 50 min was selected for label immobilization. SWV Analytical Performance. Under optimum conditions, the stripping peak current of CdTe and ZnSe QDs

EpCAM/chitosan/GCE could also specifically capture the Hep3B cells, a comparatively lower oxidation current was obtained, (Figure 2B, curve c) indicating that the presence of graphene greatly increased the density of anti-EpCAM on the biosensor surface and further enhanced the detection signal (Figure 2B, curve a). A series of control experiments were conducted. No peaks were observed after incubation of the antibodies-modified GCE in a cell-free solution, followed by incubation in Si/Cd/anti-EpCAM and Si/Zn/anti-GPC3 suspensions (Figure 2B, curve d); no peaks were observed after incubation of the antibodies-modified GCE in a solution of PP5 cells (EpCAM and GPC3 negative), followed by incubation in Si/Cd/anti-EpCAM and Si/Zn/anti-GPC3 suspensions (Figure 2B, curve e); just one peak for Cd was detected after incubation of the antibodies-modified GCE in a solution of MCF-7 cells (EpCAM positive, GPC3 negative), followed by incubation in Si/Cd/anti-EpCAM and Si/Zn/antiGPC3 suspensions (Figure 2B, curve f). These observations confirmed that the SWV responses were attributed to the attachment of QDs-coated silica nanoparticles, through the sandwiched immunoreactions. When two peaks appeared simultaneously, it confirmed the presence of Hep3B cells 3170

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Figure 5. (A) Fluorescence microscopic images of anti-EpCAM-coupled red nanobioprobes. (B) Fluorescence microscopic images of anti-GPC3coupled green nanobioprobes. (C−F) Fluorescence microscopic images of Hep3B cells incubated with EpCAM-modified GCE, followed by incubating with Si/Cd1/anti-EpCAM and Si/Cd2/anti-GPC3 nanobioprobes. (C) Blue fluorescence from Hochest 33342 localized in the nuclei. (D) Red fluorescence from Si/Cd1/anti-EpCAM nanobioprobes. (E) Green fluorescence from Si/Cd2/anti-GPC3 nanobioprobes. (F) A merged image of (C), (D), and (E). Here, each fluorescent dot came from a single nanobioprobe.

expected fluorescence. To confirm the Ab-coupled nanobioprobes maintaining the capability of recognizing the target cells, the Si/Cd1/anti-EpCAM and Si/Cd2/anti-GPC3 were incubated with Hep3B cells coated GCE as mentioned in the Experimental Section. Because the target cancer cells had been stained with fluorescent dyes and the labels contained fluorescent quantum dots, they could be visualized by their different fluorescence when excited under a lamp-house after the experiment (Figure 5, panels C−E). It is only when the blue fluorescence of nuclei and the red and green fluorescence representing the Si/Cd1/anti-EpCAM and Si/Cd2/anti-GPC3 nanobioprobes, respectively, appeared simultaneously on the cell surface of the tumor cells (Figure 5F) that the positive identification of Hep3B liver cancer cells would be indicated. As controls for specificity, MCF-7 (EpCAM positive, GPC3 negative) and PP5 (EpCAM and GPC3 negative) cells were tested. For MCF-7 cells, the blue fluorescence of the nuclei and the red fluorescence of Si/Cd1/anti-EpCAM nanobioprobes could be detected (Figure 6, panels A and B, respectively). When these images were overlaid, the blue and the red fluorescence appeared simultaneously on the cell surface of MCF-7 cells (Figure 6C), demonstrating the presence of EpCAM antigen and the absence of GPC3 antigen on its cell surface. In comparison, no fluorescent signal could be detected

immobilized on the immunosensor increased with increasing concentration of Hep3B cells in the incubation solution. The current of Cd was found to be proportional to the concentration of Hep3B cells in the incubation solution, which was within the calibration range from 5 to 1 × 106 cells mL−1 (Figure 3, panels A and B). The linear curve fits a regression equation of log (Ip/μA) = 0.31 − 0.67 log (Ccell/cells mL−1), where Ip is the oxidation current and C is the cell concentration (Figure 3B, inset). The lowest detectable concentration was 5 cells mL−1 (Figure 3A), which was much lower than those of 620 cells mL−1 by an electrochemical cytosensor for detection of BGC cells42 and 800 cells mL−1 by an electrochemiluminescent cytosensor for the detection of HepG2 cells.43 The current of the Zn was proportional to the concentration of Hep3B cells from 10 to 1 × 106 cells mL−1 (Figure 4, panels A and B). The linear curve fits a regression equation of log (Ip/μA) = 0.29 − 0.83 log (Ccell/cells mL−1), the lowest detectable concentration was 10 cells mL−1 (Figure 4B, inset). Our results demonstrated that the present method is highly sensitive, especially for the detection of low CTC levels. Fluorescent Performance. Figure 5 shows the fluorescence microscope images of Ab-coupled nanobioprobes. Both Si/Cd1/anti-EpCAM (Figure 5A) and Si/Cd2/anti-GPC3 (Figure 5B) were clearly monodispersed and retained the 3171

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Figure 6. (A−C) Fluorescence microscopic images of MCF-7 cells (EpCAM positive, GPC3 negative) incubated with EpCAM-modified GCE, followed by incubating with Si/Cd1/anti-EpCAM and Si/Cd2/anti-GPC3 nanobioprobes. (A) Blue fluorescence from Hochest 33342 localized in the nuclei. (B) Red fluorescence from Si/Cd1/anti-EpCAM nanobioprobes. (C) A merged image of (A) and (B). (D) Negative control. PP5 cells (EpCAM and GPC3 negative) incubated with EpCAM-modified GCE, followed by incubation with Si/Cd1/anti-EpCAM and Si/Cd2/anti-GPC3 nanobioprobes. No fluorescent could be detected.

of 6.5% and 8.7% was obtained, respectively, for the Cd and Zn nanobioprobes, following 6 repeated detection-regeneration cycles. This observation demonstrated that the as-synthesized Si/Cd/anti-EpCAM and Si/Zn/anti-GPC3 possesses good monodispersion characteristics to enable the consistent loading of the same amount of QDs on each microsphere. The apparent uniformity in dispersion provides an added advantage for its application in the clinical diagnosis of CTCs.

for PP5 cells, demonstrating the absence of EpCAM and GPC3 antigens on its cell surface (Figure 6D). Reproducibility, Precision, Stability, and Regeneration of Immunosensor. Both the intra-assay and interassay precisions of the immunosensor were examined with 1 × 106 Hep3B cells, five times. The relative standard deviations (RSD) were 4.6% and 6.2%, respectively, showing good precision and acceptable fabrication reproducibility. In addition, when the immunosensor was stored in dry conditions at 4 °C, over 90% of the initial response remained after a storage period of 2 weeks. These results indicated that the immunosensor had acceptable reliability and stability. The regeneration step was performed by immersion of the working electrode for 10 min in 0.1 M glycine-HCl, pH = 2.2, to interrupt the antigen−antibody immunocomplexes and offer the potential to harvest the bound tumor cells for further molecular and biochemical characterizations. After each sandwich immunoassay, the electrode was immersed in 0.1 M glycine-HCl (pH 2.2) for 10 min. No detectable SWV signals could be detected while incubation of this regenerated electrode in Hep3B cell suspension, followed by Si/Cd/antiEpCAM and Si/Zn/anti-GPC3 suspension gave comparable SWV responses as earlier described. Relative standard deviation

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CONCLUSIONS An ultrasensitive and highly specific electrochemical and fluorescent immunosensing method for the detection of circulating tumor cells was achieved using a dual signal amplification strategy based on nanobiotechnology. The introduction of graphene on the immunosensor surface efficiently accelerated the electron transfer and enhanced the detection signal. The second signal amplification came from the loading of QDs on the silica nanoparticle surface. After immunoreactions, two kinds of tracing tags were coimmobilized on the cell surface that could be conveniently detected by anodic stripping analysis and fluorescence microscopy. In addition, the regeneration of the immunosensor enables the 3172

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Analytical Chemistry

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elution and harvesting of the captured CTCs cells for further molecular analysis. The present immunosensor as design described showed high sensitivity and specificity to detect low-abundance tumor cells with excellent stability, reproducibility, and accuracy, indicating its wide potential applications for clinical diagnostics.

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ASSOCIATED CONTENT

S Supporting Information *

TEM images of the as-synthesized SiO2, Si/Cd, and Si/Zn nanoparticles, XPS spectra of QD-coated silica nanoparticles before and after further coupling with antibodies, and optimization of the immunosensor. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Y.K.: e-mail, [email protected]. K.M.H.: e-mail, [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by a start-up grant from Nanyang Technological University College of Engineering and an Academic Research Fund Tier-1 from the Ministry of Education of Singapore (RG 26/11) awarded to Y.J.K., and research grants from the SingHealth Foundation, Singapore National Medical Research Council, Biomedical Research Council of Singapore, and The Singapore Millennium Foundation awarded to K.M.H.

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REFERENCES

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dx.doi.org/10.1021/ac303398b | Anal. Chem. 2013, 85, 3166−3173