Apyrimidinic Endonuclease 1 Immunosensing

Article pubs.acs.org/ac

Ultrasensitive Apurinic/Apyrimidinic Endonuclease 1 Immunosensing Based on Self-Enhanced Electrochemiluminescence of a Ru(II) Complex Ying Zhuo,*,† Ni Liao,† Ya-Qin Chai,† Guo-Feng Gui,†,‡ Min Zhao,† Jing Han,† Yun Xiang,† and Ruo Yuan*,† †

Key Laboratory on Luminescence and Real-Time Analysis, Ministry of Education, College of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, China ‡ College of Chemistry and Chemical Engineering, Bijie University, Bijie , Guizhou 551700, China S Supporting Information *

ABSTRACT: An alternative “signal on” immunosensor for ultrasensitive detection of apurinic/apyrimidinic endonuclease 1 (APE-1) was designed utilizing the self-enhanced electrochemiluminescence (ECL) of a novel Ru(II) complex functionalized coil-like nanocomposite as signal labels. The desirable self-enhanced ECL luminophore was achieved by combining the coreactant of poly(ethylenimine) (PEI) and the luminophor of bis(2,2′-bipyridine)-5-amino-1,10-phenanthroline ruthenium(II) [Ru(bpy)2(5-NH2-1,10-phen)2+] to form one novel Ru(II) complex, which exhibited significantly enhanced ECL efficiency and stability. Moreover, the carbon nanotubes (CNTs) were employed as nanocarriers for selfenhanced Ru(II) complex loading via π−π stacking to obtain the coil-like nanocomposite to act as signal probe. Compared with traditional ECL immunoassay, our proposed strategy is simple and sensitive, avoiding the adding of any coreactant into testing solution for signal amplification, and shows a detection limit down to subfemtogram per milliliter level under the optimized experimental condition.

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efficiency of Ru(bpy)32+ using the PAMAM G1.5 dendrimers as coreactant.16 Tang et al. have developed a DNA sensor with adding tripropylamine (TPA) into working buffer solution as a coreactant.17 In our previous work, we have prepared the apoferritin-templated poly(ethylenimine) (PEI) nanoparticles as labels based on in situ releasing the coreactant of PEI to develop a sensitive ECL immunosensor.18 Subsequently, we also have constructed of a reagentless ECL immunosensor by immobilizing the coreactant of poly-L-lysine to enhance the ECL of Ru(bpy)32+ for signal amplification.19 Although the luminous intensity of the Ru(bpy)32+ is indeed enhanced by adding additives as effective coreactant, it suffers from the problems of operational complexity, which would result in the adding the coreactant in the detection solution or immobilizing the coreactant on the sensing interface. With the goal of obtaining the desirable luminophores, we recently observed the self-enhanced ECL complex by covalently binding the proper coreactant to the traditional luminescent molecules, which might obtain the highly efficient and stable ECL emitters. Consequently, we have synthesized a novel

he apurinic/apyrimidinic endonuclease 1(APE-1) is a multifunctional enzyme that acts not only as a baseless endonuclease but also as a redox-modifying factor for a variety of transcription factors.1 Additionally, researches indicate that it has multiple possible roles in the response of human cancer to radiotherapy and chemotherapy.2 Up to now, various methods and strategies have been applied for the detection of APE-1, such as enzyme-linked immunoassay (ELISA),3 stopped-flow fluorescence analysis,4 and electrophoretic mobility-shift assay (EMSA).5 However, improvements are still required, as these methods remain cumbersome, time-consuming, and harmful to the operator’s health. In this regard, electrochemiluminescence (ECL) immunosensors are competitive with conventional assays, because of their high sensitivity, controllability of ECL reaction, low background, and cost-effectiveness.6−9 Ruthenium(II) tris(2,2′-bipyridyl) (Ru(bpy)32+) and its derivatives are the most extensively studied ECL luminophore, due to their advantages in chemical stability, reversible electrochemical behavior, and luminescence efficiency over a wide range of buffer pH levels.10−12 Currently, great efforts have been made toward the enhancement of the luminous intensity of the Ru(bpy)32+-based ECL system by employing the appropriate substance as effective coreactant.13−15 The Perez-Tejeda’s group achieved the enhancement of ECL © 2013 American Chemical Society

Received: July 24, 2013 Accepted: December 13, 2013 Published: December 13, 2013 1053

dx.doi.org/10.1021/ac403019e | Anal. Chem. 2014, 86, 1053−1060

Analytical Chemistry

Article

Scheme 1. Schematic Illustration of (A) Ab2/HGNPs/PTCA−PEI−Ru(II)/CNTs Probe (Ab2 Bioconjugates) Fabrication and (B) ECL Immunosensor Preparation Process and Possible Luminescence Mechanism

detection of APE-1 using Ab2-labeled HGNPs/PTCA−PEI− Ru(II)/CNTs as probe (Scheme 1). The possible mechanism of self-enhanced ECL complex of the PTCA−PEI−Ru(II) system was proposed. This method avoided the adding of any coreactant in testing solution for signal amplification and showed a detection limit down to subfemtogram per milliliter level. Furthermore, the assay approach also had acceptable stability, precision, and accuracy, showing potential applications in clinical diagnostics.

Ru(II) complex by binding bis(2,2′-bipyridine)-5-amino-1,10phenanthroline ruthenium(II) [Ru(bpy) 2 (5-NH 2 -1,10phen)2+] to the poly(ethylenimine) (PEI), a linear structure molecule with a backbone of two methylene units followed by one tertiary amine group which has been proven to serve as an effective coreactant of Ru(bpy)32+ in our previous work. It was found that the ECL efficiency is significantly enhanced by covalently coupling the coreactant of PEI and luminophor of [Ru(bpy)2(5-NH2-1,10-phen)2+] to form one molecule, since the intramolecular ECL reaction could be more efficient as compared with the intermolecular reaction due to the shorter electron-transfer path and less energy loss. Thus, an alternative “signal on” immunosensor for ultrasensitive detection of APE-1 utilizing the self-enhanced ECL complex of PEI−Ru(II) was developed in this work. In order to achieve the high sensitivity, the aromatic compound of 3,4,9,10perylene tetracarboxylic acid (PTCA) was bound to PEI− Ru(II), acting as enhancer and linker for subsequent assembly on the carbon nanotubes (CNTs) via π−π stacking. Then the CNTs were used as nanocarriers for PTCA−PEI−Ru(II) loading via π−π stacking to obtain the coil-like PTCA−PEI− Ru(II)/CNTs composite. Furthermore, the PTCA−PEI− Ru(II)/CNTs could induce the hollow gold nanoparticles (HGNPs) assembling on the surface via residual −NH2 groups of PTCA−PEI−Ru(II). The HGNPs decorated coil-like PTCA−PEI−Ru(II)/CNTs composite (HGNPs/PTCA− PEI−Ru(II)/CNTs) showed uniform size distribution, good stability, and significant ECL intensity and could easily serve as a tracing tag to label detection antibody (Ab2). The ECL immunosensing interface was constructed by simple modification of capture antibody (Ab1) on the electrochemically deposited Au nanoparticles (AuNPs) decorated glassy carbon electrode (GCE). Further investigation indicated that the proposed sandwiched immunosensor showed ultrasensitive

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EXPERIMENTAL METHODS Reagents and Materials. Bis(2,2′-bipyridine)(5-amino1,10-phenanthroline) ruthenium(II) dichloride [Ru(bpy)2(5NH2-1,10-phen)Cl2] was from Suna Tech Inc. (Suzhou, China). Apurinic/apyrimidinic endonuclease (APE-1) and apurinic/apyrimidinic endonuclease antibody (anti-APE-1, monoclonal antibody) were purchased from Santa Cruz Biotechnology, Inc. (U.S.A.). Branched polyethylenimine (PEI, 99%, average Mn ∼10 000 by GPC), gold chloride (HAuCl4), and bovine serum albumin (BSA, 96−99%) were purchased from Sigma-Aldrich Co. (St. Louis, MO, U.S.A.). Perylene-3,4,9,10-tetracarboxylic dianhydride (PTCDA) was from Lian Gang Dyestuff Chemical Industry Co. Ltd. (Liaoning, China). The multiwalled carbon nanotubes (CNTs, >95% purity) synthesized by the CVD method were purchased from Nanjing Xianfeng Nanno Co. (Nanjing, China) and used as received. N-Hydroxy succinimide (NHS) and N(3-dimethylaminopropyl)-N′-ethylcarbodiimidehydrochloride (EDC) were received from Shanghai Medpep Co. Ltd. (Shanghai, China). Phosphate buffer solutions (PBS) with various pH values and concentrations were prepared by mixing standard stock solutions of 0.1 M K2HPO4, 0.1 M NaH2PO4, and 0.1 M KCl and adjusting the pH with 0.1 M H3PO4 or 1054



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Figure 1. Schematic diagram of the procedure used to prepare PTCA−PEI−Ru(II) compounds.

Preparation of PTCA−PEI−Ru(II) Compounds. The overall process involved in fabricating the PTCA−PEI−Ru(II) compounds is shown schematically in Figure 1. To prepare the PEI−Ru(II) molecular compounds, glutaraldehyde (GA) was used to link the PEI and [Ru(bpy)2(5-NH2-1,10-phen)2+]. Briefly, 0.5 mL of 0.1 M PEI solution was mixed with 0.5 mL of 5.0 mM [Ru(bpy)2(5-NH2-1,10-phen)2+] solution; 0.5 mL of cross-linking agent 1 wt % GA was added into above solution with the incubation time of 12 h under constant stirring. The unreacted GA and [Ru(bpy)2(5-NH2-1,10-phen)2+] were purified by dialysis (with a molecular weight cutoff of 8500) in distilled water for 1 day and PEI−Ru(II) compounds solution was obtained. The reaction was monitored by FT-IR spectroscopy (see the Supporting Information, Figure S1). The PTCA solution was made by hydrolyzing PTCDA in a minimal volume of 1.0 M sodium hydroxide. Red deposits appeared in the yellow-green solution, and then the mixture solution was treated with hydrochloric acid to neutralize the excess sodium hydroxide and the pH of the solution was maintained at slightly acidic. The PTCA were collected by centrifugation and dried under vacuum at room temperature. An amount of 2.5 mg of PTCA was dissolved in 10 mL of doubly distilled water by continuous ultrasonication. An amount of 5.0 mL of mixture solution of EDC and NHS (4:1) was added to the above solution to activate the carboxyl of PTCA, and the resulting mixture was kept under vigorous agitation for overnight at room temperature. Then, the prepared PEI−Ru(II) compound was added into the above solution with stirring at 4 °C for 6 h. The novel Ru(II)

NaOH, then diluting with doubly distilled water. All chemicals were analytical grade and used without further purification. All solutions were prepared with doubly distilled water and stored in the refrigerator (4 °C). Apparatus. The ECL emission was monitored by a model MPI-A electrochemiluminescence analyzer (Xi’An Remax Electronic Science and Technology Co. Ltd., Xi’An, China). The voltage of the photomultiplier tube (PMT) was set at 800 V, and scan rate was 100 mV/s in ECL detection. A conventional three-electrode system was used with Ag/AgCl (saturated KCl) as the reference electrode, a platinum wire as auxiliary electrode, and a modified GCE (Φ = 4 mm) as the working electrode in the experiment. Cyclic voltammetric (CV) measurements were carried out with a CHI 610A electrochemistry workstation (Shanghai CH Instruments, China). A three-electrode electrochemical cell was composed of a modified GCE (Φ = 4 mm) as the working electrode, a platinum wire as the auxiliary electrode, and a saturated calomel electrode (SCE) as the reference electrode. The morphologies of different nanocomposites were characterized by scanning electron microscopy (SEM, S-4800, Hitachi, Tokyo, Japan) at an acceleration voltage of 15−20 kV. The Fourier transform infrared (FT-IR) spectra were recorded on a Spectrum GX FTIR spectroscopy system (Perkin-Elmer, U.S.A.). X-ray photoelectron spectroscopy (XPS) measurements were carried out using a VG Scientific ESCALAB 250 spectrometer (Thermoelectricity Instruments, U.S.A.) and using Al Kα X-ray (1486.6 eV) as the light source. 1055



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Figure 2. SEM images of (A) PTCA−PEI−Ru(II)/CNTs after aging treatment, (B) HGNPs and (C) HGNPs/PTCA−PEI−Ru(II)/CNTs. The insets of panels A and C show a partially enlarged SEM image of PTCA−PEI−Ru(II)/CNTs and HGNPs/PTCA−PEI−Ru(II)/CNTs, respectively. (D) Full XPS spectrum of HGNPs/PTCA−PEI−Ru(II)/CNTs.

distilled water for three times. And then it was dispersed in 2 mL of 0.1 M PBS (pH 8.0). Preparation of Ab2/HGNPs/PTCA−PEI−Ru(II)/CNTs Probe (Ab2 Bioconjugates). First, 1.0 mg of carboxylated CNTs was dissolved in 3.0 mL of doubly distilled water by continuous ultrasonication to obtain a homogeneous suspension. Then, PTCA−PEI−Ru(II) solution was added into the above solution, and the resulting mixture was kept stirring for 8 h at room temperature via π−π stacking between CNTs and PTCA. The stirring was maintained overnight at room temperature as aging treatment. Subsequently, HGNPs solution was added into it, and the mixture was stirred at 4 °C for 8 h. Later, the solution was centrifuged at 8000 rpm for 15 min, the upper solution was removed, and the lower products were washed three times with doubly distilled water. The collected lower sediment was dispersed in 1.0 mL of PBS solution (pH 8.0). Next, 100 μL of anti-APE-1 solution was added into it, and the mixture was stirred continuously at 4 °C for overnight to obtain Ab2/HGNPs/PTCA−PEI−Ru(II)/CNTs probe (Ab2 bioconjugates). Scheme 1A shows the diagram of preparation of Ab2 bioconjugates. Fabrication of the ECL Immunosensor. To obtain a mirror-like surface, the GCE, with diameter of 4 mm, was polished with 0.3 and 0.05 μm alumina, respectively, following by rinsing thoroughly with water. After that, the electrode was sonicated successively in doubly distilled water and ethanol for 5 min; the GCE was allowed to dry in air. Before modification, the GCE was dried with nitrogen at room temperature.

derivative (PTCA−PEI−Ru(II)) was separated by centrifugation at 10 000 rpm for 15 min and dispersed in 2 mL of 0.1 M PBS (pH 8.0). Then this solution was transferred into a dialysis tube (MWCO 8500) and immersed in 300 mL of distilled water with mild shaking for 2 days. The mixture in the dialysis membrane was then vacuum-dried and redissolved in 10 mL of 0.1 M PBS (pH 8.0). The reaction yield was about 75%. Preparation of Hollow Gold Nanospheres. Hollow gold nanospheres (HGNPs) were obtained according to the literature with slightly modification.20 To ensure completely air-free solutions, whole processes were conducted in the nitrogen atmosphere. First 400 μL of a 0.1 M solution of sodium citrate was added to 100 mL of doubly distilled water with rapid magnetic stirring, And then, 400 μL of 0.1 M freshly prepared sodium borohydride solution was added. Subsequently, 100 μL of 0.5 M cobalt chloride solution was added into the above solution drop by drop with stirring, and the solution changed from pale pink to brown/gray indicating the reduction of Co(II) into cobalt nanoparticles. This solution was allowed to react for 30 min to completely hydrolyze the sodium borohydride. Upon ensuring complete hydrolysis of the sodium borohydride, the flow of nitrogen was increased and a 0.1 M solution of chloroauric acid was added at 50 μL/addition to a total volume of 300 μL. Upon completion of HAuCl4 solution addition, the nitrogen flow was stopped and the solution was opened to ambient conditions under rapid stirring to oxidize any remaining cobalt metal left in solution. The obtained HGNPs were centrifugally washed extensively with doubly 1056



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Figure 3. (A) Cyclic voltammograms (dash line) and its corresponding ECL curves (solid line) of PTCA−PEI−Ru(II) compounds modified GCE in air-saturated PBS (pH 8.0) with the potential scan of +0.2 and +1.25 V. (B) ECL curves of PTCA−PEI−Ru(II)/GCE (a) in PBS (pH 8.0). PTCA−Ru(II)/GCE with (b) and without (c) PEI in PBS (pH 8.0). Curve d shows the bare GCE in the PBS containing PEI. Scan rate: 100 mV/s.

ure 2C (the inset shows a partially enlarged SEM image of the nanocomposites) verified that the HGNPs were randomly attached outside the PTCA−PEI−Ru(II)/CNTs balls. Furthermore, Figure 2D shows the full XPS patterns of HGNPs/ PTCA−PEI−Ru(II)/CNTs nanocomposite. The band at about 286 eV can be attributed to the binding energy of Ru3d3/2, which is overlapped with the C 1s peak at about 285 eV. The wide-scan spectrum of the HGNPs/PTCA−PEI−Ru(II)/ CNTs, which was loaded with gold HGNPs (Figure 2D), also clearly shows the presence of gold (Au4d5/2 at 336 eV, Au4f5/2 at 89 eV, and Au5s at 110 eV). Possible Luminescence Mechanism of PTCA−PEI− Ru(II) Compounds. Figure 3 shows ECL (Figure 3A, solid line) and electrochemical responses (Figure 3A, dash line) obtained for PTCA−PEI−Ru(II) compounds modified GCE in air-saturated PBS (pH 8.0). First, no ECL happened at the potential domain below +1.0 V when the potential was scanned anodically from +0.2 V. Until the potential reached higher than 1.0 V, where PTCA−PEI−Ru(II) could be oxidized to PTCA− PEI−Ru(III) (see Figure 3A, dash line), a significant ECL emission was observed (with the peak intensity of 2815 au, Figure 3B, curve a), indicating that the ECL was from PTCA− PEI−Ru(II)* light emission. Compared with the ECL of PTCA−PEI−Ru(II) modified GCE, the PTCA−Ru(II) one shows less ECL peak intensity (1402 au) when it scanned in air-saturated PBS (pH 8.0) containing PEI as a coreactant in the test solution with the potential scan of +0.2 and +1.25 V (Figure 3B, curve b). Curve c in Figure 3B displays the ECL of PTCA−Ru(II) modified GCE in air-saturated PBS without PEI as a coreactant in the test solution. Only peak intensity of 601 au was obtained, which could demonstrate PEI would enhance the ECL of Ru(II). Besides, no ECL peak can be observed on the bare GCE in the PBS containing PEI (curve d, Figure 3B), due to the lack of luminophor in the system. The ECL dynamic curves of the above differently modified GCE are also displayed in Figure S3 of the Supporting Information. It can be found that the maximum intensity and the lowest luminous potential

The GCE was immersed in 2 mL of 1% HAuCl4 solution for electrochemical deposition under constant potential of −0.2 V for 30 s to obtain a porous AuNPs film modified electrode. Subsequently, 15 μL of anti-APE-1 (abbreviated as Ab1) was attached by incubating at 4 °C for 12 h. The immunosensor was then washed with doubly distilled water to remove the physically adsorbed Ab1 and then incubated for 1 h with 15 μL of 1% BSA, followed by washing with doubly distilled water. Ultimately, the obtained immunosensor was stored at 4 °C when not in use. Scheme 1B shows the schematic diagram of preparation of the ECL immunosensor. Measurement Procedure. The measurement was based on a sandwich immunoassay method. Before measurement, the immunosensor was incubated with APE-1 standard solution for 30 min at 37 °C. And then the modified electrode was incubated in Ab2 bioconjugates at 37 °C for 30 min. Finally, the obtained immunosensor was investigated with an MPI-A ECL analyzer in 3 mL of 0.1 M PBS (pH 8.0) at room temperature.

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RESULTS AND DISCUSSION Characteristics of Different Nanomaterials. The morphologies of different nanocomposites were characterized by SEM at an acceleration voltage of 15−20 kV. The typical SEM image of AuNPs by electrochemical deposition demonstrated that these AuNPs exhibit flower-like structures with a diameter of 400 ± 50 nm (Figure S2, Supporting Information). An image in Figure 2A reveals the coil-like spherical structure of PTCA−PEI−Ru(II)/CNTs after aging treatment, which also confirmed the well size distribution. This observation can be attributed to the strong π−π stacking and van der Waals interactions between PTCA−PEI−Ru(II) and CNTs. Figure 2B demonstrates the SEM of HGNPs with a well-defined shape and hollow spheres structure, indicating the successful preparation of HGNPs. The surface morphologies of HGNPs/PTCA−PEI−Ru(II)/CNTs nanocomposites in Fig1057



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[Fe(CN)6]3−/4− can be observed on the bare GCE (Figure 4, curve a). When AuNPs were electrodeposited on the electrode surface, the redox peak currents increased significantly (Figure 4, curve b), suggesting that AuNPs brought excellent conductivity and large surface area to promote the electron transfer. When the electrode was modified with anti-APE-1, an obvious decrease in redox current was observed (Figure 4, curve c). Subsequently, the redox peak currents further decreased after the modified electrode was blocked with 0.25% BSA solution (Figure 4, curve d). Finally, the redox peak currents decreased apparently (Figure 4, curve e) when incubated with APE-1 solution. The reason for this was that the anti-APE-1, BSA, and APE-1 protein layers on the electrode would retard the electron transfer. Comparison of Different Labeled Ab2 Bioconjugates. To investigate the efficiency of HGNPs/PTCA−PEI−Ru(II)/ CNTs labeled APE-1 antibody, we conducted the contrast experiment to compare their ECL responses of different probes under the same conditions. Three kinds of Ab2-functionalized probes were prepared, and the results are shown in Figure 5. The Ab2-functionalized probes are (a) Ru(II)-labeled Ab2, (b) HGNPs/PTCA−Ru(II)/CNTs labeled Ab2, and (c) HGNPs/ PTCA−PEI−Ru(II)/CNTs labeled Ab2. To control the reaction condition, the same batch of immunosensors was prepared and incubated with the 20 fg/mL APE-1, then incubated with different Ab2-functionalized probe solutions, respectively. As illustrated in Figure 5A, the ECL responses of the immunosensor with Ru(II)-labeled Ab2 was raised about 155 au compared with background values (the ECL responses of the BSA/anti-APE-1/AuNPs/GCE). Then about 790 au ECL emission was produced by the immunosensor with PTCA−Ru(II)/CNTs/HGNPs labeled Ab2 (Figure 5B). The enhancement could be attributed to the increase of the loading amount of Ru(II) on the CNTs via the π−π stacking interactions. When the immunosensor was incubated with the

was obtained by the self-enhanced PTCA−PEI−Ru(II) compounds, which suggested that the intramolecular ECL reaction could be more efficient as compared with the intermolecular reaction due to the shorter electron-transfer path and less energy loss. Thus, to gain insight into the ECL procedure of the selfenhanced PEI−Ru(II), we suppose the ECL mechanism of the self-enhanced PEI−Ru(II) system as the following equations: Electrochemical Behaviors of the Electrochemiluminescence Immunosensor. To confirm with the fabrication process of the ECL immunosensor, cyclic voltammogram measurement was employed to characterize the stepwise assembled process in 0.1 M PBS (pH 8.0) containing 5.0 mM [Fe(CN)6]3−/4− (acting as redox probe) and 0.1 M KCl. As shown in Figure 4, a pair of well-defined redox peaks of

Figure 4. Cyclic voltammograms at (a) bare glassy carbon electrode (GCE), (b) AuNPs/GCE, (c) anti-APE-1/AuNPs/GCE, (d) BSA/ anti-APE-1/AuNPs/GCE, and (e) APE-1/BSA/anti-APE-1/AuNPs/ GCE in 5.0 mM [Fe(CN)6]3−/4− containing 0.1 M KCl by scanning the potential from −0.2 to 0.6 V at a scan rate of 50 mV/s.

Figure 5. ECL−time profiles of the immunosensors by using various Ab2-functionalized probes: (A) Ru(II)-labeled Ab2, (B) HGNPs/PTCA− Ru(II)/CNTs labeled Ab2, and (C) HGNPs/PTCA−PEI−Ru(II)/CNTs labeled Ab2 based on sandwiched immunoassay toward zero analyte (green line) and 20 fg/mL APE-1 (red line) in pH 8.0 PBS. (D) Control experiments: ECL−time profiles of the immunosensors toward zero analyte (green line) and BSA (100 fg/mL) (red line) in pH 8.0 PBS in a standard sandwich format. Scan rate, 100 mV/s. 1058



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that the proposed method could be used to detect APE-1 concentration quantitatively. Stability, Selectivity, and Reproducibility of the Immunosensor. The stability of the immunosensor, which was evaluated under consecutive cyclic potential scans to various concentrations of APE-1 antigen, is presented in Figure 7A. It showed that the ECL intensity increased with the increasing of APE-1 concentration, and a relative stable curve at every concentration could be obtained. To further investigate the selectivity and specificity of the proposed immunosensor, contrast experiments were performed (see Figure 7B). Carcinoembryonic antigen (CEA), BSA, and α-1-fetoprotein (AFP) were used as interfering substance to evaluate the selectivity and specificity of the proposed immunosensor. The immunosensors were incubated with 100 fg/mL CEA, 100 fg/ mL BSA, and 200 ng/mL AFP, respectively. Almost no signal change was obtained compared with the background. The immunosensor was also incubated with 5 fg/mL APE-1 containing different interfering species; compared with the ECL response obtained from the 5 fg/mL APE-1 only, no significant difference was found. All these results indicated a good selectivity and specificity of the proposed immunosensor. Analysis of Human Serum Samples. To evaluate the applicability of this ECL immunoassay for detection in real samples, seven serum samples were diluted with the appropriate volumes of dilution solution and then were measured by the proposed ECL immunosensors. In parallel, the same sampled were also detected with chemiluminescence immunoassay (CLIA). The results are shown in Table 1. The relative deviations between the two methods were in the range of −5.3−9.1%, indicating an acceptable accuracy.

as-prepared probes of HGNPs/PTCA−PEI−Ru(II)/CNTs labeled Ab2, the ECL emission was noticeably raised about 2665 au, with which ECL efficiency is up to 17 times greater than that of Ru(II) (Figure 5C). By contrast, when a nontargeting protein (BSA, 100 fg/mL) was incubated with the as-prepared immunosensor, no significant ECL signal was observed (Figure 5D, red line), indicating the well specificity and selectivity of the proposed immunosensor. Thus, these comparison results adequately indicate that the as-prepared probes of HGNPs/PTCA−PEI−Ru(II)/CNTs labeled Ab2 could be utilized for ultrasensitive detection of APE-1 with the amplification of the ECL signal. Analytical Performance of ECL Immunosensors. To evaluate the sensitivity and quantitative range of the proposed immunoassay, the novel immunoassay format was employed to detect different concentrations of APE-1. It can be seen (Figure 6) that the ECL responses increases accordingly as the

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Figure 6. ECL−time curves of the immunosensor with different concentrations of APE-1 in 0.1 M PBS (pH 8.0). APE-1 concentration: (a) 1 fg/mL, (b) 2 fg/mL, (c) 5 fg/mL, (d) 0.01 pg/mL, (e) 0.02 pg/mL, (f) 0.1 pg/mL, (g) 0.2 pg/mL, (h) 1 pg/mL. The inset is the relationship between ECL intensity and the concentration of APE-1. The voltage of the photomultiplier tube was set at 800 V. Scan rate, 100 mV/s.

CONCLUSIONS Electrogenerated chemiluminescence of a novel self-enhanced ECL luminophore of Ru(II) complex, which combined the coreactant and the luminophor in one molecule, was demonstrated for the first time. On the basis of this selfenhanced Ru(II) complex, a “signal on” immunosensor was demonstrated for ultrasensitive detection of APE-1 to test the application of this ECL system, exhibiting significant enhancement of the ECL efficiency and stability without the adding of other coreactant. Such an ECL immunosensor also provides new opportunities for ultrasensitive detection of protein at very low concentrations, and it is expected to provide new

concentration of APE-1 is varied from 1 fg/mL to 1 pg/mL with a detection limit of 0.3 fg/mL (S/N = 3). The linear regression equation is I = 1429 log c + 9141 (where I is ECL intensity and c stands for the concentration of APE-1), and the correlation coefficient of R2 = 0.996. The results demonstrated

Figure 7. (A) ECL stability of proposed immunosensor to various concentrations of APE-1. (B) The selectivity of the proposed ECL immunosensor: CEA (100 fg/mL), BSA (100 fg/mL), AFP (100 fg/mL), blank, APE-1 (5 fg/mL), a mixture containing APE-1 (5 fg/mL), CEA (100 fg/mL), BSA (100 fg/mL), and AFP (100 fg/mL). 1059



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Table 1. Comparison of Serum APE-1 Levels Determined Using Two Methods serum samples

1

2

3

4

5

6

7

CLIA (pg/mL) immunosensor (pg/mL) relative deviation (%)

0.019 0.018 −5.3

0.029 0.031 6.9

0.084 0.089 6.0

0.11 0.12 9.1

0.26 0.25 −3.8

0.39 0.41 −5.1

0.57 0.56 −1.8

(13) Qi, H.; Qiu, X.; Xie, D.; Ling, C.; Gao, Q.; Zhang, C. Anal. Chem. 2013, 85 (8), 3886−3894. (14) Zanarini, S.; Rampazzo, E.; Ciana, L. D.; Marcaccio, M.; Marzocchi, E.; Montalti, M.; Paolucci, F.; Prodi, L. J. Am. Chem. Soc. 2009, 131, 2260−2267. (15) Duan, R.; Zhou, X.; Xing, D. Anal. Chem. 2010, 82, 3099−3103. (16) Perez-Tejeda, P.; Prado-Gotor, R.; Grueso, E. M. Inorg. Chem. 2012, 51 (20), 10825−10831. (17) Tang, X.; Zhao, D.; He, J.; Li, F.; Peng, J.; Zhang, M. Anal. Chem. 2013, 85, 1711−1718. (18) Liao, N.; Zhuo, Y.; Chai, Y.-Q.; Xiang, Y.; Cao, Y.; Yuan, R.; Han, J. Chem. Commun. 2012, 48 (7), 610−7612. (19) Liao, N.; Zhuo, Y.; Chai, Y.-Q.; Xiang, Y.; Han, J.; Yuan, R. Biosens. Bioelectron. 2013, 45, 189−194. (20) Schwartzberg, A. M.; Olson, T. Y.; Talley, C. E.; Zhang, J. Z. J. Phys. Chem. B 2006, 110, 19935−19944.

possibilities for other biomolecules diagnostics as well as for bioanalysis in general.

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ASSOCIATED CONTENT

S Supporting Information *

Supporting Information about FT-IR spectra of PTCA, Ru(II), and PTCA−PEI−Ru(II), SEM image of the electrochemically deposited AuNPs, and ECL dynamic curves of differently modified GCEs, as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*Phone: +86 23 68252277. Fax: +86 23 68253172. E-mail: [email protected]. *Phone: +86 23 68252277. Fax: +86 23 68253172. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by the NNSF of China (21275119, 21105081, 21075100), Research Fund for the Doctoral Program of Higher Education (RFDP) (20110182120010), Ministry of Education of China (Project 708073), Specialized Research Fund for the Doctoral Program of Higher Education (20100182110015), and Natural Science Foundation Project of Chongqing City (CSTC-2010BB4121, CSTC-2009BA1003), the Fundamental Research Funds for the Central Universities (XDJK2010C062, XDJK2012A004), China.

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