A graphene oxide platform for energy transfer-based detection of protease activity

Biosensors and Bioelectronics 26 (2011) 3894–3899

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A graphene oxide platform for energy transfer-based detection of protease activity Juan Li, Chun-Hua Lu, Qiu-Hong Yao, Xiao-Long Zhang, Jing-Jing Liu, Huang-Hao Yang ∗ , Guo-Nan Chen The Key Lab of Analysis and Detection Technology for Food Safety of the MOE, Fujian Provincial Key Laboratory of Analysis and Detection Technology for Food Safety, College of Chemistry and Chemical Engineering, Fuzhou University, Fuzhou 350002, PR China

a r t i c l e

i n f o

Article history: Received 9 December 2010 Received in revised form 25 February 2011 Accepted 2 March 2011 Available online 8 March 2011 Keywords: Biosensors Energy transfer Graphene Protease Quantum dots

a b s t r a c t In this article, we report the first graphene oxide (GO)-based platform to detect protease activity in a homogeneous real-time format. In designing such GO-based biosensing platform, we put a protease substrate peptide as the linker between the energy transfer donor (QDs) and the energy transfer acceptor (GO) to fabricate the GO-peptide-QDs nanoprobes. In the nanoprobes, the photoluminescence (PL) of donor QDs was strongly quenched due to the presence of GO in close proximity. The protease activity caused modulation in the efficiency of the energy transfer between the acceptor and donor, thus enabling the protease assay. The proposed GO-based platform is easy to assemble and has little background interference, yet still give superior sensitivity and rapid response. Furthermore, this GO-QDs architecture can serve as a universal platform by simply changing the types of peptide sequences for the different proteases. In this work, GO-based platform has been successfully applied in the sensitive detection of matrix metalloproteinase (MMP) and thrombin activity. Meanwhile, we also utilized this platform to monitor the protease inhibitor. The proposed GO-based platform is anticipated to find applications in the diagnosis of protease-related diseases and screening of potential drugs with high sensitivity in a high-throughput way. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Proteases make up one of the most important groups of enzymes, comprising nearly 2% of the human proteome. They catalyze the breaking of specific peptide bonds in proteins and polypeptides, and play an important role in the initiation and progression of many important diseases and medical conditions including cancer, stroke and infection (Concha and Abdel-Meguid, 2002; Overall and Kleifeld, 2006; Puente et al., 2003). Thus, sensitive, accurate and convenient detection of protease activity is of great significance in diagnosis of protease-relevant diseases and development of potential drugs. Up to now, much attention has been paid to fluorescence resonance energy transfer (FRET) method for in vitro and in vivo assay of proteases due to its rapidness, simplicity, sensitivity and reproducibility (Boeneman et al., 2009; Kainmuller et al., 2005; Lee et al., 2009; Medintz et al., 2006; Mu et al., 2010; Shi et al., 2006, 2007; Zhang et al., 2002, 2010c; Zheng et al., 2007). In the FRET-based system, quantum dots (QDs) have been widely used as effective energy transfer donor due to their intrinsic optical properties such as high quantum yield, high photobleaching threshold, good chemical stability, narrow emission peaks and size-depending emission. In addition, the size-controlled

∗ Corresponding author. Tel.: +86 591 22866135; fax: +86 591 22866135. E-mail address: hhyang@fio.org.cn (H.-H. Yang). 0956-5663/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2011.03.003

luminescence functions of the QDs make them an ideal label for multiplexed type analyses. In the previous reports, organic dyes and fluorescent proteins have been used as energy acceptors in QDsbased FRET sensors for protease activity (Boeneman et al., 2009; Medintz et al., 2006; Shi et al., 2006, 2007). Recently, nanomaterials have attracted considerable attention as the quenchers in the FRETbased system for high quenching efficiency and insensitivity to the environment. Biju and coworkers reported photoluminescence (PL) intensity and lifetime of CdSe-ZnS QDs were considerably reduced after conjugation to single-walled carbon nanotubes (SWCNT) (Biju et al., 2006). Gold nanoparticles (AuNPs) also have been applied as an energy acceptor in the FRET-based protease assay (Chang et al., 2005; Kim et al., 2008; Lee et al., 2008). Graphene is a single-atom-thick and two-dimensional carbon material that has attracted great attention because of its remarkable electronic, mechanical, and thermal properties (Novoselov et al., 2004; Geim and Novoselov, 2007; Yang et al., 2010). Of our particular interest, graphene was recently predicted through theoretical calculations to be a superquencher with the long-range nanoscale energy transfer property (Swathi and Sebastian, 2008, 2009). In the previous study, the graphene oxide (GO) can efficiently quench various fluorescent dyes over a wide wavelength range (He et al., 2010; Lu et al., 2009; Wang et al., 2010a,b). Recently, we discovered that GO also had an extraordinarily high quenching efficiency for different kinds of quantum dots, such as ZnO, CdS, CdTe and CdSe–ZnS. Inspired by this observation, we herein present a

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GO-based biosensing platform to rapid and sensitive detect protease activity and their inhibition. Recently, GO-DNA architecture has been attracting much attention for the fabrication of biosensors (He et al., 2010; Lu et al., 2009, 2010a; Wang et al., 2010b). Furthermore, it has been reported that the GO exhibits good biocompatibility and low cytotoxicity and has potential applications in biological and medical fields (Lu et al., 2010a,b; Wang et al., 2010a,b). However, there are few reports about incorporating other biomolecules into GO. Now, we incorporate peptides into GO and expect to expand the application fields of GO to a great extent. 2. Materials and methods 2.1. Materials Graphene oxide (GO) was synthesized from natural graphite powder by a modified Hummers method (Hummers and Offeman, 1958; Xu et al., 2008). All peptides were synthesized by GL Biochem (Shanghai) Ltd. The sequences of the used peptides were described in the supporting information. Thrombin and Matrix metalloproteinase-2 (MMP-2) was purchased from Sigma. Amine-functionalized-CdSe-ZnS QDs (emission at 545 and 585 nm, respectively) were purchased from Wuhan Jiayuan Quantum Dots Co. Ltd. Streptavidin was purchased from Shanghai Sangon Biotechnology Co. Sulfo-SMCC (4-(N-Maleimidomethyl) cyclohexane-1carboxylic acid 3-sulfo-N-hydroxysuccinimide ester sodium salt) and AEBSF (4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride) were purchased from Sigma.

BSA as a blocking agent to reduce the nonspecific adsorption. After incubation, the mixture was centrifuged for 10 min at 10,000 rpm at 4 ◦ C. The centrifugate was washed with deionized water several times and resuspended in the buffer to obtain streptavidin-GO complex (SA-GO). 2.5. Assay of protease activity and inhibition with GO-based nanoprobes SA-GO (about 50 ␮g/mL) was mixed with P1-QDs (about 10 nM) conjugated for 30 min at room temperature to allow specific association between SA and biotin. Then, the mixture was reacted with various concentrations of thrombin at 37 ◦ C for 60 min. Each enzyme (thrombin and MMP-2) was dissolved in 10 mM HEPES buffer (pH 7.4, 150 mM NaCl, 5 mM CaCl2 ). Thrombin activity was measured by the fluorescence changes that were recorded on an F-4600 fluorometer. Control experiments were carried out under the same conditions. MMP-2 activity was measured using the same experimental set-up as that used for thrombin. The inhibitor of thrombin was incubated at varying concentrations with 50 nM thrombin. Then, 10 nM P1-QDs and 50 ␮g/mL SA-GO were added and the resulting solution was incubated at 37 ◦ C for 60 min. The detection procedure was the same as shown in the aforementioned thrombin activity experiment. The calculation of relative activity of thrombin was defined as PLi /PL0 , where PLi and PL0 are the fluorescence intensities of with thrombin in the presence and absence of inhibitors, respectively.

2.2. Instrument

3. Results and discussion

Fluorescence profiles were obtained with a Hitachi F-4600 fluorometer (Hitachi Co. Ltd., Japan). Atomic force microscopy (AFM) images were recorded at ambient temperature on an Agilent Technologies 5500 System (Agilent Technologies, Inc., USA) in tapping mode. Raman spectra were recorded at ambient temperature on a Renishaw InVia Raman spectrometer with an excitation laser at 785 nm. Fluorescence anisotropy was examined with a FLS 920 fluorescence spectrometer (Edinburgh Instruments Ltd., UK). The temperature was maintained at 25 ◦ C. The reported anisotropy values are an average of four independent measurements.

3.1. Principle of protease activity assay based on graphene oxide platform

2.3. Synthesis of peptide-conjugated QDs (P1-QDs) (Pereira and Lai, 2008) Amine-functionalized CdSe–ZnS QDs (NH2 -QDs) were added to a 50 mM sodium phosphate (PB buffer pH 7.2) solution containing sulfo-SMCC and incubated at room temperature for 1 h with gentle mixing. The maleimide activated QDs (maleimide-QDs) were purified from unreacted cross-linker via dialysis using 5000 Da MWCO microcentrifuge tubes at room temperature with successive washings of buffer. A 10-fold molar excess of peptide (P1) and the purified maleimide-QDs were combined and incubated overnight at 4 ◦ C. Purification of P1-QDs from free peptide in solution was performed via dialysis using 5000 Da MWCO microcentrifuge tubes. The purified P1-QDs were washed several times with buffer. The preparation of P2-QDs nanoprobes was similar to that of P1-QDs. 2.4. Synthesis of streptavidin-GO complex (SA-GO) The GO sheet was firstly incubated in the solution of streptavidin (PB buffer pH 7.2) for 30 min with slightly shaking. Then, the mixture was centrifuged for 10 min at 10,000 rpm at 4 ◦ C. The centrifugate was washed with deionized water several times and resuspended in the solution of bovine serum albumin (BSA, 0.5 mg/mL) for 30 min at room temperature. It is critical to use a

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In designing such GO-based biosensing platform, we envision to put a protease substrate peptide as the linker between the energy transfer donor (QDs) and the energy transfer acceptor (GO) to fabricate the GO-peptide-QDs nanoprobes. In such nanoprobes, the biotinylated peptide substrate is conjugated to QDs to obtain biotin-peptide-QDs complex. The streptavidin (SA) is directly adsorbed on the basal plane of GO to form streptavidin-GO (SA-GO) complex. In addition, BSA is used as a blocking agent to reduce the nonspecific adsorption. Then, SA-GO is associated with biotin-peptide-QDs by SA-biotin specific binding to form the GOpeptide-QDs nanoprobes. The nanoprobes are believed to be in a strongly quenched state, because the GO serves as ultra-efficient quencher. Thus, the energy transfer process between the QDs and GO will be modulated by the protease activity. In the presence of target protease, the designated peptide is cleaved into two fragments. Subsequently, the QDs-labeled peptide fragment is released from the surface of GO. In turn, the initially quenched fluorescence of QDs is recovered (Scheme 1). 3.2. Thrombin activity assay using GO-peptide-QDs nanoprobes To demonstrate the utility of our rationale, we employed thrombin as a model protease analyte at first. Thrombin, a kind of serine protease, plays an important role in the coagulation cascade, thrombosis and haemostasis. Monitoring thrombin activity and its inhibitor have important clinical and therapeutic uses (Gurm and Bhatt, 2005; Linkins and Weitz, 2005; Steinberg, 2005). GO was synthesized according to a modified Hummers method and confirmed by Raman spectroscopy. The G band is usually assigned to the E2g phonon of C sp2 atoms, while the D band is a breathing ␬-point phonon of A1g symmetry (Tuinstra and Koenig, 1970). As shown in Fig. 1A, the natural graphite displays a strong

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Scheme 1. (A) Construction of the peptide conjugated QDs; (B) schematic representation of energy transfer-based detection of protease activity using graphene platform.

G band at 1597 cm−1 , a weak D band at 1337 cm−1 (curve a). Different from the natural graphite, GO showed the well-documented D and G bands (curve b). This phenomenon agreed well with that reported previously and indicated the formation of some sp3 carbon in GO (Shen et al., 2009). The X-ray diffraction (XRD) and Fourier-transform infrared spectroscopy (FT-IR) of GO further provided the information of the successful synthesis of GO (Supporting material, Figs. S1 and S2). Recently, it has been reported that GO is an ideal substrate for study enzyme immobilization without using any cross-linking reagents and additional surface modification (Zhang et al., 2010a). We found SA also could be directly and stably adsorbed on the basal planes of GO to form streptavidin-GO (SA-GO) complex. This adsorption was possibly fulfilled through hydrophobic and ␲–␲ stacking interactions and hydrogen bonding between the oxygen functional groups of GO and nitrogen/oxygen containing groups on SA (Liu et al., 2010). In addition, this SAGO complex was stable and maintained its bioactivity for weeks in the buffer. Fig. 1B shows the typical atomic force microscopy (AFM) image of the GO after being incubated together with SA in phosphate buffer. AFM analyses revealed the height of SA-GO complex was about 4.2 nm, which was higher than pristine GO (about 1.2 nm) (Lu et al., 2009). The increasing height was roughly consistent with the size of free SA (about 3–5 nm) (Housni et al., 2007). The incorporation of the SA molecules successfully turned the GO into a simple self-assembly platform toward biotinylated peptide. For thrombin sensing, one biotinylated peptide substrate named as P1 (Kim et al., 2008) was selected as the sensing element. P1 was conjugated to maleimide-functionalized QDs (CdSe–ZnS, emission at 545 nm) to obtain peptide-conjugated QDs (P1-QDs). Then, SA-GO was associated with P1-QDs by SA-biotin specific binding to form the GO-P1-QDs nanoprobes and the fluorescence of P1QDs was strongly quenched (Fig. 2 curve b). As a control, BSA-GO was added to the solution containing P1-QDs, and reduction in the PL intensity of QDs was very small (Supporting material, Fig. S3). These results clearly demonstrated that, in the GO-P1-QDs nanoprobes, the P1-QDs were self-assembled to the surface of GO through specific biotin-SA affinity. Furthermore, energy transfer phenomena could only effectively occur when GO and QDs were in close proximity. (Chen et al., 2010; Dong et al., 2010). The quenching efficiency (QE , [%]) of the GO was calculated by using the formula: (1 − ˇ) × 100%, where ˇ is the ratio of fluorescence of the quenchedto-completely dequenched state. The QE of GO was calculated to be 97.5 ± 1.5% (Fig. 2A). It is well known that a decrease in the noise intensity of the quenched state to an undetectable level is required to maximize the fluorescent changes and achieve high sensitive detection of target. Therefore, GO would be a promising material to assay protease activity. With the designed nanoprobes, we attempted to assay thrombin activity in solution. As shown in Fig. 2A, addition of thrombin which

specifically cleaved the Arg–Gly bond in P1 sequence resulted in the detachment of QDs from GO, and a notable increase in the PL of QDs was observed. The fluorescence changes of GO-P1-QDs nanoprobes were clearly visualized by photography under ultraviolet light (Fig. 2A, inset). Furthermore, the PL intensity gradually increased as the concentration of thrombin was increased. Fig. 2B (inset) illustrates the changes in the relative PL intensity (PL/PL0 ) of the sensing platform with respect to the concentration of thrombin. PL0 denotes the PL intensity of P1-QDs and PL is the observed PL intensity of GO-P1-QDs when reacted with the respective thrombin concentrations. The assay allowed for the detection of thrombin at concentration as low as 0.5 nM based on three times the signal-to-noise level. As a control, nonspecific protease, Matrix metalloproteinase-2 (MMP-2) was tested, but the change in the PL of nanoprobes was slight (Fig. 2A, curve d). This result suggested that our GO-based energy transfer nanoprobes were high specific. Furthermore, GO-P1-QDs nanoprobes also allow measurement of thrombin activity in real time. The thrombin-catalyzed hydrolysis of P1 as a function of time upon incubation of GO-P1-QDs with different concentrations of thrombin was investigated (Supporting material, Fig. S4). It was observed that the fluorescence recovered more quickly and more completely at high thrombin concentrations. Therefore, this GO-based platform is applicable for real-time kinetic study of protease activity. 3.3. Fluorescence anisotropy The fluorescence anisotropy of a fluorophore reflects the ability of molecule to rotate in its microenvironment. Therefore, we used anisotropy measurements to further investigate molecular interactions. As shown in Fig. 3, the fluorescence anisotropy of free P1-QDs in buffer was 0.021, and it increased 6.25-fold after addition of GO, indicating that the P1-QDs were self-assembled to the surface of SAGO through specific biotin-SA affinity. However, the fluorescence anisotropy decreased after further addition of thrombin into the mixture of GO-P1-QDs. Furthermore, the value of the fluorescence anisotropy of GO-P1-QDs with thrombin is almost the same as that of free P1-QDs upon addition of thrombin. These results primarily indicated that thrombin specifically cleaved P1 resulting in the detachment of QDs from GO. 3.4. Assay of the inhibition of thrombin This simple and sensitive assay was also applied to screen for inhibitors of protease. Protease inhibitors can prevent the recovery of PL of QDs by blocking the action of protease. The validity of our method in assaying the inhibition of thrombin was tested by using AEBSF (4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride) as a model inhibitor. Fig. 4 depicts the effect of the AEBSF on the

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Fig. 2. (A) Changes in the PL intensity of GO-P1-QDs nanoprobes: (a) P1-QDs, (b) P1QDs + SA-GO, (c) P1-QDs + SA-GO + thrombin, (d) P1-QDs + SA-GO + MMP-2. Inset: Photograph of the nanoprobes under 365 nm UV radiation; (B) fluorescence emission spectra of GO-P1-QDs nanoprobes in the presence of various concentrations of thrombin. (From bottom to top: 2, 5, 10, 20, 50, 100 nM) Inset: The calibration curve for thrombin.

Fig. 1. (A) Raman spectra of graphite (a) and GO (b). Laser wavelength = 785 nm, laser power = 40 mW, integration time = 5 s. (B) AFM height image of SA-GO complex (1 ␮m × 1 ␮m, vertical scale 0–10 nm) deposited on a freshly cleaved mica surface.

activity of thrombin. The IC50 value, the inhibitor concentration required to reduce enzyme activity by 50%, was acquired from the plot of relative activity of thrombin versus inhibitor concentrations and was found to be 0.87 mM. In addition, the rate of the fluorescence recovery of QDs significantly decreased with addition of inhibitor (Supporting material, Fig. S4). These results indicated that our method can be used to study the protease inhibitor and employed for protease inhibitors screening. 3.5. MMP-2 activity assay using GO-peptide-QDs nanoprobes To illustrate the generality of our design strategy, we applied this strategy to detect another protease, matrix metalloproteinase2 (MMP-2). MMP-2 is a member of the matrix metalloproteinase (MMP) family and involved in extracellular matrix remodeling dur-

Fig. 3. Changes of fluorescence anisotropy of (a) P1-QDs (10 nM), (b) P1-QDs (10 nM) + GO (50 ␮g/mL), (c) P1-QDs (10 nM) + GO (50 ␮g/mL) + thrombin (200 nM), (d) P1-QDs (10 nM) + thrombin (200 nM).

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Fig. 4. Assay of the inhibition of thrombin activity by the GO-P1-QDs nanoprobes. The concentrations of AEBSF were 0, 0.2, 0.5, 0.8, 1 and 2 mM, respectively. Scale bars indicate the standard deviation in quadruplicate experiments.

ing morphogenesis and tissue repair and in the processes of tumor invasion and metastasis (Sato and Seiki, 1996; Stetler-Stevenson et al., 1993). We used MMP-2 substrate P2 (Lee et al., 2008) and another color of QD (CdSe-ZnS, emission at 585 nm) to similarly make GO-P2-QDs nanoprobes. Addition of MMP-2 to GO-P2-QDs nanoprobes also led to a notable recovery in the PL of QDs (Fig. 5). As a control, nonspecific protease (thrombin) was tested, but the change in the PL of nanoprobes was very small (Fig. 5A curve d). The QE of GO for QDs with emission at 585 nm was calculated to be 96.5 ± 1.8%, which was similar to that for QDs with emission at 545 nm. As shown in Fig. 5B, a proportional relationship was observed between the MMP-2 concentration and relative PL intensity (PL/PL0 ) of the sensing platform. The assay allowed for the detection of MMP-2 at concentrations as low as 1 nM based on three times the signal-to-noise level. This result suggested that our GObased energy transfer biosensing platform can serve as a general detection platform for a variety of protease activity by using the appropriate peptide substrate spacer. In addition, the preliminary experiment suggested that the proposed platform could be used to simultaneously detect thrombin and MMP-2 (Supporting material, Fig. S5). Furthermore, we found GO could efficiently quench the different kinds and colors of QDs and had approximately equal quenching efficiency exceeding 95% (data not shown). Thus, the proposed platform could be expanded easily to multiplexed assay formats by coupling GO with multicolor QDs. 3.6. Protease activity assay using GO-peptide-fluorescent dyes nanoprobes In addition, GO can also efficiently quench various organic fluorescent dyes over a wide wavelength range. To further prove the design concept, we used a fluorescent dye tagged peptide P3-FAM, as another model for the study of thrombin activity. We mixed P3-FAM with SA-GO to obtain the GO-P3-FAM nanoprobes. The fluorescence of the FAM-tagged P3 was strongly quenched by SAGO. After thrombin cleavage of the peptide substrate, the FAM was detached from the nanoprobes and following a pronounced PL signal recovery was observed. However, the presence of inhibitor for thrombin gave rise to a negligible change in the PL intensity. Furthermore, the relative PL intensity (PL/PL0 ) of the sensing system was linearly proportional to the concentration of thrombin (Supporting material, Fig. S6). Therefore, we also can design

Fig. 5. (A) Changes in the PL intensity of GO-P2-QDs nanoprobes: (a) P2-QDs, (b) P2-QDs + SA-GO, (c) P2-QDs + SA-GO + MMP-2, (d) P2-QDs + SA-GO + thrombin; (B) The calibration curve for MMP-2. Scale bars indicate the standard deviation in quadruplicate experiments.

nanoprobes by coupling GO and organic fluorescent dyes to sensitively detect protease activity. 4. Conclusions In conclusion, we report the first GO-based biosensing platform to detect protease activity. The proposed GO-based platform is easy to be assembled and has some distinguished features. First, unlike other conventional energy transfer systems, the proposed platform does not need specific couplers of donor and acceptor, because the GO can act as a common energy acceptor toward different kinds of QDs and fluorescent dyes. Thus, the proposed platform could be expanded easily to multiplexed assay of different proteases and their inhibitors. Second, GO shows exceptionally high fluorescence quenching efficiency. Therefore, we have employed the GO-based biosensing platform to sensitively and selectively detect protease down to the nM level. These detection limits are comparable to the most sensitive methods based on energy transfer, yet they can be achieved without the involvement of complicated procedures or sophisticated instrumentation. Furthermore, due to the cellular delivery ability of GO (Liu et al., 2008; Lu et al., 2010b; Zhang et al., 2010b), it is promising to apply this GO-based energy transfer

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biosensing platform to real-time monitoring of protease activity in live cells. This work is underway now in our laboratory and will be communicated in due course. Acknowledgments This work was supported by the grants from the National Natural Science Foundation of China (Nos. 20735002, 20975023), the National Basic Research Program of China (No. 2010CB732403), the Program for New Century Excellent Talents in University of China (09-0014) and the National Science Foundation of Fujian Province (2010J06003). The authors acknowledge the State Key Laboratory of Physical Chemistry of Solid Surfaces at Xiamen University for providing AFM facilities and assistance. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bios.2011.03.003. References Biju, V., Itoh, T., Baba, Y., Ishikawa, M.J., 2006. Phys. Chem. B 110, 26068–26074. Boeneman, K.B., Mei, C., Dennis, A.M., Bao, G., Deschamps, J.R., Mattoussi, H., Medintz, I.L., 2009. J. Am. Chem. Soc. 131, 3828–3829. Chang, E., Miller, J.S., Sun, J.T., Yu, W.W., Colvin, V.L., Drezek, R., West, J.L., 2005. Biochem. Biophys. Res. Commun. 334, 1317–1321. Chen, Z.Y., Berciaud, S., Nuckolls, C., Heinz, T.F., Brus, L.E., 2010. ACS Nano 4, 2964–2968. Concha, N.O., Abdel-Meguid, S.S., 2002. Curr. Med. Chem. 9, 713–726. Dong, H., Gao, W., Yan, F., Ji, H., Ju, H., 2010. Anal. Chem. 82, 5511–5517. Geim, A.K., Novoselov, K.S., 2007. Nat. Mater. 6, 183–191. Gurm, H.S., Bhatt, D.L., 2005. Am. Heart J. 149, S43–S53. He, S., Song, B., Li, D., Zhu, C., Qi, W., Wen, Y., Wang, L., Song, S., Fang, H., Fan, C., 2010. Adv. Funct. Mater. 20, 453–459. Housni, A., Cai, H., Liu, S., Pun, S.H., Narain, R., 2007. Langmuir 23, 5056–5061. Hummers, W.S., Offeman, R.E., 1958. J. Am. Chem. Soc. 80, 1339. Kainmuller, E.K., Olle, E.P., Bannwarth, W., 2005. Chem. Commun., 5459–5461. Kim, Y.P., Oh, Y.H., Oh, E., Ko, S., Han, M.K., Kim, H.S., 2008. Anal. Chem. 80, 4634–4641. Lee, S., Cha, E.J., Park, K., Lee, S.Y., Hong, J.K., Sun, I.C., Kim, S.Y., Choi, K., Kwon, I.C., Kim, K., Ahn, C.H., 2008. Angew. Chem. Int. Ed. 47, 2804–2807.

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