Enzyme-Doped Graphene Nanosheets for Enhanced Glucose

Jul 12, 2010 - Following this, graphene nanosheets were covalently conjugated to an enzyme model glucose oxidase (GOD). The presence of various ...
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Enzyme-Doped Graphene Nanosheets for Enhanced Glucose Biosensing Subbiah Alwarappan,† Chang Liu,† Ashok Kumar,‡ and Chen-Zhong Li*,† Nanobioengineering/Bioelectronics Lab, Department of Biomedical Engineering, Florida International UniVersity, 10555 West Flagler Street, Miami, Florida 33172, and Nanomaterials Research and Education Center, UniVersity of South Florida, 4202 East Fowler AVenue, Tampa, Florida 33620 ReceiVed: April 12, 2010; ReVised Manuscript ReceiVed: June 28, 2010

In this work, we report the enhanced performance of polypyrrole-graphene-glucose oxidase based enzymatic biosensors employed for in vitro electrochemical glucose detection. Initially, graphene nanosheets were chemically synthesized and surface morphologies were characterized by several physical methods. Following this, graphene nanosheets were covalently conjugated to an enzyme model glucose oxidase (GOD). The presence of various reactive functionalities such as ketonic, quinonic, and carboxylic functional groups on the edge plane of graphene easily binds with the free amine terminals of the glucose oxidase to result in a strong covalent amide linkage. Further, this covalent conjugation of the enzyme to graphene was confirmed by FT-IR measurements. Following this, the surface of a glassy carbon electrode was modified by polypyrrole. Later, the conjugated graphene-GOD were then immobilized onto the glassy carbon electrode surface already modified with polypyrrole (Ppy) and the entire assembly was employed for glucose detection. Results indicated that the electrodes immobilized with graphene conjugated to GOD exhibited a better sensitivity and response time than the electrodes immobilized with graphene alone. The observed results indicate that the 2D graphene holds great promise to be conjugated with a variety of enzymes. Besides conjugation, the entrapment of graphene-GOD within the porous structure of Ppy will hold the enzyme in a favorable position, thereby retaining the original structure and functionality of the enzymes that account for high-performance biosensing. Introduction Graphene is a two-dimensional allotrope of carbon in which the carbon atoms are closely held together in a honeycomblike lattice.1,2 In recent years, graphene is the widely researched material among several experimental and theoretical researchers due to its unique physical and chemical properties.3-7 For example, electrons in graphene obey a linear dispersion relation and resemble a massless relativistic particle, which is the basis for all of its observed peculiar electronic properties.8 Further, electrons in a graphene layer move ballistically without collision and scattering, with mobilities as high as 15 000 m2 V-1 s-1 at room temperature.9 Owing to all of these exceptional properties, graphene finds potential application in synthesizing nanocomposites and in designing microelectrical and electronic devices, such as batteries, biofuel cells, ultrasensitive sensors, and electromechanical resonators.10,11 Electrochemical biosensors based on nanomaterials, such as metal nanoparticles, carbon nanotubes, and metal oxides, have been routinely employed for various sensing applications in medical and food industries.12-15 More specifically, the usefulness of biosensors based on nanomaterials for diagnosing diabetics has been extensively studied by various research groups.16-18,42 Though the suitability of functional graphene or carbon nanotubes for ultracapacitor and energy storage applications has been studied extensively, there are very few reports available that describe the suitability of graphene for electrochemical biosensing applications, such as biological compounds and biological molecule detection.2,17-19 * To whom correspondence should be addressed. E-mail: [email protected]. Tel: 305-348-0120. Fax: 305-348-6954. † Florida International University. ‡ University of South Florida.

Electrochemical biosensors based on enzymes are suitable for a highly selective, sensitive, and rapid analysis of various biological species in vivo and in vitro. As a result, biosensors based on enzymes are extensively researched.12-19 More specifically, enzymes belonging to the family of oxidases are often employed because these enzymes initially undergo reduction. Subsequently, this reduced form of the enzyme reacts with oxygen to yield electroactive H2O2 that will undergo oxidation at the biosensor surface to yield a measurable current signal. However, the enzymes immobilized onto the electrode surface will not remain intact with the electrode surface for a longer period, and as a result, the stability and the sensitivity of the electrode decay with time. To overcome this issue, it is necessary to choose an excellent electrode material that can favorably bind with an enzyme through a strong covalent bond. From our previous studies,2 it is evident that graphene possess a number of reactive functional groups that can easily bind with the free -NH2 terminals of the enzyme or protein to result in a strong amide covalent linkage. As a result, in this work, we employ graphene as an electrode material for glucose biosensing. Moreover, during the design of biosensors, conducting polymers, such as polypyrrole (Ppy), polyaniline (PANi), polyethylene, and polystyrene,20-22 have been employed as a transduction matrix support, which enables the desired chemical reaction to occur on the electrode surface prior to electrode reaction. Of these, Ppy exhibits better electronic conductivity, environmental stability, and biocompatibility.20-22 Further, its growth can be easily controlled by electrochemical polymerization. In addition, the porous structure of Ppy will accommodate more grapheneGOD within its structure, thereby increasing the surface-tovolume ratio. As a result, the electrodes are often modified with Ppy during the development of highly sensitive electrochemical

10.1021/jp103273z  2010 American Chemical Society Published on Web 07/12/2010

Enzyme-Doped Graphene Nanosheets biosensors. For these reasons, we have employed Ppy as a matrix for the immobilization of graphene. In this paper, we describe a method in which graphene is covalently conjugated with the enzyme glucose oxidase (GOD) and immobilized onto the electrode surface. Prior to immobilizing graphene-conjugated GOD onto the electrode surface, the working electrode was electrochemically modified with Ppy, which forms a stable matrix that will encapsulate graphene-GOD on its surface. Following this, the entire electrode assembly was employed for the detection of glucose. Further, the effect of scan rate on the performance of the biosensor, its stability, and limit of detection were evaluated systematically. Experimental Section Reagents. Graphite, hydrazine hydrate, methanol, dimethyl formamide (DMF), GOD, potassium chloride, potasium ferricyanide, sodium phosphate, potassium dihydrogen phosphate, and sodium hydroxide were all purchased from Sigma-Aldrich, U.S.A., and employed without any further purification. Phosphate buffered saline (PBS) containing 50 mM Na2HPO4, 50 mM KH2PO4, and 100 mM KCl was adjusted to the desired pH by adding 0.1 M NaOH. Electrochemical Setup. Electrodeposition of Ppy and cyclic voltammetric and chronoamperometric measurements performed in this work were carried out using a CHI-630A electrochemical analyzer (CH Instruments, Inc.). All experiments were carried out with a conventional three-electrode system. The working electrode was either a bare glassy carbon electrode (GC, 3.0 mm in diameter, BAS, West Lafayette, IN) modified with Ppy or GC modified with Ppy-graphene-GOD. The reference electrode was Ag|AgCl saturated with 3.0 M KCl (Bioanalytical Systems), and all of the potential of the working electrode was measured against this reference. A platinum wire was employed as an auxiliary electrode. All the electrochemical experiments were performed at room temperature. Synthesis of Graphene. Initially, graphitic oxide (GO) was prepared from graphite powders according to a method described by Hummers and Offeman.23 GO thus obtained was then mixed with water to yield a yellow-brown suspension. This suspension was then ultrasonicated until it became clear with no particulate matter. The sonicated mixture was then treated with hydrazine hydrate, and the mixture was heated in an oil bath at about 100 °C in a water-cooled condenser for about 24 h. This resulted in the formation of GO, and it is precipitated as a black solid. This black solid was then filtered and washed with a copious volume of deionized water (5 × 100 mL) and methanol (5 × 100 mL). Following this, the precipitate was dried using a continuous N2 flow for about 10 h. Preparation of the Graphene-GOD Conjugate. About 2 mg of graphene was mixed with 2 mg mL-1 GOD in pH 7.4 PBS buffer and mixed thoroughly in a vortex mixer for 50 s. This resulted in the covalent modification of graphene with GOD, and a homogeneous solution was obtained. Electrode Modification. Prior to electrode modification, glassy carbon electrodes were polished smoothly using an emery paper. After being rinsed with water, the electrode was polished with 1.0, 0.3, and 0.05 µm alumina powders to a mirror finish, sonicated in water for 2 min, rinsed, and allowed to dry in air. Following this, the glassy carbon electrodes were modified by performing electrochemical polymerization of pyrrole (0.1 M) in NaCl (0.05 M NaClO4). The Ppy film was directly cast onto the glassy carbon electrode surface by performing 25 continuous cycles of cyclic voltammetry between the potentials of -0.8 to

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Figure 1. SEM of the (a) porous structure of electropolymerized Ppy and (b) graphene-GOD on the electrode surface.

1.3 versus Ag|AgCl in 0.1 M pyrrole/0.05 M NaClO4 (Scan rate ) 50 mV s-1). Surface Characterization. Raman spectroscopic measurements were conducted at room temperature using a Reinshaw Raman spectrometer in the back scattering mode that employs a 514.5 nm Ar+ laser operating at 50 mW. Raman spectra were collected with a 10 min exposure time with the help of a highthroughput holographic imaging spectrograph with a volume transmission grating, holographic notch filter, and thermoelectrically cooled CCD with the resolution of 4 cm-1. Following this, a field emission scanning electron microscope, JEOL JSM6330F (model), was employed for observing the porous structure of the Ppy matrix and graphene sheets. Next, transmission electron micrographs were obtained using a Philips/FEI Technai F30 model field emission gun transmission electron microscope operating at an accelerating voltage of 300 kV. Following this, the conductance of graphene was calculated as the reciprocal of resistance using a HP 4263A LCR meter. Finally, FT-IR measurements were also made using a JASCO 4100 model FT-IR instrument. Results and Discussion To understand the surface morphology of the graphene nanosheets, such as its crystalline size, types of carbon planes, conductivity, and other functional groups present in the graphene nanosheets, we have performed a series of surface probing measurements, such as scanning electron microscopy (SEM), Raman spectroscopy, transmission electron microscopy, fourpoint probe measurements, and FT-IR measurements, and the corresponding results are discussed below: Scanning Electron Microscopy. The scanning electron micrographs of Ppy (after electropolymerization) and grapheneGOD encapsulated on the electrode surface are shown in Figure 1a,b. From Figure 1a, it is evident that the electropolymerized Ppy has numerous porous structures capable of holding enzyme or substrates conjugated to enzymes intact, thereby preventing them from easily denaturing or leeching. In comparison, it was evident from Figure 1b that the surface of the electrodes was less uniform and effective in the absence of PPy. Raman Spectroscopy. Raman spectroscopy is a powerful, nondestructive tool often employed to differentiate the ordered and disordered crystalline structure of carbon. The Raman spectra of graphene were taken in the powdered form prior to immobilizing them on the electrode (Figure 2). The Raman spectra of graphene synthesized in this work exhibited a D-band around 1350 cm-1, a G-band around 1590 cm-1, and a strong 2D-band around 2700 cm-1. Of these, the D-band is disorderinduced and caused by phonon scattering at defect sites and impurities.24 The G-band is related to phonon vibrations in sp2 carbon materials,24-28 and the 2D-band is due to a double resonance process, as explained by Ferrari et al.28 Further, as

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Alwarappan et al. SCHEME 1: Representation of Graphene-GOD Entrapped within a Porous Ppy Matrix

Figure 2. Raman spectrum of graphene sheets.

Figure 3. (a) TEM, (b) HRTEM, and (c) SAED pattern of graphene sheets.

reported in the literature,26-28 the presence of a characteristic Raman signature (i.e., a single sharp 2D-band) at 2700 cm-1 with no shoulder at 2680 cm-1 confirmed that graphene employed in this work has only a few layers. Transmission Electron Microscopy. Figure 3a is a representative TEM of graphene nanosheets prepared in this work, and Figure 3b shows the layered structures of graphene sheets. From Figure 3b, it is evident that the average number of layers in our samples was in the range of 5-8 with a d value equal to 0.40 nm. The d value was observed to be the same in all areas of the graphene sample. Further, the ordered nature of graphene is evidenced by performing selected area electron diffraction (SAED) studies along the (001) axis and is shown in Figure 3c. The SAED image indicated well-resolved (100) and (110) diffraction rings and spots that confirm the formation of crystalline graphene in our work. Conductivity Measurements. Next, we have evaluated the conductivity of the graphene nanosheets synthesized in this work using a four-point probe method. The conductance of graphene employed in our work was found to be 64 mS cm-1, which is approximately 60 times more than the conductance observed for any highly conducting CNTs.18 FT-IR Measurements. The FT-IR spectrum of GOD (Figure S1a in the Supporting Information) exhibited a peak at 1490 cm-1 and another at 1600 cm-1. The peak at 1490 cm-1 is identified as the combination of N-H in-plane bending and C-N stretching of the peptide groups. The other peak at 1600 cm-1 is the result of CdO stretching vibrations of peptide linkages in the GOD backbone.26 On the other hand, the FT-IR spectrum of graphene-GOD was also performed (Figure S1b in the Supporting Information). The FT-IR spectrum of graphene-GOD also exhibited similar peaks at 1490 and 1600 cm-1 due to the combination of N-H in-plane bending and C-N stretching of the peptide groups and CdO stretching vibrations of peptide linkages in the GOD backbone, respectively. The presence of these two distinct absorption bands at 1490 and 1600 cm-1 in the graphene-GOD conjugated sample clearly indicated that GOD retained its native confirmation even after its conjugation. As a result, GOD retains its activity and graphene-GOD can be further employed for high-performance glucose biosensing.

Next, graphene conjugated to GOD was then immobilized onto the Ppy-modified electrode surface. The porous structure of Ppy is capable of encapsulating the graphene-GOD assembly without altering the structure or denaturing the activity of the GOD conjugated to graphene. Scheme 1 is a simple sketch that describes the orientation of graphene-GOD within the Ppy matrix on a glassy carbon electrode surface. Following this, the surface coverage of GOD on the Ppy-graphene electrode, the biosensing performances of the Ppy-graphene-GOD, its stability, and the effect of scan rate and concentration on the functioning of the Ppy-graphene-GOD electrode were evaluated. Surface Coverage of GOD on the Ppy-Graphene Modified Electrode. To determine the surface coverage (τ) of GOD, the effective surface area of the Ppy-graphene-modified electrode was calculated by performing cyclic voltammetry of the Ppy-graphene electrode in 1.0 mM [Fe(CN)6]3-. For a reversible process, ip ) (2.69 × 105)n3/2AD1/2ν1/2C0. For [Fe(CN)6]3-, n ) 1 and D ) 7.6 × 10-6 cm2 s-1. On the basis of this, the effective area of the Ppy-graphene electrode was found to be 0.05 cm2. Next, cyclic voltammetry of the Ppy-graphene-GOD modified electrode was performed in 1.0 mM [Fe(CN)6]3- and the charge corresponding to the reduction of [Fe(CN)6]3- was calculated. From these values, the surface coverage (τ) of GOD was found to be (2 × 10-9) ( (0.01 × 10-9) mol cm-2. The observed value of GOD coverage on graphene is almost 100 times more than the coverage of GOD on SWCNTs reported earlier.29 This observation clearly indicates that GOD has more affinity toward graphene due to its favorable chemistry with oxygen-bearing carbonyl functionalities present in graphene. Cyclic Voltammetry of Glucose at Ppy-Graphene and Ppy-Graphene-GOD Electrodes. Cyclic voltammetry of 0.1 M β-D-glucose in pH 7.4 PBS was performed using the Ppy-graphene-GOD electrode, and the corresponding voltammogram is shown in Figure 4, curve a. As a control, a similar

Figure 4. Cyclic voltammetry of 100 mM glucose in pH 7.4 PBS buffer at (a) Ppy-graphene-GOD and (b) Ppy-graphene electrodes.

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set of experiments was also performed using Ppy-graphene electrode in the absence of GOD and is shown in Figure 4, curve b. We witnessed a significant redox current induced by GOD shown in Figure 4, curve a. On the other hand, when the same set of experiments was performed using a graphene electrode (in the absence of PPy and GOD), we witnessed a mere similar voltammogram, as shown in Figure 4, curve b. On comparison, it is evident that the Ppy-graphene-GOD modified electrode exhibited an excellent redox behavior and a current density of 0.03 ( 0.003 A cm-2 (N ) 5) than the Ppy-graphene electrode 0.008 ( 0.0002 A cm-2 (N ) 5) and graphene electrode, thereby, indicating a better performance of the former electrode. The covalent modification of graphene-GOD is expected to bring the graphene surface closer to the reactive center of GOD (i.e., to the flavin adenine dinucleotide (FAD) center of GOD) and facilitate the direct electron transfer, resulting in the enhanced detection of glucose under physiological conditions, and the mechanism is as follows:

GOD (FAD) + β-D-glucose f GOD (FADH2) + gluconolactone (1) GOD (FADH2) + O2 f GOD(FAD) + H2O2

(2)

H2O2 produced during step 2 can be then detected at the electrode surface:

H2O2 f 2H+ + O2 + 2e-

(3)

However, the peak potential and the ∆Ep values of Ppy-graphene-GOD and Ppy-graphene remain unaltered, indicating that the conjugation of the enzyme has no pronounced effect on these foretold factors, confirming that the enzyme retains its activity even after conjugation with graphene. Stability of Ppy-Graphene-GOD Electrodes. To evaluate the stability or antifouling property of the Ppy-graphene-GOD electrodes, differential pulse voltammograms of these electrodes were performed in 0.1 M β-D-glucose/pH 7.4 PBS (50 scans continuously). For each electrode, the difference in peak current between the 1st scan and the 50th scan was evaluated, and from this, the percentage of fouling was evaluated. Results indicated that the Ppy-graphene-GOD electrode possesses excellent reproducibility and exhibited only a 14% ( 1% (N ) 6) decrease in the observed signal (Figure S2 in the Supporting Information). Further, when these electrodes were stored at 4 °C for 3 weeks and employed for the detection of 0.1 M β-D-glucose in pH 7.4 PBS, we witnessed only a 15% ( 1.5% current decay. From this, it is evident that the Ppy-graphene-GOD electrode exhibited a stable and steady response, thereby opening up the possibility of employing this construct to measure the plasma glucose level for the diagnosis of diabetes. Effect of Scan Rate and Concentration on the Performance of Ppy-Graphene-GOD Electrodes. Next, a scan rate study was performed using the Ppy-graphene-GOD in 0.1 M glucose/pH 7.4 PBS buffer, and the corresponding voltammograms are shown in Figure 5a. In this study, a plot of the anodic peak current density versus the square root of the scan rate was made and is shown in Figure 5b. From Figure 5b, it is evident that the electrodes exhibited a linear response within the range of 0.01-0.25 V s-1 with a correlation coefficient of 0.9988 (N ) 6), indicating a diffusion limited process at the Ppy-graphene-GOD surface. However, when the scan rate is increased

Figure 5. (a) Effect of increasing the scan rate on the performance of the Ppy-graphene-GOD electrode. (b) Calibration plot showing the linear relation between the square root of the scan rate and peak current density.

beyond 0.25 V s-1, the anodic peak current tends to remain constant, indicating sluggish electron transfer kinetics due to insufficient diffusion of glucose toward the GOD-modified graphene surface. Following this, the amperometric response of glucose at Ppy-graphene-GOD electrodes was evaluated each time following the constant addition of 25 µL of 100 mM glucose in pH 7.4 PBS buffer at an applied potential of +200 mV (Figure S3a in the Supporting Information). Results indicated that the Ppy-graphene-GOD electrodes exhibited a good linear response in the range of 2-40 µM with a correlation coefficient of 0.9976 (Figure S3b in the Supporting Information). Further, based on three times the standard deviation of the slope, a limit of detection of about 3 µM ( 0.5 µM (N ) 6) is obtained, which is almost 15 times better than the detection limit achievable using unmodified graphene electrodes. From this observation, it is evident that the proposed Ppy-graphene-GOD electrodes readily satisfy the need for the routine clinical diagnosis of diabetes by the fasting plasma glucose or the oral glucose tolerance test suggested by the American Diabetes Association.30,31 Moreover, the limit of detection for glucose achieved in this work at the Ppy-graphene-GOD electrode (3 µM ( 0.5 µM) is better than some of the other reported carbon materials based biosensors, such as the Nafion-GOD-highly ordered mesoporous carbon modified electrode (156.5 µM),32 the GOD-carbon nanotube nanoelectrode (80 µM),33 the GOD-mesocellular carbon foam-Nafion modified electrode (70 µM),34 the GOD-CNT electrode (60 µM),35 the GOD exfoliated graphite nanoplatelets-Nafion modified electrode (10 µM),36 the PDDA/GOD/PDDA/carbon nanotube modified electrode (7 µM),37 the GOD-carbon nanofiber modified electrode (2.5 µM),38 carbon paste modified with hexacyanoferrate (100 µM),39 and the Prussian Blue modified carbon nanofiber electrode (100 µM).40 In addition, the response of the Ppy-graphene-GOD electrode generally reached a steady-state level within 11 ( 1 s after the glucose addition and the amperometric response retained 80% of the initial activity after 15 h of stirring the 1.0 mM glucose solution. However, Lu et al39 described a Pt-decorated system with a sensitivity of 1 µM (1s); they have not described the stability of their sensor after several continuous cycles of voltammograms. Though Lu et al41 described a system that is slightly more sensitive than the one reported by us, we have definitely described a system in this work that is a more stable and cost-effective system than the system described elsewhere.39 This observation demonstrates that the Ppy-graphene-GOD electrode described in this work exhibited a rapid, stable, and sensitive response toward glucose detection, thereby demonstrating the capability of the proposed biosensor toward measuring the plasma glucose level (for the diagnosis of diabetes). In addition, the results are also supportive

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of the fact that the covalently modified graphene-GOD electrodes have a wide potential application in designing electrochemical sensors, particularly for biomedical applications. Conclusions In this paper, we have successfully demonstrated the possibility of conjugating GOD to graphene and employed this as a successful platform for the advanced electrochemical sensing of or detection of glucose. Results indicated that Ppy-graphene-GOD electrodes exhibited a better performance than Ppy-graphene electrodes due to the enhanced electrocatalytic activity of GOD conjugated to the graphene. Further, the covalent modification of graphene-GOD is expected to bring the reactive center of GOD (flavine adenine dinucleotide) to a closer proximity to the graphene surface and facilitate direct electron transfer, resulting in the enhanced detection of glucose under physiological conditions. Based on three times the standard deviation of the slope, Ppy-graphene-GOD electrodes exhibited an excellent sensitivity of 3 µM (applied potential ) +200 mV) for glucose detection better than those reported earlier based on other carbon materials.32-38 Further, this research has expanded the scope of graphene-GOD applications to the field of electroanalytical chemistry and provides great promise for the routine biosensing applications that may open up new challenges and approaches to explore the electrochemical behaviors of graphene or its hybrid materials for other potential utilizations. Future studies will focus on the possibility of covalent immobilization of other enzymes to graphene and employ them for biosensing numerous biologically important compounds. Acknowledgment. This current work is partially supported by Grant FA9550-08-1-0287 of the Department of Defense/ Air Force Office of Scientific Research, the Wallace H. Coulter Foundation, the FIU CTIP program, and NSF MRI 0821582 grant. The authors extend their thanks to the Advanced Materials Engineering Research Institute (AMERI) at FIU for the technical support. Supporting Information Available: The FT-IR characterization, the differential pulse voltammograms of the modified electrode, the titration measurements, and calibration of the glucose sensor are shown in the Supporting Information. This material is available free of charge via the Internet at http:// pubs.acs.org. References and Notes (1) Novoselov, K. S.; Geim, A. K.; Morozov, S. V.; Jiang, D.; Zhang, Y.; Dubonos, S.V.; Grigorieva, I. V.; Firsov, A. A. Science 2004, 306, 666. (2) Alwarappan, S.; Erdem, A.; Liu, C.; Li, C.-Z. J. Phys. Chem. C 2009, 113, 8853. (3) Novoselov, K. S.; Geim, A. K.; Morozov, S. V.; Jiang, D.; Katsnelson, M. I.; Grigorieva, I. V.; Dubonos, S. V.; Firsov, A. A. Nature 2005, 438, 197. (4) Zhang, Y. B.; Tan, Y. W.; Stormer, H. L.; Kim, P. Nature 2005, 438, 201.

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