glucose oxidase bioanode for biofuel cell applications

i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 9 ( 2 0 1 4 ) 7 4 1 7 e7 4 2 1

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Optimization of glassy carbon electrode based graphene/ferritin/glucose oxidase bioanode for biofuel cell applications Inamuddin a,*, Khursheed Ahmad a, Mu Naushad b a

Department of Applied Chemistry, Faculty of Engineering and Technology, Aligarh Muslim University, Aligarh 202002, India b Department of Chemistry, College of Science, Building#5, King Saud University, Riyadh, Saudi Arabia

article info

abstract

Article history:

A glassy carbon (GC)/graphene/ferritin/glucose oxidase (GOx) anode was developed by

Received 6 November 2013

using graphene/ferritin biocomposite as an electron transfer enhancer and mediator,

Received in revised form

respectively. The electrode exhibited good electrocatalytic activity towards the oxidation of

19 February 2014

glucose. The electrocatalytic oxidation of glucose using GOx modified electrode increased

Accepted 28 February 2014

with increasing the concentration of glucose upto 45 mM. The results showed that the

Available online 27 March 2014

graphene/ferritin biocomposite mediator provides enhancement in electron transfer generated at the active cites of GOx to the electrode. All electrochemical measurements

Keywords:

were carried out by cyclic voltammetry (CV) and linear sweep voltammetry (LSV). A satu-

Glassy carbon electrode

ration current density of 66.5
2 mA cm2 at scan rate 100 mV s1 for the oxidation of

Ferritin

45 mM glucose was achieved.

Glucose oxidase

Copyright ª 2014, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.

Bioanode Bifuel cells

Introduction In recent years researchers have been focused on the study and development of biofuel cells (BFC’s), and their applications in various fields such as biomedical and genetic engineering and biotechnology [1]. Biofuel cells are the cells that generate electricity by an electrochemical oxidation reaction occurring at the electrodes. Biofuel cells are totally different from conventional energy systems in terms of cost effectiveness or enabling competitiveness for the small scale power supply. The biofuel cells are generating electrical energy by using renewable substances such as glucose, ethanol etc. as fuel [2e4]. Various types of enzymes are being utilized for the

biocatalytic conversion of chemical energy associated with the fuel into electrical energy [5,6]. Contrarily, conventional energy systems are using expensive metal catalysts such as Pd, Pt, or Ru [5] having high redox potential and also are not specific for the conversion of particular fuel. These are the major obstacle encountered in conventional energy systems and can be resolved by using enzyme based BFCs. Biofuel cells have the possibilities to miniaturize into small power supply devices and also work closer to the redox potential of the enzyme [7]. These advantages of biofuel cells make them suitable for implantable devices such as pacemaker, cochlear implants, insulin pump, biosensors [8], drug delivery systems, nano biobatteries [9,10] vivo biosensor [6,11,12], and also remote sensing and communication devices in bioelectronics [13].

* Corresponding author. Tel.: þ91 571 2700920x3008. E-mail addresses: [email protected], [email protected] ( Inamuddin). http://dx.doi.org/10.1016/j.ijhydene.2014.02.171 0360-3199/Copyright ª 2014, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.

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The enzyme electrode bearing oxidase/hydrogenase enzymes on its surface have been used as an effective fuel oxidizing anode. Because of complex 3-Dimensional structure of enzymes, the electron transferring units are intensely buried inside the structures which enable a poor electrical communication between the redox active cites of the enzyme and the electrode [14,15]. To overcome this barrier, enzymes can be transformed to be conductive via chemical modification or by using redox mediators with conducting polymers [16,17]. These redox active conducting polymers with electron transfer mediators facilitate the transportation of electrons by shuttling between the enzyme active sites and the surface of electrodes. Graphene is the allotropes of carbon in which sp2 bonded carbon atoms is 2D planar in structure [18]. There is no doubt that graphene has risen as a shining star [19] due to it’s extraordinary properties such as electrical, mechanical, thermal and optical. The electrical connection of the enzyme with the GC electrode is growing area of research for the advancement of biofuel cells [20]. The electrical property of the graphene is utilized for making biofuel fuel cell anode [21]. The graphene is used to provide electrical communication between GC electrode and enzyme GOx. The immobilization of the enzyme and the use of redox mediators on the graphene modified GC electrode provide an efficient path to generate electron transfer between the enzyme and conductive surface of the electrode due to the presence of redox mediator. A number of non-biocompatible redox mediators have been widely used to boost the electron transfer rate [22,23]. However, mediator molecules must also be environmentally inert so that they can be easily disposed of (with the spent cell) and/or implanted as part of a medical device with- out harming the patient. Ferritin is a redox protein which is potentially redox active, biocompatible and environmentally inert mediator and work close to the oxidation potential of enzyme [19,24]. Therefore, ferritin is utilized as electron transfer mediator to the electron generated by the oxidation of fuel from the active cites of GOx to the GC electrode surface. In this paper, GC/graphene/ferritin/GOx anode is developed in which ferritin acted as an electron transfer mediator while graphene electrons transfer enhancer and can easily provide electrical communication between GOx and GC electrode.

Experimental

Switzerland). A conventional three electrode system including a working electrode of as prepared of glassy carbon (GC) biocomposite electrode, an Ag/AgCl reference electrode and a platinum wire counter electrode were used for all electrochemical measurements in phosphate buffer solution (PBS) at pH 7.0 in the presence of glucose and in the absence of glucose at room temperature (25
3  C) in air. The electrode was cleaned with S15H ultrasonic cleaner (Elmasonic, Germany).

Preparation of graphene dispersion Graphene dispersion was prepared by mixing 2 mg of graphene with 10 ml aqueous solution of 200 ppm sodium dodecyl sulfate (SDS), a cationic surfactant. The mixture was then ultrasonicated for 40 min. The performance of dispersant was measured by a UVeVis spectrophotometer (Perkin Elmer, USA; Model- Lambda 25) and absorption spectrum between 300 and 700 nm was recorded.

Preparation of graphene/ferritin/GOx electrodes A 3 mm diameter GC electrode was polished with 0.05 mm alumina slurry using a velvet pad. The electrode was ultrasonicated for a period of 10 min and washed with deionized water and allowed to dry at room temperature (25
3  C). After drying of the electrode, a 4.5 ml of graphene dispersion (as prepared above) was deposited on the GC electrode and is allowed to dry at a room temperature (25
3  C) for 6 h. Further, a 4.5 ml of (10 mg ml1) ferritin was cast on the dried graphene modified GC electrode. The modified electrode is allowed to dry at room temperature for 30 min. GOx (10 mg ml1) was dissolved in a phosphate buffer saline (PBS) solution pH 5.0 to maintain the activity of the enzyme during immobilization process. An 8.5 ml of GOx solution was cast on the dried graphene/ferritin biocomposite and allowed to absorb at room temperature (25
3  C) for 1 h. Finally, a 10 ml of a 2% glutaraldehyde aqueous solution was drop casted to cross link the graphene/ferritin/GOx biocomposite electrode firmly and left to dry for a period of 15 min. The electrode was also dipped in deionized water for a period of 2 min to remove any nonbounded GOx and glutaraldehyde. The electrode was allowed to dry at room temperature and was placed in refrigerator until the measurements were taken. The proposed design for the

The ferritin (10 mg ml1) from horse spleen, glutaraldehyde, purified graphene used were obtained from Sigma Chemicals, India. Phosphate buffer solutions of pH 5.0 and pH 7.0 (Otto Pvt. Ltd. India), glucose oxidase (GOx), Central Drug House (CDH), India, the anionic surfactant, sodium dodecyl sulphate (SDS) (Qualigens Fine Chemicals, India) and D-(þ)-glucose anhydrous (Himedia Laboratories Pvt. Ltd. India) were used as received.

Glassy Carbon Electrode

Chemicals and reagents

e-

-

Graphene

+

Ferritin

Oxidation of fuel

-

Glucose oxidase

Glutaraldehyde

Instrumental All electrochemical measurements were performed using a computer controlled Potentiostat/Galvanostat (302N Autolab,

Scheme 1 e The design for the layer-by-layer immobilization of biomolecules and their electrostatic interaction.

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1.00x10

-4

3.00x10

Current/A cm-2

Current /Acm-2

7.50x10 -4

1.50x10

0.00

-4

-1.50x10

5.00x10

2.50x10

0.00

-2.50x10

-4

-3.00x10

-1.0

-4

-4.50x10

-1.2

-0.8

-0.4

0.0

0.4

0.8

-0.5

1.2

Potential/V Fig. 1 e CVs of (a) Graphene, (b) Graphene/Ferritin, (c) Graphene/Ferritin/GOx confined onto GC electrodes in PBS (pH 7.0) at room temperature with a potential scan rate of 100 mV sL1. layer-by-layer immobilization of biomolecules and their electrostatic interaction is shown in Scheme 1.

Results and discussion The cyclic voltammetry of graphene/ferritin/Gox bioelectrode was carried out to check the mediated electron transfer and electrical communication of generated electrons from Gox surface to the GC electrode using ferritin as mediator. The results are shown in Fig. 1. The GC electrode containing graphene as a sample showed a little electrocatalytic oxidationreduction reaction with the generation of oxidation current of 5.5  105 mA cm2 while graphene/ferritin biocomposite electrode showed a current response of 1.2  104 mA cm2 which is relatively higher than the graphene coated modified GC electrode Fig. 1. It is understood that the ferritin enhanced

0.0

0.5

1.0

Potential/V Fig. 3 e CVs of (a) Graphene/Ferritin-GOx in 0 mM glucose in PBS (pH 7.0), (b) Graphene/Ferritin/GOx in 45 mM, glucose in PBS (pH 7.0) at scan rate of 100 mV sL1.

the dispersion of graphene thereby increasing the electrocatalytic response. The graphene/ferritin/GOx modified electrode showed a current response of 2.9  104 mA cm2 which is much higher than that of the graphene/GC and graphene/ ferritin biocomposite electrodes. This is suggesting that the electrocatalytic reactions occurring at the surface of the electrodes are redox mediated by ferritin and electrical communication is provided by the adsorbed graphene and consequently enhances the efficiency of electron transfer of the biocomposite GOx to the electrode. The effect of coating of layers of biocomposite GC electrode on the performance electro catalytic reaction and electrical communication was also studied and is shown in Fig. 2. Fig. 2(a) and (b) showed that graphene/ferritin double layered modified GC electrode generate a little lower current response than the graphene/ ferritin single layered modified GC electrode. Similarly, Fig. 2(c), and (d), also showed that the current generated by the graphene/ferritin/GOx double layer modified GC electrode is

1.00x10

3.00x10

Current/A cm2

Current/A cm-2

7.50x10 1.50x10

0.00

-1.50x10

5.00x10

2.50x10

0.00

-3.00x10

-2.50x10

-1.2

-0.8

-0.4

0.0

0.4

0.8

1.2

-1.0

-0.5

0.0

0.5

1.0

Potential/V Fig. 2 e CVs of (a) Graphene/Ferritin double layers, (b) Graphene/ferritin, (c) Graphene/Ferritin/GOx double layers, (d) Graphene/Ferritin/GOx modified GC electrodes in a PBS solution pH (7.0).

Potential/V Fig. 4 e CVs of (a) Graphene/Ferritin in 45 mM glucose, (b) Graphene/GOx in 45 mM glucose, (c) Graphene/Ferritin/ GOx in 45 mM, glucose in PBS 7.0.

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i.e. GC/graphene/ferritin electrode, the oxidation current was increased to some extent. It is indicating that ferritin acted as redox mediator. However, graphene/ferritin/GOx electrode acquired highest current. The enhancement in the current may be due to the mediated electron transfer through the ferritin from the active cites of GOx at electrode electrolyte interface as shown in Fig. 4. Fig. 5 shows the effect of five different scan rates (20, 40, 60, 80 and 100 mV s1) of cyclic voltammograms on the anodic currents of the graphene/ferritin/GOx biocomposite modified GC electrode in PBS of pH 7.0. The results showed that the anodic current is increasing linearly with the increase in scan rates. The kinetics of electron transfer on the active surface of the modified GC electrode was found to be not sufficiently fast because of the difference between anodic and cathodic peak shifts increased linearly with the increase in scan rates calculated by using the following equation

1.0x10 8.0x10

4.0x10 2.0x10 0.0 -2.0x10 -4.0x10 -1.0

-0.5

0.0

0.5

1.0

Potential/V Fig. 5 e CVs of Graphene/Ferritin/GOx modified GC electrode in 45 mM glucose in PBS (pH 7.0) at scan rate (a) 20, (b) 40, (c) 60, (d) 80 and (e) 100 mV sL1.

DE ¼ ðEpa  EpcÞ

lower than that of single layer coated graphene/ferritin/GOx bio composite modified GC electrode. The double layering of the biocomposite decreases the current which may be due to the increase in tunneling distance for electron generated from the active sites of the GOx to the GC electrode. The biocatalytic activity of GOx modified electrode (GC/ Graphene-Ferritin-GOx) for the oxidation of the glucose was studied in 45 mM of glucose in PBS of pH 7.0 and in absence of the glucose i.e. in PBS of pH 7.0 (no catalytic turnover), as shown in Fig. 3. It was observed that in presence of 45 mM glucose, the graphene/ferritin/GOx modified GC electrode generated a large oxidation current whereas in absence of glucose only the oxidation-reduction peak of the mediator was observed. The three types of electrodes, viz. graphene/ferritin, graphene/GOx and graphene/ferritin/GOx were examined using cyclic voltammetry in presence of 45 mM glucose as shown in Fig. 4. In case of GC/graphene and graphene/Gox electrodes, the current was hardly observed, whereas after adding ferritin

(A)

Ip ¼

n2 F2 I AV 4RT

where n is the number of electrons to be transferred (in the present case n ¼ 2), F is the Faraday constant (96,584 C mol1), I* is the surface concentration of the graphene/ferritin/GOx biocomposite (in mol cm2) to be determined, A is the surface area of the GC electrode (0.07 cm2), V is the scan rate (100 mV s1), R is the gas constant (8.314 J1 mol K), and T is the absolute temperature. The surface concentration of the bioelectrode confined by graphene/ferritin/GOx was found to be (11.11  1010 mol cm2). Graphene/ferritin/GOx modified GC electrode was characterized using linear sweep voltammetry (LSV) in the presence of different concentration of glucose from 5 to 45 mM in PBS of pH

(B)

5.0x10

7.50x10

e

4.0x10

6.00x10

d

-2

Current/Acm

where Epa is anodic current and Epc is cathodic current. The Brown-Anson model [25] was used to estimate the surface concentration of the graphene/ferritin/GOx biocomposite on GC electrode using cyclic voltammogram by the equation given below:

3.0x10

c

2.0x10

b 1.0x10

a

Current/Acm-2

Current/Acm-2

6.0x10

4.50x10

3.00x10

1.50x10

0.0 0.0

0.2

0.4

Potential/V

0.6

0.8

1.0

0.00 0

5

10

15

20

25

30

35

40

45

Concentration of glucose (mM)

Fig. 6 e (A) LSVs of the graphene/ferritin/GOx modified GC electrode in PBS (7.0) and different concentrations of glucose (a) 5 mM (b) 15 mM (c) 25 mM (d) 35 mM and (e) 45 mM at room temperature with a potential scan rate of 100 mV sL1, (B) The calibration curve corresponding to the electrocatalytic current against variable concentration of glucose.

i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 9 ( 2 0 1 4 ) 7 4 1 7 e7 4 2 1

7.0, and the current observed was plotted against different concentrations of glucose as shown in Fig. 6(A and B). Fig. 6(A) showed that the catalytic current of bioanode is increasing with increase in the glucose concentration. It is clear that the electrode is working for the catalytic oxidation of glucose via the electron transfer mechanism as shown in Scheme 1. The calibration drawn for the glucose concentration versus oxidation current density generated by using this bioanode is shown in Fig. 6(B). It is evident form the figure that the oxidation current density increases linearly with increase in the glucose concentration and a saturation current density of 66.5
2 mA cm1 at scan rate 100 mV s1 for the oxidation of glucose in 45 mg glucose concentration was achieved. This electrode acquire high current density than other anodes reported in literature which generate only few mA cm2 of current density [26e28].

Conclusion The ferritin was used as an electron transfer mediator between the enzyme, GOx, and a GC electrode while the electrical connection was provided by graphene. The use of ferritin as a mediator also increased the dispersion of graphene thereby increasing the current response. The graphene/ferritin/GOx anode showed good electrochemical activity for the oxidation of glucose. The graphene/ferritin/GOx biocomposites anode may have the possibility to use as anode in BFCs.

Acknowledgments The Authors are thankful to the Department of Applied Chemistry, Faculty of Engineering and Technology, Aligarh Muslim University, for providing research facilities and Council of Scientific and Industrial Research (CSIR), India, for financial support vide project No. 01 (2702)/12/EMR-II.

references

[1] Tai M, Stephanopoulos G. Engineering the push and pull of lipid biosynthesis in oleaginous yeast yarrowia lipolytica for biofuel production. Metab Eng 2013;15:1e9. [2] Topcagic S, Minteer SD. Development of a membraneless ethanol/oxygen biofuel cell. Electrochim Acta 2006;51:2168e72. [3] Lin J, Trabold TA, Walluk MRD, Smith F. Bio-fuel reformation for solid oxidefuel cell applications. Part 1: fuel vaporization and reactant mixing. Int J Hydrogen Energy 2013;38:12024e34. [4] Lin J, Babbitt CW, Trabold TA. Life cycle assessment integrated with thermodynamic analysis of bio-fuel options for solid oxide fuel cells. Bioresour Technol 2013;128:495e504. [5] Bullen RA, Arnot TC, Lakeman JB, Walsh FC. Biofuel cells and their development. Biosens Bioelectron 2006;21:2015e45. [6] Inamuddin, Shin KM, Kim SI, So I, Kim SJ. A conducting polymer/ferritin anode for biofuel cell applications. Electrochim Acta 2009;54:3979e83. [7] Willner I. Biomaterials for sensors, fuel cells, and circuitry. Science 2009;298:2407e8.

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[8] Shin HJ, Shin KM, Lee JW, Kwon CH, Lee SH, Kim SI, et al. Electrocatalytic characteristics of electrodes based on ferritin/carbon nanotube composites for biofuel cells. Sens Actuators B Chem 2011;160:384e8. [9] Kim JW, Choi SH, Lillehei PT, Chu SH, King GC, Watt GD. Electrochemically controlled reconstitution of immobilized ferritin for bioelectronic applications. J Electroanal Chem 2007;601:8e16. [10] Zhang B, Harb JN, Davis RC, Choi S, Kim J, Miller T, et al. Electron exchange between Fe(II)horse spleen ferritin and Co(III)/Mn(III) reconstituted horse spleen and Azotobacter vinelandii ferritins. Biochemistry 2006;45:5766e74. [11] Davis F, Higson SPJ. Biofuel cells e recent advances and applications. Biosens Bioelectron 2007;22:1224e35. [12] Shin KM, Lee JW, Wallace GG, Kim SJ. Electrochemical properties of SWNT/ferritin composite for bio applications. Sens Actuators B Chem 2008;133:393e7. [13] Chen T, Barton SC, Binyamin G, Gao Z, Zhang Y, Kim HH, et al. A miniature biofuell cell. J Am Chem Soc 2001;123:8630e1. [14] Yazicigil Z, Ramanavicius A. 1,10-Phenanthroline derivatives as mediators for glucose oxidase. Biosens Bioelectron 2010;26:267e70. [15] Ammam M, Fransaer J. Microbiofuel cell powered by glucose/ O2 based on electrodeposition of enzyme, conducting polymer and redox mediators. Part II: influence of the electropolymerized monomer on the output power density and stability. Electrochim Acta 2014;121:83e92. _ [16] Stolarczyk K, Kizling M, Majdecka D, Zelechowska K, Biernat JF, Rogalski J, et al. Biobatteries and biofuel cells with biphenylated carbon nanotubes. J Power Sources 2014;249:263e9. [17] Cosnier S, Goff AL, Holzinger M. Towards glucose biofuel cells implanted in human body for powering artificial organs: review. Electrochem Commun 2014;38:19e23. [18] Brownson DAC, Kampouris DK, Banks CE. An overview of graphene in energy production and storage applications. J Power Sources 2011;196:4873e85. [19] Singh V, Joung D, Zhai L, Das S, Khondaker SI, Seal S. Graphene based materials: past, present and future. Prog Mater Sci 2011;56:1178e271. [20] Heller A. Electrical connection of enzyme redox centers to electrodes. J Phys Chem 1992;96:3579e87. [21] Liu C, Alwarappan S, Chen Z, Kong X, Li CZ. Membraneless enzymatic biofuel cells based on graphene nanosheets. Biosens Bioelectron 2010;25:1829e33. [22] Fenga PG, Cardoso FP, Aquino Neto S, De Andrade AR. Multiwalled carbon nanotubes to improve ethanol/air biofuel cells. Electrochim Acta 2013;106:109e13. [23] Korani A, Salimi A. Fabrication of high performance bioanode based on fruitful association of dendrimer and carbon nanotube used for design O2/glucose membrane-less biofuel cell with improved bilirubine oxidase biocathode. Biosens Bioelectron 2013;50:186e93. [24] Agne`s C, Reuillard B, Goff AL, Holzinger M, Cosnier S. A double-walled carbon nanotube-based glucose/H2O2 biofuel cell operating under physiological conditions. Electrochem Commun 2013;34:105e8. [25] Bard AJ, Faulkner LR. Electrochemical methods: fundamentals and applications. 2nd ed. New York: Wiley; 2000. [26] Ivnitski D, Branch B, Atanassov P, Apblett C. Glucose oxidase anode for biofuel cell based on direct electron transfer. Electrochem Commun 2006;8:1204e10. [27] Wang X, Li D, Watanabe T, Shigemory Y, Mikawa T. A glucose/O2 biofuel cell using recombinant thermophilic enzymes. Int J Electrochem Sci 2012;7:1071e8. [28] Yan YM, Yehezkeli O, Willner I. Integrated, electrically contacted NAD(P)þ-dependent enzyme-carbon nanotube electrodes for biosensors and biofuel cell applications. J Chem Eur 2007;13:10168e75.