ultrasensitive apurinic/apyrimidinic endonuclease 1 immunosensing based on self-enhanced...

Ultrasensitive Apurinic/Apyrimidinic Endonuclease 1Immunosensing Based on Self-Enhanced Electrochemiluminescenceof a Ru(II) ComplexYing 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 forultrasensitive detection of apurinic/apyrimidinic endonuclease1 (APE-1) was designed utilizing the self-enhanced electro-chemiluminescence (ECL) of a novel Ru(II) complexfunctionalized coil-like nanocomposite as signal labels. Thedesirable self-enhanced ECL luminophore was achieved bycombining the coreactant of poly(ethylenimine) (PEI) and theluminophor of bis(2,2′-bipyridine)-5-amino-1,10-phenanthro-line ruthenium(II) [Ru(bpy)2(5-NH2-1,10-phen)

2+] to formone novel Ru(II) complex, which exhibited significantlyenhanced ECL efficiency and stability. Moreover, the carbonnanotubes (CNTs) were employed as nanocarriers for self-enhanced Ru(II) complex loading via π−π stacking to obtain the coil-like nanocomposite to act as signal probe. Compared withtraditional ECL immunoassay, our proposed strategy is simple and sensitive, avoiding the adding of any coreactant into testingsolution for signal amplification, and shows a detection limit down to subfemtogram per milliliter level under the optimizedexperimental condition.

The apurinic/apyrimidinic endonuclease 1(APE-1) is amultifunctional enzyme that acts not only as a baseless

endonuclease but also as a redox-modifying factor for a varietyof transcription factors.1 Additionally, researches indicate that ithas multiple possible roles in the response of human cancer toradiotherapy and chemotherapy.2 Up to now, various methodsand strategies have been applied for the detection of APE-1,such as enzyme-linked immunoassay (ELISA),3 stopped-flowfluorescence analysis,4 and electrophoretic mobility-shift assay(EMSA).5 However, improvements are still required, as thesemethods remain cumbersome, time-consuming, and harmful tothe operator’s health. In this regard, electrochemiluminescence(ECL) immunosensors are competitive with conventionalassays, because of their high sensitivity, controllability of ECLreaction, 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, reversibleelectrochemical behavior, and luminescence efficiency over awide range of buffer pH levels.10−12 Currently, great effortshave been made toward the enhancement of the luminousintensity of the Ru(bpy)3

2+-based ECL system by employingthe appropriate substance as effective coreactant.13−15 ThePerez-Tejeda’s group achieved the enhancement of ECL

efficiency of Ru(bpy)32+ using the PAMAM G1.5 dendrimers

as coreactant.16 Tang et al. have developed a DNA sensor withadding tripropylamine (TPA) into working buffer solution as acoreactant.17 In our previous work, we have prepared theapoferritin-templated poly(ethylenimine) (PEI) nanoparticlesas labels based on in situ releasing the coreactant of PEI todevelop a sensitive ECL immunosensor.18 Subsequently, wealso have constructed of a reagentless ECL immunosensor byimmobilizing the coreactant of poly-L-lysine to enhance theECL of Ru(bpy)3

2+ for signal amplification.19 Although theluminous intensity of the Ru(bpy)3

2+ is indeed enhanced byadding additives as effective coreactant, it suffers from theproblems of operational complexity, which would result in theadding the coreactant in the detection solution or immobilizingthe coreactant on the sensing interface.With the goal of obtaining the desirable luminophores, we

recently observed the self-enhanced ECL complex by covalentlybinding the proper coreactant to the traditional luminescentmolecules, which might obtain the highly efficient and stableECL emitters. Consequently, we have synthesized a novel

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

Article

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© 2013 American Chemical Society 1053 dx.doi.org/10.1021/ac403019e | Anal. Chem. 2014, 86, 1053−1060

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Ru(II) complex by binding bis(2,2′-bipyridine)-5-amino-1,10-phenanthroline ruthenium(II) [Ru(bpy)2(5-NH2-1,10-phen)2+] to the poly(ethylenimine) (PEI), a linear structuremolecule with a backbone of two methylene units followed byone tertiary amine group which has been proven to serve as aneffective coreactant of Ru(bpy)3

2+ in our previous work. It wasfound that the ECL efficiency is significantly enhanced bycovalently coupling the coreactant of PEI and luminophor of[Ru(bpy)2(5-NH2-1,10-phen)

2+] to form one molecule, sincethe intramolecular ECL reaction could be more efficient ascompared with the intermolecular reaction due to the shorterelectron-transfer path and less energy loss.Thus, an alternative “signal on” immunosensor for ultra-

sensitive detection of APE-1 utilizing the self-enhanced ECLcomplex of PEI−Ru(II) was developed in this work. In order toachieve the high sensitivity, the aromatic compound of 3,4,9,10-perylene tetracarboxylic acid (PTCA) was bound to PEI−Ru(II), acting as enhancer and linker for subsequent assemblyon the carbon nanotubes (CNTs) via π−π stacking. Then theCNTs 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 groupsof PTCA−PEI−Ru(II). The HGNPs decorated coil-likePTCA−PEI−Ru(II)/CNTs composite (HGNPs/PTCA−PEI−Ru(II)/CNTs) showed uniform size distribution, goodstability, and significant ECL intensity and could easily serve asa tracing tag to label detection antibody (Ab2). The ECLimmunosensing interface was constructed by simple modifica-tion of capture antibody (Ab1) on the electrochemicallydeposited Au nanoparticles (AuNPs) decorated glassy carbonelectrode (GCE). Further investigation indicated that theproposed sandwiched immunosensor showed ultrasensitive

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

■ EXPERIMENTAL METHODS

Reagents and Materials. Bis(2,2′-bipyridine)(5-amino-1,10-phenanthroline) ruthenium(II) dichloride [Ru(bpy)2(5-NH2-1,10-phen)Cl2] was from Suna Tech Inc. (Suzhou,China). Apurinic/apyrimidinic endonuclease (APE-1) andapurinic/apyrimidinic endonuclease antibody (anti-APE-1,monoclonal antibody) were purchased from Santa CruzBiotechnology, Inc. (U.S.A.). Branched polyethylenimine(PEI, 99%, average Mn ∼10 000 by GPC), gold chloride(HAuCl4), and bovine serum albumin (BSA, 96−99%) werepurchased from Sigma-Aldrich Co. (St. Louis, MO, U.S.A.).Perylene-3,4,9,10-tetracarboxylic dianhydride (PTCDA) wasfrom Lian Gang Dyestuff Chemical Industry Co. Ltd.(Liaoning, China). The multiwalled carbon nanotubes(CNTs, >95% purity) synthesized by the CVD method werepurchased 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) withvarious pH values and concentrations were prepared by mixingstandard 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

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

Analytical Chemistry Article

dx.doi.org/10.1021/ac403019e | Anal. Chem. 2014, 86, 1053−10601054

NaOH, then diluting with doubly distilled water. All chemicalswere analytical grade and used without further purification. Allsolutions were prepared with doubly distilled water and storedin the refrigerator (4 °C).Apparatus. The ECL emission was monitored by a model

MPI-A electrochemiluminescence analyzer (Xi’An RemaxElectronic Science and Technology Co. Ltd., Xi’An, China).The voltage of the photomultiplier tube (PMT) was set at 800V, and scan rate was 100 mV/s in ECL detection. Aconventional three-electrode system was used with Ag/AgCl(saturated KCl) as the reference electrode, a platinum wire asauxiliary electrode, and a modified GCE (Φ = 4 mm) as theworking electrode in the experiment. Cyclic voltammetric (CV)measurements were carried out with a CHI 610A electro-chemistry workstation (Shanghai CH Instruments, China). Athree-electrode electrochemical cell was composed of amodified GCE (Φ = 4 mm) as the working electrode, aplatinum wire as the auxiliary electrode, and a saturated calomelelectrode (SCE) as the reference electrode. The morphologiesof different nanocomposites were characterized by scanningelectron microscopy (SEM, S-4800, Hitachi, Tokyo, Japan) atan acceleration voltage of 15−20 kV. The Fourier transforminfrared (FT-IR) spectra were recorded on a Spectrum GX FT-IR spectroscopy system (Perkin-Elmer, U.S.A.). X-ray photo-electron spectroscopy (XPS) measurements were carried outusing a VG Scientific ESCALAB 250 spectrometer (Thermo-electricity Instruments, U.S.A.) and using Al Kα X-ray (1486.6eV) as the light source.

Preparation of PTCA−PEI−Ru(II) Compounds. Theoverall process involved in fabricating the PTCA−PEI−Ru(II)compounds is shown schematically in Figure 1. To prepare thePEI−Ru(II) molecular compounds, glutaraldehyde (GA) wasused 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 of5.0 mM [Ru(bpy)2(5-NH2-1,10-phen)

2+] solution; 0.5 mL ofcross-linking agent 1 wt % GA was added into above solutionwith the incubation time of 12 h under constant stirring. Theunreacted GA and [Ru(bpy)2(5-NH2-1,10-phen)

2+] werepurified by dialysis (with a molecular weight cutoff of 8500)in distilled water for 1 day and PEI−Ru(II) compoundssolution was obtained. The reaction was monitored by FT-IRspectroscopy (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 depositsappeared in the yellow-green solution, and then the mixturesolution was treated with hydrochloric acid to neutralize theexcess sodium hydroxide and the pH of the solution wasmaintained at slightly acidic. The PTCA were collected bycentrifugation and dried under vacuum at room temperature.An amount of 2.5 mg of PTCA was dissolved in 10 mL ofdoubly distilled water by continuous ultrasonication. Anamount of 5.0 mL of mixture solution of EDC and NHS(4:1) was added to the above solution to activate the carboxylof PTCA, and the resulting mixture was kept under vigorousagitation for overnight at room temperature. Then, theprepared PEI−Ru(II) compound was added into the abovesolution with stirring at 4 °C for 6 h. The novel Ru(II)

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



derivative (PTCA−PEI−Ru(II)) was separated by centrifuga-tion at 10 000 rpm for 15 min and dispersed in 2 mL of 0.1 MPBS (pH 8.0). Then this solution was transferred into a dialysistube (MWCO 8500) and immersed in 300 mL of distilledwater with mild shaking for 2 days. The mixture in the dialysismembrane was then vacuum-dried and redissolved in 10 mL of0.1 M PBS (pH 8.0). The reaction yield was about 75%.Preparation of Hollow Gold Nanospheres. Hollow gold

nanospheres (HGNPs) were obtained according to theliterature with slightly modification.20 To ensure completelyair-free solutions, whole processes were conducted in thenitrogen atmosphere. First 400 μL of a 0.1 M solution ofsodium citrate was added to 100 mL of doubly distilled waterwith rapid magnetic stirring, And then, 400 μL of 0.1 M freshlyprepared sodium borohydride solution was added. Subse-quently, 100 μL of 0.5 M cobalt chloride solution was addedinto the above solution drop by drop with stirring, and thesolution changed from pale pink to brown/gray indicating thereduction of Co(II) into cobalt nanoparticles. This solution wasallowed to react for 30 min to completely hydrolyze the sodiumborohydride. Upon ensuring complete hydrolysis of the sodiumborohydride, the flow of nitrogen was increased and a 0.1 Msolution of chloroauric acid was added at 50 μL/addition to atotal volume of 300 μL. Upon completion of HAuCl4 solutionaddition, the nitrogen flow was stopped and the solution wasopened to ambient conditions under rapid stirring to oxidizeany remaining cobalt metal left in solution. The obtainedHGNPs were centrifugally washed extensively with doubly

distilled water for three times. And then it was dispersed in 2mL of 0.1 M PBS (pH 8.0).

Preparation of Ab2/HGNPs/PTCA−PEI−Ru(II)/CNTsProbe (Ab2 Bioconjugates). First, 1.0 mg of carboxylatedCNTs was dissolved in 3.0 mL of doubly distilled water bycontinuous ultrasonication to obtain a homogeneous suspen-sion. Then, PTCA−PEI−Ru(II) solution was added into theabove solution, and the resulting mixture was kept stirring for 8h at room temperature via π−π stacking between CNTs andPTCA. The stirring was maintained overnight at roomtemperature as aging treatment. Subsequently, HGNPs solutionwas 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, theupper solution was removed, and the lower products werewashed three times with doubly distilled water. The collectedlower sediment was dispersed in 1.0 mL of PBS solution (pH8.0). Next, 100 μL of anti-APE-1 solution was added into it,and the mixture was stirred continuously at 4 °C for overnightto obtain Ab2/HGNPs/PTCA−PEI−Ru(II)/CNTs probe(Ab2 bioconjugates). Scheme 1A shows the diagram ofpreparation of Ab2 bioconjugates.

Fabrication of the ECL Immunosensor. To obtain amirror-like surface, the GCE, with diameter of 4 mm, waspolished with 0.3 and 0.05 μm alumina, respectively, followingby rinsing thoroughly with water. After that, the electrode wassonicated successively in doubly distilled water and ethanol for5 min; the GCE was allowed to dry in air. Before modification,the GCE was dried with nitrogen at room temperature.

Figure 2. SEM images of (A) PTCA−PEI−Ru(II)/CNTs after aging treatment, (B) HGNPs and (C) HGNPs/PTCA−PEI−Ru(II)/CNTs. Theinsets 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.



The GCE was immersed in 2 mL of 1% HAuCl4 solution forelectrochemical deposition under constant potential of −0.2 Vfor 30 s to obtain a porous AuNPs film modified electrode.Subsequently, 15 μL of anti-APE-1 (abbreviated as Ab1) wasattached by incubating at 4 °C for 12 h. The immunosensorwas then washed with doubly distilled water to remove thephysically adsorbed Ab1 and then incubated for 1 h with 15 μLof 1% BSA, followed by washing with doubly distilled water.Ultimately, the obtained immunosensor was stored at 4 °Cwhen not in use. Scheme 1B shows the schematic diagram ofpreparation of the ECL immunosensor.Measurement Procedure. The measurement was based

on a sandwich immunoassay method. Before measurement, theimmunosensor was incubated with APE-1 standard solution for30 min at 37 °C. And then the modified electrode wasincubated in Ab2 bioconjugates at 37 °C for 30 min. Finally,the obtained immunosensor was investigated with an MPI-AECL analyzer in 3 mL of 0.1 M PBS (pH 8.0) at roomtemperature.

■ RESULTS AND DISCUSSION

Characteristics of Different Nanomaterials. Themorphologies of different nanocomposites were characterizedby SEM at an acceleration voltage of 15−20 kV. The typicalSEM image of AuNPs by electrochemical depositiondemonstrated that these AuNPs exhibit flower-like structureswith a diameter of 400 ± 50 nm (Figure S2, SupportingInformation). An image in Figure 2A reveals the coil-likespherical structure of PTCA−PEI−Ru(II)/CNTs after agingtreatment, which also confirmed the well size distribution. Thisobservation can be attributed to the strong π−π stacking andvan der Waals interactions between PTCA−PEI−Ru(II) andCNTs. Figure 2B demonstrates the SEM of HGNPs with awell-defined shape and hollow spheres structure, indicating thesuccessful preparation of HGNPs. The surface morphologies ofHGNPs/PTCA−PEI−Ru(II)/CNTs nanocomposites in Fig-

ure 2C (the inset shows a partially enlarged SEM image of thenanocomposites) verified that the HGNPs were randomlyattached outside the PTCA−PEI−Ru(II)/CNTs balls. Fur-thermore, Figure 2D shows the full XPS patterns of HGNPs/PTCA−PEI−Ru(II)/CNTs nanocomposite. The band at about286 eV can be attributed to the binding energy of Ru3d3/2,which is overlapped with the C 1s peak at about 285 eV. Thewide-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, solidline) and electrochemical responses (Figure 3A, dash line)obtained for PTCA−PEI−Ru(II) compounds modified GCE inair-saturated PBS (pH 8.0). First, no ECL happened at thepotential domain below +1.0 V when the potential was scannedanodically from +0.2 V. Until the potential reached higher than1.0 V, where PTCA−PEI−Ru(II) could be oxidized to PTCA−PEI−Ru(III) (see Figure 3A, dash line), a significant ECLemission 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 ofPTCA−PEI−Ru(II) modified GCE, the PTCA−Ru(II) oneshows less ECL peak intensity (1402 au) when it scanned inair-saturated PBS (pH 8.0) containing PEI as a coreactant inthe 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 ofPTCA−Ru(II) modified GCE in air-saturated PBS without PEIas a coreactant in the test solution. Only peak intensity of 601au was obtained, which could demonstrate PEI would enhancethe ECL of Ru(II). Besides, no ECL peak can be observed onthe bare GCE in the PBS containing PEI (curve d, Figure 3B),due to the lack of luminophor in the system. The ECL dynamiccurves of the above differently modified GCE are also displayedin Figure S3 of the Supporting Information. It can be foundthat the maximum intensity and the lowest luminous potential

Figure 3. (A) Cyclic voltammograms (dash line) and its corresponding ECL curves (solid line) of PTCA−PEI−Ru(II) compounds modified GCEin 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.



was obtained by the self-enhanced PTCA−PEI−Ru(II)compounds, which suggested that the intramolecular ECLreaction could be more efficient as compared with theintermolecular reaction due to the shorter electron-transferpath and less energy loss.Thus, to gain insight into the ECL procedure of the self-

enhanced PEI−Ru(II), we suppose the ECL mechanism of theself-enhanced PEI−Ru(II) system as the following equations:Electrochemical Behaviors of the Electrochemilumi-

nescence Immunosensor. To confirm with the fabricationprocess of the ECL immunosensor, cyclic voltammogrammeasurement was employed to characterize the stepwiseassembled process in 0.1 M PBS (pH 8.0) containing 5.0mM [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

[Fe(CN)6]3−/4− can be observed on the bare GCE (Figure 4,

curve a). When AuNPs were electrodeposited on the electrodesurface, the redox peak currents increased significantly (Figure4, curve b), suggesting that AuNPs brought excellentconductivity and large surface area to promote the electrontransfer. When the electrode was modified with anti-APE-1, anobvious decrease in redox current was observed (Figure 4,curve c). Subsequently, the redox peak currents furtherdecreased after the modified electrode was blocked with0.25% BSA solution (Figure 4, curve d). Finally, the redox peakcurrents decreased apparently (Figure 4, curve e) whenincubated with APE-1 solution. The reason for this was thatthe anti-APE-1, BSA, and APE-1 protein layers on the electrodewould 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 contrastexperiment to compare their ECL responses of different probesunder the same conditions. Three kinds of Ab2-functionalizedprobes 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 thereaction condition, the same batch of immunosensors wasprepared and incubated with the 20 fg/mL APE-1, thenincubated with different Ab2-functionalized probe solutions,respectively. As illustrated in Figure 5A, the ECL responses ofthe immunosensor with Ru(II)-labeled Ab2 was raised about155 au compared with background values (the ECL responsesof the BSA/anti-APE-1/AuNPs/GCE). Then about 790 auECL emission was produced by the immunosensor withPTCA−Ru(II)/CNTs/HGNPs labeled Ab2 (Figure 5B). Theenhancement could be attributed to the increase of the loadingamount of Ru(II) on the CNTs via the π−π stackinginteractions. When the immunosensor was incubated with the

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 scanningthe 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.



as-prepared probes of HGNPs/PTCA−PEI−Ru(II)/CNTslabeled Ab2, the ECL emission was noticeably raised about2665 au, with which ECL efficiency is up to 17 times greaterthan that of Ru(II) (Figure 5C). By contrast, when anontargeting protein (BSA, 100 fg/mL) was incubated withthe as-prepared immunosensor, no significant ECL signal wasobserved (Figure 5D, red line), indicating the well specificityand selectivity of the proposed immunosensor. Thus, thesecomparison results adequately indicate that the as-preparedprobes of HGNPs/PTCA−PEI−Ru(II)/CNTs labeled Ab2could be utilized for ultrasensitive detection of APE-1 with theamplification of the ECL signal.Analytical Performance of ECL Immunosensors. To

evaluate the sensitivity and quantitative range of the proposedimmunoassay, the novel immunoassay format was employed todetect different concentrations of APE-1. It can be seen (Figure6) that the ECL responses increases accordingly as the

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

that the proposed method could be used to detect APE-1concentration quantitatively.

Stability, Selectivity, and Reproducibility of theImmunosensor. The stability of the immunosensor, whichwas evaluated under consecutive cyclic potential scans tovarious concentrations of APE-1 antigen, is presented in Figure7A. It showed that the ECL intensity increased with theincreasing of APE-1 concentration, and a relative stable curve atevery concentration could be obtained. To further investigatethe 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 theselectivity and specificity of the proposed immunosensor. Theimmunosensors were incubated with 100 fg/mL CEA, 100 fg/mL BSA, and 200 ng/mL AFP, respectively. Almost no signalchange was obtained compared with the background. Theimmunosensor was also incubated with 5 fg/mL APE-1containing different interfering species; compared with theECL response obtained from the 5 fg/mL APE-1 only, nosignificant difference was found. All these results indicated agood selectivity and specificity of the proposed immunosensor.

Analysis of Human Serum Samples. To evaluate theapplicability of this ECL immunoassay for detection in realsamples, seven serum samples were diluted with theappropriate volumes of dilution solution and then weremeasured by the proposed ECL immunosensors. In parallel,the same sampled were also detected with chemiluminescenceimmunoassay (CLIA). The results are shown in Table 1. Therelative deviations between the two methods were in the rangeof −5.3−9.1%, indicating an acceptable accuracy.

■ CONCLUSIONS

Electrogenerated chemiluminescence of a novel self-enhancedECL luminophore of Ru(II) complex, which combined thecoreactant and the luminophor in one molecule, wasdemonstrated for the first time. On the basis of this self-enhanced Ru(II) complex, a “signal on” immunosensor wasdemonstrated for ultrasensitive detection of APE-1 to test theapplication of this ECL system, exhibiting significant enhance-ment of the ECL efficiency and stability without the adding ofother coreactant. Such an ECL immunosensor also providesnew opportunities for ultrasensitive detection of protein at verylow concentrations, and it is expected to provide new

Figure 6. ECL−time curves of the immunosensor with differentconcentrations of APE-1 in 0.1 M PBS (pH 8.0). APE-1concentration: (a) 1 fg/mL, (b) 2 fg/mL, (c) 5 fg/mL, (d) 0.01pg/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 theconcentration of APE-1. The voltage of the photomultiplier tubewas set at 800 V. Scan rate, 100 mV/s.

Figure 7. (A) ECL stability of proposed immunosensor to various concentrations of APE-1. (B) The selectivity of the proposed ECLimmunosensor: 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).



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

■ ASSOCIATED CONTENT*S Supporting InformationSupporting Information about FT-IR spectra of PTCA, Ru(II),and PTCA−PEI−Ru(II), SEM image of the electrochemicallydeposited AuNPs, and ECL dynamic curves of differentlymodified GCEs, as noted in text. This material is available freeof charge via the Internet at http://pubs.acs.org.

■ AUTHOR INFORMATIONCorresponding 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] authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThis research was supported by the NNSF of China(21275119, 21105081, 21075100), Research Fund for theDoctoral Program of Higher Educat ion (RFDP)(20110182120010), Ministry of Education of China (Project708073), Specialized Research Fund for the Doctoral Programof Higher Education (20100182110015), and Natural ScienceFoundation Project of Chongqing City (CSTC-2010BB4121,CSTC-2009BA1003), the Fundamental Research Funds for theCentral Universities (XDJK2010C062, XDJK2012A004),China.

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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) 0.019 0.029 0.084 0.11 0.26 0.39 0.57immunosensor (pg/mL) 0.018 0.031 0.089 0.12 0.25 0.41 0.56relative deviation (%) −5.3 6.9 6.0 9.1 −3.8 −5.1 −1.8



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