Electrochemiluminescence Resonance Energy Transfer System

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Electrochemiluminescence Resonance Energy Transfer System: Mechanism and Application in Ratiometric Aptasensor for Lead Ion Yan-Mei Lei, Wei-Xing Huang, Min Zhao, Ya-Qin Chai, Ruo Yuan,* and Ying Zhuo* The Key Laboratory of Eco-Environments in the Three Gorges Reservoir Region, College of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, China S Supporting Information *

ABSTRACT: In this paper, a novel electrochemiluminescence resonance energy transfer (ECL-RET) system from O2/S2O82− to a kind of amino-terminated perylene derivative (PTC-NH2) was demonstrated for the first time, which was then applied to construct a ratiometric aptasensor for lead ion (Pb2+) detection. First, gold-nanoparticles-functionalized fullerene nanocomposites (AuNPs@nano-C60) were coated on a glassy carbon electrode (GCE), and then thiol-modified assistant probes (APs) were attached on AuNPs@nano-C60/GCE. Then the resultant electrode was hybridized with capture probes (the aptamer of the Pb2+, abbreviated as CPs) to generate DNA duplexes, which could induce PTC-NH2 to be intercalated into the dsDNA grooves by the electrostatic adsorption. Herein, ECL dual peaks at −0.7 V (vs Ag/AgCl) and −2.0 V (vs Ag/AgCl) were obtained when the prepared aptasensor was detected in air-saturated S2O82− solution, which could be attributed to the emission of excited dimmers (π-excimers) (1(NH2−PTC)2*) and 1(O2)2*, respectively. In the presence of Pb2+, the dsDNA was unwound, and Pb2+ G-quadruplex structure was generated because of the highly specific affinity between Pb2+ and CPs, which made the PTC-NH2 release from the electrode surface. As a result, the ECL signal at −0.7 V was decreased, and the ECL signal around −2.0 V was increased. By measuring the ratio of ECL intensities at two excitation potentials, the developed aptasensor exhibited the linear response range from 1.0 × 10−12 M to 1.0 × 10−7 M with a detection limit of 3.5 × 10−13 M (S/N = 3) for Pb2+, which could offer an alternative analytical method with excellent properties of high selectivity, accuracy, and sensitivity.

R

ECL-RET donor and acceptor, are limited. Herein, a novel ECL-RET protocol involving energy transfer from peroxydisulfate/oxygen (O2/S2O82−) as an ECL donor to a kind of amino-terminated perylene derivative (PTC-NH2) as an ECL acceptor was reported for the first time. Usually, S2O82− is well-known as a coreactant in ECL systems, such as the metal complexes/S2O82− (Ru(bpy)32+, Eu(crypt)3+, [Pt(tBu3tpy)(CC−C6H4-4-carbazole-9)]+),7−9 semiconductor nanoparticles/S 2 O 8 2 − (CdSe, TiO 2 , C3N4),10−12 and the polyaromatic hydrocarbons/S2O82− (9naphthylanthracene derivatives, anthracene, rubrene)13,14 cathodic ECL system. However, the ECL signal was observed in air-saturated S2O82− solution, which demonstrated that it could be serve as novel ECL emitters with the possible mechanism of the radiative deactivation of the singlet state oxygen (1(O2)2*).15 Thus, the air-saturated S2O82− solution has come into focus as an ECL luminophore due to its fascinating characteristics of simplicity, availability, sensitivity, and cheapness. To further improve the ECL intensity of the S2O82− solution, many S2O82− based-biosensors have been developed

esonance energy transfer (RET) has often occurred between a suitably matched acceptor and donor pair, which has established a platform for fluorescence (FRET), bioluminescence (BRET) and chemiluminesence (CRET).1 Nowadays, with the development of electrochemiluminescence (ECL) technical analysis, which is a means of converting electrical energy into radiative energy, electrochemiluminescence resonance energy transfer (ECL-RET) has received much attention in analysis of ion, protein, and DNA because its advantages are that it could be sensitive and controllable ECL switches for signal amplification.2 It is reported that the critical step to obtain optimal ECL-RET efficiency is to search for the perfect energy overlapped donor−acceptor pair.3 Xu’s group reported a ECL-RET protocol between ECL donor of CdS quantum dots (QDs) and acceptor of Ru(bpy)32+, which was developed for a sensitive cytosensor construction.4 Zhu’s group constructed a label-free aptasensor for the detection of thrombin based on the ECL-RET between luminol as a donor and CdSe@ZnS QDs as an acceptor in neutral conditions.5 Wu et al. also explored the ECL-RET system with graphene oxide-Au/RuSi@Ru(bpy)32+ /chitosan composites as the ECL donor and Au@Ag2S NPs as the ECL acceptor which was successfully applied to detection of target DNA.6 Therefore, it is significant to look for a new donor−acceptor pair because energy-tunable materials, especially the appealing © 2015 American Chemical Society

Received: April 17, 2015 Accepted: July 8, 2015 Published: July 8, 2015 7787

DOI: 10.1021/acs.analchem.5b01445 Anal. Chem. 2015, 87, 7787−7794

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Analytical Chemistry

Anhydrous ethylenediamine (C2H8N4, ≥99.9%) was bought from J&K Scientific Ltd. (Chongqing, China). Hydrogen tetrachloroaurate (HAuCl4·4H2O, 99.9%), bovine serum albumin (BSA, 96−99%), hexanethiol (HT, 96%), and ascorbic acid (AA, 99.7%) were purchased from Sigma Chemical Co. (St. Louis, MO). 3,4,9,10-Perylenete-tracarboxylic dianhydride (PTCDA,) was provided by LianGang Pigment and Dyestuff Chemical Industry Co., Ltd (Liaoning, China). Oligonucleotides were purchased from Sangon Bioengineering Ltd. Company (Shanghai, China), and their sequences were shown as follows: Assistant probes (APs): 5′-SH-(CH2)6 - AAC CAC ACC AA-3′; Capture probes (CPs): 5′-GGT TGG TGT GGT TGG-3′. The underlined base sequence of APs can hybridize with the underlined base sequence of CPs. DNA oligonucleotide stock solutions were prepared with Tris-HCl buffer containing 140 mM NaCl, 5 mM KCl, and 1 mM MgCl2 and were kept frozen under refrigeration with temperatures ranging from −10 to 0 °C. All other chemicals not mentioned here were of analytical reagent grade and used as received. Deionized water was used throughout the whole experiment. Apparatus. The ECL emission and cyclic voltammetric (CV) measurements were detected using an MPI-E multifunctional electrochemical and chemiluminescent analytical system (Xi’an Remax Electronic Science and Technology Co., Ltd., Xi’an, China). All experiment were performed with the conventional three-electrode system including a modified glassy carbon electrode (GCE, Φ = 4.0 mm) as working electrode, Ag/AgCl (saturated KCl) as reference electrode, and a platinum wire as counter electrode, respectively. The morphologies of nanocomposites were obtained by scanning electron microscope (SEM, S-4800, Hitachi, Tokyo, Japan) at a voltage of 20−30 kV. The UV−vis absorption spectra of PTCNH2 were recorded by UV-2450 UV−vis spectrophotometer (Shimadzu, Tokyo, Japan). Fluorescence spectra of PTC-NH2 were obtained by RF-5301PC spectrophotometer (Shimadzu, Tokyo, Japan) in this work. Preparation of AuNPs@Nano-C60 Composite. First, the C60 nanoparticles (nano-C60) were prepared by an ultrasoundassisted solution-phase conversion process according to the literature.23 To obtain abundant −NH2 and −SH groups on the surface of nano-C60, 5.0 mL of the above nano-C60 suspension (5 mg mL−1) was mixed with 2 mL of BSA (5%) solution with stirring for 12 h. Then, the BSA functionlized product was centrifuged, washed, and redispersed with 5 mL of deionized water. Next, 0.1 mL of 1.0% HAuCl4 solution was added into the above mixture suspension with stirring for 3 h. Under vigorous stirring, 5 mL of AA (50 mM) was added into above solution with stirring overnight at room temperature. The obtained AuNPs@nano-C60 was centrifuged and washed with deionized water three times. Finally, the obtained precipitate was redispersed in 2.5 mL of deionized water and stored in the refrigerator at 4 °C when not in use. Preparation of PTC-NH2. The amino-terminated perylene derivative (PTC-NH2) was achieved by the ammonolysis reaction of 3,4,9,10-perylenetetracarboxylic dianhydride (PTCDA) as follows: First, 0.20 g PTCDA was dissolved in 5 mL of acetone under stirring. Then, 0.5 mL anhydrous ethylenediamine (C2H8N4) was added into the above solution slowly at 4 °C. Subsequently, in order to remove the residual ethylenediamine, the ammonolysis product of PTCDA (named as PTC-NH2) was centrifuged and washed until the pH was

with the aid of many kinds of signal nanoenhancers or coreactants in our previous work, such as gold nanoparticles (AuNPs), fullerene nanoparticles (C60NPs), L-cysteine (L-cys), thiosemicarbazide (TSC),16,17 and so forth. To the best of our knowledge, there have been no reports on the ECL-RET of the air-saturated S2O82− solution as a luminophore. The planar perylene derivatives with π system have been widely used as highly fluorescent molecular, electrophotographic devices and organic solar cell, which could be attributed to their outstanding photoelectron properties, good chemical/thermal stability and wide absorption and emission bands within visible light range.18−20 Due to their high photoluminescence quantum yields, these perylene derivatives are also promising candidates as light emitters in ECL devices. Bard’s group reported the ECL of perylenedicarboxylic imide (PI) and perylenetetracarboxylic diimide (PDI) in mixed organic solvent of CHCl3/CH3CN (4:1).21 Murray’s group also demonstrated the ECL of the polyether perylene derivatives in CH3CN/H2O solution containing (NH4)2S2O8.22 However, these works indicated the test solutions were dependent on organic solvents, which limited the further application in the ECL biosensor using perylene derivatives as luminophore. Herein, the ECL of the PTC-NH2 was observed when it was intercalated into the dsDNA grooves via electrostatic adsorption, but additionally, the interaction mechanism between PTC-NH2 and the airsaturated S2O82− solution was first demonstrated according to the ECL-RET, exhibiting great potential application in a sensitive and controllable ECL biosensor construction. Thus, we describe a label-free ratiometric aptasensor for detection of Pb2+ based on a novel ECL-RET system between the PTC-NH2 and O2/S2O82− in this work. Herein, the prepared AuNPs@nano-C60 were coated on a glassy carbon electrode, which not only possessed good film forming ability, high specific surface area and ECL enhancement of the O2/ S2O82− system but also could provide the active interface for thiol-modified assistant probes (APs) immobilization via Au−S bonds. Capture probes (the aptamer of the Pb2+, abbreviated as CPs) were hybridized with APs to generate DNA duplexes, which could induce PTC-NH2 to be intercalated into the dsDNA grooves by the electrostatic interaction. At this step, the two ECL signal peaks were achieved, which represented the ECL emittance of O2/S2O 82− ( 1(O2) 2*) and PTC-NH2 (1(NH2−PTC)2*), respectively. When the resultant biosensor was incubated with the target of Pb2+, the dsDNA was unwound and Pb2+ G-quadruplex structure was generated because of the highly specific binding capability between Pb2+ and CPs, which made the PTC-NH2 release from the electrode surface. The two ECL signal peaks showed opposite changes where the ECL peak from PTC-NH2 decreased and the ECL peak from O2/S2O82− increased correspondingly, which further confirmed the fact that the ECL-RET between the PTC-NH2 and the O2/S2O82− system. Herein, a ratiometric ECL biosensor for Pb2+ detection based on the ECL-RET system was designed where the ratiometric method according the ratio of ECL intensities at two excitation potentials instead of absolute values, which could eliminate false positive or negative errors and make the assay more accurate. As a result, the developed aptasensor exhibited a good linear response range from 1.0 × 10−12 M to 1.0 × 10−7 M for Pb2+ detection.

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EXPERIMENTAL METHODS Reagents and Materials. Fullerene (C60, 99.5%) was bought from Pioneer Nanotechnology Co. (Nanjing, China). 7788

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at a scan rate of 0.2 V/s, and the voltage of the photomultiplier tube (PMT) was at 800 V.

7.4. Then, the resultant product was dispersed homogeneously in 10 mL deionized water and stored in the refrigerator at 4 °C for further use. Fabrication of the Aptasensor. First, the bare GCE was polished carefully with 0.3 and 0.05 μm alumina slurry to obtain a mirror-like surface, followed by sonication in the deionized water and ethanol, respectively. Next, 5 μL of the prepared AuNPs@nano-C60 suspension was cast onto the bare GCE surface and dried in air to form a homogeneous film. Subsequently, 15 μL of APs (2.5 μM) solution was dropped on the above modified electrode (AuNPs@nano-C60/GCE) at 4 °C for 16 h. Then the modified electrode (APs/AuNPs@ nano-C60/GCE) was incubated in HT (1 mM) for 40 min at room temperature to eliminate the nonspecific binding effect. Afterward, 15 μL of 2.5 μM CPs (the aptamer of Pb2+) solution was dropped on the modified electrode (HT/APs/AuNPs@ nano-C 60 /GCE) at room temperature for about 2 h. Subsequently, the prepared modified electrode (CPs/HT/ APs/AuNPs@nano-C60/GCE) was incubated in HT (1 mM) again. The resultant modified electrode (HT/CPs/HT/APs/ AuNPs@nano-C60/GCE) was incubated in 20 μL of PTC-NH2 solution (5 mg mL−1) for 2 h at room temperature. After each step, the modified electrode was thoroughly cleaned with deionized water to remove the physically absorbed species. The finished biosensor (PTC-NH2/HT/CPs/HT/APs/AuNPs@ nano-C60/GCE) was stored in the refrigerator at 4 °C for further use. The schematic graph of the fabrication process for the ECL aptasensor was illustrated in Scheme 1.

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RESULTS AND DISCUSSION ECL-RET between O2/S2O82− and PTC-NH2/S2O82−. To verify ECL-RET between O2/S2O82− and PTC-NH2/S2O82−, ECL curves and CV curves of the different modified electrode were obtained synchronously during the cyclic potential scanning between −2.0 and 0 V, and the results are shown in Figure 1. The curve a in Figure 1A represents ECL response of the bare GCE in air-saturated S2O82− solution (0.1 M, pH = 7.4). One single peak located at −2.0 V with the ECL peak intensity about 1696 au is evident and could be assigned to the radiative deactivation of 1(O2)2* in the O2/S2O82− system.12 The corresponding CV curve of a bare GCE in S2O82− solution (Figure 1B, curve a) shows an obvious reduction peak at −1.2 V. PTC-NH2 was modified on the bare GCE surface (PTCNH2/GCE) and then detected in air-saturated PBS solution (0.1 M, pH = 7.4) during the cyclic potential scanning between −2.0 and 0 V. The CV wave (Figure 1B, curve b) shows two pairs of reversible redox peaks in which there are two reduction peaks at −0.40 V and −0.80 V, and two oxidation peaks at −0.15 V and −0.35 V. These results indicated that the PTCNH2 has a multielectron transfer reaction during the cyclic potential scanning between −2.0 and 0 V.24 Meanwhile, the corresponding ECL response of PTC-NH2/GCE in PBS solution (Figure 1A, curve b) was also recorded where the ECL peak was not observed during the cyclic potential scanning between −2.0 and 0 V. However, when the PTCNH2/GCE was studied in PBS solution with the wider potential window over the range from −2.0 to 2.0 V, a weak ECL peak (Figure 1A, curve b′) was observed with the peak potential of 2.0 V. It could be speculated that the stable radical cations or excited states could be generated at a sufficiently positive potential, which were required in the ECL reaction.25 Interestingly, when the PTC-NH2/GCE was detected in S2O82− solution during the cyclic potential scanning between −2.0 and 0 V, ECL dual peaks were observed (Figure 1A, curve c), which are located at −0.7 V with the intensity of 1661 a.u and around −2.0 V with the intensity of 6084 au, respectively. More notably, the ECL peak located at −0.7 V could not be found at the bare GCE in S2O82− solution. At this point, we could presume that this ECL peak might be related to the PTC-NH2 in S2O82− solution. Meanwhile, the corresponding CV wave of PTC-NH2/GCE in S2O82− solution (Figure 1B, curve c) showed one extremely obvious reduction peak where the reduction potential shifts positively from −1.2 V to −0.7 V and the peak current increased apparently, indicating that the strong oxygenation between PTC-NH2 and S2O82−. To prove this fact that the ECL peak located at −0.7 V was related to the PTC-NH2, ECL spectra of two ECL peaks were also recorded. As shown in Figure 2A, the maximum emission wavelength of ECL peak located around −2.0 V was at 575 nm (Figure 2A, curve a), which was obtained with scan potential from −2.0 V to −1.0 V. It is could be assigned to the emission of 1(O2)2* in the O2/S2O82− system.17 However, when the scan potential was from −1.0 to 0 V, the maximum emission wavelength of ECL peak located −0.7 V was at 675 nm (Figure 2A, curve b). Herein, the ECL peak located at 675 nm could be assigned to the PTC-NH2 in the O2/S2O82− system. In addition, as shown in the inset of Figure 2A, ECL responses of the different concentration of PTC-NH2 modified electrodes were obtained in air-saturated S2O82− solution during the cyclic

Scheme 1. Schematic Illustration of the Preparation Process of the Ratiometric Aptasensor: Inset of (A) and (B) Display the Preparation Procedure of AuNPs@Nano-C60 and PTCNH2

Measurement Procedure. The ECL responses of the proposed aptasensors were investigated in 3 mL of 0.1 M PBS (pH = 7.4) containing 0.1 M K2S2O8 and 0.1 M KCl, which are incubated with Pb2+ standard solution at room temperature for 1 h. The working potential was from −2.0 to 0 V (vs Ag/AgCl) 7789

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Figure 1. Synchronously ECL-potential curves (A) and CV curves (B) of different electrodes (scan range: −2.0 to 0 V, vs Ag/AgCl): (a) bare GCE in O2/S2O82− solution, (b) PTC-NH2/GCE in PBS solution, and (c) PTC-NH2/GCE in O2/S2O82− solution. Inset of (A): the ECL intensitypotential profiles of curve b (scan range: −2.0 to 0 V, vs Ag/AgCl) and curve b′ (scan range: −2.0 to 2.0 V, vs Ag/AgCl) in PBS solution, respectively.

Figure 2. (A) ECL spectra of the PTC-NH2/GCE in the S2O82− solution: (a) the ECL peak located at −0.7 V (scan range: −0.1 to 0 V) and (b) the ECL peak located at −2.0 V (scan range: −2.0 to −1.0 V) were obtained by the optical filter. Inset: ECL-time curves of the PTC-NH2 modified electrodes in S2O82− solution with different concentrations of PTC-NH2 from (a′) to (c′): 3.6, 7.2, and 10.8 mg mL−1, respectively. (B) UV−vis absorption spectra of the S2O82− solution (a), the PTC-NH2 solution(b), and the S2O82− solution containing PTC-NH2 (c). (C) UV−vis absorption spectra of the PTC-NH2 in the S2O82− solution (a) and ECL spectra of the S2O82− solution (b) (0.1 M, pH = 7.4). (D) Fluorescence spectra of PTC-NH2: Excitation spectra (a), Emission spectra (b).

potential scanning between −2.0 and 0 V. With the increase of the concentration of PTC-NH2, the two ECL signal peaks showed opposite changes where the ECL from PTC-NH2 was increased, and the ECL from 1(O 2 ) 2* was decreased correspondingly. It could be speculated that the ECL-RET would occur between 1(O2)2* and PTC-NH2 in this ECL system, where the 1(O2)2* emits short wavelength as an energy donor and PTC-NH2 emits long wavelength as an energy acceptor. The UV−vis absorption spectra were also obtained to further identify the ECL-RET between PTC-NH2 and 1(O2)2* system. As shown in Figure 2B, the maximum absorption peak of airsaturated S2O82− solution (curve a) could not be observed in the range of 300−800 nm. However, three characteristic absorption peaks of PTC-NH2 solution (Figure 2B, curve b) were observed in the range of 430 to 700 nm and have a similar band shape of perylene derivatives, indicating absorptions were dominated by the π−π* transition of the perylene moiety.26 After the PTC-NH2 solution was mixed with air-saturated

S2O82− solution (curve c), the UV−vis intensity decreased at 300−430 nm but increased at 430−700 nm, confirming the interaction between PTC-NH2 and O2/S2O82−. Significantly, as shown in Figure 2C, the UV−vis absorption spectra of the acceptor (curve a, PTC-NH2) overlapped perfectly with the ECL spectra of the donor (curve b, 1(O2)2*), indicating the possibility of ECL-RET between 1(O2)2* and PTC-NH2 in this ECL system. Meanwhile, the fluorescence (FL) spectra of PTC-NH2 were recorded in Figure 2D. It could be found the emission maximum fluorescence occurred at 546 nm (Figure 2D, curve b) when the excitation wavelength was held at 493 nm (Figure 2D, curve a). Unexpectedly, the UV−vis absorption spectra (Figure 2B, curve b) do not have a mirror-image relationship with the fluorescence spectra, which could be assigned to the aggregation of PTC-NH2 with strong π−π stacking resulting in the change of band shapes and the red-shift of absorption peaks.26 More interestingly, the maximum ECL emission wavelength of the PTC-NH2 was located at 675 nm, which has an obvious 129 nm red-shift compared with the FL 7790

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indicating that in situ generation of AuNPs had adsorbed successfully on nano-C60 via Au−S and Au−N chemistry. ECL and CV Characterization of the Stepwise Fabrication of the Aptasensor. In order to characterize the fabrication process of the aptasensor, the corresponding ECL responses at each immobilization step were recorded in a 0.1 M S2O82− solution (pH = 7.4), as shown in Figure 4A. First, the bare GCE produced a relatively low ECL intensity (curve a) at about 1696 au, and it could be attributed to the radiative deactivation of (1(O2)2*) in the O2/S2O82− system according to the previous studies.15 After AuNPs@nano-C60 composite was modified on the electrode surface, a remarkable ECL increase was observed in curve b. This result indicated that the AuNPs@ nano-C60 could amplify the ECL signal in O2/S2O82− system.29 After successively modified with APs (curve c), HT (curve d), CPs (curve e), and HT (curve f), the ECL signal declined accordingly, which was attributed to the fact that APs, HT, and CPs on the electrode would retard the electron transfer, respectively. However, when PTC-NH2 was intercalated into the dsDNA grooves by the electrostatic adsorption (curve g), we could observe a ECL dual-peak. One strong cathodic ECL-1 with the peak potential of −0.7 V could be assigned to the excited dimmers (1(PTC-NH2)2*). The other relatively weak cathodic ECL-2 with the peak potential of −2.0 V could be assigned to the (1(O2)2*) in the O2/S2O82− system. Finally, after incubation with Pb2+ (curve h), the ECL-1 intensity of PTC-NH2 was decreased, and the ECL-2 intensity of O2/ S2O82− was increased accordingly. The reason is that the dsDNA was unwound by generating G-quadruplex structure, resulting in the release of PTC-NH2 from the electrode surface. Because of the changed ratio of ECL-1 and ECL-2, a label-free ratiometric aptasensor for detection Pb2+ was fabricated in this work. To further confirm the fabrication process of the modified electrode at each step, the CVs were also performed in 5 mM Fe(CN)63−/4− solution. As shown in Figure 4B, a pair of welldefined redox peaks could be observed at bare GCE (curve a). When AuNPs@nano-C60 was modified on the electrode surface, the redox peak current decreased relatively (curve b). When APs were assembled on the electrode surface (curve c), the peak current decreased continuatively. This is because the phosphate backbone of APs is negatively charged, which could bring about a repulsion effect to Fe(CN)63−/4−. After successive modification with APs (curve c), HT (curve d), CPs (curve e), and HT (curve f), the ECL signal decreased, respectively. The

spectra of PTC-NH2 (546 nm). Herein, it can be inferred that the excited state of PTC-NH2 in FL emission is different from that of ECL emission, where the FL spectra were attributed to the emission of excited singlets (1PTC-NH2*) and ECL spectra were attributed to the emission of excited dimers (π-excimers) (1(PTC-NH2)2*)22,25,27,28 Consequently, the ECL-RET process could be illustrated as Scheme 2. Scheme 2. Possible Mechanism of ECL-RET between PTCNH2/S2O82− and O2/S2O82−

SEM Characterization of the Nano-C60 and AuNPs@ Nano-C60 Composite. The morphologies and sizes of the asprepared nano-C60 and AuNPs@nano-C60 composite were characterized with SEM. The typical SEM image of the nanoC60 (Figure 3A) showed a well-defined shape of sphere

Figure 3. SEM images of (A) nano-C60 and (B) AuNPs@nano-C60.

structures with a diameter of 80 ± 30 nm, indicating the successfully preparation of nano-C60. Then AuNPs were in situ generated in the present of BSA modified nano-C60 nanocomposite. As shown in Figure 3B, it could be clearly observed that the large amounts of AuNPs with a diameter of 10 ± 3 nm were distributed uniformly and tightly on the nano-C60 surfaces,

Figure 4. (A) ECL responses of the electrode at different stages in 0.1 M K2S2O8 (pH = 7.4) and (B) CV responses of the electrode at different stages in PBS buffer (pH = 7.4) containing 5.0 mM [Fe(CN)6]3−/4− as redox probe: (a) GCE, (b) AuNPs@nano-C60/GCE, (c) APs/AuNPs@nanoC60/GCE, (d) HT/APs/AuNPs@nano-C60/GCE, (e) CPs/HT/APs/AuNPs@nano-C60/GCE, (f) HT/CPs/HT/APs/AuNPs@nano-C60/GCE, (g) PTC-NH2/HT/CPs/HT/APs/AuNPs@nano-C60/GCE, (h) after incubation with 5 × 10−8 M Pb2+. 7791

DOI: 10.1021/acs.analchem.5b01445 Anal. Chem. 2015, 87, 7787−7794

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Figure 5. (A) ECL-time curves of the ratiometric aptasensor with different concentrations of Pb2+ in 0.1 M S2O82− solution (pH = 7.4) from (a) to (f): 1.0 × 10−12 M, 1.0 × 10−11 M, 1.0 × 10−10 M, 1.0 × 10−9 M, 1.0 × 10−8 M, 1.0 × 10−7 M, respectively. (B) Relationship between the ECL intensity of ECL-1 (a) and ECL-2 (b) to the concentration of the Pb2+. (C) Relationship between the lg(I1/I2) and the concentration of Pb2+.

with PTC-NH2 as the ECL donor and S2O82−/O2 as ECL the acceptor is highly sensitive and has great potential for accurate detection of Pb2+. In addition, the comparison of the sensitivity of the different test platforms of Pb2+ are listed in Table S1 (Supporting Information). It could be found that this prepared biosensor has a good sensitivity compared to other test platforms. Stability and Selectivity of the Ratiometric Aptasensor. The stability of the ratiometric aptasensor was evaluated under consecutive cyclic potential scan for 15 cycles. ECL curves and CV curves were obtained synchronously during the cyclic potential scanning between −2.0 and 0 V, as shown in Figure 6A and the inset of Figure 6A, respectively. The relative

reason is that the insulation effect of ssDNA, HT, and dsDNA could retard the electron transfer on electrode surface. However, when PTC-NH2 was intercalated into the dsDNA grooves (curve g), the redox peak current was increased significantly. This result could be attributed to the positively charged PTC-NH2, which could decrease the density of negative charges of dsDNA, resulting in the Fe(CN)63−/4− reaching electrode surface easily.30 Finally, the redox peak current apparently decreased (curve h) after incubating with Pb2+, which could be assigned to the negative charge increase by releasing PTC-NH2 from the surface of developed aptasensor. As can be seen from Figure 4B, the CVs are in accordance with those of ECL characterization. ECL Response of the Ratiometric Aptasensor to Pb2+ Concentration. The sensitivity of the ratiometric ECL aptasensor was assessed by measuring the ratio of ECL-1 and ECL-2 after incubation with Pb2+. As shown in Figure 5A, with the increase of the Pb2+ concentration, the ECL-1 intensity of PTC-NH2/S2O82− (I1) decreased, and the ECL-2 intensity of O2/S2O82− (I2) increased correspondingly. Figure 5B shows the relationship between Pb2+ concentration and ECL intensity toward I1 (a) and I2 (b), from which I was found to be linearly dependent on the logarithm of Pb2+ concentration in the range from 10−12 to 10−7 M. The regression equations were I1 = 11879.56 + 924.97 lg c (c was the value of Pb2+ concentration) with the R1 of 0.9948 and I2 = −7405.97−1244.63 lg c with the R2 of 0.9901. To improve ECL accuracy, we employed the ratiometric method to detect the Pb2+ by calculating the ratio of I1 and I2. In accordance with Figure 5C, the logarithmic value of I1/I2 linearly depended on the logarithm of Pb2+ concentration in the range of from 10−12 to 10−7 M. The regression equation was lg(I1/I2) = 2.87 + 0.28 lg c with the R of 0.9987 (R > R1> R2). Obviously, the correlation coefficient was increased, which exhibited the accuracy was improved by ratiometric assay. Meanwhile, the limit of detection (LOD) was estimated to be 3.5 × 10−13 M at S/N = 3. The results demonstrated that this ratiometric ECL strategy based on a novel ECL-RET system

Figure 6. (A) ECL-time and the CVs curve (inset) of the ratiometric aptasensor under continuous scanning for 15 cycles in 0.1 M S2O82− solution (pH = 7.4). (B) Selectivity of the ratiometric aptasensor to Pb2+ (1.0 × 10−7 M) by comparing it to the interfering metal ions (1.0 × 10−5 M).

standard deviation of ECL-1 (RSD = 4.12%) and ECL-2 (RSD = 1.79%) were used to evaluate the stability. It can be seen both of the relative standard deviations were not more than 5%, which suggested that the stability of the ratiometric aptasensor was acceptable. To further investigate the selectivity and specificity of the proposed aptasensor, contrast experiments were performed using various metal ions as interfering substance like Ni2+, Co2+, 7792

DOI: 10.1021/acs.analchem.5b01445 Anal. Chem. 2015, 87, 7787−7794

Analytical Chemistry

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Cd2+, Mg2+, Ca2+, K+, and Ag+ at a concentration of 1.0 × 10−5 M and Pb2+ at 1.0 × 10−7 M. As shown in Figure 6B, only in the presence of Pb2+ was the large ratio of I1 to I2 observed, whereas the other metal ions had no obvious effect on the ratios of ECL intensity compared with the blank. All these results indicated that the ratiometric aptasensor possessed an excellent selective response to Pb2+ against other environmentally relevant metal ions. Direct Detection of Pb2+ in Soil Solutions. We had performed an experiment to evaluate the applicability and reliability of the present ECL system. The concentrations of Pb2+ in extracted soil solutions were determined by the ratiometric aptasensor as well as atomic absorption spectrometric (AAS) method. Herein, the soil solutions were sequentially extracted by the well-known Tessier method with a sample of purple soil.30 Considering the bioavailability of Pb2+ in soil, we had detected Pb2+ concentration in extracted solutions containing four fractions: exchangeable, carbonate, Fe−Mn oxides, and organic fractions. The results, summarized in Table 1, showed good agreement with those achieved by

exchangeable bound to carbonates bound to Fe−Mn oxides bound to organic matter

ratiometric aptasensor (μM)

AAS (μM)

relative error (%)

3.435 1.706 1.168

3.427 1.718 1.144

0.233 −0.698 2.10

1.784

1.816

−1.76

*E-mail: [email protected]. Tel.: +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 work was financially supported by the NNSF of China (21275119, 51473136) and the Fundamental Research Funds for the Central Universities (XDJK2015A002, XDJK2014A012), China.

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REFERENCES

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using the standard AAS method, indicating that the ratiometric aptasensor had a capacity for determining Pb2+ in practical sample analysis.

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CONCLUSIONS In conclusion, this work has demonstrated a novel ECL-RET system between PTC-NH2 as the ECL acceptor and 1(O2)2* as the ECL donor, as well as the fabrication of a label-free ratiometric aptasensor for Pb2+ detection. The absorption spectrum of PTC-NH2 was overlapped perfectly with the ECL emission spectrum of 1(O2)2*, which could ensure obtaining highly effective ECL-RET. A sensitive ECL-RET switch was obtained where the Pb2+ dominated the amount of PTC-NH2 by generating G-quadruplex structure. In view of the ratio of PTC-NH2/S2O82− peak intensity to O2/S2O82− peak intensity regulated upon the concentrations of Pb2+ in this system, the label-free ratiometric aptasensor has exhibited excellent properties of high selectivity, accuracy, and sensitivity. Moreover, this study may provide a new ECL donor and acceptor system and broaden the application of ECL-RET for detection various targets.

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AUTHOR INFORMATION

Corresponding Authors

Table 1. Determination of the Concentrations of Pb2+ Soil Extraction Solutions Using the Proposed Ratiometric Aptasensor and AAS Method sample

Article

ASSOCIATED CONTENT

* Supporting Information S

Comparison of the sensitivity of the different test platforms of Pb2+. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.5b01445. 7793

DOI: 10.1021/acs.analchem.5b01445 Anal. Chem. 2015, 87, 7787−7794

Article

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DOI: 10.1021/acs.analchem.5b01445 Anal. Chem. 2015, 87, 7787−7794