Diagnostic Detection of Human Lung Cancer-Associated Antigen

Anal. Chem. 2010, 82, 5944–5950

Diagnostic Detection of Human Lung Cancer-Associated Antigen Using a Gold Nanoparticle-Based Electrochemical Immunosensor Ja-an Annie Ho,*,† Heng-Chia Chang,† Neng-Yao Shih,‡ Li-Chen Wu,§ Ying-Feng Chang,| Chii-Chang Chen,⊥ and Chien Chou| BioAnalytical Laboratory, Department of Chemistry, National Tsing Hua University, Hsinchu 300 Taiwan, National Institute of Cancer Research, National Health Research Institutes, Tainan 704 Taiwan, Biochemistry Laboratory, Department of Applied Chemistry, National Chi Nan University, Puli, Nantou 545 Taiwan, Department of Optics and Photonics, National Central University, Jhongli 320 Taiwan, and Institute of Biophotonics, National Yang Ming University, Taipei 112 Taiwan The development of rapid and sensitive methods for the detection of immunogenic tumor-associated antigen is important not only for understanding their roles in cancer immunology but also for the development of clinical diagnostics. r-Enolase (ENO1), a p48 molecule, is widely distributed in a variety of tissues, whereas γ-enolase (ENO2) and β-enolase (ENO3) are found exclusively in neuron/neuroendocrine and muscle tissues, respectively. Because ENO1 has been correlated with small cell lung cancer, nonsmall cell lung cancer, and head and neck cancer, it can be used as a potential diagnostic marker for lung cancer. In this study, we developed a simple, yet novel and sensitive, electrochemical sandwich immunosensor for the detection of ENO1; it operates through physisorption of anti-ENO1 monoclonal antibody on polyethylene glycol-modified disposable screen-printed electrode as the detection platform, with polyclonal secondary anti-ENO1-tagged, gold nanoparticle (AuNP) congregates as electrochemical signal probes. The immunorecognition of the sample ENO1 by the congregated AuNP@antibody occurred on the surface of the electrodes; the electrochemical signal from the bound AuNP congregates was obtained after oxidizing them in 0.1 M HCl at 1.2 V for 120 s, followed by the reduction of AuCl4- in square wave voltammetry (SWV) mode. The resulting sigmoidally shaped dose-response curves possessed a linear dynamic working range from 10-8 to 10-12 g/mL. This AuNP congregate-based assay provides an amplification approach for detecting ENO1 at trace levels, leading to a detection limit as low as 11.9 fg (equivalent to 5 µL of a 2.38 pg/mL solution). * Corresponding author. Fax: +886-3-571-1082. E-mail: [email protected]. † National Tsing Hua University. ‡ National Health Research Institutes. § National Chi Nan University. | National Central University. ⊥ National Yang Ming University.

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Analytical Chemistry, Vol. 82, No. 14, July 15, 2010

There is an urgent need to develop rapid screening methods for monitoring the levels of immunogenic tumor-associated antigen (TAAs) in biosamples for both clinical diagnostics and cancer immunological research. Enolase (ENO), also known as phosphopyruvate dehydratase, is a metalloenzyme, discovered by Lohman and Mayerhof in 1934,1 that catalyzes the transformation of 2-phosphoglycerate (2-PG) to phosphoenolpyruvate (PEP) during the process of glycolysis. Three isoforms of ENO exist in mammalian cells: R-enolase (ENO1), γ-enolase (ENO2), and β-enloase (ENO3); their distributions are developmentally regulated in a tissue-specific manner. Whereas ENO1 is widely distributed in various tissues, ENO2 and ENO3 are found exclusively in neuron/neuroendocrine and muscle tissues, respectively. The levels of ENO in physiological fluids have been diagnosed in an attempt to monitor the severity of certain disease conditions.2 Higher concentrations of ENO in cerebrospinal fluid (CSF) are significantly correlated to astrocytoma; furthermore, the fastest tumor growth rates have also been observed in patients expressing the highest levels of CSF ENO. In addition, an elevated level of ENO2 can be used as an indicative, useful tumor marker for small cell lung cancer (SCLC).3 Recent studies have also described the possibility of correlating ENO1, as a highly expressed cancer marker, with nonsmall cell lung cancer (NSCLC).4,5 Moreover, overexpressed ENO1 is often found in SCLC tissue; in addition, the ENO2 /[ENO1 + ENO2] ratio has been considered a helpful indication for SCLC determination.6 Recently, it was revealed that the level of ENO1 is highly related to cancer cell (1) Lohman, K.; Meyerhof, O. Biochem. Z. 1934, 273, 60–72. (2) Royds, J. A.; Timperley, W. R.; Taylor, C. B. J. Neurol. Neurosurg. Psychiatry 1981, 44, 1129–1135. (3) Kanemoto, K.; Satoh, H.; Ishikawa, H.; Sekizawa, K. Clin. Oncol. 2006, 18, 505–508. (4) He, P.; Naka, T.; Serada, S.; Fujimoto, M.; Tanaka, T.; Hashimoto, S.; Shima, Y.; Yamadori, T.; Suzuki, H.; Hirashima, T.; Matsui, K.; Shiono, H.; Okumura, M.; Nishida, T.; Tachibana, I.; Norioka, N.; Norioka, S.; Kawase, I. Cancer Sci. 2007, 98, 1234–1240. (5) Liu, K. J.; Shih, N. Y. J. Cancer Mol. 2007, 3, 45–48. (6) Viallard, J. L.; Caillaud, D.; Kantelip, B.; Molina, C.; Dastugue, B. Chest 1988, 93, 1225–1233. 10.1021/ac1001959  2010 American Chemical Society Published on Web 06/17/2010

migration.5 Therefore, the importance of quantifying ENO1 in biological samples has become an important challenge. Lung cancer is the uncontrolled growth of abnormal cells in one or both lungs. These abnormal cells lose their normal functionality and stop developing and differentiating into healthy lung tissue. These dysfunctional extra cells can then form a mass of tissue, known as a tumor that impedes the supply of oxygen from the lung to the body via the blood circulation system. Lung cancer ranks first among all causes of cancer death in both women and men throughout the world; for example, in 2008, it accounted for 26% of all female and 31% of male cancer deaths.7 Even under therapy, there remains an increased risk of cancer recurrence and tumor cell metastasis. According to the American Cancer Society, NSCLC is one of the two most common types of lung cancer; it accounts for ca. 80% of all new cases. Therefore, there is an urgent need for an intensive monitoring strategy for NSCLC patients and the development of suitable screening methods for assaying NSCLC marker, such as ENO1. The function of an immunosensor relies on antibodies that react specifically with their corresponding antigens of interest, with quantification generally achieved by measuring a specific activity of the label, e.g., its enzyme/chemical amplification,8 fluorescence,9 bioluminescence,10 radioactivity,11 electrochemical signal,12-15 surface resonance plasmon (SPR),16 or its impact on a quartz crystal microbalance (QCM).17 Although several methods for the detection of ENO1 have been described based on an immunobioluminescent assay,18,19 two-dimensional differential in-gel electrophoresis,20 and mass spectrometry,20,21 these techniques often require complicated and time-consuming sample pretreatment and sophisticated instrumentation. Herein, we present a simple, yet novel, electrochemical immunosensor featuring anti-ENO1-tagged gold nanoparticle (AuNP) congregates as bioprobes for the trace determination of ENO1 at picogram per milliliter levels. The variable properties of AuNPs make them versatile materials for the development of electrochemical sensors in conjunction with disposable screen-printed carbon electrodes (SPCE). We began the fabrication of our (7) Jemal, A.; Siegel, R.; Ward, E.; Hao, Y.; Xu, J.; Murray, T.; Thun, M. J. CA Cancer J. Clin. 2008, 58, 71–96. (8) Blaedel, W. J.; Boguslaski, R. C. Anal. Chem. 1978, 50, 1026–1032. (9) Ho, J. A. A.; Hung, C. H. Anal. Chem. 2008, 80, 6405–6409. (10) Ho, J. A. A.; Huang, M. R. Anal. Chem. 2005, 77, 3431–3436. (11) Hage, D. S. Anal. Chem. 1999, 71, 294–304. (12) Dequaire, M.; Degrand, C.; Limoges, B. Anal. Chem. 2000, 72, 5521–5528. (13) Ho, J. A. A.; Chiu, J. K.; Hsu, W. L.; Hong, J. C.; Lin, C. C.; Hwang, K. C.; Hwu, J. R. R. J. Nanosci. Nanotechnol. 2008, 8, 1–6. (14) Ho, J. A. A.; Lin, Y. C.; Wang, L. S.; Hwang, K. C.; Chou, P. T. Anal. Chem. 2009, 81, 1340–1346. (15) Barton, A. C.; Davis, F.; Higson, S. P. J. Anal. Chem. 2008, 80, 9411– 9416. (16) Lyon, L. A.; Musick, M. D.; Natan, M. J. Anal. Chem. 1998, 70, 5177– 5183. (17) Muratsugu, M.; Ohta, F.; Miya, Y.; Hosokawa, T.; Kurosawa, S.; Kamo, N.; Ikeda, H. Anal. Chem. 1993, 65, 2933–2937. (18) Wevers, R. A.; Jacobs, A. A. C.; Hommes, O. R. Clin. Chim. Acta 1983, 135, 159–168. (19) Viallard, J. L.; Murthy, M. R. V.; Dastugue, B. Neurochem. Res. 1985, 10, 1555–1566. (20) Katayama, M.; Nakano, H.; Ishiuchi, A.; Wu, W.; Oshima, R.; Sakurai, J.; Nishikawa, N.; Yamaguchi, S.; Otsubo, T. Surg. Today 2006, 36, 1085– 1093. (21) Wall, D. B.; Kachman, M. T.; Gong, S.; Hinderer, R.; Parus, S.; Misek, D. E.; Hanash, S. M.; Lubman, D. M. Anal. Chem. 2000, 72, 1099–1111.

sensors through physical adsorption of polyethylene glycol (PEG) onto SPCE. Concurrently, the signal amplification probes were prepared by mixing polyclonal secondary anti-ENO1 with 33.0 nm diameter AuNPs, thereby providing 151 nm AuNP congregates sensitized with secondary anti-ENO1 antibody molecules.22,23 After sequential incubation with sample ENO1, the secondary anti-ENO1-tagged AuNP congregate bioprobes formed immunocomplexes on the primary monoclonal antiENO1-modified SPCE surface. Subsequent analysis using square wave voltammetry (SWV) revealed that this electrochemical immunosensor allowed the quantification of ENO1 to levels as low as 2.38 pg/mL. EXPERIMENTAL SECTION Reagents and Materials. All chemicals were of reagent grade or better. Ammonium sulfate, ampicillin, casein, ethylenediaminetetraacetic acid (EDTA), glutathione, isopropyl-β-D-thiogalactopyranoside (IPTG), octanoic acid, phenylmethanesulphonyl fluoride (PMSF), PEG (average molecule weight: 950-1050), potassium chloride, potassium phosphate, potassium ferrocyanide, sodium chloride, sodium citrate, sodium phosphate dibasic, and the THROMBIN CleanCleave kit were purchased from Sigma-Aldrich (St. Louis, MO). A B-PER glutathione S-transferase (GST) fusion protein purification kit was obtained from Pierce (Rockford, IL). ENO1 rabbit antiserum raised by immunizing with the recombinant ENO1 protein was produced by LTK Biolaboratories (Taoyuan, Taiwan). Hydrogen tetrachloroaurate (III) was acquired from Alfa Aesar (Ward Hill, MA). LB broth medium was purchased from Becton Dickinson (Sparks, MD). E. coli, transformed by plasmid carrying recombinant GST-ENO1 fused protein gene, was kindly provided by Dr. Neng-Yao Shih at the National Health Research Institutes (Tainan, Taiwan). Monoclonal anti-ENO1 was obtained from Abnova Co. (Taipei, Taiwan). Tris (base) was purchased from J. T. Baker (Phillipsburg, NJ). All solutions were prepared using deionized water having a resistivity of not less than 18 MΩ · cm (Milli-Q, Bedford, MA). Apparatus. Cyclic voltammetric (CV) and SWV were performed using a CHI 633 electrochemical analyzer/workstation (CH Instruments, Inc., Austin, TX). Disposable electrochemical SPCE, comprising a carbon working electrode, carbon counter electrode, and silver pseudoreference electrode, were purchased from Zensor R&D (Taichung, Taiwan). The average diameters of the AuNPs and the AuNP-tagged anti-ENO1 were measured using a 90Plus dynamic light scattering nanoparticle size analyzer (Brookhaven Instruments, NY). The absorbance spectrum of the AuNPs was measured using a Cintra 10e UV-vis spectrometer (GBC Scientific Equipment, Dandenong, Victoria, Australia). Scanning electron microscopy (SEM) images of the AuNPs and the anti-ENO1-tagged AuNP congregate bioprobes were obtained using a JEOL JSM-7000F thermal-type field emission scanning electron microscope (Akishima, Tokyo, Japan). Production of Recombinant ENO1 in E. coli. A seed culture (5 mL) of E. coli was grown overnight at 37 °C in LB (22) Nagatani, N.; Yuhi, T.; Chikae, M.; Kerman, K.; Endo, T.; Kobori, Y.; Takata, M.; Konaka, H.; Namiki, M.; Ushijima, H.; Takamura, Y.; Tamiya, E. NanoBiotechnology 2006, 2, 79–86. (23) Nagatani, N.; Tanaka, R.; Yuhi, T.; Endo, T.; Kerman, K.; Yuzuru, T.; Tamiya, E. Sci. Technol. Adv. Mater. 2006, 7, 270–275.


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medium containing ampicillin (50 µg/mL). The seed culture was transferred to 2.0 L of the medium, kept at 37 °C, and left to grow until the absorbance of the bacteria culture at 600 nm reached 0.6. The culture was cooled to 25 °C, IPTG was added to a final concentration of 0.1 mM, and then, GST-ENO1 was induced through incubation at 25 °C for an additional 4 h. The bacteria culture was subjected to centrifugation at 6000 rpm for 10 min; the resulting bacteria pellet was resuspended in bacterial protein extraction reagent (10 mL; from B-PER GST fusion protein purification kit) for lysing bacteria. 2.0 mM EDTA and 0.2 mM PMSF were added for protease inhibition. After sonication for 10 min, soluble protein was isolated and separated through centrifugation at 14 000 rpm for 10 min. The supernatant was collected and passed through a column packed with glutathione-immobilized sepharose gel (from B-PER GST fusion protein purification kit). The glutathionecoated column was subsequently washed several times with 10 mM Tris buffer to remove unbound protein. After elution with glutathione (10 mg/mL), fractions containing recombinant GST-ENO1 were collected. Finally, a THROMBIN CleanCleave kit was used to separate GST from ENO1 by cleaving the site between GST and ENO1. After cleavage, the solution containing GST and ENO1 was again passed through the column packed with glutathione-immobilized sepharose gel again for ENO1 purification. Preparation of Anti-ENO1-Tagged AuNP Congregate Bioprobes as Detection Biolabels. All glassware was cleaned thoroughly with aqua regia and rinsed extensively with Milli-Q water prior to use. AuNPs were synthesized through sodium citrate-mediated reduction of HAuCl4, based on a modification of the method reported by Turkevich et al.;24 1.0 mM HAuCl4 solution (30 mL) was brought to a vigorous boil with stirring in a round-bottom flask fitted with a reflux condenser, and then, 39 mM citrate solution (1.5 mL) was added in one portion. The solution containing the synthesized AuNPs was stirred for an additional 15 min under a vigorous boil and then for 10 min at ambient temperature. Concurrently, secondary polyclonal antiENO1 antibody was purified from rabbit antiserum25 based on a modification of the octanoic acid/ammonium sulfate precipitation method reported by McKinney and Parkinson.26 Bioconjugation of the AuNPs to anti-ENO1 proceeded through the addition of purified polyclonal anti-ENO1 (50 mg/mL, 50 µL) in 0.1 mM phosphate-buffered saline (PBS) to the AuNP solution (2 mL), followed by their reaction on ice for 15 min. Casein (0.1%, 50 µL) was added to the reaction mixture for blocking purposes. Secondary polyclonal anti-ENO1 antibodytagged AuNPs were subjected to centrifugation at 8000 rpm for 10 min. Pellets of anti-ENO1 antibody-tagged AuNPs were collected and resuspended in PBS (0.5 mL). Fabrication of Immunosensors. PEG solution (2.0 mg/mL, 8.0 µL) was placed onto the surface of a disposable SPCE working electrode. After rinsing with PBS to remove unbound PEG, primary monoclonal anti-ENO1 (0.2 mg/mL, 5.0 µL) was physisorbed onto the PEG-modified SPCE, followed by drying at room (24) Turkevich, J.; Stevenson, P. C.; Hillier, J. Discuss. Faraday Soc. 1951, 11, 55–75. (25) Ho, J. A. A.; Hung, C. H.; Wu, L. C.; Liao, M. Y. Anal. Chem. 2009, 81, 5671–5677. (26) McKinney, M. M.; Parkinson, A. J. Immunol. Methods 1987, 96, 271–278.

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Scheme 1. Schematic Representation of the Operation of the Electrochemical Immunosensor for the Detection of ENO1a

a

Not to scale.

temperature to improve the immobilization yield. Subsequently, casein (0.1%, 5.0 µL) was added and left to react for 20 min to block nonspecific binding sites. ENO1 samples (5.0 µL) of various concentrations were then introduced onto the modified working electrode and incubated for 1 h, allowing immunorecognition to occur. After rinsing with PBS to wash off the unbound ENO1, AuNP-tagged polyclonal anti-ENO1 (5.0 µL) was added and reacted for another 40 min to form sandwich immunocomplexes. Finally, the unbound AuNP labels were washed off with PBS, and the electrochemical signals were acquired. Electrochemical Measurements. A schematic representation of the operation of the electrochemical immunosensor for the detection of ENO1 is provided in Scheme 1. Electrochemical detection involved oxidation of the AuNPs at 1.2 V for 120 s in 0.1 M HCl (10 µL) and subsequent reduction to Au0 at 0.35 V (vs the silver pseudoreference electrode of the SPCE) using SWV, scanning from 0.8 to 0.0 V with an amplitude of 25 mV and a step potential of 4.0 mV at 15 Hz. The CV behaviors of Fe(CN)63-/4- were investigated by placing drops (20 µL) of 1.0 mM Fe(CN)63-/4- in 0.1 M KCl solution onto various SPCEs. The scanning range varied from -0.3 to +0.5 V with a scan rate of 0.05 V/s. RESULTS AND DISCUSSION Characterization of Anti-ENO1-Tagged AuNP Congregate Bioprobes. Because the changes in the optical property of AuNPs are correlated to their size, we characterized the AuNP suspension using UV-vis absorption spectroscopy. The UV-vis absorption spectrum of the Au colloidal suspension featured a sharp peak at 529 nm [Figure 1A, red line], consistent with the literature-reported SPR peak for 33.0 nm diameter AuNPs. [Figure 1B presents the size distribution of the AuNPs.]27 Aggregation of the AuNPs after addition of secondary polyclonal anti-ENO1 caused a red shift (to 544 nm) in the characteristic SPR band, as indicated in Figure 1A (black line). These spectral changes were accompanied by a corresponding color change in the solution from red to purple. This phenomenon might be due to bonding between the AuNPs and the amino groups of the antibody molecules; i.e., simple delocalization of the lone pair of electrons of an amino nitrogen atom to coordinatively unsaturated surface Au atoms.28 Figure 1B presents SEM images and the size distribution of the AuNPs before and after (27) Khlebtsov, N. G. Anal. Chem. 2008, 80, 6620–6625.

Figure 1. (A) Localized SPR bands in UV-vis absorption spectra and (B) SEM images and size distribution of the AuNPs before and after the formation of the antibody-tagged AuNP-based bioprobes.

the formation of the anti-ENO1-tagged AuNP congregate bioprobes. Dynamic light scattering revealed that the average sized of the original AuNPs, prepared through citrate-mediated reduction of HAuCl4 in aqueous solution, and the secondary anti-ENO1-tagged AuNP congregate bioprobes were ca. 33.0 and ca. 151 nm, respectively. Surface Characteristics of the PEG-Modified SPCEs. To confer the electrode surface with desired biocompatibility and minimum nonspecific adsorption, we selected PEG for antibody immobilization onto the electrode surface because of its wellknown biocompatibility and hydrophilicity.29,30 The use of PEG to fabricate immunosensors not only assists antibody immobilization but also functions as a potential stabilizer for binding sites of the antibody structure.31 PEG, a flexible and water-soluble polymer, undergoes a variety of interactions with (28) Venkataraman, L.; Klare, J. E.; Tam, I. W.; Nuckolls, C.; Hybertsen, M. S.; Steigerwald, M. L. Nano Lett. 2006, 6, 458–462. (29) Mougin, K.; Ham, A. S.; Lawrence, M. B.; Fernandez, E. J.; Hiller, A. C. Langmuir 2005, 21, 4809–4812. (30) Sharma, S.; Johnson, R. W.; Desai, T. A. Langmuir 2004, 20, 348–356. (31) Lee, C. S.; Kim, B. G. Biotechnol. Lett. 2002, 24, 839–844.

antibodies, including hydrogen bonding to the side chains of the amino acid residues and to the main chain NH groups; multiple coordination occurs to positively charged amino acids.32,33 Therefore, we suspected that a greater number of functioning antibody molecules would adsorb onto the PEGmodified surface. Figure 2A provides a comparison of the CV behavior of the PEG-modified and unmodified electrodes toward 1.0 mM potassium ferrocyanide in 0.1 M KCl buffer at scan rate of 0.05 V/s. Incorporation of PEG had no observable negative influence on the electrochemical behavior of electrodes. We observed a pair of well-defined peaks corresponding to the reduction and oxidation of Fe(CN)63-/4-, respectively, on the bare SPCE surface; the redox reaction of Fe(CN)63-/4- was slightly less reversible on the Ab/SPCE surface, as evidenced by a minor shift in ∆Ep. This phenomenon might have been due to (1) a protein fouling effect caused by the immobilized antibody molecules and/or (2) repulsive (32) Hasˇek, J. Z. Kristallogr. Suppl. 2006, 23, 613–618. (33) Michel, R.; Pasche, S.; Textor, M.; Gastner, D. G. Langmuir 2005, 21, 12327–12332.


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Figure 2. (A) Cyclic voltammograms of the 1 mM of Fe(CN)63-/4- redox couple in 0.1 M KCl on the (a) bare SPCE (black curve), (b) the PEG-modified SPCE (PEG/SPCE, red curve), (c) the monoclonal anti-ENO1-immobilized SPCE (Ab/SPCE, green curve), and (d) the monoclonal anti-ENO1-immobilized PEG-modified SPCE (Ab/PEG/SPCE, blue curve). (B) Differences in signal intensities toward various concentration of ENO1, which were generated on the Ab/SPCE and Ab/PEG/SPCE systems by oxidation of bound AuNP congregates and subsequent reduction of AuCl4- in SWV mode.

electrostatic interactions between the anionic Fe(CN)63-/4- and the negatively charged antibody molecules at values of pH greater than its isoelectric point. Similar phenomenon occurred for both the Ab/PEG/SPCE and PEG/SPCE systems. These observations suggest that the PEG layer and the antibodycaptured probes were successfully immobilized on the SPCE. Characterization of various modified SPCE by the measurement of contact angles was carried out, and the results are shown in Table S1 (Supporting Information). We defined the antibody immobilization efficiency using the expression: (amount of anti-ENO1 coupled/amount of anti-ENO1 offered) × 100%. We calculated the amount of anti-ENO1 coupled by subtracting the total amount of anti-ENO1 found in all of the wash fractions from the amount of anti-ENO1 offered. The antibody immobilization efficiency was determined from measurements of the absorbance at 595 nm (A595) using a Bio-Rad protein assay kit. Although the antibody immobilization efficiencies for the PEG/SPCE and bare SPCE were 64 and 78%, respectively, the former system produced stronger signals (generated by oxidation of bound AuNP congregates and subsequent reduction of AuCl4- in SWV mode) in the presence of ENO1 at concentrations of 10-12, 10-10, and 10-8 g/mL (Figure 2B). This observation implies that the PEG layer on the electrode stabilized the paratopes (antibody binding sites) on the antibody, in turn preserving their higher immunobinding affinity and leading to a greater number of functional paratopes being available for recognition of the sample ENO1 and subsequent formation of sandwich complexes with the anti-ENO1-tagged AuNPs congregate bioprobes. Optimization of Immunoreaction Time. We examined the effect of the incubation time for forming the immunosandwich complexes (between the bound sample ENO1 and the anti-ENO1tagged AuNP congregate bioprobes) on the performance of the electrochemical immunosensor in the detection of ENO1 at 10-10 g/mL. Electrochemical detection involved oxidation of the AuNPs at 1.2 V for 120 s in 0.1 M HCl and subsequent 5948


Figure 3. Effect of the incubation time for the formation of the immunosandwich complex on the generation of the current signal through SWV.

reduction to Au0 at 0.35 V (vs silver pseudoreference electrode of the SPCE) using SWV. Figure 3 reveals that stronger signals were obtained after longer incubation time. After incubation for 40 min, however, we observed no further significant changes in the current generated. Thus, the optimal incubation time was 40 min. Analytical Performance for the Determination of Target ENO1. The following experiments were undertaken to develop a sandwich binding immunoassay that facilitates the detection of an unknown amount of sample ENO1 with the help of a fixed amount of anti-ENO1-tagged AuNP congregate bioprobes. Sandwich immunocomplexes were formed and observed after incubating the sensing surface with various concentration of sample ENO1, followed by the addition of the anti-ENO1-tagged AuNP congregate bioprobes. As indicated in Figure 4, we investigated the target ENO1 at concentrations ranging from 10-13 to 10-7 g/mL, performing at least triplicate analyses at each concentration. The current signals generated upon reduction of the Au bioprobes were obtained using SWV, providing a sigmoidally shaped dose-response curve exhibiting linearity over the concentration range from 10-12

Table 1. Comparison of Sample Volumes, Reagent Usages, Operation Time, Dynamic Range, and LODs for the Analyses of ENO1

sample volume usage of reagents operation time dynamic range limit of detection (LOD)

Figure 4. Dose/response curve for the ENO1 target using the PEGmodified SPCE. Each data point represents an average ((1 SD) of three replicates. SWV was performed by spotting 0.1 M HCl (10 µL) onto the surface of the SPCE featuring the formed immunocomplexes (anti-ENO1 monoclonal Ab/sample ENO1/anti-ENO1-tagged AuNP congregate bioprobes) at ambient temperature, followed by oxidation of Au bioprobes at 1.20 V for 120 s. Then, the potential was scanned from 0.8 to 0 V with a step potential of 4 mV and a pulse amplitude of 25 mV at a frequency of 15 Hz. Insets: (lower right) Square wave voltammograms for the electrochemical detection of ENO1 upon serial dilutions of the ENO1 stock from 10-8 to 10-12 g/mL; (upper left) linear fit to the central data of main curve.

to 10-8 g/mL. An increase in the concentration of the target ENO1 during immunorecognition resulted in more Au bioprobes binding to the captured sample ENO1, leading to an increase in the peak current. The current signal increased in a dose-dependent manner with respect to the amount of target within the range from 10-12 to 10-8 g/mL, an adequate wide dynamic range of 4 orders of magnitude, and provided a limit of detection (LOD) of 11.9 fg (equivalent to 5 µL of a 2.38 pg/mL solution), defined by adding three times the standard deviation of the control (free of target ENO1) from its average value and a limit of quantitation (LOQ) of 103 fg (equivalent to 5 µL of a 20.7 pg/mL solution) per assay, calculated by adding 10 times the standard deviation of the control (free of target ENO1) from its average value. The Au reduction signal responses were highly precise, as indicated by the small error bars, which corresponded to the standard deviation (SD) of at least three measurements (n g 3) under each condition. The coefficients of variation (CV%) among all of the measurements were 2.36-7.12%, confirming the acceptable reproducibility of the sensor fabrication process and the electrochemical measurements. Batch-to-batch variation effect and size effect of AuNP congregate bioprobes on the sensor performance were investigated, and the results are shown in Figure S1 and Table S2 (Supporting Information). Furthermore, the result of a nonspecific binding test is illustrated in Figure S2 (Supporting Information). Table 1 provides a comparison of the sample volumes, reagent usage, operation times, dynamic ranges, and LODs of a commercial ELISA system and of our developed method for the analysis of ENO1. The LOD (11.9 fg) and linear dynamic

electrochemical immunosensor proposed in this study

commercial ELISA kit (Uscnlife)

5 µL 10-15 µL ∼2.5 h 4 orders 11.9 fg

100 µL 50-100 µL ∼5 h 2 orders 3.9 pg

range (from 10-12 to 10-8 g/mL) of our electrochemical immunosensing system for ENO1 were much better than that of the commercially obtainable kit, and the reagent consumption and operation time were considerably lower. The level of ENO1 in healthy individuals is ca. 67 ± 24 ng/ mL, whereas an elevated level of 130 ± 150 ng/mL has been found in an NSCLC patient (an individual with squamous cell carcinoma);34 both levels should be detectable, with appropriate dilution of the sample, when our proposed sensing system was used. In comparison with the approach reported previously by Tamiya et al.,35 our sensor (i) employed PEG as a preservative to keep the biofunctionality of immobilized antibody molecules; (ii) used a bigger size of anti-ENO1-tagged AuNP congregate bioprobes as detection biolabels, which resulted in greater signal readout and assumably better sensitivity; (c) only required 2 h to complete the fabrication process, while 12 h was taken for antibody immobilization and another 12 h was spent thereafter for blocking nonspecific binding sites with BSA as described by Tamiya’s group.35 In short, the fabrication of our assay system utilized relative simple and time-effective design. CONCLUSIONS We have ascertained the feasibility of employing nanogold labels on a disposable SPCE in an immunoassay that can be sensitively detected using SWV after oxidative release of Au(III) ions. Nanogold labels possess several advantages over radioisotopic or enzyme labels because (i) they are much more chemically stable or less hazardous, (ii) their fabrication is simpler and more straightforward, and (iii) their biocompatibility allows them not to interfere with the labeled biocompound (in this case, antibody molecules). Our use of PEG, on the other hand, not only helped to immobilize the antibody on the electrode surface but also functioned as a stabilizer for the antibody binding sites (paratopes), allowing the Au bioprobes to exhibit improved electrochemical immunoassay detection. Here, we present a simple electrochemical immunosensing platform for the detection of the human lung cancer-associated antigen, ENO1, by first fabricating a PEG layer on an SPCE and subsequently using anti-ENO1-tagged AuNP congregate bioprobes as signal amplifiers to improve the sensitivity of the assay. In summary, the versatility of AuNPs enables them to (34) Fujita, K.; Haimoto, H.; Imaizumi, M.; Abe, T.; Kato, K. Cancer 1987, 60, 362–369. (35) Idegami, K.; Chikae, M.; Kerman, K.; Nagatani, N.; Yuhi, T.; Endo, T.; Tamiya, E. Electroanalysis 2008, 20, 14–21.


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be used to develop electrochemical sensors in conjunction with disposable SPCEs. Our sensor (i) required very minute amounts (several microliters) of sample, (ii) exhibited shorter response time and higher sensitivity than a commercial ELISA system, (iii) and required no sophisticated instrumentation for AuNPs synthesis or signal acquisition. Because ENO1 appears to be a potential biomarker that is highly related to cancer cell migration, analytical methods that provide higher throughput and better turnover will become increasingly important for its quantification in biological samples. In addition to being an analytical tool for studying the roles of immunogenic TAAs in cancer immunology, we suspect that this single-use, disposable SPCE-based electrochemical immunosensors have potential for further development into practical clinical lung cancer diagnosis systems, such as in-field and point-of-care quantitative tests of disease-related protein biomarkers.

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ACKNOWLEDGMENT The authors thank Dr. K. Sudhakara Prasad for his valuable suggestions on this manuscript. The authors acknowledge support from the Taiwan National Science Council under Grant Nos. 982627-B-007-001 and 98-2113-M-007-013-MY3.

SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs. org.

Received for review January 22, 2010. Accepted May 25, 2010. AC1001959

Diagnostic Detection of Human Lung Cancer-Associated Antigen

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