Comparative Study of Single-, Few-, and Multilayered Graphene

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Comparative Study of Single-, Few-, and Multilayered Graphene toward Enzyme Conjugation and Electrochemical Response Subbiah Alwarappan,†,‡ Sandhya Boyapalle,† Ashok Kumar,‡ Chen-Zhong Li,§ and Shyam Mohapatra*,† †

USF Nanomedicine Research Center and Division of Translational Medicine, Department of Internal Medicine, Morsani College of Medicine, University of South Florida, Tampa, Florida 33612, United States ‡ Nanomaterials Research and Education Center, University of South Florida, 4202 East Fowler Avenue, Tampa, Florida 33620-5350, United States § NanoBiosensors/Bioelectronics Laboratory, Department of Biomedical Engineering, Florida International University, Miami, Florida 33174, United States ABSTRACT: Graphene, with its exceptional mechanical, thermal, electrical, optical, and electronic properties may exist as single-, few-, or multilayered thin sheets. Though the number of layers may affect conductivity, its role toward enzyme immobilization and enzymatic biosensing applications is unknown. Herein, we report the electrochemical performance of electrodes of graphene comprising varying layers immobilized with the enzyme glucose oxidase for the detection of the model analyte glucose. It is interesting to note that these differentlayered graphene exhibit an identical electrochemical performance and sensitivity toward the detection of glucose. In addition, all of these electrodes exhibited a similar percentage of electrode fouling after 60 cycles. Following this, we have then calculated the amount of enzyme bound to the electrode surface. Results indicated that single-, few-, and multilayered graphene electrodes immobilize a similar amount of glucose oxidase. Thus, together, these results demonstrate that the number of layers stacked within the graphene structure have no significant role in the enzyme conjugation and subsequent electrochemical response during the electroanalysis.

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INTRODUCTION Graphene has been characterized as an atomically thin twodimensional sheet consisting of sp2-hybridized carbon1−5 exhibiting exceptional mechanical,1,6,7 thermal,8,9 electrical,10,11 optical,12,13 and electronic properties.14−16 Of these, the exceptional mechanical, thermal, and electrical properties were found to be the result of the long-range πconjugation.17−23 On the basis of the number of axes perpendicular to the basal plane, graphene materials are usually classified as single-layered graphene (SLG), few-layered graphene (FLG, consisting of 3−10 layers), and multilayered graphene (MLG, consisting of more than 10 layers). Interestingly, different-layered graphene were reported to vary in their electronic properties. For example, SLG is a semimetallic conductor, whereas MLG is a metallic conductor.11 Electrochemical biosensing or enzymatic biosensing requires bulk synthesis of graphene-based materials.23 Methods for preparing bulk quantities of single-layered graphene are very difficult, time-consuming, and cost-intensive. Moreover, it remains unclear whether SLG could outperform the biosensing performances of FLG and MLG upon enzyme conjugation. Despite a large number of reports on graphene-based biosensing,21−27 the role of the number of layers toward enzyme conjugation or the minimal number of layers essential © 2012 American Chemical Society

for enzyme conjugation for a sensitive electroanalysis has not been reported yet and remains elusive. To shed light on the usefulness of graphene for various biosensing applications and to understand the role of these layers toward enzyme conjugation and electroanalysis, in this work, we have employed single- (SLG), few- (FLG) (about 5− 10 layers), and multilayered graphene (MLG) (25−50) and conjugated them to the enzyme GOx and utilized them for the detection of the analyte glucose. The observed results demonstrate that the SLG, FLG, and MLG immobilized almost equal amounts of these enzymes. Moreover, all of these enzyme-conjugated different-layered graphene exhibited almost identical electrochemical response during the analyte detection, suggesting that the number of layers stacked within the graphene structure has no significant role during or toward the enzyme conjugation, thereby exhibiting identical electrochemical response during the electroanalysis. Received: November 21, 2011 Revised: February 14, 2012 Published: February 22, 2012 6556

dx.doi.org/10.1021/jp211201b | J. Phys. Chem. C 2012, 116, 6556−6559

The Journal of Physical Chemistry C

Article

Figure 1. Transmission electron micrographs showing (a) single-layered, (b) few-layered, and (c) multilayered graphene nanosheets (pseudo color enforced for a better presentation).

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EXPERIMENTAL SECTION Materials. Single-layered graphene was received as a gift from Angstron Materials, and few-layered graphene was purchased from Nanointegris. Multilayered graphene was synthesized according to the techniques reported earlier.21 Glucose and glucose oxidase were obtained from SigmaAldrich. The glucose oxidase assay kit was purchased from Invitrogen. Methods. Graphene Synthesis. MLG was synthesized according to the method reported by us earlier.21 Briefly, graphene oxide (GO) was obtained by the Hummer’s method.28 GO thus obtained was mixed with water and ultrasonicated until it became clear. The clear solution was then allowed to react with hydrazine hydrate and heated in an oil bath to 100 °C in a water-cooled condenser for a day. This resulted in the formation of a black solid that was then filtered and washed with DI water several times and dried in a N2 atmosphere for about 5 h. Electrode Modification. Glassy carbon (GC) electrodes were polished using an electrode polishing kit using using alumina powders to a mirror finish, then sonicated in water for 3 min, rinsed, and allowed to dry in air for 5 min. SLG-modified electrodes were obtained by immobilizing 3 μL of 0.05% SLG onto the GC electrodes and allowing them to remain at 4 °C for 3 h. A similar modification procedure was followed to obtain the FLG- and MLG-modified electrodes. The surface area of all of the GC electrodes employed in our work was 7.06 mm2. As a result, the surface area of the graphene-modified GC electrodes remains the same throughout all of the electrochemical measurements. Glucose oxidase (GOx) solution was prepared by dissolving a required amount of GOx in PBS buffer (pH 7.4). In our work, we employed 4 μg/μL of GOx for immobilization. The GOx-modified graphene electrodes were obtained by immobilizing an aliquot of 4 μg/μL of GOx solution onto the graphene-modified electrodes and storing them at 4 °C for 30 min. The electrodes were then washed with DI water three times to remove the nonspecifically bound GOx onto the electrode surface. The collected wash was then assayed to estimate the amount of GOx present in the wash. The difference between the original amount applied onto the electrode surface and the one present in the wash was the amount bound to the electrode surface. Glucose Oxidase Assay. The assay was performed using the Amplex Red Glucose/Glucose Oxidase Assay Kit (Invitrogen) as per the manufacturer’s specifications. Briefly, in this assay, a glucose oxidase standard curve was obtained using glucose oxidase concentrations between 0 and 10 mU/mL. The

graphene elctrodes (SLG, FLG, MLG) were spiked with an optimized amount of glucose oxidase and washed with water. The washes were collected and assayed to identify the amount of glucose oxidase in the wash. The washes were diluted (1:100), and the amount of glucose oxidase was estimated in the washes. The difference between the amount immobilized onto the electrode surface and the amount present in the wash will give the amount of glucose oxidase bound to the electrode surface. Electrochemical Setup. Electrochemical measurements performed in this work were carried out using the eDAQ electrochemical analyzer (eDAQ Pvt Ltd., Sydney, Australia). All experiments were carried out with a conventional threeelectrode system. The working electrode was a bare glassy carbon electrode (GC, 3.0 mm in diameter, BAS, West Lafayette, IN) modified with graphene-GOD. The reference electrode was Ag|AgCl saturated with 3.0 M KCl (Bioanalytical Systems, West Lafayette, IN), and all the potential of the working electrode was measured against this reference. A platinum wire was employed as an auxiliary electrode. All of the electrochemical experiments were performed at room temperature.

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RESULTS AND DISCUSSION Initially, we confirmed the number of layers present in each graphene sample by transmission electron microscopy measurements. Figure 1a−c represents the transmission electron micrographs of SLG, FLG, and MLG, respectively. Next, we have employed these electrodes for the electrochemical detection of 10 mM glucose in pH 7.4 PBS. Figure 2, curves a−c, represents the background-subtracted cyclic voltammograms corresponding to the oxidation of 10 mM glucose at SLG, FLG, and MLG immobilized with GOx. From these voltammograms, it is evident that the oxidation of glucose occurs at 200 ± 2, 202 ± 3.5, and 204 ± 1.5 mV (almost identical potentials) at the GOx-conjugated SLG, FLG, and MLG, and their ΔEp values are almost identical (∼8 mV). Furthermore, the current densities corresponding to the oxidation of glucose at the SLG, FLG, and MLG are identical. Following this, we have evaluated the signal-to-noise ratio of all of these three different graphene surfaces, and all of these electrodes exhibited a close agreement in their S/N value. Finally, the different-layered graphene electrodes immobilized with GOx were subjected to 60 continuous voltammetric scans in the model analyte and the percentage decrease in their peak current was evaluated. The SLG, FLG, and MLG exhibited a 2.8% ± 0.1% (N = 6), 3.5% ± 0.1% (N = 6), and 3.8% ± 0.2% 6557

dx.doi.org/10.1021/jp211201b | J. Phys. Chem. C 2012, 116, 6556−6559

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Figure 4. GOx assay confirming the amount of enzyme immobilized onto the different graphene layers.

the first set of experiments, equal amounts of GOx were immobilized to SLG, FLG, and MLG and the samples were incubated at 4 °C for 30 min. The amount of GOx bound to these SLG, FLG, and MLG was found out using a standard GOx assay. The details of the assay procedure are described in the Methods section. Results indicated that the amount of GOx bound to SLG, FLG, and MLG was the same (Figure 4). From all of these observations, we would like to report that the increase in the number of graphene sheets in the sample has no significant effect toward the enzyme conjugation or on the electron-transfer rate, sensitivity, signal-to-noise ratio, and stability. All of these observed results can be explained as follows: though the number of graphene layers is different in the SLG, FLG, or MLG electrodes employed for the enzyme conjugation, the extent of π−π interaction exerted by the individual hexagonal cells of the graphene planes onto the backbone of the dimeric protein GOx is the same. As a result, the increase in the number of planes has no significant role to either increase or decrease the amount of enzyme loading (i.e., enzymes being conjugated) to the graphene samples. Further, in this work, we have also investigated the effect of scan rate on the performance of these SLG, FLG, and MLG during the electroanalysis. Results indicated that all of these electrodes exhibited a very similar performance. Further, the SLG, FLG, and MLG exhibited a 2.8, 3.5, and 3.8% decrease in their signal, respectively, after 60 consecutive scans and a signal-to-noise ratio of about 3.0, 2.5, and 3.0, respectively. The foretold two results again indicated the “uselessness” of the number of graphene layers during the electroanalysis. On the basis of the above-mentioned experimental results and discussion, it is very evident that the number of graphene layers present in the graphene sample has “no significant effect” on the enzymatic

Figure 2. Background-subtracted cyclic voltammogramms of 10 mM glucose in (a) SLG−Gox, (b) FLG−Gox, and (c) MLG−Gox.

(N = 6) decrease in their response, as shown in Figure 3a−c, respectively. From the above two experiments, it is evident that the electrochemical performance of these enzyme-conjugated SLG, FLG, and MLG remained the same. This is possible only when there is no significant increase in the amount of enzyme bound to these different-layered graphene. To verify this, we have then evaluated the amount of GOx bound to SLG, FLG, and MLG by a standard assay. The details of the assay procedure were described in the Methods section. Results indicated that the amount of GOx bound to SLG, FLG, and MLG was the same (Figure 4). In this work, the surface areas of the SLG, FLG, and MLG selected for the enzyme conjugation and electroanalysis were the same. As a result, the amount of electroactive surface was kept identical during all of the electrochemical measurements and enzyme assay measurements. Following this, the graphene electrodes were immobilized with the enzyme GOx. Herein, the immobilized GOx is expected to bind to the graphene surface through a noncovalent π−π stacking. This noncovalent π−π stacking occurs via the interaction between the dimeric protein of the GOx with the hexagonal cells (carbon rings) on the graphene surface. Following this, the electrodes were then employed for the detection of glucose. The analyte of our interest in this work is glucose and is due to its importance in the medical field to identify blood sugar level. Initially, the amount of enzyme (GOx) essential for the optimum binding was found out by a trial-and-error method. In

Figure 3. Cyclic voltammogramms of 10 mM glucose at (a) SLG−Gox, (b) FLG−Gox, and (c) MLG−GoX modified electrodes (red voltammogram, 5th scan; blue voltammogram, 60th scan). 6558

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electrochemical biosensing performance of the material. As a result, it becomes very insignificant for the electrochemists to bother about the number of graphene layers present in the sample or to follow tedious and time-consuming procedures to prepare a single-layered graphene prior to any electrochemical analysis. However, single-layered graphene continues to exhibit several advantages in electronics and FET applications. In this work, we have compared the ability of the SLG, FLG, and MLG to be conjugated with an enzyme, namely, GOx. Results indicated that all of these different graphene samples though possess different numbers of planes; they have consumed or conjugated with an almost equal amount of enzymes. Further, the performance of these enzyme-conjugated MLG, FLG, and MLG confirmed that the number of planes in graphene has no marked effect on the electron-transfer rate, electrochemical sensitivity, and stability during the electroanalysis. Therefore, we would like to mention here by saying that the enzymatic electrochemical biosensing performance of SLG, FLG, and MLG is the same and is independent of the number of stacked graphene layers present in a sample. Finally, we conclude here by saying that FLG and MLG are sufficient for enzymatic biosensing applications and there is no need to specifically use SLG (which is often very difficult to synthesize in reality) for these enzymatic biosensing applications.

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AUTHOR INFORMATION

Corresponding Author

*Phone: (813) 974-8568. Fax: (813) 974-8907. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work is supported by Grants N00014-09-1-1008 (Office of Naval Research) and 1RO1CA152005-01 (National Cancer Institute) awarded to S.M. The authors would like to thank Dr. Gary Hellerman and Dr. Mahaswetha Das at the USF Nanomedicine Research Center, Morsani College of Medicine, University of South Florida, for their valuable comments to improve the quality of the manuscript. We would also like to thank Dr. Yusuf Emirov, Nanomaterials Research and Education Center (NREC) at USF, for helping us during TEM imaging.

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REFERENCES

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