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Oxygen Vacancy Engineering in Europia Clusters/Graphitelike Carbon Nitride Nanostructures Induced Signal Amplification for Highly Efficient Electrochemiluminesce Aptasensing Xiaojiao Du, Ding Jiang, Liming Dai, Weiran Zhu, Xiaodi Yang, Nan Hao, and Kun Wang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b00162 • Publication Date (Web): 05 Feb 2018 Downloaded from http://pubs.acs.org on February 5, 2018

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Analytical Chemistry

Oxygen Vacancy Engineering in Europia Clusters/Graphite-like Carbon Nitride Nanostructures Induced Signal Amplification for Highly Efficient Electrochemiluminesce Aptasensing Xiaojiao Du,† Ding Jiang,‡ Liming Dai,† Weiran Zhu,§ Xiaodi Yang*,§ Nan Hao,† and Kun Wang∗,† †

Key Laboratory of Modern Agriculture Equipment and Technology, School of

Chemistry and Chemical Engineering, Jiangsu University, Zhenjiang, Jiangsu 212013, P.R. China ‡

Interdisciplinary Division of Biomedical Engineering, The Hong Kong Polytechnic

University, Hung Hom, Kowloon, Hong Kong, China §

Jiangsu Collaborative Innovation Center of Biomedical Functional Materials,

Jiangsu Key Laboratory of New Power Batteries, College of Chemistry and Materials Science, Nanjing Normal University, Nanjing 210023, China

∗ Corresponding author. E-mail address: [email protected] (X. Yang), Fax: +86 25 85891767; [email protected] (K. Wang). Fax: +86 511 88791708.

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Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT: Oxygen vacancy is an intrinsic defect in metal oxide semiconductors and has a crucial influence on their physicochemical and electronic properties. To boost the electrochemiluminescence (ECL) efficiency of the graphite-like carbon nitride (g-C3N4), the wet-chemical-calcination method was developed to introduce oxygen vacancy in Eu doped g-C3N4 nanostructures for the first time. The morphology and structure characterization suggest that Eu element was present in the matrix of the europia (Eu2O3) clusters. Due to oxygen vacancy promoting catalytic activity effect, the doping of Eu caused a great positive shift of onset potential and large signal amplification in cathodic ECL signals compared to pure g-C3N4. Furthermore, a novel and ultrasensitive ECL aptasensor was realized with 17β-estradiol (E2) as a prototype target by adsorption of E2 aptamer onto the Eu2O3-doped g-C3N4 (Eu2O3-g-C3N4) surface via van der Waals force. Since the specific recognition between aptamer and E2, the ECL signal decreased with the increasing concentration of E2, because the formation of E2-aptamer complex impeded the diffusion of luminophor molecules and the electrons approaching the surface of electrode. Under the optimal cases, the as-prepared ECL aptasensor showed superior performances and also manifested outstanding selectivity towards E2. The present conceptual strategy offers a novel methodology to boost the sensitivity of ECL sensor and promote the activity of ECL reagents.


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■ INTRODUCTION Oxygen vacancy is an intrinsic defect in metal oxide semiconductor and has a substantial impact on their physicochemical and electronic properties in heterogeneous catalytic area.1 Reported by Metiu and co-workers, it is also uniquely interpreted as strong Lewis basic sites.2 That means a catalytic surface with oxygen vacancy in higher Lewis basicity represents a higher adsorption and catalytic activity.3,4 Besides, many reports demonstrated that the oxygen vacancy could act as a vital part to improve the conductivity, mediate energy transfer, and promote the charge transfer.5-8 Despite the importance of oxygen vacancy in oxides has been demonstrated in various systems such as lithium batteries, solid oxide fuel cells, and photocatalytic related fields,9-11 there has scarcely ever been reported in the context of electrochemical sensors. Since the first discovery of the electrochemiluminescence (ECL) activity of graphite-like carbon nitride (g-C3N4) in 2012,12 various g-C3N4-based ECL sensing strategies have been developed during the past five years. As a new ECL luminophore, g-C3N4 presents intriguing advantages because of its stable s-triazine ring structure and outstanding optical properties.13-15 To improve the ECL property of g-C3N4 for better sensing applications, one alternative mean is to hybridize with some other nanomaterials (noble metal such as Au and Ag nanoparticles,14,16 carbon material and polymer) to achieve signal amplification.17-19 Among them, more remarkable is that Xu’s group demonstrated that rare earth europium ions (Eu3+) doped CdS nanocrystals caused a huge enhancement in the ECL intensity.20 As such, it is conceivable that the incorporation of Eu3+ would improve ECL efficiency of g-C3N4 system. Rare earth (RE) ions doped nanocomposites have drawn keen research interest in



light-emitting devices, and solar cells owing to the fact that RE ions can combine their remarkable optical properties RE ions and the electrical excitation of the host material.21 Of various RE ions, the red luminescence arising from the 5D0→7F2 intra-4f shell transition, trivalent Eu3+ is specially promising, meanwhile europia (Eu2O3) is one of the most interesting rare earth oxides and widely used in many optical-related areas.22,23 Among them, some reports have shown that Eu2O3 holds great potential for enhancing photocatalytic activity of TiO2.24,25 They attributed this to that europium element with incompletely occupied 4f and empty 5d orbitals could change the potential energy level of TiO2 and result in the generation of oxygen vacancies due to its electrons trap effect. In our previous studies, the oxygen vacancies acted as electron traps and facilitated promoting the catalytic activity of nanocrystals.26 In this regard, it is expected that Eu2O3 with oxygen vacancies would have excellent performance in the field of catalysis and prompt the ECL efficiency of g-C3N4. To our best knowledge, this is the first demonstration of oxygen vacancies introduced into ECL fields. 17β-Estradio (E2), the most active estrogen, has been widely used to fatten animals and constituted a severe threat to human health and aquatic organisms.27,28 Studies have shown that male fish can be feminized when the content of E2 in water surroundings as low as 1 ng/L (pM).29 It could be enter into the body from outside and interfere with the human normal physiological processes via food chain even in trace concentration of E2.30,31 Until now, a variety of methods has been established, such as chromatography-based methods,32,33 colorimetric method,34,35 and electrochemical methods.27,36 Of these sensors, electrochemical aptasensors are the preferred choice for E2 assay owing to their inherent advantages including low cost, simplicity, high sensitivity and selectivity.36 As a result, it is very essential to explore more novel


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electrochemical aptasensors for E2 detection. Herein, the cathodic ECL behavior of g-C3N4 luminophore markedly enhanced by Eu2O3 with oxygen vacancies was investigated. When E2 aptamer attached on the surface of g-C3N4, the ECL intensity of Eu2O3/g-C3N4 decreased owing to the fact that the biological binding event aptamer could hinder the diffusion of luminescent reagents and electrons toward the surface of electrode.37 After a series of E2 with different concentrations were incubated with the ECL aptasensor, ECL intensity would decrease in different extent (Scheme. 1). Therefore, a novel ECL aptasensor for E2 determination was fabricated successfully. The ECL aptasensor displayed excellent performances for E2 assay and open new doors towards the fabrication of highly efficient ECL aptasensors. ■ EXPERIMENTAL SECTION Reagents. The aptamer for E2 was obtained from Shanghai Sangon Biotechnology Co. Ltd. And the sequences are as follows: 5′-GCTTCCAGCTTATTGAATTACACGCAGAGGGTAGCGGCTCTGCGCATT CAATTGCTGCGCGCTGAAGCGCGGAAGC-3′. Dicyandiamide (C2H4N4) and Eu2O3 were bought from Sinopharm Chemical Reagent Co., Ltd. E2, estriol (E3), ethinylestradiol (EE) were acquired from Aladdin Chemistry Co., Ltd. 0.1 M Eu(NO3)3 solution was obtained by using Eu2O3 to dissolve in diluted nitric acid. Phosphate buffer solution (PBS; pH=7.4) was prepared by mixing the stock standard solution of 0.1 M NaH2PO4 and Na2HPO4. All the reagents were used as purchased without further purification. The ultrapure water (18.4 MΩ) purified by a Milli-QTM system (Millipore) was used throughout the whole experiments. Apparatus. Transmission electron microscope (TEM, Hitachi H800, Tokyo) and



scanning electron microscopy (SEM, Hitachi, Tokyo) were used to measure the morphology of the samples. X-ray diffraction (XRD, Bruker D8 ADVANCE diffractometer, Germany) was performed to detect the crystal structures of the samples. The chemical composition of the sample was measured through X-ray photoelectron spectroscopy (XPS) of PHI 5000 Versa Probe. The photoluminescence (PL) spectra of the samples were recorded with spectrofluorometer (Hitachi F-4500, Tokyo). ECL studies were performed by using the MPI-A ECL analyzer system (Xi'an Remax, China), and the potential was range from 0 V ~ −1.5 V. All the ECL curves were obtained via the 3-electrode system, where a platinum wire was used as a counter electrode, a Ag/AgCl electrode (saturated KCl solution) as a reference electrode, and a glassy carbon electrode (GCE, 3 mm) as a working electrode. Electrochemical impedance spectroscopy (EIS) were tested in 5 mM Fe(CN)63−/4− with 0.1 M KCl solution with a electrochemical workstation (CHI660, Chen Hua). Preparation of Samples. 2.0 g C2H4N4 was dissolved in water and the solution was refluxed with a certain amount (0, 0.3, 0.5, 1.0, 5.0 mL) of 0.1 M Eu(NO3)3 solution at 110 oC for 100 min. After that, the solution was heated to 100 oC and maintained for 12 h to obtain the solid sample. After the sample was ground to powder by agate mortar, the power can be transferred into the alumina crucible. The temperature was heated to 550 oC and maintained for 2 hours in air. Then the resultant yellow solid was ground to fine powder by agate mortar. The prepared samples were marked as sample 1, sample 2, sample 3, sample 4, and sample 5, which respectively corresponded to samples with different addition amounts (0, 0.3, 0.5, 1.0 and 5.0 mL) of Eu(NO3)3 solution. Fabrication of the ECL Sensing Platform. The procedure for fabricating


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modified GCE electrodes was stated as follows: 6 µL of sample 1 suspension (2.0 mg/mL in water) was dropped onto the surface of pretreated GCE, and then dried at room temperature (simplified as Eu2O3/g-C3N4/GCE). Afterwards, 6 µL E2 aptamer solution was cast onto the Eu2O3/g-C3N4/GCE surface. Finally, the aptamer-modified electrodes (apt/Eu2O3/g-C3N4/GCE) were stored at room temperature and then rinsed with 0.1 M PBS (pH = 7.4) to remove the redundant aptamer. ■ RESULTS AND DISCUSSION Morphology, and Structure. The morphology analysis of Eu2O3, g-C3N4 and Eu2O3/g-C3N4 nanocomposite was with the aid of TEM. Figure 1A shows TEM micrographs of Eu2O3. It can be seen that Eu2O3 with irregular shapes were obtained with tendency to form larger and denser nanoparticle aggregates. As was shown in Figure 1B, the g-C3N4 sheet had a typically overlapped and crumpled multilayer structure.38 After Eu2O3 hybridizing with g-C3N4 sheet (Figure 1C and Figure 1D), some black and gray spots (about 10 nm) appeared, which should be that the Eu2O3 clusters attached to the surface of g-C3N4 sheets. It showed that introducing g-C3N4 sheets via the thermal-treatment method resulted in smaller sized and better distributed Eu2O3 clusters. Figure 2A displayed the XRD results of g-C3N4 and Eu2O3/g-C3N4 nanocomposite. There observed distinct diffraction peak at 27.62o (002) in the XRD patterns of the prepared samples. This is well matched to g-C3N4 sheets (JCPDS Card No.87-1526).39 There were small peaks observed at 27.60o (222), 44.79o (440) and 56.32o (622) (curve b), which were assigned to Eu2O3 (JCPDS Card No. 34-0392).40 The structured Eu2O3 reflection peak appeared, indicating that Eu3+ precipitated from the g-C3N4 sheets host as Eu2O3 clusters and Eu2O3/g-C3N4 nanocomposite formed. The weakly diffraction peaks for Eu2O3 might be due to the low abundance of Eu2O3 in the



Eu2O3/g-C3N4 nanocomposite. Composition Analysis and the Formation of Oxygen Vacancy. The element composition of the as-prepared Eu2O3/g-C3N4 nanocomposite was further studied by XPS. The XPS full spectrum of different elements was illustrated in Figure 2B. From that the O, C, N, and Eu elements had been monitored in the nanocomposite. The C 1s spectrum in high-resolution case (Figure S1A) exhibited two peaks at 287.23 eV and 284.51 eV, which corresponding to N−C−N and C−C.41 For the N 1s spectrum (Figure S1B), there displayed three N states including pyridinic-N (397.34 eV), tertiary nitrogen (399.91 eV) and triazine rings C−N−C (398.62 eV).42,43 The three N states confirmed the formation of g-C3N4. Furthermore, the XPS spectra of Eu 3d core-level doublet for Eu2O3/g-C3N4 along with the corresponding deconvolution was presented in Figure 2C. The occurrence of Eu2O3 is clearly revealed by that Eu3+ (at ~1166.2 eV) together with a satellite peak assigned to the 2+ state (at ~1157.1 eV) for Eu 3d3/2 core level, while fitting of Eu 3d5/2 exhibits Eu3+ (at ~1136.4 eV) and corresponding satellite peak at 1126.4 eV (2+).44,45 Meanwhile, the deconvolution XPS spectra of the Eu 4d spectrum is depicted in Figure 2D. It is observed that two main peaks are situated at 137.6 eV and 143.0 eV labeled as 3+ state due to multiplet structure of the trivalent 4d4f6 configuration. Similarly, owing to the divalent 4d4f7 configuration, the weak peaks at around 131.2 eV and 136.3 eV are analyzed as 2+ state.45 The presence of Eu2+ and Eu3+ states is consistent with previous reports in which they claimed the divalent Eu comes from the fast surface valence transition.44,45 Oxygen vacancy defect was formed along with the generation of Eu2+ species. As such, if the Eu2+ is determined, the presence of oxygen vacancy occurred in Eu2O3. Such oxygen deficient behavior has also been observed in Fe doped TiO2 and CeO2.26,46,47 The oxygen vacancy served as electron traps, facilitating improving the


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catalytic activity. Therefore, it is expected that Eu2O3 in the nanocomposite would have the outstanding catalytic activity in the sensing applications. Optical Properties. Figure S2 showed the PL emission spectra of pure g-C3N4 and Eu2O3/g-C3N4 nanocomposite upon excitation with 245 nm. From Figure S2A, the pure g-C3N4 peaked at about 460 nm, which could be attributed to the band–band PL phenomenon from the n-π* electronic transitions along with lone pairs of nitrogen atoms inside g-C3N4.48,49 From the emission spectra of the Eu2O3/g-C3N4, There observed simultaneously the blue emission from g-C3N4 and red emission from Eu3+ ion upon excitation with 245 nm. The typical red light emission of Eu3+ with the wavelength from 550 nm ∼ 600 nm were found in the Eu2O3/g-C3N4 (Figure S2B).23 The red emission at 610 nm is attributed to the electric dipole transition 5Do→7F2 of Eu3+, and other weak emission at 578 nm and 591 nm were attributed to the transitions 5Do→7F0 and 5Do→7F1 of Eu3+, respectively.50,51 All these indicate that Eu2O3/g-C3N4 nanocomposite was synthesized successfully. ECL Behaviors of the Modified Electrodes. The ECL behaviors of the g-C3N4-Eu2O3/GCE (curve a), g-C3N4/GCE (curve b), and Eu2O3/GCE (curve c) were recorded in 0.1 M PBS containing 10 mM K2S2O8 between 0.0 and −1.5 V (Figure 3A). It is obvious that the ECL signal of the g-C3N4-Eu2O3/GCE (curve a) was a lot higher than that of the g-C3N4/GCE (curve b) and Eu2O3/GCE (curve c), implying that Eu2O3 can effectively boost the ECL signal. In addition, the ECL intensity of g-C3N4-Eu2O3/GCE is about 2.8-fold higher of g-C3N4/GCE. The corresponding cyclic voltammograms (CVs) of the Eu2O3/g-C3N4/GCE (curve a), g-C3N4/GCE (curve b), and Eu2O3/GCE (curve c) were also studied in 0.1 M PBS with 10 mM K2S2O8 contained, as was depicted in Figure 3B. For Eu2O3/GCE, there were no apparent reduction peak, while g-C3N4/GCE and Eu2O3/g-C3N4/GCE



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showed a reduction peak potential of K2S2O8.12 Moreover, compared with the g-C3N4/GCE, the Eu2O3/g-C3N4/GCE could move the ECL onset potential more positively for ∼420 mV. The CVs results demonstrated that the cathodic peak current was greatly enhanced and the onset reduction potential of g-C3N4 also shifted positively in the Eu2O3/g-C3N4 nanocomposite than those of pure g-C3N4, suggesting that the Eu2O3 in Eu2O3/g-C3N4 nanocomposite decreased the potential barrier of g-C3N4. The obtained Eu2O3/g-C3N4 were further explored in the absence and presence of K2S2O8 as the coreactant to make the ECL process clear. The ECL behaviors (Figure S3) revealed that the g-C3N4/GCE and Eu2O3/g-C3N4/GCE showed a very faint ECL signal without K2S2O8 whereas there a strong ECL signal observed with K2S2O8 added. The results suggest that the ECL emission originates from the chemical action between Eu2O3/g-C3N4 and K2S2O8. By referencing the proposed g-C3N4 model,12,14 the ECL mechanism can be elucidated as follows: g-C3N4 + ne- → ng-C3N4•−

(1)

S2O82− + e- → S2O8•2−

(2)

S2O8•2− → SO42− + SO4•−

(3)

g-C3N4•− + SO4•− → g-C3N4* + SO42−

(4)

g-C3N4* → g-C3N4 + hν

(5)

Oxygen vacancy has a substantial influence on inherent properties of oxides.52,53 Many experiment results have revealed that the oxygen vacancy acted as a crucial part in the defect intermediary energy transport between the catalyst and the reactant.5 As a result, the easier surface reaction and enhanced redox ability in electrochemical process are obtained. As for catalysis, the catalytic capacity of Eu2O3 for the ECL


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reaction of g-C3N4 can be enhanced. More specifically, in the ECL process, the energy transport between a catalyst (Eu2O3) and the reactant (g-C3N4 and S2O82−) was accelerated by the oxygen vacancy, namely, the equal (4) is promoted. Therefore, the ECL emission of Eu2O3-g-C3N4 enhanced by 2.8-fold compared to pure g-C3N4 due to oxygen vacancy promoting catalytic activity effect. Fabrication of ECL Aptasensor. EIS is an effective tool to study the interface properties of electrodes and Figure 4A displayed the EIS data of different electrodes in [Fe(CN)6]3−/4– solution. Apparently, the electron-transfer resistance (Ret) at the Eu2O3/g-C3N4/GCE was smaller than that of apt/Eu2O3/g-C3N4/GCE, showing that the phosphate backbone of the oligonucleotide with negative charge generated electrostatic repulsion force towards the [Fe(CN)6]3−/4−. After apt/Eu2O3/g-C3N4/GCE capturing E2, a significant increase in the Ret was observed. The results suggested that the aptasensing platform has been successfully constructed. To trace the fabricating process, we recorded ECL signals of the aptasensor in different assembly stages (Figure 4B). The Eu2O3/g-C3N4/GCE produced a strong ECL signal (curve a), implying that Eu2O3/g-C3N4 nanocomposite was a desirable material for the construction of ECL sensors. After E2 aptamer on the Eu2O3/g-C3N4/GCE surface, the ECL intensity of the apt/Eu2O3/g-C3N4/GCE decreased because that the loading of aptamer onto the Eu2O3/g-C3N4/GCE blocked the ability of electron transfer, thus reducing the ECL signal (curve b).31 The ECL signal further reduced when E2 incubated with its aptamer (curve c).29 As such, the detection of E2 can be realized (Scheme 1). Optimization of Working Conditions. Before the aptasensor was constructed, the ECL behaviors of the Eu2O3/g-C3N4 nanocomposite with different ratios of Eu2O3 had been investigated to obtain optimal ECL performances (Figure S4A). The results



suggested that the ECL intensity increased as the Eu2O3 increased from 0 wt% to 0.3 wt% and then decreased dramatically with the further increment of Eu2O3, implying that Eu2O3 played a crucial role in improving ECL activity of g-C3N4. Thus, 0.3 wt% ratio of Eu2O3 was selected for the aptasensor throughout the work. As displayed in Figure S4B, the effect of scan rates on the ECL response was explored. The ECL emission went up gradually at the increasing scan rate of 25~100 mV/s and then trended toward a maximum value with scan rate range of 100~200 mV/s. Therefore, in this work, we selected 100 mV/s as its optimal scan rate. To obtain the maximum sensitivity, several experimental conditions were optimized. The effects of the binding time between the aptamer and E2 on the electrodes surface were evaluated. As exhibited in Figure S4C, the ∆I increased for the 20 min and then leveled off when the reaction time exceeded 20 min. Consequently, 20 min binding time was applied for E2 detection. Finally, the influence of aptamer concentration was also studied by measuring the relative ECL intensity changes (∆I) before and after addition aptamer were obtained with different concentrations (Figure S4D). The best conditions for assays were determined according to the sensitivity (maximum ∆I) of the proposed aptasensor. Therefore, 2 µM aptamer was used in further experiments. Sensitive Detection of E2 Based on the ECL Aptasensor. In order to evaluate the analytical performance of the ECL aptasensor, the variation of the ECL intensity was performed in various concentrations of E2 under the optimal conditions in Figure 5A. The ECL intensity steadily declined with the E2 concentration increasing from 10 fM to 10 nM, implying that an increasing number of E2-aptamer complexes forming increased the steric hindrance for the apt/Eu2O3/g-C3N4/GCE. When the E2 concentration was over 10 nM the ECL intensity remained almost invariable,


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demonstrating that the quantity of E2-aptamer complexes on the sensing interface reached saturation. Besides, the calibration curve for the E2 assay was presented in Figure 5B. The linear relation between the ECL intensity (I) and the logarithm of the E2 concentration was built up with a correction coefficient of 0.9895. The detection limit is estimated to be 3.33 fM (S/N=3). As listed in Table S1, the analytical performances of the ECL aptasensor for E2 detection were compared with other methods. The sensitivity of the proposed method was higher than the their results.29,30,54,55 Reproducibility, Stability and Specificity. To evaluate the reproducibility of the prepared ECL aptasensor, an intra-assay and an inter-assay relative standard deviation (RSD) was examined. The intra-assay RSD is 5.37 % for detecting 10 pM E2 with 5 replicate tests, and the inter-assay RSD is 7.13 % by testing 10 pM E2 with 5 similar ECL aptasensor, implying the ECL aptasensor having eminent reproducibility. For practical application of a aptasensor, operational stability is one of the major focus of attention. Figure S5A displays the ECL curve of the Eu2O3/g-C3N4/GCE under 25 cycles of consecutive scans in 10 mM K2S2O8 solution. A robust ECL signal was observable, signifying the excellent stability of the ECL response. The selectivity of the ECL aptasensor for E2 assay was measured by recording the ECL intensity variation (∆I) of the aptasensor with other interferent components added, such as EE, E3, bisphenol-A (BPA), and hydroquinone (HQ),. The results were shown in Figure S5B. It was found that 100-fold concentration of the other interferent components solely slightly affected on the ECL intensity. These verified that the as-prepared aptasensor would be utilized to monitor E2 with high selectivity. Milk Powder Samples Analysis. 5 g of milk powder and 30 mL ethanol were mixed by ultrasonic treatment for 30 min. Then the suspension was treated by



centrifugation at 12000 rpm for 5 min to collect the supernatant and dried in vacuum. The residues were dissolved in 2 mL ethanol to obtain the sample solution. Finally, the obtained milk sample was spiked with three different concentrations of E2 standard solution for use. The samples without adding E2 were used as negative control. The subsequent ECL detection was performed as the conventional ECL aptasensing. The mean quantity of E2 in milk powder samples was 13.97 ng g−1 (n=3), meanwhile the detailed detection results were depicted in Table S2. With the RSD of 3.7~6.9 %, the recoveries were 83.87~105.26 %. These indicated that the developed aptasensor could be used to E2 practical assays in real samples. ■ CONCLUSION In summary, the oxygen vacancy was engineered into Eu2O3/g-C3N4 nanocomposite for establishing a sensitive and selective ECL sensing plateau for E2 detection. The results demonstrated that Eu2O3 exhibited attractive sensitizing effects for the g-C3N4/S2O82− ECL system due to oxygen vacancy promoting the catalytic activity effect. The novel ECL method had good performances for E2 determination. It could provide a promising guideline for developing highly efficient ECL aptasensors. ■ ASSOCIATED CONTENT

Supporting Information More experimental details and supplementary figures of the whole detection system. This material is available free of charge via the Internet at http://pubs.acs.org. ■ AUTHOR INFORMATION Corresponding Author * Address: Jiangsu Collaborative Innovation Center of Biomedical Functional


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Materials, Jiangsu Key Laboratory of New Power Batteries, College of Chemistry and Materials Science, Nanjing Normal University, Nanjing 210023, China; E-mail: [email protected], Fax: +86 25 85891767; Key Laboratory of Modern Agriculture Equipment and Technology, School of Chemistry and Chemical Engineering, Jiangsu University, Zhenjiang, Jiangsu 212013, P.R. China. E-mail: [email protected]. Tel.: +86 511 88791800. Fax: +86 511 88791708. Author Contributions The manuscript was written through the contributions of all the authors. All the authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest. ■ ACKNOWLEDGMENTS The present work was supported by the National Natural Science Foundation of China (Nos. 21175061, 21375050, 21575067 and 21675066), Research Foundation of Zhenjiang Science and Technology Bureau (No. NY2016011) and Innovation Project of Science and Technology for Graduates of Jiangsu University (No. KYLX16_0907).



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Page 20 of 26

Scheme 1

SO42−−

ECL signal

SO4•−

S2O82−−

hv 1

g-C3N4 •−

2

g-C3N4 3

g-C3N4∗

1

e−

e− e− GCE

Eu2O3

e− Oxygen Vacancy Binding

2

e−

e− e− GCE

e−

3

e−

e− e− GCE

e−

Aptamer E2

Scheme 1. The illustration of the fabrication and mechanism of the ECL aptasensor.


Page 21 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 1

A

B

0.2 µm

100 nm

C

D

100 nm

0.5 µm

Figure 1. TEM images of (A) Eu2O3, (B) g-C3N4, (C) Eu2O3/g-C3N4, and (D) SEM image of Eu2O3/g-C3N4.



Figure 2 B

b

Intensity (a.u.)

Intensity (a.u.)

A

Survey

Eu 3d

N 1s O 1s C 1s Eu 4d

a

C 3+ Eu 3d5/2 2+

2+ Multiplet 1170 1155 1140 1125 Binding Energy (eV)

900 600 300 Binding Energy (eV)

0

Eu 4d

D Intensity (a.u.)

3+ Eu 3d3/2

1200

72 Eu 3d

3+ Eu 4d5/2

36 54 2θ (degree)

Eu 4d3/2

18

Intensity (a.u.)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 26

2+

150 145 140 135 130 125 Binding Energy (eV)

Figure 2. (A) XRD patterns of g-C3N4 (a) and Eu2O3/g-C3N4 (b). The XPS spectra of the Eu2O3/g-C3N4: (B) the survey spectrum of Eu2O3/g-C3N4 and high-resolution spectra of (C) Eu 3d, and (D) Eu 4d.


Page 23 of 26

Figure 3 a

0.0

B

-0.3

c

-4

A

Current (10 A)

ECL Intensity (a.u.)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


b c Time (s)

-0.6 -0.9 -1.2

b a -1.6

-1.2 -0.8 -0.4 Potential (V)

0.0

Figure 3. (A) ECL curves and (B) cyclic voltammograms of Eu2O3/g-C3N4/GCE (a), g-C3N4/GCE (b), and Eu2O3/GCE (c) in 0.1 M PBS containing 10 mM K2S2O8.



Figure 4

A


600 Z'' (Ohm)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 26

400

200

B

a

b c

ab c

0 0

300

600 900 1200 1500 Z' (Ohm)

Figure 4. (A) EIS of Eu2O3/g-C3N4/GCE (a), apt/Eu2O3/g-C3N4/GCE (b), and after the incubation with 10 pM E2 on the apt/Eu2O3/g-C3N4/GCE (c). (B) ECL curves of Eu2O3/g-C3N4/GCE (a), apt/Eu2O3/g-C3N4/GCE (b), and after the incubation with 10 pM E2 on the apt/Eu2O3/g-C3N4/GCE (c) in 0.1 M PBS (pH=7.4) containing 10 mM K2S2O8.


Page 25 of 26

Figure 5

j


B

a ECL Intensity (a.u.)

A ECL Intensity (a.u.)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0.0

14

13

4.3 8.6 C E2 (nM)

12 11 10 -Log (CE2)

9

8

Figure 5. (A) The relationship between the ECL intensity and different E2 concentration: 1×10−14 (a), 5×10−13 M (b), 1×10−12 M (c), 5×10−12 M (d), 1×10−11 (e), 5×10−11 (f), 1×10−10 M (g), 5×10−10 M (h), 1×10−9 M (i), and 1×10−8 M (j). (B) The corresponding linear calibration curve for E2 detection. Error bars: ±S.D., n=5.



For TOC Only:

SO42−−

SO4•−

S2O82−−

e−

g-C3N4 •−

hv

ECL signal

g-C3N4∗ e−

e−

e−

Signal Amplification

GCE g-C3N4

Eu2O3

Oxygen Vacancy


Page 26 of 26

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