Electrochemiluminescent (ECL) is a kind of luminescence produced from electron transfer reactions of electrochemically generated species on electrode surfaces through an applied potential ^[1]. Because of its versatility,good temporal and spatial control,it has attracted considerable attention as an important and valuable detection method during the past several decades ^{[2, 3]}. Among all ECL systems,Ru(bpy)₃²⁺ is one of the most extensively studied compounds,due to its ability to undergo recycled ECL reactions in aqueous solutions and its good chemical,electrochemical, and photochemical stability ^{[4, 5]}. However,its ECL application in solution phase is also limited by the consumption of the expensive reagent and use of extra pump to deliver the reagent to the electrochemical cell continuously. Up to now,considerable efforts have been focused on the immobilization of Ru(bpy)₃²⁺ on the electrode surface to develop a cost-effective,regenerable and sensitive ECL sensors ^{[6, 7, 8, 9]}. Among them,nanomaterials based immobilization methods acquire the advantages with high sensitivity and good stability ^{[10, 11, 12, 13]}.

Grapheneoxide(GO),a derivative of graphene,is a kindof layered nanomaterial consisting of hydrophilic oxygenated graphene sheets bearing various oxygen functional groups (e.g.,carboxyl,hydroxyl and epoxy groups) on their basal planes and edges ^{[14, 15]}. Therefore,GO is a good matrix for the immobilization of ruthenium complex to develop solid-state ECL sensors by virtue of its high surface area,high π-conjunction,excellent optical transmittance and oxygen functional groups ^{[16, 17, 18, 19]}. For example,Cui et al. developed a reagent-free ECL method for the detection of tripropylamine (TPA) by the preparation of GO covalently functionalized with ruthenium(Ⅱ) complex (Ru-GO),the Ru(Ⅱ) complexwas first designed with a long alkyl amino functional group ^[16]. Xu et al. immobilized Ru(phen)₃²⁺ with GO for ECL analysis based on the electrostatic interactionand π-π stacking interaction^[17]. Although these reported Ru(Ⅱ) complex functionalized GO based ECL sensors showed good ECL activity,and outstanding long-term stability,they displayed the disadvantage of relative complexity for designing Ru(Ⅱ) complex with functional group or the limited amounts of Ru(Ⅱ) complex immobilized on the GO. Therefore,it is still necessary to develop simpleGObasedimmobilizationmethod for ECL analysis with high sensitivity. Recently,silica coated GO composite material has been reported and applied in many fields ^{[20, 21, 22, 23, 24]}. Kou and Gao ^[20] reported the silica nanoparticle coated GO as general building blocks for large-area superhydrophilic coatings. Shi et al. used silica coated GO composite and molecularly imprinted polymers for electrochemical sensing dopamine ^[24]. In consideration of that silica is also a good matrix for immobilizing Ru(bpy)₃²⁺ with the advantages: hydrophilic,non-toxic,chemically stable,and transparent to visible light ^{[25, 26]}. Herein,we reported a simple and novel approach to immobilize Ru(bpy)₃²⁺ in GO-silica composite material for ECL analysis. This strategy was carried out by directly doping Ru(bpy)₃²⁺ in the GO-silica composite film (Ru/GO-SiCF) using one pot hydrolysis and condensation of tetraethylorthosilicate (TEOS) in the water-alcohol solution of GO and Ru(bpy)₃²⁺ at room temperature. TEOS can hydrolyze and condense on the GO surface through Si-O-Si bond to form the SiCF ^[23]. Ru(bpy)₃²⁺ can be immobilized in the composite film through electrostatic interactions. This strategy displayed the advantages compared with GO or silica nanoparticle immobilization method as follows. On one hand, large amounts of Ru(bpy)₃²⁺ can be directly immobilized with high stability in silica,which does not need relative complex procedures for preparing the functional group modified Ru(Ⅱ) complex. On the other hand,the composite film was characterized by high surface- volume ratio and can offer large numbers of reactive sites for the existence of GO,which will improve the charge transfer and mass transport efficiency compared with silica nanoparticle immobilization method. After directly modifying this Ru/GO-SiCF on the GC electrode,it showed excellent ECL performance in sensitivity and stability for the determination of TPA. 2. Experimental 2.1. Apparatus and reagents

Scanning electron microscopy (SEM) images were determined with Hitachi S-4800. The accelerating voltage was 15 kV. Transmission electron microscopy (TEM) was performed on JEM-3010 (JEOL,Japan). The Fourier transform infrared (FT-IR) spectra were recorded with TENSOR27 FT-IR spectrometer (Bruker, Germany). Cyclic voltammetry experiments were made on a CHI660A electrochemical workstation (Shanghai Chenhua Instrument Corporation,China). The ECL emission was monitored with a model MPI-E electrochemiluminescence analyzer (Xi’an Remax Analyses Instrument Co. Ltd.) with the voltage of the photomultiplier tube (PMT) set at 600 V. The working electrode was glassy carbon (GC) electrode. An Ag/AgCl (sat. KCl) reference electrode was used for all measurements. A platinum wire was used as a counter electrode.

GO was purchased from XFNano Material Tech Co.,Ltd. (Nanjing,China),tris(2,2'-bipyridyl)dichlororuthenium(Ⅱ) hexahydrate (Ru(bpy)₃Cl₂ ⋅ 6H₂O) was purchased from Beijing Aipoo Huamei Biotechnology Co.,Ltd. (Beijing,China). Tetraethyl orthosilicate (TEOS) was obtained from Shantou Xilong Chemical Factory (Guangdong,China). Tripropylamine (TPA) was obtained from Sinopharm Chemical Reagent Co.,Ltd. All other chemicals were obtained from Reagent & Glass Apparatus Corporation of Changsha and were of analytical grade used without further purification. All solutions were prepared and diluted using ultrapure water (18.2 MΩ cm) from the Millipore Milli-Q system. 2.2. The preparation of Ru/GO-SiCF and Ru(bpy)₃²⁺ doped SiO₂ nanoparticle (Ru/SiNP)

The Ru/GO-SiCF was prepared by the following procedure. In brief,in a typical synthesis with small modification ^[22],2.5 mL of GO aqueous solution was added into 20 mL of ethanol solution, followed by addition of 1 mL of NH₃ ⋅ H₂O. After stirring at room temperature for 15 min,50 mL of TEOS was added into the mixture. Then 300 mL 0.05 mol/L Ru(bpy)₃²⁺ aqueous solution was added. After that,the mixture was ultrasonicated for 12 h and kept overnight at room temperature. Finally,the product was centrifuged and further washed with ethanol and water three times, respectively. The resulting precipitates were dried in a vacuum oven at 60 8C for 24 h to obtain Ru(bpy)₃²⁺ doped GO-silica composite and dispersed in water (mild ultrasonic) when used. The method for the preparation of Ru/SiNP was the same as the composite just without GO. 3. Results and discussion 3.1. Characterization of Ru/GO-SiCF

Fig. 1A shows the TEM image of GO. Obviously,the GO sheet is a flat thin layer. To provide further evidence that the oxygencontaining groups,the FTIR spectra of GO was demonstrated. As shown in Fig. 1B,the appearance of characteristic absorption peaks at 3400,1730,1600 and 1050 cm^-1 (stretching vibrations of -OH, C=O,C=C,and C-O,respectively) revealed the presence of -OH, C=O,C=C and C-O functional groups in GO. In order to prove that the Ru/GO-SiCF can be successfully prepared as the proposed method,the morphology of GO,Ru/GO-SiCF,and Ru/SiNP were characterized by SEM. Fig. 2A shows the SEM image of the GO, many pristine GO sheets (several micrometers in size) stack tightly with relatively flat surface,the wrinkled structure exhibited in the image may be due to the multiplicity of oxygen functionalities in thin GO layers. In comparison,the morphology of Ru/GO-SiCF was shown in Fig. 2B,many puffy thin sheets distributed in random direction can be observed,and the GO sheets exhibits deeper color than Ru/GO-SiCF,suggesting that GO sheets coated by silica layers homogeneously and the silica layers can act as spacers to prevent the re-stacking of the composite sheet. Fig. 2C shows the SEM image of Ru/SiNP,which indicates that TEOS are prone to hydrolyze and condense to form spherical silica nanoparticle without GO template under the same reaction condition of Ru/GOSiCF. The above observations further validate that TEOS can hydrolyze and condense on the GO surface through Si-O-Si bond to form the GO-SiCF ^[21]. The chemical composition of the film was determined by the energy-dispersive X-ray spectra (EDX). From the EDX image (Fig. 2D),the peaks of Ru,C,Si,and O elements are observed,demonstrating the composite thus formed consist of Ru(bpy)₃²⁺,GO and SiO₂.

Fig. 1

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Fig. 1.TEM image (A) and FTIR spectra (B) of GO.

Fig. 2

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Fig. 2.SEM image of GO (A),Ru/GO-SiCF (B),Ru/SiNP (C) and EDX image of the Ru/GO-SiCF (D).

The electrochemical and ECL behavior of Ru/GO-SiCF modified GC electrode has been investigated with TPA,since the Ru(bpy)₃²⁺-TPA system has been well studied and gave much higher ECL compared with other commonly used reductants ^[27]. The ECL sensor was prepared by directly coating 5 mL Ru/GO-SiCF on the GC electrode and allowed drying in air. The electrochemical behavior of Ru/GO-SiCFmodified electrode was studied using cyclic voltammetry. Fig. 3A shows the cyclic voltammograms (CVs) of the as-formed film on a GC electrode in the absence (red curve) and presence (black curve) of 1 mmol/L TPA at a scan rate of 100 mV/s in 10 mmol/L phosphate buffer (PBS). In the absence of TPA,the modified electrode has obvious anodic and cathodic peaks of Ru(bpy)₃²⁺. This phenomenon indicates that Ru(bpy)₃²⁺ is successfully immobilized by the composite. However,after the addition of 1 mmol/L TPA, the oxidation current of Ru(bpy)₃²⁺ increased clearly while the reduction current decreased,which is consistent with the electrocatalytic reaction mechanism. At the same time,the significant enhanced ECL signal is observed with 1 mmol/L TPA (Fig. 3B),which is consistent with the Ru(bpy)₃²⁺-TPA reaction mechanism described before. The strong ECL signal demonstrated that GO-silica composite film can be a good platform to immobilize a great deal of Ru(bpy)₃²⁺,and facilitate the permeation of TPA,improve the charge transfer and mass transport efficiency,resulting in the increase of sensitivity. In comparison,the electrochemical behavior of the Ru/SiNP modified GC electrode was investigated (note that the quantity of SiO2 and Ru(bpy)₃²⁺ was the same as in the Ru/GO-SiCF). Fig. 3A shows the CV of corresponding modified electrode (blue curve),the significant peak currents decreasing for both oxidation and reduction processes of Ru(bpy)₃²⁺ can be observed,indicating that GO play an important role in improving the charge transfer and mass transport efficiency of the composite,which can be further proved in Fig. 3B,the ECL intensity was also much lower than the Ru/GO-SiCF modified electrode under the same condition (blue curve).

Fig. 3

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Fig. 3.(A) Cyclic voltammograms (CV) of Ru/GO-SiCF modified GC electrode in the absence (red curve) and presence (black curve) of 1 mmol/L TPA in PBS (pH 7.5) at the scan rate of 100 mV/s; CV of Ru/SiNP modified GC electrode in PBS (pH 7.5) at the scan rate of 100 mV/s (blue curve). (B) ECL-potential curve for Ru/GO-SiCF modified GC electrode in PBS (pH 7.5) with (black curve) and without (red curve) 1 mmol/L TPA at the scan rate of 100 mV/s; ECL-potential curve for Ru/SiNP modified GC electrode in PBS (pH 7.5) with 1 mmol/L TPA at the scan rate of 100 mV/s (blue curve).

For sensitivity studies,calibration curves for TPA have been constructed using the present ECL sensor based on the Ru/GO-SiCF modified GC electrode (Fig. 4A.). Each point represents a mean of three ECL signals obtained by consecutively cyclic potential scanning (100 mV/s) in PBS (pH 7.5). Compared with GO or silica ECL sensor,the detection limit is about two orders of magnitude lower ^{[14, 16, 22, 23]},which may be attributed to the following two facts: (1) more Ru(bpy)₃²⁺ molecules can be immobilized in the composite film through electrostatic attraction and physical entrapment. (2) Due to the high surface-volume ratio of GO, the reaction activity of the Ru(bpy)₃²⁺-TPA ECL system can be enhanced,the charge transfer and mass transport will become faster.Under the selected conditions,the ECL intensity (noted as I) varies linearlywith TPA concentration (I-log) over the range from 1 × 10^-11 mol/L to 1 × 10^-5 mol/L,and the linear equation is I = 45.25logC_TPA + 523.50 with a linear correlation coefficient of 0.9920. The detection limit is 14.58 pmol/L at a signal-to-noise ratio of 3. The stability of Ru/GO-SiCF ECL sensor has also been examined. To study the stability initially,the electrode was immersed in 10 mMPBS buffer solution all the time. Fig. 4B shows the ECL-time profile of Ru(bpy)₃²⁺ immobilized GO-silica composite modified electrode in the present of l μmol/L TPA under continuous potential scanning for 10 cycles. The relative standard deviation (RSD) was less than 2%,suggesting good reproducibility and stability of the ECL signal at the composite film modified electrode. The long-term storage stability of the present sensor was studied over a 7-day period by monitoring its ECL response to l μmol/L TPA with intermittent usage (every 1-2 days) and by storing in the air at ambient environment when not in use. The ECL changed slightly and the coating composite film does not come off during this period,The result was likely that charge transfer in composite films was very fast,and the composite films were stable due to the strong electrostatic interaction between positively charged Ru(bpy)₃²⁺ and negatively charged silica and GO,which can avoid the leakage from the composite.

Fig. 4

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Fig. 4.(A) Calibration of TPA at the Ru/GO-SiCF modified GC electrode in PBS (pH 7.5) at the scan rate of 100 mV/s. (B) ECL emission of the composite film modified GC electrode in the present of l mmol/L TPA under continuous CVs for 10 cycles at the scan rate of 100 mV/s.

In conclusion,we have developed a simple method to prepare the Ru/GO-SiCF solid-state ECL material. The advantages can be listed as follow: First,the present method is very simple since Ru(bpy)₃²⁺ is doped just by adding the reagents in one pot to form the composite at room temperature. Second,the composite film modified electrode show high sensitivity,large dynamic concentration range and good stability for ECL sensor. Furthermore,owing to the excellent surface chemistry of silica,we can easily immobilize some important biomolecules on the surface of silica. This novel material will find further application in ECL based bioanalysis and biosensor.

This work was supported in part by the Key Project of Natural Science Foundation of China (Nos. 21175039,21322509, 21305035,21190044,and 21221003),Research Fund for the Doctoral Program of Higher Education of China (No. 20110161110016) and the project supported by Hunan Provincial Natural Science Foundation and Hunan Provincial Science and Technology Plan of China (No. 2012TT1003).