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Biosensors and Bioelectronics 25 (2010) 1829–1833

Contents lists available at ScienceDirect

Biosensors and Bioelectronics

journa l homepage: www.e lsev ier .com/ locate /b ios

hort communication

embraneless enzymatic biofuel cells based on graphene nanosheets

hang Liua, Subbiah Alwarappana, Zhongfang Chenb, Xiangxing Konga, Chen-Zhong Lia,∗

Nanobioengineering/Bioelectronics Laboratory, Department of Biomedical Engineering, Florida International University, 10555 W Flagler Street, Miami, FL 33174, United StatesDepartment of Chemistry, Institute for Functional Nanomaterials, University of Puerto Rico, San Juan, PR 00931, United States

r t i c l e i n f o

rticle history:eceived 3 August 2009eceived in revised form 29 October 2009ccepted 11 December 2009vailable online 22 December 2009

eywords:

a b s t r a c t

The possibility of employing graphene sheets as a potential candidate for the construction of biofuelcells is reported in this paper. Initially, graphene sheets were chemically synthesized and characterizedby surface characterization techniques. Following this, graphene was employed to fabricate the anodeand cathode in the biofuel cell. The anode of the biofuel cell consists of a gold electrode on which weco-immobilized graphene – glucose oxidase using silica sol–gel matrix. Voltammetric measurementswere conducted to quantitatively evaluate the suitability of employing graphene sheets as an electrode

raphene nanosheetsiofuel cellol–gelnzymelucoseiniaturized power source

iomedical device

dopant and its performance was compared with single walled carbon nanotubes (SWCNTs). The cathodeof the biofuel cell was constructed in a similar method except that graphene was co-immobilized withbilirubin oxidase. Finally, two membraneless enzymatic biofuel cells, one using graphene sheets andthe other using SWCNTs, were constructed and their performances were compared. Upon comparison,graphene based biofuel cell exhibited a maximum power density of about 24.3 ± 4 �W (N = 3), whichis nearly two times greater than that of the SWCNTs biofuel cell, and the performance of the graphene

ys.

biofuel cell lasted for 7 da

. Introduction

In recent years, there has been considerable interest towardshe development of enzymatic biofuel cells (EBFC) as they cane employed as an in vivo power source for implantable medicalevices such as pacemakers, micro drug pumps, deep brain stim-lators, etc. (Gao et al., 2007; Barton et al., 2004; Bullen et al.,006; Kim et al., 2006; Ikeda and Kano, 2003). The most attrac-ive feature of this EBFC is that they can utilize glucose or otherarbohydrates abundantly present in the human body as a fuel. Toate, there have been considerable efforts among many researcherso fabricate practical EBFC devices out of different theoretical con-epts (Mano et al., 2002; Willner, 2002; Service, 2002; Katz et al.,005; Katz and Willner, 2003). Glucose, a major component of theuman serum, is most widely used as the fuel for theses EBFCs.owever, low power density and poor stability of the EBFC are the

wo major challenges to be rectified in the upcoming days. A non-ompartmentalized glucose|O2 biofuel cell possessing a maximumower of 4 �W cm−2 and a life time of 48 h was reported (Katz et

l., 1999), while an abiotically catalyzed glucose fuel cell exhibitedmaximum power density of 3.3 �W cm−2 with a life time of 224ays (Kerzenmachera et al., 2008). In another study, a cell lifetimef up to 45 days was reported with enzymes entrapped in a modi-

∗ Corresponding author. Tel.: +1 305 348 0120; fax: +1 305 348 6954.E-mail address: licz@fiu.edu (C.-Z. Li).

956-5663/$ – see front matter © 2009 Elsevier B.V. All rights reserved.oi:10.1016/j.bios.2009.12.012

© 2009 Elsevier B.V. All rights reserved.

fied Nafion membrane (Moore et al., 2004). Furthermore, an EBFCemploying glucose oxidase (GOx) and laccase as biocatalysts gen-erated a maximum power density of 5.49 �W cm−2 using glucoseas a fuel (Liu and Dong, 2007).

The low power density of the EBFC in comparison with conven-tional inorganic fuel cells is due to location of the active site of theenzyme buried deep under the protein shell hindering the electrontransfer pathway between the enzyme’s active site and the elec-trode (Liu and Dong, 2007). In order to overcome this issue, mostresearchers employ carbon nanotubes (CNTs) to decrease the elec-tron transfer resistance and increase the electrode surface area (Gaoet al., 2007; Li et al., 2008; Lim et al., 2007; Liu and Dong, 2007).The covalent binding of the enzyme with CNTs has resulted in afaster electron transfer rate. However, a complex chemical treat-ment process of CNTs has to be performed in order to create activebinding sites on the edge of the CNTs. Such a process hinders themass production of this electrode (Li et al., 2005; Imamura et al.,1995; Degani and Heller, 1988). Furthermore, a number of redoxmediators are widely used to boost the electron transfer rate. Theredox potential of the mediator used should lie between the redoxpotential of the enzyme and that of the electrode. As a result, theelectrons are gradually shuttled from the enzyme to the mediator

and then to the electrode (Lim et al., 2007).

Graphene, a two-dimensional (2D) nanostructure of carbon dis-covered in 2004, possesses a very large surface area of about2630 m2 g−1, which is about the size of a football stadium (Stolleret al., 2008). Further, the electrons on the graphene surface move

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allistically over the sheet without any collisions with mobili-ies as high as 10,000 cm2 V−1 s−1 at room temperature (Geim and

acDonald, 2007; Novoselov et al., 2004). In addition, grapheneas found to exhibit an excellent conductivity. In our previousork, a four-point probe method was used to measure the conduc-

ance of graphene and it was calculated to be 64 mS cm−1, which ispproximately 60 times more than that of SWCNTs (Alwarappan etl., 2009; Dai et al., 2007). It is also worth mentioning that grapheneossesses a number of surface active functional moieties such asarboxylic, ketonic, quinonic and C C. Of these, the carboxylic andetonic groups are reactive and can easily bind covalently withOx. The presence of extended C C conjugation in graphene is alsoxpected to shuttle electrons. Nonetheless, to the best of our knowl-dge, there are no reports available in literature which employsraphene as an electrode material for EBFC.

The biocompatible sol–gel encapsulation method is widely pre-erred to immobilize biomolecules such as proteins, nucleic acidsnd cells. The microstructured porous sol–gel matrix prevents theiomolecules from being denatured by pH or temperature and thusetains their long-term bioactivity (Kandimalla et al., 2006). On thether hand, the porous structure of the sol–gel matrix allows diffu-ion of the fuel towards the electrode surface for the redox reactiono occur.

In the present work, we describe an EBFC system essen-ially based on silica sol–gel immobilized graphene sheets/enzymeomposite electrodes. Further, we employ glucose oxidase (GOx)nd bilirubin oxidase (BOD) as the anodic and cathodic enzyme,espectively (Komaba et al., 2008; Kuwahara et al., 2008, 2009;

illner et al., 2009;). In addition, we employ ferrocenemethanolFM) and 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)iammonium salt (ABTS) (Lim et al., 2007) as anodic and cathodicediators, respectively. Due to the specific catalytic activity of

hese enzymes, the proton exchange membrane is eliminated toacilitate the fabrication process. After the fabrication of grapheneased membraneless EBFC, the maximum power density is foundo be 24.3 ± 4 �W (N = 3). After 7 days, the power output droppedo 50% of its original power output. For comparison, a similarBFC system was constructed using single walled carbon nan-tubes (SWCNTs) and the power output was measured by the sameethod.

. Experimental

.1. Chemicals and instruments

GOx (E.C. 1.1.3.4, from Aspergillus niger) and BOD (E.C. 1.3.3.5,rom Myrothecium verrucaria) were purchased from MP Biomedi-als (Solon, OH). FM, ABTS and glucose were all obtained from VWRnternational Inc. (West Chester, PA). The stock glucose solution

as left at room temperature for 24 h to mutarotate before use.edox mediators FM and ABTS were dissolved in 0.1 M PBS (pH.4). SWCNTs were purchased from STREM Chemicals (Newbury-ort, MA), Polyethylene Glycol (PEG) from Promega Co. (Madison,I), Tetramethoxysilane (TMOS) from Alfa Aesar (Ward Hill, MA)

nd Hydrazine Hydrate from Aldrich (St. Louis, MO) were used as-eceived.

Electrochemical measurements were performed using a CHI-30A electrochemical analyzer (CH Instruments Inc.). Cyclicoltammetric measurements were performed in a 10 mL cell withonventional three electrode set up consisting of a modified gold

lectrode as the working electrode, an Ag|AgCl reference electrode3 M KCl) and a platinum counter electrode.

Raman spectroscopic measurements were conducted at roomemperature using a Raman spectrometer in the back-scatteringonfiguration. The 514.5 nm Ar+ laser was operating at 50 mW. Fol-

tronics 25 (2010) 1829–1833

lowing this, a field emission scanning electron microscope (SEM)JEOL JSM6330F (model) was employed for observing the surface ofgraphene sheets and the porous structure of silica sol–gel matrices.

2.2. Preparation of graphene based EBFC and SWCNT based EBFC

2.2.1. Synthesis of graphene sheetsInitially, graphene was synthesized as follows: Graphitic oxide

(GO) was prepared from graphite powder as described by Hummersand Offeman (1958). GO thus obtained was then subjected to a seri-ous of chemical steps to yield graphene (Alwarappan et al., 2009).

2.2.2. Preparation of anode and cathode and fabrication of EBFCThe TMOS sol–gel employed in this study is obtained by the

hydrolysis followed by the condensation of TMOS (Lim et al., 2007).Briefly, we prepare TMOS sol as follows: 7.5 mL of TMOS was mixedwith 1.7 mL of DI water and 200 �L of 0.04 M HCl. In order to initi-ate hydrolysis, the mixture was sonicated in an ice bath for 15 minand stored at 4 ◦C for 24 h. Following this, 2 mg of graphene wasmixed with 50 �L of PEG and sonicated for 15 min to yield uniformdispersion of graphene in PEG.

The mechanism of the graphene based membraneless EBFCassembly employed in this work is shown in (Fig. 1A). The anodeand cathode of the graphene based EBFC was designed by mixing100 �L of 8 mM FM PBS solution with 2.5 mg of GOx (for cathodeGOx is replaced by BOD and FM by ABTS). The resulting mixturewas then added to the foretold dispersion of graphene in PEG andthoroughly vortexed for a minute followed by the addition of 50 �Lof TMOS sol and vortexed for another 40 s. Soon after gelation, itwas casted on a gold plate electrode (0.5 cm × 2.0 cm) into a 1 mmthin layer. Both the anode and cathode were glued onto two self-designed Teflon holders which were then clamped together andseparated using spacers in between them (Fig. 1B). Prior to use, thebiofuel cell assembly was stored in pH 7.4 PBS (0.1 M) at 4 ◦C for24 h for further gelation and mechanical stability of the sol–gel thinlayer. Moreover, the SWCNT based EBFC was designed and testedusing the same strategy. Finally, biofuel cell experiments were car-ried out in a 25 mL beaker containing 100 mM glucose PBS solutionin which the previously assembled EBFC and Ag|AgCl referenceelectrode (not shown) were immersed (Fig. 1C). After a stable opencircuit voltage (Voc) was achieved, varying external loads (500 � to500 k�) were applied across the anode and cathode and the poweroutputs were obtained.

3. Results and discussion

3.1. Surface characterization of graphene sheets and silica sol–gelmatrices

The stability of the enzymes and the diffusion of fuel towardsthe electrode surface were two crucial factors to achieve high powerdensity and long-term activity of the EBFC. The microporous struc-ture of the sol–gel can act as cages to protect immobilized enzymesfrom being denatured and leaching out while also providing bothglucose and oxygen sufficient access to the enzymes. In this case,the size of the sol–gel cages should be slightly larger than that ofthe graphene-enzyme complexes. To illustrate and compare thesizes of the sol–gel cages and graphene-enzyme complexes, SEMwas used to observe the microstructure of the sol–gel matrices sur-faces (Fig. 2A) and a single graphene sheet (Fig. 2B). The length of agraphene sheet was found to be approximately 5 �m, which is sim-

ilar to the size of a sol–gel cage. Further, the SEM micrographs of asingle graphene sheet looks like a “petal of a flower” and indicatesthat graphene possess an ordered and soft texture.

Raman spectrum of graphene exhibited a D-band peak at1363 cm−1 due to the breathing mode of �-point phonons of A1g

C. Liu et al. / Biosensors and Bioelectronics 25 (2010) 1829–1833 1831

Fig. 1. (A) Graphene based membraneless EBFC components; (B) EBFC test setup; (C) schematic configuration of the graphene based membraneless EBFC employing GOx|FMand BOD|ABTS functionalized electrodes as biocatalytic anode and cathode, respectively.

Fig. 2. (A) SEM picture of the porous structure of the silica sol–gel film; (B) SEM picture of a single graphene sheet; (C) Raman spectra of (a) graphene nanosheets and (b)SWCNTs [the inset is the RBM of SWCNTs]; (D) cyclic voltammogram of (a) graphene based anode (b) SWCNT based anode in 100 mM glucose solution and (c) graphenebased anode in PBS (PH 7.4) without glucose (scan rate: 500 mV s−1).

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ymmetry, a G-band peak at 1596 cm−1 that corresponds to therst-order scattering of the E2g phonons (Choi et al., 2006) as shown

n Fig. 2C(a). The Raman features are consistent with a previouseport (Choi et al., 2006), indicating that the resulting product athe end of our synthesis is graphene. On the other hand, the spec-rum of SWCNTs exhibited characteristic peaks centered at 220,335, and 1590 cm−1 due to the radial breathing mode, disordered-band and tangential G-band respectively as shown in Fig. 2C(b).pon calculation, the intensity ratios ID/IG of graphene and SWCNTsere found to be 0.55 and 0.35 respectively indicating graphene’s

reater sp2 character. This enhanced sp2 character of graphene isesponsible for shuttling the electrons and assisting in the bettererformance of the EBFC.

.2. Comparison of electrochemical performances of grapheneased anode and SWCNT based anode

To demonstrate the suitability of graphene as an electrode mate-ial for the construction of EBFC, initially we performed a cyclicoltammetric experiment by employing a graphene based anodes the working electrode and is shown in Fig. 2D(a). For compar-son, a similar experiment was performed using a SWCNT basednode and the corresponding voltammogram is shown in Fig. 2D(b).ll these electrochemical measurements were performed using00 mM air saturated glucose solution in PBS (PH 7.4) as an elec-rolyte at room temperature. Results indicated that the grapheneased anode exhibited almost two times higher current than theWCNT based anode. Further, another control experiment was alsoerformed using a graphene based anode in PBS solution (PH 7.4)

n the absence of glucose and is shown in Fig. 2D(c). Upon com-arison of Fig. 2D (a) and (c), the catalyzed current generated fromhe glucose can be easily observed, which indicates that grapheneas retained the bioactivity of the GOx. Moreover, a similar set ofxperiments were performed to evaluate the electrochemical per-ormances of the graphene based cathode. The catalyzed current ofhe graphene based cathode was found to be approximately twoimes higher than that of the SWCNT cathode (Fig. S1a and b), andhe bioactivity of the graphene-BOD complex was also evidenced byomparing the cyclic voltammograms with and without oxygen inhe solution (Fig. S1a and c) (supplementary materials). From theseesults, it is evident that enzymatic electrodes based on graphenexhibited a larger current density than those based on SWCNTs.he observed performance of the graphene based electrode cane attributed to its larger surface area than its counterpart (SWC-Ts). Moreover, the larger number of dislocations and electroactive

unctional groups in graphene than in SWCNTs can covalently bindith more GOx or BOD, thereby catalysing the redox reactionsore efficiently. In addition, the large amount of defects induced

y reduction process of graphene oxide also provides ideal activesites for further chemical modification. From the above compari-

ig. 3. (A) Current–voltage behaviors of (�) graphene based EBFC and (�) SWCNT based Et different cell voltage for (�) graphene based EBFC and (�) SWCNT based EBFC in 100 mMf time. The external load in the test was 15 k�. Other conditions are the same as those in

tronics 25 (2010) 1829–1833

son, it is evident that graphene is an appropriate electrode materialwhich can be employed to design the EBFC reported in this work.

3.3. Evaluation of the performance of graphene based EBFC

In literature, there are numerous reports available that describethe development of EBFC by adding mediator together with the fuel(Lim et al., 2007; Liu and Dong, 2007). However, such configura-tions are not feasible for future in vivo applications since humanplasma does not contain any redox mediator. To overcome thisdifficulty, we designed a novel, robust and membraneless EBFCin which the redox mediators (FM and ABTS) are co-immobilizedtogether with graphene and enzyme (GOx and BOD) onto the anodeand the cathode, respectively. After assembling the graphene basedmembraneless EBFC (as described in the experimental section), theassembly was placed inside 100 mM air saturated glucose solution(fuel) taken in a 25 mL beaker. The Voc and the maximum currentdensity of this graphene based EBFC were found to be 0.58 ± 0.05 V(N = 3) and 156.6 ± 25 �A cm−2 (N = 3), respectively. Similarly, theassembly of SWCNT based membraneless EBFC was tested underthe same conditions, the Voc and maximum current density werefound to be 0.39 ± 0.04 V (N = 3) and 86.8 ± 13 �A cm−2 (N = 3),respectively. The current–voltage behaviors at different externalloads of the foretold two membraneless EBFC systems are shownin Fig. 3A. Although the ideal cell voltage is determined by the dif-ference in the formal potentials of the fuel substrate and oxidizer,graphene based EBFC exhibited a 0.19 V higher Voc than its counter-part with the same fuel and oxidizer. The observed 0.19 V decreasein the Voc of the SWCNT based EBFC can be attributed to the leach-ing of the enzymes GOx and BOD out of the sol–gel. Such leaching ispossibly due to the unfavorable interaction of 3D SWCNTs with theenzymes as a result of the lack of active binding sites. In addition, theobserved decrease in the Voc of the SWCNT based EBFC is also due tothe kinetic limitation of electron transfer in the SWCNTs and higherohmic resistance (Katz et al., 1999; Alwarappan et al., 2009). Simi-larly, the observed decrease in the maximum current density of theSWCNT based EBFC is caused by the diminished electron transferrate of the SWCNTs. On the other hand, graphene possess a greaterdensity of reactive functional groups such as –C O, O–C O, C C,–O– and results in the favorable interaction with the free terminalsof GOx and BOD such as –NH2, giving rise to a strong covalent bond.As a result, the discrepancies of SWCNTs were not observed in 2Dgraphene based EBFC which exhibits a higher Voc and maximumcurrent density than its SWCNT counterpart.

Further, the power densities of these two systems were calcu-

lated and shown in Fig. 3B. The maximum power density of thegraphene based EBFC was found to be 24.3 ± 4 �W (N = 3) at 0.38 V(load 15 k�), while the power density of the SWCNT based EBFCwas found to be 7.8 ± 1.1 �W (N = 3) at 0.25 V (load 15 k�). Inter-estingly, the Voc and the maximum current density of the graphene

BFC with different external loads in 100 mM glucose solution; (B) power densitiesglucose solution; (C) stability of the assembled graphene based EBFC as a function(A) and (B).

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ased EBFC are twice that of the SWCNT based EBFC. Moreover, thealculated maximum power density of the graphene based EBFC isound to be three times that of its counterpart. In order to evaluatehe stability of the graphene based EBFC, the system was stored inH 7.4 PBS solution at 4 ◦C and tested every day with a 15 k� exter-al load. After the first 24 h, it had lost 6.2% of its original powerutput. Later, the power output was found to decay slowly andecomes 50% of its original power output after 7 days as shown inig. 3C.

In modern EBFC development, the biostability of the enzymes ishe main factor to retain the long-term performance of the mem-raneless EBFC. However, no significant breakthrough has beenchieved on the EBFC’s lifetime in our work. Thus, our futureesearch focuses on the study of different enzyme immobilizationethods to lengthen our EBFC’s lifetime (Habrioux et al., 2008). On

he other hand, the improvement of power output can be achievedy optimizing the electrode geometry. Microporous gold or carbonased electrodes can be used as current collectors instead of theold plate electrode used in this work (Imamura et al., 1995).

. Conclusion

In the present work, we successfully demonstrated the possibil-ty of employing graphene as a potential candidate for designing thenode and cathode of the membraneless EBFC. Further, our elec-rochemical results demonstrated that the catalytic efficiency ofraphene based anodes is twice that of SWCNT based anodes. Asresult, the graphene based EBFC yields a maximum power den-

ity of 24.3 ± 4 �W cm−2 (N = 3) with a lifetime of 7 days, which ishree times larger than the maximum power density generated byhe SWCNT based EBFC. Another novelty of this design is that the

ediators were entrapped within the sol–gel along with graphene-nzyme complexes, thereby offering a practical strategy for futuren vivo study of the power generation device.

cknowledgements

We would like to thank Dr. Srinivas Kulkarni and the Advancedaterial Engineering Research Institute (AMERI) at FIU for helping

s with the SEM. This current work is partially supported underrant FA9550-07-1-0344 of the Department of Defense/Air Forceffice of Scientific Research, NSF MRI 0821582 NSF Grant CHE-

tronics 25 (2010) 1829–1833 1833

0716718, the Institute for Functional Nanomaterials (NSF Grant0701525), and the US EPA Grant RD-83385601 and the 2008 FIUFaculty Research Award to Dr. Chenzhong Li.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.bios.2009.12.012.

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