Dual-Wavelength Electrochemiluminescence Ratiometry Based on

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Dual-wavelength electrochemiluminescence ratiometry based on resonance energy transfer between Au nanoparticles functionalized g-C3N4 nanosheet and Ru(bpy)32+ for microRNA detection Qiu-Mei Feng, Yi-Zhong Shen, Mei-Xing Li, Zhuo-Lei Zhang, Wei Zhao, Jing-Juan Xu, and Hong-Yuan Chen Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b03670 • Publication Date (Web): 02 Dec 2015 Downloaded from http://pubs.acs.org on December 4, 2015

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Analytical Chemistry

Dual-wavelength electrochemiluminescence ratiometry based on resonance energy transfer between Au nanoparticles functionalized g-C3N4 nanosheet and Ru(bpy)32+ for microRNA detection Qiu-Mei Feng,1 Yi-Zhong Shen,1 Mei-Xing Li,1 Zhuo-Lei Zhang, Wei Zhao*, Jing-Juan Xu*

and Hong-Yuan Chen

State Key Laboratory of Analytical Chemistry for Life Science and Collaborative Innovation Center of Chemistry for Life Sciences, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023, China.

* Corresponding author. Tel/Fax: +86-25-89687294;

E-mail address: [email protected] (J.J. Xu); [email protected] (W. Zhao)

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ABSTRACT:

Here, a dual-wavelength ratiometric electrochemiluminescence (ECL) approach is reported based on resonance energy transfer (RET) from graphite-like carbon nitride nanosheet (g-C3N4 NS) to Ru(bpy)32+ for sensitive detection of microRNA (miRNA). In this approach, Au nanoparticles (Au NPs) functionalized g-C3N4 NS nanohybrid (Au-g-C3N4 NH) coated on glassy carbon electrode (GCE) could exhibit strong and stable ECL emissions with emission peak centered at 460 nm. The ECL emission at such wavelength matched well with the absorption peak of Ru(bpy)32+ as well as impeccably stimulate the emission of Ru(bpy)32+ at the wavelength of 620 nm, producing ECL-RET with high efficiency. Thus, based on the ECL signals quenching at 460 nm and increasing at 620 nm, a dual-wavelength ratiometric ECL-RET system was achieved. This system was then utilized for determination of target miRNA. With the attachment of thiol-modified molecular beacon on Au-g-C3N4 NH, target miRNA hybridized with the molecular beacon to form DNA-RNA duplex. The obtained DNA-RNA duplex could be cleaved by duplex-specific nuclease to release target miRNA which would take part in the next cycle for further hybridization. Finally, the introducing of Ru(bpy)32+ was through the probe DNARu(bpy)32+ complementary with the rest single-strand DNA on electrode. By measuring the ratio of ECL460 nm/ECL620 nm, we could accurately quantify the concentration of miRNA-21 in a wide range from 1.0 fM to 1.0 nM. This work provides an important reference for the study of dualwavelength ECL ratiometry and also exhibits potential capability in the detection of nucleic acids.

KEYWORDS: dual-wavelength ratiometry, electrochemiluminescence, resonance energy transfer, Au-g-C3N4 nanohybrid, Ru(bpy)32+, enzyme-assisted cycle amplification.

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INTRODUCTION Recently, ratiometric assay, in which the quantification depends on the ratio of two signals instead of absolute values, has attracted widespread attention. This double-signal ratiometric technique could effectively normalize the variation in environmental changes, such as path length, background light and scattering, and thus offer more precise measurement in analytical application than single-signal measurement.1-4 Up to now, ratiometric detection has extensively developed in fluorescence,5-7 electrochemiluminescence (ECL)8-11 and electrochemical12-14 analysis. Among them, the ratiometric fluorescence is mainly based on the use of dualwavelength signal ratio and the ratiometric electrochemical method is based on dual-potential signal ratio. For ECL, undergoing a highly exergonic reaction, it produces an electronically excited state caused by electrogenerated species and emits light upon relaxation to a lower-level state. Thus, ratiometric ECL should include both dual-potential and dual-wavelength signal ratiometric assays. A few works addressed on the dual-potential ratiometric ECL systems in biological and chemical analysis. For example, the dual-potential ratiometric ECL systems were performed in CdS QDs-luminol for DNA,4 miRNA15 and Mg2+ detection,8 g-C3N4-luminol for cancer cells detection16 and O2-amino terminated perylene derivative for lead ion determination.9 However, restricted by the luminescence intensity and wavelengths of commonly used ECL emitters, dual-wavelength ratiometric ECL has not been developed in analytical detection. In luminescence ratiometric detection, resonance energy transfer (RET) has been widely applied in the construction of biosensors. Under the luminophores donor and acceptor at close proximity, this RET system could give rise to the quench of donor emission through nonradiative energy dissipation in the acceptor. For ratiometric fluorescence, many Förster resonance energy transfer (FRET) systems were applied in for pH determination,17 H2S sensing in cells,18 Fe3+



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detection19 and so on. Inspired by ratiometric fluorescence, our group firstly developed the dualpotential ratiometric ECL resonance energy transfer (ECL-RET) sensing approaches for nucleic acid detection by measuring the ratio of ECLluminol/ECLCdS at two excitation potentials.4,15 Recently, Yuan’s group further designed a dual-potential ratiometric ECL-RET system for the determination of lead ion.9 For dual-wavelength ratiometric ECL-RET system, this strategy should integrate dual independent emissions of two ECL emitters with the spectral overlap between donor’s ECL spectrum and acceptor’s absorption spectrum. As a conventional ECL reagent, due to its high ECL efficiency, good chemical stability and superior biocompatibility, Ru(bpy)32+ has been widely used in ECL bioanalytical system. Our group reported the effective ECL-RET systems in CdS nanoparticles-Ru(bpy)32+ donor-acceptor pair for immunosensing and cytosensing.20,21 However, the ECL intensity disparity between Ru(bpy)32+ and CdS QDs was evident and thus caused the decrement of ECL intensity from CdS QDs far less than the increment of ECL intensity from Ru(bpy)32+. The CdS QDs-Ru(bpy)32+ pair was not the best for the dual-wavelength ratiometric ECL-RET system. Therefore, a key point in dual-wavelength ratiometric ECL-RET is to find a suitable luminescent reagent whose ECL intensity is comparable to that of Ru(bpy)32+. Graphite-like carbon nitride nanosheet (g-C3N4 NS), as a metal-free semiconductor material has recently been proved to be an effective, strong and stable ECL emitter. This layered material was widely used in ECL sensors for sensitive determination of carcinoembryonic antigen (CEA),22,23 DNA,24 concanavalin A,25 metal ions,26 dopamine,27 lactate28 and rutin.29 Despite showing superior luminescence and excellent biocompatibility, less attention was paid to its ECL-RET application. G-C3N4 NS has a broad ECL emission peaked at ca. 460 nm in the ECL spectrum. Coincidentally, Ru(bpy)32+, just possesses an obvious excitonic absorption peak at


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460-470 nm owning to metal-to-ligand charge transfer absorption.30 Therefore, g-C3N4 NSRu(bpy)32+ pair with strong ECL intensity and suitable spectral overlap between ECL spectrum of g-C3N4 NS and absorption spectrum of Ru(bpy)32+ may break the bottleneck of the foundation and application of the dual-wavelength ratiometric ECL system. Herein, an ECL approach is reported based on an efficient Au NPs-g-C3N4 NH/Ru(bpy)32+ RET pair. It is a pioneering work of dual-wavelength ECL ratiometric assay. Compared with single wavelength ECL detection, it is more reliable. We firstly utilized the spectral overlap between g-C3N4 NS and Ru(bpy)32+ to build ECL sensor for highly sensitive target miRNA detection. A hairpin structure molecular beacon and enzyme-assisted cycle amplification were used to improve the sensitivity and for efficient energy transfer between Au-g-C3N4 NH and Ru(bpy)32+. Only after hybridization with target miRNA did the hairpin structure open up, then probe DNA-Ru(bpy)32+ incubated to be located at close proximity of Au-g-C3N4 NH, resulting in an energy transfer from Au-g-C3N4 NH to Ru(bpy)32+. Finally, the decline of ECL intensity from Au-g-C3N4 NH at 460 nm and the increase of ECL intensity from Ru(bpy)32+ at 620 nm both reflect the amount of target miRNA. EXPERIMENTAL SECTION Table 1. DNA sequences employed in this work. name

Sequences (5′ to 3′)



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DNA and RNA sequences employed in ECL sensing Molecular beacon

SH-TTT CGC ACT AGC TTA TCA GAT TTT TCA ACA TCA GTC TGA TAA GCT A

Probe DNA

TAA GCT AGT GCG TTT-NH2

miRNA-2131,32

UAG CUU AUC AGA CUG AUG UUG A

SM miRNA-21

UAG CUU AUC AGA AUG AUG UUG A

TM miRNA-21

UAG CAU AUC CGA CCG AUG UUG A

DNA sequences employed in purification of probe DNA-Ru(bpy)32+ Capture DNA

CGC ACT AGC TTA ATC CAG AGT GAC GCA GCG TTTNH2

Substituent DNA

CGC TGC GTC ACT CTG GAT TAA GCT AGT GCG

The underlined region of the molecular beacon identifies the stem sequence. The structure of the molecular beacon: the loop is made of DNA and the stem is modified with 2-OMe-RNA. SM: single-based mismatched miRNA-21; TM: three-base mismatched miRNA-21. The mismatched bases for miRNA-21 are highlighted in the boxes. Synthesis of g-C3N4 NS. The bulk g-C3N4 material was prepared following the previously reported literatures.22,33,34 In detail, 20 g of melamine was placed in an alumina crucible with a cover and then heated at 600 °C for 2 h with a heating rate of 3 °C min-1, leading to yellow powder. Although highly water-dispersible g-C3N4 nanoﬂake particles can be prepared by chemically oxidizing the bulk g-C3N4 with nitric acid, a liquid exfoliating method with minor modiﬁcation was used to obtain ultrathin g-C3N4 NS in this present study. In brief, 1 mg of the bulk g-C3N4 powder was dispersed in 6 mL of water, and an ultrasound was performed on the mixture consecutively for 16 h. Then, the final formed suspension was centrifuged at 5000 rpm to remove the residual unexfoliated g-C3N4 and the mass concentration of the g-C3N4 NS suspension was calculated by weighing the power dried from a certain volume of the suspension.


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In addition, the control acidified g-C3N4 NS was prepared according to a previously reported work.35 Briefly, 1 mg of bulk g-C3N4 material was treated with 5 M HNO3 and refluxed for 24 h at 125 °C. Then, the obtained product was centrifuged and washed three times with water. The ultimate product was dried in vacuum oven at 35 °C for 12 h. Preparation of the Au-g-C3N4 NH. Au-g-C3N4 NH was prepared according to the literature with a slight modification.22 In brief, 30 µL of 0.01 M HAuCl4 solution was added to g-C3N4 NS suspension (0.15 mg mL-1) under stirring. The obtained suspension was sonicated for 10 min and then stirred for 2 h at room temperature. This process was repeated three times. Afterwards, 75 µL of fresh NaBH4 solution (0.01 M) was injected quickly to the suspension and the reaction mixture was stirred for 20 min in ice bath. Then, 30 µL of sodium citrate solution (0.01 M) was dropped into the above suspension and the stirring reaction was further kept for 30 min at 25 °C. In order to remove excess NaBH4, sodium citrate and unbound Au NPs, the obtained nanohybrid material was centrifuged and washed with water for three times. The final deposition was redispersed into 6 mL of water and stored at 4 °C for further use. Synthesis of probe DNA-Ru(bpy)32+. The fabrication and purification process of probe DNARu(bpy)32+ was shown in Figure S1. Figure S1A displayed the formed process of probe DNARu(bpy)32+. Briefly, 200 µL of Ru(bpy)32+-NHS (0.5 mM) was added into 200 µL of aminogroup modified probe DNA (1.5×10-6 M) and the mixture was shaken at low speed over light at 25 °C to form probe DNA-Ru(bpy)32+. To remove excess Ru(bpy)32+ molecule and unbound probe DNA, the purification of probe DNA-Ru(bpy)32+ was carried out by magnetic separation technique (Figure S1B). Firstly, 100 µL EDC (20 mg/mL) and NHS (10 mg/mL) were added to 300 µL of carboxylated magnetic beads solution and the mixture was incubated at 37 °C for 1 h to activate the carboxyl groups on the magnetic beads. After being rinsed with 0.1 M PBS buffer



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(pH 7.4), 200 µL of amino-modified capture DNA (1.5×10-6 M) were added into the activated magnetic beads and the resultant mixture incubated for 1 h with gentle mixing, followed by rinsing with PBS. After that, 2 wt% BSA was added to the magnetic beads-capture DNA solution for 1 h to block the nonspecific active binding sites of magnetic beads. Then, the obtained magnetic beads-capture DNA conjugates were resuspended in 200 µL PBS. At this time, the prepared probe DNA-Ru(bpy)32+ solution (Figure S1A) was added into the magnetic beadscapture DNA solution and incubated with 1 h at 37 °C to gain magnetic beads-capture DNAprobe DNA-Ru(bpy)32+. Excess Ru(bpy)32+ were removed by magnetic force. Finally, the acquired magnetic beads-capture DNA-probe DNA-Ru(bpy)32+ was incubated with 200 µL of substituent DNA (1.5×10-6 M) with 1 h at 37 °C. Due to the totally complementary between capture DNA and substituent DNA, a part complementary probe DNA-Ru(bpy)32+ would be released from magnetic beads-capture DNA-probe DNA-Ru(bpy)32+ and the probe DNARu(bpy)32+supernatant was got under a magnetic field. Preparation of cellular extracts. The miRNA-21 extracts from HeLa cells were prepared according to a previously reported work.15 Human cervical cells (HeLa) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, GIBCO) supplemented with 10% fetal calf serum, streptomycin (100 µg mL-1) and penicillin (100 µg mL-1). These cells were maintained at 37 °C in a humidified atmosphere (95% air and 5% CO2). In the RNase-free environment, after washed twice by PBS buffer, cells were lysed with RNA extractor reagent and then chloroform was added. The obtained mixture was centrifuged for 15 min at 12000 rpm in 4 °C. Then, isopropanol was added into the supernatant to precipitate RNA. After washed with 75% ethanol, miRNA-21 was dissolved in RNase-free buffer.


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Fabrication of the ECL biosensor. The preparation of ECL biosensor is shown in Scheme 1. Firstly, a glassy carbon electrode (GCE) was applied as a substrate for immobilizing Au-g-C3N4 NH. The GCE was polished in sequential order with 1.0, 0.3 and 0.05 nm of alumina and thoroughly rinsed with distilled water. Subsequently, 10 µL of Au-g-C3N4 NH suspension was drop-cast on the surface of GCE and dried to get Au-g-C3N4 NS film modified GCE at room temperature. The acquired Au-g-C3N4 NH-GCE was immersed in 100 µL of the stem-loop structure molecular beacon solution (0.5×10-6 M) activated by 10 mM TCEP to cut S-S bond and incubated for 2 h at 4 °C. Subsequently, the electrode was rinsed with PBS buffer and the resulting molecular beacon modified electrode was immersed in 60 µL of MCH (100 µM) for 1 h to block the nonspecific active binding sites and force the modified DNA probe to adopt an upright surface orientation that favor further hybridization. Finally, the electrode surface was rinsed with PBS buffer (pH 7.4) to remove the unbounded substance and stored for the further use. Analysis procedure. The gaining electrode was immerged in 100 µL of PBS buffer containing 5.0 mM Mg2+, different concentrations of miRNA, 0.1 U µL-1 DSN at 37 °C for 60 min. And DSN stop solution containing 5 mM EDTA was used to end the enzyme amplification reaction. After washing with PBS buffer containing 5.0 mM Mg2+ thoroughly, the resulting electrode incubated with probe DNA-Ru(bpy)32+ at 37 °C for 60 min. ECL detection was accomplished with the electrodes in 0.1 M PBS (pH 8.3) containing 0.1 M K2S2O8 and scanned from 0 to -1.5 V. RESULTS AND DISSCUSSION Characteristics of g-C3N4 NS. Recently, the reported fabrication methods for g-C3N4 NS mainly involved thermal oxidizing acid etching and liquid ultrasonic exfoliation.22,33-38 As we



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know, introducing oxygen-containing functional groups (such as hydroxyls, epoxides, diols, ketones and carboxyls) through strong acid oxidizing in the graphite was for the formation of graphene oxide nanosheets.39 Although the hydrophilic performance of graphite was improved, graphene oxide possessed poor conductivity. Similarly, acidified g-C3N4 NS has good hydrophilicity and weak electron transfer ability. XPS is a powerful tool for examining the elements of g-C3N4 NS. As demonstrated in Figure 1A, it could be found that ultrasonic exfoliation g-C3N4 NS and acidified g-C3N4 NS are both mainly composed of carbon and nitrogen elements, while a small amount of oxygen element was detected in acidified g-C3N4 NS, indicating the formation of oxygen-containing functional groups in the g-C3N4 NS layers. Further difference between ultrasonic exfoliation g-C3N4 NS and acidified g-C3N4 NS comes from the FT-IR spectra in Figure 1B. The typical absorption peaks of the vibration of triazine ring at 810 cm-1, stretching vibration of connected units of C-N(-C)-C (full condensation) or CNH-C (partial condensation) at 1000-1800 cm-1 and N-H stretching and hydrogen-bonding interactions absorption bands at 3000-3300 cm-1 were obtained in both exfoliation g-C3N4 NS and acidified g-C3N4 NS.37 It can be noted that a weak absorption peak at 1132 cm-1 corresponding to the stretching vibration of C-O appears in the FT-IR spectra of acidified g-C3N4 NS. Curve a and b in Figure C are the ECL signals from acidified and ultrasonic exfoliation g-C3N4 NS, respectively. In the presence of coreactant K2S2O8, when the potential scanned from 0 to 1.5 V, the semiconductor g-C3N4 NS and the coreactant S2O82- were reduced to g-C3N4•− and SO4•−, respectively. The strong oxidant SO4•− could react with g-C3N4•− to produce excited state g-C3N4* which decayed to the ground state with light emitting.40 Although chemically oxidized bulk g-C3N4 with nitric acid or sulfuric acid could bring highly water-dispersible, compared with


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the ultrasonic exfoliation g-C3N4 NS, weak ECL signal from acidified g-C3N4 NS was obtained. Thus, in this study, the g-C3N4 NS as the ECL emitter was directly prepared by ultra-sonicationassisted liquid exfoliation of bulk g-C3N4. Besides, transmission electron microscopy (TEM) and X-Ray nanoparticle diffraction (XRD) further demonstrated the crystal structure and chemical states of the as-prepared ultrasonic exfoliation g-C3N4 NS in Figure S2. Characteristics of Au-g-C3N4 NH. To further improve the conductivity of g-C3N4 NS and enhance bonding sites of molecular beacon modified with SH, Au NPs were introduced in the gC3N4 NS. Figure 2A shows the TEM image of the hybridized Au-g-C3N4 NH. We can see that a large number of Au NPs are distributed on the surface of g-C3N4 NS. –NH– and –NH2 groups on the surface of g-C3N4 NS brought about dozens of anchoring sites to coordinate with gold ions and promoted the reduction of AuCl4-, resulting in the formation of stable Au NPs on the surface of g-C3N4 NS.22 From the magnified TEM image of Au-g-C3N4 NH, the average size of Au NPs was approximately 5 nm. The weight percentage (wt %) of Au in the assembled Au-g-C3N4 NH is examined as 3.82 % via ICP-AES. Figure 2B shows the results of energy dispersive X-ray (EDX) analysis. The presence of C (0.28 Kev), N (0.39 Kev) and Au (2.22 Kev) elements verified the existence of Au-g-C3N4 NH, while Au (2.22 Kev) elements cannot be observed in gC3N4 NS (inset of Figure 2B). Besides, compared with the UV-vis spectra of g-C3N4 NS (Figure 2C, curve a), Au-g-C3N4 NH (Figure 2C, curve b) exhibited a new characteristic absorption band at 520 nm (highlighted in a blank rectangle) which was attributed to Au NPs. Figure 2D displayed the effect of Au NPs on the electrochemical and ECL signal responses of g-C3N4 NS. Compared with g-C3N4 NS (blank, curve a, a’), Au-g-C3N4 NH possessed stronger electrochemical current and ECL emission (red, curve b, b’), which was attributed to the excellent electronic transmission ability of noble metal nanoparticles. Meanwhile, the stability of



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Au-g-C3N4 NH film modified GCE was evaluated by monitoring the ECL response in Figure S3, which did not appear obvious changes in the ECL intensity under continuous potential scanning for fifteen cycles. Construction and characterization of the dual-wavelength ratiometric ECL-RET sensor. As illustrated in Scheme 1, a dual-wavelength ratiometric ECL-RET sensor was designed to detect miRNA. The hairpin molecular beacon probe labeled with a thiol at its 5’ end was immobilized on a GCE modified with Au-g-C3N4 NH. When the immobilized molecular beacon probe met target miRNA, the hairpin structure changed to rodlike through complementary hybridization and a DNA-RNA duplex was formed. To improve detection sensitivity, enzymeassisted cycle amplification was applied. DSN could selectively cleave double-stranded DNA or DNA in DNA-RNA heteroduplex and has little role on the single-stranded DNA, single- or double-stranded RNA. To avoid the digestion of the stem of molecular beacon by DSN, the hairpin molecular beacon is modified with 2-OMe-RNA on its stem.41 Thus, the formed DNARNA duplex on the modification electrode was cleaved off by DSN and the released target miRNA can be reused in subsequent reaction cycles. Then, the obtained electrode was incubated with solution of probe DNA-Ru(bpy)32+ which would hybridize with the remaining part of molecular beacon. If there was no target miRNA, the immobilized molecular beacon probe would be in a “closed” state. The remaining part of molecular beacon was too short to form a stable duplex with probe DNA-Ru(bpy)32+. While, in the existing of miRNA, the formed DNARNA duplex was split by DSN and probe DNA-Ru(bpy)32+ could hybridize with the rest singlestrand DNA. The closely contacted Ru(bpy)32+ quenched the ECL signal from Au-g-C3N4 NH at 460 nm and brought a new ECL signal from Ru(bpy)32+ at 620 nm, because the base ground state of Ru(bpy)32+ molecule was excited to (Ru(bpy)32+)* which decayed to the ground state with


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light emitting under the energy absorption from (g-C3N4)*. Therefore, the increase of target miRNA resulted in the quenching of ECL response from Au-g-C3N4 NH at 420 nm and meanwhile the enhancement of ECL signal from Ru(bpy)32+ at 620 nm. Figure 3A (curve a) presents the typical absorption spectrum of Ru(bpy)32+. Ru(bpy)32+ has an obvious excitonic absorption peak at about 460 nm. The ECL spectrum of the Au-g-C3N4 NH modified GCE had a strong ECL emission centered at about 460 nm (Figure 3A, curve b). Correspondingly, an excitonic emission peak of g-C3N4 NH centered at 445 nm was observed in the fluorescence spectrum. Obviously, the ECL emission of the Au-g-C3N4 NH film had a considerable spectral overlap with the absorption spectrum of Ru(bpy)32+, implying the efficient ECL-RET from Au-g-C3N4 NH to Ru(bpy)32+. In the presence of coreactant S2O82-, when the electrode potential became sufficiently negative, the ECL-potential curve of Au-g-C3N4 NH was displayed in Figure 3B (curve a). However, no cathodic ECL emission of Ru(bpy)32+ in this potential range from 0 to -1.5 V was shown (curve b), implying that Ru(bpy)32+ could be used as a sensitive energy acceptor and bring interference free from its own ECL emission. Figure 4A displays ECL responses of different modified electrodes after each step. When the bare GCE was modified with Au-g-C3N4 NH, a distinguished and stable ECL signal appeared (curve a) at 460 nm. Subsequently, with the grafting of stem-loop structure molecular beacon, the ECL signal obviously decreased (curve b), ascribed to the formation of less conductive layers on the surface of electrode. After the incubation of MCH (curve c), target miRNA and DSN (curve d), ECL intensity further decreased. While, after the hybridization of probe DNARu(bpy)32+, the ECL signal of Au-g-C3N4 NH is distinctly reduced at 460 nm and meanwhile a new emission peak occurred at 620 nm (Figure 4B, curve e), which was attributed to the emission of Ru(bpy)32+ molecule, confirming the successful assembly of probe DNA-Ru(bpy)32+



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and effective interactions of ECL-RET between g-C3N4 NH and Ru(bpy)32+. All above results were consistent with the EIS characterization of miRNA ECL sensing system (Figure S4). Figure 4C shows the ECL spectra of the modified electrode in the absence and presence of target miRNA-21. In the absence of miRNA-21, only Au-g-C3N4 NH emission peak at 460 nm (Figure 4C, curve a) exhibited. While, with the increase of miRNA-21 concentration, the ECL emission peak of Ru(bpy)32+ at 620 nm increased and that of Au-g-C3N4 NH at 460 nm decreased greatly (Figure 4C, curve b-d), indicating the effective ECL-RET between Au-g-C3N4 NH and Ru(bpy)32+. Analytical performance. On the basis of above results, the effective dual-wavelength ratiometric ECL-RET system between Au-g-C3N4 NH and Ru(bpy)32+ was applied to sensitively detect different concentrations of miRNA-21. At the beginning, we optimized the modification time for molecular beacon on the GCE and the duration of hybridization between molecular beacon and target miRNA (Figure S5). 2 h of modification time and 60 min of hybridization time were selected. Under the optimal experimental conditions, the sensing behavior of the proposed dual-wavelength ratiometric ECL-RET system to miRNA-21 concentration was studied with the concentrations of molecular beacon and probe DNA-Ru(bpy)32+ both fixed at 0.5 µM. As shown in Figure 5A, with the increase of miRNA-21 concentration, the ECL intensity at 460 nm decreased and the ECL signal at 620 nm increased, correspondingly. Furthermore, the logarithmic value of ECL460 nm/ECL620 nm linearly depended on the logarithm of the miRNA-21 concentration in the range of 1.0 fM to 1.0 nM with a correlation coefficient of 0.9914 (Figure 5B). The detection limit was 0.5 fM at the signal to noise ratio of 3. In addition, the concentration of miRNA-21 in HeLa cell extracts was measured and the average copy number of miRNA-21 in single cell was about (2.7±0.31)×103, which was consistent with the reported


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results of miRNA-21 expression.42 The recovery rates of standard addition were in a range considered to be acceptable (95% to 106%), indicating that the proposed dual-wavelength ratiometric ECL-RET biosensor is applicable for microRNA detection in real samples. Compared with some existing miRNA-21 detection methods, this dual-wavelength ratiometric ECL-RET biosensor provides a more effective approach with wider dynamic concentration response range and lower detection limit for the analysis of miRNA-21 (Table S1). In this dual-wavelength ratiometric ECL-RET system, the selectivity was evaluated using three types of RNA sequences at the same concentration (100 pM) which included completely complementary miRNA-21, the single-base mismatched miRNA-21 (SM miRNA-21) and threebase mismatched miRNA-21 (TM miRNA-21), respectively. As shown in Figure 5C, significant differences in the ECL460 nm/ECL620 nm were obtained between complementary miRNA-21 and other RNAs. The RNAs with single-base-mismatch and three-base-mismatch showed small changes compared to the blank. These results indicated that the developed dual-wavelength ratiometric ECL-RET sensing system exhibited an acceptable sequence specificity. CONCLUSION In conclusion, we have demonstrated an efficient dual-wavelength ratiometric ECL-RET system between Au-g-C3N4 NH as a donor and Ru(bpy)32+ as an acceptor for highly sensitive and excellent specific detection of target miRNA. Au-g-C3N4 NH on the electrode showed a stable and strong ECL emission in the presence of coreactant S2O82-, implying good potential in ECL detection. The ECL intensity of Au-g-C3N4 NH (460 nm) decreased with the increase of target miRNA and meanwhile that of Ru(bpy)32+ (620 nm) enhanced gradually, suggesting that ECLRET efficiently take place between Au-g-C3N4 NH and Ru(bpy)32+. Our ECL-RET system



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would open a new vista to process dual-wavelength ECL ratiometric assay for much broader analytical applications. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] (W. Zhao), [email protected] (J.J. Xu) Tel: +86-25-89687294 Author Contributions 1: these authors contributed equally. Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS This work was supported by the 973 Program (Grant 2012CB932600, 2013CB933802), the National Natural Science Foundation (21327902, 21535003) of China. This work was also supported by a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions. Supporting Information Available. Materials used in all experiments, preparation of probe DNA-Ru(bpy)32+, characterization of g-C3N4 NS via TEM, XRD, ECL, EIS, optimization of detection conditions, comparison of current work with existing method. This material is available free of charge via the Internet at http://pubs.acs.org.


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Figure 1 (A) XPS spectrum, (B) FT-IR spectra and (C) ECL signals of acidified g-C3N4 NS (curve a) and ultrasonic exfoliation g-C3N4 NS (curve b) in 0.1 M PBS (pH 8.3) containing 0.1 M K2S2O8. PMT was set at -300 V.

Figure 2 (A) TEM image of the synthetic Au-g-C3N4 NH. Inset is the magnified version. (B) The EDX of Au-g-C3N4 NH. Inset is the EDX of g-C3N4 NS. (C) UV-vis absorption spectra of (a) gC3N4 NS and (b) Au-g-C3N4 NH. (D) Corresponding cyclic voltammograms (CVs) and ECL signal responses of (blank, curve a, a’) g-C3N4 NS modified on the GCE and (red, curve b, b’) Au-g-C3N4 NH modified on the GCE in 0.1 M PBS (pH 8.3) containing 0.1 M K2S2O8. PMT was set at -300 V.



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Scheme 1 Schematic illustration of the dual-wavelength ratiometric ECL-RET biosensor configuration strategy.

Figure 3 (A) UV-vis absorption spectrum of Ru(bpy)32+ (a) and ECL spectrum of the Au-g-C3N4 NH modified GCE (b). Inset is the fluorescence emission of g-C3N4 NS. The ECL spectrums were gained through a series of optical filter (spaced 20 nm) and measured in 0.1 M PBS (pH 8.3) containing 0.1 M S2O82-. PMT was set at -500 V. (B) Cyclic ECL curves of Au-g-C3N4 NH (curve a) and Ru(bpy)32+ (curve b). PMT was set at -300 V.


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Figure 4 (A) The ECL responses of (a) Au-g-C3N4 NH/GCE, (b) molecular beacon/Au-g-C3N4 NH/GCE, (c) MCH/molecular beacon/Au-g-C3N4 NH/GCE, (d) MCH/molecular beacon/Au-gC3N4 NH/GCE after hybridization with 10 pM miRNA-21 and 0.1 U µL-1 DSN, (e) further incubation with probe DNA-Ru(bpy)32+. PMT was set at -300 V. (B) The corresponding ECL responses of (d) MCH/molecular beacon/Au-g-C3N4 NH/GCE after hybridization with 10 pM miRNA-21 and 0.1 U µL-1 DSN and (e) further incubation with probe DNA-Ru(bpy)32+ were individually measured through optical filter (at 460 nm and 620 nm) and PMT was set at -500 V. (C) The ECL spectrum of molecular beacons/MCH/Au-g-C3N4 NH modified electrode recorded in the incubation of probe DNA-Ru(bpy)32+ with different concentrations of miRNA-21 (from a to d: 0, 10 fM, 1 pM, 100 pM). The ECL spectra were gained through a series of optical filter (spaced 20 nm) and measured in 0.1 M PBS (pH 8.3) containing 0.1 M S2O82-. PMT was set at 500 V.



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Figure 5 (A) The relationship between the ECL intensity of (a) Au-g-C3N4 NH at 460 nm or (b) Ru(bpy)32+ at 620 nm and the logarithmic of concentration of miRNA-21 (from 1.0 fM to 1.0 nM). (B) Relationship between the ratio of ECL signal at 460 nm (Au-g-C3N4 NH) to ECL signal at 620 nm (Ru(bpy)32+) and the concentration of miRNA-21. (C) Bar graph of the ECL460 nm/ECL620 nm

for different RNA sequences (100 pM): (a) blank, (b) three-base mismatched

miRNA-21 (TM), (c) single-base mismatched miRNA-21 (SM) and (d) miRNA-21. The ECL is measured in 0.1 M PBS (pH 8.3) containing 0.1 M S2O82-. The ECL responses of Au-g-C3N4 NH were obtained through optical filter of 460 nm and ECL responses of Ru(bpy)32+ gained through optical filter of 620 nm. PMT was set at -500 V.


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For TOC only


Dual-Wavelength Electrochemiluminescence Ratiometry Based on

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