Enhanced electrochemiluminescence of CdSe quantum dots composited with graphene oxide and chitosan for sensitive sensor

Biosensors and Bioelectronics 31 (2012) 369–375

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Enhanced electrochemiluminescence of CdSe quantum dots composited with graphene oxide and chitosan for sensitive sensor Teng Wang, Shengyi Zhang ∗ , Changjie Mao, Jiming Song, Helin Niu, Baokang Jin, Yupeng Tian Department of Chemistry, Key Laboratory of Inorganic Materials Chemistry of Anhui Province, Anhui University, Hefei 230039, China

a r t i c l e

i n f o

Article history: Received 24 August 2011 Received in revised form 12 October 2011 Accepted 24 October 2011 Available online 31 October 2011 Keywords: Electrochemiluminescence Sensor CdSe quantum dots Graphene oxide Chitosan

a b s t r a c t A novel strategy for the enhancement of electrochemiluminescence (ECL) was developed by combining CdSe quantum dots (QDs) with graphene oxide-chitosan (GO-CHIT). The ECL sensor fabricated with CdSe QDs/GO-CHIT composite exhibited high ECL intensity, good biocompatibility and long-term stability, and was used to detect of cytochrome C (Cyt C). The results show that the ECL sensor has high sensitivity for Cyt C with the linear range from 4.0 to 324 ␮M and the detection limit of 1.5 ␮M. Furthermore, the ECL sensor can selectively sense Cyt C from glucose and bovine serum albumin (BSA). © 2011 Elsevier B.V. All rights reserved.

1. Introduction As well known, electrochemiluminescence (ECL) is a light emission process in which the species generated at electrodes undergo high-energy electron transfer reactions to form excited states that emit light. Due to its high sensitivity, fast detection, good temporalspatial control and low cost, ECL detection methods have attracted much attention (Bertoncello and Forster, 2009; Liu and Ju, 2008; Miao, 2008). According to the luminous categories, generally, ECL systems are classified into inorganic system, organic system and quantum dots (QDs) system. Since Bard’s group reported the ECL of Si QDs (Ding et al., 2002), ECL systems based on QDs have been extensively studied for biological applications, due to their controllable size and emission wavelength, high-yield photoluminescence (PL) and good chemical stability (Jie et al., 2010; Liu and Ju, 2008; Shan et al., 2010). In this field, CdSe QDs have always been the subject of intense ECL study because of their unique luminescent properties and relatively low cost. In spite of QDs’ potential and success in biological applications, however, the QDs themselves have lower ECL signals and poor biocompatibility comparing with conventional luminescent reagents, which limits the applications of QDs in bioanalyses. Therefore, it is urgent to explore effective methods to improve the ECL behavior and biocompatibility of QDs.

∗ Corresponding author. Fax: +86 551 5107342. E-mail address: [email protected] (S. Zhang). 0956-5663/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2011.10.048

The carbon nanotubes (CNTs) that possess fast electron mobility and excellent electrocatalytic activity, have been combined with QDs to construct sensitive ECL biosensor (Jie et al., 2008b). Recently, as a new kind of carbon-based materials, graphene or graphene oxide (GO) with two-dimensional (2D) nanostructures has received a growing research interest (Eda and Chhowalla, 2010; Park and Ruoff, 2009; Segal, 2009). Considering its low manufacturing costs, large specific surface area, fast electron transferring rate, long-term stability and friendly biocompatibility, graphene or GO is expected to be a perfect alternative biosensor material to CNTs. Therefore, after Bard and his coworkers found the ECL property of GO (Fan et al., 2009), GO amplified ECL in the presence of QDs and the ECL of graphene–QDs composites were respectively studied (Li et al., 2011b; Wang et al., 2009). Our group reported the photoelectrochemical performance of graphene–QDs composite film (Li et al., 2011a). In addition, chitosan (CHIT), a natural polysaccharide biopolymer with amino and hydroxy groups, has been used to disperse QDs and construct biosensors due to its good water permeability and excellent biocompatibility (Kang et al., 2009; Zhang and Gorski, 2005). In this paper, we have successfully designed a novel ECL sensor based on the CdSe QDs enhanced by GO and CHIT. Since the GO-CHIT composite has porous structure that can load much more CdSe QDs and provide large interface for electrode reactions, the ECL sensor as-prepared exhibited high ECL intensity and longterm stability. Significantly, the ECL sensor has high sensitivity for cytochrome C (Cyt C) and can selectively sense Cyt C from glucose and bovine serum albumin (BSA).

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2. Experimental

2.4. Preparation and test of ECL sensors

2.1. Chemicals and instruments

First, a glass carbon disk electrode (GCE, 4 mm) was carefully polished with 1.0, 0.3, and 0.05 ␮m ␣-Al2 O3 powder on fine abrasive paper in turn, and washed ultrasonically with water. Prior to modification, the clean GCE electrode was scanned between 0.5 and 1.6 V in 0.5 M H2 SO4 solution until a reproducible cyclic voltammogram (CV) was obtained. After the electrode was rinsed thoroughly with water and dried at room temperature, 10 ␮L of CdSe QDs/GOCHIT composite (or other products) solution was dropped on the surface of the electrode. The modified electrodes (i.e., ECL sensors) as-prepared were dried in air, and then used in all kinds of electrochemical and ECL experiments in which three-electrode system was used: a Ag/AgCl reference electrode (Ag/AgCl), a Pt counter electrode and a GCE work electrode modified with given product. The ECL tests including detection for Cyt C were performed in 0.1 M PB (pH 7.4) solution containing 0.1 M K2 S2 O8 and 0.1 M KCl by CV scanning between 0 and −1.5 V at 100 mV s−1 . Nyquist plots of EIS were obtained in 10 mM PB (pH 7.4) solution containing 5 mM Fe(CN)6 4−/3− and 0.1 M KCl, by using the same three-electrode system as that in the ECL detection. Scheme 1 outlines the fabricating procedure for ECL sensor made up of CdSe QDs/GO-CHIT composite.

Chemicals. Graphite powder (99.995%), sulfuric acid (95%), potassium permanganate (99%), potassium nitrate (99%), lcysteine (98.5%) and d-glucose were purchased from Shanghai Chemical Reagent Co., Ltd. (Shanghai, China). Chitosan was obtained from Haidebei Marine Biological Engineering Co., Ltd. (Jinan, China). Cytochrome C (molecular weight 12,588) and Bovine serum albumin (98%) were provided by Shanghai Sangon Biological Engineering Technology Co., Ltd. (Shanghai, China). All reagents were of analytical reagent grade and used without further purification. The stock phosphate buffer (PB, pH 7.4, 0.1 M) solution was prepared by mixing NaH2 PO4 with Na2 HPO4 . Doubly distilled water was used throughout experiments. Instruments and conditions. X-ray diffractometer (XRD, Rigaku D/max-RA, graphite monochromatized CuK␣1 radiation,  = 0.15406 nm); field emission scanning electron microscopy (SEM, S4800, Hitachi, Japan, at 5.0 kV); transmission electron microscopy (TEM, JEM-2100, JEOL, Japan, at 200 kV); UV–vis absorption spectrometer (UV-3600, Shimadzu, Japan); photoluminescence (PL) spectrometer (F-2500, Hitachi, Japan); electrochemistry work station (LK2005A, Tianjin, China); electrochemiluminescence analyzer (MPI-A, Xi’An Remax Electronic Science & Technology Co. Ltd., China. The spectral width and voltage of the photomultiplier tube were set at 200–800 nm and 700 V, respectively); electrochemistry work station for electrochemical impedance spectroscopy (EIS) measurements (ZAHNER-elektrik GmbH & Co. KG). 2.2. Preparation of GO-CHIT composite Graphene oxide was prepared from graphite powder by a modified Hummers method. In detail, graphite (1.0 g), NaNO3 (1.0 g) and 46 mL of H2 SO4 (98%) were added into a flask under stirring in an ice bath. Then, 6.0 g KMnO4 was slowly added to the mixture solution that was vigorously stirring at below 20 ◦ C. After stirred at room temperature for 1 h, the resulting solution was diluted with 70 mL of water and then stirred at 95 ◦ C for 2 h. Soon after, the mixture solution was further diluted with 100 mL of water and deoxidized with 30 mL of 30% H2 O2 . Finally, the product formed in mixture solution was separated out and washed with water for several times. The graphene oxide, a gray powder, was obtained by drying the product under vacuum. The GO-CHIT composite solution containing 0.50 mg mL−1 GO and 0.50 wt% CHIT, was prepared by dispersing graphene oxide in chitosan solution whose acidity had been adjusted to pH 4.5. 2.3. Preparation and characterization of CdSe QDs/GO-CHIT composite First, CdSe QDs capped by l-cysteine (Cys) were synthesized by a modified procedure reported (Park et al., 2009). The mixture solution containing 2 mL of 0.15 M CdSO4 and 2.64 mL of 1 M Cys was adjust to pH 10 by 1 M NaOH, and diluted to 200 mL with water. Then, 0.75 mL of 0.1 M Na2 SeSO3 was added into the mixture solution. After resulting solution was stirred at room temperature for 1 h, the solution of CdSe QDs capped by Cys was formed. Finally, the CdSe QDs/GO-CHIT composite solution was obtained by ultrasonicmixing GO-CHIT composite solution with CdSe QDs solution (pH 5.5) in the volume ratio of 1:3. The morphology, structure and spectra of the products as-obtained were respectively characterized by instrumental methods.

3. Results and discussion 3.1. Characterization The characterization results of the products are shown in Fig. 1. From curve (a) in Fig. 1A, it can be seen that there is a sharp diffraction peak at 25.2◦ that corresponds to (0 0 2) facet of hexagonal crystalline graphite (Zhou et al., 2010a). Compared with the XRD pattern of graphite, the disappearance of the peak at 25.2◦ and the appearance of the peak at 11.6◦ (curve (b) in Fig. 1A) indicate the successful oxidation of the graphite and the formation of GO (Zhou et al., 2010b). From the TEM image (insert in Fig. 1A), it is observed that monolayer GO sheets as-formed have a transparent flakelike shape with wrinkles and corrugated parts that result from electronic repulsion between the soft and flexible layers. Fig. 1B shows that the GO-CHIT composite has typical crumpled and wrinkled graphene sheet structure with the rough surface, which is a favorable construction for loading QDs. As reported (Schniepp et al., 2006; Stankovich et al., 2006), in chemical exfoliation process, the plane edge of graphene sheets yielded chemical functional groups (such as C–OH and –COOH) that make GO hydrophile and easy to interact with chitosan that has amino and hydroxy functional groups. Therefore, the stable GO-CHIT composite was formed by chemical force produced by reactions between functional groups. From Fig. 1C, it is observed that there is homogeneous and dense coverage of CdSe QDs on the surface of the GO-CHIT composite. High-resolution transmission electron microscopy (HRTEM) image (insert in Fig. 1C) displays the lattice image of a CdSe QD whose grain size is about 2 nm. The SEM image (Fig. 1D) reveals that the CdSe QDs/GO-CHIT composite has a porous structure whose frameworks are evenly and densely interspersed with CdSe QDs. Fig. 2A shows the UV–vis absorption spectra of the products. On the spectrum of GO (curve (a)), there are two absorption features: a peak at 226 nm originated from the ␲→␲* transition of C C, and a shoulder at about 298 nm corresponding to the n→␲* transition of the C O bond (Luo et al., 2009). For GO-CHIT composite (curve (b)), the GO absorption peak at 226 nm was weakened due to the interaction between GO and CHIT. From the spectrum of CdSe QDs (curve (c)), the size of QD particles and molar concentration of QD solution are respectively estimated to be 1.72 nm and 6.04 ␮M, based on strong excitonic adsorption peak at 420 nm and

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Scheme 1. Fabricating procedures for ECL sensor.

Fig. 1. (A) XRD patterns of graphite (a) and GO (b), insert: TEM image of GO; (B) TEM image of GO-CHIT composite; (C) TEM image of CdSe QDs/GO-CHIT composite, insert: HRTEM image of a CdSe QD; (D) SEM image of CdSe QDs/GO-CHIT composite.

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Fig. 2. (A) UV–vis absorption spectra: (a) GO, (b) GO-CHIT composite, (c) CdSe QDs, (d) CdSe QDs/GO-CHIT composite; (B) Photoluminescence spectra obtained with excitation wavelength 420 nm at room temperature: (a) CdSe QDs, (b) CdSe QDs/GO-CHIT composite, (c) the supernatant obtained by separating out CdSe QDs/GO-CHIT composite.

the empirical equations reported (Yu et al., 2003). Here, it is worthy of pointing out that the calculated size of QDs is consistent with that of HRTEM observation. In addition, for CdSe QDs, the weak peak at about 360 nm is attributed to the absorption of the particles with smaller size (Park et al., 2009). Obviously, the absorption peak (at 420 nm on curve (d)) of the CdSe QDs/GO-CHIT composite is much weaker than that of CdSe QDs themselves, which means that the CdSe QDs have been strongly combined with GO-CHIT sheets. The room-temperature photoluminescence (PL) spectra of the products are shown in Fig. 2B. For CdSe QDs, the PL emission peak at 515 nm (curve a), as well as UV–vis absorption peak at 420 nm, indicates the consequence of quantum confinement (Jie et al., 2009). The CdSe QDs/GO-CHIT composite has a similar PL emission spectrum (curve (b)) to pure CdSe QDs, except for little decrease in emission intensity and very little red shift (5 nm) in peak location. According to literatures (Cao et al., 2010; Grzelczak et al., 2006; Nethravathi et al., 2011), the decrease of emission intensity is attributed to the electron transfer from excited CdSe QDs to GO-CHIT sheets, and very little red shift of peak location means that the change in size distribution of CdSe QDs is neglectable after adsorption onto the GO-CHIT sheets. The faint PL emission of the supernatant solution obtained by separating out the CdSe QDs/GO-CHIT composite, as shown on curve (c) in Fig. 2B, reveals that a majority of CdSe QDs in system have combined with GO-CHIT sheets. 3.2. ECL behavior The ECL–potential curves obtained by the electrodes modified with different products are shown in Fig. 3A. Obviously, GCE itself and the electrodes modified with CHIT and GO-CHIT composite have hardly ECL emission (curves (a)–(c)). However, all electrodes containing CdSe QDs have good ECL emission peak (curves (d)–(f)), which means that the ECL emissions result from CdSe QDs. The comparison between curve (d) and curve (e) reveals that the introduction of CHIT enhanced the ECL emission and made the ECL peak potential positively shifted. The phenomenon is explained as that the amine groups on CHIT molecules can facilitate the radical generation and decrease the excitation energy of the ECL reaction of CdSe QDs (Jie et al., 2009, 2008b). As shown in Fig. 3A (curve (f)), the ECL peak intensity of the CdSe QDs/GO-CHIT composite is over 2.5fold higher than that of pure CdSe QDs, and is higher than that of the CdSe QDs/CNT composite (Jie et al., 2009) or CdSe QDs/CNT-CHIT composite (Jie et al., 2008b). Furthermore, the strong ECL emission produced by the CdSe QDs/GO-CHIT composite is very stable along with successive CV scans (insert in Fig. 3A). Presumably, the excellent ECL behavior of the CdSe QDs/GO-CHIT composite results from three points: (1) the GO has good conductibility and stability;

(2) the GO-CHIT composite sheets have large rough specific surface that can load much more CdSe QDs and provide large interface of electrode reaction; (3) the porous structure of the GO-CHIT composite can facilitate the diffusion of coreactant K2 S2 O8 into the membrane, resulting in the occurrence of ECL signal not only at the interface but also in the inside of porous film (Li et al., 2011b). In the interest of examining the conductivity of the GO, the electrochemical impedance spectroscopy (EIS) that is known as an effective method for exploring the features of surfacemodified electrodes, was performed and the results are shown in Fig. 3B. According to literatures (Chen et al., 2008; Li et al., 2009; Ramanavicius et al., 2010), the semicircle diameter at higher frequencies corresponds to the electron transfer resistance (Ret), and the linear part at lower frequencies corresponds to the solution diffusion resistance (Rs). Here, from the EIS spectra, it is obtained that for GCE itself, CHIT electrode, GO-CHIT composite electrode and CdSe QDs/GO-CHIT composite electrode, the electron transfer resistances are 800 , 4800 , 1600  and 3100 , respectively, and the solution diffusion resistances are 179 , 91 , 129  and 84 , respectively. Clearly, CHIT and CdSe QDs have nonconductive property, and the GO can enhance the electrical conductivity of the composites. Noteworthy, the CdSe QDs/GO-CHIT composite electrode has least solution diffusion resistance among electrodes, since the diffusion of the electroactive species in solution can take place not only at the interface but also in the inside of porous film. Fig. 3C shows the relationship of ECL curve with CV curve for the CdSe QDs/GO-CHIT composite. Obviously, there is a wide cathodic peak at −1.14 V on CV curve and an emission peak at −1.4 V on ECL curve. However, there is no ECL emission produced at scanning from 0 to −1.2 V. Fig. 3D shows the ECL spectrum of the CdSe QDs/GO-CHIT composite, which is analogous to PL spectrum (Fig. 2B) and just the ECL peak (555 nm) red-shifted, comparing with PL peak (520 nm). Generally, the ECL emission is considered as the release of the energy from the hole–electron recombination on the surface of the nanoparticles, whereas the PL emission is known as the transition of the states in the core of nanoparticles. Since the energy gaps among the hyperfine states on the surface are normally smaller than those in the core, for our CdSe QDs/GO-CHIT composite, a red shift occurred in the ECL wavelength compared with the corresponding PL wavelength (Ding et al., 2002). Based on above experiment results and consulting literatures (Jie et al., 2008a,b; Li et al., 2011b; Myung et al., 2002; Wang et al., 2009), the possible ECL mechanism is proposed as follows. Basically, the ECL emission originated from electrochemically oxidized and reduced of CdSe QDs reacting with coreactant K2 S2 O8 . On potential scan with an initial negative direction, the CdSe QDs immobilized on the electrode were reduced (or electron-injected) to negatively charged species (CdSe•− ). Meanwhile, the S2 O8 2− in

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Fig. 3. (A) ECL–potential curves obtained by different GCE electrodes modified with: (a) bare electrode, (b) CHIT, (c) GO-CHIT composite, (d) CdSe QDs, (e) CdSe QDs-CHIT composite, (f) CdSe QDs/GO-CHIT composite, insert: repeated ECL emissions along with successive CV scans on the electrode modified with CdSe QDs/GO-CHIT composite; (B) Nyquist plots of EIS obtained by different GCE electrodes modified with: (a) bare electrode, (b) CHIT, (c) GO-CHIT composite, (d) CdSe QDs/GO-CHIT composite; (the frequency range is between 0.01 and 100,000 Hz with signal amplitude of 5 mV); (C) ECL–potential curve (a) and cyclic voltammogram (b) of the electrode modified with CdSe QDs/GO-CHIT composite; (D) ECL spectrum obtained by the electrode modified with CdSe QDs/GO-CHIT composite.

solution diffused to the surface of electrode and produced strong oxidant SO4 •− by electrode reaction. In the reaction duration of scanning from 0 to −1.2 V, a wide cathodic peak on CV curve was formed, but there is no ECL emission produced (Fig. 3C). Thereafter, the CdSe•− reacted with the SO4 •− and produced excited state species (CdSe*) that emitted light. In reaction process, the GO in the composite can improve the radical species stability and promote the formation of CdSe•− and SO4 •− . In addition, the GO acted as a good intermedium for electron transfer between CdSe QDs and GCE electrode. The equations corresponding to each step of the emission reactions are formulated as follows: CdSe + e− → CdSe•− S2 O8

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The effect factors on ECL emission were studied by a series of experiments and the results are shown in Fig. 4. When the wire connecting the work electrode was removed at −1.4 V (marked by OFF in Fig. 4A) in negatively scanning process, the intensity of ECL emission quickly dropped to zero, along with the quick fall of the electrode current, which means that the ECL emission correlated intimately with the electrode reaction process (Zhu et al., 2011). Fig. 4B shows the effect of GO amount in the CdSe QDs/GO-CHIT

composite on ECL emission. Clearly, when the GO concentration in synthesis solution of GO-CHIT composite is 0.5 mg mL−1 , the CdSe QDs/GO-CHIT composite as-prepared has a maximum ECL emission. The effects of the electrolyte solution concentration and acidity on ECL emission are shown in Fig. 4C and D. For ECL emission, the optimum electrolyte solution is 0.1 M K2 S2 O8 in 0.1 M PB (pH 7.4) solution. 3.3. ECL detection As discussed above, the electrode modified with CdSe QDs/GOCHIT composite is favorable for the ECL sensor, due to its strong and stable ECL emission. Here, the ECL sensor as-prepared was used to detect cytochrome C (Cyt C), and the results are shown in Fig. 5A. Clearly, the ECL intensity decreased gradually along with the increase of Cyt C concentrations. The quenching mechanism of Cyt C to ECL is explained as the energy transfer between the excited CdSe QDs and quencher Cyt C, and as the Cyt C inhibition to the ECL reaction between CdSe QDs and K2 S2 O8 . Generally, the relationship of quencher concentration with ECL intensity is treated by the fluorescence quenching principle described as Stern–Volmer equation, I0 /I = 1 + K[Q]. Here, I0 is the initial ECL intensity, I is the ECL intensity at a given quencher concentration [Q], and K is the quenching constant. Based on this quenching principle, our calibration curve for Cyt C concentrations is drawn out (insert of Fig. 5A). The test results show that the linear range for Cyt C is from 4.0 to 324 ␮M (R = 0.9946, n = 7) and the detection limit for Cyt C is 1.5 ␮M at ECL

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Fig. 4. The experiment results for the electrode modified with CdSe QDs/GO-CHIT composite: (A) ECL-potential curve (a) and cyclic voltammogram (b) obtained by breaking off circuit at −1.40 V; (B) the effect of GO concentration in synthesis solution on ECL intensity; (C) the effect of K2 S2 O8 concentration in electrolyte solution on ECL intensity; (D) the effect of electrolyte solution acidity on ECL intensity.

Fig. 5. (A) ECL intensity changes vs. Cyt C concentrations (␮M): (a) 0, (b) 4, (c) 14, (d) 24, (e) 44, (f) 84, (g) 164, and (h) 324, insert: calibration curve for Cyt C determination; (B) interference test results with ECL sensor.

intensity decrease degree of 1.93% (three times noise). The value of quenching constant K is calculated to be 0.01961 ␮M−1 , and the relative standard deviation (RSD) is 6.3% for six ECL sensors in Cyt C solution (100 ␮M). To probe the specificity of the ECL sensor, the interference test was performed by adding glucose and bovine serum albumin (BSA) to detection solution. Comparing with the effect of Cyt C on ECL intensity, as shown in Fig. 5B, the effect of two interferents is neglectable. Therefore, it is concluded that the ECL sensor as-prepared has fair specificity for the determination of Cyt C.

4. Conclusions In this work, a novel composite consisting of the CdSe quantum dots, graphene oxide and chitosan has been designed to construct sensitive electrochemiluminescence sensor. In this system, the GO has large specific surface, good conductibility and high stability, and can activate the electrode reactions and promote the formation of the radical species. The CHIT with good biocompatibility has amine groups that can decrease the excitation energy of the ECL reaction of CdSe QDs. In addition, the GO-CHIT composite has typical porous structure that can load much more CdSe QDs and provide

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large interface for electrode reactions. Therefore, the ECL sensor as-prepared exhibited high ECL intensity and long-term stability. The results for bioassay show that the ECL sensor has high sensitivity and good selectivity to cytochrome C. Significantly, based on the proposed strategy, a series of ECL sensors made up of QDs composites can be easily fabricated. Acknowledgments Support for this work from the National Natural Science Foundation of China (Nos. 20875001, 21175001, 20905001, 21071002, 21071001) and the, Anhui Research Project (Nos. 2006KJ007TD, KJ2010A030) is gratefully acknowledged. References Bertoncello, P., Forster, R.J., 2009. Biosens. Bioelectron. 24, 3191–3200. Cao, A., Liu, Z., Chu, S., Wu, M., Ye, Z., Cai, Z., Chang, Y., Wang, S., Gong, Q., Liu, Y., 2010. Adv. Mater. 22, 103–106. Chen, X., Wang, Y., Zhou, J., Yan, W., Li, X., Zhu, J.-J., 2008. Anal. Chem. 80, 2133–2140. Ding, Z., Quinn, B.M., Haram, S.K., Pell, L.E., Korgel, B.A., Bard, A.J., 2002. Science 296, 1293–1297. Eda, G., Chhowalla, M., 2010. Adv. Mater. 22, 2392–2415. Fan, F.R.F., Park, S., Zhu, Y.W., Ruoff, R.S., Bard, A.J., 2009. J. Am. Chem. Soc. 131, 937–939. Grzelczak, M., Correa-Duarte, M.A., Salgueirino-Maceira, V., Giersig, M., Diaz, R., LizMarzan, L.M., 2006. Adv. Mater. 18, 415–420. Jie, G.F., Huang, H.P., Sun, X.L., Zhu, J.J., 2008a. Biosens. Bioelectron. 23, 1896–1899. Jie, G.F., Li, L.L., Chen, C., Xuan, J., Zhu, J.J., 2009. Biosens. Bioelectron. 24, 3352– 3358. Jie, G.F., Liu, P., Zhang, S.S., 2010. Chem. Commun. 46, 1323–1325.

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